Split (1)

train · ~9.11M rows (showing the first 93.5k)

identifier stringlengths 11 32	pdf_url stringlengths 17 4.62k ⌀	lang stringclasses 120 values	error stringclasses 1 value	title stringlengths 2 500 ⌀	source_name stringlengths 1 435 ⌀	publication_year float64 1.9k 2.02k	license stringclasses 3 values	word_count int64 0 1.64M	text stringlengths 1 9.75M
https://openalex.org/W2986289242	https://applbiolchem.springeropen.com/track/pdf/10.1186/s13765-019-0477-6	English	null	Simultaneous biosorption of nickel and cadmium by the brown algae Cystoseria indica characterized by isotherm and kinetic models	Applied biological chemistry	2,019	cc-by	8,543	© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Abstract Biosorption is an effective way of extracting heavy metal ions from aqueous solutions of various compositions. The brown algae, Cystoseria indica, when treated with sodium chloride, demonstrates significant capacity to extract cadmium and nickel, simultaneously, from aqueous solutions. The batch system was running over wide ranges of initial metal ion concentrations (5–150 mg/L), pH (2–6), adsorbent mass (1–4 g/L), and contact times (20–300 min), at a temperature of 25 °C. The results obtained when applying the system in these conditions exhibit higher removal capacities for cadmium than nickel. The optimal conditions of the biosorption process were found as the adsorbent mass of 1 g/L, initial concentration of adsorbates of 100 mg/L and pH of 6. The equilibrium data obtained are better described by the extended-Freundlich isotherm for nickel and cadmium. The maximum biosorption of nickel and cadmium in binary-metal-component system were 18.17 and 55.34 mg/g, respectively. The kinetic data derived from these experiments were evaluated with pseudo-first-order, pseudo-second-order and intra-particle-diffusion kinetic models. Kinetic examination of the equilibrium data derived from these models suggest that the adsorption of nickel and cadmium both follow the intra-particle-diffusion kinetic model. Keywords: Brown algae, Cadmium nickel extraction, Biosorption not biodegradable and they tend to accumulate in liv- ing organisms as well as oceans, lakes, and rivers [4]. Common methods used to separate heavy metals from industrial wastewaters include chemical precipitation, filtration, reverse osmosis, membranes, ion exchange and adsorption [5]. There are a variety of treatment processes deployed to remove heavy metal ions from the environ- ment, but these, typically, are associated with expen- sive running costs [6]. Consequently, there is an urgent requirement to develop more-effective and low-cost pro- cesses to remove heavy metals from aqueous solutions [7]. Simultaneous biosorption of nickel and cadmium by the brown algae Cystoseria indica characterized by isotherm and kinetic models Mohammad Khajavian1, David A. Wood2, Ahmad Hallajsani3 and Nasrollah Majidian1 Correspondence: mohamadkhajavian@gmail.com 1 Department of Chemical Engineering, Islamic Azad University Branch of North Tehran, Tehran, Iran Full list of author information is available at the end of the article Khajavian et al. Appl Biol Chem (2019) 62:69 https://doi.org/10.1186/s13765-019-0477-6 Batch biosorption Bioadsorption experiments were conducted over wide ranges of initial metal ion concentrations (5–150 mg/L), pH (2–6), adsorbent mass (1–4 g/L), and contact times (10–180 min), at a temperature of 25 °C, with the algae added to a series of Erlenmeyer flasks. Previous research has shown that the cadmium and nickel biosorption abil- ity was excellent using brown algae (Cystoseria indica) at the pH of 6 [10]. The flasks contained a 100-mL solution of nickel and cadmium simultaneously. The flasks were agitated at 175 rpm in an incubator shaker at 25 °C for 5 h. Algae were separated from the metal solutions by filtration. After each experiment, the remaining metal ions concentrations in solutions after the adsorption experiments were determined using atomic absorption spectroscopy (PinAcle 500, Perkin Elmer, USA). The con- centrations of the metals adsorbed (mg/L) by the treated algae was calculated using Eq. (1): The Gulf of Oman is considered as one of the main ecological capitals with the highest biological diversity. Macroalgae is found in the northern coast of the Gulf of Oman is known as the wealthiest sources of macroal- gae. Brown algae (Cystoseria indica) are vital groups of bioactive complexes in the northern coast of the Gulf of Oman. These algae (C. indica) are reputation due to their high density and richness in the region, growth in large sizes and valuable combinations such as alginic acid, iodine vitamins, and minerals. The Gulf of Oman is used for fisheries, aquaculture activities, import and export, economy, urbanization, and industries Heavy metals discharging into the marine ecosystem of the Gulf of Oman have possible to negatively affect marine organism [15]. Here, the surface of brown algae (Cystoseria indica), derived from the Oman Sea is treated with sodium chlo- ride to increase the stability of its biosorbent compo- nents. The effects of the initial concentrations of nickel and cadmium on the biosorbent performance of Cystose- ria indica are established and the optimal conditions of the biosorption process were found. Biosorption equilib- rium data are evaluated using extended-Langmuir and extended-Freundlich isotherms. The biosorption mecha- nism is also investigated in terms of its kinetic reaction characteristics at optimum concentrations over a range of contact times. Batch biosorption (1) q = V Ci −Cf m (1) where: Ci and Cf (mg/L) are the initial and final metal-ion concentrations in the solution, respectively; V is the solu- tion volume in liters; and, m is the mass of biosorbent. Unlike continuous system, the batch adsorption pro- cess is running without input and output. Methods yeast (Saccharomyces cerevisiae), algae, industrial wastes, agricultural wastes and other polysaccharide materials [3]. Biosorption technology is well known for providing practically-feasible methods for removing heavy metal from the environment [8]. Introduction Heavy metals constitute significant contaminants in the oceans, in terrestrial water courses and industrial wastewaters. Typically, pollution related to heavy met- als is associated with various industries such as batteries, mining, nuclear reactors, oil refineries, and agriculture. Nickel and cadmium are two of the most common heavy- metal contaminants, derived particularly from oil refin- ing, textiles manufacture, batteries, paints, and coatings [1]. The presence of heavy metals in the environment causes damage to many species in the biosphere, includ- ing adverse human health impacts and ecosystem dis- ruption [2, 3]. Soluble heavy metal components are Several biomass-based heavy-metal extraction meth- ods have been proposed. A large quantity of materials has been investigated as biosorbents for the removal of metals or organics extensively. The tested biosorbents can be basically classified into the following categories: bacteria (Bacillus subtilis), fungi (Rhizopus arrhizus), *Correspondence: mohamadkhajavian@gmail.com 1 Department of Chemical Engineering, Islamic Azad University Branch of North Tehran, Tehran, Iran Full list of author information is available at the end of the article Khajavian et al. Appl Biol Chem (2019) 62:69 Page 2 of 12 Khajavian et al. Appl Biol Chem (2019) 62:69 Materials and preparations A set of aqueous metal solutions displaying various con- centrations of cadmium (Cd) and Nickel (Ni) were estab- lished by diluting cadmium nitrate (Cd (NO3)2·4H2O) and nickel nitrate (Ni (NO3)2·6H2O) with distilled water. The pH of the solutions was adjusted using HCl (0.1 N) and NaOH (0.1 N) to achieve the desired values. A number of studies have demonstrated that among biosorbents, marine algae, especially the brown algae, are excellent biosorbent for heavy metals [9]. The car- boxylic group is known to be the key acidic functional group in the brown algae [10]. This is because of the carboxylic groups constitute the highest percentage of titratable sites in dried brown algae biomass. It is the existence of these active sites that provides the capacity of brown algae to adsorb heavy metals [11]. As brown algae occur in abundance in the biosphere there is the potential to exploit them as cost-effective biosorbents [12]. Despite their valuable biosorbent properties for extracting and recovering heavy metals, brown algae have the potential to cause secondary pollution [13]. Organic substances (e.g., alginate) released as the brown algae biosorption processes proceed are poten- tial pollutants that can hinder their use industrial appli- cations. Moreover, there is the potential for leaching to occur as biosorption proceeds, thereby removing key adsorptive components of brown algae causing degra- dation their biosorption capacity [14].h Cystoseria indica samples were gathered from the coastal area of the Oman Sea, Iran. The algal samples were initially washed with deionized water to remove surface contaminants and impurities (e.g. sand and salt). Clean algal samples were then dried by exposure to the sun for 24 h. Dried algal samples were then crushed and subsequently sieved to a particle size of 0.5–1.0 mm. The sieved samples were then washed with deionized water and desiccated in an oven at 70 °C for 24 h. The sam- ples were then subjected to surface alkaline treatment with sodium chloride (NaCl), which, as demonstrated by Brierley [16], disrupts the cell walls of algae potentially exposing additional functional groups to which metal ions are able to bind (Brierley protocol) [16, 17]. Biosorption isothermfl Industrial effluents typically contain a combination of sev- eral metal contaminants [18]. Consequently, it is usually Khajavian et al. Appl Biol Chem (2019) 62:69 Khajavian et al. Appl Biol Chem (2019) 62:69 Khajavian et al. Appl Biol Chem (2019) 62:69 Page 3 of 12 (5) qe2 = KF2C 1 n2 +X2 e2 Cx2 e2 + y2Cz2 e1 necessary to remove more than one metal contaminant during bioadsorption. It is for this reason that we investi- gate here the efficiency of Cystoseria indica in simultane- ously removing for nickel and cadmium from aqueous solutions. Published experiments suggest that many adsor- bents are less effective in systems contaminated with multiple-metal components than those contaminated with single-metal components [19]. This effect is likely to be caused by metal ions hindering each other during the adsorption process in multi-component systems. The mathematical models considered to best describe biosorp- tion phenomena, are the Langmuir and Freundlich iso- therms [20, 21]. The Langmuir isotherm is based on three basic theoretical assumptions: (5) where: x1, y1, z1, x2, y2, z2 are the Freundlich isotherm constants; Ce1 and Ce2 are the unadsorbed concentra- tions of nickel and cadmium, respectively; the unit of Ce1 and Ce2 are mg/L. qe1 and qe2 are the equilibrium adsorp- tion values of nickel and cadmium, respectively; qe1 and qe2 are equal to mg/L. Suffix 1 indicates nickel and suf- fix 2 signifies cadmium; and, KF1, KF2, n1 and n2 are the Freundlich isotherm constants [25]. These are obtained from the equilibrium data of the single metal compo- nents and are listed in Table 1 [26, 27]. These data come from experiments in which nickel and cadmium were adsorbed by the Cystoseria indica separately. All the model parameters were evaluated by non-linear regres- sion using MATLAB software. Coefficient of determina- tion (R2) is used to measure the goodness of fit between the measured data and the theoretical models. • Adsorption is monolayer; • All the active sites are equivalent and the adsorption process is uniform; and, • The adsorption of a molecule by a free site does not depend on the occupied neighboring sites. The coefficient of determination (R2) is defined by Eq. (6): The Langmuir equation is applicable to homogenous adsorption while the Freundlich equation is employed to describe heterogeneous systems and reversible adsorp- tion and is not restricted to the formation of monolayers. Biosorption isothermfl The mathematic expression of the extended-Langmuir iso- therm can be expressed by Eqs. (2) and (3): (6) R2 = 1 − m i=1(qexp i −qcal i )2 m i=1(qexp ave −qcal i )2 (6) A higher R2 (i.e. closer to 1.0) indicates a better fitting curve. (2) qe1=qm,1 KL1Ce1 1 + KL1Ce1 + KL2Ce2 (3) qe2=qm,2 KL2Ce2 1 + KL1Ce1 + KL2Ce2 (2) Biosorption kinetics Experimental observations indicate that the adsorp- tion processes for both cadmium and nickel using Cys- toseria indica reached equilibrium after approximately 180 min of contact. For batch systems, the adsorption process kinetics can be described by a number of mod- els [28, 29]. These models include: pseudo-first-order (Eq. 7), pseudo-second-order (Eq. 8), and intra-particle diffusion (Eq. 9) [30]. The linearized pseudo-first-order, (3) where: KL1, KL2, qm1, qm2 are the Langmuir isotherm con- stants; Ce1 (mg/L) and Ce2 (mg/L) are the unadsorbed concentration of nickel and cadmium, respectively; and qe1 (mg/L) and qe2 (mg/L) are the equilibrium adsorption values for nickel and cadmium, respectively [22, 23]. On the other hand, for multi-component systems, exper- imental equilibrium data were difficult to obtain. Therefore, several methods were proposed to estimate multi-compo- nent adsorption equilibrium in a mixture on the basis of single-component adsorption isotherms [24]. For exam- ple, Freundlich extended model was proposed to predict binary component adsorption systems using known single adsorption isotherms the Freundlich isotherm assumes that the stronger binding sites are occupied first. The math- ematic expression of extended-Freundlich isotherm can be expressed as Eqs. (4) and (5): Table 1 Optimized adsorption isotherm parameters of nickel and cadmium calculated for single-component system with algae Cystoseria indica at 25 °C Isotherms Parameters Nickel Cadmium Langmuir qm (mg/g) 38.601 70.887 KL (L/mg) 0.111 0.287 R2 0.980 0.90 Freundlich Kf (mg/g) (L/mg) 17.142 21.302 n 2.739 1.277 R2 0.980 0.980 Table 1 Optimized adsorption isotherm parameters of nickel and cadmium calculated for single-component system with algae Cystoseria indica at 25 °C (4) qe1 = KF1C 1 n1 +X1 e1 Cx1 e1 + y1Cz1 e2 qe1 = KF1C 1 n1 +X1 e1 Cx1 e1 + y1Cz1 e2 (4) Khajavian et al. Appl Biol Chem (2019) 62:69 Page 4 of 12 pseudo-second-order, and intra-particle diffusion kinetic models are defined by Eqs. (7), (8) and (9), respectively [30, 31]: Oxygen-containing acidic surface functional groups (hydroxyl, phenolic, aldehydic, ketonic, and carboxylic) on the surface of biochar, which are active binding sites for the adsorption process, were derived from macromol- ecules in macroalgae [32]. The FTIR spectra of C. Indica (Fig. 2) showed the intense bonds at ~ 3400 1/cm (O–H stretching vibration), which can be allocated to hydroxyl functional groups [32]. Biosorption kinetics The absorbance bonds at ~ 2900 1/cm, ~ 1640 1/cm, and ~ 1100 1/cm were assigned to vibration modes of C–H, aromatic CC and, COC= bonds, respectively [33, 34]. Furthermore, the intense peak at 1037 1/cm (Si-OS-i stretching vibration) is due to high silica content of the biomasses and consequently the ash content of the biochars [35]. The following indi- ces obtained from the integration of absorbance peak regions were utilized to evaluate the chemical proper- ties of biochars, aromaticity, the degree of the aromatic ring, aliphatic chain length, oxygen function groups, and ‘C’ factor. The ratio of the C=O to CC= stretching group (‘C’ factor) was used as a criterion for describing oxygen- containing groups in samples [33, 35]. (7) dq dt = K1 qe −qt (8) dqt dt = K2(qe −qt)2 (9) qt = Kpt 1 2 + C (7) dq dt = K1 qe −qt (7) (8) (9) qt = Kpt 1 2 + C qt = Kpt 1 2 + C (9) where: qt (mg/L) and qe (mg/L) are the metal adsorbed after specific exposure times and at equilibrium conditions, respectively; and K1 (min−1) and K2 (g/mg min) are the first-order reaction rate equilibrium constant and the second-order reaction rate equilibrium constant, respec- tively; and, in Eq. 9, C (mg/g) is adsorption constant and Kp (mg/g min0.5) is the rate of intra-particle diffusion. Characterization of C. indica SEM micrographs were obtained using a scanning elec- tron microscope (Hitachi SU-70, Japan). Figure 1 shows SEM images of the C. indica at the different magnifica- tions. C. indica had a honeycomb structure. Pores greater than 200 mm were observed at surface of C. indica. SEM image of C. indica showed no uniform pore distribution and high roughness. One of the most important factors affecting the biosorp- tion of metal ions is acidity of solution. Solution pH affects the cell wall metal binding sites and the metal ion chemistry in water. In the present study, the effect of pH and biosorbent pretreatment on two metals biosorp- tion was understandable (Fig. 3). At low pH, metal cati- ons and protons contested for binding sites, resulting in Fig. 1 SEM Image of C. indica with different magnifications, 20.0 k.x (a), 25.0 k.x (b) Khajavian et al. Appl Biol Chem (2019) 62:69 Page 5 of 12 low adsorption of metal ions. Moreover, the number of positively charged surface sites increased, which did not favor the adsorption of positively charged metal cati- ons due to electrostatic repulsion. With an increase in pH, the number of negatively charged active sites also increased, which would attract more positively charged metal ions for binding. The pH of 6 is the best pH in which both metals reach their highest adsorption capac- ity. Consequently, this pH (6) was selected for further investigations. Fig. 2 ATR-FTIR of C. indica 0 5 10 15 20 25 30 35 40 45 50 55 0 1 2 3 4 5 6 7 qe (mg /g ) pH Nickel Cadmium Fig. 3 Effect of pH on Ni (II) and Ca(II) biosorption onto the modified C. indica biosorbent Fig. 2 ATR-FTIR of C. indica 0 5 10 15 20 25 30 35 40 45 50 55 0 1 2 3 4 5 6 7 qe (mg /g ) pH Nickel Cadmium Fi 3 Eff f H Ni (II) d C (II) bi i h difi d C i di bi b Fig. 3 Effect of pH on Ni (II) and Ca(II) biosorption onto the modified C. indica biosorbent increased, which would attract more positively charged metal ions for binding. The pH of 6 is the best pH in which both metals reach their highest adsorption capac- ity. Consequently, this pH (6) was selected for further investigations. Characterization of C. indica increased, which would attract more positively charged metal ions for binding. The pH of 6 is the best pH in which both metals reach their highest adsorption capac- ity. Consequently, this pH (6) was selected for further investigations. low adsorption of metal ions. Moreover, the number of positively charged surface sites increased, which did not favor the adsorption of positively charged metal cati- ons due to electrostatic repulsion. With an increase in pH, the number of negatively charged active sites also Khajavian et al. Appl Biol Chem (2019) 62:69 Page 6 of 12 Effect of temperatureh The biosorption studies were carried out for both heavy metal at four different temperatures 20, 25, 30 and 35 °C in binary component systems, and the obtained results were presented in Fig. 6. It can be observed that the biosorption capacity of cadmium increased with an increase of temperature from 20 to 25 °C, over 180 min of contact time. Also, the biosorption capacity of cadmium was constant after 25 °C. On the other hand the biosorp- tion capacity of nickel was constant with increasing of temperature. Effect of biosorbent dosageh 180 min was selected for all the equilibrium tests. Yinghui and Fang studied the biosorption of Cu(II), Cd(II), Ni(II) and Zn(II) onto pretreated biomass of brown algae [34] and Ibrahim, investigate the biosorption of Co(II), Cd(II), Cr(II) and Pb(II) by red macro-algae [35] and reported similar contact time behavior for Cd(II) and Ni(II) metal ions as observed in current study. The biosorbent dosage is an important factor because it can determine the capacity of biosorbent for a given ini- tial metal ion concentration. The effect of biomass dosage in the range of 1–4 g/L on the Ni and Ca uptake by NaCl- pretreated C. indica in the initial metal concentration of 100 mg/L and temperature of 25 °C at pH 6 is shown in Fig. 4. According to this Fig. 4, the biosorption capacity of Ni(II) and Ca(II) from an aqueous solution decreased with the increase of the biosorbent dosage, because some of the active sites stayed unsaturated during the biosorp- tion process while the number of sites available on the NaCl-pretreated C. indica biomass increased by increas- ing the biosorbent dosage. Consequently, the dosage of 1 g/L was selected for further investigations. Effect of contact timeh The rate of adsorption is of high importance in modeling and designing the adsorption process in industry. There- fore, the effect of contact time on nickel and cadmium sorption onto the C. indica is revealed in (Fig. 5) in the initial concentration of 100 mg/L and 25 °C at pH 6 with the contact time varies from 10 to 180 min. According to Fig. 3, biosorption capacity increased with increasing contact time. More than 95% of the total adsorption of nickel and cadmium occurs within the first 60 min. This behavior might be due to the fact that initially all active sites on the adsorbents surface are vacant and the solu- tion concentration is high. After that period, few surface active sites will be available, so only a very low increase in the metal uptake is observed. Therefore, a contact time of Effect of initial concentration Published research [27, 28, 36] indicates that nickel and cadmium achieve their highest capacities of algal adsorp- tion in aqueous solutions with concentrations ranging from 5 to 130 mg/L when they are in the two-compo- nent system. The effects of interfering metal ions on the adsorption of cadmium and nickel ions by Cystoseria indica are illustrated in Figs. 7 and 8. Tests were conducted to compare the adsorption per- formance of Cystoseria indica with respect to nickel and cadmium. An initial concentration of nickel of 60 mg/L 0 5 10 15 20 25 30 35 40 45 50 55 60 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 qe (mg /g ) Biomass Dosage (g/L) Nickel Cadmium Fig. 4 Effect of biomass dosage on biosorption of Ni(II) and Ca(II) onto NaCl-pretreated C. indica biomass Fig. 4 Effect of biomass dosage on biosorption of Ni(II) and Ca(II) onto NaCl-pretreated C. indica biomass Khajavian et al. Appl Biol Chem (2019) 62:69 Page 7 of 12 was set in three containers. In one of those containers, the initial concentration of cadmium was set at 30 mg/L, in the second container it was set at 60 mg/L, and in the third container it was set at 130 mg/L. The meas- ured adsorption of nickel by Cystoseria indica was 17.5, 9 6 and 5 mg/L, respectively in the first, second and concentration of cadmium of 60 mg/L was set in another three containers. In one of those containers the initial concentration of nickel was set at 30 mg/L, in the second container was set at 60 mg/L, and in the third container it was set at 130 mg/L. The measured adsorption of cad- mium by Cystoseria indica was 35, 20 6 and 10 1 mg/L, 0 5 10 15 20 25 30 35 40 45 50 55 0 20 40 60 80 100 120 140 160 180 200 qt (mg /g ) Contact me (min) Nickel Cadmium Fig. 5 Effect of contact time on biosorption of Ni (II) and Ca(II) onto C. indica 0 5 10 15 20 25 30 35 40 45 50 55 60 15 20 25 30 35 40 qe (mg /g ) Temperature (°C) Nickel Cadmium Fig. Effect of initial concentration 6 Effect of temperature on bio sorption of Ni (II) and Ca(II) in binary component systems 0 5 10 15 20 25 30 35 40 45 50 55 0 20 40 60 80 100 120 140 160 180 200 qt (mg /g ) Contact me (min) Nickel Cadmium Fi 5 Eff t f t t ti bi ti f Ni (II) d C (II) t C i di Fig. 5 Effect of contact time on biosorption of Ni (II) and Ca(II) onto C. indica 0 5 10 15 20 25 30 35 40 45 50 55 60 15 20 25 30 35 40 qe (mg /g ) Temperature (°C) Nickel Cadmium Fig. 6 Effect of temperature on bio sorption of Ni (II) and Ca(II) in binary component systems 0 5 10 15 20 25 30 35 40 45 50 55 60 15 20 25 30 35 40 qe (mg /g ) Temperature (°C) Nickel Cadmium Fig. 6 Effect of temperature on bio sorption of Ni (II) and Ca(II) in binary component systems Fig. 6 Effect of temperature on bio sorption of Ni (II) and Ca(II) in binary component systems concentration of cadmium of 60 mg/L was set in another three containers. In one of those containers the initial concentration of nickel was set at 30 mg/L, in the second container was set at 60 mg/L, and in the third container it was set at 130 mg/L. The measured adsorption of cad- mium by Cystoseria indica was 35, 20.6 and 10.1 mg/L, respectively in the first, second and third container. These was set in three containers. In one of those containers, the initial concentration of cadmium was set at 30 mg/L, in the second container it was set at 60 mg/L, and in the third container it was set at 130 mg/L. The meas- ured adsorption of nickel by Cystoseria indica was 17.5, 9.6 and 5 mg/L, respectively in the first, second and third containers. For comparison purposes, an initial Khajavian et al. Appl Biol Chem (2019) 62:69 Page 8 of 12 observations suggest that Cystoseria indica clearly has a tendency to adsorb significantly more cadmium than nickel. Adsorption is believed to occur at several func- tional groups within algae, including carboxyl, sulfonic acid and alginate [37]. Effect of initial concentration There may also be other func- tional groups in Cystoseria indica that are involved in the synergistic effect in the binary system by which cadmium is more successful adsorbed at active sites by Cystoseria indica. The alginic acid component, present in alginate within the cell walls of Cystoseria indica and other brown algae, has been suggested as the reason for the higher cadmium adsorption potential [29 38] Metallic behav 20 30 40 50 60 70 80 90 100 Initial concentration of Ni 0 5 10 15 20 25 qe(mg Ni/gr biosorbent) 30ppm Cd 60ppm Cd 130ppm Cd Fig. 7 The effect of interfering nickel ions on cadmium adsorption by Cystoseria indica 10 20 30 40 50 60 70 80 90 100 Initial concentration of Cd 0 10 20 30 40 50 qe(mg Cd/gr biosorbent) 30ppm Ni 60ppm Ni 130ppm Ni Fig. 8 The effect of interfering cadmium ions on nickel adsorption by Cystoseria indica 20 30 40 50 60 70 80 90 100 Initial concentration of Ni 0 5 10 15 20 25 qe(mg Ni/gr biosorbent) 30ppm Cd 60ppm Cd 130ppm Cd Fi 7 Th ff f i f i i k l i d i d i b C i i di Fig. 7 The effect of interfering nickel ions on cadmium adsorption by Cystoseria indica 10 20 30 40 50 60 70 80 90 100 Initial concentration of Cd 0 10 20 30 40 50 qe(mg Cd/gr biosorbent) 30ppm Ni 60ppm Ni 130ppm Ni Fig. 8 The effect of interfering cadmium ions on nickel adsorption by Cystoseria indica 10 20 30 40 50 60 70 80 90 100 Initial concentration of Cd 0 10 20 30 40 50 qe(mg Cd/gr biosorbent) 30ppm Ni 60ppm Ni 130ppm Ni Fi 8 Th ff t f i t f i d i i i k l d ti b C t i i di Fig. 8 The effect of interfering cadmium ions on nickel adsorption by Cystoseria indica synergistic effect in the binary system by which cadmium is more successful adsorbed at active sites by Cystoseria indica. The alginic acid component, present in alginate within the cell walls of Cystoseria indica and other brown algae, has been suggested as the reason for the higher cadmium adsorption potential [29, 38]. Effect of initial concentration On the other hand, the biosor Table 2 Optimum isotherm parameters of binary-m component systems for adsorption of nickel and cad using Cystoseria indica treated with NaCl (pH = 6, co time 4 h, temperature 25 °C, impeller speed 175 rpm) Isotherms Parameters Nickel Cadm Extended-Langmuir qm (mg/g) 18.170 55.34 KL (L/mg) 0.166 0.20 R2 0.920 0.90 Extended-Freundlich x 0.004 0.02 y 1.090 2.88 z 0.010 0.19 R2 0.940 0.97 Fig. 9 The molecular structure of alginic acid including glucuron equilibrium data are better described (i.e., higher R2) using the extended-Freundlich isotherm for cadmium. depend on the physical and chemical characteristics of the adsorbent that, in turn, affects the equilibrium behav- ior of the biosorption processes. The number and type of ions, the concentration of each component ion, pH, and the isotherm models considered will determine the equi- librium constants [39–41]. Alginate is considered to play a pivotal role in the abil- ity of different brown algae species to adsorb heavy metal ions [42]. Alginate has a non-uniform macromolecular structure providing it with the ability to form a selec- tive bond with cadmium ions. Because of its orientation, shape and the space between dents, glucuronic acid cre- ate better complex with cadmium than mannuronic acid in terms of allowing ions to permeate in the structure [31]. The molecular structure of alginate is illustrated in Fig. 9.i Adsorption isothermsh The isotherm constants and coefficient of determination (R2) calculated from the tests for the Cystoseria indica tests with the extended-Langmuir and extended-Fre- undlich isotherms are listed in Table 2. The optimized conditions were obtained from above section following: Initial concentration 100 mg/L, pH 6, biosorbent dos- age 1 g/L. Also, all experiments were lasted 180 min at 25 °C. The appropriate parameter values for the isotherm models considered for Cystoseria indica adsorption of nickel and cadmium are derived from the experimental data by maximizing the objective function (i.e., R2). These values are listed in Table 1 for single-metal-component systems and Table 2 for binary-metal-component system. For the nickel and cadmium binary system, the biosorp- tion equilibrium data for Cystoseria indica are better described (i.e., higher R2) using the extended- Freundlich isotherm for nickel. On the other hand, the biosorption Figures 10, 11, 12 and 13 show curve-fitting for the extended-Freundlich and extended-Langmuir iso- therms based on the experimental data. The extended- Freundlich isotherm data demonstrates positive correlations between experimental data (q exp) and cal- culated-adsorption (q cal) for both cadmium and nickel. The extended-Langmuir isotherm data shows low-confi- dent correlation between experimental data (q exp) and calculated-adsorption (q cal) for nickel and cadmium data because of the low value of R2. It is apparent that 0 5 10 15 20 25 30 0 5 10 15 20 25 30 qe Ni calc (mg/L) qe Ni exp (mg/L) qcal=qexp Fig. 10 Parity plot of calculated versus experimental values for Extended-Langmuir isotherm of nickel in the adsorption process of Cystoseria indica at different concentrations of the metal ions 0 5 10 15 20 25 30 0 5 10 15 20 25 30 qe Ni calc (mg/L) qe Ni exp (mg/L) qcal=qexp Table 2 Optimum isotherm parameters of binary-metal- component systems for adsorption of nickel and cadmium using Cystoseria indica treated with NaCl (pH = 6, contact time 4 h, temperature 25 °C, impeller speed 175 rpm) Isotherms Parameters Nickel Cadmium Extended-Langmuir qm (mg/g) 18.170 55.341 KL (L/mg) 0.166 0.203 R2 0.920 0.90 Extended-Freundlich x 0.004 0.028 y 1.090 2.880 z 0.010 0.190 R2 0.940 0.970 Table 2 Optimum isotherm parameters of binary-metal- component systems for adsorption of nickel and cadmium using Cystoseria indica treated with NaCl (pH = 6, contact time 4 h, temperature 25 °C, impeller speed 175 rpm) Fig. Effect of initial concentration Metallic behav- ior in multi-component systems is believed to strongly synergistic effect in the binary system by which cadmium is more successful adsorbed at active sites by Cystoseria indica. The alginic acid component, present in alginate within the cell walls of Cystoseria indica and other brown algae, has been suggested as the reason for the higher cadmium adsorption potential [29, 38]. Metallic behav- ior in multi-component systems is believed to strongly observations suggest that Cystoseria indica clearly has a tendency to adsorb significantly more cadmium than nickel. Adsorption is believed to occur at several func- tional groups within algae, including carboxyl, sulfonic acid and alginate [37]. There may also be other func- tional groups in Cystoseria indica that are involved in the adsorption of both metal ions. Figures 7 and 8 display the Khajavian et al. Appl Biol Chem (2019) 62:69 Page 9 of 12 Khajavian et al. Appl Biol Chem Khajavian et al. Appl Biol Chem (2019) 62:69 depend on the physical and chemical characterist the adsorbent that, in turn, affects the equilibrium b ior of the biosorption processes. The number and ty ions, the concentration of each component ion, pH the isotherm models considered will determine the librium constants [39–41]. Adsorption isotherms The isotherm constants and coefficient of determin (R2) calculated from the tests for the Cystoseria i tests with the extended-Langmuir and extended undlich isotherms are listed in Table 2. The optim conditions were obtained from above section follo Initial concentration 100 mg/L, pH 6, biosorbent age 1 g/L. Also, all experiments were lasted 180 m 25 °C. The appropriate parameter values for the isot models considered for Cystoseria indica adsorpti nickel and cadmium are derived from the experim data by maximizing the objective function (i.e., R2). h values are listed in Table 1 for single-metal-comp systems and Table 2 for binary-metal-component sy For the nickel and cadmium binary system, the bio tion equilibrium data for Cystoseria indica are b described (i.e., higher R2) using the extended- Freun isotherm for nickel. Adsorption isothermsh 13 Parity plot of calculated versus experimental values for Extended-Freundlich isotherm of cadmium in the adsorption process of Cystoseria indica at different concentrations of the metal ions 0 5 10 15 20 25 30 0 5 10 15 20 25 30 qe Ni calc (mg/L) qe Ni exp (mg/L) qcal=qexp Fig. 12 Parity plot of calculated versus experimental values for Extended-Freundlich isotherm of nickel in the adsorption process of Cystoseria indica at different concentrations of the metal ions 0 5 10 15 20 25 30 0 5 10 15 20 25 30 qe Ni calc (mg/L) qe Ni exp (mg/L) qcal=qexp Fig. 12 Parity plot of calculated versus experimental values for Extended-Freundlich isotherm of nickel in the adsorption process of Cystoseria indica at different concentrations of the metal ions the extended-Freundlich isotherm was a better fit than the extended-Langmuir isotherm equation for nickel and cadmium sorption according to the values of R2 and, the highest value of R2 was 0.97 for cadmium in extended- Freundlich isotherm. Also, the highest value of R2 was 0.94 for nickel in extended-Freundlich isotherm. Two best values suggested that the extended-Freundlich is the best model for nickel and cadmium sorption. It indicates that the sorption of nickel and cadmium ions are similar a multilayer sorption. the extended-Freundlich isotherm was a better fit than the extended-Langmuir isotherm equation for nickel and cadmium sorption according to the values of R2 and, the highest value of R2 was 0.97 for cadmium in extended- Freundlich isotherm. Also, the highest value of R2 was 0.94 for nickel in extended-Freundlich isotherm. Two best values suggested that the extended-Freundlich is the best model for nickel and cadmium sorption. It indicates that the sorption of nickel and cadmium ions are similar a multilayer sorption. Fig. 12 Parity plot of calculated versus experimental values for Extended-Freundlich isotherm of nickel in the adsorption process of Cystoseria indica at different concentrations of the metal ions Adsorption isothermsh 10 Parity plot of calculated versus experimental values for Extended-Langmuir isotherm of nickel in the adsorption process of Cystoseria indica at different concentrations of the metal ions Fig. 10 Parity plot of calculated versus experimental values for Extended-Langmuir isotherm of nickel in the adsorption process of Cystoseria indica at different concentrations of the metal ions Fig. 9 The molecular structure of alginic acid including glucuronic acid (G) mannuronic acid (M) as it exists within alginate [43] Fig. 9 The molecular structure of alginic acid including glucuronic acid (G) mannuronic acid (M) as it exists within alginate [43] g. 9 The molecular structure of alginic acid including glucuronic acid (G) mannuronic acid (M) as it exists within alginate [43 Khajavian et al. Appl Biol Chem (2019) 62:69 Khajavian et al. Appl Biol Chem Page 10 of 12 Table 3 Comparison between adsorption rate constant, the estimated qe and the coefficients of determination associated with pseudo-first-order, pseudo-second- order and intra-particle diffusion kinetic models applied to nickel and cadmium adsorption by Cystoseria indica at 25 °C 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 qe Cd calc (mg/L) qe Cd exp (mg/L) qcal=qexp Kinetic models Parameters Nickel Cadmium Kinetic models Parameters Nickel Cadmium Pseudo-first-order qe (mg/g) 18.70 39.201 k1 (1/min) 0.112 0.212 R2 0.831 0.741 Pseudo-second-order qe (mg/g) 24.592 45.272 k2 (g/mg.min) 0.003 0.002 R2 0.864 0.811 Intra-particle diffusion C (mg/g) 15.542 32.031 kid (mg/g.min0.5) 1.726 2.942 R2 0.90 0.930 Fig. 11 Parity plot of calculated versus experimental values for Extended-Langmuir isotherm of cadmium in the adsorption process of Cystoseria indica at different concentrations of the metal ions Fig. 11 Parity plot of calculated versus experimental values for Extended-Langmuir isotherm of cadmium in the adsorption process of Cystoseria indica at different concentrations of the metal ions 0 5 10 15 20 25 30 0 5 10 15 20 25 30 qe Ni calc (mg/L) qe Ni exp (mg/L) qcal=qexp Fig. 12 Parity plot of calculated versus experimental values for Extended-Freundlich isotherm of nickel in the adsorption process of Cystoseria indica at different concentrations of the metal ions 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 qe Cd calc (mg/L) qe Cd exp (mg/L) qcal=qexp Fig. References 1. Kim DJ, Shin HJ, Ahn BK, Lee Jh (2016) Competitive adsorption of thallium in different soils as influenced by selected counter heavy metals. J Appl Biol Chem 59(5):695–701. https://doi.org/10.1007/s13765-016-0215-2 1. Kim DJ, Shin HJ, Ahn BK, Lee Jh (2016) Competitive adsorption of thallium in different soils as influenced by selected counter heavy metals. J Appl Biol Chem 59(5):695–701. https://doi.org/10.1007/s13765-016-0215-2 22. Putro JN, Santoso SP, Ismadji S, Ju YH (2017) Investigation of heavy metal adsorption in binary system by nanocrystalline cellulose e Bentonite nanocomposite: improvement on extended Langmuir isotherm model. J Microporous Mesoporous Mater 246:166–177 p g 2. Luo Z, Wang Z, Yan Y, Li J, Yan C, Xing B (2018) Titanium dioxide nanopar- ticles enhance inorganic arsenic bioavailability and methylation in two freshwater algae species. J Environ Pollut 238(5):631–637 2. Luo Z, Wang Z, Yan Y, Li J, Yan C, Xing B (2018) Titanium dioxide nanopar- ticles enhance inorganic arsenic bioavailability and methylation in two freshwater algae species. J Environ Pollut 238(5):631–637 23. Gaikwad MS, Balomajumder C (2017) Simultaneous electrosorptive removal of chromium(VI) and fluoride ions by capacitive deionization (CDI): multicomponent isotherm modeling and kinetic study. J Sep Purif Technol 182:272–281. https://doi.org/10.1016/j.seppur.2017.06.017 3. Xu L, Shen J, Marinova D, Guo X, Sun F, Zhu F (2013) Changes of public environmental awareness in response to the Taihu blue-green algae bloom incident in China. J Environ Dev Sustain 15(5):1281–1302 24. Wang C, Boithias L, Ning Z, Hana Y, Sauvage S, Sanchez-Perez JM, Kuramochi K, Hatano R (2016) Comparison of Langmuir and Freundlich adsorption equations within the SWAT-K model for assessing potassium environmental losses at basin scale. J Agric Water Manag 180(PartB):205– 211. https://doi.org/10.1016/j.agwat.2016.08.001 4. Park JH, Ok YS, Kim SH et al (2015) Characteristics of biochars derived from fruit tree pruning wastes and their effects on lead adsorption. J Korean Soc Appl Biol Chem 58(5):751–760. https://doi.org/10.1007/s1376 5-015-0103-1 5. Erdoğdular AO, Apar DK (2019) Bioremoval of reactive dye Remazol Navy by kefir grains. J Appl Biol Chem 62:22. https://doi.org/10.1186/s1376 5-019-0429-1 25. Fan Huan HJ, Tsai YS, Furuya E (2008) Prediction of individual Freundlich isotherms from binary and ternary phenolic compounds mixtures. Chem- osphere 71:886–893 25. Fan Huan HJ, Tsai YS, Furuya E (2008) Prediction of individual Freundlich isotherms from binary and ternary phenolic compounds mixtures. Chem- osphere 71:886–893 6. Received: 20 September 2019 Accepted: 8 November 2019 20. Negm NA, Wahed MGAbdE, Hassan ARA, Abou Kana MTH (2018) Feasibility of metal adsorption using brown algae and fungi: effect of biosorbents structure on adsorption isotherm and kinetics. J Mol liq 264:292–306. https://doi.org/10.1016/j.molliq.2018.05.027 21. Gustafsson JP, Akram M, Tiberg C (2015) predicting sulphate adsorption/ desorption in forest soils: evaluation of an extended Freundlich equation. J Chemosphere 119:83–89 Acknowledgements g This research was supported by Islamic Azad University branch of North Tehran (2018–2019). This research was supported by Islamic Azad University branch of North Tehran (2018–2019). 13. Montazer-Rahmatia MM, Rabbania P, Abdolalia A, Keshtkarb AR (2011) Kinetics and equilibrium studies on biosorption of cadmium, lead, and nickel ions from aqueous solutions by intact and chemically modified brown algae. J Hazard Mater 185(1):401–407. https://doi.org/10.1016/j. jhazmat.2010.09.047 13. Montazer-Rahmatia MM, Rabbania P, Abdolalia A, Keshtkarb AR (2011) Kinetics and equilibrium studies on biosorption of cadmium, lead, and nickel ions from aqueous solutions by intact and chemically modified brown algae. J Hazard Mater 185(1):401–407. https://doi.org/10.1016/j. jhazmat.2010.09.047 Competing interests h h d l h The authors declare that they have no competing interests. 18. Hadi P, Barford J, McKay GM (2013) Synergistic effect in the simultaneous removal of binary cobalt–nickel heavy metals from effluents by a novel e-waste-derived material. J Chem Eng 228(2013):140–146 18. Hadi P, Barford J, McKay GM (2013) Synergistic effect in the simultaneous removal of binary cobalt–nickel heavy metals from effluents by a novel e-waste-derived material. J Chem Eng 228(2013):140–146 Adsorption process kinetics Table 3 shows that the experimental data using Cystose- ria indica conform more closely to the intra-particle dif- fusion model for both nickel and cadmium. However, the fit with that model is only approximate (i.e. R2 ≤ 0.93), with the lower-exposure-time data responsible for much of the data spread. On the other hand, the data fit for the pseudo-first-order and pseudo-second-order models is very poor, R2 ≤ 0.741 and ≤ 0.811, respectively. Table 3 lists the coefficient of determination (R2) values of 0.90 for nickel and 0.93 for cadmium, for the intra-particle diffusion model, values that are significantly higher than those achieved by pseudo-first-order and pseudo-sec- ond-order models. 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 qe Cd calc (mg/L) qe Cd exp (mg/L) qcal=qexp Fig. 13 Parity plot of calculated versus experimental values for Extended-Freundlich isotherm of cadmium in the adsorption process of Cystoseria indica at different concentrations of the metal ions 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 qe Cd calc (mg/L) qe Cd exp (mg/L) qcal=qexp The consequences achieved in this study can be related with to some others reported in the works Comparing uptake capacities of brown marine algae obtained for metal concentrations in the liquid phase similar to that used in the present study, have shown that In spite of some differences in the experimental conditions, cad- mium, and nickel uptake capacities are close to those obtained by other researchers. The accumulation profiles obtained for the different brown algae species show that Fig. 13 Parity plot of calculated versus experimental values for Extended-Freundlich isotherm of cadmium in the adsorption process of Cystoseria indica at different concentrations of the metal ions Page 11 of 12 Page 11 of 12 Khajavian et al. Appl Biol Chem (2019) 62:69 Khajavian et al. Appl Biol Chem the metal uptake is rather fast, and more than 75% of the total uptake occurs within the first 20 min and then no further significant adsorption is observed [31, 44]. 11. Chu WL, Dang NL, Kok YY, Yap KSI, Phang SM, Convey P (2018) Heavy metal pollution in Antarctica and its potential impacts on algae. J Polar Sci 20(1):75–83. https://doi.org/10.1016/j.polar.2018.10.004 12. Shanura Fernandoa IP, Nahb JW, Jeona YJ (2016) Potential anti-inflamma- tory natural products from marine algae. J Environm Toxicol Pharmacol 48:22–30 Funding 16. Pinnola A, Formighieri C, Bassi R (2019) Algae: a new biomass resource. In: Kaltschmitt M (ed) Energy from organic materials (biomass), pp 165–197 https://doi.org/10.1007/978-1-4939-7813-7_436 16. Pinnola A, Formighieri C, Bassi R (2019) Algae: a new biomass resource. In: Kaltschmitt M (ed) Energy from organic materials (biomass), pp 165–197 https://doi.org/10.1007/978-1-4939-7813-7_436 Availability of data and materials Not applicable. 17. Sari A, Tuzen M (2008) Biosorption of cadmium (II) from aqueous solution by red argue (Ceramium virgatum): equilibrium, kinetic and thermody- namic studies. J Hazard Mater 157(2–3):448–454 17. Sari A, Tuzen M (2008) Biosorption of cadmium (II) from aqueous solution by red argue (Ceramium virgatum): equilibrium, kinetic and thermody- namic studies. J Hazard Mater 157(2–3):448–454 Authors’ contributions MK carried out heavy metal sampling, heavy metal solution analyses, and data organization. AH, MK, DAW and NM participated in interpreting the obtained results and organizing the manuscript. All authors read and approved the final manuscript. 14. Davis TA, Volesky B, Mucci A (2003) A review of the biochemistry of heavy metal biosorption by brown algae. Water Res 37:4311–4330 14. Davis TA, Volesky B, Mucci A (2003) A review of the biochemistry of heavy metal biosorption by brown algae. Water Res 37:4311–4330 15. Sinaei M, Loghmani M, Bolouki M (2011) Application of biomarkers in brown algae (Cystoseria indica) to assess heavy metals (Cd, Cu, Zn, Pb, Hg, Ni, Cr) pollution in the northern coasts of the Gulf of Oman. J Ecotoxicol Environ Saf 164:675–680 15. Sinaei M, Loghmani M, Bolouki M (2011) Application of biomarkers in brown algae (Cystoseria indica) to assess heavy metals (Cd, Cu, Zn, Pb, Hg, Ni, Cr) pollution in the northern coasts of the Gulf of Oman. J Ecotoxicol Environ Saf 164:675–680 Author details 1 1 Department of Chemical Engineering, Islamic Azad University Branch of North Tehran, Tehran, Iran. 2 DWA Energy Limited, Lincoln, UK. 3 Department of Chemical Engineer, Tehran University, Tehran, Iran. 19. Villar da Gama BM, do Nascimento GE, Cesar D, Sales S, Rodríguez-Díaz JM, Bezerra de Menezes Barbosa CMB, Duarte MMMB (2018) Mono and binary component adsorption of phenol and cadmium using adsorbent derived from peanut shells. J Clean Prod 201:219–228 Received: 20 September 2019 Accepted: 8 November 2019 Received: 20 September 2019 Accepted: 8 November 2019 References Kim SU, Owens VN, Kim SY, Hong CO (2017) Effect of different way of bottom ash and compost application on phytoextractability of cadmium in contaminated arable soil. Appl Biol Chem 60(4):353–362. https://doi. org/10.1007/s13765-017-0287-7 26. Chen Z, Ma W, Han M (2008) Biosorption of nickel and copper and to treated algae (Undria pinnatifida): application of isotherm and kinetics study. J Hazard Mater 155:327–333 26. Chen Z, Ma W, Han M (2008) Biosorption of nickel and copper and to treated algae (Undria pinnatifida): application of isotherm and kinetics study. J Hazard Mater 155:327–333 27. Yalçın S, Sezer S, Apak R (2012) Characterization and lead(II), cadmium(II), nickel(II) biosorption of dried marine brown macro algae Cystoseira bar- bata. Environ Sci Pollut Res 19(18):3118–3125. https://doi.org/10.1007/ s11356-012-0807-2 7. Lee JH, Hur HG (2014) Microbially facilitated incorporation of As(III) into bioreduced Fe-(hydr)oxide minerals. J Appl Biol Chem 57(1):123–128. https://doi.org/10.1007/s13765-014-4026-z 8. Li J, Chen J, Chen S (2018) Supercritical water treatment of heavy metal and arsenic metalloid-bioaccumulating-biomass. J Ecotoxicol Environ Saf 157:102–110 28. Nuhoglu Y, Malkoc E, Gurses A, Canpolat N (2002) The removal of cu (II) from aqueous solutions by Ulothrix zonata. J Bioresour Technol 85:331–333 9. Zhimiao Z, Xinshan S, Wei W, Yanping X, Zhijie G, Yuhui W, Yufeng Z, Yu C, Mengyuan M (2016) Influences of iron and calcium carbonate on wastewater treatment performances of algae based reactors. J Bioresour Technol 216:1–11 29. Mudhoo A, Garg VK, Wang S (2012) Heavy metals: toxicity and removal by biosorption. In: Lichtfouse E, Schwarzbauer J, Robert D (eds) Environmen- tal chemistry for a sustainable world, pp 379–442 30. Volesky B (2001) Detoxification of metal-bearing effluents: biosorption for the next century. J Hydrometall 59:203–216 30. Volesky B (2001) Detoxification of metal-bearing effluents: biosorption for the next century. J Hydrometall 59:203–216 10. Akbari M, Hallajisani A, Keshtkar AR, Shahbeig H, Ghorbanian SA (2015) Equilibrium and kinetic study and modeling of Cu(II) and Co(II) synergistic biosorption from Cu(II)-Co(II) single and binary mixtures on brown algae C. indica. J Environ Chem Eng 3:140–149 31. Hamdy A (2000) Biosorption of heavy metals by marine algae. Curr Micro- biol 4:232. https://doi.org/10.1007/s002840010126 31. Hamdy A (2000) Biosorption of heavy metals by marine algae. Curr Micro- biol 4:232. https://doi.org/10.1007/s002840010126 Page 12 of 12 Page 12 of 12 Khajavian et al. Appl Biol Chem (2019) 62:69 Khajavian et al. Appl Biol Chem (2019) 62:69 32. References Leng L, Yuan X, Huang H, Shao J, Wang H, Chen X, Zeng G (2015) Bio-char derived from sewage sludge by liquefaction: characterization and appli- cation for dye adsorption. J Appl Surf Sci 346:223–231 39. Volesky B, Naja GM (2011) Biosorption for industrial application. Biosorp- tion process fundamentals and a pilot design. Biosorption Ind Appl 6:25. https://doi.org/10.1016/B978-0-08-088504-9.00399-8 40. Davisa TA, Volesky B, Mucci A (2003) A review of the biochemistry of heavy metal bio sorption by brown algae. J Water Res 37:4311–4330 40. Davisa TA, Volesky B, Mucci A (2003) A review of the biochemistry of heavy metal bio sorption by brown algae. J Water Res 37:4311–4330 33. Xiao B, Dai Q, Yu X, Yu P, Zhai S, Liu R, Guo X, Liu J, Chen H (2018) Effects of sludge thermal-alkaline pretreatment on cationic red X-GRL adsorption onto pyrolysis biochar of sewage sludge. J Hazard Mater 343:347–355 41. Senthilkumar R, Vijayaraghavan K, Thilakavathi M, Iyer PVR, Velan M (2006) Seaweeds for the remediation of waste waters contaminated with zinc(II) ions. J Hazard Mater 136:791–799 41. Senthilkumar R, Vijayaraghavan K, Thilakavathi M, Iyer PVR, Velan M (2006) Seaweeds for the remediation of waste waters contaminated with zinc(II) ions. J Hazard Mater 136:791–799 34. Liu Y, Cao Q, Luo F, Chen J (2009) Biosorption of Cd2+, Cu2+, Ni2+ and Zn2+ ions from aqueous solutions by pretreated biomass of brown algae. J Hazard Mater 163:931–938. https://doi.org/10.1016/j.jhazm at.2008.07.046.18755544 42. Kumar M, Singh AK, Sikandar M (2018) Study of sorption and desorption of Cd (II) from aqueous solution using isolated green algae Chlorella vul- garis. J Appl Water Sci 8:225. https://doi.org/10.1007/s13201-018-0871-y 42. Kumar M, Singh AK, Sikandar M (2018) Study of sorption and desorption of Cd (II) from aqueous solution using isolated green algae Chlorella vul- garis. J Appl Water Sci 8:225. https://doi.org/10.1007/s13201-018-0871-y 35. Abdulrazzaq H, Jol H, Husni A, Abu-Bakr R (2014) Characterization and stabilisation of biochars obtained from empty fruit bunch, wood, and rice husk. J Bioresour 9:2888–2898 43. Pereira RF, Sousa A, Barrias CC, Bayat A, Granja PL, Bártolo PJ (2017) Advances in bioprinted cell-laden hydrogels for skin tissue engineering. J Biomanufacturing Rev 2:1. https://doi.org/10.1007/s40898-017-0003-8 43. Pereira RF, Sousa A, Barrias CC, Bayat A, Granja PL, Bártolo PJ (2017) Advances in bioprinted cell-laden hydrogels for skin tissue engineering. J Biomanufacturing Rev 2:1. https://doi.org/10.1007/s40898-017-0003-8 36. References De France FP, Tavares APM, Da Costa ACA (2002) Calcium interference with continuous biosorption of zinc by Sargassum sp. (Phaeophyta) in tubular laboratory reactors. J Bioresour Technol 83:159–163 44. Freitas OMM, Martins RJE, Delerue-Matos CM, Boaventura RAR (2008) Removal of Cd(II), Zn(II) and Pb(II) from aqueous solutions by brown marine macro algae: kinetic modelling. J Hazard Mater 153:493–501 44. Freitas OMM, Martins RJE, Delerue-Matos CM, Boaventura RAR (2008) Removal of Cd(II), Zn(II) and Pb(II) from aqueous solutions by brown marine macro algae: kinetic modelling. J Hazard Mater 153:493–501 37. Emami Z, Ehsani M, Zandi M, Foudazi R (2018) Controlling alginate oxida- tion conditions for making alginate-gelatin hydrogels. J Carbohydr Polym 198:509–517 39. Volesky B, Naja GM (2011) Biosorption for industrial application. Biosorp- tion process fundamentals and a pilot design. Biosorption Ind Appl 6:25. https://doi.org/10.1016/B978-0-08-088504-9.00399-8 Publisher’s Note 38. Xiong Y, Xu J, Shan W, Lou Z, Fang D, Zang S, Han G (2013) A new approach for rhenium(VII) recovery by using modified brownalgae Lami- naria japonica adsorbent. J Bioresour Technol 127:464–472 Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations.
https://openalex.org/W4304699916	https://www.researchsquare.com/article/rs-2164430/latest.pdf	English	null	A fairness assessment of mobility-based COVID-19 case prediction models	arXiv (Cornell University)	2,022	cc-by	6,241	A fairness assessment of mobility-based COVID-19 case prediction models Abdolmajid Erfani (  erfani@umd.edu ) University of Maryland Introduction The interactions between human mobility and epidemic spread have been studied unprecedentedly during the COVID-19 pandemic 1–8. With these efforts, nonpharmaceutical interventions (such as national lockdowns) have been evaluated for their effectiveness and socio-economic impact on different groups 9–11, models have been developed to predict disease spatial diffusion 12,13, and scenarios have been modeled to assess their outcomes 14–17. Studies have demonstrated that mobility data are a meaningful proxy measure of social distancing 18, affect viral spreading 19,20, and are useful for predicting the spread of COVID-19 21–23. In particular, to control the spread of new cases and plan efficiently for hospital needs and capacities during an epidemic, public health decision-makers require accurate predictions of future case numbers 7. For example, a study by Ilin et al. showed that changes in mobility can be used to predict COVID-19 cases. Their study demonstrated that public mobility data can be used to develop reduced-form and simple models that mimic the behavior of more sophisticated epidemiological models for predicting COVID-19 cases on a 10-day basis 21. Another study examined several state-of-the-art machine learning models and statistical methods and demonstrated how mobility data can improve prediction trends when used as exogenous information in models 22. As discussed, mobility data from anonymized smartphones has been shown to improve COVID-19 case prediction models. However, mobility data bias has received little attention in this predictive context. There exist only just a handful of papers reporting demographic bias in mobility data due to differences in smartphone ownership and use 24–26; and since data providers are not transparent about how mobility data is collected, or about the socio-economic and demographic groups represented in them, directly measuring and correcting bias in mobility data is difficult. In this study, we hypothesize that the presence of socio-economic and demographic bias in the mobility data used to train the COVID-19 case predictive models, might result in unfairly less accurate predictions for particular socio-economic and demographic groups. Unfair predictions provided to decision makers e.g., predictions of COVID-19 cases for minority groups that are lower than reality, could in turn be used to unfairly assign more resources to population groups that do not necessarily need them. To test our hypothesis, we evaluated the performance of two types of mobility-based COVID-19 case prediction models highly used by decision makers due to its interpretability: linear regressions and time series models. Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/13 Abstract In light of the outbreak of COVID-19, analyzing and measuring human mobility has become increasingly important. A wide range of studies have explored spatiotemporal trends over time, examined associations with other variables, evaluated non-pharmacologic interventions (NPIs), and predicted or simulated COVID-19 spread using mobility data. Despite the benefits of publicly available mobility data, a key question remains unanswered: are models using mobility data performing equitably across demographic groups? We hypothesize that bias in the mobility data used to train the predictive models might lead to unfairly less accurate predictions for certain demographic groups. To test our hypothesis, we applied two mobility-based COVID infection prediction models at the county level in the United States using SafeGraph data, and correlated model performance with sociodemographic traits. Findings revealed that there is a systematic bias in models’ performance toward certain demographic characteristics. Specifically, the models tend to favor large, highly educated, wealthy, young, urban, and non-black-dominated counties. We hypothesize that the mobility data currently used by many predictive models tends to capture less information about older, poorer, non-white, and less educated regions, which in turn negatively impacts the accuracy of the COVID-19 prediction in these regions. Ultimately, this study points to the need of improved data collection and sampling approaches that allow for an accurate representation of the mobility patterns across demographic groups. Human mobility We used SafeGraph’s publicly-available human mobility data at the county level in the US. SafeGraph uses location information extracted from smartphones to provide aggregate data characterizing mobility in terms of visit volumes to types of places and volumes of origin-destination (OD) flows 32. For this study specifically, we used the data publicly available in the origin-destination- time (ODT) platform 33, that computes OD flows between counties as the aggregation of trips that start at an individual’s home county location (origin), with a destination defined as a stay location within a county for longer than a minute. OD flows between all counties in the US were collected throughout all days of the year 2020. Figure 1.a illustrates how the average number of trips across the US changed over the year 2020. According to various studies in the US using mobility data, the dataset collected in Fig. 1.a also shows similar trends of mobility change 34,35. Data In our study, we use mobility data from SafeGraph to build COVID-19 case prediction models; and we explore model performance across socio-economic and demographic features to potentially identify unfair results for specific groups i.e., differences in error distributions across social groups. We next describe these three types of datasets, with all being publicly available. Introduction In contrast to more complex epidemiological models that are hard to tune due to its parametric nature, and deep learning models that are difficult to interpret, linear models and time series are easy to train and test 21,27−29. The models were trained using SafeGraph’s mobility data, and performance was measured via predictive errors. To assess the fairness of the predictions, we analyzed the relationship between the model prediction errors and specific socio-economic and demographic features at the county level in the United States and across the two model types. Evaluating the performance of two diverse interpretable models allowed us to account for potential algorithmic bias i.e., bias introduced by the algorithm itself 30,31. If unfair predictions are pervasive across types of models trained and tested with the same data, we can partially attribute the unfairness to the mobility data itself. Page 2/13 Page 2/13 Socio-economic and demographic data Data on socio-economic and demographic characteristics at the county level was also collected from public databases 37. Studies have shown that sociodemographic factors such as age, race, income, educational level, and area of residence can influence smartphone ownership and usage, which may have an impact on mobility data biases 38,39. Therefore, we collected a wide range of information, including the population, income, education, age, ownership of smartphones, and the percentage of Black Americans. Also, we used the US National Center for Health Statistics Urban-Rural Classification Scheme for Counties 40, which assigns each county an ordinal code ranging from 1 (most urban) to 6 (most rural). Methods In this section we will describe (i) the two types of models used in the COVID-19 case prediction; (ii) the training and evaluation of these models; and (iii) the process proposed to evaluate the fairness of the predictions across socio-economic and demographic groups, as well as across models. COVID‑19 cases In order to obtain the cumulative and daily confirmed cases of COVID-19 for each county unit, we refer to the data repository compiled by the Johns Hopkins Center for Systems Science and Engineering (CSSE) 36. As shown in Fig. 1.b, the weekly number of new COVID infections has been increasing over the year 2020. Models Model 1: Linear regression (Ilin et al. 21, Wang et al. 41, Ayan et al. 42, and Sahin 43). Several papers have suggested that linear regressions that combine mobility data with historical COVID-19 cases can successfully predict future cases 21,41−43. These models generally use different lags between mobility rates and COVID-19 cases to account for the infection period i.e., the period between the person’s movement – and potentially interaction with others and infection – and the person testing positive for COVID-19. For this study we use Ilin at al. linear model 21 because rather than picking one lag, they propose to consider multiple lags within the model encompassing the plethora of linear regressions that have been tested in the literature. Specifically, Ilin at al. use a distributed-lag model to estimate log confirmed infections as the dependent variable, with average mobility over lags 1–7, 8–14, and 15–21 days to predict the number of COVID-19 cases at a given day: log = β1m1−7,it + β2m8−14,it + β3m15−21,it + ϵit# (1) Iit Ii,t−1 Page 3/13 Page 3/13 where i is the unit of analysis, is the first difference of log confirmed cases at time t, and m represents mobility measures averaged over lags 1–7, 8–14 and 15–21, respectively. log Iit Ii,t−1 Model 2: Time series forecasting (Aji et al. 28, Zhao et al. 29, Zeng et al. 44, and Klein et al. 45). The Autoregressive Integrated Moving Average (ARIMA) model is a statistical method that considers both past and present data for forecasting. An ARIMAX model, also known as ARIMA with multiple regressors, extends the basic ARIMA model to include other external variables for prediction. In the COVID-19 setting, mobility data and other sources of information have been used as regressors to potentially improve the predictive models 29,44,45. For example, in their study Zhao et al. 29 conclude that with mobility data, time series forecasting provides accurate predictions with mobility data lags of between 8–10 days for dense or sparse populations respectively. Models In this study, we consider an ARIMAX (p, d, q) model that can be expressed as: yt = βxt + ∑p j=1 ∅jyt−j + ϵt + ∑q j=1 θjϵt−j# (2) where y is the number of confirmed infections, x is the mobility change as exogenous variable lagged by 21 days (similar to Model 1's selection of lag), p is the Autoregressive (AR) parameter, q is the Moving Average (MA) parameter, d is the degree of first differencing to make data stationarity, and Ɛ is the error. By using the Python package Auto Arima, we were able to generate the best p, d, and q values based on the data set, thus providing better forecasts 46. To summarize, the lag of mobility, historic number of COVID cases can be used to predict future cases at unit of analysis. Training and model evaluation To train the models, we used both historical COVID-19 data and mobility OD flows from mid-April to December 2020. Rather than using the raw mobility OD flows, we used a measure of mobility change over a baseline, which was calculated by dividing the daily mobility by the average daily mobility in February 2020, a non-holiday month before the COVID-19 pandemic. This is a common approach in prior COVID-19 case predictive models that use mobility data 21,29. Different training lengths were evaluated for both models, and the ones with the best accuracies were selected. To train the linear regression (Model 1), we used a 21-day training set with a 1-day shifting window from April 14th to December 30th. The time series model (Model 2), was trained over a period of 90 days, resulting in predictions available from early August to the end of the year. Once trained, each model was used to predict the number of COVID-19 cases for two lookaheads: 1-day (next day) and 7-days time (week) intervals at the county level, as predictions on a daily and weekly basis are a common theme in previous studies 21,28,29,41−44. In this process, thousands of regressions and ARIMAX models are trained at the county level on a daily basis to be able to predict 1 day and 7 days later how many COVID-19 cases will occur. Finally, the model performance was evaluated via the error rate, which was calculated on a daily basis based on the difference between the actual number of COVID cases and predictions: Errorratet = ∣∣ ∣∣# (3) Predictionvaluet−ActualnumberofCOV IDcasest ActualnumberofCOV IDcasest Fairness Analysis. We analyzed the fairness of the predictions for each model by computing the average weekly error rate per lookahead (1-day and 7- day) at the county level, followed by a Pearson’s correlation analysis between the average weekly error rate across counties in the US and their socio-economic and demographic characteristics presented in the data section: household income (average household income), smartphone ownership (percentage of households owning smartphones), population, education level (bachelor's degree), urbanity-rurality level (NCHS classification), age (median age), ethnicity (black percentage). Ultimately, the correlation analysis allows us to assess if performance is similar (fair) or not (unfair) across social groups. Model Performance Page 4/13 First, we discuss the COVID-19 case prediction performance of the two models presented (see Fig. 2). Fairness Performance For Model 1, we observe a moderate and negative correlation between the prediction error rate and income (R (1-day) = -0.22, R (7- days) = -0.17, p-value < 0.01), smartphone ownership (R (1-day) = -0.24, R (7-days) = -0.20, p-value < 0.01), population (R (1-day) = -0.20, R (7-days) = -0.17, p-value < 0.01), bachelor degree (R (1-day) = -0.23, R (7-days) = -0.21, p-value < 0.01). The results suggest that Model 1 – a regression model of COVID-19 cases with mobility – performs better (has fewer errors) in counties with higher incomes, higher smartphone ownership, larger populations, and higher educational levels. On the other hand, correlation analysis indicates a moderate and positive relationship between NCHS code and error rate (R (1-day) = + 0.30, R (7-days) = + 0.23, p-value < 0.01), median age (R (1-day) = + 0.21, R (7-days) = + 0.17, p-value < 0.01), black percentage (R (1-day) = + 0.23, R (7-days) = + 0.18, p- value < 0.01). Therefore, as rurality, age, and black population percentage increased, the model's error rate increased, suggesting it performs worse in rural areas and among older and/or black communities (Fig. 3 represents the weekly correlations for some of these features). For Model 2, we observe a moderate and negative correlation between the prediction error rate and the income (R (1-day) = -0.31, R (7-days) = -0.27, p-value < 0.01), smartphone ownership (R (1-day) = -0.31, R (7-days) = -0.28, p-value < 0.01), population (R (1-day) = -0.33, R (7-days) = -0.29, p-value < 0.01), bachelor degree (R (1-day) = -0.32, R (7-days) = -0.28, p-value < 0.01). These results reveal that Model 2 – an ARIMAX with mobility data added as exogenous variable – performs better (i.e., has lower errors) in counties that share higher income, higher smartphone ownership, larger populations, and higher educational levels (see Fig. 4 for weekly representations of the weekly correlations for some of these features). On the other hand, the correlation analysis also reveals a moderate and positive relationship between the error rate and the NCHS code (R (1-day) = + 0.35, R (7-days) = + 0.39, p-value < 0.01), median age (R (1-day) = + 0.29, R (7-days) = + 0.28, p-value < 0.01), black percentage (R (1-day) = + 0.27, R (7-days) = + 0.27, p-value < 0.01). Page 4/13 First, we discuss the COVID-19 case prediction performance of the two models presented (see Fig. 2). Page 4/13 As we can observe, both models predict the number of next day cases (1-day) with an average weekly error rate of 10–20%, and the number of cases in a week (7-days) with an average weekly error rate of 30–40%. Models' performance is in a comparable range to previous studies 21,28,29,41−44, but with the difference that we reported the results for the entire year of 2020 and the US, not specific regions or COVID waves. regions or COVID waves. Fairness Performance In other words, the model's error rate increased as rurality, age, and black percentage increased, revealing a model that performs worse in rural environments, and among older and/or black populations. Given that these findings are replicated across models 1 and 2, thus controlling for algorithmic bias, we posit that the model is unfair in part due to the bias in the mobility data used in the model, although bias in the way COVID-19 case data is collected, could also influence the outcome. To summarize the fairness analysis across models, Tables 1 and 2 provide the monthly correlation averages between the sociodemographic factors at the county level and the error rates for Models 1 and 2 1-day and 7-day predictions, respectively. As discussed, due to the diverse size of the optimal training windows, Model 1 predictions run from April till December, while Model 2 predictions are produced from August till December. With a few fluctuations, and as discussed with in the weekly analyses in Figs. 3 and 4, both models show the same pattern of results throughout 2020: lower prediction errors in large, highly educated, wealthy, young, urban, and non-black-dominated counties. Page 5/13 Page 5/13 Page 5/13 Page 5/13 Table 1 County-level correlations between model 1 error rate and sociodemographic features. Income smartphone Population Education NCHS Age Black% 1day 7day 1day 7day 1day 7day 1day 7day 1day 7day 1day 7day 1day 7day April -0.30 -0.19 -0.30 -0.18 -0.24 -0.16 -0.35 -0.21 0.27 0.15 0.14 0.11 0.15 0.12 May -0.21 -0.15 -0.22 -0.15 -0.18 -0.14 -0.22 -0.16 0.26 0.15 0.18 0.13 0.14 0.11 Jun -0.27 -0.18 -0.23 -0.15 -0.18 -0.14 -0.25 -0.17 0.29 0.19 0.15 0.14 0.14 0.13 July -0.24 -0.16 -0.18 -0.15 -0.17 -0.13 -0.24 -0.16 0.23 0.18 0.14 0.14 0.16 0.13 August -0.25 -0.17 -0.25 -0.13 -0.21 -0.14 -0.25 -0.14 0.30 0.16 0.22 0.12 0.21 0.13 September -0.22 -0.19 -0.29 -0.26 -0.23 -0.20 -0.23 -0.23 0.36 0.29 0.28 0.25 0.27 0.23 October -0.20 -0.17 -0.26 -0.21 -0.24 -0.19 -0.21 -0.16 0.35 0.27 0.26 0.19 0.36 0.25 November -0.21 -0.19 -0.28 -0.19 -0.21 -0.18 -0.21 -0.18 0.39 0.28 0.28 0.24 0.36 0.26 December -0.16 -0.16 -0.19 -0.20 -0.15 -0.14 -0.17 -0.17 0.22 0.18 0.21 0.22 0.27 0.23 Table 2 County-level correlations between model 2 error rate and sociodemographic features. Fairness Performance Income smartphone Population Education NCHS Age Black% 1day 7day 1day 7day 1day 7day 1day 7day 1day 7day 1day 7day 1day 7day August -0.25 -0.27 -0.27 -0.23 -0.28 -0.24 -0.28 -0.26 0.24 0.30 0.28 0.26 0.24 0.26 September -0.30 -0.28 -0.25 -0.27 -0.29 -0.26 -0.31 -0.29 0.30 0.33 0.24 0.26 0.27 0.27 October -0.30 -0.30 -0.30 -0.34 -0.33 -0.35 -0.31 -0.29 0.31 0.42 0.27 0.25 0.24 0.28 November -0.32 -0.26 -0.35 -0.28 -0.34 -0.31 -0.34 -0.26 0.46 0.50 0.30 0.36 0.28 0.31 December -0.38 -0.27 -0.41 -0.28 -0.35 -0.28 -0.37 -0.28 0.46 0.42 0.29 0.26 0.30 0.22 Discussion Table 1 County-level correlations between model 1 error rate and sociodemographic features. Income smartphone Population Education NCHS Age Black% 1day 7day 1day 7day 1day 7day 1day 7day 1day 7day 1day 7day 1day 7day April -0.30 -0.19 -0.30 -0.18 -0.24 -0.16 -0.35 -0.21 0.27 0.15 0.14 0.11 0.15 0.12 May -0.21 -0.15 -0.22 -0.15 -0.18 -0.14 -0.22 -0.16 0.26 0.15 0.18 0.13 0.14 0.11 Jun -0.27 -0.18 -0.23 -0.15 -0.18 -0.14 -0.25 -0.17 0.29 0.19 0.15 0.14 0.14 0.13 July -0.24 -0.16 -0.18 -0.15 -0.17 -0.13 -0.24 -0.16 0.23 0.18 0.14 0.14 0.16 0.13 August -0.25 -0.17 -0.25 -0.13 -0.21 -0.14 -0.25 -0.14 0.30 0.16 0.22 0.12 0.21 0.13 September -0.22 -0.19 -0.29 -0.26 -0.23 -0.20 -0.23 -0.23 0.36 0.29 0.28 0.25 0.27 0.23 October -0.20 -0.17 -0.26 -0.21 -0.24 -0.19 -0.21 -0.16 0.35 0.27 0.26 0.19 0.36 0.25 November -0.21 -0.19 -0.28 -0.19 -0.21 -0.18 -0.21 -0.18 0.39 0.28 0.28 0.24 0.36 0.26 December -0.16 -0.16 -0.19 -0.20 -0.15 -0.14 -0.17 -0.17 0.22 0.18 0.21 0.22 0.27 0.23 Table 2 County-level correlations between model 2 error rate and sociodemographic features. Income smartphone Population Education NCHS Age Black% 1day 7day 1day 7day 1day 7day 1day 7day 1day 7day 1day 7day 1day 7day August -0.25 -0.27 -0.27 -0.23 -0.28 -0.24 -0.28 -0.26 0.24 0.30 0.28 0.26 0.24 0.26 September -0.30 -0.28 -0.25 -0.27 -0.29 -0.26 -0.31 -0.29 0.30 0.33 0.24 0.26 0.27 0.27 October -0.30 -0.30 -0.30 -0.34 -0.33 -0.35 -0.31 -0.29 0.31 0.42 0.27 0.25 0.24 0.28 November -0.32 -0.26 -0.35 -0.28 -0.34 -0.31 -0.34 -0.26 0.46 0.50 0.30 0.36 0.28 0.31 December -0.38 -0.27 -0.41 -0.28 -0.35 -0.28 -0.37 -0.28 0.46 0.42 0.29 0.26 0.30 0.22 Discussion To combat the COVID-19 pandemic, governments and private companies around the world were promoting the use of digital public health technologies for data collection and processing 47–49. Through the use of GPS, cellular networks and Wi-Fi, smartphones can collect and aggregate location data in real-time to monitor population flows, identify transmission hotspots, determine the effectiveness of NPIs 50, and predict future COVID-19 cases 7,20,25. Using SafeGraph's mobility data, we examined whether two popular predictive models that use mobility data to predict COVID-19 cases over time, performed fairly across social groups. Our findings revealed a correlation between a county's socio-economic and demographic characteristics and the models’ error rates. In particular, we observed that the prediction errors were lower in large, highly educated, wealthy, young, urban, and non-black-dominated counties. Given that the findings were similar across models, thus controlling for algorithmic bias, we posit that the presence of bias in the mobility data negatively impacts the model predictions by unfairly outputting case numbers with higher errors for specific social groups. Furthermore, our results show that mobility data appears to be less likely to capture older, poorer, non-white, and less educated users. Thus, allocating public health resources based on such mobility data could disproportionately harm seniors and minorities at high risk. We acknowledge that these unfair results could also be in part due to bias in COVID-19 data collection processes, which might suffer from higher under-reporting by certain social groups, and our future work will look into this. To generalize smartphone-derived insights over a population, the mobility data must reveal information about the population without bias i.e., information that is representative across socio-economic and demographic groups. However, due to the lack of Page 6/13 ground truth data about the socio-economic and demographic characteristics of the population whose mobility data is collected, this study has also shown that investigating performance fairness can provide valuable insights into potential mobility data biases. ground truth data about the socio-economic and demographic characteristics of the population whose mobility data is collected, this study has also shown that investigating performance fairness can provide valuable insights into potential mobility data biases. Finally, as the research community moves forward with the use of mobility data in COVID-19 case prediction models, we think it is important to consider the following set of recommendations. Data availability All data we use in this study are all publicly available. These datasets include the SafeGraph mobility data (. https://docs.safegraph.com/docs/social-distancing-metrics), COVID-19 confirmed cases (https://github.com/CSSEGISandData/COVID-19), and sociodemographic information at US counties level (https://www.census.gov/data/datasets.html). Author contributions The authors confirm contribution to the paper as follows: study conception and design: V.F and A.E; data collection: A.E; analysis and interpretation of results: A.E and V.F; draft manuscript preparation: A.E and V. F. Both authors reviewed and approved the final version of the manuscript. Availability of Data and Materials Correspondence and requests for materials should be addressed to A.E. Discussion First, and whenever possible, we strongly suggest applying sampling bias mitigation approaches to correct for under-represented groups in the data. Second, mitigation approaches might not always be possible, due to the lack of demographic information about the individuals whose cell phone data is being collected. For that reason, we encourage the research community working with mobility data to report fairness analyses together with the performance of the predictive models proposed. We hope that these practices will enhance pandemic management via case prediction models that are more transparent and fairer, and that will allow for more equitable decision making. References 1. Alessandretti, L. What human mobility data tell us about COVID-19 spread. Nature Reviews Physics, 4(1), 12-13 (2022). 1. Alessandretti, L. What human mobility data tell us about COVID-19 spread. Nature Reviews Physics, 4(1), 12-13 (2022). 2. Rutten, P., Lees, M. H., Klous, S., Heesterbeek, H., & Sloot, P. Modelling the dynamic relationship between spread of infection and observed crowd movement patterns at large scale events. Scientific Reports, 12(1), 1-16 (2022). 2. Rutten, P., Lees, M. H., Klous, S., Heesterbeek, H., & Sloot, P. Modelling the dynamic relationship between spread of infection and observed crowd movement patterns at large scale events. Scientific Reports, 12(1), 1-16 (2022). 3. Hu, S., Xiong, C., Yang, M., Younes, H., Luo, W., & Zhang, L. A big-data driven approach to analyzing and modeling human mobility trend under non-pharmaceutical interventions during COVID-19 pandemic. Transportation Research Part C: Emerging Technologies, 124, 102955 (2021). 4. Hu, T. et al. Human mobility data in the COVID-19 pandemic: characteristics, applications, and challenges. International Journal of Digital Earth, 14(9), 1126-1147 (2021). 4. Hu, T. et al. Human mobility data in the COVID-19 pandemic: characteristics, applications, and challenges. International Journal of Digital Earth, 14(9), 1126-1147 (2021). 5. Nouvellet, P. et al. Reduction in mobility and COVID-19 transmission. Nature communicat 5. Nouvellet, P. et al. Reduction in mobility and COVID-19 transmission. Nature communications, 12(1), 1-9 (2021). 5. Nouvellet, P. et al. Reduction in mobility and COVID-19 transmission. Nature communications, 12(1), 1-9 (2021). 6. Kartal, M. T., Depren, Ö., & Depren, S. K. The relationship between mobility and COVID-19 pandemic: Daily evidence from an emerging country by causality analysis. Transportation Research Interdisciplinary Perspectives, 10, 100366 (2021). 6. Kartal, M. T., Depren, Ö., & Depren, S. K. The relationship between mobility and COVID-19 pandemic: Daily evidence from an emerging country by causality analysis. Transportation Research Interdisciplinary Perspectives, 10, 100366 (2021). 7. Wellenius, G. A. et al. Impacts of social distancing policies on mobility and COVID-19 case growth in the US. Nature communications, 12(1), 1-7 (2021). 7. Wellenius, G. A. et al. Impacts of social distancing policies on mobility and COVID-19 case growth in the US. Nature communications, 12(1), 1-7 (2021). 8. Gutiérrez-Jara, J. P., Vogt-Geisse, K., Cabrera, M., Córdova-Lepe, F., & Muñoz-Quezada, M. T. Effects of human mobility and behavior on disease transmission in a COVID-19 mathematical model. Scientific Reports, 12(1), 1-18 (2022). 8. Gutiérrez-Jara, J. References P., Vogt-Geisse, K., Cabrera, M., Córdova-Lepe, F., & Muñoz-Quezada, M. T. Effects of human mobility and behavior on disease transmission in a COVID-19 mathematical model. Scientific Reports, 12(1), 1-18 (2022). 9. Coleman, N., Gao, X., DeLeon, J. et al. Human activity and mobility data reveal disparities in exposure risk reduction indicators among socially vulnerable populations during COVID-19 for five U.S. metropolitan cities. Sci Rep 12, 15814 (2022). https://doi.org/10.1038/s41598-022-18857-7 Page 7/13 Page 7/13 10. Gozzi, N., Tizzoni, M., Chinazzi, M., Ferres, L., Vespignani, A., & Perra, N. Estimating the effect of social inequalities on the mitigation of COVID-19 across communities in Santiago de Chile. Nature communications, 12(1), 1-9 (2021). 11. Chang, S. et al. Mobility network models of COVID-19 explain inequities and inform reopening. Nature, 589(7840), 82-87 (2021) 12. Canino, M. P., Cesario, E., Vinci, A., & Zarin, S. Epidemic forecasting based on mobility patterns: an approach and experimental evaluation on COVID-19 Data. Social Network Analysis and Mining, 12(1), 1-15 (2022). 13. Chinazzi, M., et al. The effect of travel restrictions on the spread of the 2019 novel coronavirus (COVID-19) outbreak. Science, 368(6489), 395-400 (2020). 14. Hu, S., et al. Do racial and ethnic disparities in following stay-at-home orders influence COVID-19 health outcomes? A mediation analysis approach. PloS one, 16(11), e0259803 (2021). 15. Mahmoudi, J., & Xiong, C. How social distancing, mobility, and preventive policies affect COVID-19 outcomes: Big data-driven evidence from the District of Columbia-Maryland-Virginia (DMV) megaregion. Plos one, 17(2), e0263820 (2022). 16. Aleta, A. et al. Modelling the impact of testing, contact tracing and household quarantine on second waves of COVID-19. Nature Human Behaviour, 4(9), 964-971 (2020). 17. Beigi, P., Haque, M., Rajabi, M. S., & Hamdar, S. Bike Share's Impact on COVID-19 Transmission and Bike Share's Responses to COVID-19: A case study of Washington DC. arXiv preprint arXiv:2205.05011 (2022). 18. Pan, Y., et al. Quantifying human mobility behaviour changes during the COVID-19 outbreak in the United States. Scientific Reports, 10(1), 1-9 (2020). 19. Levin, R., Chao, D. L., Wenger, E. A., & Proctor, J. L. Insights into population behavior during the COVID-19 pandemic from cell phone mobility data and manifold learning. Nature Computational Science, 1(9), 588-597 (2021). 20. Badr, H. S., & Gardner, L. M. Limitations of using mobile phone data to model COVID-19 transmission in the USA. The Lancet Infectious Diseases, 21(5), e113 (2021). 21. Ilin, C. et al. References Public mobility data enables COVID-19 forecasting and management at local and global scales. Scientific reports, 11(1), 1-11 (2021). 22. García-Cremades, S., et al. Improving prediction of COVID-19 evolution by fusing epidemiological and mobility data. Scientific Reports, 11(1), 1-16 (2021). 23. Alali, Y., Harrou, F., & Sun, Y. A proficient approach to forecast COVID-19 spread via optimized dynamic machine learning models. Scientific Reports, 12(1), 1-20 (2022). 24. Coston, A. et al. Leveraging administrative data for bias audits: Assessing disparate coverage with mobility data for COVID-19 policy. In Proceedings of the 2021 ACM Conference on Fairness, Accountability, and Transparency (pp. 173-184) (2021). 25. Milusheva, S., Bjorkegren, D., & Viotti, L. Assessing Bias in Smartphone Mobility Estimates in Low Income Countries. In ACM SIGCAS Conference on Computing and Sustainable Societies (pp. 364-378) (2021). 26. Schlosser, F., Sekara, V., Brockmann, D., & Garcia-Herranz, M. Biases in human mobility data impact epidemic modeling. arXiv preprint arXiv:2112.12521 (2021). 27. Khan, F. M., & Gupta, R. ARIMA and NAR based prediction model for time series analysis of COVID-19 cases in India. Journal of Safety Science and Resilience, 1(1), 12-18 (2020). 28. Aji, B. S., & Rohmawati, A. A. Forecasting number of COVID-19 cases in Indonesia with ARIMA and ARIMAX models. In 2021 9th International Conference on Information and Communication Technology (ICoICT) (pp. 71-75). (2021). 29. Zhao, J., Han, M., Wang, Z., & Wan, B. Autoregressive count data modeling on mobility patterns to predict cases of COVID-19 infection (2022). 30. Kordzadeh, N., & Ghasemaghaei, M. Algorithmic bias: review, synthesis, and future research directions. European Journal of Information Systems, 31(3), 388-409 (2022). 31. Johnson, I., McMahon, C., Schöning, J., & Hecht, B. The effect of population and" structural" biases on social media-based algorithms: A case study in geolocation inference across the urban-rural spectrum. In Proceedings of the 2017 CHI conference on Human Factors in Computing Systems (pp. 1167-1178) (2017). 32. SafeGraph. Social Distancing Metrics. https://docs.safegraph.com/docs/social-distancing- 32. SafeGraph. Social Distancing Metrics. https://docs.safegraph.com/docs/social-distancing-metrics (2020). Page 8/13 Page 8/13 Page 8/13 33. Li, Z. et al. ODT FLOW: Extracting, analyzing, and sharing multi-source multi-scale human mobility. Plos one, 16(8), e0255259 (2021). 34. Xiong, C., Hu, S., Yang, M., Luo, W., & Zhang, L. Mobile device data reveal the dynamics in a positive relationship between human mobility and COVID-19 infections. Proceedings of the National Academy of Sciences, 117(44), 27087-27089 (2020). 35. Lee, M. et al. Human mobility trends during the early stage of the COVID-19 pandemic in the United States. PLoS One, 15(11), e0241468 (2020). 35. Lee, M. et al. Human mobility trends during the early stage of the COVID-19 pandemic in the United States. PLoS One, 15(11), e0241468 (2020). 36. COVID-19 Data Repository by the Center for Systems Science and Engineering (CSSE). Johns Hopkins University (2020). https://github.com/CSSEGISandData/COVID-19 36. COVID-19 Data Repository by the Center for Systems Science and Engineering (CSSE). Johns Hopkins University (2020). https://github.com/CSSEGISandData/COVID-19 37. U.S. Census Bureau. Annual Estimates of the Resident Population for Selected Age Groups by Sex for the United States, States, Counties and Puerto Rico Commonwealth and Municipios. (2019) https://www.census.gov/data/datasets.html). 37. U.S. Census Bureau. Annual Estimates of the Resident Population for Selected Age Groups by Sex for the United States, States, Counties and Puerto Rico Commonwealth and Municipios. (2019) https://www.census.gov/data/datasets.html). 38. Kim, Y., Briley, D. A., & Ocepek, M. G. Differential innovation of smartphone and application use by sociodemographics and personality. Computers in Human Behavior, 44, 141-147 (2015). 38. Kim, Y., Briley, D. A., & Ocepek, M. G. Differential innovation of smartphone and application use by sociodemographics and personality. Computers in Human Behavior, 44, 141-147 (2015). 39. Rahmati, A., Tossell, C., Shepard, C., Kortum, P., & Zhong, L. Exploring iPhone usage: the influence of socioeconomic differences on smartphone adoption, usage and usability. In Proceedings of the 14th international conference on Human-computer interaction with mobile devices and services (pp. 11-20) (2012). 39. Rahmati, A., Tossell, C., Shepard, C., Kortum, P., & Zhong, L. Exploring iPhone usage: the influence of socioeconomic differences on smartphone adoption, usage and usability. In Proceedings of the 14th international conference on Human-computer interaction with mobile devices and services (pp. 11-20) (2012). 40. Ingram, D. D., & Franco, S. J. 2013 NCHS urban-rural classification scheme for counties. US Department of Health and Human Services, Centers for Disease Control and Prevention, National Center for Health Statistics (2014). 40. Ingram, D. D., & Franco, S. J. Page 8/13 Anatomy into the battle of supporting or opposing reopening amid the COVID-19 pandemic on Twitter: A temporal and spatial analysis. Plos one, 16(7), e0254359 (2021). 48. Li, L., Erfani, A., Wang, Y., & Cui, Q. Anatomy into the battle of supporting or opposing reopening amid the COVID-19 pandemic on Twitter: A temporal and spatial analysis. Plos one, 16(7), e0254359 (2021). 49. Whitelaw, S., Mamas, M. A., Topol, E., & Van Spall, H. G. Applications of digital technology in COVID-19 pandemic planning and response. The Lancet Digital Health, 2(8), e435-e440 (2020). 49. Whitelaw, S., Mamas, M. A., Topol, E., & Van Spall, H. G. Applications of digital technology in COVID-19 pandemic planning and response. The Lancet Digital Health, 2(8), e435-e440 (2020). 50. Budd, J. et al. Digital technologies in the public-health response to COVID-19. Nature medicine, 26(8), 1183-1192 (2020). 50. Budd, J. et al. Digital technologies in the public-health response to COVID-19. Nature medicine, 26(8), 1183-1192 (2020). Figures Page 8/13 2013 NCHS urban-rural classification scheme for counties. US Department of Health and Human Services, Centers for Disease Control and Prevention, National Center for Health Statistics (2014). 41. Wang, R., Ji, C., Jiang, Z., Wu, Y., Yin, L., & Li, Y. A short-term prediction model at the early stage of the COVID-19 pandemic based on multisource urban data. IEEE Transactions on Computational Social Systems, 8(4), 938-945 (2021). 41. Wang, R., Ji, C., Jiang, Z., Wu, Y., Yin, L., & Li, Y. A short-term prediction model at the early stage of the COVID-19 pandemic based on multisource urban data. IEEE Transactions on Computational Social Systems, 8(4), 938-945 (2021). 42. Ayan, N., Chaskar, S., Seetharam, A., Ramesh, A., & Antonio, A. D. A. Mobility-aware COVID-19 Case Prediction using Cellular Network Logs. In 2021 IEEE 46th Conference on Local Computer Networks (LCN) (pp. 479-486). IEEE (2021). 42. Ayan, N., Chaskar, S., Seetharam, A., Ramesh, A., & Antonio, A. D. A. Mobility-aware COVID-19 Case Prediction using Cellular Network Logs. In 2021 IEEE 46th Conference on Local Computer Networks (LCN) (pp. 479-486). IEEE (2021). 43. Şahin, M. Forecasting COVID-19 cases based on mobility. MANAS Journal of Engineering, 8 44. Zeng, C. et al. Spatial-temporal relationship between population mobility and COVID-19 outbreaks in South Carolina: time series forecasting analysis. Journal of medical Internet research, 23(4), e27045 (2021). 44. Zeng, C. et al. Spatial-temporal relationship between population mobility and COVID-19 outbreaks in South Carolina: time series forecasting analysis. Journal of medical Internet research, 23(4), e27045 (2021). 45. Klein, B. et al. Forecasting hospital-level COVID-19 admissions using real-time mobility data. medRxiv (2022). 45. Klein, B. et al. Forecasting hospital-level COVID-19 admissions using real-time mobility data 46. Januschowski, T., Gasthaus, J., & Wang, Y. Open-Source Forecasting Tools in Python. Foresight: The International Journal of Applied Forecasting, 55, (2019). 46. Januschowski, T., Gasthaus, J., & Wang, Y. Open-Source Forecasting Tools in Python. Foresight: The International Journal of Applied Forecasting, 55, (2019). 47. Gasser, U., Ienca, M., Scheibner, J., Sleigh, J., & Vayena, E. Digital tools against COVID-19: taxonomy, ethical challenges, and navigation aid. The Lancet Digital Health, 2(8), e425-e434 (2020). 47. Gasser, U., Ienca, M., Scheibner, J., Sleigh, J., & Vayena, E. Digital tools against COVID-19: taxonomy, ethical challenges, and navigation aid. The Lancet Digital Health, 2(8), e425-e434 (2020). 48. Li, L., Erfani, A., Wang, Y., & Cui, Q. 50. Budd, J. et al. Digital technologies in the public-health response to COVID-19. Nature medici Correlation analysis with selected factors on a weekly basis for Model 1. (a) NCHS code (b) Age, and (c) education. Correlation analysis with selected factors on a weekly basis for Model 2. (a) Income (b) Smartphone ownership, and (c) education. Figures Page 9/13 Page 9/13 e 1 on mobility measures, COVID-19 infections. (a) Weekly average number of trips across the U.S. (b D infections across the U.S. Figure 1 Data on mobility measures, COVID-19 infections. (a) Weekly average number of trips across the U.S. (b) Weekly new number of COVID infections across the U.S. Page 10/13 re 2 iction error rate on a weekly basis. (a) Regression model (b) Time series model ure 2 Figure 2 Prediction error rate on a weekly basis. (a) Regression model (b) Time series model Page 11/13 ure 3 relation analysis with selected factors on a weekly basis for Model 1. (a) NCHS code (b) Age, and (c) education. Figure 3 Correlation analysis with selected factors on a weekly basis for Mode Figure 3 Correlation analysis with selected factors on a weekly basis for Model 1. (a) NCHS code (b) Age, and (c) education. Correlation analysis with selected factors on a weekly basis for Model 1. (a) NCHS code (b) Age, and (c) education. Page 12/13 Page 12/13 Figure 4 Correlation analysis with selected factors on a weekly basis for Model 2. (a) Income (b) Smartphone ownership, and (c) education. Figure 4 Correlation analysis with selected factors on a weekly basis for Mo Figure 4 Correlation analysis with selected factors on a weekly basis for Model 2. (a) Income (b) Smartphone ownership, and (c) education. Page 13/13
https://openalex.org/W4200325438	https://ccforum.biomedcentral.com/track/pdf/10.1186/s13054-021-03864-3	English	null	Predictors for extubation failure in COVID-19 patients using a machine learning approach	Critical care	2,021	cc-by	8,905	Fleuren et al. Critical Care (2021) 25:448 https://doi.org/10.1186/s13054-021-03864-3 Fleuren et al. Critical Care (2021) 25:448 https://doi.org/10.1186/s13054-021-03864-3 RESEARCH Predictors for extubation failure in COVID‑19 patients using a machine learning approach Lucas M. Fleuren1* , Tariq A. Dam1, Michele Tonutti2, Daan P. de Bruin2, Robbert C. A. Lalisang2, Diederik Gommers3, Olaf L. Cremer4, Rob J. Bosman5, Sander Rigter6, Evert‑Jan Wils7, Tim Frenzel8, Dave A. Dongelmans9, Remko de Jong10, Marco Peters11, Marlijn J. A. Kamps12, Dharmanand Ramnarain13, Ralph Nowitzky14, Fleur G. C. A. Nooteboom15, Wouter de Ruijter16, Louise C. Urlings‑Strop17, Ellen G. M. Smit18, D. Jannet Mehagnoul‑Schipper19, Tom Dormans20, Cornelis P. C. de Jager21, Stefaan H. A. Hendriks22, Sefanja Achterberg23, Evelien Oostdijk24, Auke C. Reidinga25, Barbara Festen‑Spanjer26, Gert B. Brunnekreef27, Alexander D. Cornet28, Walter van den Tempel29, Age D. Boelens30, Peter Koetsier31, Judith Lens32, Harald J. Faber33, A. Karakus34, Robert Entjes35, Paul de Jong36, Thijs C. D. Rettig37, Sesmu Arbous38, Sebastiaan J. J. Vonk2, Mattia Fornasa2, Tomas Machado2, Taco Houwert2, Hidde Hovenkamp2, Roberto Noorduijn Londono2, Davide Quintarelli2, Martijn G. Scholtemeijer2, Aletta A. de Beer2, Giovanni Cinà2, Adam Kantorik2, Tom de Ruijter39, Willem E. Herter2, Martijn Beudel40, Armand R. J. Girbes1, Mark Hoogendoorn41, Patrick J. Thoral1, Paul W. G. Elbers1 and the Dutch ICU Data Sharing Against Covid-19 Collaborators Open Access Predictors for extubation failure in COVID‑19 patients using a machine learning approach Lucas M. Fleuren1* , Tariq A. Dam1, Michele Tonutti2, Daan P. de Bruin2, Robbert C. A. Lalisang2, Diederik Gommers3, Olaf L. Cremer4, Rob J. Bosman5, Sander Rigter6, Evert‑Jan Wils7, Tim Frenzel8, Dave A. Dongelmans9, Remko de Jong10, Marco Peters11, Marlijn J. A. Kamps12, Dharmanand Ramnarain13, Ralph Nowitzky14, Fleur G. C. A. Nooteboom15, Wouter de Ruijter16, Louise C. Urlings‑Strop17, Ellen G. M. Smit18, D. Jannet Mehagnoul‑Schipper19, Tom Dormans20, Cornelis P. C. de Jager21, Stefaan H. A. Hendriks22, Sefanja Achterberg23, Evelien Oostdijk24, Auke C. Reidinga25, Barbara Festen‑Spanjer26, Gert B. Brunnekreef27, Alexander D. Cornet28, Walter van den Tempel29, Age D. Boelens30, Peter Koetsier31, Judith Lens32, Harald J. Faber33, A. Karakus34, Robert Entjes35, Paul de Jong36, Thijs C. D. Rettig37, Sesmu Arbous38, Sebastiaan J. J. Vonk2, Mattia Fornasa2, Tomas Machado2, Taco Houwert2, Hidde Hovenkamp2, Roberto Noorduijn Londono2, Davide Quintarelli2, Martijn G. Scholtemeijer2, Aletta A. de Beer2, Giovanni Cinà2, Adam Kantorik2, Tom de Ruijter39, Willem E. Herter2, Martijn Beudel40, Armand R. J. Girbes1, Mark Hoogendoorn41, Patrick J. Thoral1, Paul W. G. Elbers1 and the Dutch ICU Data Sharing Against Covid-19 Collaborators Abstract Introduction: Determining the optimal timing for extubation can be challenging in the intensive care. In this study, we aim to identify predictors for extubation failure in critically ill patients with COVID-19. Methods: We used highly granular data from 3464 adult critically ill COVID patients in the multicenter Dutch Data Warehouse, including demographics, clinical observations, medications, fluid balance, laboratory values, vital signs, and data from life support devices. All intubated patients with at least one extubation attempt were eligible for analy‑ sis. Transferred patients, patients admitted for less than 24 h, and patients still admitted at the time of data extraction were excluded. Potential predictors were selected by a team of intensive care physicians. The primary and secondary outcomes were extubation without reintubation or death within the next 7 days and within 48 h, respectively. We trained and validated multiple machine learning algorithms using fivefold nested cross-validation. Predictor impor‑ tance was estimated using Shapley additive explanations, while cutoff values for the relative probability of failed extubation were estimated through partial dependence plots. Results: A total of 883 patients were included in the model derivation. The reintubation rate was 13.4% within 48 h and 18.9% at day 7, with a mortality rate of 0.6% and 1.0% respectively. The grandient-boost model performed best (area under the curve of 0.70) and was used to calculate predictor importance. Ventilatory characteristics and settings Correspondence: l.fleuren@amsterdamumc.nl 1 Department of Intensive Care Medicine, Laboratory for Critical Care Computational Intelligence, Amsterdam Medical Data Science, Amsterdam UMC, Vrije Universiteit, Amsterdam, The Netherlands Full list of author information is available at the end of the article Correspondence: l.fleuren@amsterdamumc.nl 1 Department of Intensive Care Medicine, Laboratory for Critical Care Computational Intelligence, Amsterdam Medical Data Science, Amsterdam UMC, Vrije Universiteit, Amsterdam, The Netherlands Full list of author information is available at the end of the article *Correspondence: l.fleuren@amsterdamumc.nl 1 Department of Intensive Care Medicine, Laboratory for Critical Care Computational Intelligence, Amsterdam Medical Data Science, Amsterdam UMC, Vrije Universiteit, Amsterdam, The Netherlands Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Patients All critically ill patients extubated after more than 24 h of invasive mechanical ventilation were eligible for inclu- sion. Transferred patients were included if the transfer destination data were available. We excluded patients transferred before extubation or within 1 day after extu- bation in case the transfer destination data were not available. Patients transferred more than 24 h after extu- bation were assumed to be fit for transport and classified as successful extubations. Patients still admitted at the time of data collection were excluded. The collection of large intensive care datasets that span the entire intensive care admission paves the way for machine learning models to capture this complex inter- play of predictors by using machine learning models. Pre- vious non-COVID-19 machine learning work has aimed to predict simple and difficult weaning [7] and extubation failure [8–15]. However, data was frequently from over a decade ago, mechanical ventilator data was usually lack- ing, and no data was included from COVID-19 patients. Taken together, we identify an opportunity for machine learning models to predict unsuccessful extubation in critically ill COVID-19 patients. Data source All data came from the DDW, a large, multicenter, full- admission, electronic health record data warehouse with data from critically ill COVID-19 patients in the Nether- lands [16]. The data warehouse currently contains 3464 patients admitted between the beginning of the crisis in March 2020 and March 2021. Data spans both the first and second wave of ICU admissions from 25 hospitals in the Netherlands. The institutional review board of Amsterdam University Medical Center location VUmc waived the need for informed consent from individual patients and approved of an opt out procedure. An understanding of the risk factors for extubation fail- ure will aid the clinician in determining the optimal time point for extubation. Previous studies in non-COVID-19 patients have investigated numerous factors related to extubation outcome, including age, maximum inspira- tory pressure, and the rapid shallow breathing index [4]. However, given the complex interplay of many patient and treatment related characteristics in extubation suc- cess, a single parameter rarely provides sufficient accu- racy to guide decision making [5]. Moreover, it remains largely unclear whether these parameters are similar for COVID-19 patients [6].h © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Fleuren et al. Critical Care (2021) 25:448 Page 2 of 10 were the most important predictors. More specifically, a controlled mode duration longer than 4 days, a last fraction of inspired oxygen higher than 35%, a mean tidal volume per kg ideal body weight above 8 ml/kg in the day before extubation, and a shorter duration in assisted mode (< 2 days) compared to their median values. Additionally, a higher C-reactive protein and leukocyte count, a lower thrombocyte count, a lower Glasgow coma scale and a lower body mass index compared to their medians were associated with extubation failure. Conclusion: The most important predictors for extubation failure in critically ill COVID-19 patients include ventilatory settings, inflammatory parameters, neurological status, and body mass index. These predictors should therefore be routinely captured in electronic health records. Keywords: Extubation, Prediction, Risk factors, Extubation failure the bedside. In this study, we aim to identify and validate the most important predictors for extubation failure in COVID-19 patients. Introductionh The decision to extubate a COVID-19 patient can be challenging and a delicate trade-off between early and postponed extubation. In non-COVID patients, extu- bation failure occurs in 10–20% of intensive care cases and is associated with increased mortality [1]. While postponing extubation and waiting for further clinical improvement appears sensible, unnecessary extubation delays may lead to more ventilator-associated complica- tions and inefficient use of scarce intensive care resources [2, 3]. Methodsh This study follows the transparent reporting of a multi- variable prediction model for individual prognosis or diagnosis (TRIPOD) guidelines [17]. Outcomesh We created the Dutch Data Warehouse (DDW), a mul- ticenter database with critically ill COVID-19 patients [16]. All structured electronic health record (EHR) data for these patients have been combined and cleaned for research purposes. These data therefore represent the structured EHR data readily available to the intensivist at The primary outcome was unsuccessful separation from invasive mechanical ventilation defined according to the WIND criteria [18], which mandate an extubation without reintubation or death within the next 7 days, or discharge from the ICU without invasive mechanical ventilation within 7 days [18]. The use of non-invasive Fleuren et al. Critical Care (2021) 25:448 Fleuren et al. Predictors and scoping literature search Potential predictors for modeling were selected by a team of intensivists. Notably, the list included medication and fluid balance. To facilitate the selection process, machine learning studies that predict extubation failure were identified in the literature. Each of the identified arti- cles was scanned full-text and included predictors were extracted. The total list of studies can be found in Addi- tional file 1: Table S1. In addition, to account for the wide variety of ventilator settings in the DDW, the parameters from the landmark paper by Amato et al. on the associa- tion between ventilator parameters and outcome were included in the selection [19]. The mean or last value from the last 24 h before extubation as specified by the team of intensivists were included to facilitate interpre- tation of the model. The total dose in the last 24 h was included for the medications. For any predictor pair with an interpredictor correlation higher than 0.5, the most clinically insightful predictor was selected. The full list of predictors can be found in Table 1. Outcomesh Critical Care (2021) 25:448 Page 3 of 10 Table 1 Included parameters Overview of included parameters and their aggregation FiO2: Fraction of inspired oxygen, PEEP: positive end expiratory pressure, P/F ratio: PaO2/FiO2 ratio, PCO2: partial pressure of carbon dioxide, RASS score: Richmond Agitation and Sedation Scale Predictor Aggregation Age Apache-II score Mean last 24 h Body mass index (BMI) C-reactive protein Mean last 24 h Creatinine Mean last 24 h FiO2 Mean last 24 h, Last value Fluid balance Sum Glasgow coma scale Mean last 24 h Glucose Mean last 24 h Cardiac comorbidity Diabetes comorbidity Respiratory comorbidity Renal comorbidity Heart rate Mean last 24 h Hematocrit Mean last 24 h Gender Duration of mechanical ventilation Leukocyte count Mean last 24 h Benzodiazepine dose Given in last 24 h Clonidine dose Given in last 24 h, Total dose last 24 h Dexmedetomidine dose Given in last 24 h, Total dose last 24 h Fentanyl dose Given in last 24 h, Total dose last 24 h Haloperidol dose Given in last 24 h, Total dose last 24 h Midazolam dose Given in last 24 h, Total dose last 24 h Propofol dose Given in last 24 h, Total dose last 24 h Quetiapine dose Given in last 24 h, Total dose last 24 h p0.1 Mean last 24 h, Last value P/F ratio Mean last 24 h PCO2 arterial Last value PEEP Last value pH Last value Hours since last proning session Pressure above PEEP Mean last 24 h, Last value Respiratory rate Mean last 24 h RASS score Mean last 24 h, Last value Thrombocyte count Mean last 24 h Tidal volume per kg ideal body weight Mean last 24 h, Last value Duration of controlled mode Hours since last controlled mode Ventilatory ratio Mean last 24 h Table 1 Included parameters ventilation is disregarded in this definition. As second- ary outcomes, we applied the same criteria to a 48 h’ time window after extubation. The definition of extubation in EHR data has been published previously and reasonably excludes palliative care patients [16]. We did not distin- guish between accidental and elective extubations as the reason for extubation is not routinely recorded. Modeling A ll Across all 25 hospitals, a nested cross validation was per- formed to assess model performance. First, the data was split into five equally large sets called outer folds. These outer folds were then each split into a train and test set. Each of the train sets was again divided into five subsets called the inner folds. A model was trained on these 5 inner folds with a randomized hyperparameter search. Model performance after training on these inner folds was then tested on the corresponding outer fold test set. Importantly, observations belonging to the same patient were always kept in the same split to prevent leakage of information. The overall model performance was the average of all outer fold test set performances. We trained a logistic regression model, decision trees, and an XGBoost algorithm. These models were selected for their ease of determining predictor importance. Model performance was gauged with the area under the receiver operating characteristic (AUROC), Brier score, average precision, and calibration loss. Data imputa- tion, standardization and automated feature selection were carried out on each outer fold separately. Missing values were imputed with the median and predictors were standardized to have a mean of 0 and a standard Fleuren et al. Critical Care (2021) 25:448 Page 4 of 10 ventilatory characteristics, inflammation markers, neuro- logical status and body mass index. deviation of 1. Lasso regression was performed for auto- matic feature selection, and the L1 regularization term was optimized together with the other hyperparameters [20]. Population and outcome A total of 2.421 patients were mechanically ventilated during their ICU stay. In case of a patient transfer, data from the transferring and receiving hospital were merged when available. We excluded 517 transfers for which out- come or admission data were lacking, 123 patients that were still intubated when data were extracted, and 139 patients that were intubated less than 24 h. 568 patients died on the mechanical ventilator before their first extu- bation attempt and 191 patients received a tracheostomy. As a result, a total of 883 patients were included in the modeling. The reintubation rate in this COVID-19 pop- ulation was 18.9% within 7 days and 13.4% within 48 h. The mortality rate was 1.0% within 7 days and 0.6% in the first 48 h after extubation. Patient characteristics are out- lined in Table 2. Modeling Model performance for the primary outcome is shown in Additional file 1: Table S2 for each of the models. The XGBoost algorithm yielded the highest performance with an AUROC of 0.70, outperforming logistic regression (AUROC 0.67) and a decision tree (AUROC 0.59). Model performance for the prediction of unsuccessful extuba- tion 48 h after extubation is presented in Additional file 1: Table S2. All algorithms, XGBoost (AUROC 0.67), logis- tic regression (0.66), and a decision tree (AUROC 0.54), performed worse compared to the primary outcome. Inflammation markers, neurological scores and body mass index Both a higher CRP, an elevated leukocyte count and higher thrombocyte count in the 24 h preceding extu- bation are predictors of an unsuccessful extubation attempt, while temperature was not in the top predict- ing features. For neurological scores, on the other hand, low EMV scores predict unsuccessful extubation. Lastly, BMI showed an inverse relationship with extubation fail- ure; patients with a higher BMI had a lower probability of extubation failure. An increase in the chances of unsuc- cessful extubation is observed below 28 kg/m2 compared to the median in the PD-plot (shown in Additional file 1: Fig. S1). Ventilatory characteristics Predictor importance was estimated with the Shap- ley additive explanation (SHAP) framework. SHAP val- ues represent a predictor’s marginal contribution to the overall prediction [21] and are state of the art in machine learning explainability. Moreover, Partial Dependence Plots (PDPs) were created to visualize the average change in probability of successful extubation for all values of a predictor while keeping all other predictors constant [22]. Standard deviations represent the distribution of the data. All analyses were carried out in Python 3.8 (Python software foundation). Ventilatory characteristics are shown in Table 2. A short time-period between the last controlled mode and extubation, and a longer duration in controlled mode throughout the course of mechanical ventilation were associated with unsuccessful extubation. The PD-plots depict the difference in predicted probability of extuba- tion failure compared to the median value for all of the observed values. The PD-plot shows a time since the last controlled mode shorter than 2 days and a controlled mode duration longer than 4 days are associated with increased chances of unsuccessful extubation compared to the median value. For the ventilator settings, a higher fraction of inspired oxygen and a higher average tidal volume in the last 24 h are predictive of extubation failure. The PD-plot in Fig. 2 shows that an FiO2 above 35% or a tidal volume per kg ideal body weight above 8 ml/kg compared to their median values increases the probability of unsuccessful extubation. The median PEEP was 8 cmH2O (IQR 5–8 cmH2O) before extubation, with a median pressure sup- port of 6 cmH2O (IQR 5–9 cmH2O). No patients received PEEP levels below 5 cmH2O, while pressure above PEEP was below 5 cmH2O in 7.3% of patients. Discussion To the best of our knowledge, this is the first study that identifies predictors for extubation failure in critically ill COVID-19 patients from a large and multicenter cohort that contains a wide variety of routinely collected clini- cal predictors. The most important predictors of extuba- tion failure are ventilatory characteristics, inflammatory parameters, GCS score, and body mass index. These risk factors may aid intensive care professionals in selecting the optimal time point for extubation. Predictor importance Predictor importance was calculated with the XGBoost model since it yielded the highest performance. The SHAP values for the highest predictors are shown in Fig. 1. The most important predictive feature of extuba- tion failure was the last FiO2 value before extubation. The majority of important predictors can be grouped into Fleuren et al. Critical Care (2021) 25:448 Page 5 of 10 Table 2 Patient characteristics Table 2 Patient characteristics Overview of patient characteristics, lab characteristics and ventilatory characteristics before extubation. Predictor importance All values are medians with an interquartile range, unless otherwise indicated FiO : Fraction of inspired oxygen PEEP: positive end expiratory pressure P/F ratio: PaO /FiO ratio IBW: ideal body weight PO : partial pressure of oxygen PCO : Total patients (N = 883) Successful extubation (N = 707) Unsuccessful extubation (N = 176) Male 71.5% (N = 883) 70.4% (N = 707) 75.6% (N = 176) Age, years 63 (55–70, N = 883) 63 (55–69, N = 707) 65 (57–72, N = 176) Age, years < 60 360 (40.8%) 301 (42.6%) 59 (33.5%) 60–70 314 (35.6%) 249 (35.2%) 65 (36.9%) 70–80 199 (22.5%) 150 (21.2%) 49 (27.8%) > 80 10 (1.1%) 7 (1.0%) 3 (1.7%) Body mass index, kg/m2 27.9 (25.1–31.6, N = 809) 28.4 (25.4–32.0, N = 642) 26.8 (24.2–30.1, N = 167) Body mass index, kg/m2 < 25 200 (24.7%) 145 (22.6%) 55 (32.9%) 25–30 336 (41.5%) 267 (41.6%) 69 (41.3%) 30–35 174 (21.5%) 145 (22.6%) 29 (17.4%) > 35 98 (12.1%) 84 (13.1%) 14 (8.4%) Lab values (last 24 h before extubation) C-reactive protein, mg/L 57 (23–114, N = 731) 53 (21–108, N = 583) 72 (33–146, N = 148) Creatinine, micromol/L 66 (52–96, N = 820) 65 (52–90, N = 657) 66 (53–108, N = 163) Leukocyte count, 109/L 11.3 (8.8–14.2, N = 817) 11.2 (8.8–13.9, N = 653) 11.7 (8.9–15.5, N = 164) Thrombocyte count, 109/L 350 (269–457, N = 824) 356 (269–462, N = 664) 340 (259–419, N = 160) Respiratory characteristics (last measured) Time since last controlled mode, hours 74 (35–130, N = 773) 77 (37–137, N = 603) 64 (27–115, N = 170) FiO2, % 36 (30–41, N = 866) 35 (30–41, N = 691) 40 (33–45, N = 175) Pressure above PEEP, cmH2O 6 (5–9, N = 773) 6 (5–10, N = 617) 7 (5–8, N = 156) PEEP, cmH2O 8 (5–8, N = 867) 8 (6–9, N = 693) 7 (5–8, N = 174) Respiratory rate, /min 22 (18–26, N = 883) 22 (18–26, N = 707) 23 (18–27, N = 176) Tidal volume, ml/kg IBW 7.5 (6.4–9.0, N = 857) 7.5 (6.3–8.9, N = 682) 7.6 (6.6–9.1, N = 175) Ventilatory ratio 1.8 (1.4–2.3, N = 722) 1.8 (1.4–2.3, N = 577) 1.9 (1.5–2.4, N = 145) P/F ratio 206 (168–258, N = 861) 209 (171–262, N = 690) 192 (163–242, N = 171) pO2 arterial, mmHg 74 (67–84, N = 792) 75 (68–85, N = 634) 72 (65–83, N = 158) pCO2 arterial, mmHg 41 (37–46, N = 732) 41 (37–46, N = 579) 40 (36–46, N = 153) Bicarbonate arterial, mmol/L 29 (26–31, N = 861) 29 (26–32, N = 688) 28 (25–31, N = 173) Airway occlusion pressure (P0.1), cmH2O 2.1 (1.3–3.8, N = 450) 2.1 (1.2–3.7, N = 356) 2.3 (1.4–4.0, N = 94) erview of patient characteristics, lab characteristics and ventilatory characteristics before extubation. Predictor importance All values are medians with an interq erwise indicated FiO2: Fraction of inspired oxygen, PEEP: positive end expiratory pressure, P/F ratio: PaO2/FiO2 ratio, IBW: ideal body weight, PO2: partial pressure of oxygen, PCO2: partial pressure of carbon dioxide This study is unique as it provides predictive modeling of extubation failure across twenty-five hospitals. All pre- vious machine learning studies in non-COVID patients for predicting extubation failure have been single center [7–15]. Model performance was higher in these studies, presumably due to overfitting resulting from the sole use of local data. Algorithms may be biased towards local extubation practices and extubation readiness assess- ments, making these models less generalizable to other clinical settings. extubation failure. These factors are systematically and frequently recorded by the ventilators, and are poten- tially modifiable. Two of the most important predictors associated with higher chances of failed extubation are the duration of the controlled and assisted ventilation modes prior to extubation. A longer time in a controlled mode was a stronger predictor than the total duration of mechanical ventilation. Moreover, a longer time in assisted mode was associated with improved chances of successful extubation. A possible explanation may be the reduced activity and consequent atrophy of the dia- phragm or other skeletal muscles in controlled modes In our study, ventilatory characteristics, including ven- tilator settings, are the most important risk factors for Fleuren et al. Critical Care (2021) 25:448 Page 6 of 10 Fig. 1 SHAP values for most important predictors of extubation failure. Overview of SHAP values for the top 20 predictors of successful extubation (negative SHAP values) or unsuccessful extubation (positive SHAP values). Features are ordered according to importance. FiO2: fraction of inspired oxygen, IBW: ideal body weight, PEEP: positive end expiratory pressure, P/F ratio: PaO2/FiO2 ratio Fig. 1 SHAP values for most important predictors of extubation failure. Overview of SHAP values for the top 20 predictors of successful extubation (negative SHAP values) or unsuccessful extubation (positive SHAP values). Features are ordered according to importance. FiO2: fraction of inspired oxygen, IBW: ideal body weight, PEEP: positive end expiratory pressure, P/F ratio: PaO2/FiO2 ratio Fig. 1 SHAP values for most important predictors of extubation failure. Overview of SHAP values for the top 20 predictors of successful extubation (negative SHAP values) or unsuccessful extubation (positive SHAP values). Features are ordered according to importance. Predictor importance FiO2: fraction of inspired oxygen, IBW: ideal body weight, PEEP: positive end expiratory pressure, P/F ratio: PaO2/FiO2 ratio before an extubation attempt. In addition, we observed that higher mean tidal volumes corrected for the ideal body weight in the last day before extubation were an important predictor of extubation failure. Patients with high average tidal volumes may suffer from more lung injury that may increase the risk of unsuccessful extu- bation [25]. While most of the ventilator settings are readily available, relevant respiratory system maneuvers such as spontaneous breathing trials, tracheobronchial suctioning and maximum inspiratory pressure that would ideally be included, were inconsistently recorded in the EHR systems and therefore not included in modelling. To evaluate their predicting importance in [23, 24]. Of note, none of the previous machine learning studies included the duration of controlled ventilation as a predictor. Our results show that the duration of venti- lation modes should be recorded and taken into account when assessing extubation readiness. For the ventilator settings, a higher FiO2 before extu- bation was associated with an increased risk of extu- bation failure. A higher FiO2 may indicate incomplete resolution of pulmonary pathology. Higher PEEP levels, on the other hand, were associated with better extu- bation success. The interquartile ranges of PEEP are low, however, indicating low PEEP is common practice Fleuren et al. Critical Care (2021) 25:448 Page 7 of 10 ependence plots. PD-plot for the last FiO2 recording, mean glasgow coma score and tidal volume per kg ideal body weight in the uration of the controlled mode Fig. 2 Partial dependence plots. PD-plot for the last FiO2 recording, mean glasgow coma score and tidal volume per kg ideal body weight in the Fig. 2 Partial dependence plots. PD-plot for the last FiO2 recording, mean glasgow coma score and tidal volume per kg ideal body weight in the last 24 h, and duration of the controlled mode Fleuren et al. Critical Care (2021) 25:448 Page 8 of 10 Page 8 of 10 extubation failure, data of these maneuvers need to be incorporated systematically in the EHR. as the study population as a whole [31]. Lastly, the rela- tionships identified in this study are associations and do not equal causation. As with any clinical observational dataset, we cannot observe counterfactual states; once a patient is extubated we irretrievably lose the outcome in case the patient would have been kept on mechanical ventilation. Abbreviations APACHE II A APACHE II: Acute physiology and chronic health evaluation iI; AUROC: Area under the receiver operating characteristics curve; BMI: Body mass index; CRP: C-reactive protein; DDW: Dutch data warehouse; EHR: Electronic health record; EMV score: Eye motor verbal score; FiO2: Fraction of inspired oxygen; GCS: Glasgow coma scale; IQR: Interquartile range; PDP: Partial dependence plot; PEEP: Positive end expiratory pressure; RASS: Richmond agitation and sedation scales; SHAP: Shapley additive explanation; SOFA: Sequential organ failure assessment. Our study has several limitations. We aim to apply a holistic set of predictors across centers to assess extu- bation readiness. In routine practice, however, indi- vidualized treatment and diagnostic decisions result in variation of available parameters [29], and predictors may be unavailable in the 24 h prior to extubation. For example, it is not possible to conclude that cardiac mark- ers like NT-pro-BNP or troponin do not aid in the pre- diction of extubation failure, because these markers were not routinely determined. Along the same line, we had to merge groups of medications, because individual drugs may not be administered frequently enough to be use- ful in the modeling. To truly exploit the predictive power of machine learning models, we should strive to system- atically record the predictors of interest and determine which algorithms work in what clinical circumstances [30]. Predictor importance While many of the ventilatory settings are predictive of extubation failure, we would ultimately be interested in the effects of continuing mechanical venti- lation for another day on extubation success. We believe that these results will provide a crucial step for other study designs to investigate the causal relation between modifiable predictors and successful extubation. Other important predictors included signals of ongo- ing or developing inflammation, poorer neurological sta- tus, and body mass index. Inflammation parameters are routinely determined in most intensive care units when extubation decisions are made. Conversely, neurological scores can be ambivalently scored in the intensive care unit. The Glasgow Coma Scale was originally designed for brain damage patients [26], but is used for the gen- eral intensive care patient. Unequivocal interpretation of sedated states, however, may hamper the use of this scale in the context of extubation readiness. Based on these results, we would recommend systematically recording and evaluating the predictive value of other scores like the Richmond agitation sedation scales. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s13054-021-03864-3. Additional file 1. Table S1 Overview of machine learning studies that investigate extubation readiness. Table S2 Model performance. Figure S1 PD-plot for BMI Conclusionhi This is the first study to identify risk factors of extuba- tion failure in a large multi-center cohort of critically ill COVID-19 patients. The large number of hospitals included limits the risk of overfitting due to specific local practices. From a large set of clinically important predic- tors, ventilatory characteristics, inflammatory markers, neurological status and BMI were most important pre- dictors for failed extubation. These predictors should be taken into account to determine extubation readiness. Lastly, body mass index upon admission had an inverse relationship with extubation failure. Apart from one small study that found an association between BMI and post extubation stridor [27], no other studies were identi- fied that found BMI to be an important predictor. As in any predictive study, the effect of BMI may be explained by an unmeasured predictor or a selection bias. That means, a low-BMI patient would have to be sicker to be admitted to the ICU. A negligible correlation was found between BMI and SOFA score, however, as an indicator of illness severity. Previous studies have also shown that BMI is uncorrelated with immunological responses or adverse outcomes [28]. Overall, once in the ICU, BMI is not related to higher chances of unsuccessful extubation and may not be a valid reason to postpone extubation. Acknowledgements The Dutch ICU Data Sharing Against COVID-19 Collaborators: From collaborat‑ ing hospitals having shared data: Julia Koeter, MD, Intensive Care, Canisius Wilhelmina Ziekenhuis, Nijmegen, The Netherlands. Roger van Rietschote, Business Intelligence, Haaglanden MC, Den Haag, The Netherlands. M.C. Reuland, MD, Department of Intensive Care Medicine, Amsterdam UMC, Universiteit van Amsterdam, Amsterdam, The Netherlands. Laura van Manen, MD, Department of Intensive Care, BovenIJ Ziekenhuis, Amsterdam, The Netherlands. Leon Montenij, MD, PhD, Department of Anesthesiology, Pain Management and Intensive Care, Catharina Ziekenhuis Eindhoven, Eindhoven, The Netherlands. Jasper van Bommel, MD, PhD, Department of Intensive Care, Erasmus Medical Center, Rotterdam, The Netherlands. Roy van den Berg, Department of Intensive Care, ETZ Tilburg, Tilburg, The Netherlands. Ellen van Geest, Department of ICMT, Haga Ziekenhuis, Den Haag, The Netherlands. Anisa Hana, MD, PhD, Intensive Care, Laurentius Ziekenhuis, Roermond, The Netherlands. B. van den Bogaard, MD, PhD, ICU, OLVG, Amsterdam, The A further limitation is the missing outcome data because of patient transfers to centers not included in this project. The potential bias is considered small, as we connected all patients’ stays whenever available and transferred patients had similar baseline characteristics Page 9 of 10 Page 9 of 10 Fleuren et al. Critical Care (2021) 25:448 Fleuren et al. Critical Care (2021) 25:448 Netherlands. Prof. Peter Pickkers, Department of Intensive Care Medicine, Radboud University Medical Centre, Nijmegen, The Netherlands. Pim van der Heiden, MD, PhD, Intensive Care, Reinier de Graaf Gasthuis, Delft, The Nether‑ lands. Claudia (C.W.) van Gemeren, MD, Intensive Care, Spaarne Gasthuis, Haar‑ lem en Hoofddorp, The Netherlands. Arend Jan Meinders, MD, Department of Internal Medicine and Intensive Care, St Antonius Hospital, Nieuwegein, The Netherlands. Martha de Bruin, MD, Department of Intensive Care, Franciscus Gasthuis & Vlietland, Rotterdam, The Netherlands. Emma Rademaker, MD, MSc, Department of Intensive Care, UMC Utrecht, Utrecht, The Netherlands. Frits H.M. van Osch, PhD, Department of Clinical Epidemiology, VieCuri Medisch Centrum, Venlo, The Netherlands. Martijn de Kruif, MD, PhD, Department of Pulmonology, Zuyderland MC, Heerlen, The Netherlands. Nicolas Schroten, MD, Intensive Care, Albert Schweitzerziekenhuis, Dordrecht, The Netherlands. Klaas Sierk Arnold, MD, Anesthesiology, Antonius Ziekenhuis Sneek, Sneek, The Netherlands. J. W. Fijen, MD, PhD, Department of Intensive Care, Diakonessen‑ huis Hospital, Utrecht, The Netherland. Jacomar J. M. van Koesveld, MD, ICU, IJsselland Ziekenhuis, Capelle aan den IJssel, The Netherlands. Koen S. Simons, MD, PhD, Department of Intensive Care, Jeroen Bosch Ziekenhuis, Den Bosch, The Netherlands. Author details Author details 1 Department of Intensive Care Medicine, Laboratory for Critical Care Com‑ putational Intelligence, Amsterdam Medical Data Science, Amsterdam UMC, Vrije Universiteit, Amsterdam, The Netherlands. 2 Pacmed, Amsterdam, The Netherlands. 3 Department of Intensive Care, Erasmus Medical Center, Rotter‑ dam, The Netherlands. 4 Department of Intensive Care, UMC Utrecht, Utrecht, The Netherlands. 5 ICU, OLVG, Amsterdam, The Netherlands. 6 Department of Anesthesiology and Intensive Care, St. Antonius Hospital, Nieuwegein, The Netherlands. 7 Department of Intensive Care, Franciscus Gasthuis and Vliet‑ land, Rotterdam, The Netherlands. 8 Department of Intensive Care Medicine, Radboud University Medical Center, Nijmegen, The Netherlands. 9 Department of Intensive Care Medicine, Amsterdam UMC, Amsterdam, The Netherlands. 10 Intensive Care, Bovenij Ziekenhuis, Amsterdam, The Netherlands. 11 Intensive Care, Canisius Wilhelmina Ziekenhuis, Nijmegen, The Netherlands. 12 Intensive Care, Catharina Ziekenhuis Eindhoven, Eindhoven, The Netherlands. 13 Depart‑ ment of Intensive Care, ETZ Tilburg, Tilburg, The Netherlands. 14 Intensive Care, HagaZiekenhuis, Den Haag, The Netherlands. 15 Intensive Care, Laurentius Ziekenhuis, Roermond, The Netherlands. 16 Department of Intensive Care Med‑ icine, Northwest Clinics, Alkmaar, The Netherlands. 17 Intensive Care, Reinier de Graaf Gasthuis, Delft, The Netherlands. 18 Intensive Care, Spaarne Gasthuis, Haarlem en Hoofddorp, The Netherlands. 19 Intensive Care, VieCuri Medisch Centrum, Venlo, The Netherlands. 20 Intensive Care, Zuyderland MC, Heerlen, The Netherlands. 21 Department of Intensive Care, Jeroen Bosch Ziekenhuis, Den Bosch, The Netherlands. 22 Intensive Care, Albert Schweitzerziekenhuis, Dordrecht, The Netherlands. 23 ICU, Haaglanden Medisch Centrum, Den Haag, The Netherlands. 24 ICU, Maasstad Ziekenhuis Rotterdam, Rotterdam, The Netherlands. 25 ICU, SEH, BWC, Martiniziekenhuis, Groningen, The Netherlands. 26 Intensive Care, Ziekenhuis Gelderse Vallei, Ede, The Netherlands. 27 Depart‑ ment of Intensive Care, Ziekenhuisgroep Twente, Almelo, The Netherlands. 28 Department of Intensive Care, Medisch Spectrum Twente, Enschede, The Netherlands. 29 Department of Intensive Care, Ikazia Ziekenhuis Rotterdam, Rotterdam, The Netherlands. 30 Antonius Ziekenhuis Sneek, Sneek, The Netherlands. 31 Intensive Care, Medisch Centrum Leeuwarden, Leeuwarden, The Netherlands. 32 ICU, IJsselland Ziekenhuis, Capelle Aan Den IJssel, The Netherlands. 33 ICU, WZA, Assen, The Netherlands. 34 Department of Intensive Care, Diakonessenhuis Hospital, Utrecht, The Netherlands. 35 Department of Intensive Care, Adrz, Goes, The Netherlands. 36 Department of Anesthesia and Intensive Care, Slingeland Ziekenhuis, Doetinchem, The Netherlands. 37 Department of Anesthesiology, Intensive Care and Pain Medicine, Amphia Ziekenhuis, Breda, The Netherlands. 38 Department of Intensive Care, LUMC, Leiden, The Netherlands. 39 BigData Republic, Nieuwegein, The Netherlands. Acknowledgements Joost Labout, MD, PhD, ICU, Maasstad Ziekenhuis Rotterdam, The Netherlands. Bart van de Gaauw, MD, Martiniziekenhuis, Groningen, The Netherlands. Michael Kuiper, Intensive Care, Medisch Centrum Leeuwarden, Leeuwarden, The Netherlands. Albertus Beishuizen, MD, PhD, Department of Intensive Care, Medisch Spectrum Twente, Enschede, The Netherlands. Dennis Geutjes, Department of Information Technology, Slingeland Ziekenhuis, Doet‑ inchem, The Netherlands. Johan Lutisan, MD, ICU, WZA, Assen, The Nether‑ lands. Bart P. Grady, MD, PhD, Department of Intensive Care, Ziekenhuisgroep Twente, Almelo, The Netherlands. Remko van den Akker, Intensive Care, Adrz, Goes, The Netherlands. Tom A. Rijpstra, MD, Department of Anesthesiology, Intensive Care and Pain Medicine, Amphia Ziekenhuis, Breda, The Netherlands. W.G. Boersma, MD, PhD, Department of Pulmonology, Northwest Clinics, Alkmaar, the Netherlands. From collaborating hospitals having signed the data sharing agreement: Daniel Pretorius, MD, Department of Intensive Care Medi‑ cine, Hospital St Jansdal, Harderwijk, The Netherlands. Menno Beukema, MD, Department of Intensive Care, Streekziekenhuis Koningin Beatrix, Winterswijk, The Netherlands. Bram Simons, MD, Intensive Care, Bravis Ziekenhuis, Bergen op Zoom en Roosendaal, The Netherlands. A.A. Rijkeboer, MD, ICU, Flevoz‑ iekenhuis, Almere, The Netherlands. Marcel Aries, MD, PhD, MUMC+, Univer‑ sity Maastricht, Maastricht, The Netherlands. Niels C. Gritters van den Oever, MD, Intensive Care, Treant Zorggroep, Emmen, The Netherlands. Martijn van Tellingen, MD, EDIC, Department of Intensive Care Medicine, afdeling Intensive Care, ziekenhuis Tjongerschans, Heerenveen, The Netherlands. Annemieke Dijkstra, MD, Department of Intensive Care Medicine, Het Van Weel-Bethesda Ziekenhuis, Dirksland, The Netherlands. Rutger van Raalte, Department of Intensive Care, Tergooi hospital, Hilversum, The Netherlands. Funding Partially funded by grants from ZonMw (Project 10430012010003, file 50-55700-98-908), Zorgverzekeraars Nederland and the Corona Research Fund. The sponsors had no role in any part of the study. Availability of data and materials The Dutch Data Warehouse is available for global collaboration, within the restrictions imposed by privacy laws and ethics. Information on how to access the data warehouse may be found at www.amsterdammedicaldatascience.nl. Author details 53 Martiniziekenhuis, Groningen, The Netherlands. 54 Department of Information Technology, Slingeland Ziekenhuis, Doetinchem, The Netherlands. 55 Intensive Care, Adrz, Goes, The Netherlands. 56 Department of Pulmonology, Northwest Clinics, Alkmaar, The Netherlands. 57 Department of Intensive Care Medicine, Hospital St Jansdal, Harderwijk, The Netherlands. 58 Department of Intensive Care, Streekziekenhuis Koningin Bea‑ trix, Winterswijk, The Netherlands. 59 Intensive Care, Bravis Ziekenhuis, Bergen Op Zoom en Roosendaal, The Netherlands. 60 ICU, Flevoziekenhuis, Almere, The Netherlands. 61 MUMC+, University Maastricht, Maastricht, The Netherlands. 62 Intensive Care, Treant Zorggroep, Emmen, The Netherlands. 63 Department f I t i C M di i Afd li I t i C Zi k h i Tj h Authors’ contributions LF drafted the manuscript. TD, DB, RL, MF, TM, MS, SV, AB, DQ, RN, TH, PT, WH and PE were involved in data processing and analytics. All authors contributed to data collection and critically reviewed the manuscript. All authors have full access to the data. All authors read and approved the final manuscript. Competing interests Competing interests The authors declare that they have no competing interests. Author details 40 Department of Neurology, Amsterdam UMC, Universiteit Van Amsterdam, Amsterdam, The Netherlands. 41 Quantitative Data Analytics Group, Depart‑ ment of Computer Science, Faculty of Science, Vrije Universiteit, Amsterdam, The Netherlands. 42 Business Intelligence, Haaglanden MC, Den Haag, The Netherlands. 43 Department of Intensive Care Medicine, Amsterdam UMC, Universiteit Van Amsterdam, Amsterdam, The Netherlands. 44 Department of Intensive Care, BovenIJ Ziekenhuis, Amsterdam, The Netherlands. 45 Depart‑ ment of Anesthesiology, Pain Management and Intensive Care, Catharina Ziekenhuis Eindhoven, Eindhoven, The Netherlands. 46 Department of ICMT, Haga Ziekenhuis, Den Haag, The Netherlands. 47 Department of Intensive Care Medicine, Radboud University Medical Centre, Nijmegen, The Netherlands. 48 Department of Internal Medicine and Intensive Care, St Antonius Hospital, Nieuwegein, The Netherlands. 49 Department of Clinical Epidemiology, VieCuri Medisch Centrum, Venlo, The Netherlands. 50 Department of Pulmonol‑ ogy, Zuyderland MC, Heerlen, The Netherlands. 51 Department of Intensive Care, Diakonessenhuis Hospital, Utrecht, The Netherlands. 52 ICU, Maasstad Ziekenhuis, Rotterdam, The Netherlands. 53 Martiniziekenhuis, Groningen, The Netherlands. 54 Department of Information Technology, Slingeland Ziekenhuis, Doetinchem, The Netherlands. 55 Intensive Care, Adrz, Goes, The Netherlands. 56 Department of Pulmonology, Northwest Clinics, Alkmaar, The Netherlands. 57 Department of Intensive Care Medicine, Hospital St Jansdal, Harderwijk, The Netherlands. 58 Department of Intensive Care, Streekziekenhuis Koningin Bea‑ trix, Winterswijk, The Netherlands. 59 Intensive Care, Bravis Ziekenhuis, Bergen Op Zoom en Roosendaal, The Netherlands. 60 ICU, Flevoziekenhuis, Almere, The Netherlands. 61 MUMC+, University Maastricht, Maastricht, The Netherlands. 62 Intensive Care, Treant Zorggroep, Emmen, The Netherlands. 63 Department of Intensive Care Medicine, Afdeling Intensive Care, Ziekenhuis Tjongerschans, 1 Department of Intensive Care Medicine, Laboratory for Critical Care Com‑ putational Intelligence, Amsterdam Medical Data Science, Amsterdam UMC, Vrije Universiteit, Amsterdam, The Netherlands. 2 Pacmed, Amsterdam, The Netherlands. 3 Department of Intensive Care, Erasmus Medical Center, Rotter‑ dam, The Netherlands. 4 Department of Intensive Care, UMC Utrecht, Utrecht, The Netherlands. 5 ICU, OLVG, Amsterdam, The Netherlands. 6 Department of Anesthesiology and Intensive Care, St. Antonius Hospital, Nieuwegein, The Netherlands. 7 Department of Intensive Care, Franciscus Gasthuis and Vliet‑ land, Rotterdam, The Netherlands. 8 Department of Intensive Care Medicine, Radboud University Medical Center, Nijmegen, The Netherlands. 9 Department of Intensive Care Medicine, Amsterdam UMC, Amsterdam, The Netherlands. 10 Intensive Care, Bovenij Ziekenhuis, Amsterdam, The Netherlands. 11 Intensive Care, Canisius Wilhelmina Ziekenhuis, Nijmegen, The Netherlands. 12 Intensive Care, Catharina Ziekenhuis Eindhoven, Eindhoven, The Netherlands. 13 Depart‑ ment of Intensive Care, ETZ Tilburg, Tilburg, The Netherlands. Author details 14 Intensive Care, HagaZiekenhuis, Den Haag, The Netherlands. 15 Intensive Care, Laurentius Ziekenhuis, Roermond, The Netherlands. 16 Department of Intensive Care Med‑ icine, Northwest Clinics, Alkmaar, The Netherlands. 17 Intensive Care, Reinier de Graaf Gasthuis, Delft, The Netherlands. 18 Intensive Care, Spaarne Gasthuis, Haarlem en Hoofddorp, The Netherlands. 19 Intensive Care, VieCuri Medisch Centrum, Venlo, The Netherlands. 20 Intensive Care, Zuyderland MC, Heerlen, The Netherlands. 21 Department of Intensive Care, Jeroen Bosch Ziekenhuis, Den Bosch, The Netherlands. 22 Intensive Care, Albert Schweitzerziekenhuis, Dordrecht, The Netherlands. 23 ICU, Haaglanden Medisch Centrum, Den Haag, The Netherlands. 24 ICU, Maasstad Ziekenhuis Rotterdam, Rotterdam, The Netherlands. 25 ICU, SEH, BWC, Martiniziekenhuis, Groningen, The Netherlands. 26 Intensive Care, Ziekenhuis Gelderse Vallei, Ede, The Netherlands. 27 Depart‑ ment of Intensive Care, Ziekenhuisgroep Twente, Almelo, The Netherlands. 28 Department of Intensive Care, Medisch Spectrum Twente, Enschede, The Netherlands. 29 Department of Intensive Care, Ikazia Ziekenhuis Rotterdam, Rotterdam, The Netherlands. 30 Antonius Ziekenhuis Sneek, Sneek, The Netherlands. 31 Intensive Care, Medisch Centrum Leeuwarden, Leeuwarden, The Netherlands. 32 ICU, IJsselland Ziekenhuis, Capelle Aan Den IJssel, The Netherlands. 33 ICU, WZA, Assen, The Netherlands. 34 Department of Intensive Care, Diakonessenhuis Hospital, Utrecht, The Netherlands. 35 Department of Intensive Care, Adrz, Goes, The Netherlands. 36 Department of Anesthesia and Intensive Care, Slingeland Ziekenhuis, Doetinchem, The Netherlands. 37 Department of Anesthesiology, Intensive Care and Pain Medicine, Amphia Ziekenhuis, Breda, The Netherlands. 38 Department of Intensive Care, LUMC, Leiden, The Netherlands. 39 BigData Republic, Nieuwegein, The Netherlands. 40 Department of Neurology, Amsterdam UMC, Universiteit Van Amsterdam, Amsterdam, The Netherlands. 41 Quantitative Data Analytics Group, Depart‑ ment of Computer Science, Faculty of Science, Vrije Universiteit, Amsterdam, The Netherlands. 42 Business Intelligence, Haaglanden MC, Den Haag, The Netherlands. 43 Department of Intensive Care Medicine, Amsterdam UMC, Universiteit Van Amsterdam, Amsterdam, The Netherlands. 44 Department of Intensive Care, BovenIJ Ziekenhuis, Amsterdam, The Netherlands. 45 Depart‑ ment of Anesthesiology, Pain Management and Intensive Care, Catharina Ziekenhuis Eindhoven, Eindhoven, The Netherlands. 46 Department of ICMT, Haga Ziekenhuis, Den Haag, The Netherlands. 47 Department of Intensive Care Medicine, Radboud University Medical Centre, Nijmegen, The Netherlands. 48 Department of Internal Medicine and Intensive Care, St Antonius Hospital, Nieuwegein, The Netherlands. 49 Department of Clinical Epidemiology, VieCuri Medisch Centrum, Venlo, The Netherlands. 50 Department of Pulmonol‑ ogy, Zuyderland MC, Heerlen, The Netherlands. 51 Department of Intensive Care, Diakonessenhuis Hospital, Utrecht, The Netherlands. 52 ICU, Maasstad Ziekenhuis, Rotterdam, The Netherlands. Consent for publication Consent for publication Not applicable. Ethics approval and consent to participate pp p p The Medical Ethics Committee at Amsterdam UMC, location VUmc waived the need for patient informed consent and approved of an opt-out procedure for the collection of COVID-19 patient data during the COVID-19 crisis. Publisher’s Note S i N i 11. Lin M-Y, Li C-C, Lin P-H, Wang J-L, Chan M-C, Wu C-L, et al. Explainable machine learning to predict successful weaning among patients requir‑ ing prolonged mechanical ventilation: a retrospective cohort study in central Taiwan. Front Med. 2021;8:663739. Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. 12. Jia Y, Kaul C, Lawton T, Murray-Smith R, Habli I. Prediction of weaning from mechanical ventilation using convolutional neural networks. Artif Intell Med. 2021;117:102087. 13. Zhao Q-Y, Wang H, Luo J-C, Luo M-H, Liu L-P, Yu S-J, et al. Development and validation of a machine-learning model for prediction of extubation failure in intensive care units. Front Med. 2021;8:676343. 14. Kuo H-J, Chiu H-W, Lee C-N, Chen T-T, Chang C-C, Bien M-Y. Improvement in the prediction of ventilator weaning outcomes by an artificial neural network in a medical ICU. Respir Care. 2015;60:1560–9. 15. Otaguro T, Tanaka H, Igarashi Y, Tagami T, Masuno T, Yokobori S, et al. Machine learning for the prediction of successful extubation among patients with mechanical ventilation in the intensive care unit: a retro‑ spective observational study. J Nippon Med Sch Nippon Ika Daigaku Zasshi. 2021. https://doi.org/10.1272/jnms.JNMS.2021_88-508. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: 16. Fleuren LM, Dam TA, Tonutti M, de Bruin DP, Lalisang RCA, Gommers D, et al. References g j 24. Mechanical Ventilation–induced Diaphragm Atrophy Strongly Impacts Clinical Outcomes \| American Journal of Respiratory and Critical Care Medicine [Internet]. [cited 2021 Sep 21]. Available from: https://doi.org/ 10.1164/rccm.201703-0536OC 1. Thille AW, Richard J-CM, Brochard L. The decision to extubate in the intensive care unit. Am J Respir Crit Care Med. 2013;187:1294–302. 1. Thille AW, Richard J-CM, Brochard L. The decision to extubate in the intensive care unit. Am J Respir Crit Care Med. 2013;187:1294–302. 2. Slutsky AS, Ranieri VM. Ventilator-induced lung injury. N Engl J Med. 2013;369:2126–36. 2. Slutsky AS, Ranieri VM. Ventilator-induced lung injury. N Engl J Med. 2013;369:2126–36. 25. Yoshida T, Amato MBP, Kavanagh BP, Fujino Y. Impact of spontaneous breathing during mechanical ventilation in acute respiratory distress syndrome. Curr Opin Crit Care. 2019;25:192–8. 3. Ventilator-Associated Events: Prevalence, Outcome, and Relat ionship With Ventilator-Associated Pneumonia. Critical Care Medicine [Internet]. [cited 2021 Jun 17]. Available from: https://journals.lww.com/ccmjournal/ Abstract/2015/09000/Ventilator_Associated_Events__Prevalence,_Outco me,.3.aspx 26. Teasdale G, Jennett B. Assessment of coma and impaired consciousness: a practical scale. The Lancet. 1974;304:81–4. 27. Erginel S, Ucgun I, Yildirim H, Metintas M, Parspour S. High body mass index and long duration of intubation increase post-extubation stridor in patients with mechanical ventilation. Tohoku J Exp Med. 2005;207:125–32. 4. Baptistella AR, Sarmento FJ, da Silva KR, Baptistella SF, Taglietti M, Zuquello RÁ, et al. Predictive factors of weaning from mechanical ventilation and extubation outcome: a systematic review. J Crit Care. 2018;48:56–62. 4. Baptistella AR, Sarmento FJ, da Silva KR, Baptistella SF, Taglietti M, Zuquello RÁ, et al. Predictive factors of weaning from mechanical ventilation and extubation outcome: a systematic review. J Crit Care. 2018;48:56–62. 4. Baptistella AR, Sarmento FJ, da Silva KR, Baptistella SF, Taglietti M, Zuquello RÁ, et al. Predictive factors of weaning from mechanical ventilation and extubation outcome: a systematic review. J Crit Care. 2018;48:56–62. 5. Heunks LM, van der Hoeven JG. Clinical review: The ABC of weaning failure—a structured approach. Crit Care. 2010;14:245. 6. Ionescu F, Zimmer MS, Petrescu I, Castillo E, Bozyk P, Abbas A, et al. Extu‑ bation failure in critically ill COVID-19 patients: risk factors and impact on in-hospital mortality. J Intensive Care Med. 2021;36:1018–24. y 5. Heunks LM, van der Hoeven JG. Clinical review: The ABC of weaning failure—a structured approach. Crit Care. 2010;14:245. 28. Kooistra EJ, de Nooijer AH, Claassen WJ, Grondman I, Janssen NAF, Netea MG, et al. Received: 21 October 2021 Accepted: 13 December 2021 22. Friedman JH. Greedy function approximation: a gradient boosting machine. Ann Stat. 2001;29:1189–232. machine. Ann Stat. 2001;29:1189–232. 23. Rapid Disuse Atrophy of Diaphragm Fibers in Mechanically Ventilated Humans \| NEJM [Internet]. [cited 2021 Sep 21]. Available from: https://doi. org/10.1056/nejmoa070447 References A higher BMI is not associated with a different immune response and disease course in critically ill COVID-19 patients. Int J Obes. 2021;45:687–94. 6. Ionescu F, Zimmer MS, Petrescu I, Castillo E, Bozyk P, Abbas A, et al. Extu‑ bation failure in critically ill COVID-19 patients: risk factors and impact on in-hospital mortality. J Intensive Care Med. 2021;36:1018–24. 29. Qian Z, Alaa AM, van der Schaar M, Ercole A. Between-centre differences for COVID-19 ICU mortality from early data in England. Intensive Care Med. 2020. https://doi.org/10.1007/s00134-020-06150-y. y 7. Hsieh MH, Hsieh MJ, Cheng A-C, Chen C-M, Hsieh C-C, Chao C-M, et al. Predicting weaning difficulty for planned extubation patients with an artificial neural network. Medicine (Baltimore). 2019;98:e17392. 7. Hsieh MH, Hsieh MJ, Cheng A-C, Chen C-M, Hsieh C-C, Chao C-M, et al. Predicting weaning difficulty for planned extubation patients with an artificial neural network. Medicine (Baltimore). 2019;98:e17392. 30. Eaneff S, Obermeyer Z, Butte AJ. The case for algorithmic stewardship for artificial intelligence and machine learning technologies. JAMA. 2020. https://doi.org/10.1001/jama.2020.9371. 8. Fabregat A, Magret M, Ferré JA, Vernet A, Guasch N, Rodríguez A, et al. A machine learning decision-making tool for extubation in Intensive care unit patients. Comput Methods Programs Biomed. 2021;200:105869. 8. Fabregat A, Magret M, Ferré JA, Vernet A, Guasch N, Rodríguez A, et al. A machine learning decision-making tool for extubation in Intensive care unit patients. Comput Methods Programs Biomed. 2021;200:105869. 31. Fleuren LM, de Bruin DP, Tonutti M, Lalisang RCA, Elbers PWG, Gommers D, et al. Large-scale ICU data sharing for global collaboration: the first 1633 critically ill COVID-19 patients in the Dutch Data Warehouse. Inten‑ sive Care Med. 2021. https://doi.org/10.1007/s00134-021-06361-x. p p g 9. Hsieh M-H, Hsieh M-J, Chen C-M, Hsieh C-C, Chao C-M, Lai C-C. An artificial neural network model for predicting successful extubation in intensive care units. J Clin Med. 2018;7:240. 10. Tsai T-L, Huang M-H, Lee C-Y, Lai W-W. Data science for extubation predic‑ tion and value of information in surgical intensive care unit. J Clin Med. 2019;8:1709. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: Consent for publication Not applicable. Consent for publication Not applicable. Not applicable. Fleuren et al. Critical Care (2021) 25:448 Fleuren et al. Critical Care (2021) 25:448 Fleuren et al. Critical Care (2021) 25:448 Page 10 of 10 Page 10 of 10 Heerenveen, The Netherlands. 64 Department of Intensive Care Medicine, Het Van Weel-Bethesda Ziekenhuis, Dirksland, The Netherlands. 65 Department of Intensive Care, Tergooi Hospital, Hilversum, The Netherlands. 20. Tibshirani R. Regression Shrinkage and Selection via the Lasso. J R Stat Soc Ser B Methodol. 1996;58:267–88. 21. Chen H, Janizek JD, Lundberg S, Lee S-I. True to the Model or True to the Data? ArXiv200616234 Cs Stat [Internet]. 2020 [cited 2021 Jan 28]; Avail‑ able from: http://arxiv.org/abs/2006.16234 Received: 21 October 2021 Accepted: 13 December 2021 Received: 21 October 2021 Accepted: 13 December 2021 Publisher’s Note S i N i The Dutch data warehouse, a multicenter and full-admission elec‑ tronic health records database for critically ill COVID-19 patients. Crit Care. 2021;25:304. 17. Collins GS, Reitsma JB, Altman DG, Moons KGM. Transparent reporting of a multivariable prediction model for individual prognosis or diagnosis (TRIPOD): the TRIPOD statement. BMJ. 2015;350:g7594. 18. Béduneau G, Pham T, Schortgen F, Piquilloud L, Zogheib E, Jonas M, et al. Epidemiology of weaning outcome according to a new definition. The WIND study. Am J Respir Crit Care Med. 2016;195:772–83. y p 19. Amato MBP, Meade MO, Slutsky AS, Brochard L, Costa ELV, Schoenfeld DA, et al. Driving pressure and survival in the acute respiratory distress syndrome. N Engl J Med. 2015;372:747–55.
https://openalex.org/W2557689343	https://europepmc.org/articles/pmc5234545?pdf=render	English	null	Preparation of MS2 Phage-Like Particles and Their Use As Potential Process Control Viruses for Detection and Quantification of Enteric RNA Viruses in Different Matrices	Frontiers in microbiology	2,016	cc-by	9,676	ORIGINAL RESEARCH published: 01 December 2016 doi: 10.3389/fmicb.2016.01911 Citation: Mikel P, Vasickova P, Tesarik R, Malenovska H, Kulich P, Vesely T and Kralik P (2016) Preparation of MS2 Phage-Like Particles and Their Use As Potential Process Control Viruses for Detection and Quantiﬁcation of Enteric RNA Viruses in Different Matrices. Front. Microbiol. 7:1911. doi: 10.3389/fmicb.2016.01911 Preparation of MS2 Phage-Like Particles and Their Use As Potential Process Control Viruses for Detection and Quantiﬁcation of Enteric RNA Viruses in Different Matrices Pavel Mikel1,2, Petra Vasickova1, Radek Tesarik1, Hana Malenovska1, Pavel Kulich1, Tomas Vesely1 and Petr Kralik1 Pavel Mikel1,2, Petra Vasickova1, Radek Tesarik1, Hana Malenovska1, Pavel Kulich1, Tomas Vesely1 and Petr Kralik1 1 Veterinary Research Institute, Department of Food and Feed Safety, Brno, Czechia, 2 Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czechia The detection and quantiﬁcation of enteric RNA viruses is based on isolation of viral RNA from the sample followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR). To control the whole process of analysis and in order to guarantee the validity and reliability of results, process control viruses (PCV) are used. The present article describes the process of preparation and use of such PCV– MS2 phage-like particles (MS2 PLP) – in RT-qPCR detection and quantiﬁcation of enteric RNA viruses. The MS2 PLP were derived from bacteriophage MS2 carrying a unique and speciﬁc de novo-constructed RNA target sequence originating from the DNA of two extinct species. The amount of prepared MS2 particles was quantiﬁed using four independent methods – UV spectrophotometry, ﬂuorimetry, transmission electron microscopy and a speciﬁcally developed duplex RT-qPCR. To evaluate the usefulness of MS2 PLP in routine diagnostics different matrices known to harbor enteric RNA viruses (swab samples, liver tissue, serum, feces, and vegetables) were artiﬁcially contaminated with speciﬁc amounts of MS2 PLP. The extraction efﬁciencies were calculated for each individual matrix. The prepared particles fulﬁll all requirements for PCV – they are very stable, non-infectious, and are genetically distinct from the target RNA viruses. Due to these properties they represent a good morphological and physiochemical model. The use of MS2 PLP as a PCV in detection and quantiﬁcation of enteric RNA viruses was evaluated in different types of matrices. Edited by: Edited by: Jean-christophe Augustin, Ecole Nationale Vétérinaire d’Alfort, France Edited by: Jean-christophe Augustin, Ecole Nationale Vétérinaire d’Alfort, France Reviewed by: Tineke H. Jones, Agriculture and Agri-Food Canada, Canada Sandra Martin-Latil, Administración Nacional de la Seguridad Social (ANSES), France *Correspondence: Pavel Mikel mikel@vri.cz Specialty section: This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology Specialty section: This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology Received: 13 July 2016 Accepted: 15 November 2016 Published: 01 December 2016 Received: 13 July 2016 Accepted: 15 November 2016 Published: 01 December 2016 Keywords: RT-qPCR, RNA virus, process control virus, isolation, detection, quantiﬁcation, extraction efﬁciency calculation, MS2 phage-like particle Preparation of Insert The fragment encoding the control sequence derived from thylacine and the moa bird was obtained by PCR using a de novo template and the primer pairs TM-BlpI F and TM-HindIII R, which included BlpI and HindIII restriction enzyme sites (Table 1). The composition of the reaction mixture was 12.5 µl of Fast start PCR master (Roche Molecular Diagnostics, Germany), 7.5 pmol of each primer and 3.3 pmol of template DNA. The assay was run in a total volume of 25 µl under the following conditions: 95◦C for 3 min, followed by 25 cycles of 94 ◦C for 30 s, 56◦C for 30 s and 72◦C for 30 s and a ﬁnal extension of 72◦C for 5 min. PCR products were examined using agarose gel electrophoresis (1%) staining with ethidium bromide, and were puriﬁed using the QIAquick PCR puriﬁcation kit (Qiagen, Germany) and subsequently digested at 37◦C for 2 h. The composition of the restriction enzyme digest reaction was 500 ng of PCR product, 2 µl NEBuﬀer 2 (New England Biolabs, UK; NEB), 100 U HindIII and 10 U BlpI endonucleases (both NEB) in a ﬁnal volume of 20 µl. The cleaved PCR product was further puriﬁed using the QIAquick PCR puriﬁcation kit (Qiagen). Based on these recommendations, MS2 phage-like particles (MS2 PLP) that could be used as PCV for RT-qPCR detection of enteric RNA viruses were prepared. The technology for production of MS2 PLP is theoretically well established and uses the knowledge gained from the study of the familiar bacteriophage MS2 (Leviviridae, +ssRNA) (Pickett and Peabody, 1993; DuBois et al., 1997; Pasloske et al., 1998; Cheng et al., 2006; Wei B.J. et al., 2008; Yu et al., 2008). The use of wild- type bacteriophage MS2 instead of MS2 PLP has two major disadvantages. First, wild-type MS2 bacteriophage has the ability to proliferate – theoretically, in a speciﬁc samples such as those with fecal contamination that naturally contain Escherichia coli (E. coli), MS2 bacteriophage can proliferate and exceed the number of detected pathogenic RNA viruses present in the sample. Second, the wild-type MS2 bacteriophage cannot be used as a highly speciﬁc PCV because its genome does not contain the speciﬁc target sequences. In contrast with wild- type bacteriophage MS2, MS2 PLP cannot replicate and allow packaging of the speciﬁc control RNA sequence into their capsid. INTRODUCTION Furthermore, the PCV should be of a similar size to the target virus to provide a good morphological and physicochemical model, should be genetically distinct from the target virus to avoid cross-reactivity, and should not be naturally present in the analyzed sample. and their stability during long-term storage. The particles have similar physiochemical properties as the wild-type bacteriophage MS2, which has been used many times as a PCV in the detection of enteric RNA viruses (Dreier et al., 2006; Rolfe et al., 2007; Blaise-Boisseau et al., 2010; Shulman et al., 2012) and as the surrogate virus in enteric RNA virus environmental stability studies (Shin and Sobsey, 2003; Bae and Schwab, 2008; Park and Sobsey, 2011). The present article is a follow- up study of a previously published theoretical concept (Mikel et al., 2015) and describes a method of preparation of MS2 PLP carrying a speciﬁc control sequence and their use as a PCV in RT-qPCR detection and quantiﬁcation of enteric RNA viruses in swab, liver tissue, serum, feces, and vegetable samples. INTRODUCTION The quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is nowadays considered as the gold standard method for detection and quantiﬁcation of enteric RNA viruses such as hepatitis A virus (HAV), hepatitis E virus (HEV) or human noroviruses (NoV) (Mattison et al., 2009; Blaise-Boisseau et al., 2010; Di Pasquale et al., 2010; Vasickova et al., 2012; Hennechart- Collette et al., 2014). These enteric viruses have a signiﬁcant impact on human health throughout December 2016 \| Volume 7 \| Article 1911 Frontiers in Microbiology \| www.frontiersin.org Preparation of MS2 Phage-Like Particles Mikel et al. the world. Worldwide, HAV infections account for 1.4 million cases annually and about 102 million asymptomatic and symptomatic cases occurred all together in 2013 (WHO, 2012; Vos et al., 2015), HEV infections account for 20 million cases annually (Lozano et al., 2012; Rein et al., 2012) and NoV is responsible for approximately 90% of epidemic non-bacterial outbreaks of gastroenteritis around the world (Lindesmith et al., 2003). RT-qPCR is a fast and sensitive method capable of detecting as few as 10 genome copies of viral nucleic acid in a sample (Puig et al., 2002). Because of the drawbacks of RT-qPCR including the necessity of monitoring the eﬃciency of concentration and RNA extraction steps, the removal of reverse transcription (RT) and PCR inhibitors there is a need of developing an entire RT-qPCR assay with a system of controls. Eﬀective control of all the analytical steps is now required for diagnostic assays and generally involves the utilization of a non- pathogenic virus – process control virus (PCV) – which is added in a deﬁned amount to the sample prior to the processing. Viral concentration and isolation procedures from complex matrices (e.g., food matrices or environmental samples) are often laborious and time-intensive, which increases the likelihood of mistakes that may lead to the failure of the analysis. Therefore, in 2013 the European Committee for Standardization (CEN) released ISO technical speciﬁcations (ISO/TS) ISO/TS 15216-1 and ISO/TS 15216-2 (The methods for determination of HAV and NoV in food using RT-qPCR), which require the use of PCV together with external control RNA (EAC) in RT-qPCR detection of these viruses in such complex matrices. According to these technical speciﬁcations, a cultivable non-enveloped positive- sense single stranded RNA (+ssRNA) virus shall be used as such a control. MATERIALS AND METHODS Construction of Control Sequence The speciﬁc control sequence was derived from mitochondrial DNA (mtDNA) sequences of two extinct species – thylacine (Thylacinus cynocephalus, GenBank accession No. FJ515781.1), and the moa bird (Dinornis struthoides, GenBank accession No. AY326187.1). Due to the fact that the sequence was derived from mDNA of two extinct species, its natural occurrence in the analyzed samples is highly unlikely. The control sequence with a total length of 150 bp was synthesized de novo (Elisabeth Pharmacon, Czech Republic). The speciﬁcity of the control sequence was veriﬁed using BLAST1. It was also analyzed using OligoAnalyzer 3.1 software2 for the ability to form secondary structures, such as hairpins, which could result in unsuitability for RT or/and qPCR. 1http://blast.ncbi.nlm.nih.gov/Blast.cgi 2http://eu.idtdna.com/calc/analyzer Frontiers in Microbiology \| www.frontiersin.org Ligation of Insert and Vector, Transformation of Competent Cells and Veriﬁcation of Construct p Ligation of insert and AU2 vector DNA was performed using a Quick-ligation kit (NEB) according to the manufacturer’s instructions. Transformation of E. coli TOP10 chemically competent cells (Life Technologies, USA) was performed according to the manufacturer’s recommendations. E. coli TOP10 cells were chosen for their high transformation eﬃciency, which provides for a large number of successfully transformed colonies. Transformed cells were grown on Luria Bertani (LB; Sigma–Aldrich, Czech Republic) agar plates containing 30 µg/ml of kanamycin (Sigma-Aldrich) overnight. Plasmid DNA was puriﬁed (NucleoSpin Plasmid kit; Macherey-Nagel) from twenty randomly selected colonies and the presence of speciﬁc inserts was conﬁrmed by PCR using vector-speciﬁc (M13 uni and M13 rev) and insert-speciﬁc (TM F and TM R) primers (Table 1) as well as by sequencing (Euroﬁns MWG Operon, Germany). Preparation of Vector Escherichia coli BL21 (DE3) carrying the AU2 plasmid, which contains a fragment encoding MS2 maturase and coat protein was obtained from the Belgian coordinated collections of micro- organisms (BCCM, collection No. LMBP 7766). Puriﬁed plasmid AU2 (NucleoSpin Plasmid kit; Macherey-Nagel, Germany) was cleaved with BlpI and HindIII restriction endonucleases at 37◦C for 2 h. The composition of the restriction enzyme digest reaction was 500 ng of plasmid DNA, 5 µl NEBuﬀer 2, 100 U HindIII and 10 U BlpI endonucleases in a ﬁnal volume of 50 µl. The AU2 plasmid was dephosphorylated using 0.25 U calf intestinal alkaline phosphatase (NEB) added to the restriction mixture. The dephosphorylation reaction was run at 37◦C for 1 h. Subsequently, plasmid DNA was puriﬁed using the QIAquick PCR puriﬁcation kit (Qiagen). Intact MS2 PLP were veriﬁed by transmission electron microscopy (TEM; electron microscope Philips EM 208; FEI, Czech Republic) at 18,000 × magniﬁcation and an accelerating voltage of 80 kV. Intact MS2 PLP were veriﬁed by transmission electron microscopy (TEM; electron microscope Philips EM 208; FEI, Czech Republic) at 18,000 × magniﬁcation and an accelerating voltage of 80 kV. Intact MS2 PLP were veriﬁed by transmission electron microscopy (TEM; electron microscope Philips EM 208; FEI, Czech Republic) at 18,000 × magniﬁcation and an accelerating voltage of 80 kV. Preparation of Insert The advantages of MS2 PLP include their ability to protect the control RNA contained in their capsid from degradation by ubiquitous ribonucleases, their non-pathogenicity to humans, December 2016 \| Volume 7 \| Article 1911 Frontiers in Microbiology \| www.frontiersin.org Frontiers in Microbiology \| www.frontiersin.org 2 Preparation of MS2 Phage-Like Particles Mikel et al. TABLE 1 \| Speciﬁc oligonucleotides used in the present study. Name Target Sequence TM-BlpI F 5′-GATACAGCTCAGCCGCTTCCGTCAAACCCCTAA-3′ TM-HindIII R 5′-TAGGAAGCTTGGGTTTAGAATGTTTTCTCCCGT-3′ TM F PCV 5′-CGCTTCCGTCAAACCCCTAA-3′ TM R PCV 5′-GGGTTTAGAATGTTTTCTCCCGT-3′ TM P PCV 5′-FAM-TGCTAATGTGGTGATTGCGTGTG-BHQ1-3′ NP F IAC 5′-AGAGGACCGGGATATTCGAC-3′ NP R IAC 5′-AGGTAGTCCGAGGAAAACTCTAAAC-3′ NP P IAC 5′-Cy5-AGGCTCTTCTATGTTCTGACCTTGTTGGA- BHQ2-3′ M13 uni 5′-TGTAAAACGACGGCCAGT-3′ M13 rev 5′-CAGGAAACAGCTATGACC-3′ Underlined sequences are BlpI or HindIII restriction enzyme sites. PCV, process control virus; IAC, internal ampliﬁcation control. TABLE 1 \| Speciﬁc oligonucleotides used in the present study. transformed into E. coli BL21 (DE3) cells (NEB) according to the manufacturer’s instructions and the bacterial cells were grown in LB broth containing 30 µg/ml of kanamycin at 37◦C until the culture reached an optical density of 600 nm (OD600) = 1.7. Two milliliter of the culture were transferred to 200 ml of LB broth containing 30 µg/ml of kanamycin, cultivated at 37◦C until OD600 = 0.8 and centrifuged at 600 × g for 10 min at room temperature. Subsequently, the pellet was resuspended in 200 ml of LB broth containing 30 µg/ml of kanamycin. Protein expression was induced by addition of 1 mM isopropyl- L-thio-D-galactopyranoside (IPTG; Sigma–Aldrich) at 37◦C for 16 h. The cell suspension was centrifuged at 600 × g for 10 min at 4◦C and cells were lyzed by ultrasonic disruption (Bandelin VW3100 sonicator, probe MS73, Bandelin, Germany) at amplitude 50%, and with four pulses for a total length of 2 min at 4◦C. To verify the production of MS2 coat protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) and immunoblot analysis was carried out using a phage MS2 Coat Protein polyclonal antibody (Merck Millipore, USA) as primary antibody and Goat Anti-Rabbit IgG, Fc speciﬁc fragment (Jackson ImmunoResearch, UK) as secondary antibody. Puriﬁcation of MS2 PLP To eliminate free nucleic acids the suspension containing lyzed cells and MS2 PLP was brieﬂy centrifuged at 6700 × g for 15 min at 4◦C and the supernatant (1 ml) was incubated with 100 U of DNase I (NEB) and 50 U of RNase A (Qiagen) at 37◦C for 40 min. The particles were subsequently puriﬁed using sucrose density gradient ultracentrifugation. Brieﬂy, 1 ml of supernatant was applied to the ultracentrifugation tubes, where two sucrose density layers of 25 and 45% were layered. Ultracentrifugation was conducted at 195000 × g (Rotor SW 55 Ti, Beckman, USA) for 3 h at 5◦C. A strong source of light – LED – was used to distinguish three opalescent layers inside the ultracentrifugation tube. The three layers were checked for the presence of MS2 PLP by agarose gel electrophoresis (1%) staining with ethidium bromide. The top layer was removed by a needle and 1 ml syringe and the obtained MS2 PLP were dialyzed using a Float-A-Lyzer G2 membrane (molecular weight cut oﬀ 1000 kDa; Spectrum Laboratories, Netherlands) against 10 mM Tris containing 100 mM NaCl and 1 mM EDTA for 20 h at 4◦C with three buﬀer changes. The purity of obtained MS2 PLP was controlled using agarose gel electrophoresis and TEM as described above. Transformation of E. coli BL21 (DE3) Cells and Induction of MS2 PLP Expression The number of MS2 PLP was determined by UV spectrophotometry using the Avogadro constant, extinction coeﬃcient of 0.125 mg/ml of MS2 bacteriophage per absorbance unit at 260 nm and a molecular weight of 3.0 × 106 as was previously described (Cheng et al., 2006). Sample absorbance Recombinant plasmid DNA (pAU2-TM) carrying the speciﬁc control sequence derived from thylacine and the moa bird was December 2016 \| Volume 7 \| Article 1911 Frontiers in Microbiology \| www.frontiersin.org 3 Preparation of MS2 Phage-Like Particles Mikel et al. at 260 nm was measured six times and mean and standard deviation values were calculated. On the basis of these data the quantity of MS2 PLP was calculated as follows: the fragment encoding the speciﬁc control sequence derived from thylacine and the moa bird was performed as was previously described Vasickova et al. (2012). In vitro transcripts were stored at −80◦C. Number of MS2 PLP =x × y z Reverse transcription was carried out using PrimeScript Reverse Transcriptase (Takara, Shiga, Japan) with slight modiﬁcations to the manufacturer’s protocol. The RT mixture (20 µl) contained 0.5 nmol of dNTP mix (Serva, Heidelberg, Germany), 20,000 molecules of IAC RNA, 2 pmol of both reverse primers (TM R and NP R; Table 1), 4 µl of PrimeScript reaction buﬀer, 5 U of reverse transcriptase, 1 U of RNase inhibitor (NEB) and 5 µl of isolated RNA. The reaction was performed at 50◦C for 1 h followed by 75◦C for 15 min and a cooling step at 10◦C. where x = Avogadro constant (6.023 × 1023/mol), y = extinction coeﬃcient of MS2 bacteriophage (0.125 × 10−3 g/ml) and z = molecular weight of MS2 bacteriophage (3.0 × 106 g/mol). The result of this calculation revealed that the concentration of MS2 PLP in a suspension of OD260 was 1 is 2.5 × 1013 particles/ml. As a second method for MS2 PLP quantiﬁcation TEM was used according to (Malenovska, 2013) with small modiﬁcations; the grid was stained with 2% ammonium molybdate (pH = 7.0) for 2 min, the latex standard (Agar Scientiﬁc, Stansted, England) had a concentration of 1.2 × 1012 particles/ml and the used magniﬁcation was 28,000 × to 36,000 × (Malenovska, 2013). MS2 PLP was counted in 300 randomly selected ﬁelds of view. The optimized duplex qPCR assay was run in duplicate for each analyzed sample in a total volume of 20 µl. Transformation of E. coli BL21 (DE3) Cells and Induction of MS2 PLP Expression The reaction mix contained 10 µl of LightCycler 480 Probes Master (Roche), 5 pmol of the NP F and NP R primers, 10 pmol of the TM F and TM R primers, 4 pmol of the IAC probe and 1 pmol of the NP P probe, and 5 µl template cDNA. 1 U of Uracil DNA Glycosylase (Roche) was used in each qPCR reaction to avoid possible carry-over contamination. qPCR was performed using the Roche LightCycler 480 under the following reaction conditions: initial denaturation at 95◦C for 10 min, followed by 45 cycles at 95◦C for 10 s, 60◦C for 30 s and 72◦C for 10 s. The subsequent analysis of results (quantiﬁcation) was carried out using the “Fit point analysis” option of the LightCycler 480 Software release 1.5.0 (version 1.5.0.39). A third method for MS2 PLP was quantiﬁcation by ﬂuorimetric measurement of protein concentration using the Qubit Protein Assay Kit (Life Technologies) and Qubit 3.0 ﬂuorometer (Life Technologies). The suspension containing MS2 PLP was diluted 10-fold in water and ﬂuorimetric calculation of protein concentration in the samples was done according to manufacturer instructions. The samples were analyzed in hexaplicates and the mean and standard deviation values were calculated. As a fourth method for MS2 PLP quantiﬁcation RT- qPCR was used as described below. Quantiﬁcation standards were prepared from a 10-fold dilution of in vitro-prepared RNA transcripts of the fragment encoding the speciﬁc control sequence derived from thylacine and the moa bird in the range of 5 × 109 genome copies/5 µl to 5 × 101 genome copies /5 µl. RNA in vitro transcripts were quantiﬁed by ﬂuorimetry using the Qubit RNA BR Assay Kit (Life Technologies) and Qubit 3.0 ﬂuorometer (Life Technologies) according to the manufacturer’s instructions. Stability of MS2 PLP was veriﬁed by their incubation with 100 U of DNase I (NEB) and 50 U of RNase A (Qiagen) at 37◦C for 1 h. As controls for the reaction, DNA (500 ng of plasmid pAU-TM) and RNA (500 ng of IAC in vitro transcript, see below) were incubated under the same conditions. The ability of MS2 PLP to resist the action of nucleases was controlled using agarose gel electrophoresis (1%). Transformation of E. coli BL21 (DE3) Cells and Induction of MS2 PLP Expression MS2 PLP were lyzed in triplicate by heating at 95◦C for 5 min (Cheng et al., 2006) and 5 µl of undiluted, ten-fold and hundred-fold diluted (RNase-free H2O, 40 U/ml RNase inhibitor, NEB; 50 ng/µl carrier RNA, Life Technologies) lyzed MS2 PLP were used as template RNA for the RT-qPCR reaction. RT was carried out in triplicate for each dilution and qPCR was done in triplicated for each sample. Prepared MS2 PLP were diluted with 10 mM Tris containing 100 mM NaCl and 1 mM EDTA supplemented with 0.05 mg/ml of bovine serum albumin (BSA; Life Technologies) to a ﬁnal concentration of 1 × 106/µl, veriﬁed by TEM, aliquoted (70 µl) and stored at −80◦C. RT qPCR for Detection and Quantiﬁcation of MS2 PLP A two-step, duplex, RT-qPCR was optimized for MS2 PLP and internal ampliﬁcation control (IAC) detection and quantiﬁcation. To distinguish false and truly negative results in vitro transcribed RNA (IAC) was included in the RT-qPCR assay. The IAC- speciﬁc sequence was derived from the genomic sequences of two plants – nepenthes (Nepenthes ampullaria, GenBank accession No. GQ338261.1), and potato (Solanum tuberosum, GenBank accession No. AF483209) and from Mycobacterium avium subsp. paratuberculosis (GenBank accession No. X70277). The DNA-containing IAC sequence and DNA-containing fragment encoding the speciﬁc control sequence were prepared according to Vojkovska et al. (2015). In vitro transcription of the IAC and Swab, liver tissue, serum, feces, and vegetable samples were artiﬁcially contaminated with the 5 µl of MS2 PLP (5 × 106 particles). RNA from liver tissue (50 mg aseptically taken from three diﬀerent inner-liver locations) was isolated using the RNeasy Mini kit (Qiagen), while RNA from serum (200 µl) and feces (200 µl from 250 mg of feces resuspended in 2.25 ml of phosphate-buﬀered saline buﬀer – PBS) was isolated using the QIAamp Viral RNA kit (Qiagen) with slight modiﬁcations as described previously (Vasickova et al., 2011, 2012). RNA from December 2016 \| Volume 7 \| Article 1911 Frontiers in Microbiology \| www.frontiersin.org 4 Preparation of MS2 Phage-Like Particles Mikel et al. FIGURE 1 \| Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE – left part of the picture) and Western blot (right part of the picture) results. 1,6, E. coli BL21 (DE3) negative control; 2,5, IPTG-non-induced culture; 3,4, IPTG-induced culture. 13 kDa MS2 coat protein was massively produced in the IPTG-induced culture. M, marker Spectra multicolor broad range protein ladder (Fermentas), the marker values are in kDa. leafy green vegetables (25 g) and swabs (100 cm2, cotton swab rinsed in 5 ml of PBS) was isolated using NucliSENS Magnetic extraction reagents (bioMérieux, Durham, NC, USA) according to ISO/TS 15216-1. Elution volumes of isolated RNA were 100 µl for swab and vegetable samples and 60 µl for liver tissue, serum and feces samples. RT-qPCR for detection of MS2 PLP was performed as described above. Subsequently, 15 µl of MS2 PLP from aliquots which were used for artiﬁcial contamination of each sample were thermally lyzed (95◦C for 5 min) and 5 µl of lyzed solution was directly added in duplicate to RT-qPCR. This approach allowed calculation of the exact z-value (see below). Production of MS2 PLP The pAU-TM expression vector carrying all the necessary information for the production of MS2 PLP together with a unique control sequence was successfully prepared. The sequence of the expression vector was veriﬁed by sequencing (data not shown). The production of MS2 coat protein in cell culture after induction was controlled by SDS-PAGE and Western blot analysis (Figure 1). The results of these analyzes showed that the 13 kDa MS2 coat protein was produced in massive amounts in the IPTG-induced cell culture carrying the pAU2-TM expression vector. In contrast, only basal production of MS2 coat protein was observed in the control IPTG non-induced cell culture and no MS2 coat protein production was observed in the control E. coli BL21 (DE3) without the pAU-TM plasmid. The formation of intact MS2 PLP in the supernatant was inspected by TEM after ultrasonic disruption of collected E. coli cells (Figure 2). These results clearly showed spontaneous assembly of MS2 PLP in the induced cell culture. FIGURE 2 \| Transmission electron microscopy (TEM) photograph of the intact MS2 phage-like particles (MS2 PLP) present in the supernatant after ultrasonic disruption of E. coli production cells. MS2 PLP are about 27 nm in diameter. The scale is 100 nm. RT qPCR for Detection and Quantiﬁcation of MS2 PLP The extraction eﬃciency of each sample was calculated as follows: Extraction eﬃciency (%) = x 5×y z × 100 where x = number of MS2 PLP in the sample quantiﬁed in RT- qPCR, y = volume in which the isolated RNA was eluted and z = number of MS2 PLP added to the sample prior to nucleic acid isolation. To determine the quantity of isolated RNA molecules, RT-qPCR quantity value (x) is divided by 5 to obtain the quantity of RNA molecules in 1 µl (RT-qPCR uses 5 µl of template RNA), followed by multiplication by elution volume (y). where x = number of MS2 PLP in the sample quantiﬁed in RT- qPCR, y = volume in which the isolated RNA was eluted and z = number of MS2 PLP added to the sample prior to nucleic acid isolation. To determine the quantity of isolated RNA molecules, RT-qPCR quantity value (x) is divided by 5 to obtain the quantity of RNA molecules in 1 µl (RT-qPCR uses 5 µl of template RNA), followed by multiplication by elution volume (y). FIGURE 1 \| Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE – left part of the picture) and Western blot (right part of the picture) results. 1,6, E. coli BL21 (DE3) negative control; 2,5, IPTG-non-induced culture; 3,4, IPTG-induced culture. 13 kDa MS2 coat protein was massively produced in the IPTG-induced culture. M, marker Spectra multicolor broad range protein ladder (Fermentas), the marker values are in kDa. FIGURE 1 \| Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE – left part of the picture) and Western blot (right part of the picture) results. 1,6, E. coli BL21 (DE3) negative control; 2,5, IPTG-non-induced culture; 3,4, IPTG-induced culture. 13 kDa MS2 coat protein was massively produced in the IPTG-induced culture. M, marker Spectra multicolor broad range protein ladder (Fermentas), the marker values are in kDa. 2,5, IPTG-non-induced culture; 3,4, IPTG-induced culture. 13 kDa MS2 coat protein was massively produced in the IPTG-induced culture. M, marker Spectra multicolor broad range protein ladder (Fermentas), the marker values are in kDa. Puriﬁcation of MS2 PLP FIGURE 2 \| Transmission electron microscopy (TEM) photograph of the intact MS2 phage-like particles (MS2 PLP) present in the supernatant after ultrasonic disruption of E. coli production cells. MS2 PLP are about 27 nm in diameter. The scale is 100 nm. Three opalescent layers were distinguishable inside the ultracentrifugation tube after ultracentrifugation. All layers were removed and the presence of MS2 PLP in these layers was controlled by agarose gel electrophoresis (1%) and staining with ethidium bromide (Figure 3). It was found that the lower layer contained no MS2 PLP, the middle layer only a trace amount of MS2 PLP and that the top layer contained the highest amount of MS2 PLP. Collected MS2 PLP were further puriﬁed by dialysis. Dialyzed MS2 PLP was also controlled by agarose gel electrophoresis (1%) and staining with ethidium bromide. The band containing puriﬁed MS2 PLP was clearly visible (data not shown). The integrity of puriﬁed MS2 PLP was inspected by TEM (Figure 4). December 2016 \| Volume 7 \| Article 1911 Frontiers in Microbiology \| www.frontiersin.org 5 Mikel et al. Preparation of MS2 Phage-Like Particles FIGURE 3 \| Result of agarose gel electrophoresis (1%) detection of MS2 phage-like particles (MS2 PLP) in three layers removed after ultracentrifugation from the tube. 1, top layer; 2, middle layer; 3, lower layer. M, marker 2-log DNA ladder (New England Biolabs, UK), the marker values are in kb. The top layer contained the highest amount of MS2 PLP (the band on the gel around 1.5 kb). FIGURE 5 \| Transmission electron microscopy photograph from TEM MS2 phage-like particles (MS2 PLP) quantiﬁcation against latex standard. The large black “dot” is the latex standard and the white dots are MS2 PLP. The scale is 500 nm. FIGURE 5 \| Transmission electron microscopy photograph from TEM MS2 phage-like particles (MS2 PLP) quantiﬁcation against latex standard. The large black “dot” is the latex standard and the white dots are MS2 PLP. The scale is 500 nm. FIGURE 3 \| Result of agarose gel electrophoresis (1%) detection of MS2 phage-like particles (MS2 PLP) in three layers removed after ultracentrifugation from the tube. 1, top layer; 2, middle layer; 3, lower layer. M, marker 2-log DNA ladder (New England Biolabs, UK), the marker values are in kb. The top layer contained the highest amount of MS2 PLP (the band on the gel around 1.5 kb). Puriﬁcation of MS2 PLP 10-fold and 100-fold diluted suspensions of thermally lyzed MS2 PLP were tested in order to detect possible inhibitory eﬀects of heat denatured proteins on RT-qPCR results. This dilution was done only in RT-qPCR quantiﬁcation of MS2 PLP experiment in which high quantity of MS2 PLP was expected. Because described RT-qPCR system prefers to amplify control sequence before IAC sequence, in case that the quantity of MS PLP was very high no IAC was detected. When the sample was diluted, IAC could be detected and the results were therefore valid. In calculation of extraction eﬃciency of MS2 PLP from spiked matrices no dilution was done because the MS2 PLP quantity was not so high and the IAC was reliably detected even in undiluted samples. Ampliﬁcation of IAC had to be successful otherwise the results were not valid. FIGURE 4 \| Transmission electron microscopy (TEM) photograph of the intact MS2 phage-like particles (MS2 PLP) after puriﬁcation steps. The scale is 100 nm. RT-qPCR quantiﬁcation method was chosen as the most accurate method because it was the only method capable of identifying a speciﬁc control RNA encapsulated inside the MS2 PLP. Veriﬁcation of the Stability of MS2 PLP in the Presence of Nucleases FIGURE 4 \| Transmission electron microscopy (TEM) photograph of the intact MS2 phage-like particles (MS2 PLP) after puriﬁcation steps. The scale is 100 nm. The stability of MS2 PLP was checked by agarose gel electrophoresis (1%) and staining with ethidium bromide (data not shown). These analyzes clearly showed that only the MS2 PLP were stable in the solution with high concentrations of DNase and RNase whereas naked DNA and RNA molecules were quickly degraded in this suspension. Quantiﬁcation of MS2 PLP Besides MNV-1, other PCV –Turnip crinkle virus (TCV), Mengovirus, Tulane virus and bacteriophage MS2 were also tested and bacteriophage MS2 and Tulane virus performed relatively well across all spiked levels (Gentry-Shields and Jaykus, 2015). Moreover, evaluation of two commercially available Mengovirus suspensions showed signiﬁcantly diﬀerent RNA extraction eﬃciency results. It was clear that diﬀerences in virus source (e.g., method of propagation) can drastically impact the eﬃciency of RNA extraction (Gentry-Shields and Jaykus, 2015). Also in this respect, MS2 PLP have an additional advantage, because they are produced strictly in E. coli BL21 (DE3) cells, which excludes the diﬀerences in virus source and provides for high quality, standardized PCV stocks. FIGURE 6 \| Transmission electron microscopy photograph of homogenous and non-aggregated MS2 phage-like particles (MS2 PLP). The scale is 200 nm. FIGURE 6 \| Transmission electron microscopy photograph of homogenous and non-aggregated MS2 phage-like particles (MS2 PLP). The scale is 200 nm. Quantiﬁcation of MS2 PLP The number of MS2 PLP was determined by UV spectrophotometry, TEM (Figure 5), ﬂuorimetric measurement of protein concentration and RT-qPCR. The results of MS2 PLP quantiﬁcation experiments are summarized in Table 2. According to results of RT-qPCR quantiﬁcation prepared MS2 PLP were diluted to a ﬁnal concentration of 1 × 106/µl. The prepared solution was veriﬁed by TEM. TEM inspection revealed that prepared MS2 PLP did not form aggregates and that the suspension was homogeneous (Figure 6). Values of MS2 PLP concentrations obtained by UV spectrophotometry, TEM and ﬂuorimetry were comparable. In the case of RT-qPCR quantiﬁcation of MS2 PLP, undiluted, December 2016 \| Volume 7 \| Article 1911 Frontiers in Microbiology \| www.frontiersin.org 6 Preparation of MS2 Phage-Like Particles Mikel et al. TABLE 2 \| The results of MS2 phage-like particles (MS2 PLP) quantiﬁcation experiments. Quantiﬁcation method UV spectrophotometry TEM Fluorimetry RT-qPCR Concentration of MS2 PLP/ml ±SD 2.5 × 1013 ± 2.24 × 1011 1.57 × 1013 ± not applicable 7.28 × 1013 ± 1.35 × 1013 2.89 × 1012 ± 2.39 × 1011 TEM, transmission electron microscopy; RT-qPCR, reverse transcription polymerase chain reaction. SD, standard deviation. TABLE 2 \| The results of MS2 phage-like particles (MS2 PLP) quantiﬁcation experiments. Quantiﬁcation method UV spectrophotometry TEM Fluorimetry RT-qPCR Concentration of MS2 PLP/ml ±SD 2.5 × 1013 ± 2.24 × 1011 1.57 × 1013 ± not applicable 7.28 × 1013 ± 1.35 × 1013 2.89 × 1012 ± 2.39 × 1011 TEM, transmission electron microscopy; RT-qPCR, reverse transcription polymerase chain reaction. SD, standard deviation. (ISO/TS 15216-1, 2013). However, recent studies have utilized MNV-1 instead of Mengovirus as the most appropriate virus for validation of HAV detection (Hennechart-Collette et al., 2015). Ideally, each genus of enteric RNA virus should have its own PCV that most closely matches its speciﬁc properties. However, such an approach is impossible in the case of routine detection of a broad spectrum of RNA viruses in diﬀerent matrices. For example it was suggested that the most suitable PCV for NoV would be MNV-1, despite the recovery rates of MNV-1 were signiﬁcantly diﬀerent in bottled water and tap water (Hennechart-Collette et al., 2014). On the other hand, some recent studies have shown that MNV-1 virus mimicked the behavior of NoV during sample processing to the least extent and with the most dissimilar recovery to NoV (Gentry- Shields and Jaykus, 2015). Artiﬁcial Contamination and RT-qPCR Detection of MS2 PLP from Different Matrices MS2 PLP were added in the amount of 5 × 106 particles to the diﬀerent types of matrices (swabs, liver tissue, serum, feces, and leafy green vegetables) to reveal their ability to serve as PCV in the RT-qPCR detection of enteric RNA viruses. According to results of RT-qPCR the extraction eﬃciencies were calculated for each individual matrix. The obtained eﬃciencies from swabs, liver tissue, serum, feces and leafy green vegetables were 41.60, 5.28, 77.04, 8.77, and 4.29%, respectively (Table 3). MS2 PLP are appropriate candidates for PCV only in the case of detection of structurally similar RNA viruses – small, non-enveloped ssRNA viruses with icosahedral structure, among which the majority of common enteric viruses belong – to closely mimic their physiochemical properties during the nucleic acid extraction step. Therefore, MS2 PLP can be good PCV in the detection of HAV, HEV or NoV from food matrices. Although their production is not a trivial task, once they can be produced, a high amount is obtained. In comparison with commercially available PCV the price of self-producing MS2 PLP is signiﬁcantly lower. Moreover, MS2 PLP can be stored for a long time without degradation (WalkerPeach et al., 1999; Beld et al., 2004; Hietala and Crossley, 2006; Wei Y.X. et al., 2008; Yu et al., 2008; Zhan et al., 2009; Song et al., 2011). Another positive feature of MS2 PLP is that they are not infectious and thus working with them is safe for laboratory personnel. Their advantage is the possibility to pack any ssRNA sequence into a capsid which can then serve as a speciﬁc control sequence. DISCUSSION Nucleic acid isolation efﬁciency was calculated according to the formula described within the text. SD, standard devia RNA extraction eﬃciency and also allows detection of potential inhibition in each analyzed sample. Then, additional RT-qPCR assays can be performed to detect various types of enteric RNA viruses. Based on this approach, it is possible to obtain reliable results for RT-qPCR detection of enteric RNA viruses. may be determined using the Avogadro constant, extinction coeﬃcient of 1 OD260 = 0.125 mg/ml of MS2 bacteriophage and the molecular weight of 3 × 106 (DuBois et al., 1997; Cheng et al., 2006; Wei B.J. et al., 2008). This indirect method of enumeration of MS2 PLP was used together with ﬂourimetry and with direct calculation of viral particles by TEM. The TEM quantiﬁcation has not been used in any previous publications dealing with MS2 PLP. This was probably due to their relatively small size (around 27 nm) because quantitation of virus particles with TEM was used mostly for viruses larger than 40 nm (Stinski et al., 1979; Kwon et al., 2003; Wei et al., 2007; Weidmann et al., 2011). The concentrations of MS2 PLP obtained using UV spectrophotometry, ﬂuorimetry and TEM were consistent. q To demonstrate the usefulness of MS2 PLP as PCV, MS2 PLP were added to diﬀerent types of matrices, which commonly harbor viruses. The exact number of MS2 PLP added to samples (5 × 106 particles) was chosen according to ISO/TS 15216-1 and ISO/TS 15216-2. Nucleic acid was isolated from diﬀerent types of matrices using commercially available isolation kits. All isolation procedures were able to isolate viral RNA (PCV RNA) with an eﬃciency of higher than 1%, which was recognized as the threshold of successful isolation (ISO/TS 15216-1). When the extraction eﬃciency is lower than 1%, sample results are not valid and the sample must be retested. Although the purpose of this study was not to evaluate or to compare the performance of various RNA isolation procedures, some interesting ﬁndings were obtained. First, the extraction eﬃciency does not depend on the used isolation kit, as is obvious from the extraction eﬃciency of swab samples (41.60%) and vegetable samples (4.29%) both isolated with NucliSENS Magnetic extraction reagents (bioMérieux), or serum samples (77.04%) and fecal samples (8.77%), both isolated with QIAamp Viral RNA kit (Qiagen). DISCUSSION The RNA extraction protocols are therefore well suited and the resulting extraction eﬃciency is much more dependent on concentration steps, which precede the isolation of viral RNA. For example, in the case of vegetable matrix, samples undergo a relatively complicated, time-consuming rinsing and concentration procedure prior to the nucleic acid isolation step, resulting in relatively low extraction eﬃciency. Identical isolation applied to swab samples without any pre-processing steps results in higher extraction eﬃciency of RNA (Table 3). Second, the composition of the analyzed sample has a crucial impact on extraction eﬃciency. The extraction eﬃciency from the serum matrix (77.04%), which is a relatively homogenous matrix, is almost 10-fold higher than extraction eﬃciency from feces (8.77%), which are not as homogeneous as serum samples and are expected to contain a higher concentration of inhibitory substances. A similar impact of sample composition is also obvious in the case of swab and vegetable samples (Abu Al-Soud and Radstrom, 2000; Radstrom et al., 2004; Schrader et al., 2012). Using an RT-qPCR method, the measured number of MS2 PLP was about 1 log10 lower (2.89 × 1012 MS2 PLP/ml) than by previously described methods. This indicates that the ratio of MS2 PLP carrying speciﬁc control RNA sequence in a population of prepared MS2 PLP is only around 10%. This result can be explained on the basis of the latest ﬁndings regarding the capsid assembling mechanism of bacteriophage MS2 which use a sophisticated coassembly process where the RNA genome interacts at multiple sites with the capsid proteins during assembly (Dykeman et al., 2013). Therefore, it is probable that the majority of the prepared MS2 PLP contain non-speciﬁc cellular RNA rather than speciﬁc control RNA, which is in concordance with previous ﬁndings (Pickett and Peabody, 1993). Based on these ﬁndings, the RT-qPCR method was selected for quantiﬁcation of prepared MS2 PLP because it was the only method capable of identifying a speciﬁc control RNA encapsulated inside the particle. Addition of a speciﬁc number of MS2 PLP – PCV – to each analyzed sample should provide information about the process of the sample analysis and therefore, it is possible to determine the extraction eﬃciency of RNA from each sample. Frontiers in Microbiology \| www.frontiersin.org DISCUSSION RT-qPCR-based detection and quantiﬁcation of RNA viruses for diagnostic purposes requires strict control to avoid inaccurate results. Bacteriophage MS2 and its derivatives (including MS2 PLP) are not the only possible useful PCV for the RT-qPCR detection of enteric RNA viruses. Many diﬀerent viruses were used as PCV for the RT-qPCR detection of HAV and NoV from food matrices, e.g., Murine norovirus 1 (MNV-1) (Martin- Latil et al., 2012; Coudray et al., 2013; Hennechart-Collette et al., 2014), Feline calicivirus (FCV) (Mattison et al., 2009; Di Pasquale et al., 2010), Mengovirus, San Miguel sea lion virus serogroup 17 (SMSV-17) (DePaola et al., 2010) or echo type 9 virus (Nishida et al., 2007). According to ISO/TS 15216 recommendation, Mengovirus shall be used as a PCV in RT-qPCR determination of HAV and NoV in food matrices On the other hand, a negative feature of MS2 PLP is that they do not form plaques because they do not carry a gene for lysis. Therefore, their number cannot be easily determined using a conventional plaque assay. For this reason, indirect methods for MS2 PLP quantiﬁcation are used. The number of MS2 PLP December 2016 \| Volume 7 \| Article 1911 Frontiers in Microbiology \| www.frontiersin.org 7 Preparation of MS2 Phage-Like Particles Mikel et al. MS2 phage-like particles (MS2 PLP) isolated from different types of matrices artiﬁcially contaminated with 5 × 106 TABLE 3 \| Extraction efﬁciency of MS2 phage-like particles (MS2 PLP) isolated from different types of matrices artiﬁcially contaminated with 5 × 106 MS2 PLP per sample. Matrix Number of MS2 phage-like particles isolated from samples1 Positive samples/analyzed samples Extraction efﬁciency Mean SD Mean SD Swab 2,66 × 106 7,74 × 105 20/20 41,60% 12,09 Liver tissue 1,45 × 106 7,72 × 105 20/20 5,28% 2,63 Serum 6,10 × 106 1,76 × 106 20/20 77,04% 22,22 Feces 7,40 × 105 1,80 × 105 20/20 8,77% 2,14 Vegetable 2,99 × 105 1,89 × 105 18/18 4,29% 2,71 1Total amount of MS2 PLP from each sample was determined according to the calibration curve included in each RT-qPCR experiment. RT-qPCR was run in duplicate for each sample. Nucleic acid isolation efﬁciency was calculated according to the formula described within the text. SD, standard deviation. al amount of MS2 PLP from each sample was determined according to the calibration curve included in each RT-qPCR experime each sample. ACKNOWLEDGMENTS The authors would like to thank Neysan Donnelly (Max-Planck- Institute of Biochemistry, Germany) for grammatical corrections of the manuscript. FUNDING The results of the project LO1218 were obtained with ﬁnancial support from the MEYS of the CR under the NPU I program and project RO 0516 from the MA of the CR. This work was supported by Grant No. NT13884-4/2012 of the Ministry of Health of the Czech Republic. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. This work took some generally accepted recommendations used in RT-qPCR detection of RNA viruses from food matrices and applied them also for clinical samples. This work does not represent a competitive approach for ISO/TS 15216. In case of food samples, using of MS2 PLPs in RT-qPCR detection exactly according to ISO/TS would require comparison of the PCV used in ISO/TS (Mengovirus) with the MS2 PLPs and also discussion regarding additional speciﬁc requirements for PCV with technical committee that developed the standard. DISCUSSION Because false negative results may be caused not only by the failure of viral concentration steps and the nucleic acid extraction step but also by the inhibition of enzymatic reactions steps or failures in the elution, another control RNA molecule (IAC) was added to the RT-qPCR to detect possible inhibition on the level of RT-qPCR. Therefore, each isolated RNA sample should be ﬁrst subjected to a RT-qPCR assay for detection and quantiﬁcation of MS2 PLP with IAC. This ﬁrst assay allows exact determination of the December 2016 \| Volume 7 \| Article 1911 Frontiers in Microbiology \| www.frontiersin.org 8 Preparation of MS2 Phage-Like Particles Mikel et al. CONCLUSION The use of PCV in RT-qPCR detection of RNA viruses in complex matrices is highly beneﬁcial, since further increases the validity of the results. ISO/TS 15216 emphasizes the use of PCV (Mengovirus) in RT-qPCR detection of RNA viruses from food matrices (ISO/TS 15216-1, 2013; ISO/TS 15216-2, 2013). The use of appropriate PCV in RT-qPCR detection of RNA viruses from clinical samples is also necessary, but so far there is no ISO/TS. Therefore, the potential of MS2 PLPs as PCVs was tested in RT-qPCR detection of RNA viruses from food matrices together with clinical samples. Since there is still no universally accepted standard for clinical samples, some requirements (exact number of MS2 PLPs added to the samples, threshold of successful isolation) were adopted from standard applied in the analysis of food matrices (ISO/TS 15216-1). Moreover, MS2 PLPs also fulﬁll some additional requirements of ISO/TS 15216 – are non-enveloped, carry +ssRNA, have a similar size to the target viruses, are genetically distinct from the target viruses and their natural occurrence in tested sample is highly unlikely. On the other hand, MS2 PLPs do not meet the requirement of ISO/TS 15216-1 that PCV shall be a cultivable virus. This requirement is based on need to be able to maintain a suﬃcient supply of PCV in the laboratory by cultivating the PCV in cell culture and get more in-house prepared supplies of it. This can be a problem in laboratories where cultivation of viruses is restricted or in laboratories which do not have experience with the cultivation of viruses in cell cultures. In these cases, MS2 PLPs represents an alternative approach because they can be easily produces through bacterial cultures in high quantity. Moreover, this production approach of MS2 PLPs excludes the diﬀerence in virus source and provides for high quality, standardized PCV stocks. The described RT-qPCR system including MS2 PLP represents a potent tool for the complex monitoring of sample analysis. The design of the system with the inclusion of control RNA and IAC sequences allows their usage in any diagnostic procedure for the molecular detection of non-enveloped RNA viruses from diﬀerent types of matrices. The testing of the developed RT-qPCR system on diﬀerent matrices of clinical and food origin artiﬁcially contaminated with MS2 PLP showed that it is robust enough to provide reliable and precise data. AUTHOR CONTRIBUTIONS Conception and design of the work: PM, PV, and PeK; Acquisition of data: PM, RT, HM, PK, and TV; Interpretation of data: PM, PV, and PeK; Drafting the work: PM; Revision of the manuscript: PV and PeK; All authors approved the version to be published in Frontiers in Microbiology and agreed to be accountable for all aspects of the work. REFERENCES Reduction of Norwalk virus, poliovirus 1, and bacteriophage MS2 by ozone disinfection of water. Appl. Environ. Microbiol. 69, 3975–3978. doi: 10.1128/aem.69.7.3975-3978. 2003 ISO/TS 15216-2 (2013). Horizontal Method for Determination of Hepatitis A Virus and Norovirus in Food Using Real-Time RT-PCR Part 2: Method for Qualitative Detection, 1st Edn, Corrected Version 2013-05-01. Geneva: International Organization for Standardization. Shulman, L. M., Hindiyeh, M., Muhsen, K., Cohen, D., Mendelson, E., and Sofer, D. (2012). Evaluation of four diﬀerent systems for extraction of RNA from stool suspensions using MS-2 coliphage as an exogenous control for RT-PCR inhibition. PLoS ONE 7:e39455. doi: 10.1371/journal.pone. 0039455 Kwon, Y. J., Hung, G., Anderson, W. F., Peng, C. A., and Yu, H. (2003). Determination of infectious retrovirus concentration from colony-forming assay with quantitative analysis. J. Virol. 77, 5712–5720. doi: 10.1128/jvi.77.10. 5715-5720.2003 Song, L. Q., Sun, S. P., Li, B., Pan, Y., Li, W. L., Zhang, K., et al. (2011). External Quality assessment for Enterovirus 71 and Coxsackievirus A16 detection by reverse transcription-PCR using armored RNA as a virus surrogate. J. Clin. Microbiol. 49, 3591–3595. doi: 10.1128/JCM. 00686-11 Lindesmith, L., Moe, C., Marionneau, S., Ruvoen, N., Jiang, X., Lindbland, L., et al. (2003). Human susceptibility and resistance to Norwalk virus infection. Nat. Med. 9, 548–553. doi: 10.1038/nm860 Lozano, R., Naghavi, M., Foreman, K., Lim, S., Shibuya, K., Aboyans, V., et al. (2012). Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet 380, 2095–2128. doi: 10.1016/S0140-6736(12)61 728-0 Stinski, M. F., Mocarski, E. S., and Thomsen, D. R. (1979). DNA of human cytomegalovirus: size heterogeneity and defectiveness resulting from serial undiluted passage. J. Virol. 31, 231–239. Vasickova, P., Kralik, P., Slana, I., and Pavlik, I. (2012). Optimisation of a triplex real time RT-PCR for detection of hepatitis E Virus RNA and validation on biological samples. J. Virol. Methods 180, 38–42. Malenovska, H. (2013). Virus quantitation by transmission electron microscopy, TCID50, and the role of timing virus harvesting: a case study of three animal viruses. J. Virol. Methods 191, 136–140. doi: 10.1016/j.jviromet.2013.04.008 Vasickova, P., Slany, M., Chalupa, P., Holub, M., Svoboda, R., and Pavlik, I. (2011). Detection and phylogenetic characterization of human hepatitis E Virus strains. Czech Republic. Emerg. Infect. Dis. 17, 917–919. doi: 10.3201/eid1705. 101205 Martin-Latil, S., Hennechart-Collette, C., Guillier, L., and Perelle, S. (2012). REFERENCES Preparation of MS2 Phage-Like Particles Dykeman, E. C., Stockley, P. G., and Twarock, R. (2013). Building a viral capsid in the presence of genomic RNA. Phys. Rev. 87:022717. doi: 10.1103/PhysRevE.87. 022717 Pickett, G. G., and Peabody, D. S. (1993). Encapsidation of heterologous RNAs by bacteriophage-MS2 coat protein. Nucleic Acids Res. 21, 4621–4626. doi: 10.1093/nar/21.19.4621 Gentry-Shields, J., and Jaykus, L.-A. (2015). Comparison of process control viruses for use in extraction and detection of human norovirus from food matrices. Food Res. Int. 77, 320–325. doi: 10.1016/j.foodres.2015.05.027 Puig, M., Mihalik, K., Yu, M. Y. W., Feinstone, S. M., and Major, M. E. (2002). Sensitivity and reproducibility of HCV quantitation in chimpanzee sera using TaqMan real-time PCR assay. J. Virol. Methods 105, 253–263. doi: 10.1016/ s0166-0934(02)00119--112 Hennechart-Collette, C., Martin-Latil, S., Guillier, L., and Perelle, S. (2014). Multiplex real-time RT-qPCR for the detection of Norovirus in bottled and tap water using murine norovirus as a process control. J. Appl. Microbiol. 116, 179–190. doi: 10.1111/jam.12345 Radstrom, P., Knutsson, R., Wolﬀs, P., Lovenklev, M., and Lofstrom, C. (2004). Pre-PCR processing - strategies to generate PCR-compatible samples. Mol. Biotechnol. 26, 133–146. j Hennechart-Collette, C., Martin-Latil, S., Guillier, L., and Perelle, S. (2015). Determination of which virus to use as a process control when testing for the presence of hepatitis A virus and norovirus in food and water. Int. J. Food Microbiol. 202, 57–65. doi: 10.1016/j.ijfoodmicro.2015. 02.029 Rein, D. B., Stevens, G. A., Theaker, J., Wittenborn, J. S., and Wiersma, S. T. (2012). The global burden of hepatitis E virus genotypes 1 and 2 in 2005. Hepatology 55, 988–997. doi: 10.1002/hep.25505 Rolfe, K. J., Parmar, S., Mururi, D., Wreghitt, T. G., Jalal, H., Zhang, H., et al. (2007). An internally controlled, one-step, real-time RT-PCR assay for norovirus detection and genogrouping. J. Clin. Virol. 39, 318–321. doi: 10.1016/j.jcv.2007. 05.005 Hietala, S. K., and Crossley, B. M. (2006). Armored RNA as virus surrogate in a real-time reverse transcriptase PCR assay proﬁciency panel. J. Clin. Microbiol. 44, 67–70. doi: 10.1128/jcm.44.1.67-70.2006 Schrader, C., Schielke, A., Ellerbroek, L., and Johne, R. (2012). PCR inhibitors - occurrence, properties and removal. J. Appl. Microbiol. 113, 1014–1026. doi: 10.1111/j.1365-2672.2012.05384.x ISO/TS 15216-1 (2013). Microbiology of Food and Animal Feed – Horizontal Method for Determination of Hepatitis A Virus and Norovirus in Food Using Real-Time RT-PCR Part 1: Method for Quantiﬁcation, 1st Edn, Corrected Version 2013-05-01. Geneva: International Organization for Standardization. Shin, G. A., and Sobsey, M. D. (2003). REFERENCES Comparison of two extraction methods for the detection of hepatitis A virus in semi-dried tomatoes and murine norovirus as a process control by duplex RT-qPCR. Food Microbiol. 31, 246–253. doi: 10.1016/j.fm.2012. 03.007 Vojkovska, H., Kubikova, I., and Kralik, P. (2015). Evaluation of DNA extraction methods for PCR-based detection of Listeria monocytogenes from vegetables. Lett. Appl. Microbiol. 60, 265–272. doi: 10.1111/lam. 12367 Mattison, K., Brassard, J., Gagne, M. J., Ward, P., Houde, A., Lessard, L., et al. (2009). The feline calicivirus as a sample process control for the detection of food and waterborne RNA viruses. Int. J. Food Microbiol. 132, 73–77. doi: 10.1016/j.ijfoodmicro.2009.04.002 Vos, T., Barber, R. M., Bell, B., Bertozzi-Villa, A., Biryukov, S., Bolliger, I., et al. (2015). Global, regional, and national incidence, prevalence, and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet 386, 743–800. doi: 10.1016/S0140-6736(15) 60692-4 Mikel, P., Vasickova, P., and Kralik, P. (2015). Methods for preparation of MS2 phage-like particles and their utilization as process control viruses in RT-PCR and qRT-PCR detection of RNA viruses from food matrices and clinical specimens. Food Environ. Virol. 7, 96–111. doi: 10.1007/s12560-015- 9188-2 WalkerPeach, C. R., Winkler, M., DuBois, D. B., and Pasloske, B. L. (1999). Ribonuclease-resistant RNA controls (armored RNA) for reverse transcription- PCR, branched DNA, and genotyping assays for hepatitis C virus. Clin. Chem. 45, 2079–2085. Nishida, T., Nishio, O., Kato, M., Chuma, T., Kato, H., Iwata, H., et al. (2007). Genotyping and quantitation of noroviruses in oysters from two distinct sea areas in Japan. Microbiol. Immunol. 51, 177–184. doi: 10.1111/j.1348-0421.2007. tb03899.x Wei, B. J., Wei, Y. X., Zhang, K., Yang, C. M., Wang, J., Xu, R. H., et al. (2008). Construction of armored RNA containing long-size chimeric RNA by increasing the number and aﬃnity of the pac site in exogenous RNA and sequence coding coat protein of the MS2 bacteriophage. Intervirology 51, 144–150. doi: 10.1159/000141707 Park, G. W., and Sobsey, M. D. (2011). Simultaneous comparison of Murine Norovirus, Feline Calicivirus, coliphage MS2, and GII.4 Norovirus to evaluate the eﬃcacy of sodium hypochlorite against human Norovirus on a fecally soiled stainless steel surface. Foodborne Pathog. Dis. 8, 1005–1010. doi: 10.1089/fpd. 2010.0782 Wei, Y. X., Yang, C. M., Wei, B. J., Huang, J., Wang, L. N., Meng, S., et al. (2008). REFERENCES transcription-PCR detection of severe acute respiratory syndrome coronavirus. J. Clin. Microbiol. 44, 3557–3561. doi: 10.1128/JCM.00713-06 transcription-PCR detection of severe acute respiratory syndrome coronavirus. J. Clin. Microbiol. 44, 3557–3561. doi: 10.1128/JCM.00713-06 Abu Al-Soud, W., and Radstrom, P. (2000). Eﬀects of ampliﬁcation facilitators on diagnostic PCR in the presence of blood, feces, and meat. J. Clin. Microbiol. 38, 4463–4470. Coudray, C., Merle, G., Martin-Latil, S., Guillier, L., and Perelle, S. (2013). Comparison of two extraction methods for the detection of hepatitis A virus in lettuces using the murine norovirus as a process control. J. Virol. Methods 193, 96–102. doi: 10.1016/j.jviromet.2013.05.003 Bae, J., and Schwab, K. J. (2008). Evaluation of murine norovirus, feline calicivirus, poliovirus, and MS2 as surrogates for human norovirus in a model of viral persistence in surface water and groundwater. Appl. Environ. Microbiol. 74, 477–484. doi: 10.1128/AEM.02095-06 DePaola, A., Jones, J. L., Woods, J., Burkhardt, W., Calci, K. R., Krantz, J. A., et al. (2010). Bacterial and viral pathogens in live oysters: 2007 United States market survey. Appl. Environ. Microbiol. 76, 2754–2768. doi: 10.1128/AEM.02590-09 Di Pasquale, S., Paniconi, M., Auricchio, B., Oreﬁce, L., Schultz, A. C., and De Medici, D. (2010). Comparison of diﬀerent concentration methods for the detection of hepatitis A virus and calicivirus from bottled natural mineral waters. J. Virol. Methods 165, 57–63. doi: 10.1016/j.jviromet.2010. 01.003 Beld, M., Minnaar, R., Weel, J., Sol, C., Damen, M., van der Avoort, H., et al. (2004). Highly sensitive assay for detection of enterovirus in clinical specimens by reverse transcription-PCR with an armored RNA internal control. J. Clin. Microbiol. 42, 3059–3064. doi: 10.1128/jcm.42.7.3059-3064.2004 Blaise-Boisseau, S., Hennechart-Collette, C., Guillier, L., and Perelle, S. (2010). Duplex real-time qRT-PCR for the detection of hepatitis A virus in water and raspberries using the MS2 bacteriophage as a process control. J. Virol. Methods 166, 48–53. doi: 10.1016/j.jviromet.2010.02.017 Dreier, J., Stoermer, M., Maede, D., Burkhardt, S., and Kleesiek, K. (2006). Enhanced reverse transcription-PCR assay for detection of norovirus genogroup I. J. Clin. Microbiol. 44, 2714–2720. doi: 10.1128/JCM.00443-06 DuBois, D. B., Winkler, M. M., and Pasloske, B. L. (1997). Ribonuclease resistant viral RNA standards. U.S. Patent 5:124. Cheng, Y. J., Niu, J. J., Zhang, Y. Y., Huang, J. W., and Li, Q. G. (2006). Preparation of His-tagged armored RNA phage particles as a control for real-time reverse December 2016 \| Volume 7 \| Article 1911 Frontiers in Microbiology \| www.frontiersin.org 9 Mikel et al. Frontiers in Microbiology \| www.frontiersin.org December 2016 \| Volume 7 \| Article 1911 REFERENCES RNase-resistant virus-like particles containing long chimeric RNA sequences produced by two-plasmid coexpression system. J. Clin. Microbiol. 46, 1734–1740. doi: 10.1128/JCM.02248-07 Pasloske, B. L., Walkerpeach, C. R., Obermoeller, R. D., Winkler, M., and DuBois, D. B. (1998). Armored RNA technology for production of ribonuclease- resistant viral RNA controls and standards. J. Clin. Microbiol. 36, 3590–3594. December 2016 \| Volume 7 \| Article 1911 Frontiers in Microbiology \| www.frontiersin.org 10 Mikel et al. Preparation of MS2 Phage-Like Particles Wei, Z., McEvoy, M., Razinkov, V., Polozova, A., Li, E., Casas-Finet, J., et al. (2007). Biophysical characterization of inﬂuenza virus subpopulations using ﬁeld ﬂow fractionation and multiangle light scattering: correlation of particle counts, size distribution and infectivity. J. Virol. Methods 144, 122–132. doi: 10.1016/j.jviromet.2007.04.008 Zhan, S., Li, J. M., Xu, R. H., Wang, L. A., Zhang, K., and Zhang, R. (2009). Armored long RNA controls or standards for branched DNA assay for detection of human immunodeﬁciency virus type 1. J. Clin. Microbiol. 47, 2571–2576. doi: 10.1128/jcm.00232-09 Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Weidmann, M., Sall, A. A., Manuguerra, J.-C., Koivogui, L., Adjami, A., Traore, F. F., et al. (2011). Quantitative analysis of particles, genomes and infectious particles in supernatants of haemorrhagic fever virus cell cultures. Virol. J. 8:81. doi: 10.1186/1743-422X-8-81 Copyright © 2016 Mikel, Vasickova, Tesarik, Malenovska, Kulich, Vesely and Kralik. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. WHO (2012). Prevention and Control of Viral Hepatitis Infection: Framework for Global Action. Geneva: World Health Organization. Yu, X. F., Pan, J. C., Ye, R., Xiang, H. Q., Kou, Y., and Huang, Z. C. (2008). Preparation of armored RNA as a control for multiplex real-time reverse transcription-PCR detection of inﬂuenza virus and severe acute respiratory syndrome coronavirus. J. Clin. Microbiol. 46, 837–841. doi: 10.1128/JCM. 01904-07 December 2016 \| Volume 7 \| Article 1911 Frontiers in Microbiology \| www.frontiersin.org 11
https://openalex.org/W4323664867	https://dergipark.org.tr/tr/download/article-file/2302704	Turkish	null	AISI 316L Alaşımın Tel Erozyon Yöntemi ile İşlenmesinde Kesme Parametrelerinin Yüzey Kalitesine Etkisi	Osmaniye Korkut Ata Üniversitesi Fen Bilimleri Enstitüsü dergisi (Online)	2,023	cc-by	4,412	Kutay AYDIN1, Hakan KAZAN2 Kutay AYDIN1, Hakan KAZAN2 1,2Amasya Üniversitesi, Mühendislik Fakültesi, Makine Mühendisliği Bölümü, 05100, Amasya 1https://orcid.org/0000-0003-3614-4877 2https://orcid.org/0000-0001-7745-8974 Sorumlu yazar: kutay.aydin@amasya.edu.tr 1,2Amasya Üniversitesi, Mühendislik Fakültesi, Makine Mühendisliği Bölümü, 05100, Amasya The Impact of the Cutting Parameters on the Surface Quality of AISI 316L with WEDM The Impact of the Cutting Parameters on the Surface Quality of AISI 316L with WEDM Research Article ABSTRACT Article History: Received: 10.03.2022 Accepted: 02.08.2022 Published online: 10.03.2023 Wire electrical discharge machining (WEDM) which is widely used in the industry has an important place in the manufacturing process. WEDM is a preferred manufacturing method, especially in terms of the production of parts that are difficult to produce with traditional manufacturing methods and low energy consumption. This focused on the effects of different cutting parameters on the surface quality of AISI 316L austenitic stainless steel alloy during wire erosion processing. In addition, it is aimed to increase the surface quality without extending the production period. In this context, experimental studies were carried out in full factorial design with different cutting parameters, and then the behavior of the cutting parameters on the surface quality was investigated with variance and regression analyses. As a result, an increasing trend was observed in the surface roughness values with the increase in the feed rate and pulse on time and the decrease in the pulse off time. The lowest surface roughness value was measured as 3.095 µm at 2 mm/min feed rate, 16 µs pulse on time, and 480 µs pulse off time parameters. As a result of statistical data analysis, it was determined that the most effective cutting parameter in terms of surface quality was the pulse time with a rate of 83.96%. Keywords: Wire cutting method WEDM AISI 316L ANOVA Surface roughness To Cite: Aydın K., Kazan H. AISI 316L Alaşımın Tel Erozyon Yöntemi ile İşlenmesinde Kesme Parametrelerinin Yüzey Kalitesine Etkisi. Osmaniye Korkut Ata Üniversitesi Fen Bilimleri Enstitüsü Dergisi 2023; 6(1): 575-584. OKU Fen Bilimleri Enstitüsü Dergisi 6(1): 575-584, 2023 OKU Journal of The Institute of Science and Technology, 6(1): 575-584, 2023 Osmaniye Korkut Ata Üniversitesi Fen Bilimleri Enstitüsü Dergisi Osmaniye Korkut Ata University Journal of The Institute of Science and Technology AISI 316L Alaşımın Tel Erozyon Yöntemi ile İşlenmesinde Kesme Parametrelerinin Yüzey Kalitesine Etkisi Kutay AYDIN1, Hakan KAZAN2 1,2Amasya Üniversitesi, Mühendislik Fakültesi, Makine Mühendisliği Bölümü, 05100, Amasya OKU Fen Bilimleri Enstitüsü Dergisi 6(1): 575-584, 2023 OKU Journal of The Institute of Science and Technology, 6(1): 575-584, 2023 Osmaniye Korkut Ata Üniversitesi Fen Bilimleri Enstitüsü Dergisi Osmaniye Korkut Ata University Journal of The Institute of Science and Technology AISI 316L Alaşımın Tel Erozyon Yöntemi ile İşlenmesinde Kesme Parametrelerinin Yüzey Kalitesine Etkisi Kutay AYDIN1*, Hakan KAZAN2 1,2Amasya Üniversitesi, Mühendislik Fakültesi, Makine Mühendisliği Bölümü, 05100, Amasya OKU Fen Bilimleri Enstitüsü Dergisi 6(1): 575-584, 2023 OKU Journal of The Institute of Science and Technology, 6(1): 575-584, 2023 Osmaniye Korkut Ata Üniversitesi Fen Bilimleri Enstitüsü Dergisi Osmaniye Korkut Ata University Journal of The Institute of Science and Technology AISI 316L Alaşımın Tel Erozyon Yöntemi ile İşlenmesinde Kesme Parametrelerinin Yüzey Kalitesine Etkisi OKU Fen Bilimleri Enstitüsü Dergisi 6(1): 575-584, 2023 O 6 Osmaniye Korkut Ata Üniversitesi Fen Bilimleri Enstitüsü Dergisi Osmaniye Korkut Ata University Journal of The Institute of Science and Technology Araştırma Makalesi Makale Tarihçesi: Geliş tarihi: 10.03.2022 Kabul tarihi:02.08.2022 Online Yayınlanma: 10.03.2023 Endüstride kullanımı oldukça yaygınlaşan tel erozyonla işleme teknolojisi, özellikle geleneksel imalat yöntemleri ile üretilmesi zor parçaların imalatı ve düşük enerji tüketimi bakımından imalat sektöründe önemli bir yere sahiptir. Bu çalışmada, AISI 316L östenitik paslanmaz çelik alaşımın tel erozyon yöntemi ile işlenmesi sırasında farklı kesme parametrelerinin yüzey kalitesi üzerinde oluşturduğu etkilere odaklanılmıştır. Bununla birlikte üretim süresini uzatmadan yüzey kalitesinin arttırılması amaçlanmıştır. Bu kapsamda, farklı kesme parametreleri ile tam faktöriyel tasarımda deneysel çalışmalar yapılmış, devamında varyans ve regresyon analizleri ile kesme parametrelerinin yüzey kalitesi üzerindeki davranışları araştırılmıştır. Sonuç olarak, ilerleme hızı ve bombardıman süresinin artması ve bekleme süresinin azalması ile yüzey pürüzlülük değerlerinde artış eğilimi görülmüştür. En düşük yüzey pürüzlülük değeri, 3,095 µm olarak 2 mm/dk ilerleme hızı, 16 µs bombardıman süresi ve 480 µs bekleme süresi parametrelerinde ölçülmüştür. İstatistiksel veri analizleri sonucunda, yüzey kalitesi açısından en etkin kesme parametresinin %83,96 oranla bombardıman süresi olduğu tespit edilmiştir. Endüstride kullanımı oldukça yaygınlaşan tel erozyonla işleme teknolojisi, özellikle geleneksel imalat yöntemleri ile üretilmesi zor parçaların imalatı ve düşük enerji tüketimi bakımından imalat sektöründe önemli bir yere sahiptir. Bu çalışmada, AISI 316L östenitik paslanmaz çelik alaşımın tel erozyon yöntemi ile işlenmesi sırasında farklı kesme parametrelerinin yüzey kalitesi üzerinde oluşturduğu etkilere odaklanılmıştır. Bununla birlikte üretim süresini uzatmadan yüzey kalitesinin arttırılması amaçlanmıştır. Bu kapsamda, farklı kesme parametreleri ile tam faktöriyel tasarımda deneysel çalışmalar yapılmış, devamında varyans ve regresyon analizleri ile kesme parametrelerinin yüzey kalitesi üzerindeki davranışları araştırılmıştır. Sonuç olarak, ilerleme hızı ve bombardıman süresinin artması ve bekleme süresinin azalması ile yüzey pürüzlülük değerlerinde artış eğilimi görülmüştür. En düşük yüzey pürüzlülük değeri, 3,095 µm olarak 2 mm/dk ilerleme hızı, 16 µs bombardıman süresi ve 480 µs bekleme süresi parametrelerinde ölçülmüştür. İstatistiksel veri analizleri sonucunda, yüzey kalitesi açısından en etkin kesme parametresinin %83,96 oranla bombardıman süresi olduğu tespit edilmiştir. The Impact of the Cutting Parameters on the Surface Quality of AISI 316L with WEDM The Impact of the Cutting Parameters on the Surface Quality of AISI 316L with WEDM Research Article ABSTRACT Article History: Received: 10.03.2022 Accepted: 02.08.2022 Published online: 10.03.2023 Wire electrical discharge machining (WEDM) which is widely used in the industry has an important place in the manufacturing process. WEDM is a preferred manufacturing method, especially in terms of the production of parts that are difficult to produce with traditional manufacturing methods and low energy consumption. AISI 316L Alaşımın Tel Erozyon Yöntemi ile İşlenmesinde Kesme Parametrelerinin Yüzey Kalitesine Etkisi AISI 316L Alaşımın Tel Erozyon Yöntemi ile İşlenmesinde Kesme Parametrelerinin Yüzey Kalitesine Etkisi To Cite: Aydın K., Kazan H. AISI 316L Alaşımın Tel Erozyon Yöntemi ile İşlenmesinde Kesme Parametrelerinin Yüzey Kalitesine Etkisi. Osmaniye Korkut Ata Üniversitesi Fen Bilimleri Enstitüsü Dergisi 2023; 6(1): 575-584. Araştırma Makalesi This focused on the effects of different cutting parameters on the surface quality of AISI 316L austenitic stainless steel alloy during wire erosion processing. In addition, it is aimed to increase the surface quality without extending the production period. In this context, experimental studies were carried out in full factorial design with different cutting parameters, and then the behavior of the cutting parameters on the surface quality was investigated with variance and regression analyses. As a result, an increasing trend was observed in the surface roughness values with the increase in the feed rate and pulse on time and the decrease in the pulse off time. The lowest surface roughness value was measured as 3.095 µm at 2 mm/min feed rate, 16 µs pulse on time, and 480 µs pulse off time parameters. As a result of statistical data analysis, it was determined that the most effective cutting parameter in terms of surface quality was the pulse time with a rate of 83.96%. Keywords: Wire cutting method WEDM AISI 316L ANOVA Surface roughness To Cite: Aydın K., Kazan H. AISI 316L Alaşımın Tel Erozyon Yöntemi ile İşlenmesinde Kesme Parametrelerinin Yüzey Kalitesine Etkisi. Osmaniye Korkut Ata Üniversitesi Fen Bilimleri Enstitüsü Dergisi 2023; 6(1): 575-584. 1. Giriş Tel erozyonla işleme, yüksek mekanik ve ısıl özelliklerinden dolayı işlenmesi zor malzemeler olarak bilinen süper alaşımlar, metal ve seramik matrisli kompozitler gibi malzemelerin işlenmesinde sıklıkla başvurulan geleneksel olmayan imalat metotlarından biridir (Tosun ve ark., 2003; Marelli ve ark., 2020; Işık ve ark., 2021; Eksilmez ve ark., 2022). Tel erozyonla işleme yöntemi, yüksek sertlik ve tokluğa sahip malzemelerden karmaşık şekiller üretebilme kabiliyeti sebebiyle savunma, havacılık, otomotiv, kalıp ve kalıp yapım vb. endüstrilerde yaygın olarak kullanılmaktadır (Chiang ve ark., 2007; Tilekar ve ark., 2014; Cebeci ve ark., 2020). Tel erozyon yönteminin çalışma prensibi temel olarak iletken iş parçasından, servo motor yardımıyla pirinç, tungsten, bakır veya molibden tel kullanılarak temas yüzeyi oluşturulmadan talaş kaldırılması işlemidir. Burada, ısınma ve soğumanın tekrarlandığı karmaşık bir elektro termal süreç gerçekleşir. Telden (anode) iş parçasına (cathode) doğru akan elektrik arkı ile iş parçası eriyip buharlaşır; talaş, yalıtkan sıvı vasıtası ile soğutularak iş parçasından uzaklaştırılır (Snoeys ve ark., 1986; Esme ve ark., 2013; Özlü, 2021). Endüstrinin sürekli artış gösteren seri ve kaliteli ürün talebinin karşılanabilmesi için tel erozyon kesme parametreleri hem endüstri hem de akademide önemli bir araştırma konusudur. Bu bağlamda çok sayıda çalışmada bombardıman süresi, bekleme süresi, tel ilerleme oranı, akım, voltaj vb. birçok giriş parametresinin talaş kaldırma oranı, yüzey pürüzlülüğü gibi çıktılara etkisi farklı malzemeler için deneysel ve istatistiksel olarak araştırılmıştır (Koklu, 2012; Li ve ark., 2013; Azam ve ark., 2016; Aydın ve ark., 2022). Bakır tel kullanılarak, bombardıman süresi ve tepe akımının AISI 316 alaşımından tel erozyon yöntemiyle talaş kaldırma oranı ve yüzey pürüzlülüğü üzerindeki etkileri Taguchi L9 istatistiksel yaklaşımıyla incelenmiştir (Ramachandra, 2017). Bu çalışma sonucuna göre hem yüzey pürüzlülüğü hem de talaş kaldırma oranı üzerinde tepe akımının etken faktör olduğu gözlemlenmiştir. Benzer şekilde AISI 316L üzerinde tungsten tel malzeme kullanılarak yapılan kesme işleminde voltaj ve akımın talaş kaldırma oranına etkileri sunulmuştur (Singh ve ark., 2017). Aynı malzeme üzerinde yapılan bir diğer çalışmada ise bombardıman zamanı, tepe akımı, servo voltajı ve tel gerilmesinin yüzey pürüzlülüğü üzerindeki etkileri incelenmiştir. Çalışma sonucuna göre bombardıman zamanı diğer parametrelerle kıyaslanınca yüzey pürüzlülüğü üzerinde önemli bir rol oynamaktadır (Prathipati ve ark., 2020). Başka bir çalışmanın konusu ise AISI 316L çeliğinin tel erozyonla kesme işleminde oluşan yüzey pürüzlülüğüne bombardıman süresi, bekleme süresi ve voltaj parametrelerinin etkilerinin Taguchi yöntemiyle incelenmesi olmuştur (Babu ve ark., 2018). Bu araştırmadan elden edilen bulgular sonuncunda bombardıman süresinin yüzey pürüzlülüğü üzerinde önemli bir etkiye sahip olduğu sonucuna ulaşılmıştır. ABSTRACT Wire electrical discharge machining (WEDM) which is widely used in the industry has an important place in the manufacturing process. WEDM is a preferred manufacturing method, especially in terms of the production of parts that are difficult to produce with traditional manufacturing methods and low energy consumption. This focused on the effects of different cutting parameters on the surface quality of AISI 316L austenitic stainless steel alloy during wire erosion processing. In addition, it is aimed to increase the surface quality without extending the production period. In this context, experimental studies were carried out in full factorial design with different cutting parameters, and then the behavior of the cutting parameters on the surface quality was investigated with variance and regression analyses. As a result, an increasing trend was observed in the surface roughness values with the increase in the feed rate and pulse on time and the decrease in the pulse off time. The lowest surface roughness value was measured as 3.095 µm at 2 mm/min feed rate, 16 µs pulse on time, and 480 µs pulse off time parameters. As a result of statistical data analysis, it was determined that the most effective cutting parameter in terms of surface quality was the pulse time with a rate of 83.96%. 575 1. Giriş Buna ilaveten, lazer eritme ve döküm ile elde edilen AISI 316L çeliğinin yüzey pürüzlülükleri üzerindeki tepe akımı etkileri araştırmacılar tarafından karşılaştırılmıştır (Machno ve ark., 2022). AISI 316L çeliğinin yüzey pürüzlülüğü ve talaş kaldırma çıktılarının incelendiği güncel bir çalışmada ise, düşük hızlardaki tel ilerleme oranı, voltaj, 576 bombardıman süresi, bekleme süresi ve tepe akım değerleri Taguchi ve gri ilişki analiz yöntemleri kullanılarak karşılaştırılmıştır (Soota ve Rajput, 2022). Bu çalışma sonuçlarına göre işlem parametrelerinin yüzey pürüzlüğüne etkileri sırasıyla tel ilerleme oranı, tepe akımı, bombardıman süresi, voltaj ve bekleme süresi olarak hesaplanmıştır. Ayrıca, araştırmacılar düşük ilerleme çalışma koşullarında bombardıman süre ve tepe akımındaki artışların yüzey kalitesini arttırdığını gözlemlemişlerdir. Literatürde, islenmesi zor malzemeler ve daha spesifik olarak kimya, sağlık, nükleer, elektronik vb. birçok endüstride tercih edilen AISI 316L çeliğinin tel erozyonla işleme parametreleri detaylandırılmıştır. Ancak üretim süresinin kısaltılması ve yüzey kalitesinin arttırılması için kritik rol oynayan yüksek ilerleme oranı ve bekleme sürelerinin yüzey pürüzlülüğü üzerine etkileri incelenmemiştir. Üretim süresinin kısaltılması ve yüzey pürüzlülüğünün azaltılması sürdürülebilir bir seri imalatın anahtar faktörlerindendir. Bu sebeple, bu çalışmada 3 farklı seviyedeki bombardıman süresi ve bekleme sürelerinin yine 3 farklı ilerleme oranında yüzey pürüzlülüğü üzerine etkileri deneysel olarak incelenmiş ve deney sonuçları istatistiksel olarak karşılaştırılmıştır. 2. Materyal ve Metot Çalışmada iş parçası olarak AISI 316L östenitik paslanmaz çelik kullanılmıştır. Tablo 1’de iş parçası olarak kullanılan AISI 316L östenitik paslanmaz çeliğin kimyasal bileşimi verilmiştir. İş parçasına uygulanmış bir ısıl işlem bulunmamaktadır. Deneyler, Hightech DK-7732 CNC WEDM tezgahında yapılmıştır. Kesici tel olarak 0,18 mm Guangming Molibden alaşım tel, yalıtkan sıvı olarak saf su ve JR3A Ointment Coolant, 1/30-40 (kg/kg) (JR3A/pure water) oranında emülsiyonu kullanılmıştır. Molibden tel kullanan tezgahlarda tel bir tambura sarılı olarak devamlı kullanılır. Tel hızı 11 m/s, yalıtkan emülsiyon sirkülasyon basıncı ise 1 bar seviyesinde sabit olarak uygulanmıştır. Deneysel kurulum şematik olarak Şekil 1’de gösterilmiştir. Tablo 1. AISI 316L alaşımın kimyasal bileşimi % Ağırlık Ana C Si Mn S P Cr Mo Ni AISI 316L Fe 0,30 1 2 0,03 0,05 16-18 2-3 10-14 Şekil 1. Deneysel süreç şeması Şekil 1. Deneysel süreç şeması Şekil 1. Deneysel süreç şeması 577 Deneysel olarak elde edilen yüzey pürüzlülük değerleri ile optimum kesme parametrelerinin anlaşılması mümkündür. Ancak, kesme parametrelerinin yüzey pürüzlülüğü üzerindeki davranışlarının detaylı bir şekilde tespit edilebilmesi ve en etkin kesme parametresinin belirlenmesi için varyans ve regresyon analizleri yapılmıştır. İstatistiksel veri analizi, Minitab® R19 yazılımı ile yapılmıştır. Deneyler ve veri analizleri, tam faktöriyel deney düzeni ile gerçekleştirilmiştir. Kesme parametreleri ve bağımsız kontrol faktörleri olarak ilerleme hızı, bombardıman süresi ve bekleme süresi seçilmiştir. Her faktör için üç seviye belirlenmiştir (Tablo 2). Veri analizleri ile kontrol faktörleri ve seviyelerinin çıktı (yüzey pürüzlülüğü) üzerindeki etkinlik seviyeleri tespit edilmiştir. Regresyon analizleri ile kesme parametrelerinin yüzey pürüzlülüğü üzerindeki etkisi ampirik bir matematiksel model ile tanımlanmıştır. Yüzey pürüzlülük değerleri Mitutoyo Surfest SJ-210 cihazı ile ölçülmüştür. Ölçümler, ISO 4288 standardına göre yapılmış olup kesim uzunluğu (λc) 0,8 mm ve travers uzunluğu (Lt) 5,6 mm seçilmiştir. Tablo 2. Kontrol faktörleri, seviyeler ve çıktılar Faktörler İlerleme Hızı (mm/dk) Bombardıman Süresi (µs) Bekleme Süresi (µs) Seviye 1 2 16 45 Seviye 2 3 32 144 Seviye 3 4 64 480 Çıktılar Yüzey Pürüzlülüğü (µm) 3. Bulgular ve Tartışma Tablo 2. Kontrol faktörleri, seviyeler ve çıktılar Faktörler İlerleme Hızı (mm/dk) Bombardıman Süresi (µs) Bekleme Süresi (µs) Seviye 1 2 16 45 Seviye 2 3 32 144 Seviye 3 4 64 480 Çıktılar Yüzey Pürüzlülüğü (µm) 3. Bulgular ve Tartışma Tablo 2. Kontrol faktörleri, seviyeler ve çıktılar 3. Bulgular ve Tartışma 3. Bulgular ve Tartışma Deneyler sonucunda, yüzey pürüzlülük değerlerinin değişimleri ilerleme hızı için Şekil 2’de, bombardıman süresi için Şekil 3’te ve bekleme süresi için ise Şekil 4’te gösterilmiştir. Tam faktöriyel deney düzeni ve elde edilen bulgular Tablo 3’te verilmiştir. Şekil 2 incelendiğinde, ilerleme hızı artışının yüzey pürüzlülüğünü artırdığı görülmektedir. İlerleme hızının artışı ile tel ve iş parçası arasındaki etkileşim süresi azalır. Bu durum sonucunda yüzey pürüzlülük değerlerinde artış görülmüştür. Şekil 3 incelendiğinde, elektron bombardıman süresi artışının yüzey pürüzlülük değerlerinin belirgin bir şekilde artırdığı gözlenmektedir. Bombardıman süresi artışı ile telden iş parçasına doğru elektron akışı artar. Artan elektron bombardımanı sonucunda, iş parçası üzerinde oluşan krater büyüklüğü artar. Bu durum, yüzey kalitesinin azalmasına neden olmaktadır (Azam ve ark., 2016). Şekil 4’te bekleme süresi artışı ile yüzey pürüzlülüğü azalmaktadır. Elektron bombardımanları arasındaki bekleme süresi boyunca yalıtkan sıvı, talaşları kesme bölgesinden uzaklaştırır. Bekleme süresi artışı ile kesme bölgesinin kararlılığı artar ve talaşların uzaklaştırılması kolaylaşır. Bu durum yüzey pürüzlülüğünü azalmasına neden olur (Sharma ve ark., 2013; Soota ve Rajput, 2022). En düşük yüzey pürüzlülük değeri 2 mm/dk ilerleme hızı, 16 µs bombardıman süresi ve 480 µs bekleme süresi parametrelerinde elde edilmiştir. 578 Tablo 3. Deney kurulumu ve sonuçlar Tablo 3. Deney kurulumu ve sonuçlar Deney no Kesme parametreleri Sonuçlar İlerleme Hızı (mm/min) Bombardıman Süresi (µs) Bekleme Süresi (µs) Yüzey Pürüzlülüğü Ra (µm) 1 2 16 45 3,345 2 2 16 144 3,267 3 2 16 480 3,095 4 2 32 45 4,531 5 2 32 144 4,379 6 2 32 480 4,305 7 2 64 45 5,721 8 2 64 144 5,516 9 2 64 480 5,452 10 3 16 45 3,687 11 3 16 144 3,547 12 3 16 480 3,411 13 3 32 45 4,823 14 3 32 144 4,617 15 3 32 480 4,523 16 3 64 45 6,138 17 3 64 144 6,049 18 3 64 480 5,881 19 4 16 45 4,439 20 4 16 144 4,225 21 4 16 480 4,047 22 4 32 45 5,398 23 4 32 144 5,207 24 4 32 480 4,972 25 4 64 45 6,875 26 4 64 144 6,731 27 4 64 480 6,685 Şekil 2. İlerleme hızına bağlı olarak yüzey pürüzlülüğü değişimleri Tablo 3. 3. Bulgular ve Tartışma Deney kurulumu ve sonuçlar Deney no Kesme parametreleri Sonuçlar İlerleme Hızı (mm/min) Bombardıman Süresi (µs) Bekleme Süresi (µs) Yüzey Pürüzlülüğü Ra (µm) 1 2 16 45 3,345 2 2 16 144 3,267 3 2 16 480 3,095 4 2 32 45 4,531 5 2 32 144 4,379 6 2 32 480 4,305 7 2 64 45 5,721 8 2 64 144 5,516 9 2 64 480 5,452 10 3 16 45 3,687 11 3 16 144 3,547 12 3 16 480 3,411 13 3 32 45 4,823 14 3 32 144 4,617 15 3 32 480 4,523 16 3 64 45 6,138 17 3 64 144 6,049 18 3 64 480 5,881 19 4 16 45 4,439 20 4 16 144 4,225 21 4 16 480 4,047 22 4 32 45 5,398 23 4 32 144 5,207 24 4 32 480 4,972 25 4 64 45 6,875 26 4 64 144 6,731 27 4 64 480 6,685 Şekil 2. İlerleme hızına bağlı olarak yüzey pürüzlülüğü değişimleri Şekil 2. İlerleme hızına bağlı olarak yüzey pürüzlülüğü değişimleri 579 Şekil 3. Bombardıman süresine bağlı olarak yüzey pürüzlülüğü değişimleri Şekil 4. Bekleme süresine bağlı olarak yüzey pürüzlülüğü değişimleri Şekil 3. Bombardıman süresine bağlı olarak yüzey pürüzlülüğü değişimleri Şekil 4. Bekleme süresine bağlı olarak yüzey pürüzlülüğü değişimleri Şekil 4. Bekleme süresine bağlı olarak yüzey pürüzlülüğü değişimleri Ölçülen yüzey pürüzlülük bulgularının, bağımsız kontrol faktörleri olarak seçilen ilerleme hızı, bombardıman süresi ve bekleme süresi parametrelerine göre değiştiği kabulü ve %95 güven seviyesi ile varyans ve regresyon veri analizleri yapılmıştır. Varyans analizi sonuçları ve % etki oranları Tablo 4’te verilmiştir. Sonuçlar incelendiğinde, tüm P değerlerinin %5 (P<0,05) oranından küçük çıktığı görülmektedir. Buna ek olarak faktörlerin F değerleri ile kritik F değerleri de karşılaştırılmıştır. Tüm faktörler için 0,05'e eşit anlamlılık veya %95 güven seviyesindeki kritik F değeri (F.05,2,26) 3,369’dur. Faktörlerin F değerleri, kritik F değerinden büyük çıkmıştır. Düşük P değerleri ve kritik F değeri karşılaştırması sonucunda kontrol faktörleri ve seviyelerin seçimleri istatiksel açıdan anlamlı seviyededir (Bolar ve ark., 2018). Yüzey kalitesi açısından en etkili kesme parametresi, %83,96 oranla bombardıman süresi olarak elde edilmiştir. 580 Tablo 4. Varyans analizleri Tablo 4. 4. Sonuçlar Bu çalışmada, AISI 316L östenitik paslanmaz çelik alaşımın tel erozyon yöntemi ile işlenerek, farklı kesme parametrelerinin yüzey kalitesine etkileri araştırılmıştır. Bu kapsam dahilinde, farklı ilerleme hızı, bombardıman süresi ve bekleme süre parametreleri seçilerek tam faktöriyel deneysel çalışma ve istatiksel analizler yapılmıştır. Elde edilen sonuçlar şu şekilde özetlenmiştir: • Yüzey pürüzlülüğü açısından; ilerleme hızı ve bombardıman süresi arttıkça ve bekleme süresi azaldıkça pürüzlülük değerlerinde artış gözlenmiştir. • Yüzey pürüzlülüğü açısından; ilerleme hızı ve bombardıman süresi arttıkça ve bekleme süresi azaldıkça pürüzlülük değerlerinde artış gözlenmiştir. • En düşük yüzey pürüzlülük değeri, 2 mm/dk ilerleme hızı, 16 µs bombardıman süresi ve 480 µs bekleme süresi parametrelerinde elde edilmiştir. • Varyans analizleri açısından; yüzey pürüzlülüğü üzerindeki en etkin kesme parametresinin %83,96 oranla bombardıman süresi olduğu anlaşılmıştır. • Regresyon analizleri açılarından; modelin belirlilik katsayısı %99,34 oranında elde edilmiştir. Bu oran ile kurulan istatistiksel modelin ve deneysel bulguların birbirleri ile uyumlu olduğu sonucuna varılmıştır. • Regresyon analizleri açılarından; modelin belirlilik katsayısı %99,34 oranında elde edilmiştir. Bu oran ile kurulan istatistiksel modelin ve deneysel bulguların birbirleri ile uyumlu olduğu sonucuna varılmıştır. Sonuç olarak, işlenmesi zor malzemeler grubunda bulunan AISI 316L alaşımın tel erozyon yöntemi ile işlenmesinde, üretim süresini uzatmadan yüzey kalitesinin artırmak amacıyla farklı kesme parametrelerinin yüzey pürüzlülüğü üzerindeki etkileri belirlenmiştir. 3. Bulgular ve Tartışma Model özeti S R2 R2(adj) R2(pred) 0,090564 99,49% 99,34% 99,07% Tablo 5. Model özeti S R2 R2(adj) R2(pred) 0,090564 99,49% 99,34% 99,07% Tablo 5. Model özeti 𝑌ü𝑧𝑒𝑦 𝑃ü𝑟ü𝑧𝑙ü𝑙üğü 𝑧𝑒𝑦 𝑃ü𝑟ü𝑧𝑙ü𝑙üğü = 4,8469 −(0,4457 × İ𝑙𝑒𝑟𝑙𝑒𝑚𝑒 ℎ𝚤𝑧𝚤_1) −(0,1051 × İ𝑙𝑒𝑟𝑙𝑒𝑚𝑒 ℎ𝚤𝑧𝚤_2) + (0,5508 × İ𝑙𝑒𝑟𝑙𝑒𝑚𝑒 ℎ𝚤𝑧𝚤_3) −(1,1732 × 𝐵𝑜𝑚𝑏𝑎𝑟𝑑𝚤𝑚𝑎𝑛 𝑠ü𝑟𝑒𝑠𝑖_1) −( 0,0963 × 𝐵𝑜𝑚𝑏𝑎𝑟𝑑𝚤𝑚𝑎𝑛 𝑠ü𝑟𝑒𝑠𝑖_2) + (1,2696 × 𝐵𝑜𝑚𝑏𝑎𝑟𝑑𝚤𝑚𝑎𝑛 𝑠ü𝑟𝑒𝑠𝑖_3) + (0,1483 × 𝐵𝑒𝑘𝑙𝑒𝑚𝑒 𝑠ü𝑟𝑒𝑠𝑖_1) −( 0,0093 × 𝐵𝑒𝑘𝑙𝑒𝑚𝑒 𝑠ü𝑟𝑒𝑠𝑖_2) −(0,1390 × 𝐵𝑒𝑘𝑙𝑒𝑚𝑒 𝑠ü𝑟𝑒𝑠𝑖_3) (1) (1) Çıkar Çatışması Beyanı Makale yazarları aralarında herhangi bir çıkar çatışması olmadığını beyan ederler. 3. Bulgular ve Tartışma Varyans analizleri Kaynak (Source) Serbestlik Derecesi (DF) Kareler Toplamı (Adj SS) Kareler Ortalaması (Adj MS) F değeri P değeri Etki (%) (Contribution) Model 6 31,9674 5,3279 649,6 0,000 99,49 Lineer 6 31,9674 5,3279 649,6 0,000 99,49 İlerleme hızı, mm/dk 2 4,6172 2,3086 281,47 0,000 14,37 Bombardıman süresi, µs 2 26,9775 13,4888 1644,6 0,000 83,96 Bekleme süresi, µs 2 0,3727 0,1863 22,72 0,000 1,16 Hata 20 0,164 0,0082 0,51 Toplam 26 32,1315 100,00 Kontrol faktörleri değişiminin yüzey pürüzlülüğü üzerindeki yarattığı davranışın belirgin olarak görülebilmesi için etki grafiği çizilmiştir (Şekil 5). Grafikte, tüm faktör ve seviyeler için yüzey pürüzlülük değerleri ve standart hata barları verilmiştir. Etki grafiği incelendiğinde, ilerleme hızı ve bombardıman süresi artışı ile yüzey pürüzlülük değerlerinin arttığı, bekleme süresi artışı ile yüzey pürüzlülük değerlerinin azaldığı görülmektedir. Bu durum, deneysel bulgular ile uyum göstermektedir. Grafikte, standart hata değerleri incelendiğinde ise ilerleme hızı ve bombardıman süresi faktörlerinin bütün seviyeleri için bağımsız etki alanları elde edilmiştir. Bekleme süresinin tüm seviyelerinde hata değerlerinde çakışma mevcuttur. Bu durum deneysel bulgularda da görüldü gibi, bekleme süresi faktörünün yüzey kalitesi üzerindeki etkisinin belirgin olmadığını göstermektedir. Tablo 5’te regresyon analizi sonucu elde edilen model özeti gösterilmiştir. Belirlilik katsayıları (R2) incelendiğinde, yüzey pürüzlülüğü modelinin deneysel sonuçlar ile %99,34 oranında uyumlu olduğu görülmektedir. Regresyon analizi sonucu elde edilen ampirik matematiksel modeli Eşitlik 1’de gösterilmiştir. Kontrol faktörleri değişiminin yüzey pürüzlülüğü üzerindeki yarattığı davranışın belirgin olarak görülebilmesi için etki grafiği çizilmiştir (Şekil 5). Grafikte, tüm faktör ve seviyeler için yüzey pürüzlülük değerleri ve standart hata barları verilmiştir. Etki grafiği incelendiğinde, ilerleme hızı ve bombardıman süresi artışı ile yüzey pürüzlülük değerlerinin arttığı, bekleme süresi artışı ile yüzey pürüzlülük değerlerinin azaldığı görülmektedir. Bu durum, deneysel bulgular ile uyum göstermektedir. Grafikte, standart hata değerleri incelendiğinde ise ilerleme hızı ve bombardıman süresi faktörlerinin bütün seviyeleri için bağımsız etki alanları elde edilmiştir. Bekleme süresinin tüm seviyelerinde hata değerlerinde çakışma mevcuttur. Bu durum deneysel bulgularda da görüldü gibi, bekleme süresi Kontrol faktörleri değişiminin yüzey pürüzlülüğü üzerindeki yarattığı davranışın belirgin olarak görülebilmesi için etki grafiği çizilmiştir (Şekil 5). Grafikte, tüm faktör ve seviyeler için yüzey pürüzlülük değerleri ve standart hata barları verilmiştir. Etki grafiği incelendiğinde, ilerleme hızı ve bombardıman süresi artışı ile yüzey pürüzlülük değerlerinin arttığı, bekleme süresi artışı ile yüzey pürüzlülük değerlerinin azaldığı görülmektedir. Bu durum, deneysel bulgular ile uyum göstermektedir. Şekil 5. Yüzey pürüzlülüğü açısından etki grafiği Şekil 5. Yüzey pürüzlülüğü açısından etki grafiği 581 Tablo 5. Araştırmacıların Katkı Oranı Beyan Özeti Yazarlar makaleye eşit oranda katkı sağlamış olduklarını beyan ederler. 582 Kaynakça Aydın K., Uğur L., Güvercin S., Gül F. Investigation of the machining performance of ferritic ductile cast Iron in WEDM using response surface methodology. Sigma Journal of Engineering and Natural Sciences 2022; 40(1): 95-107. Azam M., Jahanzaib M., Abbasi JA., Wasim A. Modeling of cutting speed (CS) for HSLA steel in wire electrical discharge machining (WEDM) using moly wire. Journal of the Chinese Institute of Engineers 2016; 39(7): 802-808. Babu RR., Subhair S., Siva S. Optimization of process parameter in WEDM for Stainless Steel 316 by Using Taguchi Method. International Journal of Engineering Research & Technology 2018; 7(9): 188-192. Bolar G., Das A., Joshi SN. Measurement and analysis of cutting force and product surface quality during end-milling of thin-wall components. Measurement 2018; 121: 190-204. Cebeci İ., Özlü B., Demir H. AISI 310 kalite östenitik paslanmaz sac malzemenin lazerle kesilmesinde kesme parametrelerinin kesim kalitesine etkisinin incelenmesi. Journal of the Institute of Science and Technology 2020; 10(4): 2791-2799. Chiang KT., Chang FP., Tsai DC. Modeling and analysis of the rapidly resolidified layer of SG cast iron in the EDM process through the response surface methodology. Journal of Materials Processing Technology 2007; 182(1-3): 525-533. Eksilmez N., Özlü B., Demir H. Hardox 500 Çeliğinin lazerle işlenmesinde kesme geometrisine ve işleme parametrelerine bağlı kesim kalitesinin değerlendirilmesi. Osmaniye Korkut Ata Üniversitesi Fen Bilimleri Enstitüsü Dergisi 2022; 5(1): 301-314. Esme U., Kulekci MK., Akkurt A., Seker U., Ozkul I. Mathematical modeling of diameter and circularity deviation in wire electrical discharge machining of a hot work tool steel. Materials Testing 2013; 55(6): 472-477. Işık R., Özlü B., Demir H. St-37 Malzemesinin lazer ile kesme işleminde seçilen parametrelerin etkisinin deneysel ve istatiksel olarak incelenmesi. Fırat University Journal of Engineering 2021; 33(1): 161-171. Koklu U. Optimization of kerf and surface roughness of AL 7 475-T 7 351 alloy machined with WEDM process using the grey-based Taguchi method. Metalurgija/Metallurgy 2012; 51(1): 47- 50. Li L., Guo Y., Wei X., Li W. Surface integrity characteristics in wire-EDM of Inconel 718 at different discharge energy. Procedia CirP 2013; 6: 220-225. Machno M., Franczyk E., Bogucki R., Matras A., Zebala W. A comparative study on the structure and quality of SLM and Cast AISI 316L samples subjected to WEDM processing. Materials 2022; 15(3): 701. Marelli D., Singh S., Nagari S., Subbiah R. Optimisation of machining parameters of wire-cut EDM on super alloy materials–A review. Materials Today: Proceedings 2020; 26: 1021-1027. 583 Özlü B. Kaynakça Experimental and statistical investigation of the effects of cutting parameters on kerf quality and surface roughness in laser cutting of Al 5083 alloy. Surface Review and Letters 2021; 28(10): 2150093. Prathipati R., Dora SP., Chanamala R. Wear behavior of wire electric discharge machined surface of 316L stainless steel. SN Applied Sciences 2020; 2(3): 1-8. Ramachandra R. Optimization of MRR and SR for AISI 316 Stainless steel material by using RSM Technique in EDM machining. International Journal of Engineering, Science and Mathematics 2017; 6(6): 314-321. Sharma N., Khanna R., Gupta R., Multi quality characteristics of WEDM process parameters with RSM. Procedia Engineering 2013; 64: 710-719. Singh G., Bhui AS., Sidhu SS. Influence of input parameters on MRR of AISI-316L using tungsten electrode machined by EDM. Редакционная коллегия 2017: 15. Snoeys R., Staelens F., Dekeyser W. Current trends in non-conventional material removal processes. CIRP Annals-Manufacturing Technology 1986; 35(2): 467-480. Soota T., Rajput S. Optimization and measurement of kerf width and surface roughness of AISI 316L. Forces in Mechanics 2022; 6: 100071. Tilekar S., Das SS., Patowari P. Process parameter optimization of wire EDM on Aluminum and mild steel by using Taguchi method. Procedia Materials Science 2014; 5: 2577-2584. Tosun N., Cogun C., Inan A. The effect of cutting parameters on workpiece surface roughness in wire EDM. Machining Science and Technology 2003; 7(2): 209-219. 584
https://openalex.org/W2111362364	https://yedion.yvc.ac.il/info/Course/Teacher_000000491_Z61545792809536185.pdf	English	null	Role of Mental Retardation-Associated Dystrophin-Gene Product Dp71 in Excitatory Synapse Organization, Synaptic Plasticity and Behavioral Functions	PloS one	2,009	cc-by	14,653	Abstract Background: Duchenne muscular dystrophy (DMD) is caused by deficient expression of the cytoskeletal protein, dystrophin. One third of DMD patients also have mental retardation (MR), likely due to mutations preventing expression of dystrophin and other brain products of the DMD gene expressed from distinct internal promoters. Loss of Dp71, the major DMD-gene product in brain, is thought to contribute to the severity of MR; however, the specific function of Dp71 is poorly understood. Methodology/Principal Findings: Complementary approaches were used to explore the role of Dp71 in neuronal function and identify mechanisms by which Dp71 loss may impair neuronal and cognitive functions. Besides the normal expression of Dp71 in a subpopulation of astrocytes, we found that a pool of Dp71 colocalizes with synaptic proteins in cultured neurons and is expressed in synaptic subcellular fractions in adult brains. We report that Dp71-associated protein complexes interact with specialized modular scaffolds of proteins that cluster glutamate receptors and organize signaling in postsynaptic densities. We then undertook the first functional examination of the brain and cognitive alterations in the Dp71-null mice. We found that these mice display abnormal synapse organization and maturation in vitro, altered synapse density in the adult brain, enhanced glutamatergic transmission and reduced synaptic plasticity in CA1 hippocampus. Dp71- null mice show selective behavioral disturbances characterized by reduced exploratory and novelty-seeking behavior, mild retention deficits in inhibitory avoidance, and impairments in spatial learning and memory. Conclusions/Significance: Results suggest that Dp71 expression in neurons play a regulatory role in glutamatergic synapse organization and function, which provides a new mechanism by which inactivation of Dp71 in association with that of other DMD-gene products may lead to increased severity of MR. Citation: Daoud F, Candelario-Martı´nez A, Billard J-M, Avital A, Khelfaoui M, et al. (2009) Role of Mental Retardation-Associated Dystrophin-Gene Product Dp71 in Excitatory Synapse Organization, Synaptic Plasticity and Behavioral Functions. PLoS ONE 4(8): e6574. doi:10.1371/journal.pone.0006574 Editor: Antoni L. Andreu, Hospital Vall d’Hebron, Spain Editor: Antoni L. Andreu, Hospital Vall d’Hebron, Spain Received April 14, 2009; Accepted June 25, 2009; Published August 10, 2008 Received April 14, 2009; Accepted June 25, 2009; Published August 10, 2008 Copyright: 2009 Daoud et al. This is an open-access article distributed under the terms of the Creative Commons Attributi unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Funding: This work was supported by grants from AFM (Association Francaise contre les Myopathies, France) to C.V., to D.Y and U.N., and to J.C., from FRM (Fondation pour la Recherche Medicale, France), ANR (Agence Nationale de Recherche, ANR 05-Neuro-040-01, France) and FJL to J.C., from MDA (Muscular Dystrophy Association), ISF (Israel Science Foundation) and the Nella and Leon Benoziyo Center for Neurosciences to U.N. and D.Y., and by CNRS (Centre National de la Recherche Scientifique, France) and INSERM (Institut National de la Sante et de la Recherche Medicale, France). F.D. had a fellowship from Institut Francais de Cooperation (Tunisie). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: cyrille.vaillend@u-psud.fr Role of Mental Retardation-Associated Dystrophin-Gene Product Dp71 in Excitatory Synapse Organization, Synaptic Plasticity and Behavioral Functions Fatma Daoud1,2, Aurora Candelario-Martı´nez3, Jean-Marie Billard4, Avi Avital5,6, Malik Khelfaoui1,2, Yael Rozenvald7, Maryvonne Guegan8,9, Dominique Mornet10, Danielle Jaillard11, Uri Nudel7, Jamel Chelly1,2, Dalila Martı´nez-Rojas3, Serge Laroche8,9, David Yaffe7, Cyrille Vaillend8,9* 1 Institut Cochin, Universite´ Paris-Descartes, CNRS, UMR 8104, Paris, France, 2 INSERM U 567, Paris, France, 3 CINVESTAV, Physiology, Biophysics and Neurosciences, Mexico City, Mexico, 4 Centre de Psychiatrie et Neurosciences, INSERM, UMR 894, Universite´ Paris Descartes, Faculte´ de Me´decine Rene´ Descartes, Paris, France, 5 Department of Psychology and The Center for Psychobiological Research, Yezreel Valley College, Emek Yezreel, Israel, 6 Neurobiology, Weizmann Institute of Science, Rehovot, Israel, 7 Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel, 8 CNRS, Neurobiologie de l’Apprentissage, de la Me´moire et de la Communication, UMR 8620, Orsay, France, 9 Univ Paris-Sud, Orsay, France, 10 INSERM, Equipe ESPRI 25 Muscle et Pathologies, Universite´ de Montpellier I, UFR de Me´decine, EA 4202, Montpellier, France, 11 Univ Paris-Sud, CNRS, UMR 8080, Centre Commun de Microscopie Electronique (CCME), Orsay, France Citation: Daoud F, Candelario-Martı´nez A, Billard J-M, Avital A, Khelfaoui M, et al. (2009) Role of Mental Retardation-Associated Dystrophin-Gene Product Dp71 in Excitatory Synapse Organization, Synaptic Plasticity and Behavioral Functions. PLoS ONE 4(8): e6574. doi:10.1371/journal.pone.0006574 Competing Interests: The authors have declared that no competing interests exist. * E-mail: cyrille.vaillend@u-psud.fr Introduction its spatio-temporal expression in muscles, brain structures and cell types. Its loss in all DMD patients leads to muscle degeneration. At least two shorter non-muscle products, Dp140 and Dp71, that in common with dystrophin harbor the cystein-rich and carboxy- terminus domains, are also expressed in brain [1] and might be involved in MR [3]. As Dp71 is the most abundant dystrophin- gene product in adult brain [4,5] and as DMD patients with mutations located in Dp71 genomic region display severe MR, Dp71 loss-of-function, mostly due to translation-terminating mutations, has emerged as a major contributing factor [2,6]. Mental retardation (MR) is a clinical feature in a third of patients suffering from Duchenne muscular dystrophy (DMD), an X-linked genetic disease caused by mutations in the dystrophin gene [1]. The etiology of MR in DMD is complex and likely relies on cumulative effects of inactivation of dystrophin and other DMD-gene products expressed from distinct internal promoters (Dp260, Dp140, Dp116, Dp71) [2]. Full-length dystrophin (Dp427) is derived from three independent promoters that regulate PLoS ONE \| www.plosone.org August 2009 \| Volume 4 \| Issue 8 \| e6574 1 Dp71 and Brain Function Dp71 is expressed around brain blood vessels, most likely in perivascular astrocyte endfeet [7], and is detected in cultured astrocytes [8] and glial Mu¨ller cells in retina [9], suggesting a role for Dp71 in blood-brain barrier function. However, Dp71 mRNA has also been detected in olfactory bulb and hippocampal neurons [10], and the protein shows expression in cultured neurons [8] and in postsynaptic densities in vivo [11]. Yet, studies of mouse lines lacking Dp427 or all dystrophins only showed mild cognitive impairments and failed to unveil the specific contribution of Dp71 to neuronal function and to the genesis of MR [12–14]. normally expressed in hippocampus. No expression was observed in hippocampus from KO mice, indicating that both Dp71d and Dp71f are lacking in this Dp71-null model. We also explored expression of Dp71 using immunofluores- cence on sections from mouse and rat hippocampus (Figure S2). A strong immunoreactive signal was present along the walls of blood vessels and in some astrocyte processes (Figure S2, A), which likely reflects the major expression of Dp71 in perivascular astrocyte end-feet [7]. Introduction However, both H4 and DYS2 antibodies confirmed Dp71 neuronal expression in both CA1-3 and dentate gyrus in WT and dystrophin-deficient mdx mice, but not in Dp71-null mice (Figure S2, B and C1-3), which is consistent with the distribution of Dp71 mRNA [10] and the X-gal staining described above. Punctate immunoreactivity corresponding to synaptic expression of Dp71 could not be clearly demonstrated on brain sections. Many X-linked developmental disorders associated with MR are related to proteins involved in signaling pathways that regulate cytoskeleton organization at excitatory synapses [15]. Here, to explore the specific function of Dp71, we examined its expression in neurons in vivo and in vitro, and characterized its localization in excitatory synaptic compartments. Because Dp71, as Dp427, is a key component of dystrophin-associated protein complexes (DAPC) formed by transmembrane (dystroglycans) and cytoplas- mic (syntrophins, dystrobrevins) proteins that link the extracellular matrix to the actin cytoskeleton, and because DAPC may regulate signaling pathways involved in structural organization of special- ized membrane-contact zones, in particular clustering of mem- brane receptors or ion channels [16], we characterized the Dp71- DAPC in brain and its interactions with postsynaptic proteins. Dp71-null mice [17] enabled us to examine for the first time the effects of selective Dp71-DAPC disruption on synapses organiza- tion in cultured neurons and on neurotransmission, synaptic plasticity, synapse distribution and ultrastructure in CA1 hippo- campal area in the adult brain. Finally, we performed the first behavioral characterization of Dp71-null mice. Western blotting of subcellular fractions of adult mouse cortex and hippocampus, validated by anti-synaptophysin and anti-PSD- 95 antibodies as markers of the pre- and postsynaptic structures, was used to further detail Dp71 expression in synaptic compart- ments. The 71 kDa product (Dp71) was detected in total homogenate (H) and in most of the collected fractions (Figure S3), and it was clearly expressed in postsynaptic density fractions (PSD1, PSD2) (Figure 1D). The PSD2 fraction was selectively associated with expression of the postsynaptic marker, PSD-95, but not synaptophysin, confirming the presence of Dp71 in the postsynaptic densities in the adult brain. Dp71 is Expressed in Excitatory Synapses of Cultured Neurons Dp71 is Expressed in Excitatory Synapses of Cultured Neurons To probe Dp71 expression in synapses of pyramidal cells and examine its putative role in synapse organization and maturation, we used immunofluorescence in primary cultured cells from mouse cortex and hippocampus. After two days in vitro, both H4 and 5F3 antibodies revealed immunoreactivity in the soma and along neurites in neurons from WT mice (Figure S4 A–B). H4 staining, characterized by a fine continuous, tiny and diffuse labeling (Figure 2 A1–A3), was still present in neurons from KO mice (Figure S4 C–D) and therefore did not reflect the sole expression of Dp71. In contrast, the 5F3 antibody detected specifically Dp71, as a discontinuous staining in neuritic processes of cultured pyramidal neurons from WT (Figure S4, A vs C), but not from KO mice. This discontinuous but strong staining revealed by 5F3 at varicosities and growth cones (Figure 2 A2, A3) was confirmed after 21 days of differentiation, when cultured cells show extensive sprouting of neuritic processes and acquisition of neuronal and synaptic phenotypes. At this stage, Dp71 had a synaptic pattern of expression, with immunoreactivity showing discontinuous granu- lar or clustered structures along dendrites (Figure 2 B1–B3). Neuronal subpopulation expressing Dp71 was characterized by double-staining using presynaptic markers of mature excitatory (Type-1 vesicular glutamate transporter, VGLUT1) or inhibitory synapses (GAD-65/67). Neurons intensely labeled with GAD-65/ 67, likely corresponding to inhibitory neurons, did not express 5F3 immunoreactivity (Figure 2 C1–C3), in contrast to VGLUT1- positive excitatory neurons that clearly showed staining for Dp71f (Figure 2 C4–C6). As 2464% of VGLUT1 clusters co-localized with Dp71 clusters, whereas only 763% of GAD-65/67 clusters were Dp71-positive, Dp71f isoforms thus appear comparatively more expressed in excitatory synapses. Regional, Cellular and Subcellular Expression of Dp71 Expression in Brain First, replacement of the Dp71 first and unique exon by the b- gal reporter gene in Dp71-null mice (KO) was used to estimate Dp71-promoter activity by b-gal-enzyme histochemistry (X-gal staining) on brain sections. We found that the Dp71 promoter drives high and widespread expression of the transgenic b-gal gene in both cortical and subcortical brain structures, including hippocampal subfields (Figure 1A). This confirms previous studies of Dp71 mRNA distribution in hippocampal formation [10,18], and extends the putative expression of Dp71 to pyramidal neurons of CA1. We then analyzed the expression of the Dp71 protein, first by immunoblotting protein extracts from adult rat and mouse brains. Using polyclonal H4 and monoclonal DYS2 antibodies, both directed against the carboxy-terminus domain common to all dystrophin-gene products, we detected the major short dystrophin- gene product of 71 kDa, Dp71, in the two main hippocampal sub- regions, the CA1 area and dentate gyrus (DG) (Fig. 1B) Transcripts encoding Dp71 are known to be alternatively spliced for exon 71–74 and/or 78 to generate multiple protein products in various tissues, which bear (Dp71d) or lack (Dp71f) specific protein-binding regions [19]. To determine whether these splice variants are expressed in hippocampus, we examined Dp71d expression with H4 and DYS2 that only react with Dp71 isoforms bearing exon 78, and Dp71f expression with the 5F3 monoclonal antibody [20,21] that specifically recognizes variants lacking exon 78 (Figure S1, for Dp71 isoforms and antibody epitopes). As expected, all antibodies detected the 71 kDa product in WT mice (Figure 1C), indicating that Dp71d and Dp71f, the two major forms of Dp71 products resulting from alternative splice events are In control neurons co-stained for Dp71 and specific synaptic markers, we found clusters of Dp71 closely in apposition to, and sometimes co-localized with both VGLUT1 (Figure 2C) and Shank (Figure 2D), another major post-synaptic density scaffolding protein interacting with glutamate receptors. Co-localization was PLoS ONE \| www.plosone.org August 2009 \| Volume 4 \| Issue 8 \| e6574 August 2009 \| Volume 4 \| Issue 8 \| e6574 2 Dp71 and Brain Function Dp71 and Brain Function Figure 1. Regional and subcellular expression of Dp71 in brain. A. Hippocampal Dp71 promoter activity in CA1-3 and dentate gyrus (DG) revealed by X-gal staining in brain section from a Dp71-null mouse. B. Immunoblots showing dystrophin-gene products detected by polyclonal (H4) and monoclonal (DYS2) antibodies in dissected CA (1) and DG (2) regions of the rat hippocampal formation. Regional, Cellular and Subcellular Expression of Dp71 Expression in Brain Note that in addition to Dp71, the H4 antibody also revealed Dp427, Dp140 and Dp116. Molecular weight markers are indicated on the left. C. Immunoblots of hippocampal extracts from WT and Dp71-null (KO) mice. Dp71 isoforms bearing exon 78 were detected by H4 and DYS2 antibodies, and those lacking exon 78 by 5F3. The doublet of bands detected by 5F3 likely reflects presence of glycosylated and non-glycosylated forms of Dp71f [5,21]. D. Detection of Dp71 in the postsynaptic densities. Protein extracts from subcellular fractions obtained from control mouse brains were probed with the anti-Dp71 (H4) antibody (top panel) and with the presynaptic and postsynaptic markers synaptophysin (central panel) and PSD-95 (bottom panel), respectively. H: total homogenate; PSD1/PSD2: isolated PSD fractions (Detailed distribution in subcellular fractions is shown in Fig. S3). doi:10.1371/journal.pone.0006574.g001 Figure 1. Regional and subcellular expression of Dp71 in brain. A. Hippocampal Dp71 promoter activity in CA1-3 and dentate gyrus (DG) revealed by X-gal staining in brain section from a Dp71-null mouse. B. Immunoblots showing dystrophin-gene products detected by polyclonal (H4) and monoclonal (DYS2) antibodies in dissected CA (1) and DG (2) regions of the rat hippocampal formation. Note that in addition to Dp71, the H4 antibody also revealed Dp427, Dp140 and Dp116. Molecular weight markers are indicated on the left. C. Immunoblots of hippocampal extracts from WT and Dp71-null (KO) mice. Dp71 isoforms bearing exon 78 were detected by H4 and DYS2 antibodies, and those lacking exon 78 by 5F3. The doublet of bands detected by 5F3 likely reflects presence of glycosylated and non-glycosylated forms of Dp71f [5,21]. D. Detection of Dp71 in the postsynaptic densities. Protein extracts from subcellular fractions obtained from control mouse brains were probed with the anti-Dp71 (H4) antibody (top panel) and with the presynaptic and postsynaptic markers synaptophysin (central panel) and PSD-95 (bottom panel), respectively. H: total homogenate; PSD1/PSD2: isolated PSD fractions (Detailed distribution in subcellular fractions is shown in Fig. S3). doi:10.1371/journal.pone.0006574.g001 found in 4764% of Shank-positive clusters and in 24% of VGLUT1-positive clusters, suggesting a main localization of Dp71 in postsynaptic compartments. The percent of synapses (defined by co-localized VGLUT1 and PSD-95 clusters) positive for Dp71 was 4864.25% and 5764.6% of Dp71 clusters were located at synapses (n = 9 control neurons), suggesting that a significant pool of Dp71 clusters is located at mature excitatory synapses in neurons of control mice. Regional, Cellular and Subcellular Expression of Dp71 Expression in Brain NMDAr-associated adapter protein, PSD-95, and the G-protein- activated inwardly rectifying potassium channel 1 (GIRK1) (Figure 3C). Two non-receptor tyrosine kinases involved in neurite outgrowth, dendritic spine morphogenesis and synaptic plasticity, the focal adhesion kinase (FAK) and C-Src, also co-immunopre- cipitated with Dp71. In contrast, phosphatidylinositol-3 kinase (PI3K) was not detected in the bound fraction, suggesting absence of nonspecific binding. Other control experiments (not shown), such as IP with unspecific whole-molecule IgG followed by detection with the specific primary and secondary antibodies used in this study, and IP of protein extracts from KO mice using DYS2, did not reveal co-IP of the synaptic proteins mentioned above, which strengthens the conclusion of a specific association of Dp71, but not other dystrophin-gene products, with signaling proteins in excitatory synapses. PLoS ONE \| www.plosone.org The Dp71-DAPC Interacts with Synaptic Proteins To identify Dp71-associated proteins (DAPs) [11], we carried out immunoprecipitation (IP) experiments from rat hippocampal extracts using DYS2 antibody. We found that a1 and a2 dystrobrevins, a and c1-syntrophins, b-dystroglycan and actin co-immunoprecipitate with Dp71 (Figure 3A). These results were confirmed by reciprocal IP using a monoclonal antibody directed against a-dystrobrevins. Here, Dp71 was the only dystrophin-gene product detected in the bound fraction probed with DYS2, suggesting that the other dystrophins did not take part in this specific complex. Reciprocal IP using anti-NR2A-B antibody allowed us to detect Dp71d and Dp71f, thus showing that both isoforms interact with the scaffold proteins associated with postsynaptic glutamate receptors in PSDs (Figure 3D). Another IP using anti-PSD-95 antibody revealed that binding of NMDAr, PSD-95, nNOS and FAK was not affected in brains from KO mice (Figure 3E), suggesting that Dp71 may be a partner, or modulator, of the core scaffold of proteins involved in the organization of glutamatergic synapses, although its congenital loss does not seem to prevent expression of these protein complexes. Components of the DAPC were still expressed in brains of Dp71-null mice (Figure 3B). However, b-dystroglycan expression was drastically reduced in KO mice (,0.5% of WT). Expression of a-syntrophins was also reduced (,58% of WT), while c1- syntrophin was overexpressed by more than 200%. In contrast, no difference was observed in the expression of a-dystrobrevins, suggesting that Dp71 is not required for membrane targeting of these DAPs. Dp71 Loss Alters Glutamatergic Synapse Maturation In cultured neurons from Dp71-null mice, changes in the number of VGLUT1 clusters and large PSD-95 clusters were observed, suggesting that Dp71 loss may affect the clustering and distribution of these synaptic proteins. To quantify this, we used neuronal cell cultures from WT and KO mice triple immuno- Next we found that key neuronal and synaptic proteins can be co-immunoprecipitated with Dp71. These include AMPA gluta- mate receptor (AMPAr) subunits GluR1–3, the NR2A-B subunits of the NMDA subtype of glutamate receptors (NMDAr), the August 2009 \| Volume 4 \| Issue 8 \| e6574 PLoS ONE \| www.plosone.org 3 Dp71 and Brain Function Figure 2. Expression of Dp71 in mouse excitatory neurons in vitro. A and B. Primar Figure 2. Expression of Dp71 in mouse excitatory neurons in vitro. A and B. Primary cultured neurons labeled with H4 (red) and 5F3 (green) antibodies after two (A) and 21 days (B) of differentiation. The Dp71-DAPC Interacts with Synaptic Proteins White box indicates selected neuritic segments shown below at higher magnification. Note continuous staining with H4 contrasting with discontinuous staining of 5F3 in neurites. C. Dp71 product detected with 5F3 (green, C1, C4) in subpopulations of inhibitory (C1-3) and excitatory (C4-6) neurons characterized by GAD-67 and VGLUT1 immunostaining. Overlays show co- expression of Dp71 and VGLUT1 (C6), with juxtaposition of the markers and presence of VGLUT1-positive Dp71 clusters (arrowheads), but very few co-expression with GAD-67 (C3). D. Representative images of control neurons double-labeled with 5F3 (green, D1) and the postsynaptic marker Shank (red, D2). Overlay (D3) and higher magnification of a selected dendritic segments (D3, bottom) shows juxtaposition of the two markers and the presence of Shank-positive Dp71 clusters (arrowhead). Scale bars, 10 mm. doi:10.1371/journal.pone.0006574.g002 Figure 2. Expression of Dp71 in mouse excitatory neurons in vitro. A and B. Primary cultured neurons labeled with H4 (red) and 5F3 (green) antibodies after two (A) and 21 days (B) of differentiation. White box indicates selected neuritic segments shown below at higher magnification. Note continuous staining with H4 contrasting with discontinuous staining of 5F3 in neurites. C. Dp71 product detected with 5F3 (green, C1, C4) in subpopulations of inhibitory (C1-3) and excitatory (C4-6) neurons characterized by GAD-67 and VGLUT1 immunostaining. Overlays show co- expression of Dp71 and VGLUT1 (C6), with juxtaposition of the markers and presence of VGLUT1-positive Dp71 clusters (arrowheads), but very few co-expression with GAD-67 (C3). D. Representative images of control neurons double-labeled with 5F3 (green, D1) and the postsynaptic marker Shank (red, D2). Overlay (D3) and higher magnification of a selected dendritic segments (D3, bottom) shows juxtaposition of the two markers and the presence of Shank-positive Dp71 clusters (arrowhead). Scale bars, 10 mm. doi:10.1371/journal.pone.0006574.g002 August 2009 \| Volume 4 \| Issue 8 \| e6574 PLoS ONE \| www.plosone.org 4 Dp71 and Brain Function Figure 3. Characterization of the brain Dp71-DAPC. A. Immunoprecipitation of Dp71 with DYS2 or of aDB with aDB antibodies in rat whole- hippocampus extracts. Co-immunoprecipitated proteins were analyzed on western blots. B. Immunoblots of hippocampal extracts from WT and KO mice using antibodies directed against a-DB, a-syn, c1-syn, b-DG and actin. C. Immunoprecipitation of Dp71 with DYS2 and westernblot analysis of interacting proteins in rat hippocampus. Co-immunoprecipitated proteins were AMPAr subunits Glu-R1-3, NMDAr subunits NR2A-B, PSD-95, FAK, C- Src and GIRK1, not PI3K. D. Immunoprecipitation using the NMDAR2A&B antibody in rat hippocampal extracts. The Dp71-DAPC Interacts with Synaptic Proteins The proteins interacting with the NMDAr complex were PSD-95, GRB2, Dp71f and Dp71d, a-DB and a-syn. E. Immunoprecipitation of the PSD-95 protein from hippocampal extracts of KO mice; NMDAr, FAK, a-DB, nNOS and c1-syn were pulled down in the bound fraction. a-DB: a-dystrobrevins, a-syn: a-syntrophin, c1-syn: c1- syntrophin, b-DG: b-dystroglycan. T: Hippocampus total homogenate; B: Bound fraction; U: Unbound fraction. Proteins molecular mass (left) are indicated on the right. doi:10.1371/journal.pone.0006574.g003 Figure 3. Characterization of the brain Dp71-DAPC. A. Immunoprecipitation of Dp71 with DYS2 or of aDB with aDB antibodies in rat whole- hippocampus extracts. Co-immunoprecipitated proteins were analyzed on western blots. B. Immunoblots of hippocampal extracts from WT and KO mice using antibodies directed against a-DB, a-syn, c1-syn, b-DG and actin. C. Immunoprecipitation of Dp71 with DYS2 and westernblot analysis of interacting proteins in rat hippocampus. Co-immunoprecipitated proteins were AMPAr subunits Glu-R1-3, NMDAr subunits NR2A-B, PSD-95, FAK, C- Src and GIRK1, not PI3K. D. Immunoprecipitation using the NMDAR2A&B antibody in rat hippocampal extracts. The proteins interacting with the NMDAr complex were PSD-95, GRB2, Dp71f and Dp71d, a-DB and a-syn. E. Immunoprecipitation of the PSD-95 protein from hippocampal extracts of KO mice; NMDAr, FAK, a-DB, nNOS and c1-syn were pulled down in the bound fraction. a-DB: a-dystrobrevins, a-syn: a-syntrophin, c1-syn: c1- syntrophin, b-DG: b-dystroglycan. T: Hippocampus total homogenate; B: Bound fraction; U: Unbound fraction. Proteins molecular mass (left) are indicated on the right. doi:10 1371/journal pone 0006574 g003 doi:10.1371/journal.pone.0006574.g003 stained with 5F3, VGLUT1 and PSD-95 (Figure 4A). In neurons lacking Dp71, the mean area of PSD-95 clusters was larger than in controls (p,0.001; Figure 4B, arrows in A5), whereas there was only a slight, non-significant reduction in the number of PSD-95 puncta (p = 0.09, NS; Figure 4C). This suggests that each cluster in Dp71-deficient neurons contains more PSD-95 protein. Using the same biochemical fractions as in Figure 1D, we compared PSD-95 expression between genotypes by autoradiography (Figure 4D). A drastic overexpression of PSD-95 was found in KO mice in the PSD1 (WT: 32326652, KO: 1010261021; p,0.03) and PSD2 fractions (WT: 18176109, KO: 11025.256802.75; p,0.01). The ratio of PSD-95 mRNA expression in KO vs WT mice was close to 1, as assessed by quantitative RT-PCR on whole-brain extracts (normalized to that of the housekeeping-gene, cyclophylin), indicating no quantitative variation of PSD-95 message between genotypes (not shown). This suggests that Dp71 loss selectively increases stabilization or local translation of PSD-95 [22]. The Dp71-DAPC Interacts with Synaptic Proteins We also found overexpression of PSD-93 in the PSD2 fraction (WT: 63066537, KO: 1500362478, p = 0.07), whereas expression of another synaptic protein, synaptophysin, was not altered, suggesting that the increased amounts of PSD-95/93 were not due to a general increase in protein expression. In all, these results suggest major alterations in the expression, organization and/or localization of PSD proteins in neurons of Dp71-null mice. stained with 5F3, VGLUT1 and PSD-95 (Figure 4A). In neurons lacking Dp71, the mean area of PSD-95 clusters was larger than in controls (p,0.001; Figure 4B, arrows in A5), whereas there was only a slight, non-significant reduction in the number of PSD-95 puncta (p = 0.09, NS; Figure 4C). This suggests that each cluster in Dp71-deficient neurons contains more PSD-95 protein. Using the same biochemical fractions as in Figure 1D, we compared PSD-95 expression between genotypes by autoradiography (Figure 4D). A drastic overexpression of PSD-95 was found in KO mice in the PSD1 (WT: 32326652, KO: 1010261021; p,0.03) and PSD2 fractions (WT: 18176109, KO: 11025.256802.75; p,0.01). The ratio of PSD-95 mRNA expression in KO vs WT mice was close to 1, as assessed by quantitative RT-PCR on whole-brain extracts (normalized to that of the housekeeping-gene, cyclophylin), indicating no quantitative variation of PSD-95 message between genotypes (not shown). This suggests that Dp71 loss selectively increases stabilization or local translation of PSD-95 [22]. We also found overexpression of PSD-93 in the PSD2 fraction (WT: 63066537, KO: 1500362478, p = 0.07), whereas expression of Quantification of VGLUT1 clusters revealed a different pattern of distribution (Figure 4, A2 vs A6), with an increased number of VGLUT1-positive puncta (p,0.001; Figure 4C) and a decrease in their size (p,0.001; Figure 4B). We then quantified the colocalization of PSD-95 clusters with VGLUT1 as an indicator of the proportion of functional synapses in cultured neurons (e.g., [23]), which was estimated 56.4466.8% in neurons from WT mice. In neurons from Dp71-null mice, however, colocalization only reached 27.964.48% and the number of clusters double- stained with VGLUT1 and PSD-95 (Figure 4, A3 and A7) was significantly reduced (11.7761.39 per 50 mm) compared to WT mice (32.6262.22) (p,0.0001), which likely reflected a reduced density of functional synaptic junctions. Although part of PSD-95 expression in Dp71-null mice may correspond to mislocated August 2009 \| Volume 4 \| Issue 8 \| e6574 5 PLoS ONE \| www.plosone.org Dp71 and Brain Function Figure 4. The Dp71-DAPC Interacts with Synaptic Proteins Dp71 loss alters the organization of glutamatergic synapses in vitro. A. Triple-immunofluorescence staining of dendrite segments from mature cultured neurons of WT (Top) and KO mice (Bottom), using antibodies directed against PSD-95 (green, A1, A5), VGLUT1 (red, A2, A6), and Dp71 (blue on overlay in A4). Dp71-deficient neurons show abnormally larger PSD-95 clusters on excitatory synapses (arrows in A5), a scarcity of synapses double-stained for PSD95 and VGLUT1 (A7) compared to controls (A3). Dp71 clusters positive for both PSD95 and VGLUT1 were present in WT neurons only (arrow in A4). Scale bars, 10 mm. B–C. Quantitative analyses of PSD-95 and VGLUT1 clusters. Histograms show the size of the clusters (B, puncta area) and the number of clusters per 50-mm dendritic length (C, puncta/50 mm), in WT (white) versus Dp71-null neurons (black). p,0.001. D. Immunoblot analysis of PSD95 expression in total homogenate (H) and postsynaptic density fractions (PSD1, PSD2) from WT and KO mouse brain extracts (n = 15 mice per genotype distributed in three independent immunoblots). doi:10.1371/journal.pone.0006574.g004 Figure 4. Dp71 loss alters the organization of glutamatergic synapses in vitro. A. Triple-immunofluorescence staining of dendrite segments from mature cultured neurons of WT (Top) and KO mice (Bottom), using antibodies directed against PSD-95 (green, A1, A5), VGLUT1 (red, A2, A6), and Dp71 (blue on overlay in A4). Dp71-deficient neurons show abnormally larger PSD-95 clusters on excitatory synapses (arrows in A5), a scarcity of synapses double-stained for PSD95 and VGLUT1 (A7) compared to controls (A3). Dp71 clusters positive for both PSD95 and VGLUT1 were present in WT neurons only (arrow in A4). Scale bars, 10 mm. B–C. Quantitative analyses of PSD-95 and VGLUT1 clusters. Histograms show the size of the clusters (B, puncta area) and the number of clusters per 50-mm dendritic length (C, puncta/50 mm), in WT (white) versus Dp71-null neurons (black). p,0.001. D. Immunoblot analysis of PSD95 expression in total homogenate (H) and postsynaptic density fractions (PSD1, PSD2) from WT and KO mouse brain extracts (n = 15 mice per genotype distributed in three independent immunoblots). doi:10.1371/journal.pone.0006574.g004 morphological alteration of the postsynaptic active zone in Dp71- null mice. proteins in non-synaptic zones, we however found that 62.0765.12% of the abnormally large PSD-95 clusters (.0.4 mm2) observed in KO mice were VGLUT1-positive, thus indicating that accumulation of PSD-95 also largely occurs in synaptic regions. The Dp71-DAPC Interacts with Synaptic Proteins Triple-labeling for Dp71, VGLUT1 and PSD-95 (Figure 4 A4) revealed that 4864.25% of excitatory synapses were Dp71-positive in controls, suggesting that at least half of these synapses would be affected by Dp71 loss. Hippocampal Neurotransmission and Synaptic Plasticity The basic neuronal architecture of the hippocampus appeared normal in KO mice (Figure S5). Presynaptic fiber volleys (PFVs) and AMPA/kainate receptor-mediated field excitatory postsynap- tic potentials (fEPSPs) were recorded in CA1 in hippocampal slices from WT (40 slices, 10 mice) and KO mice (37 slices, 9 mice). Whereas comparable PFVs were measured in KO and WT mice (p.0.5, NS; Figure S6 A), fEPSP slopes were significantly higher in KO mice (p,0.001; Figure S6 B), resulting in enhanced coupling between PFV and fEPSPs (Figure 6A), i.e., enhanced synaptic transmission. When the NMDAr component of glutama- tergic transmission was isolated (6 slices from 3 mice of each genotype), no significant group difference was found in PFVs’ slopes (p.0.7, Figure S6 C), but NMDAr-mediated fEPSPs (Figure S6 D) and PFV-fEPSP coupling (Figure 6B) were again significantly enhanced in mutant mice (p,0.05). Analyzing PFVs in the absence of EPSPs (with 10 mM bicuculline, 10 mM DNQX, and 30 mM APV in bathing medium) showed no difference between KO (n = 6) and WT mice (n = 5) (PFV slope: p.0.8; PFV amplitude: p.0.8, data not shown), which confirms that the increased neurotransmission in mutants was not due to changes in afferent fiber density or excitability. Behavioral Analysis of Dp71-Null Mice Motor Capacities. KO and WT mice showed comparable fall latencies in the inverted grid test (44.2564.53 s and 41.3864.82 s, respectively) and comparable latencies to grip the wire in the suspension test (2.5460.43 s and 2.3660.49 s) (p.0.6). Although KO mice had reduced locomotor activity during the first 10 min of open-field exploration (p,0.05), they displayed fast habituation and similar baseline locomotion compared to controls from approximately 15 min until the end of testing (Figure 7A). KO mice also showed normal avoidance of the center zone of the arena compared to controls (not shown). Both genotypes showed comparable locomotor activity in a cross-maze (number of arms visited; KO: 19.4461.64; WT: 22.0561.73; p.0.3) and similar rearing frequency in both experiments (not shown). This indicates that basal motor functions were not affected by the loss of Dp71, in agreement with the lack of overt muscle histopathology in this model (not shown) and the absence of Dp71 expression in differentiated skeletal muscles [17]. Figure 5. Dp71 loss affects density and morphology of axospinous asymmetric synapses in CA1 hippocampus in vivo. A. Electron micrograph showing the CA1 neuropil (stratum radiatum) with typical non-perforated (NP) and perforated (P) subtypes of excitatory synapses. Scale bar, 1 mm. B. Relative density of axospinous asymmetric synapses expressed as synapses per 100 mm2. p,0.05. C. Frequency histogram distributions of the PSD length of sampled CA1 spine synapses in 25 nm bins. Insert shows measures of PSD length in NP and P synapses, with the total length of a perforated PSD estimated as the sum of the individual electron-dense traces. Scale bar: 0.5 mm. doi:10.1371/journal.pone.0006574.g005 Exploration and Emotional Reactivity. The initial reduction of activity shown by KO mice during forced exploration of the open-field was confirmed when mice were repeatedly exposed to the open-field during habituation sessions preceding object recognition testing (number of squares crossed, p,0.002; crossings in central area, p,0.05; rearings, p,0.05). A clear reduction of object exploration was also detected during both acquisition and retention sessions of this task: Object investigations were comparable between genotypes on the first acquisition session, whereas they were significantly reduced in KO mice when exposed to new sets of objects on successive sessions (Figure 7B). A significant decrease in the number of contacts with objects (p,0.007) and in the duration of object exploration (p,0.03) was observed during both acquisition and retention. Behavioral Analysis of Dp71-Null Mice In contrast, no reduction of exploratory behavior was observed when KO mice were exposed to a novel arena that offered a retreat opportunity into a small familiar home-box (free-choice exploration, all parameters p.0.5, not shown). Finally, no difference was found between genotypes in the plus-maze anxiety test (% entries in open arms: WT: 16.6163.82; KO: 13.1464.94, p.0.2). Thus, KO mice exhibited reduced exploratory and novelty-seeking behaviors, which could not be directly attributed to enhanced emotional reactivity. magnitude of LTP expressed after 30 min was significantly lower in KO than in WT mice (last 15 min, p,0.04). When LTP was induced by a stronger HFS (26100 Hz; WT: 11 slices; 6 mice; KO: 11 slices, 5 mice, Figure 6D), the magnitude of LTP was significantly reduced in KO mice (128.4061% of baseline) compared to WT mice (160.1861.04%) during 1 h post-HFS (p,0.02). Immediately post-tetanus (30 s), fEPSP potentiation was already significantly smaller in KO (185.08615.88%) than in WT mice (289.26634.64%) (p,0.02), and remained significantly reduced for at least 30 min (p,0.01). During the last 10 min of recording, LTP levels were comparable in KO (125.1662.76%) and WT mice (143.6861.25%) (p.0.2), indicating that this stronger HFS protocol induced stable and comparable LTP maintenance in the two genotypes. magnitude of LTP expressed after 30 min was significantly lower in KO than in WT mice (last 15 min, p,0.04). When LTP was induced by a stronger HFS (26100 Hz; WT: 11 slices; 6 mice; KO: 11 slices, 5 mice, Figure 6D), the magnitude of LTP was significantly reduced in KO mice (128.4061% of baseline) compared to WT mice (160.1861.04%) during 1 h post-HFS (p,0.02). Immediately post-tetanus (30 s), fEPSP potentiation was already significantly smaller in KO (185.08615.88%) than in WT mice (289.26634.64%) (p,0.02), and remained significantly reduced for at least 30 min (p,0.01). During the last 10 min of recording, LTP levels were comparable in KO (125.1662.76%) and WT mice (143.6861.25%) (p.0.2), indicating that this stronger HFS protocol induced stable and comparable LTP maintenance in the two genotypes. Learning and Memory. In the object recognition task, the percent time spent exploring each of the two objects during acquisition was comparable in WT (49.3 and 50.6961.89%) and KO mice (50.69 and 49.3162.74%). Following a 10 min, 24 h or 48 h delay, one of the familiar objects was replaced by a novel object and the time spent exploring the novel object (recognition index) was measured. Dp71 Loss Alters Excitatory Synapse Density and Morphology in the Adult Brain To determine possible morphological alteration of excitatory synapses in Dp71-null mice in vivo, we compared axospinous asymetric synapse density and morphology in both genotypes using quantitative electron microscopy (EM). The volume of the anterodorsal hippocampus calculated from vibratome sections used for EM was comparable in WT (3.0160.04 mm3) and KO mice (2.9660.41 mm3) (p.0.91, not shown), suggesting no differential tissue shrinkage between genotypes. A total of 665 synapses in KO (n = 4) and 533 synapses in control mice (n = 3) were analyzed in the distal dendritic layer of CA1 anterodorsal hippocampus (Figure 5). The majority of axospinous asymetric synapses were non-perforated (92–94%), whereas perforated synapse subtype (5–7%; sample image in Fig. 5A) and multiple spine boutons (MSBs) (1–2%) were less represented. The number of axospinous excitatory synapses was reduced by ,11% in KO mice compared to controls (Figure 5B; p,0.029). The density of the non-perforated subtype was also reduced by ,13% (KO: 2.3261.05, WT: 26.7860.43 per 100 mm2; p,0.045), whereas the densities of perforated synapses and of MSBs were not affected (not shown). The distribution of PSD lengths in non-perforated synapses was slightly skewed toward the larger sizes in the KO mice (Figure 5C) (D = 0.087, p,0.024, KS test), which suggests We then examined long-term potentiation (LTP) using two protocols of high-frequency stimulation (HFS, 2650 Hz, 26100 Hz). Stimulus intensities were adjusted to obtain compa- rable fEPSP slopes in the two genotypes before the induction of LTP (WT: 0.10760.01 V.s21, KO: 0.11060.001 V.s21, p.0.5). When induced by two trains at 50 Hz (WT: 12 slices, 6 mice; KO: 8 slices; 5 mice, Figure 6C), the level of LTP was comparable between genotypes during the first 15 min after HFS, but the August 2009 \| Volume 4 \| Issue 8 \| e6574 PLoS ONE \| www.plosone.org 6 Dp71 and Brain Function Figure 5. Dp71 loss affects density and morphology of axospinous asymmetric synapses in CA1 hippocampus in vivo. A. Electron micrograph showing the CA1 neuropil (stratum radiatum) with typical non-perforated (NP) and perforated (P) subtypes of excitatory synapses. Scale bar, 1 mm. B. Relative density of axospinous asymmetric synapses expressed as synapses per 100 mm2. p,0.05. C. Frequency histogram distributions of the PSD length of sampled CA1 spine synapses in 25 nm bins. Dp71 Loss Alters Excitatory Synapse Density and Morphology in the Adult Brain Insert shows measures of PSD length in NP and P synapses, with the total length of a perforated PSD estimated as the sum of the individual electron-dense traces. Scale bar: 0.5 mm. doi:10.1371/journal.pone.0006574.g005 both KO (8 slices, 5 mice) and WT mice (7 slices, 5 mice), was statistically comparable between genotypes (Figure S6 E). This confirmed that LTP in both genotypes is NMDAr-dependent and suggests that LTP impairment in Dp71-null mice is not due to reduced presynaptic efficiency. Finally, we examined LTD induced by LFS (900 pulses at 1 Hz) in area CA1 in 9 slices from WT (n = 5) and 8 slices from KO mice (n = 4). The time course and magnitude of LTD were comparable between genotypes during 1 h post-LFS (p.0.8; Figure 6F), suggesting that reduced LTP in KO mice is not due to facilitation of LTD mechanisms. PLoS ONE \| www.plosone.org Behavioral Analysis of Dp71-Null Mice Time- course of LTP induced by HFS (time 0) at 2650 Hz (C) and 26100 Hz (D). Synaptic strength is expressed as percent change of fEPSP slopes from baseline taken as 100% (dotted line) and plotted against time. Sample traces show averaged fEPSPs recorded in response to constant test stimuli in stratum radiatum of CA1 area before (a) and 45–60 min after induction (b). E. PPF induced by stimulus pairs (50 ms inter-pulse interval) and expressed as the ratio of the second over the first fEPSP slopes. p,0.02.F. Time course of LTD of the fEPSP after low frequency stimulation (900 pulses at 1 Hz). Sample traces show averaged fEPSPs recorded before (a) and 60 min after induction (b) (scale bars: 5 ms, 0.5 mV). doi:10.1371/journal.pone.0006574.g006 Dp71 and Brain Function Figure 6. Dp71 loss alters synaptic transmission and plasticity in CA1 hippocampal area. KO: Fil glutamatergic transmission mediated via AMPAr. Sample traces (Top) show examples of AMPAr-mediat (stimulation intensity: 300 mA); neurotransmission (bottom) is expressed as AMPAr-mediated fEPSP slopes (PFV) slopes (PFV-fEPSP coupling). B. NMDAr component of glutamatergic transmission. Sample traces of N intensity: 500 mA) and NMDAr-mediated PFV-fEPSP coupling (bottom). The fEPSPs were isolated in the presen course of LTP induced by HFS (time 0) at 2650 Hz (C) and 26100 Hz (D). Synaptic strength is expressed baseline taken as 100% (dotted line) and plotted against time. Sample traces show averaged fEPSPs recorde d f b f d f d b d d b l Figure 6. Dp71 loss alters synaptic transmission and plasticity in CA1 hippocampal area. KO: Filled symbols; WT: Open symbols. A. Basal glutamatergic transmission mediated via AMPAr. Sample traces (Top) show examples of AMPAr-mediated fEPSPs in response to single pulses (stimulation intensity: 300 mA); neurotransmission (bottom) is expressed as AMPAr-mediated fEPSP slopes plotted against presynaptic fiber volley (PFV) slopes (PFV-fEPSP coupling). B. NMDAr component of glutamatergic transmission. Sample traces of NMDAr-mediated fEPSPs (Top, stimulation intensity: 500 mA) and NMDAr-mediated PFV-fEPSP coupling (bottom). The fEPSPs were isolated in the presence of bicuculline and DNQX. C–D. Time- course of LTP induced by HFS (time 0) at 2650 Hz (C) and 26100 Hz (D). Synaptic strength is expressed as percent change of fEPSP slopes from baseline taken as 100% (dotted line) and plotted against time. Behavioral Analysis of Dp71-Null Mice No statistical difference was detected between groups: The recognition index differed significantly from chance in both WT and KO mice at the 10 min and 24 h delays, but not at the 48 h delay (Figure 7C), both genotypes showing good and comparable short and long-term recognition memory, and a similar rate of forgetting. Learning and Memory. In the object recognition task, the percent time spent exploring each of the two objects during acquisition was comparable in WT (49.3 and 50.6961.89%) and KO mice (50.69 and 49.3162.74%). Following a 10 min, 24 h or 48 h delay, one of the familiar objects was replaced by a novel object and the time spent exploring the novel object (recognition index) was measured. No statistical difference was detected between groups: The recognition index differed significantly from chance in both WT and KO mice at the 10 min and 24 h delays, but not at the 48 h delay (Figure 7C), both genotypes showing good and comparable short and long-term recognition memory, and a similar rate of forgetting. We also analyzed forms of short-term plasticity, paired-pulse facilitation (PPF) and posttetanic potentiation (PTP). The slopes of the first fEPSP were adjusted to similar levels in KO (n = 5, 14 slices) and WT mice (n = 5, 12 slices) before induction of PPF (p.0.6). The PPF ratio was however significantly decreased in slices from KO compared to WT mice (p,0.02, Fig. 6E), suggesting alteration in presynaptic mechanisms of glutamate release. PTP was induced by delivering HFS (26100 Hz) in the presence of 80 mM APV. The transient PTP, which decayed to baseline within a few minutes in August 2009 \| Volume 4 \| Issue 8 \| e6574 PLoS ONE \| www.plosone.org 7 Dp71 and Brain Function Figure 6. Dp71 loss alters synaptic transmission and plasticity in CA1 hippocampal area. KO: Filled symbols; WT: Open symbols. A. Basal glutamatergic transmission mediated via AMPAr. Sample traces (Top) show examples of AMPAr-mediated fEPSPs in response to single pulses (stimulation intensity: 300 mA); neurotransmission (bottom) is expressed as AMPAr-mediated fEPSP slopes plotted against presynaptic fiber volley (PFV) slopes (PFV-fEPSP coupling). B. NMDAr component of glutamatergic transmission. Sample traces of NMDAr-mediated fEPSPs (Top, stimulation intensity: 500 mA) and NMDAr-mediated PFV-fEPSP coupling (bottom). The fEPSPs were isolated in the presence of bicuculline and DNQX. C–D. Behavioral Analysis of Dp71-Null Mice Sample traces show averaged fEPSPs recorded in response to constant test stimuli in stratum radiatum of CA1 area before (a) and 45–60 min after induction (b). E. PPF induced by stimulus pairs (50 ms inter-pulse interval) and expressed as the ratio of the second over the first fEPSP slopes. p,0.02.F. Time course of LTD of the fEPSP after low frequency stimulation (900 pulses at 1 Hz). Sample traces show averaged fEPSPs recorded before (a) and 60 min after induction (b) (scale bars: 5 ms, 0.5 mV). doi:10.1371/journal.pone.0006574.g006 In the cross-maze, mice of both genotypes showed a comparable number of different arm choices within every set of 4 arm entries (WT: 3.2860.04; KO: 3.1960.06; p.0.3) and their performance was not affected by stereotyped response patterns (% chaining: WT: 19.9963.65; KO: 24.3765.05, p.0.4), suggesting that spontaneous alternation is not impaired in KO mice. In contrast, KO mice showed impaired performance when spatial delayed alternation was assessed in a T-maze (Figure 7D). Only WT mice showed significant alternation rates at 6 h and 24 h, suggesting impaired spatial memory performance at long delays in KO mice. During spatial learning in the water-maze, acquisition of the navigation task was clearly impaired in Dp71-null mice, which, in contrast to WT mice, showed no significant improvement of performance over training (Figure 7E), as expressed by the latency (last 4 blocks of trials, genotype effect: p,0.01) and distance swum (p,0.01) to find the platform. While mice of both genotypes had PLoS ONE \| www.plosone.org August 2009 \| Volume 4 \| Issue 8 \| e6574 8 Dp71 and Brain Function Figure 7. Selective alterations of exploratory behavior and learning and memory performance in Dp71-null mice. KO: Filled symbols; WT: Open symbols. A. Spontaneous locomotor activity during exploration of an empty open-field (9 KO, 10 WT). The number of squares crossed along the walls for 50 min is shown by bins of 5 min recorded every 10 min. p,0.02. B. Object investigations expressed as the number of contacts made with three different sets of objects used during successive acquisition sessions in the object recognition task (13 KO, 16 WT). p,0.01. C. Object-recognition memory performance at retention delays of 10 min, 24 h and 48 h. significantly different from chance (50%), p,0.05. D. Spatial alternation in a T-maze. Data are % of mice alternating (14 KO, 24 WT) at retention delays of 1, 6 and 24 h. Behavioral Analysis of Dp71-Null Mice doi:10.1371/journal.pone.0006574.g007 August 2009 \| Volume 4 \| Issue 8 \| e6574 PLoS ONE \| www.plosone.org 9 Dp71 and Brain Function in perisynaptic regions. In brains of Dp71-null mice, loss of Dp71 resulted in reduced levels of b-dystroglycan and a-syntrophin. This, however, did not prevent the formation of NMDAr complexes, which also maintained stable associations with a-dystrobrevins and a-syntrophin, suggesting that distinct mechanisms are involved in postsynaptic membrane targeting of Dp71, DAPs and members of glutamate-receptor complexes. comparable swim speed and thigmotaxis (both parameters p.0.2), KO mice displayed higher accelerations during progression segments (p,0.05) and a high rate of 360u-rotation of the animal’s body (p,0.0001), suggesting darting or impulsive-like responses and increased path tortuosity. When training was performed in a smaller pool with a larger platform and with one more trial per day (see Materials and Methods S1), conditions that likely reduce task difficulty, WT mice showed faster learning (Figure S7), but KO mice were still impaired, showing delayed acquisition of the task (Escape latency: p,0.05; day 2: p,0.008). In these conditions, however, their performance progressively improved and reached similar scores as that of the controls on the last two training days (p.0.2). The KO mice also displayed slightly longer latencies during a reversal test (p.0.05, NS), which was reminiscent of the performance deficit during acquisition. g p p Emerging notion conceptualize DAPC as a scaffold for proteins involved in membrane stabilization and transmembrane signaling [16]. DAPC stabilizes large clusters of acetylcholine receptors at the neuromuscular junction in association with utrophin [33] and modulates GABAA-receptor clustering in association with Dp427 in central inhibitory synapses [34] without being required for membrane anchoring of these receptors. Here, we found that cultured primary neurons from Dp71-null mice have abnormally large clusters of PSD-95 protein and a reduced number of mature synapses. Hence, whereas Dp71-DAPC seems dispensable for postsynaptic targeting of glutamate receptor complexes, it may be a key modulator of their organized distribution. At the ultrastructural level in the adult brain, we found that KO mice have a reduced number of excitatory synapses (,11–13%) with larger cross- sectional PSD length, which parallels our findings in cultured neurons and suggests reduced synapse density and altered morphology of the postsynaptic active zone in Dp71-null mice. Associative learning was assessed using three different fear- conditioning protocols: The hippocampus-dependent contextual and amygdala-dependent cued-fear conditioning, and inhibitory avoidance. Behavioral Analysis of Dp71-Null Mice Retention performance was evaluated 24 h post- training by measuring freezing to the context alone (context test) and to the auditory conditioned stimulus (CS, tone) delivered in a new context (cue-CS test), and by the latency to enter the electrified zone in inhibitory avoidance. KO and WT mice expressed comparable freezing behavior during habituation, acquisition and retention of both contextual and auditory-cued fear conditioning (Figure 7F). Both groups also displayed comparable performance during acquisition of inhibitory avoid- ance learning (not shown), but the latency to enter the electrified zone was shorter in KO than in WT mice at the 24 h retention delay (Fig. 7G). p gy p y p p Using the hippocampal slice preparation, we found that both AMPAr and NMDAr-mediated components of CA1 glutamatergic transmission were drastically enhanced in KO mice. This was associated with reduced LTP levels, yet larger magnitude of LTP could be achieved in KO mice with stronger tetanization protocols, whereas LTD was not affected. Given the range of proteins found to interact with Dp71-DAPC, such alterations could be attributed to several pre- and postsynaptic mechanisms. A presynaptic hypothesis involving increased glutamate release is suggested by the overall increase in neurotransmission and reduced paired-pulse facilitation observed in KO mice. Moreover, in cultured neurons congenital loss of Dp71 was associated with an altered distribution of the presynaptic vesicular transporter VGLUT1. A purely presynaptic account seems however unlikely as PTP under NMDAr blockade was comparable between genotypes. On the other hand, the contribution of postsynaptic mechanisms to enhanced synaptic transmission in Dp71-KO is suggested by the increased PSD length, which may correlate with aggregation of receptors and signaling molecules at the active zone [35–37], and by the abnormal accumulation of PSD-95 in Dp71-deficient synapses, which could increase NMDAr gating [38], promote the delivery of AMPAr to synapses and increase AMPAr currents [39–41]. Such changes could explain that LTP expression was partially occluded in the Dp71 mutant mice, as suggested in mice overexpressing PSD-95 [40,41]. At this stage, however, the primary mechanisms linking Dp71 loss to altered synaptic function remain uncertain. The observed synaptic alterations indicate that the congenital loss of Dp71 significantly impinges synapse organization and maturation, but does not imply that Dp71 takes part actively to experience- driven synaptic plasticity in the adult brain. Behavioral Analysis of Dp71-Null Mice Moreover, other factors related to Dp71 function in other cell types might also contribute to the phenotype: The Dp71-DAPC appears to endorse a major role in the clustering of aquaporin (AQP4) channels in perivascular astrocytes [7] and its disruption may alter the blood-brain barrier and water and K+homeostasis, which could indirectly affect neuronal function [42]. Behavioral Analysis of Dp71-Null Mice significantly different from 50%, p,0.05. E. Spatial learning in the water-maze (12 KO, 10 WT). Escape latencies (left panel) and distance swum to reach the platform (right panel) monitored over 7 days (1 block of 4 trials per day). p,0.05. F. Contextual and cued-fear memory. Graph shows percent freezing (12 KO, 13 WT) before (pre-CS) and immediately after administration of the unconditioned stimulus (US, footshock) during learning (CS-US), and during retention trials 24 h later in the same context (context test), or in a new context before (new context) and after presentation of the auditory CS (cue-CS test). G. Inhibitory avoidance learning (17 Dp71-null, 12 WT). Retention performance at 24 h is expressed as the latency to step into the electrified zone. p,0.05. doi:10.1371/journal.pone.0006574.g007 Figure 7. Selective alterations of exploratory behavior and learning and memory performance in Dp71-null mice. KO: Filled symbols; WT: Open symbols. A. Spontaneous locomotor activity during exploration of an empty open-field (9 KO, 10 WT). The number of squares crossed along the walls for 50 min is shown by bins of 5 min recorded every 10 min. p,0.02. B. Object investigations expressed as the number of contacts made with three different sets of objects used during successive acquisition sessions in the object recognition task (13 KO, 16 WT). p,0.01. C. Object-recognition memory performance at retention delays of 10 min, 24 h and 48 h. significantly different from chance (50%), p,0.05. D. Spatial alternation in a T-maze. Data are % of mice alternating (14 KO, 24 WT) at retention delays of 1, 6 and 24 h. significantly different from 50%, p,0.05. E. Spatial learning in the water-maze (12 KO, 10 WT). Escape latencies (left panel) and distance swum to reach the platform (right panel) monitored over 7 days (1 block of 4 trials per day). p,0.05. F. Contextual and cued-fear memory. Graph shows percent freezing (12 KO, 13 WT) before (pre-CS) and immediately after administration of the unconditioned stimulus (US, footshock) during learning (CS-US), and during retention trials 24 h later in the same context (context test), or in a new context before (new context) and after presentation of the auditory CS (cue-CS test). G. Inhibitory avoidance learning (17 Dp71-null, 12 WT). Retention performance at 24 h is expressed as the latency to step into the electrified zone. p,0.05. Discussion The function of Dp140 in brain is at present unknown [1]. Loss of Dp427 in mdx mice alters GABAA-receptor clustering [34], which has been associated with abnormal enhancement of hippocampal LTP [13,46], reorganization of inhibitory synapses and morphological alterations of hippocampal excitatory-synapse subtypes [47], and behavioral impairments mainly characterized by mild memory retention deficits [13,48]. Here we show that Dp71, in addition to its potential role in the control of the blood-brain barrier in relation to its expression in perivascular astrocytes [7], plays a role in glutamatergic synapse organization and function and that its loss also results in cognitive impairments. The present results suggest stronger learning impairments in Dp71-null mice than in mdx mice, which supports the hypothesis that Dp71 loss has a major role in the genesis of cognitive deficits in DMD. However, the two models display distinct synaptic and behavioral alterations and it would thus be interesting in future studies to examine the consequences of mutations preventing the expression of both proteins. This could be tentatively addressed with the mdx3CV mutant mouse that lacks all dystrophin-gene products, but the few studies to date show that this model does not replicate the behavioral and neurophysiolog- ical alterations reported in mdx [12,14] and Dp71-null mice. The possibility that residual expression or compensatory mechanisms may attenuate the loss of dystrophins in this model has not been resolved. The generation of new mouse models with brain-specific mutations and combined loss-of-function of several dystrophin- gene products will thus be an important step to determine the respective involvement of GABAergic and glutamatergic functions in the neural basis of MR in DMD. Biochemistry A ib di Antibodies. The polyclonal H4 antibody directed against the C-terminal part of dystrophins and recognizing Dp71, the specific monoclonal 5F3 antibody directed against the last 31 residues of Dp71 isoforms lacking exon 78 (Figure S1), and the antibodies directed against b-dystroglycan, a- and c1-syntrophins were all generated by D. Mornet and characterized in detail. The monoclonal DYS2 antibody (Novocastra Laboratories, Newcastle, U.K.) is directed against the C-terminal part of dystrophins, in the same region of the protein as H4. All antibodies used and sources are described in Table S1. Fluorescein- or Cyanine 3-conjugated goat anti-rabbit, donkey anti-rabbit and goat anti-mouse secondary antibodies were from Jackson ImmunoResearch (West Grove, PA, USA). Western blot analyses. Brains were removed immediately after decapitation and whole hippocampi were dissected out. In some experiments the dentate gyrus and CA1-3 hippocampal subfield were dissected and analyzed as separate samples. Mouse dissected brain structures were homogenized with a polytron (30 s, 4uC, full speed) in 5 volumes (wt/vol) of extraction buffer. Protein concentration was evaluated by nanodrop. Equivalent amounts of protein were resolved on 10% (rat) or 8% (mouse) SDS-PAGE and electrotransferred to a nitrocellulose membrane. For the subcellular and PSD fractionation, five mouse brains were used in a procedure adapted from Huttner et al. [49]. On the last step of the protocol, the PSD fractions PSD1 and PSD2 were resuspended as described elsewhere [50,51] (see Materials and Methods S1 and Figure S3 for a detailed description). For the subcellular and PSD fractionation, five mouse brains were used in a procedure adapted from Huttner et al. [49]. On the last step of the protocol, the PSD fractions PSD1 and PSD2 were resuspended as described elsewhere [50,51] (see Materials and Methods S1 and Figure S3 for a detailed description). For immunoprecipitation assays, one mg-protein of rat hippocampus was homogenized in RIPA 1 buffer (50 mM Tris- HCl pH 8, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, and 2.5 mM EDTA) containing protease inhibitors and incubated overnight at 4uC with appropriate antibody. Protein-antibody complexes were mixed with Protein A/G Plus Agarose (Santa Cruz Biotechnology, USA) and incubated overnight at 4uC. After extensive washing with buffer Animals Male Wistar rats, dystrophin-deficient mdx mice and Dp71-null mice (KO) and their control WT littermates (.3 mo old) were used. Rats were purchased from Charles River (France); mdx mice and their controls of the C57BL/10ScSn strain were originally purchased from Jackson Laboratories (USA) and Harlan (France), respectively, and then bred in the mouse transgenic facility in Orsay (France) as described [13]; Dp71-null and littermate controls were produced in the animal facility at Weizmann Institute (Israel). Animals were kept under a 12-h light-dark cycle (light on: 7.00 a.m.) with food and water ad libitum. Generation of Dp71-null mice by homologous recombination has been described previously [17]. Briefly, most of the first and unique exon and a small part of the first intron of Dp71 have been replaced by the promoter-less gene encoding a b-gal-neomycin resistance chimeric protein, which specifically abolished the expression of Dp71 without interfering with the expression of other DMD-gene products. Females heterozygous for the mutation were backcrossed for at least 9–10 generations to balb/c males to generate the KO and littermate WT mice. Genotype was determined by PCR analysis of tail DNA. Functional analyses were conducted blind to the genotype. Discussion However, the two models display distinct synaptic and behavioral alterations and it would thus be interesting in future studies to examine the consequences of mutations preventing the expression of both proteins. This could be tentatively addressed with the mdx3CV mutant mouse that lacks all dystrophin-gene products, but the few studies to date show that this model does not replicate the behavioral and neurophysiolog- ical alterations reported in mdx [12,14] and Dp71-null mice. The possibility that residual expression or compensatory mechanisms may attenuate the loss of dystrophins in this model has not been resolved. The generation of new mouse models with brain-specific mutations and combined loss-of-function of several dystrophin- gene products will thus be an important step to determine the respective involvement of GABAergic and glutamatergic functions in the neural basis of MR in DMD. maturation, architecture and function provides valuable insights into identifying some of the complex functional pathways underlying MR in DMD. Special interest should focus on factors and signals that coordinate the interplay between the pre- and postsynaptic compartments during synapse morphogenesis and plasticity, which appear involved in several types of mental disorders [15]. The behavioral characterization of Dp71-null mice revealed selective disturbances characterized by reduced explor- atory behavior, slight retention deficits in inhibitory avoidance, and impaired spatial learning and memory. In Dp71-null mice, exploration deficits may partly relate to emotional reactivity, neglect and/or withdrawal from novelty-seeking behavior; disor- ders that have been associated with enhanced glutamatergic transmission [43]. Such deficiencies that may compromise the patients’ autonomy and social rehabilitation have not as yet been systematically assessed in DMD (but see [44,45]). Impaired navigation in the water maze and spatial alternation in the T- maze suggest an alteration in hippocampus-dependent encoding and/or consolidation of spatial information, in line with the synaptic and neurophysiological alterations observed in Dp71-null mice in this structure. However, the ability of Dp71-null mice to reach a normal level of performance in the navigation task when task difficulty is reduced, the mild deficit in inhibitory avoidance and unaffected retention performance in object recognition and contextual fear conditioning suggest that certain hippocampal functions are preserved. Although the Dp71-null mouse constitutes a unique model to explore the specific contribution of congenital Dp71 loss in brain, it is important to note that the genesis of MR in DMD likely encompass cumulative inactivation of Dp427, Dp71 and Dp140 [2,3]. Discussion In DMD, Dp71 deficiency is associated with MR in a subpopulation of patients [6] and is thought to contribute to the severity of MR phenotypes. Dp71 function is however unclear and complex, as the protein appears to be expressed in both perivascular astrocyte end-feet and neurons. Here, we further characterized this complex pattern of expression and show that Dp71 interacts with specialized modular scaffolds that organize signaling complexes at glutamatergic synapses. We also found that Dp71 loss affects synaptic maturation and function, and results in deficits in selective cognitive functions. The data provide evidence for an association of Dp71-DAPC with the multi-protein scaffolds that cluster glutamate receptors and organize signaling proteins required for synaptic transmission and plasticity, including NMDAr and AMPAr subunits, and other PDZ- containing proteins such as PSD-95 and nNOS. This specific Dp71- DAPC contains the two main splice variants, Dp71d and Dp71f, along with b-dystroglycan, a- and c1-syntrophins and a-dystro- brevins. Dp71-DAPC is also associated with key signaling proteins such as FAK, c-Src and Grb2, proteins known to contribute to neurite outgrowth, dendritic spine morphogenesis and synaptic plasticity [24–26]. It has been shown that in the nervous system DAPC binds nNOS [27], ErbB4 neuregulin receptors [28] and transmembrane channels [29,30] through the PDZ domain of syntrophins, whereas the adaptor protein Grb2 mediates FAK/b- dystroglycan interactions [31]. Future studies will determine whether such mechanisms can explain how Dp71-DAPC binds to synaptic proteins. Interestingly, we found that Dp71-DAPC also interacts with GIRK1 (Kir3.1) potassium channels typically detected in perisynaptic lipid rafts [32]. This suggests cross-linkage between organizing molecules of the PSD and lipid rafts and the possible existence of two distinct pools of Dp71-DAPC in PSD and The diversity of mechanisms presumably modified as a consequence of Dp71 loss underscores the complex cellular and subcellular distribution of Dp71 in brain. The finding that a pool of Dp71 may act as a regulator of glutamatergic synapse PLoS ONE \| www August 2009 \| Volume 4 \| Issue 8 \| e6574 August 2009 \| Volume 4 \| Issue 8 \| e6574 PLoS ONE \| www.plosone.org 10 Dp71 and Brain Function tation and were conducted following the guidelines of the animal facilities in Orsay (France) approved by the national direction of veterinary services (Direction des Services Ve´te´rinaires, DSV- France, agreement # B91-429 for the rats and B91-471-104 for the mice). Discussion maturation, architecture and function provides valuable insights into identifying some of the complex functional pathways underlying MR in DMD. Special interest should focus on factors and signals that coordinate the interplay between the pre- and postsynaptic compartments during synapse morphogenesis and plasticity, which appear involved in several types of mental disorders [15]. The behavioral characterization of Dp71-null mice revealed selective disturbances characterized by reduced explor- atory behavior, slight retention deficits in inhibitory avoidance, and impaired spatial learning and memory. In Dp71-null mice, exploration deficits may partly relate to emotional reactivity, neglect and/or withdrawal from novelty-seeking behavior; disor- ders that have been associated with enhanced glutamatergic transmission [43]. Such deficiencies that may compromise the patients’ autonomy and social rehabilitation have not as yet been systematically assessed in DMD (but see [44,45]). Impaired navigation in the water maze and spatial alternation in the T- maze suggest an alteration in hippocampus-dependent encoding and/or consolidation of spatial information, in line with the synaptic and neurophysiological alterations observed in Dp71-null mice in this structure. However, the ability of Dp71-null mice to reach a normal level of performance in the navigation task when task difficulty is reduced, the mild deficit in inhibitory avoidance and unaffected retention performance in object recognition and contextual fear conditioning suggest that certain hippocampal functions are preserved. Although the Dp71-null mouse constitutes a unique model to explore the specific contribution of congenital Dp71 loss in brain, it is important to note that the genesis of MR in DMD likely encompass cumulative inactivation of Dp427, Dp71 and Dp140 [2,3]. The function of Dp140 in brain is at present unknown [1]. Loss of Dp427 in mdx mice alters GABAA-receptor clustering [34], which has been associated with abnormal enhancement of hippocampal LTP [13,46], reorganization of inhibitory synapses and morphological alterations of hippocampal excitatory-synapse subtypes [47], and behavioral impairments mainly characterized by mild memory retention deficits [13,48]. Here we show that Dp71, in addition to its potential role in the control of the blood-brain barrier in relation to its expression in perivascular astrocytes [7], plays a role in glutamatergic synapse organization and function and that its loss also results in cognitive impairments. The present results suggest stronger learning impairments in Dp71-null mice than in mdx mice, which supports the hypothesis that Dp71 loss has a major role in the genesis of cognitive deficits in DMD. Electrophysiology Acute hippocampal slices from mice (3–4 mo) were used as previously described [13]. Extracellular synaptic responses were recorded in CA1 stratum radiatum to monitor PFVs and fEPSPs evoked by electrical stimulation of the Schaffer collateral/commis- sural fibers at 0.1 Hz. Input/output curves were constructed by measuring the slopes of fEPSPs mediated by AMPAr and NMDAr at different stimulus intensities (100–500 mA; 60 ms duration, averages of 3 responses). NMDAr-mediated responses were isolated in the presence of the GABAA receptor antagonist, bicuculline (10 mM), and the AMPA/kainate receptor antagonist 6,7-Dinitroquinoxaline- 2,3-dione (DNQX, 10 mM). Addition of D-APV (30 mM) was used to analyze PFVs in the absence of EPSPs. PPF was induced by stimulation with pairs of pulses with a 50-ms interstimulus interval and quantified as the ratio of the second over the first fEPSP slopes. To investigate LTP, the initial slope of 3 averaged fEPSPs was measured for 15 min (baseline) and tetanic stimulation consisted of two trains, 20 s apart, of HFS of either 50 Hz or 100 Hz (duration: 1 s at test intensity). LTD was induced by low-frequency stimulation (900 pulses at 1 Hz). Testing with single pulses was resumed for 60 min after induction of synaptic plasticity. Cell culture. Cortex from individual mice at embryonic day 15.5 (E15.5) were dissected, dissociated by trypsin treatment and mechanical trituration, and plated onto polylysine-coated glass coverslips (14 mm in diameter) (SIGMA-Aldrich) at a density of ,5000 neurons/cm2. Cultures were maintained in neurobasal medium complemented with B27 and glutamate (Invitrogen) as described elsewhere [52]. Immunocytochemistry. Cultured cells were fixed with 100% methanol at 220uC for 10 min, permeabilized with 0.1% triton X- 100, blocked in PBS plus 3% BSA, and then incubated with appropriate primary and secondary antibodies. For triple-staining assays, Zenon labeling technology (Molecular probes, Invitrogen) was applied to enable the use of two monoclonal antibodies on mouse tissues. For quantification of immunostained puncta, images were taken with a 40X oil-objective, automatically threshold and analyzed with the ImageJ software (NIH, USA, http://rsb.info.nih.gov/ij/). A total of 35 primary dendrites from 7 neurons were analyzed for each genotype. For each dendrite, a 50-mm segment was analyzed (250 mm per neuron). Each experiment was repeated at least twice with independent neuronal cultures. Electrophysiology Neurons from each genotype were analyzed using identical imaging conditions to quantify Dp71 co-localization with presynaptic (VGLUT1) and postsynaptic (SHANK) markers of mature excitatory synapses and with a presynaptic marker of inhibitory synapses (GAD-65/67). A synapse was defined by juxtaposition of the pre- and postsynaptic markers. Ethics Statement Animal studies complied with the European Communities Council Directive (CEE 86/609) for animal care and experimen- PLoS ONE \| www.plosone.org August 2009 \| Volume 4 \| Issue 8 \| e6574 August 2009 \| Volume 4 \| Issue 8 \| e6574 11 Dp71 and Brain Function RIPA 2 (50 mM Tris-HCl pH 8, 150 mM NaCl, 0.2% Triton X- 100, 0.02% SDS and 0.2% sodium deoxycholate) the resin containing the protein-antibody complexes was suspended in sample buffer (1/3 of initial volume), boiled and analyzed by western blot (bound fraction). The buffer of the crude extract used for immunoprecipitation was diluted 1:10 and analyzed in parallel (unbound fraction). were estimated from 15 counting frames per animal (total sampled neuropil area: 663.33 mm2). When the PSD was perforated, the sum of the individual trace lengths was considered for PSD length (Figure 5C, insert) [36]. Behavioral testing Subjects. Eighty-six male mice on a main balb/c background (40 KO and 46 WT littermates), aged 3 to 8 month, were used. Because mice with inbred balb/c background are considered poor learners in spatial tasks, outcrosses between female balb/c- heterozygous for Dp71 and C57BL/6 male mice were undertaken to obtain CB6F1 hybrids tested in the water maze. The behavioral paradigms have been conducted as described [13,48,55,56] with few adaptations. Detailed protocols are provided as supporting information in Materials and Methods S1. Immunohistochemistry. Fresh frozen brains were processed as described [53]. Sections were fixed for 3 min in acetone/ methanol (1:1) at 220uC, stained for 24 h at 4uC with primary antibody and then incubated 45 min with secondary antibodies. In some experiments the tissue was fixed by transcardiac perfusion with 4% paraformaldehyde in 0.1M-phosphate buffer (pH 7.4). Controls were prepared by omitting the primary antibody; in these controls, no specific staining could be detected. Immunohistochemistry. Fresh frozen brains were processed as described [53]. Sections were fixed for 3 min in acetone/ methanol (1:1) at 220uC, stained for 24 h at 4uC with primary antibody and then incubated 45 min with secondary antibodies. In some experiments the tissue was fixed by transcardiac perfusion with 4% paraformaldehyde in 0.1M-phosphate buffer (pH 7.4). Controls were prepared by omitting the primary antibody; in these controls, no specific staining could be detected. Statistical analyses Data are reported as means6SEM. They were analyzed using ANOVA with genotype as the between-subject factor, comple- mented by a within-subject factor analysis for time or place dependence of effects (Statview 5.0). One set of data, the latencies recorded in inhibitory avoidance, did not pass the normality and equal-variance tests (SigmaStat 2.0, SPSS Inc.) and was therefore analyzed with non-parametric Mann-Whitney U test. In object recognition, comparison of recognition indexes to chance was performed using univariate t-tests. In delayed alternation, a chi- square analysis was used to compare alternation rates to chance. The distribution of PSD length in the EM study was analyzed with the Kolmogorov-Smirnov (KS) test. X-gal staining. Beta-galactosidase enzyme histochemistry was performed on both fresh-frozen and formalin-fixed brains from Dp71-null mice. Sections were washed in 0.1M PBS, stained for 1 h at 40uC with X-gal (Promega France), dehydrated and then cleared in Xylene. Dimethyl Formamide (0.4%), MgCl2 (1M), potassium ferrocyanide (5%) and ferricyanide (5%) were added to the staining buffer. X-gal staining. Beta-galactosidase enzyme histochemistry was performed on both fresh-frozen and formalin-fixed brains from Dp71-null mice. Sections were washed in 0.1M PBS, stained for 1 h at 40uC with X-gal (Promega France), dehydrated and then cleared in Xylene. Dimethyl Formamide (0.4%), MgCl2 (1M), potassium ferrocyanide (5%) and ferricyanide (5%) were added to the staining buffer. Electron microscopy Brain tissue was processed as previously described [47] (see Materials and Methods S1). Ultrathin sections (70-nm thick) were visualized at 614000 using a Philips EM208 transmission electron microscope (Philips Electron Optics, Eindhoven, NL) and photographed with a CCD AMT digital camera (Hamamatsu, Japan). Synapses were examined in the distal stratum radiatum (,150 mm from the CA1 pyramidal cell bodies). Axospinous symmetric (excitatory) synapses were identified by a thick PSD, synaptic cleft and presynaptic component with synaptic vesicles [54]. Those with one or more perforations in the PSD were classified as perforated and those showing a continuous PSD as non-perforated (Figure 5A). Single presynaptic terminals contact- ing more than one postsynaptic spine were defined as multiple spine boutons (MSBs). The relative density of asymmetric synapses (synapses per mm2) and the cross-sectional length of their PSDs PLoS ONE \| www.plosone.org Found at: doi:10.1371/journal.pone.0006574.s004 (0.66 MB TIF) amino acids in the Dp71 protein. Variants of Dp71 protein with minor expression in brain have also been described (Austin et al., Neuromuscul Disord 10(3):187–93, 2000): Dp71D110m is derived from Dp71 transcripts deleted for exons 71–74 and contains the C-terminal sequence of muscle dystrophin, while Dp71D110a contains the alternative 31-amino acid C-terminal sequence due to the splicing of exon 78 (Austin et al., J Biol Chem 277:47106– 47113, 2002). The location of the epitopes recognized by antibodies H4, DYS2 and 5F3 are indicated. Found at: doi:10.1371/journal.pone.0006574.s001 (1.01 MB TIF) Found at: doi:10.1371/journal.pone.0006574.s004 (0.66 MB TIF) Found at: doi:10.1371/journal.pone.0006574.s004 (0.66 MB TIF) Figure S5 Gross hippocampal anatomy is normal in Dp71-null mice. Formalin-fixed brain sections of WT and KO mice were immunostained with various cell markers: NeuN antibody was used to stain neuronal nuclei in CA1-4 and DG regions. Tuj-1 antibody immunostained pyramidal neurons and basal dentrites in CA1. The anti-parvalbumin antibody was used to reveal parvalbumin-containing interneurons. Found at: doi:10.1371/journal.pone.0006574.s005 (3.70 MB TIF) Figure S6 Basal neurotransmission and PTP in Dp71-null and WT mice. (A–B) Basal glutamatergic transmission mediated through AMPAr: (A) presynaptic fibre volley (PFV) slopes plotted against intensity; (B) AMPAr-mediated fEPSP slopes plotted against intensity. (C–D). NMDAr component of glutamatergic transmission: (C) PFV and (D) NMDAr-mediated fEPSP against intensity. WT(open symbols) and KO mice (black symbols). (E) Time-course of the post-tetanic potentiation (PTP) induced by a 26100 Hz HFS delivered in the presence of the NMDAr antagonist APV (80 mM). Genotype effect: p.0.37, NS. Found at: doi:10.1371/journal.pone.0006574.s006 (0.62 MB TIF) Figure S2 Immunofluorescence detection of Dp71 in brain sections. Hippocampal expression of Dp71 isoforms revealed by H4 on rat brain sections. Representative images show Dp71 expression in (A) walls of blood vessels and perivascular astrocytes (arrowhead), and (B) in the granule-cell layer (gcl) of dentate gyrus as incomplete rings of immunoreactivity circling granule cell bodies and small to large dots around or within cell bodies. C. Immunolabeling of Dp71 with H4 antibody in the gcl and walls of blood vessels (arrowheads) in WT (C1), mdx (C2) and Dp71-null mice (C3). F d t d i 10 1371/j l 0006574 002 (2 61 MB TIF) Found at: doi:10.1371/journal.pone.0006574.s002 (2.61 MB TIF) Figure S7 Spatial learning and reversal in the water maze. In this experiment, the pool diameter was 1.30 m and mice were submitted to 5 daily trials (Dark symbols, 10 KO, Open symbols, 16 WT). Acknowledgments The authors are grateful to S. Vandergeenst, N. Samson and P. Veyrac for animal care. We thank J.C. Garcı´a Torres for technical assistance, R. Robichon and S. Marty for advice on immunohistochemistry, R. Miranda for advice and kind help for EM study, and M. Segal (Weizmann Institute) for helpful discussions and suggestions. Found at: doi:10.1371/journal.pone.0006574.s004 (0.66 MB TIF) Acquisition was followed by a reversal task (arrow) on day 5. Escape latencies show delayed learning in KO mice during the first two days (p,0.05), with improved performance by day 3–4 and intact reversal. Found at: doi:10.1371/journal.pone.0006574.s007 (0.43 MB TIF) Figure S3 Fractionation study. A. Schematic representation of the fractionation protocol (detailed description in Materials and Methods S1). B. Protein extracts from subcellular fractions obtained from control mouse brains probed with the anti-Dp71 (H4) antibody (top panel). Bottom panels show expression of the presynaptic and postsynaptic markers synaptophysin and PSD-95, respectively. Fractions as follows: H, total homogenate; S1, cell soma; P1, dense nuclei-associated membrane; S2, supernatant, P2: crude synaptosomal membrane, S3, cytosolic; P3, light membrane compartment; LS1, soluble; LP1, synaptosomal membrane; LP2, synaptic-vesicle enriched; SPM, synaptic plasma membrane; PSD1/PSD2, postsynaptic density fractions. Found at: doi:10.1371/journal.pone.0006574.s003 (1.84 MB TIF) Table S1 Primary antibodies used in this study. *It could be the immunogen. Found at: doi:10.1371/journal.pone.0006574.s008 (0.05 MB DOC) Materials and Methods S1 Supplemental Materials and Methods. Materials and Methods S1 Supplemental Materials and Methods. Figure S4 Expression of Dp71 in cultured neurons of control (WT) and Dp71-null (KO) mice. Immunofluorescence assays were performed using the H4 antibody (red) that binds all dystrophin- gene products and the 5F3 antibody (green) directed against Dp71 isoforms lacking exon 78. Representative images of cultured neurons from WT (A, B) and KO mice (C, D). Both antibodies labeled neurites and perinuclear regions of control neurons. H4 immunolabeling was present in both control and Dp71-deficient cells, reflecting its binding to several dystrophin-gene products. In contrast, 5F3 antibody labeled neurites of control neurons, but not that of Dp71-deficient neurons. This staining pattern confirms that 5F3 detects specifically Dp71 isoforms lacking exon 78 and expressed in neurites. 5F3 also revealed a basal discontinuous granular labeling in the Golgi complex in both control and Dp71- deficient neurons, which may reflect binding to another dystro- phin-like protein in the perinuclear region (Cha´vez et al., Biochem Biophys Res Commun 274:275–280, 2000). Nuclei stained with DAPI (blue). Found at: doi:10.1371/journal.pone.0006574.s009 (0.04 MB DOC) Author Contributions Conceived and designed the experiments: JMB AA MK YR UN JC DMR DY CV. Performed the experiments: FD ACM JMB AA YR MG DJ CV. Analyzed the data: FD JMB YR DMR CV. Contributed reagents/ materials/analysis tools: DM CV. Wrote the paper: CV. Discussion of results and paper revision: CV FD DM UN JC DMR SL DY. Mouse breeding and genotyping: YR UN DY. 2. Desguerre I, Christov C, Mayer M, Zeller R, Becane HM, et al. (2009) Clinical heterogeneity of duchenne muscular dystrophy (DMD): definition of sub- phenotypes and predictive criteria by long-term follow-up. PLoS ONE 4(2): e4347. 4. Lederfein D, Levy Z, Augier N, Mornet D, Morris G, Fuchs, et al. (1992) A 71- kilodalton protein is a major product of the Duchenne muscular dystrophy gene 5. Jung D, Filliol D, Metz-Boutigue MH, Rendon A (1993) Characterization and subcellular localization of the dystrophin-protein 71 (Dp71) from brain. Neuromuscul Disord 3: 515–518. 3. Moizard MP, Billard C, Toutain A, Berret F, Marmin N, et al. (1998) Are Dp71 and Dp140 brain dystrophin isoforms related to cognitive impairment in Duchenne muscular dystrophy? Am J Med Genet 80: 32–41. in brain and other nonmuscle tissues. Proc Natl Acad Sci U S A 89(12): 5346–50. 1. Lidov HG (1996) Dystrophin in the nervous system. Brain Pathol 6: 63–77. 6. Moizard MP, Toutain A, Fournier D, Berret F, Raynaud M, et al. (2000) Severe cognitive impairment in DMD: obvious clinical indication for Dp71 isoform point mutation screening. Eur J Hum Genet 8: 552–556. 7. Haenggi T, Soontornmalai A, Schaub MC, Fritschy JM (2004) The role of utrophin and Dp71 for assembly of different dystrophin-associated protein in brain and other nonmuscle tissues. Proc Natl Acad Sci U S A 89(12): 5346–50. in brain and other nonmuscle tissues. Proc Natl Acad Sci U S A 89(12): 5346–50. 5. Jung D, Filliol D, Metz-Boutigue MH, Rendon A (1993) Characterization and subcellular localization of the dystrophin-protein 71 (Dp71) from brain. Neuromuscul Disord 3: 515–518. 6. Moizard MP, Toutain A, Fournier D, Berret F, Raynaud M, et al. (2000) Severe cognitive impairment in DMD: obvious clinical indication for Dp71 isoform point mutation screening. Eur J Hum Genet 8: 552–556. 7. Haenggi T, Soontornmalai A, Schaub MC, Fritschy JM (2004) The role of utrophin and Dp71 for assembly of different dystrophin-associated protein PLoS ONE \| www.plosone.org Supporting Information Figure S1 Schematic representation of Dp71 isoforms. (A) Dp71 is transcribed from a promoter located between exons 62 and 63 of the DMD gene; it has a unique N-terminus of seven amino acids and contains the cysteine-rich and common C-terminal domain of dystrophins, a region for binding to DAPs complexes. (B) Dp71 alternative spliced transcripts. Dp71d isoform lacks exon 71 and Dp71f isoform lacks exons 71 and 78 (Marquez et al., Neuroscience 118:957–966, 2003). Deletion of exon 78 changes the reading frame, resulting in the replacement of the last 13 hydrophilic amino acids of dystrophin with 31 new hydrophobic PLoS ONE \| www.plosone.org August 2009 \| Volume 4 \| Issue 8 \| e6574 12 Dp71 and Brain Function Dp71 and Brain Function Stein V, House DR, Bredt DS, Nicoll RA (2003) Postsynaptic density-95 mimics and occludes hippocampal long-term potentiation and enhances long-term depression. J Neurosci 23: 5503–5506. y p ( ) 15. Vaillend C, Poirier R, Laroche S (2008) Genes, plasticity and mental retardation. Behav Brain Res 192(1): 88–105. p 41. Ehrlich I, Malinow R (2004) Postsynaptic density 95 controls AMPA receptor incorporation during long-term potentiation and experience-driven synaptic plasticity. J Neurosci 24: 916–927. ( ) 16. Albrecht DE, Froehner SC (2002) Syntrophins and dystrobrevins: defining the dystrophin scaffold at synapses. Neurosignals 11: 123–129. y p y p g 17. Sarig R, Mezger-Lallemand V, Gitelman I, Davis C, Fuchs O, et al. (1999) Targeted inactivation of Dp71, the major non-muscle product of the DMD gene: differential activity of the Dp71 promoter during development. Hum Mol Genet 8: 1–10. p y J 42. Nicchia GP, Srinivas M, Li W, Brosnan CF, Frigeri A, et al. (2005) New possible roles for aquaporin-4 in astrocytes: cell cytoskeleton and functional relationship with connexin43. FASEB J 19(12): 1674–6. 43. Granger R, Staubli U, Davis M, Perez Y, Nilsson L, et al. (1993) A drug that facilitates glutamatergic transmission reduces exploratory activity and improves performance in a learning dependent task. Synapse 15(4): 326–9. 18. Gorecki DC, Abdulrazzak H, Lukasiuk K, Barnard EA (1997) Differential expression of syntrophins and analysis of alternatively spliced dystrophin transcripts in the mouse brain. Eur J Neurosci 9(5): 965–76. 44. Donders J, Taneja C (2009) Neurobehavioral Characteristics of Children with Duchenne Muscular Dystrophy. Child Neuropsychol 22: 1–10. 44. Donders J, Taneja C (2009) Neurobehavioral Characteristics 19. Austin RC, Howard P, D’Souza VN, Klamut HJ, Ray PN (1995) Cloning and characterization of alternatively spliced isoforms of Dp71. Hum Mol Genet 4: 1475–1483. Duchenne Muscular Dystrophy. Child Neuropsychol 22: 1–10. 45. Hendriksen JG, Vles JS (2008) Neuropsychiatric disorders in males with duchenne muscular dystrophy: frequency rate of attention-deficit hyperactivity disorder (ADHD), autism spectrum disorder, and obsessive–compulsive disorder. J Child Neurol 23(5): 477–81. 20. Fabbrizio E, Leger J, Anoal M, Leger JJ, Mornet D (1993) Monoclonal antibodies targeted against the C-terminal domain of dystrophin or utrophin. FEBS Lett 322: 10–14. 46. Vaillend C, Billard JM (2002) Facilitated CA1 hippocampal synaptic plasticity in dystrophin-deficient mice: role for GABAA receptors? Hippocampus 12(6): 713–7. 21. Fabbrizio E, Nudel U, Hugon G, Robert A, Pons F, et al. Dp71 and Brain Function complexes (DPCs) in the choroid plexus and microvasculature of the brain. Neuroscience 129: 403–413. 33. Banks GB, Fuhrer C, Adams ME, Froehner SC (2003) The postsynaptic submembrane machinery at the neuromuscular junction: requirement for rapsyn and the utrophin/dystrophin-associated complex. J Neurocytol 32: 709–726. 8. Alema´n V, Osorio B, Cha´vez O, Rendon A, Mornet D, et al. (2001) Subcellular localization of Dp71 dystrophin isoforms in cultured hippocampal neurons and forebrain astrocytes. Histochem Cell Biol 115: 243–254. 34. Fritschy JM, Schweizer C, Brunig I, Luscher B (2003) Pre- and post-synaptic mechanisms regulating the clustering of type A gamma-aminobutyric acid receptors (GABAA receptors). Biochem Soc Trans 31: 889–892. y 9. Dalloz C, Sarig R, Fort P, Yaffe D, Bordais A, et al. (2003) Targeted inactivation of dystrophin gene product Dp71: phenotypic impact in mouse retina. Hum Mol Genet 12: 1543–54. 35. Cheung HH, Takagi N, Teves L, Logan R, Wallace MC, et al. (2000) Altered association of protein tyrosine kinases with postsynaptic densities after transient cerebral ischemia in the rat brain. J Cereb Blood Flow Metab 20: 505–512. 10. Gorecki DC, Barnard EA (1995) Specific expression of G-dystrophin (Dp71) in the brain. Neuroreport 6: 893–896. 36. Desmond NL, Levy WB (1986a) Changes in the postsynaptic density with long- term potentiation in the dentate gyrus. J Comp Neurol 253: 476–482. p 11. Blake DJ, Hawkes R, Benson MA, Beesley PW (1999) Different dystrophin-like complexes are expressed in neurons and glia. J Cell Biol 147: 645–658. 37. Martone ME, Hu BR, Ellisman MH (2000) Alterations of hippocampal postsynaptic densities following transient ischemia. Hippocampus 10: 610–616. 12. Vaillend C, Billard JM, Claudepierre T, Rendon A, Dutar P, et al. (1998) Spatial discrimination learning and CA1 hippocampal synaptic plasticity in mdx and mdx3cv mice lacking dystrophin gene products. Neuroscience 86(1): 53–66. 38. Lin Y, Jover-Mengual T, Wong J, Bennett MV, Zukin RS (2006) PSD-95 and PKC converge in regulating NMDA receptor trafficking and gating. Proc Natl Acad Sci U S A 103: 19902–19907. 13. Vaillend C, Billard JM, Laroche S (2004) Impaired long-term spatial and recognition memory and enhanced CA1 hippocampal LTP in the dystrophin- deficient Dmd(mdx) mouse. Neurobiol Dis 17: 10–20. 39. El-Husseini AE, Schnell E, Chetkovich DM, Nicoll RA, Bredt DS (2000) PSD- 95 involvement in maturation of excitatory synapses. Science 290: 1364–1368. ( ) 14. Vaillend C, Ungerer A (1999) Behavioral characterization of mdx3cv mice deficient in C-terminal dystrophins. Neuromuscul Disord 9(5): 296–304. 40. Dp71 and Brain Function (1994) Characterization and localization of a 77 kDa protein related to the dystrophin gene family. Biochem J 299(Pt 2): 359–365. 47. Miranda R, Se´brie´ C, Degrouard J, Gillet B, Jaillard D, et al. (2009) Reorganization of Inhibitory Synapses and Increased PSD Length of Perforated Excitatory Synapses in Hippocampal Area CA1 of Dystrophin Deficient mdx Mice. Cereb Cortex 19(4): 876–88. 22. Lee CC, Huang CC, Wu MY, Hsu KS (2005) Insulin stimulates postsynaptic density-95 protein translation via the phosphoinositide 3-kinase-Akt-mammalian target of rapamycin signaling pathway. J Biol Chem 280(18): 18543–50. 23. Kim S, Burette A, Chung HS, Kwon SK, Woo J, et al. (2006) NGL family PSD- 95-interacting adhesion molecules regulate excitatory synapse formation. Nat Neurosci 9(10): 1294–301. 48. Vaillend C, Rendon A, Misslin R, Ungerer A (1995) Influence of dystrophin- gene mutation on mdx mouse behavior. I. Retention deficits at long delays in spontaneous alternation and bar-pressing tasks. Behav Genet 25: 569–579. 24. Lauri SE, Taira T, Rauvala H (2000) High-frequency synaptic stimulation induces association of fyn and c-src to distinct phosphorylated components. Neuroreport 11: 997–1000. 49. Huttner WB, Schiebler W, Greengard P, De Camilli P (1983) Synapsin I (protein I), a nerve terminal-specific phosphoprotein. III. Its association with synaptic vesicles studied in a highly purified synaptic vesicle preparation. J Cell Biol 96: 1374–1388. 25. Moeller ML, Shi Y, Reichardt LF, Ethell IM (2006) EphB receptors regulate dendritic spine morphogenesis through the recruitment/phosphorylation of focal adhesion kinase and RhoA activation. J Biol Chem 281: 1587–1598. 50. Carlin RK, Grab DJ, Cohen RS, Siekevitz P (1980) Isolation and characterization of postsynaptic densities from various brain regions: enrichment of different types of postsynaptic densities. J Cell Biol 86: 831–845. J 2 26. Robles E, Go´mez TM (2006) Focal adhesion kinase signaling at sites of integrin- mediated adhesion controls axon pathfinding. Nat Neurosci 9: 1274–1283. 51. Cho KO, Hunt CA, Kennedy MB (1992) The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs-large tumor suppressor protein. Neuron 9: 929–942. 27. Brenman JE, Chao DS, Gee SH, McGee AW, Craven SE, et al. (1996) Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and alpha1-syntrophin mediated by PDZ domains. Cell 84: 757–767. 52. Brewer GJ, Torricelli JR, Evege EK, Price PJ (1993) Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J Neurosci Res 35: 567–576. 28. References PLoS ONE \| www.plosone.org August 2009 \| Volume 4 \| Issue 8 \| e6574 13 August 2009 \| Volume 4 \| Issue 8 \| e6574 Dp71 and Brain Function Dp71 and Brain Function Garcia RA, Vasudevan K, Buonanno A (2000) The neuregulin receptor ErbB-4 interacts with PDZ-containing proteins at neuronal synapses. Proc Natl Acad Sci U S A 97(7): 3596–601. 53. Knuesel I, Bornhauser BC, Zuellig RA, Heller F, Schaub MC, et al. (2000) Differential expression of utrophin and dystrophin in CNS neurons: an in situ hybridization and immunohistochemical study. J Comp Neurol 422: 594–611. 29. Gee SH, Madhavan R, Levinson SR, Caldwell JH, Sealock R, et al. (1998) Interaction of muscle and brain sodium channels with multiple members of the syntrophin family of dystrophin-associated proteins. J Neurosci 18: 128–137. y y p 54. Mayhew TM (1996) How to count synapses unbiasedly and efficiently at the ultrastructural level: proposal for a standard sampling and counting protocol. J Neurocytol 25: 793–804. 30. Connors NC, Kofuji P (2002) Dystrophin Dp71 is critical for the clustered localization of potassium channels in retinal glial cells. J Neurosci 22: 4321–4327. 55. Poirier R, Jacquot S, Vaillend C, Soutthiphong AA, Libbey M, et al. (2007) Deletion of the Coffin-Lowry syndrome gene Rsk2 in mice is associated with impaired spatial learning and reduced control of exploratory behavior. Behav Genet 37: 31–50. 31. Cavaldesi M, Macchia G, Barca S, Defilippi P, Tarone G, et al. (1999) Association of the dystroglycan complex isolated from bovine brain synapto- somes with proteins involved in signal transduction. J Neurochem 72: 1648–1655. 32. Koyrakh L, Lujan R, Colon J, Karschin C, Kurachi Y, et al. (2005) Molecular and cellular diversity of neuronal G-protein-gated potassium channels. J Neurosci 25: 11468–11478. 56. Paylor R, Tracy R, Wehner J, Rudy JW (1994) DBA/2 and C57BL/6 mice differ in contextual fear but not auditory fear conditioning. Behav Neurosci 108: 810–817. PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org August 2009 \| Volume 4 \| Issue 8 \| e6574 14
https://openalex.org/W1557801781	https://europepmc.org/articles/pmc4491405?pdf=render	English	null	Influence of Selenium Content in the Culture Medium on Protein Profile of Yeast Cells<i>Candida utilis</i>ATCC 9950	Oxidative medicine and cellular longevity	2,015	cc-by	4,866	Department of Biotechnology, Microbiology and Food Evaluation, Faculty of Food Sciences, Warsaw University of Life Sciences-SGGW, Nowoursynowska 159C, 02-776 Warsaw, Poland Department of Biotechnology, Microbiology and Food Evaluation, Faculty of Food Sciences, Warsaw University of Life Sciences-SGGW, Nowoursynowska 159C, 02-776 Warsaw, Poland Correspondence should be addressed to Marek Kieliszek; marek-kieliszek@wp.pl Received 26 March 2015; Revised 25 May 2015; Accepted 31 May 2015 Academic Editor: Angel Catal´a Copyright © 2015 Marek Kieliszek et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Selenium is an essential trace element for human health and it has been recognized as a component of several selenoproteins with crucial biological functions. It has been identified as a component of active centers of many enzymes, as well as integral part of biologically active complexes. The aim of the study was to evaluate the protein content and amino acid profile of the protein of fodder yeast Candida utilis ATCC 9950 cultured in media control and experimental enriched selenium. Protein analysis was performed using SDS-PAGE method consisting of polyacrylamide gel electrophoresis in the presence of SDS. The highest contents of soluble protein (49,5 mg/g) were found in yeast cells after 24-hour culture conducted in control (YPD) medium. In the presence of selenium there were determined small amounts of protein content. With increasing time of yeast culture (to 72 hours) the control and experimental media were reported to reduce soluble protein content. In electropherogram proteins from control cultures was observed the presence of 10 protein fractions, but in all the experimental cultures (containing 20, 30, and 40 mg/L selenium) of 14 protein fractions. On the basis of the molecular weights of proteins, it can be concluded that they were among others: selenoprotein 15 kDa and selenoprotein 18 kDa. Hindawi Publishing Corporation Oxidative Medicine and Cellular Longevity Volume 2015, Article ID 659750, 6 pages http://dx.doi.org/10.1155/2015/659750 Hindawi Publishing Corporation Oxidative Medicine and Cellular Longevity Volume 2015, Article ID 659750, 6 pages http://dx.doi.org/10.1155/2015/659750 Marek Kieliszek, StanisBaw BBahejak, and Anna Bzducha-Wróbel Marek Kieliszek, StanisBaw BBahejak, and Anna Bzducha-Wróbel Department of Biotechnology, Microbiology and Food Evaluation, Faculty of Food Sciences, Warsaw University of Life Sciences-SGGW, Nowoursynowska 159C, 02-776 Warsaw, Poland Oxidative Medicine and Cellular Longevity Oxidative Medicine and Cellular Longevity Selenoproteins were first discovered in 1973, GPx being one of them. What is more, in 1973, two additional proteins such as glycine reductase present in the cells of Clostridium sticklandii bacteria and formate dehydrogenase occurring in Clostridium thermoaceticum were discovered [5]. Selenium occurring in yeast cell is transformed to selenoamino acids, which are more absorbed from the gastrointestinal tract and additionally exhibit reduced toxicity compared to the inorganic forms. 2.3. Isolation and Determination of the Amount of Proteins. Isolation of proteins from the control and experimental yeast cell cultures was performed using Y-PER formulation pur- chased from Thermo Scientific (USA). Protein concentration was determined by the Lowry method [9]. 2.4. Dialysis and Electrophoretic Separation. The resulting supernatant obtained from the isolation of proteins was subjected to dialysis (dialysis bags, MWCO 12400, 99.99%, Sigma-Aldrich, Germany) to remove all impurities (excess of salts, ions). The process of dialysis was carried out using a magnetic stirrer at 4∘C for 24 hours. Polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) was conducted according to the Laemmli method [10] in 4% stacking gel and 20% separating gel (Mini-PROTEAN TGX Precast Gels, Bio-Rad). To determine the molecular weight, an appropriate molecular weight marker (Precision Plus Protein Dual Xtra Standards, Bio-Rad) was used. To visualize protein bands, the gels were stained with Coomassie Brilliant Blue R-250. Electrophoretically separated proteins were documented with GelDoc 2000 Gel Documentation System (Bio-Rad, France). Change of environmental conditions and occurrence of different stress factors, such as the presence of selenium in the culture medium, may lead to the expression of genes associated with the biosynthesis of new proteins in yeast cells. It is believed the expression of selenoproteins in organisms reaches a maximum value at low amounts of selenium, while, at higher doses, it may cause lipid peroxidation of cell membranes [6]. According to McKenzie et al. [7], the rate of genes transcription responsible for the biosynthesis of selenoproteins is conditioned by other abiotic factors, for example, temperature, pH, hydrogen peroxide, and hydroxyl radicals. The metabolism of selenium compounds in cells is based on a series of transformations leading to a reduction of the degree of oxidation followed by the formation of selenide (H2Se). 3. Results and Discussion In all cultures carried out using the experimental media, with increasing doses of selenium (>20 mg/L), the content of soluble protein slightly decreased. The highest protein content (51.7 mg/g) was found during 24 hours of cultivation in the control medium. During yeast cultivation in medium supplemented with selenium at doses of 20 and 30 mg/L, after 24 hours of cultivation, the protein content was 49.5 and 48.5 mg/g, respectively (Table 1). The obtained results did not differ significantly when comparing the content of soluble proteins in control culture biomass. Moreover, in yeast biomass obtained from the 24-hour cultivation, in the medium with the highest selenium supplementation (40 mg/L), no significant reduction in the protein content compared to the culture from the YPD control medium was observed. The objective of the undertaken study was to evaluate the content of amino acid protein and the protein profile of fodder yeast cells of Candida utilis ATCC 9950 cultivated in control and experimental media enriched with selenium. Oxidative Medicine and Cellular Longevity This is a common intermediate metabolite and, depending on the demand, may be used for the synthesis of selenoproteins or may be converted into methylated forms followed by their elimination from the organism in the same form [8]. Another possibility is the formation of elemental selenium in yeast cell structures [1]. 2.5. Statistical Methods. The obtained results were subjected to statistical analysis using the Statgraphics Plus 4.1. program. Significant differences between mean values were evaluated by Tukey’s test at significance level 𝛼= 0.05. 1. Introduction an extensive research on the role it plays in organisms. Despite the discovery of most of its functions, the role of selenoproteins, however, is still ambiguous [2]. Selenium is one of those elements that determine the proper functioning of an organism by exhibiting antioxidant prop- erties and protecting the organism against free radicals and carcinogenic factors. The primary source of selenium is a properly balanced diet, in which the food products provide the organism’s need for this element. Although the content of selenium occurring in food and fodder is diverse, preparations of selenium yeasts enriched with organic forms of selenium are the most valuable and the safest methods of dietary supplements [1].hi Selenium is involved in the metabolism of hydrogen peroxide and lipid hydroperoxides. It constitutes an integral part of such enzymes as glutathione peroxidase (GPx), thiore- doxin reductase (TR), and deiodinase iodotyrosine, which protect cells from the harmful effects of free radicals formed during oxidation processes. In these processes, selenium plays a role similar to tocopherol (vitamin E) [3]. In addition, selenium exhibits antioxidant and antitumor activity. Selenium is a constituent of approximately 25 known selenoproteins commonly found in human organisms and 12 selenoproteins occurring in yeast cells [4]. They are involved in many metabolic processes at the cellular level. The most important biological role of selenium is associated with its presence in the active centers of many enzymes and proteins. Due to its ability to permanently bind macronutrients and trace elements into its cellular structure, yeast is not only a source of protein, but also a source of deficit microelements. Their ingestion provides a multidirectional and beneficial effect on human health. For many years, studies on the enrichment of yeast cells with selenium have been carried out using the ability of cell biomass to bind this element. Yeast can bind ions of the elements from the environment, permanently incorporating them into their cellular structures. Develop- ment of analytical methods and the studies conducted by Schwarz and Foltz led, in 1957, to including selenium into the list of elements essential for life. This discovery initiated 2 2 2. Materials and Methods (kDa) 250 100 150 75 50 25 37 20 15 22 15 43 18 60 70 M 1 2 3 4 M 5 6 7 8 M 48h 72h Table 1: Changes of protein content (mg/g) isolated from yeast cells C. utilis grown in control (YPD) and experimental media enriched with selenium. Selenium contents in medium (mg/L) Cultivation time (h) 24 48 72 Protein content (mg/g) 0 (YPD) 51,77 ± 2,58d 47,53 ± 1,60abcd 46,77 ± 1,57abc 20 49,52 ± 2,00cd 46,95 ± 2,00abc 46,02 ± 0,93abc 30 48,57 ± 1,91bcd 44,44 ± 1,07ab 43,79 ± 1,22a 40 48,08 ± 0,64abcd 43,73 ± 0,74a 43,51 ± 1,76a Values marked by the same letter are not different at 𝛼= 0,05. Table 1: Changes of protein content (mg/g) isolated from yeast cells C. utilis grown in control (YPD) and experimental media enriched with selenium. Values marked by the same letter are not different at 𝛼= 0,05. 15 24 hours. For the control culture, this value was estimated at approximately 10%.h Figure 1: Electropherogram of proteins derived from C. utilis yeast biomass cultivated for 48 and 72 hours in the control and experimental media enriched in the form of Na2SeO3 (M: marker, 1: 40 mg/L, 2: 30 mg/L, 3: 20 mg/L, 4: YPD, 5: 40 mg/L, 6: 30 mg/L, 7: 20 mg/L, and 8: YPD). The presence of selenium in experimental media (20, 30, and 40 mg/L) and extension of the cultivation time up to 48 and 72 hours resulted in decreased protein content in yeast cell biomass in relation to the medium without any selenium supplementation. These correlations may be explained by the mechanism of the toxic effect of selenium on yeast cells. The exposure of yeast cells to selenium for a greater length of time may lead to morphological changes at the level of the nucleus (chromatin condensation followed by its fragmentation) and the whole cell. Changes were also observed in the structure of cell membranes, resulting in their increased permeability, which in consequence disrupts the functioning of channels and membrane transporters. One of the common causes of cellular protein damage is the formation of carbonyl groups caused by oxidation of amino acid residues under oxidative stress conditions. The process of protein carbonylation based on the reaction of the external amino acids with the aldehyde groups of lipids leads to the loss of cell membrane integrity [11]. 2. Materials and Methods The lower protein content in yeast biomass could also be the result of the detoxification processes of selenium in yeast cells: formation of the elemental form of selenium. These processes which occur in yeast cells could also be responsible for the induction of conformational changes in the polymers of the yeast cell wall [12].t structures, using membrane vesicles, carrying small selenium complexes across the cytoplasmic membrane [13].h The next goal of this study was aimed at a qualitative evaluation of the protein bands isolated from the yeast cell biomass of C. utilis in polyacrylamide gel (Figure 1). After the analysis of electropherograms, a relevant contribution of protein fraction characterized by a molecular weight of about 43 kDa in all protein extracts under study, isolated from C. utilis ATCC 9950 strain, was reported. According to McDermott et al. [14] and C´assio et al. [15], protein of a molecular weight of 43 kDa (Jen1p monocarboxylic transporter) is responsible for the absorption of acids, among others, lactic, acetic, and pyruvic acids. Simultaneously, this protein may be involved in the intracellular transport of selenium [14]. In the electropherogram of proteins derived from the control culture, the presence of nine protein fractions was reported, while, for proteins from the experimental culture (with the addition of 20, 30, and 40 mg/L of selenium), the number of protein fractions was equal to 14. Electrophoretic separation of yeast proteins from the control and experimen- tal cultures did not reveal any presence of a fraction char- acterized by a molecular weight greater than 100 kDa. The results obtained indicate that the number of protein fractions on electropherograms was dependent on the availability of selenium in the culture medium. The resulting differences could probably be observed as a result of biochemical and physiological changes that occurred during the process of growth and aging of yeast cells. y Based on the studies conducted, it was found that after 24 hours of culture (after completion of the logarithmic growth phase), yeast cells began to die. As a result of the above- mentioned processes, decrease in the content of soluble pro- tein after 48 and 72 hours of culture was observed. Probably, this phenomenon was observed as a result of the higher activ- ity of lytic enzymes while decomposing cellular structures after completion of the logarithmic growth phase. Studies conducted by Parrondo et al. 2. Materials and Methods 2.1. Yeast Strain. The strain of Candida utilis ATCC 9950 fodder yeast obtained from the collection of pure cultures of the Department of Biotechnology and Food Microbiology, Warsaw University of Life Science-SGGW, constituted the biological material used in the study. The strain was stored in YPD medium at pH 5.0 and at 4∘C, with fresh medium being provided every 4 weeks. In the following two days of cultivation (i.e., after 48th and 72nd hours), protein content remained at a slightly lower concentration (47.5–43.5 mg/g). Statistical analysis showed that the observed differences in the protein content obtained from a 48-hour of yeast cultivation in media supplemented with selenium at doses of 20, 30, and 40 mg/L were not significant. Similar pattern was found after 72 hours of cultivation for similar doses of selenium. 2.2. Media and Culture Conditions. Liquid YPD medium composed of 20 g/L glucose, 20 g/L peptone, and 10 g/L yeast extract at pH 4.5–5.0 was used as the control medium. The experimental medium consisted of YPD liquid medium additionally enriched with selenium salts (Na2SeO3) in such volumes that the final selenium content was 20, 30, and 40 mg/L. Yeast inoculum was prepared by inoculating liquid YPD medium from the slant. The resulting inoculum consti- tuted an initial material for the inoculation of the control and experimental media, 10% (v/v). Yeast cultures in the control and experimental media were cultivated at 28∘C for 24, 48, and 72 hours on a reciprocating shaker with amplitude of 200 cycles/min. The obtained results showed that longer duration of the cultivation of experimental culture (i.e., up to 72nd hour) resulted in a reduction by about 9% of the amount of soluble protein isolated from a cellular biomass of C. utilis strain tested, compared to the cultivation carried out during Oxidative Medicine and Cellular Longevity 3 (kDa) 250 100 150 75 50 25 37 20 15 22 15 43 18 60 70 M 1 2 3 4 M 5 6 7 8 M 48h 72h Figure 1: Electropherogram of proteins derived from C. utilis yeast biomass cultivated for 48 and 72 hours in the control and experimental media enriched in the form of Na2SeO3 (M: marker, 1: 40 mg/L, 2: 30 mg/L, 3: 20 mg/L, 4: YPD, 5: 40 mg/L, 6: 30 mg/L, 7: 20 mg/L, and 8: YPD). 2. Materials and Methods [13] showed that the longer the time of culture, the greater the decrease in the content of soluble protein in the cellular structures of Kluyveromyces marxianus yeasts. It was associated with progressive processes of autolysis in yeast cells in the stationary growth phase and dying yeast cells.i Literature data [11, 16, 17] indicates that the presence of selenium in the environment regulates the expression of most genes involved in the response of yeast cells to oxidative stress (mainly via Yap1p transcription factor, glutathione reductase, and glutaredoxin), which in consequence could affect the expression of genes involved in protein synthesis. A progressive detoxification process occurring in the yeast could constitute another mechanism, which resulted in a reduction of soluble protein content. This process was based on the removal of protein-selenium complexes from yeast cell 4 Oxidative Medicine and Cellular Longevity it can be concluded that they involved 15, 18, and 22 kDa selenoproteins, respectively [22].f The occurrence of four new fractions on the electro- pherogram of proteins derived from yeast cells cultivated in media supplemented with selenium indicated a significant influence of this element on the biosynthesis of selenium proteins in yeast cells. It is worth noting that the presence of new protein fractions did not affect the increase in the total content of soluble protein in yeast cells cultivated in media supplemented with selenium. Such correlation may result from the small content and the molecular weight of protein fractions obtained as well as from progressive detoxification processes in yeast cells. According to Tastet et al. [23] and Schauml¨offel [24], the protein characterized by molecular weight of 18 kDa is SIP18 (salt induced protein 18). The literature data [25] indicates that biosynthesis of this protein is activated under environmental stress conditions in the experimental culture in the presence of salt (in this case, an excess of selenium IV ions). Extension of the duration of yeast culture up to 72 hours in all media (control and supplemented with selenium) did not affect the number of protein fractions isolated from C. utilis ATCC 9950 yeast biomass. In the studies conducted by Chassaigne et al. [26] on commercial lyophilisates of Saccharomyces cerevisiae selenium yeast containing 1300 𝜇g/g of selenium (Pharma Nord, Vejle, Denmark), the presence of 15 intensive protein fractions was demonstrated. The highest intensity was observed for molecular bands of 30 kDa. 2. Materials and Methods Using ETV-ICP-MS (ICP-MS with electrothermal evaporation), the authors were able to determine the concentration of selenium in the individual protein bands.h Studies presented by Bła˙zejak [18] demonstrated the presence of seven protein fractions isolated from yeast cell biomass of C. utilis cultured in control YPD medium. Differences in the number of protein fractions obtained in the above-mentioned study in the control YPD medium (14 fractions), when compared to the data presented by Bła˙zejak [18], resulted from the additional dialysis of protein extracts isolated from yeast biomass. The use of commercial polyacrylamide gels (20%) was also reflected in the higher amounts of protein fractions that were obtained. The general pictures of the proteins extracted from yeast cell biomass originating from all cultures supplemented with selenium, in principle, did not differ from each other. The only observ- able differences in the number of the fractions obtained could be observed between the proteins isolated from yeast cell cultured in the control (YPD) and in the selenium- supplemented media. The highest content of selenium was found in the protein fractions characterized by molecular weights of 14 kDa (10 ng/cm gel), 25 kDa (approximately 5 ng/cm gel), and 43 kDa (12 ng/cm gel). Above the molecular weight of 100 kDa, no protein fractions were reported. Different results were presented by Bryszewska et al. [27], who demonstrated that high accumulation of selenium in S. cerevisiae yeast cells occurred in protein fractions above 75 kDa. Analysis of electrophoretic separation of cellular proteins obtained after 24 hours of experimental culture revealed the presence of new fractions of molecular weight 22 and 60 kDa, which were not found in proteins from the control culture. According to the literature data [19], the first protein present in yeast cells of similar weight is the complex of GPx enzyme (glutathione peroxidase). GPx, one of the first selenoproteins to be characterized, usually exhibits a tetrameric structure with one selenium atom in each subunit. So far, eight types of GPx have been identified, of which five occur in selenocysteine. In terms of a protein characterized by a mass of 60 kDa, it could be selenoprotein P, which contains up to 10 selenocysteine residues [20]. Increased activity of this enzyme complex in the cells of the C. 2. Materials and Methods utilis strain under study could result from the presence of selenium ions, which in the experimental medium are responsible for the occurrence of stress conditions, among others, oxidative stress [10, 21]. Interesting observations in this regard have been made based on the results of the studies conducted by Zhang et al. [28]. In proteins of Bifidobacterium animalis 01 from the control MRS medium, as well as from medium supplemented with selenium at a dose of 2.5 mg/L, the presence of 16 protein fractions was demonstrated. It was found that selenium was present in all protein fractions isolated. The highest content of this element was observed in bands that corresponded to a molecular weight between 10 and 20 kDa (24 ng/cm gel). The analysis of the results showed that more selenium was involved in interactions with proteins of low molecular weight.h The results of electrophoretic separation of proteins isolated from C. utilis yeast cells cultivated in media sup- plemented with selenium, in relation to those obtained from the control media, exhibited the presence of four new protein fractions characterized by weights of 60, 22, 18, and 15 kDa. It proved the activation of processes associated with metabolism of this element. Several authors [29, 30] suggest that the presence of selenium in the culture medium effected the changes in the antioxidant system of yeast cells, which resulted in an increased protein biosynthesis of selenium- dependent proteins.f In addition, the presence of the protein fraction of 70 kDa was observed in yeast cells from both the control and the experimental media, regardless of the duration of culture time and selenium concentration. According to the literature data [22], it could be TR (thioredoxin reductase) that contains a selenocysteine sequence in its catalytic center. Proteins isolated from C. utilis yeast cells cultivated in media supplemented with selenium (20, 30, and 40 mg/L) contained more protein fractions of molecular weights below 30 kDa when compared to cells from the control YPD medium. It proved the occurrence of transport processes and bioaccumulation of selenium yeast cell structures. Based on the obtained results on molecular weights of proteins, In summary, the observed differences in the protein profile obtained from yeast biomass derived from both the control and the experimental cultures supplemented with selenium (20, 30, and 40 mg/L) indicate the possibility of expression of genes responsible for the synthesis of seleno- proteins, among others, GPx (22 kDa). References [18] S. Bła˙zejak, “Biomass of Candida utilis ATCC 9950 yeast as a source of magnesium bioplexes,” Polish Journal of Food and Nutrition Sciences, vol. 15/56, no. 1, pp. 9–16, 2006. [1] M. Kieliszek and S. Bła˙zejak, “Selenium: significance, and outlook for supplementation,” Nutrition, vol. 29, no. 5, pp. 713– 718, 2013. [19] T. Tamura and T. C. Stadtman, “A new selenoprotein from human lung adenocarcinoma cells: purification, properties, and thioredoxin reductase activity,” Proceedings of the National Academy of Sciences of the United States of America, vol. 93, no. 3, pp. 1006–1011, 1996. [2] J. L. Barger, T. Kayo, T. D. Pugh et al., “Gene expression profiling reveals differential effects of sodium selenite, selenomethionine, and yeast-derived selenium in the mouse,” Genes and Nutrition, vol. 7, no. 2, pp. 155–165, 2012. [20] L. V. Papp, J. Lu, A. Holmgren, and K. K. Khanna, “From selenium to selenoproteins: synthesis, identity, and their role in human health,” Antioxidants and Redox Signaling, vol. 9, no. 7, pp. 775–806, 2007. [3] M. Kieliszek, S. Bła˙zejak, and R. Jędrzejczak, “Wiązanie selenu przez dro˙zd˙ze paszowe Candida utilis ATCC 9950,” Bromatolo- gia i Chemia Toksykologiczna, vol. 45, no. 3, pp. 628–633, 2012. [4] S. J. Fairweather-Tait, R. Collings, and R. Hurst, “Selenium bioavailability: current knowledge and future research require- ments,” American Journal of Clinical Nutrition, vol. 91, no. 5, pp. 1484S–1491S, 2010. [21] U. K. Laemmli, “Cleavage of structural proteins during the assembly of the head of bacteriophage T4,” Nature, vol. 227, no. 5259, pp. 680–685, 1970. [22] J. Gromadzi´nska, B. A. Zachara, and W. Wąsowicz, “Seleno- białka,” in Selen—Pierwiastek Wa˙zny dla Zdrowia, Fascynujący dla Badacza, M. Wierzbicka, E. Bulska, A. Pyrzy´nska, I. Wysocka, and B. A. Zachara, Eds., pp. 172–196, Wydawnictwo Malamut, Warsaw, Poland, 2007. [5] L. Flohe, W. A. Gunzler, and H. H. Schock, “Glutathione perox- idase: a selenoenzyme,” FEBS Letters, vol. 32, no. 1, pp. 132–134, 1973. [6] N. Dilsiz, S. C¸elik, ¨O. Yilmazo, and M. Digrak, “The effects of selenium, vitamin E and their combination on the composition of fatty acids and proteins in Saccharomyces cerevisiae,” Cell Biochemistry and Function, vol. 15, no. 4, pp. 265–259, 1997. [23] L. Tastet, D. Schauml¨offel, B. Bouyssiere, and R. Lobinski, “Iden- tification of selenium-containing proteins in selenium-rich yeast aqueous extract by 2D gel electrophoresis, nanoHPLC- ICP MS and nanoHPLC-ESI MS/MS,” Talanta, vol. 75, no. 4, pp. 1140–1145, 2008. [7] R. C. McKenzie, J. R. Arthur, and G. J. 2. Materials and Methods The contribution of selenium to the metabolism of C. utilis ATCC 9950 yeasts 5 Oxidative Medicine and Cellular Longevity is associated with protection against oxidative stress in cells. The presence of selenium in the culture medium induces the activity of many enzymes associated with intracellular transport of selenium; however, these processes did not affect the increase in the content of soluble protein in yeast cells. The analysis of electrophoretic separations demonstrated qualitative differences between the protein bands obtained from yeast cells cultivated in the control and the experimental media. In protein fractions of yeasts obtained from media supplemented with selenium (20–40 mg/L), the presence of new protein fractions characterized by molecular weights of 60, 22, 18, and 15 kDa was revealed. The presence of selenium in the medium affected the occurrence of oxidative stress, which in turn induced the expression of genes responsible for the biosynthesis of selenoproteins in yeast cells. [12] M. Kieliszek, S. Bła˙zejak, I. Gientka, and A. Bzducha-Wr´obel, “Accumulation and metabolism of selenium by yeast cells,” Applied Microbiology and Biotechnology, 2015. [13] J. Parrondo, L. A. Garc´ıa, and M. D´ıaz, “Nutrient balance and metabolic analysis in a kluyveromyces marxianus fermentation with lactose-added whey,” Brazilian Journal of Chemical Engi- neering, vol. 26, no. 3, pp. 445–456, 2009.fi [14] J. R. McDermott, B. P. Rosen, and Z. Liu, “Jen1p: a high affinity selenite transporter in yeast,” Molecular Biology of the Cell, vol. 21, no. 22, pp. 3934–3941, 2010. [15] F. C´assio, C. Le˜ao, and F. Baltazar, “Plasma membrane transport of monocarboxylates in yeasts: overview and emerging issues,” in Mycology in the New Millennium—Prospects and Outlook, K. R. Sridhar, Ed., pp. 248–263, IK International Publishing House Pvt. Ltd., New Delhi, India, 2009. [16] G. Peyroche, C. Saveanu, M. Dauplais et al., “Sodium selenide toxicity is mediated by O2-dependent DNA breaks,” PLoS ONE, vol. 7, no. 5, Article ID e36343, 2012. The authors declare that they have no conflict of interests. The authors declare that they have no conflict of interests. Conflict of Interests [17] K. Schwarz and C. M. Foltz, “Selenium as an integral part of factor 3 against dietary necrotic liver degeneration,” Journal of the American Chemical Society, vol. 79, no. 12, pp. 3292–3293, 1957. References Beckett, “Selenium and the regulation of cell signaling, growth, and survival: molecular and mechanistic aspects,” Antioxidants and Redox Signaling, vol. 4, no. 2, pp. 339–351, 2002. [24] D. Schauml¨offel, “New ways in qualitative and quantitative protein analysis: nano chromatography coupled to element mass spectrometry,” Journal of Trace Elements in Medicine and Biology, vol. 21, no. 1, pp. 18–22, 2007. [8] S. J. Fairweather-Tait, J. Y. Bao, M. R. Broadley et al., “Selenium in human health and disease,” Antioxidants and Redox Signaling, vol. 14, no. 7, pp. 1337–1383, 2011. [25] V. J. Miralles and R. Serrano, “A genomic locus in Saccharomyces cerevisiae with four genes up-regulated by osmotic stress,” Molecular Microbiology, vol. 17, no. 4, pp. 653–662, 1995. [9] O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, “Protein measurements with the Folin phenol reagent,” The Journal of Biological Chemistry, vol. 193, no. 1, pp. 267–275, 1951. [26] H. Chassaigne, C. C. Ch´ery, G. Bordin, and A. R. Rodriguez, “Development of new analytical methods for selenium speci- ation in selenium-enriched yeast material,” Journal of Chro- matography A, vol. 976, no. 1-2, pp. 409–422, 2002. [10] K. Rajashree and T. Muthukumar, “Preparation of selenium tolerant yeast Saccharomyces cerevisiae,” Journal of Microbiology and Biotechnology Research, vol. 3, no. 3, pp. 46–53, 2013. [27] M. Bryszewska, B. Pęczkowska, J. Rudzi´nski, A. Diowksz, and W. Ambroziak, “Biologiczne ´zr´odła organicznych form selenu,” in XXXVI Zjazd Polskiego Towarzystwa Biochemicznego, P11-03, p. 289, Pozna´n, Poland, 2000. [11] A. Izquierdo, C. Casas, and E. Herrero, “Selenite-induced cell death in Saccharomyces cerevisiae: protective role of glutaredox- ins,” Microbiology, vol. 156, no. 9, pp. 2608–2620, 2010. Oxidative Medicine and Cellular Longevity 6 [28] B. Zhang, K. Zhou, J. Zhang et al., “Accumulation and species distribution of selenium in Se-enriched bacterial cells of the Bifidobacterium animalis 01,” Food Chemistry, vol. 115, no. 2, pp. 727–734, 2009. [29] V. Mapelli, P. R. Hillestrøm, K. Patil, E. H. Larsen, and L. Olsson, “The interplay between sulphur and selenium metabolism influences the intracellular redox balance in Saccharomyces cerevisiae,” FEMS Yeast Research, vol. 12, no. 1, pp. 20–32, 2012. [30] M. P. Bansal and T. Kaur, “Growth characteristics and selenium status changes of yeast cells with inorganic and organic sele- nium supplementation: selenium, a chemopreventive agent,” Journal of Medicinal Food, vol. 5, no. 2, pp. 85–90, 2002.
https://openalex.org/W2017936124	https://www.scielo.br/j/rbeaa/a/dqNgZ8MrZFmpk3RVGxLcZRL/?lang=pt&format=pdf	Portuguese	null	Fatores meteorológicos sobre a atividade reprodutiva de cabras leiteiras na época seca do semiárido pernambucano	Revista Brasileira de Engenharia Agrícola e Ambiental	2,014	cc-by	5,525	R E S U M O Palavras-chave: caprinos estresse calórico involução uterina retorno ao estro Objetivou-se, neste trabalho, avaliar o efeito do estresse calórico sobre a atividade reprodutiva de cabras SPRD de aptidão leiteira, paridas no período seco no semiárido pernambucano. Foram utilizados 24 animais divididos em 4 tratamentos: grupo controle, formado por cabras que tiveram acesso apenas ao pasto e à palma in natura e 3 grupos com suplementação feita por concentrados isoproteicos (20% proteína bruta (PB), variando o teor de nutrientes digestíveis totais (NDT) em 65, 75 e 85%. Os dados foram expressos em média ± desvio padrão. Os fatores meteorológicos foram observados pelo índice de temperatura de globo negro e umidade (ITGU) e os fisiológicos: frequências cardíaca (FC) e respiratória (FR); temperaturas cutânea (TC) e retal (TR) analisados em esquema fatorial. Foram encontradas diferenças estatísticas nas frequências respiratória e cardíaca que, por sua vez, mostraram uma interação entre os meses e momentos de observação. A involução uterina não apresentou diferença entre os tratamentos (p > 0,05). O retorno ao estro tendeu a ser mais precoce nos grupos 85 e 75%. Em conclusão, o estresse calórico não afetou a atividade reprodutiva de cabras SPRD de aptidão leiteira, paridas na época seca e manejadas de forma semi-intensiva, no semiárido pernambucano. Fatores meteorológicos sobre a atividade reprodutiva de cabras leiteiras na época seca do semiárido pernambucano1 Thiago V. C. Nascimento2, Fabiano A. de Oliveira3, Sílvia H. N. Turco4, Mabel F Cordeiro5 & Edilson S Lopes Júnior6 1 Parte da Dissertação de Mestrado do primeiro autor apresentado para o Programa de Pós-Gradução em Ciência Animal da UNIVASF, financiada pela FACEPE 2 UFBA. Salvador, BA. E-mail: thiagovcn_vet@hotmail.com (Autor correspondente) 3 UFBA. Salvador, BA. E-mail: agronomoca@gmail.com 4 CENAMB/UNIVASF. Juazeiro, BA. E-mail: silvia.turco@univasf.edu.br 5 LAFIBRA/UNIVASF. Petrolina, PE. E-mail: mabel.cordeiro@univasf.edu.br 6 LAFIBRA/UNIVASF. Petrolina, PE. E-mail: edilson.lopes@univasf.edu.br Metereological factors on reproductive activity of goats in dry season of semi-arid region of Pernambuco Key words: goats heat stress uterine involution estrus return A B S T R A C Th The heat stress conditions with reference to meteorological and individual factors were investigated, and the influence of this condition on the reproductive activity of undefined breed of goats (SPRD), calved during the dry period. In all, 24 animals were used, divided into four treatments: control group, consisting of goats that had access only to the pasture and palm in nature and 3 groups through supplementation with concentrated isoproteic 20% crude protein (CP), varying the total digestible nutrients (TDN) content of 65, 75 and 85%. Data were expressed as mean ± standard deviation. The meteorological factors were observed by index of temperature of black globe and humidity (ITGU) and the physiological through respiratory (RF) and cardiac frequencies (CF); cutaneous (CT) and rectal temperatures (RT) and analysed in factorial scheme. Statistical differences were found in the respiratory and cardiac frequencies, showed an interaction between the month and times of observation. The uterine involution, did not show differences between treatments (p > 0.05). The return to estrus tended to be faster in groups 85 and 75% than in other groups. There was no negative influence of mild heat stress, indicated by climatic factors (ITGU) and the individual factors on the reproductive activity of goats SPRD. No Semiárido brasileiro é comum atribuir, à diminuição da atividade reprodutiva de caprinos e ovinos, apenas a pouca disponibilidade de alimentos durante o período seco (Torreão et al., 2008). Nogueira et al. (2011) relataram que durante a época seca no semiárido pernambucano, cabras da raça Canindé e sem padrão racial definido (SPRD) e mesmo recebendo suplementação, foram susceptíveis à influência da temperatura e umidade do ar não apresentando comportamento de estro nos meses de setembro e outubro. Utilizam-se, para observar como Revista Brasileira de Engenharia Agrícola e Ambiental Campina Grande, PB, UAEA/UFCG – http://www.agriambi.com.br Protocolo 164.13 – 22/05/2013 • Aprovado em 29/11/2013 ISSN 1807-1929 v.18, n.5, p.539–544, 2014 Revista Brasileira de Engenharia Agrícola e Ambiental Campina Grande, PB, UAEA/UFCG – http://www.agriambi.com.br Protocolo 164.13 – 22/05/2013 • Aprovado em 29/11/2013 ISSN 1807-1929 v.18, n.5, p.539–544, 2014 Introdução O Semiárido nordestino abrange uma área de aproximadamente 95 milhões de hectares, os quais representam cerca de 70% do território desta região. Climaticamente, o Semiárido se caracteriza por possuir clima quente e seco, com duas estações, a seca e a úmida, com pluviosidade de aproximadamente 300-800 mm. A maior parte das chuvas se concentra em três a quatro meses dentro da estação úmida (Araújo Filho, 1995). Thiago V. C. Nascimento et al. Thiago V. C. Nascimento et al. 540 os fatores meteorológicos afetam os animais, índices de conforto térmico animal em que um dos mais utilizados é o índice de temperatura de globo negro e umidade (ITGU) que também leva em consideração a irradiação solar; Morais et al. (2008) afirmam que elevados valores de ITGU promovem desconforto térmico nos animais. grupos de seis animais cada um. O grupo controle era formado por cabras que tiveram acesso apenas aos pastos cultivados de capim Tifton 85 (Cynodon spp.), das 8 às 15 h e após retornarem do pasto receberam, em instalações cobertas, uma média de 4 kg animal-1 de palma (Opuntia ficus-indica Mill.) in natura, triturada, além de água e suplementação mineral ad libitum; os animais dos outros três grupos foram submetidos ao mesmo manejo do grupo controle, além de terem recebido 400 g de um concentrado isoproteico contendo 20% de proteína bruta (PB) e variando o NDT (nutrientes digestíveis totais) de acordo com o tratamento: Grupo 65% recebeu concentrado contendo 65% NDT, atendendo à necessidade energética mínima das cabras em puerpério, conforme preconizado pelo NRC (2007); Grupo 75% recebeu concentrado contendo 75% NDT e o grupo 85% recebeu um concentrado formulado com 85% NDT. Com relação ao seu manejo, as crias permaneceram em período integral com as mães até o 15° dia pós-parto; a partir do 16° dias elas foram separadas das cabras, durante a noite, até completarem 30 dias após o parto quando foram completamente separadas das mães. Garcia-Ispierto et al. (2006) também observaram que os fatores climáticos relacionados ao desconforto calórico podem interferir na reprodução já que o estresse promovido pelo calor aumenta a liberação de cortisol que, por sua vez, diminui a síntese de hormônios gonadotróficos. em que: Tgn - temperatura de globo negro Tpo - temperatura de ponto de orvalho Por outro lado foram coletados, em todos os animais, os seguintes dados fisiológicos: frequências respiratória (FR) e cardíaca (FC), além das temperaturas superficial cutânea (TC) e retal (TR). Os dados fisiológicos também foram obtidos duas vezes por semana, pela manhã (8 h) e à tarde (15 h). Para determinação da FR foi quantificado o número de respirações por minuto aferido através da auscultação nas primeiras costelas, do lado direito utilizando-se, para tal, um estetoscópio, durante 15 s, cujo valor obtido foi multiplicado por quatro obtendo-se também a frequência por minuto. A FC foi aferida da mesma forma, porém se auscultou as costelas do lado esquerdo; já na TR utilizou-se um termômetro clínico digital introduzido-o, a uma distância de cinco cm, no reto de cada animal, por dois minutos enquanto para a TC foi utilizado um termômetro de infravermelho, a 10 cm de distância da pele, em quatro locais distintos (fronte, dorso e flancos), com os quais se calculou a TC média. Desta forma, o trabalho tem por objetivo avaliar o efeito do estresse calórico sobre a atividade reprodutiva de cabras SPRD de aptidão leiteira, paridas no período seco, no semiárido pernambucano. R. Bras. Eng. Agríc. Ambiental, v.18, n.5, p.539–544, 2014. Introdução Após o parto as fêmeas tendem, também, a ter redução da síntese e liberação de hormônios gonadotróficos gerando um período de ausência de comportamento de estros e ovulações, ou seja, entrando em anestro puerperal durante o qual a fêmea se encontra em uma situação de balanço energético negativo, quando há mobilização das reservas corporais (Costa et al., 2007; Mahdi & Khallili, 2008). Este anestro é o período de recuperação do trato reprodutivo da fêmea para que ela possa abrigar uma nova gestação (Salmazo et al., 2008). Quando esta parição ocorre no período seco pode-se somar, a este anestro fisiológico, o anestro nutricional ocasionado pela nutrição inadequada durante o puerpério tal como o anestro por estresse térmico. Devido a isto o retorno ao estro de fêmeas paridas no período seco é mais tardio quando comparado com o das fêmeas paridas no período chuvoso (Eloy et al., 2003; Torreão et al., 2008). Os dados meteorológicos coletados foram: temperaturas de bulbo seco, bulbo úmido e de globo negro; umidade relativa do ar e a velocidade do vento, registrados na frequência de duas vezes por semana, durante todo o experimento, nos horários das 8 h (antes dos animais serem conduzidos ao pasto) e às 15 h (quando os animais eram novamente estabulados). Através desses dados foi calculado o Índice de Temperatura de globo negro e umidade (ITGU), de acordo com a equação desenvolvida por Buffington et al. (1981): Na tentativa de amenizar o desconforto térmico promovido pelo estresse calórico, o corpo dos animais tenta dissipar, através de alterações fisiológicas e comportamentais, o calor com elevação das frequências respiratória e cardíaca, modificações no comportamento geral do animal, como a redução de movimentos ruminais e menor liberação de hormônios metabólicos (Gomes et al., 2008; Morais et al., 2008; Pereira et al., 2011; Roberto & Souza, 2011); entretanto, a literatura ainda apresenta limitação nos estudos quanto à influência do estresse térmico sobre os parâmetros reprodutivos durante o puerpério, de cabras mestiças com aptidão leiteira. ITGU Tgn 0,36 Tpo 41,5 = + × + (1) (1) em que: Material e Métodos O experimento foi realizado na Fazenda Umburana localizada em Santa Maria da Boa Vista, Pernambuco. O município está localizado a 8° 48’ de latitude Sul, 39º 49’ de longitude Oeste, a uma altitude de 447 m e com temperatura média anual de 25,5 ºC, com clima que se enquadra no tipo BSh da classificação de Köppen, definido como semiárido (chuva anual inferior a 750 mm), sem excesso hídrico. A estação chuvosa ocorre no período de novembro a abril e a estação seca ocorre no período de maio a outubro. O período experimental foi de julho a outubro de 2011, ou seja, durante o período seco do ano (Miolo, 2003). Foram utilizadas 24 fêmeas caprinas SPRD de aptidão leiteira recém-paridas homogeneamente e distribuídas em quatro A atividade reprodutiva foi avaliada através da involução uterina, do retorno da atividade ovariana puerperal, observada através de exame ultrassonográfico e do retorno ao estro, através da avaliação de comportamento estral, ou seja, da observação de R. Bras. Eng. Agríc. Ambiental, v.18, n.5, p.539–544, 2014. 541 Tabela 1. Indice de temperatura, de umidade e de globo negro (ITGU), ao longo dos meses, de acordo com o horário de observação Horário de observação ITGU Meses Julho Agosto Setembro Outubro 08 h 70,00 cB 77,24 bcB 78,49 abB 83,48 aB 15 h 86,90 bA 95,80 aAa 95,66 aAa 96,11 aA Letras minúsculas diferentes na mesma linha, bem como letras maiúsculas diferentes na mesma coluna, indicam diferença significativa (P < 0,01) Tabela 1. Indice de temperatura, de umidade e de globo negro (ITGU), ao longo dos meses, de acordo com o horário de observação aceitação da monta. A avaliação da involução uterina foi iniciada a partir de 48 h após o parto, a cada três dias, até a completa involução uterina. Com relação à observação do retorno da atividade ovariana folicular, as imagens ultrassonográficas dos ovários foram obtidas de todos os animais visando acompanhar a dinâmica folicular no pós-parto até a primeira ovulação, a cada sete dias (Eloy et al., 2003). O dia do retorno da atividade ovariana puerperal foi marcado com a emergência da primeira onda folicular no dia em que foram observados um ou mais folículos maior(es) que 2 mm de diâmetro, sendo o diâmetro folicular obtido pela maior distância (mm) entre dois pontos da cavidade antral do folículo (Uribe-Velásquez et al., 2010). Material e Métodos O diâmetro, posição e características dos folículos antrais, com no mínimo 2 mm de diâmetro, foram registrados como descrito por Castro et al. (1999). Horário de observação ITGU Meses Julho Agosto Setembro Outubro 08 h 70,00 cB 77,24 bcB 78,49 abB 83,48 aB 15 h 86,90 bA 95,80 aAa 95,66 aAa 96,11 aA Letras minúsculas diferentes na mesma linha, bem como letras maiúsculas diferentes na mesma coluna, indicam diferença significativa (P < 0,01) Letras minúsculas diferentes na mesma linha, bem como letras maiúsculas diferentes na mesma coluna, indicam diferença significativa (P < 0,01) Letras minúsculas diferentes na mesma linha, bem como letras maiúsculas diferentes na mesma coluna, indicam diferença significativa (P < 0,01) variações deste índice durante o período experimental observando-se que os valores de ITGU do horário das 8 h eram inferiores aos do horário das 15 h (p < 0,05). O ITGU leva em consideração a irradiação solar; trata-se de um achado normal, isto é, que ITGU seja, à tarde, superior ao ITGU pela manhã. Morais et al. (2008) mostraram que os valores de ITGU, acima de 85,00, promovem desconforto térmico nos animais acarretando, inicialmente, um aumento na FR e FC. Pinho et al. (2010) afirmaram que os caprinos são mais tolerantes ao calor que outras espécies de ruminantes domésticos; todavia, Uribe-Velasquéz et al. (2001) ressaltaram que a tolerância ao calor pode variar com outros fatores, como a raça e a produção de leite. Esses autores constataram, ainda, que os animais mestiços tendem a ser mais adaptados devido à sua rusticidade e, como consequência, à maior adaptabilidade às condições adversas, embora possuam uma produção menor de leite, quando comparados com animais de linhagens mais puras e produtivas. O comportamento estral das cabras foi observado por meio do uso de um reprodutor caprino, uma vez ao dia, a partir do 20° dia após o parto até a manifestação do estro, o qual foi admitido quando a fêmea permitiu a monta do macho (Eloy et al., 2003). O delineamento experimental foi inteiramente casualizado, com quatro tratamentos e seis repetições por tratamento. Os dados foram expressos em média ± desvio padrão. Material e Métodos Os parâmetros meteorológicos e fisiológicos foram analisados em um esquema fatorial sendo: um fatorial duplo (2 x 4) para os parâmetros meteorológicos, ou seja, dois períodos do dia (8 e 15 h), quatro meses do ano (julho, agosto, setembro e outubro) e para os parâmetros fisiológicos utilizou-se um esquema de fatorial triplo, 4 x 2 x 4, com 6 repetições levando-se em consideração os quatro tratamentos (85; 75; 65% e controle), dois períodos do dia (8 h e 15 h), quatro meses do ano (julho, agosto, setembro e outubro). Visando à comparação dos diversos parâmetros entre os grupos estudados previamente, testou-se a normalidade dos dados, após o que se utilizou a Análise de Variância e a homogeneidade foi testada pelo teste de Bartlett. As médias foram comparadas pelo Teste de Tukey e todas as análises foram realizadas por meio do programa Assistat v.7.6 Beta (Silva & Azevedo, 2011). Os valores foram considerados estatisticamente significativos quando apresentaram nível de significância menor que 0,05 de probabilidade (p < 0,05). Roberto & Souza (2011) destacaram que os valores de ITGU acima de 85,00 em alguns trabalhos com raças caprinas (Roberto et al., 2010; Silva et al., 2010) mesmo considerados altos, não foram capazes de promover o estresse de forma acentuada concordando com os dados deste trabalho. Silva et al. (2010) encontraram valores de ITGU variando entre 80,11 e 92,17 e afirmaram que caprinos adaptados toleram este alto ITGU sem que tais índices representem uma situação perigosa de desconforto térmico. Os resultados encontrados revelam que a frequência respiratória e cardíaca foi influenciada pelos horários de observação e pelos meses, conforme descrito na Tabela 2. As FR encontradas em todos os meses e em ambos os períodos foram superiores aos valores considerados fisiológicos, de 20 a 30 movimentos respiratórios por minuto (mpm) (Gonçalves, 2008). Os dados de FR obtidos foram inferiores tanto no período da manhã quanto no da tarde, aos encontrados por Pereira et al. (2011) que observaram, em cabras Saanen, uma FR variando de 77 mpm a 111 mpm. Durante o mês de R. Bras. Eng. Agríc. Ambiental, v.18, n.5, p.539–544, 2014. Letras minúsculas diferentes na mesma linha, bem como letras maiúsculas diferentes na mesma coluna, indicam diferença significativa (p < 0,01) Resultados e Discussão Na Tabela 1 se encontram os dados relativos ao desconforto térmico dos animais expressos através do índice de temperatura, de umidade e de globo negro (ITGU); foram observadas Letras minúsculas diferentes na mesma linha, bem como letras maiúsculas diferentes na mesma coluna, indicam diferença significativa (p < 0,01) Tabela 2. Médias das frequências respiratória (FR) e cardíaca (FC), de acordo com os meses e os horários de observação e com os tratamentos Parâmetro Horário de observação Meses Tratamentos Julho Agosto Setembro Outubro 85% 75% 65% Controle FR* 8 034,17 aa 030,00 Baa 029,76 Baa 033,21 Baa 030,94 035,92 29,44 028,57 15 035,44 ba 045,85 aAa 050,10 aAa 049,62 aAa 044,06 051,91 41,55 043,82 FC 8 091,50 aB 085,97 abB 084,10 bBa 085,98 abB 086,66 091,56 84,21 082,59 15 102,14 bA 101,39 bAa 103,95 abA 108,27 aAa 102,93 110,30 99,59 103,08 uências respiratória (FR) e cardíaca (FC), de acordo com os meses e os horários de observação R. Bras. Eng. Agríc. Ambiental, v.18, n.5, p.539–544, 2014. Thiago V. C. Nascimento et al. 542 Figura 1. Temperatura retal de acordo com os tratamentos, os meses e os horários de observação (8 e 15 h) julho foi observada uma FR menor durante o período da tarde quando comparado com os outros meses (P < 0,01) e, quando comparadas com as FR da manhã e da tarde neste mesmo mês, não foram encontradas diferenças significativas (P > 0,05). Isto pode ter ocorrido devido à tentativa dos animais em aumentar a produção de calor por meio de uma movimentação maior, a qual também proporciona aumento da FR a fim de reduzir a sensação de frio já que, na manhã de julho, foram encontradas as menores temperaturas tanto pela manhã quanto à tarde (Tabela 1). No horário das 15 h foi observado menor valor de FR no mês de julho quando comparado com os outros meses (P < 0,01). Quando comparados os valores de FR no horário das 8 h vs. das 15 h neste mês, não foram encontradas diferenças significativas (P > 0,05). Figura 1. Temperatura retal de acordo com os tratamentos, os meses e os horários de observação (8 e 15 h) i A FC do mês de outubro no horário das 15 h foi superior à dos meses de julho e agosto (P < 0,01). Resultados e Discussão Este fato, assim como o fato, dos valores de FR e FC terem sido superiores, às 15 h quando comparados ao horário das 8 h, na maioria dos meses, podem ser justificados devido ao mecanismo de termorregulação do animal que promove o aumento dessas frequências de forma a favorecer a perda de calor reduzindo o desconforto da elevação de temperatura do horário das 15 h estando de acordo com os dados encontrados por Pinho et al. (2010) e por Gomes et al. (2008). Não foram verificadas diferenças estatísticas (P > 0,05) das interações dos tratamentos com os horários de observação, em ambos os parâmetros avaliados. que consumiam este concentrado. Para todos os tratamentos o horário das 15 h apresentou maiores valores de TR que o horário das 8 h (P < 0,01). Ainda foi observada uma interação entre os horários de observação e os meses; no mês de julho o horário das 15 h apresentou maiores valores de TR que nos meses de agosto e setembro (P < 0,05). Gomes et al. (2008) afirmaram que animais que recebem maior nível de suplementação tendem a apresentar também maior TR, corroborando com os dados encontrados neste trabalho. Os autores afirmaram que o aumento de TR no horário da tarde reflete um acúmulo de calor no corpo dos animais. Os maiores valores de ITGU no horário das 15 h também favorecem este acúmulo de calor ao longo do dia. Roberto et al. (2010) relataram que a TR mais alta no período da tarde é uma estratégia evolutiva que tem por princípio a dissipação do calor interno já que a TR maior que a temperatura média ambiente faz com que o corpo troque calor com o ambiente de menor temperatura. A TR, durante todo o período experimental, se manteve dentro da temperatura fisiológica da espécie caprina, que varia de 38,5 a 40,0 °C (Gonçalves, 2008). A Tabela 3 mostra os valores médios de temperatura cutânea (TC), em relação aos meses e o horário de observação, assim como entre os grupos de tratamento e o horário de observação, sendo observadas diferenças estatísticas nessas interações (P < 0,01). Durante o mês de outubro a TC foi maior (P < 0,01) em ambos os horários de observação que os demais meses. Resultados e Discussão No horário das 15 h os valores de TC foram superiores aos das 8 h durante todos os meses, o que se deve aos maiores valores de ITGU neste horário. Na Tabela 4 é resumida a atividade reprodutiva das cabras durante o período experimental, em que os meses de julho e agosto foi onde ocorreram as parições, agosto e setembro, os meses de ocorrência da involução uterina e do surgimento do 1° folículo ≥ 2 mm e, finalmente, setembro e outubro os meses de retorno do estro. A Figura 1 mostra a distribuição das temperaturas retais (TR) durante os meses e horários de observação. Houve diferença estatística entre os horários de observação (P < 0,01), sendo que o horário das 15 h apresentou maiores valores de TR em relação ao horário das 8 h. Foi observada interação entre as dietas e o horário durante o horário das 8 h, em que o concentrado com 85% de NDT teve maior valor de TR que os tratamentos 65% e controle (P < 0,05), porém durante o horário da tarde não houve diferença entre os tratamentos o que pode estar associado à maior geração de calor proporcionado pelo alto índice de energia no concentrado com 85% de NDT gerando, assim, maior irradiação de calor por parte dos animais O período de involução uterina encontrado corrobora com os citados por Simplício et al. (2000) ao observarem que as cabras têm sua involução uterina completa, de 35 a 45 dias. Da mesma forma Salmito-Vanderley & Marques Júnior (2004) trabalharam com cabras sem padrão racial definido (SPRD) e afirmaram que a involução uterina foi concluída por volta dos 30 dias pós-parto. A Tabela 4 aponta, ainda, tendência de Letras minúsculas diferentes na mesma linha, bem como letras maiúsculas diferentes na mesma coluna, indicam diferença significativa (p < 0,01) Tabela 3. Literatura Citada superioridade de precocidade de involução uterina para os grupos com suplementação com maior teor de energia (Grupos 85 e 75%). Mesmo os animais suplementados com os maiores teores de energia apresentando TC e TR maiores nos meses correspondentes à involução uterina, esses grupos tiveram sua involução ocorrendo numericamente mais cedo que os outros grupos, o que mostra que as temperaturas cutânea e retal, mais elevadas, não tiveram efeito deletério. A mesma observação pode ser feita acerca do retorno da atividade ovariana puerperal, marcada pelo surgimento do 1° folículo. Araújo Filho, J. A. Manipulação da vegetação lenhosa da caatinga para fins pastoris. Sobral: Embrapa CNPC, 1995. 18p. Circular Técnica, n.11fi Buffington, D. E.; Collazo-Arocho, A.; Canton, G. H. Black globe-humidity index (BGHI) as comfort equation for dairy cows. Transaction of the ASAE, v.24, p.711-714, 1981. Castro, T. De; Rubianes, E.; Menchaca, A.; Rivero, A. Ovarian dynamics, serum estradiol and progesterone concentrations during the interovulatory interval in goats. Theriogenology, v.52, p.399-411, 1999. Todas as cabras do experimento apresentaram estro até o 105º dia cujos resultados diferem daqueles relatados por Freitas et al. (2004), que também acompanharam cabras leiteiras com parição na época seca do ano e relataram que até o 200° dia pós-parto 81,36% das cabras havia apresentado estro; entretanto, a maior precocidade dos dados deste experimento em comparação com os de Freitas et al. (2004), pode ser devida à diferença racial visto que se tratava de cabras leiteiras de raça pura enquanto os animais deste trabalho eram mestiços e, portanto, mais adaptados e com menor produção de leite. A maior adaptabilidade dos animais do presente estudo acarretou- lhes menor sofrimento nas condições adversas do período seco. Já a baixa produção de leite decorre dos baixos teores plasmáticos de prolactina desses animais quando comparados àqueles de raças especializadas para a produção leiteira. Gaafar et al. (2005) relataram haver uma relação inversa entre a concentração de prolactina e a aparição de estro, o que explica, então, a maior precocidade do retorno ao estro pós-parto de animais de raças não especializadas para a produção leiteira. Costa, R. L. D. da; Cunha, E. A. da; Fontes, R. da S.; Quirino, C. R.; Santos, L. E. dos; Bueno, M. S.; Otero, W. G.; Veríssimo, C. J. Desempenho reprodutivo de ovelhas Santa Inês submetidas à amamentação contínua ou controlada. Boletim de Indústria Animal, v.64, p.51-59, 2007. Eloy, A. M. X.; Andrioli, A.; Simplício, A. A. Resultados e Discussão Médias da temperatura cutânea (TC), em °C, de acordo com a interação dos meses com os horários de observação e dos tratamentos com o horário de observação Horário de observação Meses Tratamentos Julho Agosto Setembro Outubro 85% 75% 65% Controle 08 33,05 cB 34,00 bcB 35,36 bB 40,78 aA 37,01 Aa 35,75 abB 35,96 abB 34,47 bB 15 34,93 cA 38,96 bAa 38,90 bA 41,24 aA 38,21 Aa 38,02 Aaa 37,95 Aaa 39,86 Aa Letras minúsculas diferentes na mesma linha, bem como letras maiúsculas diferentes na mesma coluna, indicam diferença significativa (p < 0,01) ç ç Horário de observação Meses Tratamentos Julho Agosto Setembro Outubro 85% 75% 65% Controle 08 33,05 cB 34,00 bcB 35,36 bB 40,78 aA 37,01 Aa 35,75 abB 35,96 abB 34,47 bB 15 34,93 cA 38,96 bAa 38,90 bA 41,24 aA 38,21 Aa 38,02 Aaa 37,95 Aaa 39,86 Aa R. Bras. Eng. Agríc. Ambiental, v.18, n.5, p.539–544, 2014. 543 Tabela 4. Períodos médios (± desvio padrão), em dias, para a ocorrência da involução uterina para o surgimento do 1° folículo ≥ 2 mm e para o momento Tabela 4. Períodos médios (± desvio padrão), em dias, para a ocorrência da involução uterina para o surgimento do 1° folículo ≥ 2 mm e para o momento Os dados apontam que, mesmo com os valores de ITGU considerados elevados, eles não foram suficientes para promover estresse térmico acentuado nos animais, a ponto de interferir negativamente na atividade reprodutiva. Os dados apontam que, mesmo com os valores de ITGU considerados elevados, eles não foram suficientes para promover estresse térmico acentuado nos animais, a ponto de interferir negativamente na atividade reprodutiva. de retorno ao estro em cabras leiteiras de retorno ao estro em cabras leiteiras Tratamento Involução uterina Surgimento 1° folículo ≥2 mm Tempo de retorno ao estro Grupo 85% NDT 32,00 ± 3,40 32,33 ± 7,03 70,00 ± 09,63 Grupo 75% NDT 29,50 ± 8,40 33,00 ± 4,16 73,17 ± 12,12 Grupo 65% NDT 36,30 ± 3,00 33,40 ± 7,23 82,40 ± 10,78 Controle 36,80 ± 5,80 35,20 ± 7,78 84,60 ± 11,84 Média 33,65 ± 5,15 33,41 ± 8,69 77,00 ± 19,90 Não houve diferença estatística entre os tratamentos (p > 0,05) Conclusão O estresse calórico não afeta a atividade reprodutiva de cabras SPRD de aptidão leiteira, paridas na época seca e manejadas de forma semi-intensiva, no semiárido pernambucano. Não houve diferença estatística entre os tratamentos (p > 0,05) Literatura Citada Atividade ovariana no pós-parto de cabras SRDs (sem raça definida) no nordeste do Brasil. Ars Veterinária, v.19, p.166-171, 2003. Freitas, V. J. F.; Rondina, D.; Nogueira, D. M.; Simplício, A. A. Post-partum anoestrus in Anglo-nubian and Saanen goats raised in the semi-arid region of Northeast of Brazil. Livestock Production Science, v.90, p.219-226, 2004.hi Gaafar, K. M.; Gabr, M. K.; Teleb, D. F. The hormonal profile during the estrous cycle and gestation in Damascus goats. Small Ruminant Research, v.57, p.85-93, 2005. Garcia-Ispierto, I.; López-Gatius, F; Santolaria, P. Relationship between heat stress durant the peri-implantation period and early fetal loss in dairy cattle. Theriogenology, v.65, p.799-807, 2006. Os dados encontrados para retorno da atividade estral são inferiores aos mencionados por Eloy et al. (2003) que, trabalhando com cabras SPRD no período seco, encontraram uma média de 109,40 ± 8,71 dias para o retorno da atividade estral. A maior precocidade do retorno ao estro, neste trabalho, pode ser explicada pela suplementação que promoveu um aporte maior de energia reduzindo o efeito deletério do balanço energético negativo no puerpério. Pereira et al. (2011) afirmaram que o estresse térmico pode intensificar o balanço energético negativo o que não foi verificado neste trabalho tendo em vista o trabalho de Eloy et al. (2003) que apontaram que a intensificação do balanço energético negativo puerperal é o que torna o anestro mais prolongado durante o período seco, quando comparado com o período chuvoso o qual foi relatado, por eles, como o período de maiores quantidade e qualidade de forragem. Gomes, C. A. V.; Furtado, D. A.; Medeiros, A. N.; Silva, D. S.; Pimenta Filho, E. C.; Lima Júnior, V. Efeito do ambiente térmico e níveis de suplementação nos parâmetros fisiológicos de caprinos Moxotó Revista Brasileira de Engenharia Agrícola e Ambiental, v.12, p.213-219, 2008. Gonçalves, R. C. Semiologia do sistema respiratório. In: Gonçalves, R. C.; Feitosa, F. L. F. Semiologia veterinária: A arte do diagnóstico. 2.ed., São Paulo: Rocca, 2008. p.313-331. Mahdi, D.; Khallili, K. Relationship between follicle growth and circulating gonadotrophin levels during postnatal development of sheep. Animal Reproduction Science v.106, p.100-112, 2008. R. Bras. Eng. Agríc. Ambiental, v.18, n.5, p.539–544, 2014. Thiago V. C. Nascimento et al. 544 Salmazo, R.; Mizubuti, I. Y.; Moreira, F. B.; Rocha, M. A. da; Ribeiro, E. L. A.; Seneda, M. M.; Hiroki, P. T.; Krawulski, C. C.; Rigo, A. G.; Schroeder, R. V. Literatura Citada Efeito de diferentes níveis de suplementação concentrada sobre o desempenho reprodutivo de vacas leiteiras mantidas a pasto. Ciências Agrárias, v.29, p.731-740, 2008. Morais, D. A. E. F.; Lima, F. R. G.; Vasconcelos, A. M. De; Lima, P. O.; Morais, J. H. G. Manifestação de estro, fertilidade e desempenho reprodutivo de cabras leiteiras de diferentes raças em ambiente quente. Revista Caatinga, v.21, p.62-67, 2008. Miolo, A. Novas regiões: Vinho de clima tropical. In: Congresso Brasileiro de Viticultura e enologia, 10, 2003, Bento Gonçalves, Anais...Bento Gonçalves: Embrapa Uva e Vinho, 2003. p.141-144. Salmito-Vanderley, C. S. B.; Marques Júnior, A. P. Involução uterina em cabras sem raça definida Revista Brasileira de Reprodução Animal, v.28, p.278-281, 2004. Nogueira, D. M.; Voltolini, T. V.; Moreira, J. N.; Lopes Júnior, E. S.; Oliveira, V. G. de. Efeito de regimes alimentares sobre o peso corporal e parâmetros reprodutivos de cabras nativas. Archivos de Zootecnia, v.60, p.339-1342, 2011. Silva, E. M. N.; Souza, B. B.; Sousa, O. B.; Silva, G. A.; Freitas, M. M. S. Avaliação da adaptabilidade de caprinos ao semiárido através de parâmetros fisiológicos e estruturas do tegumento. Revista Caatinga, v.23, p.142-148, 2010. NRC - National Research Council. Nutrient requeriments of small ruminants. 7.ed. Washington: National Academic Press, 2007. 408p. Silva, F. de A. S. e; Azevedo, C. A. V. de. Assistat, versão 7.6 Beta, Campina Grande, 2011. Simplício, A. A.; Santos, D. O.; Salles, H. O. Manejo de caprinos para a produção de leite em regiões tropicais Ciência Animal, v.10, p.13-27, 2000. Pereira, G. M.; Souza, B. B.; Silva, A. M. A.; Roberto, J. V. B.; Silva, C. M. B. A. Avaliação do comportamento fisiológico de caprinos da raça Saanen no semiárido paraibano. Revista Verde, v.6, p.83-88, 2011. Torreão, J. N. da C.; Pimenta Filho, E. C.; Medeiros, A. N. de; Gonzaga Neto, S.; Catanho, M. T. J. de A.; Barreto, L. M. G.; Silva, J. O. da. Retorno da atividade cíclica reprodutiva em ovelhas da raça Morada Nova submetidas a diferentes níveis de energia metabolizável. Revista Brasileira de Saúde e Produção Animal, v.9, p.621-630, 2008. Pinho, R. O.; Guimaraes, J. D.; Martins, L. F.; Castilho, E. F.; Barros, M. H. C.; Freitas, B. W.; Rodrigues, B. L. F.; Borges, M. C. B.; Guimaraes, S. E. F. Parâmetros reprodutivos de cabras leiteiras submetidas a condições bioclimáticas artificiais semelhantes à região amazônica oriental. Revista Científica Eletrônica de Medicina Veterinária, v.6, p.1-19, 2010. Uribe-Velásquez, L. R. Bras. Eng. Agríc. Ambiental, v.18, n.5, p.539–544, 2014. Literatura Citada F.; Oba, E.; Brasil, L. H. A.; Sousa, F. N.; Wechsler, F. S. Efeitos do estresse térmico nas concentrações plasmáticas de progesterona (P4) e estradiol 17-b (E2) e temperatura retal em cabras da raça pardo alpina Revista Brasileira de Zootecnia, v.30, p.388-393, 2001. Roberto, J. V. B.; Souza, B. B. Fatores ambientais, nutricionais e de manejo e índices de conforto térmico na produção de ruminantes no semiárido. Revista Verde de Agroecologia e Desenvolvimento Sustentável, v.6, p.8-13, 2011. Uribe-Velásquez, L. F.; Souza, M. I. L.; Osorio, J. H. Resposta ovariana de cabras submetidas a implantes de progesterona seguidos de aplicações de gonadotrofina coriônica eqüina Revista Brasileira de Zootecnia, v.39, p.1214-1222, 2010. Roberto, J. V. B.; Souza, B. B.; Silva, A. L. N.; Justiniano, S. V.; Freitas, M. M. S. Parâmetros hematológicos de caprinos de corte submetidos a diferentes níveis de suplementação no semi-árido paraibano. Revista Caatinga, v.23, p.127-132, 2010.
https://openalex.org/W3204697809	https://kops.uni-konstanz.de/bitstream/123456789/55444/3/Rathcke_2-m6uiw1fmzg7t8.pdf	English	null	Towards a Comprehensive Account of Rhythm Processing Issues in Developmental Dyslexia	Brain sciences	2,021	cc-by	21,484	Tamara Rathcke 1,2,* and Chia-Yuan Lin 2,3 1 Department of Linguistics, Faculty of Humanities, University of Konstanz, 78464 Konstanz, German 2 Modern Languages and Linguistics School of Cultures and Languages University of Kent 1 Department of Linguistics, Faculty of Humanities, University of Konstanz, 78464 Konstanz, Germany 2 Modern Languages and Linguistics, School of Cultures and Languages, University of Kent, 1 Department of Linguistics, Faculty of Humanities, University of Konstanz, 78464 Konst p g y y 2 Modern Languages and Linguistics, School of Cultures and Languages, University of Kent, Canterbury CT2 7NR UK; c lin@kent ac uk y , ; 3 Department of Psychology, School of Humanities and Health Sciences, University of Huddersﬁeld, Huddersﬁeld HD1 3DH, UK * Correspondence: tamara.rathcke@uni-konstanz.de Abstract: Developmental dyslexia is typically deﬁned as a difﬁculty with an individual’s command of written language, arising from deﬁcits in phonological awareness. However, motor entrain- ment difﬁculties in non-linguistic synchronization and time-keeping tasks have also been reported. Such ﬁndings gave rise to proposals of an underlying rhythm processing deﬁcit in dyslexia, even though to date, evidence for impaired motor entrainment with the rhythm of natural speech is rather scarce, and the role of speech rhythm in phonological awareness is unclear. The present study aimed to ﬁll these gaps. Dyslexic adults and age-matched control participants with variable levels of previ- ous music training completed a series of experimental tasks assessing phoneme processing, rhythm perception, and motor entrainment abilities. In a rhythm entrainment task, participants tapped along to the perceived beat of natural spoken sentences. In a phoneme processing task, participants moni- tored for sonorant and obstruent phonemes embedded in nonsense strings. Individual sensorimotor skills were assessed using a number of screening tests. The results lacked evidence for a motor impairment or a general motor entrainment difﬁculty in dyslexia, at least among adult participants of the study. Instead, the results showed that the participants’ performance in the phonemic task was predictive of their performance in the rhythmic task, but not vice versa, suggesting that atypical rhythm processing in dyslexia may be the consequence, but not the cause, of dyslexic difﬁculties with phoneme-level encoding. No evidence for a deﬁcit in the entrainment to the syllable rate in dyslexic adults was found. Rather, metrically weak syllables were signiﬁcantly less often at the center of rhythmic attention in dyslexic adults as compared to neurotypical controls, with an increased tendency in musically trained participants. Citation: Rathcke, T.; Lin, C.-Y. Towards a Comprehensive Account of Rhythm Processing Issues in Developmental Dyslexia. Brain Sci. 2021, 11, 1303. https://doi.org// 10.3390/brainsci11101303 Academic Editors: Anja Lowit, Sónia Frota and Marina Vigário Received: 31 July 2021 Accepted: 21 September 2021 Published: 30 September 2021 Keywords: speech rhythm; developmental dyslexia; phonological awareness; sensorimotor synchronization; phoneme monitoring; rhythm perception; motor entrainment Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Citation: Rathcke, T.; Lin, C.-Y. Towards a Comprehensive Account of Rhythm Processing Issues in Developmental Dyslexia. Brain Sci. 2021, 11, 1303. https://doi.org// 10.3390/brainsci11101303 brain sciences brain sciences brain sciences Tamara Rathcke 1,2,* and Chia-Yuan Lin 2,3 This ﬁnding could not be explained by an auditory deﬁcit in the processing of acoustic-prosodic cues to the rhythm structure, but it is likely to be related to the well-documented auditory short-term memory issue in dyslexia. Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-2-m6uiw1fmzg7t8 1. Introduction 1.1. The Role of Phonology in Dyslexia 1.1. The Role of Phonology in Dyslexia Towards a Comprehensive Account of Rhythm Processing Issues in Developmental Dyslexia Tamara Rathcke 1,2,* and Chia-Yuan Lin 2,3 https://www.mdpi.com/journal/brainsci 1.1. The Role of Phonology in Dyslexia Dyslexia is considered a neurodevelopmental disorder [1], typically characterized by difﬁculties with an individual’s command of written language [2–6]. The issues with reading, (hand-)writing and spelling cannot be accounted for by an intellectual deﬁcit [7], though other linguistic abilities may also be delayed in children with dyslexia [8]. The de- lays concern a slower speech production development (speciﬁcally the pronunciation of longer, more complex words), as compared to age-matched children without read- ing or writing difﬁculties [3]. The native language of the child is known to inﬂuence the severity of the disorder [9]. Less severe reading problems occur amongst children whose native languages have a more transparent grapheme-to-phoneme mapping [10]. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/brainsci https://www.mdpi.com/journal/brainsci Brain Sci. 2021, 11, 1303. https://doi.org/10.3390/brainsci11101303 Brain Sci. 2021, 11, 1303 2 of 31 Conversely, languages with a less transparent grapheme-to-phoneme mapping (such as En- glish) pose more problems for children with dyslexia, and lead to more severe cases [2,9,10]. Conversely, languages with a less transparent grapheme-to-phoneme mapping (such as En- glish) pose more problems for children with dyslexia, and lead to more severe cases [2,9,10]. g p p y Whether or not a phonological deﬁcit might be the underlying issue in dyslexia has been a matter of long-standing debates [11–14], with a large body of evidence docu- menting a tight relationship between phonological awareness and reading ability [15–18]. ‘Phonological awareness’ is an umbrella term used to describe different aspects of audi- tory processing of structural phonological units—such as phonemes, syllables, words or sentences—within one’s native or ambient language [19–22], and is thought to lay a crucial foundation to the general language processing system [23]. A well-developed phonological awareness involves an efﬁcient encoding of an entire phonological system and indicates that an individual has the ability to recognize, discriminate, retain and manipulate the sound structure of their language(s) [22,24]. This awareness has been widely reported as impaired in dyslexic listeners, as indicated by a reduced ability to segment lexical items into discrete phonological units such as syllables and phonemes [9,25,26], poor phoneme awareness [27,28], a reduced short-term memory capacity for verbal information and a slow retrieval of lexical information [14,29]. 1.1. The Role of Phonology in Dyslexia Overall, the deﬁcit of input representations is assumed to be more severe than the deﬁcit of phonological output representations [29]. The mapping of variable acoustic signals onto discrete phonological representations of the listener’s native language(s) is one of the major challenges in speech perception [30]. Children with dyslexia seem to have a deﬁcit in perceiving speech sounds in a categorical fashion like their age-matched or reading-level matched peers, i.e., they tend to make insufﬁcient use of acoustic cues to phonemic contrasts while extensively attending to the acoustics of allophonic variation [31–34], (see [35] for a meta-analysis). Such allophonic perception is not speciﬁc to dyslexia. It has generally been documented in preschoolers and is known to give way to categorical perception with increased reading experience as the schooling progresses [36], though at the physiological level of neural activation, the tendency to allophonic (instead of categorical) perception may persist even in well- compensated adults with developmental dyslexia [37]. 1.2. Motor Entrainment Deﬁcits in Dyslexia Individuals diagnosed with developmental dyslexia have shown a number of mo- tor deﬁcits indicative of issues with rhythmic entrainment and potentially suggestive of an underlying rhythm processing impairment. For example, Wolff [38] applied a ﬁnger tapping paradigm to the study of rhythmic sensorimotor synchronization (SMS) abili- ties in dyslexia. Twelve adolescents diagnosed with developmental dyslexia were asked to tap in time with auditory prompts of varied complexity and at different time scales. The results demonstrated that in comparison to a neurotypical control group, dyslexic participants tended to produce larger negative mean asynchronies while synchronizing with a metronome, and experienced increased difﬁculties with the phase correction when tapping to a metronome as well as with the motor reproduction when exposed to sim- ple auditory rhythms. Wolff et al. [39] tested dyslexic and control participants from two age groups—adolescents and young adults—and asked them to perform a series of non- linguistic SMS tasks. Accordingly, the study groups differed in their tapping stability, with dyslexic adolescents showing greater motor instability than all other participants. Thomson et al. [40] investigated the SMS performance of dyslexic adults and used a ﬁnger tapping paradigm following the methodology by [38,39], and additionally including an unpaced tapping task that measured participants’ spontaneous tapping rate. No group differences were found for negative mean asynchronies with a metronome (contra [38]). Instead, dyslexic adults showed greater variability of their inter-tap intervals during both paced and unpaced tapping. This ﬁnding was only partially in line with the results dis- cussed in Wolff et al. [39] who identiﬁed a signiﬁcant tapping instability for the adolescent but not the adult group of dyslexic participants. More broadly, motor synchronization skills seem to be interrelated with the acqui- sition of literacy. As shown by Tierney and Kraus [41], individual variability in tapping Brain Sci. 2021, 11, 1303 3 of 31 with a metronome correlates with reading skills in normally developing adolescents. However, previous results based on dyslexic participants’ performance with non-linguistic stimuli have provided somewhat inconclusive evidence for a generally impaired motor entrainment which might be indicative of the fact that dyslexia is primarily a language- speciﬁc impairment [42]. Thus, the involved rhythm processing difﬁculties might be more readily amenable to an empirical observation in a linguistic rather than a non-linguistic setting. To date, only a limited number of studies have employed SMS to investigate dyslexic listeners’ perception of rhythm in spoken language. 1.2. Motor Entrainment Deﬁcits in Dyslexia Wood and Terrel [43] asked children with and without a reading impairment to tap or clap along with the syllables in individual spoken words, as a means of counting how many syllables each word con- tained. Results showed that among three groups of readers with different abilities (poor readers, age-matched controls and reading age controls), poor readers scored low while the age-matched controls achieved the highest scores for their ability to correctly clap out the number of syllables in a word; however, the group difference in the scores was not signiﬁcant. Leong and Goswami [22] studied SMS with short, metrically regular speech in dyslexic and neurotypical adults. The experimental sentences of the study included four traditional nursery rhymes that contained either a trochee (SW, e.g., ‘Mary Mary quite contrary’) or an iamb (WS, e.g., ‘The Queen of Hearts she made some tarts’). Each rhyme was presented three times (with short silent breaks in-between) while participants tapped along to the perceived rhythm of each rhyme, maintaining their tapping during the silent intervals between repetitions. The results showed that neither the inter-tap interval (ITI) duration nor the asynchronies measured with the onset of lexically stressed vowels (a proxy of syllables’ perceptual centers, [44,45]) differed across the two groups of participants. Instead, Leong and Goswami [22] argue that the SMS performance of the two participant groups could be best described as divergent at the level of the syllabic entrainment while the stress- and phoneme-level entrainment was comparable in participants with dyslexia and the neurotypical controls. The conclusion was based on the acoustic analyses of amplitude envelopes obtained from the nursery rhymes, and a subsequent decomposition of amplitude modulations into instantaneous frequencies using a Hilbert transform ([46], cf. [47]). Such analyses aim at representing speech at multiple timescales: thought to correspond to phoneme, syllable and stress ﬂuctuations inherent to spoken language. Following a signal decomposition, the accompanying SMS behaviors can be then described as being variably phase-locked to the different timescales of speech ([22], cf. [48]). As our research has shown [48], signal decomposition and its derivatives do not represent consistent SMS anchors in speech. In comparison to manually identiﬁed vowel onsets, any amplitude derivative is signiﬁcantly worse at predicting the location of ﬁnger taps collected during SMS with natural speech, even in healthy participants. 1.2. Motor Entrainment Deﬁcits in Dyslexia In contrast, vowel onsets constitute stable SMS anchors in simple verbal stimuli [49] as well as in complex natural sentences [48,50]. While decompositional approaches seem to excel at tracking temporal ﬂuctuations pertaining to different layers of prosodic hierarchy and occurring within large timescales of longer speech samples [47], the local time resolution of such approaches is, however, relatively poor, but is ultimately required for an adequate temporal representation of perceptually relevant rhythmic events (cf. [44,45]). An alternative approach to the study of SMS with natural speech was developed in our previous research [49,50]. It combines the necessary temporal precision at the local, intra- syllabic level [44,45] with the hierarchical metrical representation of linguistic units [51–53]. Accordingly, SMS is collected while participants synchronize with what they perceive as the beat of sentences played back to them on a loop (e.g., 20, not just three, repetitions of a sentence as in previous work [22,54]). Individual and group SMS performance is then described as probability functions for the time course of a sentence, specifying local time points that are most/least likely to attract a tap and comparing them to the metrical representation of the sentence. We have repeatedly demonstrated that vowel onsets serve as consistent anchors of SMS in English [48,50], and that SMS-likelihood is also shaped by Brain Sci. 2021, 11, 1303 4 of 31 the properties of the prosodic hierarchy. Vowels in metrically strong positions are more likely to attract a ﬁnger tap than vowels in metrically weak positions, at least in English [50]. Moreover, our approach allows for the calculation of SMS accuracy which we also showed to be inﬂuenced by the metrical weight of a vowel within the prosodic hierarchy: En- glish participants tend to tap more precisely with metrically strong than with metrically weak vowels [50]. Overall, the results obtained with our version of the SMS paradigm resemble a series of well-established SMS ﬁndings collected for temporally more regular types of auditory signals, such as metronome and music (see [55,56] for an overview). Importantly, individual variations in SMS can be quite large (cf. [57]) and are known to be inﬂuenced by the degree of musical training of the participants ([50] cf. [58,59]). y g g The study of the effects that music training has on the human brain and cognitive development is a burgeoning ﬁeld (see [60] for an overview of the key ﬁndings). 1.2. Motor Entrainment Deﬁcits in Dyslexia Musical training is often seen as “a resource that tones the brain for auditory ﬁtness” [60], with a number of auditory perception beneﬁts that extend to language. For example, music training is related to an improved speech perception in challenging listening conditions [61– 63], increased verbal working memory [61,64–66] and enhanced speech segmentation skills [67,68]. Even non-professional musicians show such beneﬁts (e.g., [69,70]). As far as rhythm perception and time-keeping skills are concerned, musically trained non-expert participants show fewer errors and less variability when synchronizing with a metronome than musically untrained participants (e.g., [71–74]). Our previous work has shown that these abilities also transfer to SMS with language [50]. Accordingly, participants with higher levels of musical training (which included playing an instrument, singing and dancing) also produced smaller asynchronies when synchronizing with natural speech. g p y y g p The idea that musical training might be associated with an enhancement of reading abilities during childhood has been investigated in a number of studies (e.g., [75–79]). Music-based interventions in (pre-)schoolers have also been tested as a means of mitigating and treating developmental dyslexia (e.g., [80–84]). Even though not all studies have been able to demonstrate that music training leads to a signiﬁcant improvement of individual difﬁculties with reading and writing, a major gain in phonological awareness skills has been documented in most previous research (see [85] for a meta-analysis). The facilitating effect of music training on phonological awareness has been demonstrated even before the onset of literacy acquisition, in 3 y.o. toddlers [84]. Improved phonological awareness is thought to moderate the impact of music training on literacy development, via changes in auditory processing mechanisms during music practice (e.g., [60,85–87]). Interestingly, a recent study with adult dyslexic and control participants showed that individual differences in musical rhythm ability captured the variability in perception of rhythmic grouping of spoken units better than the individual’s dyslexia status did [88]. 1.3. Processing of Rhythm and Its Acoustic Correlates as the Underlying Issue in Dyslexia j g q p g The question of whether or not an impairment of basic auditory processing mecha- nisms might cause such deﬁcits in individuals with dyslexia, and which speciﬁc acoustic properties of speech signals might be processed insufﬁciently, has been addressed in a number of studies (see [117] for a review). Prosodically relevant acoustic cues include the fundamental frequency (F0), duration and intensity (e.g., [118,119]), though lexical stress in English relies less on F0 [120,121] and more on vowel quality and the corresponding formant frequencies [122,123]. Interestingly, the perception of formant frequencies does not seem to cause any difﬁculties to dyslexic participants [124,125]. In a perception study with manipulated format trajectories that distinguished between /ba/ and /wa/, Goswami et al. [125] found that dyslexic children had an increased sensitivity to the changes in vowel formants which encode /ba/ vs. /wa/, and in fact outperformed the age-matched controls on this task. The ﬁnding points toward a speciﬁc auditory processing strength in dyslexic listeners [124,125], and is somewhat at odds with their weak perception of lexical stress, given that one of its main phonetic correlates is vowel quality which is encoded by formant frequencies ([122,123], cf. [120] for a cross-linguistic review). In a series of experiments, Goswami and colleagues have demonstrated that the indi- vidual sensitivity to stress patterns (d’) is correlated with the sensitivity to acoustic signal rise-time, but not with the sensitivity to signal intensity or frequency (e.g., [110,115,125,126]). Similarly, a meta-analysis by Hämäläinen et al. [117] reported that all previous experiments that tested rise-time perception in dyslexic participants identiﬁed a relationship between individual rise-time sensitivity and reading ability. In contrast, existing ﬁndings with regards to the role of other prosodically relevant cues such as duration, frequency and intensity are rather mixed [117]. According to some proposals (e.g., [127–129]), an efﬁcient perceptual encoding of the acoustic properties of signal rise-time is key to the perception of metrical structure in speech, and prosodic processing difﬁculties observed in dyslexic listeners arise as a consequence of a lack of such encoding. However, previous research into the relationship between acoustic signal rise-time and metrical encoding difﬁculties in dyslexia has employed purely psychoacoustic tests to measure rise-time sensitivity in listeners (e.g., [40,110,115,129–131]) or perception tests involving simple /ba/-/wa/ monosyllables [125]. In natural speech, the role of syllable rise-time for the perception of rhythm is less clear-cut, and existing evidence is less conclusive ([48] cf. 1.3. Processing of Rhythm and Its Acoustic Correlates as the Underlying Issue in Dyslexia Auditory and motor systems are known to be tightly connected during the pro- cessing of rhythmic patterns across a diverse range of acoustic signals (e.g., [89–95]). Given the importance that rhythm, or more broadly prosody, plays in speech process- ing, dyslexic difﬁculties might stem from the insufﬁcient processing of prosody and its acoustic correlates. Prosody is known to guide speech segmentation at various levels of linguistic structure, including phonemes [96–100], morphemes [101] and words [102–105]. In a language like English, speech segmentation is facilitated by the presence of lexical stress which directs perceptual attention to potential word onsets [106,107], given that word-initial stress prevails in the English lexicon [108]. Alternations of strong and weak syllables cuing the distribution of lexically stressed syllables in sentences create unique rhythmic templates that shape spoken word recognition and facilitate lexical access [109]. Individual sensitivity to speech prosody is known to correlate with reading abilities and the acquisition of phonology [22]. In particular, sensitivity to rhythmic alternations of strong (S) and weak (W) syllables that encode metrical representations of words and sen- Brain Sci. 2021, 11, 1303 5 of 31 tences shows processing deﬁcits in individuals with dyslexia as compared to age-matched controls [22,43,110–115]. A series of production and perception studies have produced compelling evidence that dyslexic individuals (both children and adults) do not efﬁciently encode metrical templates in English. For example, Goswami et al. [110] implemented a ‘Dee Dee’ task [114,116] and asked 12-year-old children to replace syllables of highly recognizable personal names with a metrical template consisting of strong and weak versions of the syllable ‘dee’ (e.g., ‘Harry Potter’ would be ‘DEEdee-DEEdee’). The results showed that dyslexic children performed poorly in this production task, compared to their age-matched controls. In a perceptual discrimination task, Leong and colleagues [115] presented their dyslexic participants with a series of English four-syllable words that were produced with either a SWWW or a WSWW pattern (e.g., MAternity or maTERnity, DIf ﬁculty or diFFIculty) and then paired to either have the same or a different stress pattern. Compared to age-matched neurotypical controls, adult participants with diagnosed developmental dyslexia were signiﬁcantly less accurate in judging stress templates across pairs of words, irrespective of the SWWW or WSWW stress judgement required or the same/different lexical pairings involved. 1.4. Open Questions and Hypotheses 1.4. Open Questions and Hypotheses The main aim of the present study is to investigate rhythm perception and motor entrainment in dyslexia using natural speech, and to deepen the understanding of the underlying deﬁcits in adults with dyslexia. y g y Most of the existing research that documented metrical encoding difﬁculties in dyslexia tested rhythm perception in simple words and phrases (see [117] for an overview) or studied the motor entrainment to metrically regular nursery rhymes [22]. However, nat- ural speech is syntactically complex and lacks such regularities of strong and weak syl- lables, or any acoustic isochrony to support the temporal prediction of rhythmically rel- evant events [135,136]. Thus, we can expect natural speech to cause more difﬁculties to prosodic processing in dyslexic participants. To study rhythm perception and entrainment in complex auditory signals such as natural speech, the present study implements an SMS paradigm that we developed previously [50]. In the methodologically motivated study [50], we investigated SMS with looped spoken sentences and tested several acoustic anchors that might attract SMS in natural speech. Our ﬁndings showed that spoken sen- tences can reliably entrain movement when looped, and that (neurotypical) participants move in synchrony with vowel onsets when asked to synchronize to the sentence beat. Crucially, the paradigm is sensitive to the metrical structure of sentences as strong syllables tend to evoke more taps and increase the synchronization accuracy. The present research applies the established paradigm to test rhythm perception and entrainment in dyslexia, and uses a larger number of sentences with syllabic nuclei being occupied not only by vow- els but also by consonants. Nasals and laterals can constitute the nuclei of weak syllables in natural speech in English [137]. These sounds are relatively high on the sonority scale and are acoustically distinguished by the presence of a formant structure similar to vowels, though with an overall lower amplitude [138–140]. g p If dyslexic listeners have rhythm processing difﬁculties due to their issues with the syllabic entrainment as Leong and Goswami [22] suggest, we would expect dyslexic participants to produce fewer and less precise taps for all syllables, regardless of the syllable’s metrical weight. 1.3. Processing of Rhythm and Its Acoustic Correlates as the Underlying Issue in Dyslexia [132]). y g Importantly, rise-time modulations depend on a large number of both acoustic and linguistic factors in natural connected speech. The duration of the syllable rise-time co- varies with its duration and intensity [48], and encodes a number of linguistic functions beyond prosody, including the manner of consonant articulation [133,134] and complexity of syllable onsets [48]. Given this polyfunctionality of amplitude rise-time modulations, linguistic consequences of an impaired rise-time perception cannot be clearly deﬁned, thus impeding causality in the discussion of the underpinnings of dyslexia. Brain Sci. 2021, 11, 1303 6 of 31 1.4. Open Questions and Hypotheses If the rhythm processing difﬁculty arises from an auditory issue with the encoding of lexical stress and the hierarchical relationships between stressed and unstressed syllables, we would expect adult participants with dyslexia to perform worse than the age-matched controls in their SMS with the vowels of strong syllables only which would be indicative of an entrainment deﬁcit with longer time-scales. g Moreover, the role of prosody in phonological awareness is unclear. Previous research has not conclusively demonstrated if, and how, the ability to segment and identify con- crete phonemes in variable, continuous acoustic speech signals is interrelated with the ability to perceive metrical hierarchies of lexical and phrasal prominences. The two aspects of auditory speech processing can be linked in three ways. First, prosody can support the bootstrapping of segmental phonology in natural speech (cf. [141]), and thus be the origin of the dyslexic deﬁcits reported above [22,43,110–115,125]. Second, the inability to perceive metrical relationships between strong and weak syllables might be a conse- quence of a limited ability to attend to the relevant acoustic cues encoding segmental contrasts in connected speech. The third possibility is that the two aspects of auditory speech processing play an independent role in phonological awareness, and their deﬁcits are unrelated in dyslexia. To test the nature of the relationship between phoneme aware- ness and rhythmic skills, we additionally asked our participants to perform a phoneme monitoring task by listening to nonsense strings containing target obstruents and sonorants. Phoneme monitoring taps phonological awareness by testing the ability to attend to vari- able acoustic cues, to segment a continuous speech stream into phoneme-sized units, and to map those units onto corresponding phonological representations. Since nonsense strings contain no real words that could facilitate phoneme access via lexical boost, the task’s demands are purely acoustic. Given that consonant contrasts encoded in formant dynam- ics rarely cause processing issues for dyslexic listeners [124,125], we expected dyslexic listeners to show little difﬁculties in identifying sonorants but perform worse than the Brain Sci. 2021, 11, 1303 7 of 31 control participants (i.e., with lower accuracy and longer reaction times) when monitoring for obstruents (cf. [35,142]). To understand the relationship between phoneme perception and prosodic processing, participants’ performance in each task is used as a predictor of their performance in the other task. 1.4. Open Questions and Hypotheses That is, individual sensitivity to variable acoustic representations of target phonemes embedded in nonsense strings (d’) is ﬁt as a predictor of participants’ SMS, and vice versa, individual SMS rates with strong and weak syllables in the SMS task are ﬁt as predictors of participants’ phoneme monitoring performance. Depending on the predictive power of these individual measures in the best-ﬁt models, conclusions can be drawn on the relationships between phoneme perception and rhythm processing. p p p p y p g Moreover, whether or not dyslexia results from a general auditory deﬁcit of linguis- tically relevant aspects of signal acoustics has been controversially debated (see [132] for an overview). Previous research into the underlying causes of the deﬁcits in phonological awareness and prosodic processing has frequently relied on correlational data obtained from substantially different types of tasks, both linguistic and non-linguistic. Therefore, the discussion of causality among the involved processes and the scope of the derived conclu- sions are to be considered limited (cf. [132]). Studies of the auditory processing deﬁcits in dyslexia have mostly employed psychoacoustic tests to measure individual sensitivity to the acoustic properties of rise-time, duration or intensity, without attesting their direct links to the prosodic and segmental categories at hand [40,110,115,129–131]. However, acoustic cues such as rise-time, duration and intensity encode a multitude of linguistic functions and categories in connected speech [133,143–145]. If an auditory processing deﬁcit of (any of) these acoustic parameters was at the heart of dyslexia, their impact should be measurable, especially in verbal tasks. Therefore, the present study seeks to estimate the effects of variable acoustic rise-time, duration and intensity that are simultaneously present in the stimuli, on participants’ performance in the two experimental tasks: phoneme monitoring and SMS. Finally, given that music practice changes general auditory processing mechanisms that are also pertinent to speech and language (e.g., [60,85–87]), we expect levels of musical training to play a role in the two tasks. Musically trained participants, regardless of their dyslexia status, are likely to show better performance in the experiments (i.e., faster RTs, lower asynchronies, higher accuracy and SMS rates), potentially highlighting the higher importance of musical training over dyslexic difﬁculties among well-compensated adults [88]. 2.1. Materials and the Phoneme Monitoring Task The stimuli for the phoneme monitoring task were selected from the materials used in previous research [105]. The materials of this study comprised short nonsense sequences (e.g., 3-syllable [bl@‘kinIm] or 4-syllable [SIkl@‘tiðIZ]) whose metrical structure was either WSW or SWSW. In all sequences, the penultimate syllable bore primary stress, and the weak vowel quality was either a schwa or a high front lax vowel [I]. The original study implemented a word-spotting paradigm to investigate the role of cross-dialectal timing cues in lexical segmentation and access. However, the task turned out to be quite difﬁcult for listeners, with average spotting rates around 30% or below. For the purposes of the present study, 40 experimental stimuli and 40 distractors were identiﬁed in the materials. The target phonemes contained either an obstruent (/p/, /k/), or a sonorant (/m/, /l/). The distractor sequences did not contain the target phonemes. The 80 stimuli were grouped into eight blocks of 10, each comprising of ﬁve targets and ﬁve distractors in a random order. Each target phoneme was monitored for in a total of two blocks. The order of testing blocks was pseudo-randomized, prohibiting one phoneme from being the target of two consecutive blocks. A practice block containing ﬁve /s/- targets and ﬁve non-targets was given before a formal testing session. Brain Sci. 2021, 11, 1303 8 of 31 8 of 31 At the start of each block, participants were instructed to monitor for one of the target phonemes and tap on a tambourine as soon as they heard the speciﬁed target. The tambourine was placed at a comfortable distance in front of the participants. It was connected to a piezoelectric sensor. An Arduino microcontroller linked the sensor to a Dell Latitude 7390 laptop running the PsychoPy software which recorded and extracted the timing of participants’ taps. The rise-time, duration and intensity were measured for each target. The rise-time of the syllables containing the target was calculated following the same routines that we employed in our previous research [50]. Accordingly, amplitude envelopes were derived by the envelope function in Matlab (version 9.5 (R2018b), The MathWorks Inc., Natick, MA, USA), which operated on the absolute signal amplitude, smoothing it by applying a spline interpolation with an 11 ms window. The rise-time measured the temporal distance between a local amplitude minimum and a maximum identiﬁed in the smoothed envelope contour around each syllable onset [21,129]. 2.1. Materials and the Phoneme Monitoring Task The duration and the intensity were measured from sentence spectrograms in Praat [146]. The duration of stops included both the closure and the VOT (e.g., [147]). The average intensity of syllables containing targets was normal- ized with reference to the mean intensity of the whole sentence. These measurements are summarized in Table 1, along with the results of an analysis of variance testing whether the acoustic difference between the obstruent and the sonorant targets was consistent and signiﬁcant in these stimuli. Table 1. Means (and standard deviations) of the acoustic measurements taken from the experimental stimuli, along with the corresponding statistical results. Acoustic Factors Obstruents /p/, /k/ Sonorants /m/, /l/ ANOVA F p Rise-time 66.21 (26.69) 69.28 (42.95) F(1, 38) = 0.07 0.79 Duration 50.17 (17.71) 87.95 (22.71) F(1, 38) = 32.76 <0.001 Relative intensity 0.89 (0.04) 1.00 (0.05) F(1, 38) = 62.21 <0.001 Table 1. Means (and standard deviations) of the acoustic measurements taken from the experimental stimul corresponding statistical results. standard deviations) of the acoustic measurements taken from the experimental stimuli, along with the and standard deviations) of the acoustic measurements taken from the experimental stimuli, along with the tatistical results. To capture individual differences in the sensitivity to the acoustic cues of the tar- get’s phonemes embedded in a nonsense string, individual d’-values (MacMillan and Creelman, 2005) were calculated for each participant and used as a predictor in the SMS models. All target phonemes were included in the d’-calculation, given that no signiﬁcant differences in spotting sonorants vs. obstruents were observed across the two groups of participants (see Section 3.2). 2.2. Materials and the SMS Task Twenty English sentences were spoken by a 30-year-old female native speaker of Standard British English and recorded at 44,100 Hz sampling rate in a sound-attenuated booth. The sentences varied in the overall number of syllables (ranging from 7 to 13) and their duration (ranging from 1.2 to 2.3 s). Each sentence contained one or two syllables with sonorant nuclei (see Appendix A for the full sentence list). For each syllable, rise- time, nucleus duration and intensity were measured as described above. Similar to the preparation of the materials for the phoneme monitoring task, the average intensity of each nucleus was normalized with reference to the mean intensity of the corresponding test sentence. The acoustic measurements are summarized in Table 2, broken down by the metrical weight of the syllable containing the corresponding nucleus (strong vs. weak) and the type of the nucleus in the weak syllable (vowel vs. sonorant). Brain Sci. 2021, 11, 1303 9 of 31 Table 2. Means (and standard deviations) of the acoustic measurements taken from the experimental stimuli, along with the corresponding statistical results. Acoustic Factors S W ANOVA W (Vowel) W (Sonorant) ANOVA F p F p Rise-time 50.41 (18.58) 37.68 (15.47) F(1, 194) = 26.93 <0.001 34.14 (14.15) 50.47 (13.28) F(1, 118) = 27.84 <0.001 Duration 92.98 (43.48) 60.82 (33.02) F(1, 194) = 34.38 <0.001 51.53 (28.96) 94.39 (23.91) F(1, 118) = 47.82 <0.001 Relative intensity 1.09 (0.05) 1.04 (0.05) F(1, 192) 1 = 35.88 <0.001 1.04 (0.05) 1.06 (0.05) F(1, 116) 1 = 1.35 0.25 1 The intensity measurements could not be taken for two syllables out of a total of 196 syllables tested in the present study (see Appendix A). Table 2. Means (and standard deviations) of the acoustic measurements taken from the experimental stimuli, along with the corresponding statistical results. Sentence loops were then created, following previously established procedures of mea- suring SMS with natural linguistic stimuli [50]. Accordingly, each sentence was repeated 10 times, with a 400 ms silent pause between repetitions. The order of the experimental sentences was randomly created for each individual session. Participants were instructed to start synchronizing the movement of the index ﬁnger of their dominant hand in time with the beat of the spoken sentence from its third repetition until the end of the loop. 2.3. Other Motor Tasks General individual SMS abilities were captured by four additional motor tasks. In two unpaced tapping tasks, participants were instructed to tap at their most comfortable speed for one minute, and then to tap at their fastest possible speed for 30 s. No auditory prompts were given in the unpaced tapping tasks. In two paced tapping tasks, participants were instructed to tap in synchrony with two auditory sequences consisting of 25 repetitions of the syllable [bi:] (“bee”, cf. [49]). The ﬁrst sequence contained the syllable presented at an inter-onset-interval (IOI) of 600 ms while the second sequence had an IOI of 300 ms. Note that instead of a metronome or pure tones, we used a simple verbal prompt in the paced tapping task as our previous research demonstrated that the SMS performance in the paced tapping task is comparable across a range of verbal and non-verbal stimuli as long as they share the temporal frame of occurrence ([49], see also [135]). 2.2. Materials and the SMS Task Please note that participants were not explicitly advised to synchronize their taps with every syllable nucleus (though this tapping pattern best reﬂects the subjective experience of the speech beat that we previously documented in neurotypical participants using the SMS task, [50]). Prior to the formal testing, participants had an opportunity to practice their un- derstanding of the SMS task with ﬁve sentence loops containing 20 repetitions and no instructions given as to when to start synchronizing. Participants’ ﬁnger taps were recorded on the tambourine that was also used in all other tasks throughout the experiment. To capture the individual differences in the entrainment to syllable nuclei, individual SMS rates were calculated as the number of syllables with which a participant synchronized, divided by the total number of syllables in the materials. If , in the course of sentence repetitions, participants did not consistently produce a tap within a ±120 ms window around a nucleus onset [50,55], the syllable was identiﬁed as lacking SMS (e.g., the third syllable in the example shown in Figure 1). Thus, the SMS rate expressed the proportion of nuclei with which a participant synchronized, and was ﬁt as a predictor in the models of the phoneme monitoring data. 10 of 31 Brain Sci. 2021, 11, 1303 Figure 1. Aggregated densities of SMS with the test sentence “The couple watched the stars twinkle”. (A) displays the performance of the control group, and (B) that of the dyslexic group. Vertical blue lines indicate onsets of vowels, and pink lines indicate onsets of sonorant nuclei. Figure 1. Aggregated densities of SMS with the test sentence “The couple watched the stars twinkle”. (A) displays the performance of the control group, and (B) that of the dyslexic group. Vertical blue lines indicate onsets of vowels, and pink lines indicate onsets of sonorant nuclei. 2.5. Participants Twenty-ﬁve adults (6 M) participated in the current study. Attention was paid to matching the age and the gender of dyslexic and control participants during recruitment. All participants were British monolinguals without any known hearing or motor disabilities. All participants gave informed written consent to participate in the experiments, and were compensated for their time. The study received ethical approval (reference number: 0291920, received 6 January 2020) from the Faculty of Humanities Research Ethics Advisory Group for Human Participants at the University of Kent. One female participant was excluded from further analyses because her taps were too soft to be detected by the tambourine. Thirteen (of the remaining twenty-four) participants self-reported to have been ofﬁcially diagnosed with developmental dyslexia (3 M, mean age = 21.5 years, SD = 1.2 years) while eleven participants self-reported as having normal reading and writing abilities (3 M, mean age = 21.6 years, SD = 1.2 years). Individual dyslexia scores were calculated for each participant based on their answers to a standard- ized checklist [149]. Previous research established that the scores obtained from the adult checklist were correlated with objective measures of literacy skills [150]. As expected, the control group had a low average score indicative of no dyslexic difﬁculties (mean: 35.0, SD: 3.9) while the dyslexic group had a higher average score consistent with symptoms of mild dyslexia (mean: 58.2, SD: 9.5; Welch’s two sample t-test: t(16.7) = 8.14, p < 0.001). There was no overlap in the dyslexia scores between the two groups. In keeping with the checklist guidelines [149], the control participants of the present study scored 41 or below while the dyslexic participants scored 45 or above. No trained or professional musicians participated in the present study, though the musicality scores of the sample varied greatly, from 0 (no musical training received) to 21 (a relatively high level of musical experience). The majority of the participants had some music training: ten (out of thirteen) dyslexic participants and seven (out of eleven) controls reported to have learned an instrument or taken singing classes. Overall, the dyslexic participants of the study (mean: 7.4, SD: 6.4) engaged with music training more than the participants of the non-dyslexic control group (mean: 4.0, SD: 3.7). However, there was a lot of individual variability and the difference between the two groups of participants was not signiﬁcant (t(19.8) = 1.573, p = 0.13). 2.4. Experimental Procedure Participants were invited to a quiet room of the Linguistics Laboratory at the Uni- versity of Kent and gave written consent prior to taking part in the experiment. At the beginning of an experimental session, they completed a short 5-item musicality question- naire. Numerical codes were assigned to each answer and summed up in an individual musicality score ([50], cf. [148]). The questionnaire asked participants to self-report whether Brain Sci. 2021, 11, 1303 11 of 31 11 of 31 they had any musical training (0 for “no”, 1 for “yes”) and were still actively practicing their hobby (0 for “no”, 1 for “yes”). Further questions collected information about the age they started their training (2 for 0–10 y.o., 1 for 10–20 y.o., 0 for 20+ y.o.), the number of years they engaged with playing an instrument or singing (0–14 in the present sample), and how many instruments they played (0–4 in the sample, including singing). The derived musicality score was a numerical composite of the questionnaire answers, with higher scores indicating a higher level of musical training and experience ([50], cf. [148]). Upon completion of the questionnaires, participants engaged with a series of ﬁnger- tapping tasks presented to them in a ﬁxed order, consisting of (1) SMS with the linguistic stimuli, (2) two unpaced tapping tasks, (3) two paced tapping tasks, one at an IOI of 600 ms and one at an IOI of 300 ms. An experimental session concluded with the phoneme monitoring task, followed by a standardized 15-item checklist screening for dyslexia [149]. The playback of all experimental stimuli and the recording of participants’ taps were administered in PsychoPy. Good quality headphones (Sennheiser HD 380) were used in the experiment, with participants being free to adjust the volume to an individually comfortable level. The whole experiment took approximately 40 min to complete. 2.6. Data Preparation The timing of raw tapping data collected by PsychoPy was ﬁrst corrected by subtract- ing the delay of the Arduino microcontroller device (1 ms). Subsequently, the individual taps of each participant were aggregated across eight repetitions of each experimental sentence. The aggregated data were then subject to a Gaussian kernel density estima- tion, performed using the ggplot2 library in R [152]. This procedure created a smoothed representation of tapping distributions for each participant and sentence. The resulting density displays are reﬂective of the participants’ preference to synchronize with speciﬁc anchor points in the sentence [50]. A higher density peak at a certain timepoint within a sentence indicated that this timepoint consistently attracted taps throughout sentence repe- titions, thus highlighting the foci of rhythmic attention during speech perception [59,153]. Examples of the density distributions describing the SMS performance of the dyslexic vs. the control group recorded in the sentence “The couple watched the stars twinkle” are given in Figure 1. Subsequently, the ﬁndpeaks-function from the R-package pracma [154] was applied to each sentence, in order to identify the SMS peaks with a 40% threshold of the maximal peak value that occurred within a 100 ms distance from each other. Given that vowel onsets have been shown to constitute stable SMS anchors in different types of verbal stimuli [48–50] (Lin & Rathcke, 2020; Rathcke et al., 2019, 2021), the present study analyzed SMS with manually deﬁned nucleus onsets, by calculating distances between the identiﬁed SMS peaks and the nearby (±120 ms) nucleus onsets [50]. Temporal distances between the temporal locations of tapping peaks and the nearby onsets of syllable nuclei were calculated as an index of participants’ SMS performance. Absolute asynchronies represented the synchronization accuracy with syllable nuclei while signed asynchronies reﬂected the tendency to anticipate, or to lag behind, a nucleus. Onsets of syllable nuclei were manually annotated by an expert phonetician (the ﬁrst author). The presence of voicing, formant structure and the time course of signal intensity guided the segmentation. The acoustic and auditory criteria were combined in the segmentation of vowel onsets in post-sonorant contexts. Similarly, asynchronies between the vowel onset in [bi:] and the nearby tap were calculated in the paced tapping tasks. 2.6. Data Preparation Finally, to characterize participants’ performance in the unpaced tapping task, the mean duration of inter-tap- intervals (ITIs) and the coefﬁcient of their variability (CV, calculated as SD(ITI)/mean(ITI)) were measured for each participant in the spontaneous and fast task condition. 2.5. Participants A series of post-hoc power analyses were run in Rstudio (R-version 4.1.0), using the simr package [151]. The power to detect group differences in ﬁve planned mixed-effects models was estimated, given the sample size. Based on 10,000 simulations of the best-ﬁt models, the power to detect a group difference of 50 ms response time at α = 0.05 was above 60% in both tasks, the phoneme monitoring (72.15%) and the SMS (absolute asynchrony: 65.80%; signed asynchrony: 100%). Above 90% was the power to detect a group-level Brain Sci. 2021, 11, 1303 12 of 31 12 of 31 difference at α = 0.05 in the logistic mixed-effects models comparing the hit/miss responses during the phoneme monitoring (99.92%), and the likelihood of the synchronization with syllable nuclei during SMS (100.0%). 2.7. Data Analyses All analyses reported below were conducted in Rstudio running R-version 4.1.0., using the packages lme4 [155], emmeans [156], ggplot2 [152], and sjPlot [157]. 3.1. Group Differences in Spontaneous Movement and Time-Keeping 3.1. Group Differences in Spontaneous Movement and Time-Keeping Neither the two unpaced tapping tasks (measuring the movement ability at a fast or a comfortable rate) nor the two paced tapping tasks (measuring the synchronization accuracy with targets presented at the rate of 300 or 600 ms) showed any signiﬁcant group- level differences. Both participant groups had a non-clinical proﬁle of their general SMS abilities [57], as shown in Table 3. 13 of 31 Brain Sci. 2021, 11, 1303 Table 3. Summary of group means (and standard deviations) measured in spontaneous and paced motor tasks, along with the corresponding statistical results. Measurements Taken Dyslexic Group Control Group Welch’s Two Sample t-Test Cohen’s d t p Spontaneous tapping at a comfortable rate ITI (ms) 498 (152) 427 (151) t (21.39) = 1.14 0.27 0.47 CV 0.15 (0.16) 0.07 (0.03) t(13.02) = 1.74 0.10 0.69 Spontaneous tapping at a fast rate ITI (ms) 185 (28) 195 (52) t(14.74) = 0.56 0.58 −0.24 CV 0.15 (0.13) 0.22 (0.24) t(14.95) = 0.90 0.38 −0.38 Paced tapping at 300 ms IOI ITI (ms) 294 (22) 291 (26) t(19.42) = 0.28 0.78 0.12 CV 0.09 (0.07) 0.08 (0.04) t(20.85) = 0.29 0.77 0.12 Signed asynchrony (ms) 3 (19) −11 (20) t(20.96)= 1.76 0.09 0.72 Signed asynchrony (proportion IOI) 0.01 (0.06) −0.04 (0.07) t(20.96)= 1.76 0.09 0.72 Absolute asynchrony (ms) 32 (24) 33 (20) t(21.99) = 0.04 0.97 −0.02 Absolute asynchrony (proportion IOI) 0.11 (0.08) 0.11 (0.07) t(21.99) = 0.04 0.97 −0.02 Paced tapping at 600 ms IOI ITI 687 (327) 593 (19) t(12.10) = 1.03 0.32 0.41 CV 0.13 (0.24) 0.07 (0.06) t(13.85) = 0.89 0.39 0.35 Signed asynchrony (ms) −25 (37) −23 (34) t(21.81) = 0.15 0.88 −0.06 Signed asynchrony (proportion IOI) −0.04 (0.06) −0.04 (0.06) t(21.81) = 0.15 0.88 −0.06 Absolute asynchrony (ms) 49 (20) 45 (23) t(20.00) = 0.40 0.69 0.17 Absolute asynchrony (proportion IOI) 0.08 (0.03) 0.07 (0.03) t(20.00) = 0.40 0.69 0.17 Table 3. Summary of group means (and standard deviations) measured in spontaneous and paced motor tasks, along with the corresponding statistical results. 3.2. The Phoneme Monitoring Task A logistic mixed regression (estimated using ML and the Nelder-Mead optimizer) was ﬁt to hit/miss responses collected during the phoneme monitoring task. A linear mixed regression analysis was performed on the reaction time data (log-transformed, [158]). In both analyses, predictors included individual SMS rates collected in the SMS task and participants’ music training (both scaled and centered around the mean), rise-time duration of the consonant-to-vowel transition (on a logarithmic scale), relative intensity of the syllable (scaled and centered around the mean), syllable weight (strong/weak), target type (sonorant/obstruent), target position in the syllable structure (onset/coda) and the target being part of a consonant cluster or not (1/0). An interaction of these predictors with group (dyslexic/control) was tested in both models. Participant and sentence were ﬁtted as crossed random effects. Maximal random effect structure was retained if the models converged and did not produce a singular ﬁt [159]. The total explanatory power of the best-ﬁt models Brain Sci. 2021, 11, 1303 14 of 31 14 of 31 was quite high (conditional R2 of the logistic model = 0.27, that of the linear model = 0.48), with a smaller contribution of the ﬁxed effects alone (marginal R2 of the logistic model = 0.10, that of the linear model = 0.04). Despite large individual variability in the data, no predictor entered an interac- tion with group. The two groups of participants did not differ in their accuracy or re- action times when performing the phoneme monitoring task, on either class of the target phonemes; therefore, individual sensitivity d’ included responses to all targets. There was no differential inﬂuence of the three acoustic parameters (rise-time, duration, intensity). Individual SMS rates or music training did not affect the group performance signiﬁcantly. These results are summarized in Tables 4 and 5. Table 4. Summary of the logistic mixed-effects model best ﬁtting the accuracy data. Table 4. Summary of the logistic mixed-effects model best ﬁtting the accuracy data. 3.2. The Phoneme Monitoring Task Factor AIC df χ2 p Cluster 1110.0 1 10.73 <0.01 Target type 1109.7 1 6.94 <0.01 Syllable position 1110.5 1 7.71 <0.01 Relative intensity 1116.4 1 13.66 <0.001 Group 1104.7 1 0.12 0.73 Group × target type 1106.6 1 0.58 0.44 Group × rise-time 1105.8 1 3.02 0.08 Group × target duration 1106.4 1 0.09 0.76 Group × relative intensity 1106.6 1 0.05 0.82 Group × SMS rate 1108.2 1 0.35 0.56 Group × musicality 1108.3 1 0.05 0.82 Table 5. Summary of the linear mixed-effects model best ﬁtting the RT data. Factor Sum Sq. df F p Target type 1.41 1 7.15 <0.05 Relative intensity 2.98 1 15.08 <0.001 Group 0.41 1 2.09 0.16 Group × target type 0.13 1 0.66 0.42 Group × rise-time 0.11 1 0.54 0.46 Group × target duration 0.09 1 0.48 0.49 Group × relative intensity 0.26 1 1.30 0.25 Group × SMS rate 0.19 1 0.97 0.33 Group × musicality 0.40 1 2.01 0.17 3.3. SMS with Linguistic Stimuli 3.3.1. SMS Likelihood A logistic mixed-effects regression was performed to test for the likelihood of a tapping peak being present (1) or absent (0) in the proximity of a nucleus onset (±120 ms around the onset, [50]). Predictors included the metrical weight of the syllable (strong/weak), the rise-time of the amplitude envelope around the nucleus onset, the nucleus duration (both on a logarithmic scale), nucleus intensity (scaled and centered around the mean), Table 5. Summary of the linear mixed-effects model best ﬁtting the RT data. Factor Sum Sq. df F p Target type 1.41 1 7.15 <0.05 Relative intensity 2.98 1 15.08 <0.001 Group 0.41 1 2.09 0.16 Group × target type 0.13 1 0.66 0.42 Group × rise-time 0.11 1 0.54 0.46 Group × target duration 0.09 1 0.48 0.49 Group × relative intensity 0.26 1 1.30 0.25 Group × SMS rate 0.19 1 0.97 0.33 Group × musicality 0.40 1 2.01 0.17 Table 5. Summary of the linear mixed-effects model best ﬁtting the RT data. g 3.3.1. SMS Likelihood A logistic mixed-effects regression was performed to test for the likelihood of a tapping peak being present (1) or absent (0) in the proximity of a nucleus onset (±120 ms around the onset, [50]). Predictors included the metrical weight of the syllable (strong/weak), the rise-time of the amplitude envelope around the nucleus onset, the nucleus duration (both on a logarithmic scale), nucleus intensity (scaled and centered around the mean), A logistic mixed-effects regression was performed to test for the likelihood of a tapping peak being present (1) or absent (0) in the proximity of a nucleus onset (±120 ms around the onset, [50]). Predictors included the metrical weight of the syllable (strong/weak), the rise-time of the amplitude envelope around the nucleus onset, the nucleus duration (both on a logarithmic scale), nucleus intensity (scaled and centered around the mean), Brain Sci. 2021, 11, 1303 15 of 31 15 of 31 participants’ sensitivity to acoustic cues of phonemes (d’) and their musicality scores (both individual measurements were scaled and centered around the group mean). Two-way interactions of these predictors with group (dyslexic/control) were tested and removed if an interaction did not help to improve the model ﬁt or caused model convergence issues. Participant and sentence were ﬁtted as random effects. We started with a maximal random effect structure and retained those random effects that allowed the models to converge (Barr et al., 2013). We changed the default optimizer (to “bobyqa”) and increased the number of iterations from default 10,000 to 100,000, to combat model convergence issues. The total explanatory power of the best-ﬁt model was substantial (conditional R2 = 0.28), with a relatively large contribution of the ﬁxed effects alone (marginal R2 = 0.20). Pairwise comparisons of the group performance report estimated marginal means (or least-squares means), obtained using the emmeans package in R [156]. The relevant results are summarized in Table 6. The best-ﬁt model contained three two-way interactions. The interaction of group and metrical weight indicated that dyslexic participants were less likely to synchronize with metrically weak syllables than non- dyslexic controls (β = 0.61, SE = 0.26, z = 2.36, p < 0.05) but did not differ in their SMS with metrically strong syllables (β = 0.08, SE = 0.32, z = 0.24, p = 0.81, see Figure 2A). Individual variability in d’ also interacted with group. g 3.3.1. SMS Likelihood While individual sensitivity to acoustic cues of a phoneme in a nonsense string (d’) did not affect the SMS in dyslexic participants (β = 0.19, SE = 0.15, z = 1.23, p = 0.22), such individual sensitivity of the control partici- pants was predictive of their SMS. Participants who were better able to correctly segment and identify phonemes in nonsense strings also synchronized with more syllable nuclei (β = 0.50, SE = 0.21, z = 2.44, p < 0.05, see Figure 2B). Moreover, an interaction of individual musicality scores with group showed that higher levels of musical training led to a higher SMS likelihood in the controls (β = 0.72, SE = 0.26, z = 2.77, p < 0.01) but to a lower SMS likelihood in dyslexic participants (β = −0.38, SE = 0.15, z = −2.50, p < 0.05, Figure 2C). Table 6. Summary of the logistic mixed-effects model best ﬁtting the SMS probability data. Factor AIC df χ2 p Group × metrical weight 4217.1 1 4.34 <0.05 Group × individual d’ 4219.4 1 6.64 <0.01 Group × musicality 4223.6 1 10.79 <0.01 Group × rise-time 4216.2 1 0.86 0.35 Group × nucleus duration 4204.6 1 0.65 0.42 Group × relative intensity 4204.8 1 0.07 0.78 An additional logistic mixed-effects regression was ﬁt to a subset of the data contain- ing SMS with weak syllables only. The structure of the model mirrored the above, with the only replacement of the factor metrical weight by the factor nucleus type (vowel/sonorant). The best-ﬁt model is given in Table 7. The model’s explanatory power was moderate (con- ditional R2 = 0.19) with a smaller contribution of the ﬁxed effects alone (marginal R2 = 0.10). The corresponding effects are plotted in Figure 3. Accordingly, both groups were equally more likely to synchronize with vocalic than sonorant nuclei (β = 0.42, SE = 0.13, z = 3.29, p < 0.01, Figure 3A) while they differed with regard to the individual effects of musical training (Figure 3C) and sensitivity (d’, Figure 3B) on their SMS. More speciﬁcally, similar interactions of group with the individual abilities were found in the subset of data as well as in the whole dataset (cf. Figure 2B,C). Table 6. Summary of the logistic mixed-effects model best ﬁtting the SMS probability data. g 3.3.1. SMS Likelihood Factor AIC df χ2 p Group × metrical weight 4217.1 1 4.34 <0.05 Group × individual d’ 4219.4 1 6.64 <0.01 Group × musicality 4223.6 1 10.79 <0.01 Group × rise-time 4216.2 1 0.86 0.35 Group × nucleus duration 4204.6 1 0.65 0.42 Group × relative intensity 4204.8 1 0.07 0.78 Table 6. Summary of the logistic mixed-effects model best ﬁtting the SMS probability data. An additional logistic mixed-effects regression was ﬁt to a subset of the data contain- ing SMS with weak syllables only. The structure of the model mirrored the above, with the only replacement of the factor metrical weight by the factor nucleus type (vowel/sonorant). The best-ﬁt model is given in Table 7. The model’s explanatory power was moderate (con- ditional R2 = 0.19) with a smaller contribution of the ﬁxed effects alone (marginal R2 = 0.10). The corresponding effects are plotted in Figure 3. Accordingly, both groups were equally more likely to synchronize with vocalic than sonorant nuclei (β = 0.42, SE = 0.13, z = 3.29, p < 0.01, Figure 3A) while they differed with regard to the individual effects of musical training (Figure 3C) and sensitivity (d’, Figure 3B) on their SMS. More speciﬁcally, similar interactions of group with the individual abilities were found in the subset of data as well as in the whole dataset (cf. Figure 2B,C). An additional logistic mixed-effects regression was ﬁt to a subset of the data contain- ing SMS with weak syllables only. The structure of the model mirrored the above, with the only replacement of the factor metrical weight by the factor nucleus type (vowel/sonorant). The best-ﬁt model is given in Table 7. The model’s explanatory power was moderate (con- ditional R2 = 0.19) with a smaller contribution of the ﬁxed effects alone (marginal R2 = 0.10). The corresponding effects are plotted in Figure 3. Accordingly, both groups were equally more likely to synchronize with vocalic than sonorant nuclei (β = 0.42, SE = 0.13, z = 3.29, p < 0.01, Figure 3A) while they differed with regard to the individual effects of musical training (Figure 3C) and sensitivity (d’, Figure 3B) on their SMS. More speciﬁcally, similar interactions of group with the individual abilities were found in the subset of data as well as in the whole dataset (cf. Figure 2B,C). 16 of 31 Brain Sci. 2021, 11, 1303 Figure 2. g 3.3.1. SMS Likelihood Likelihood of SMS estimated in adult participants with dyslexia (red lines) vs. controls (blue lines), depending on (A) metrical weight of a syllable, (B) individual sensitivity to acoustic cues d’ and (C) individual musicality scores. Figure 2. Likelihood of SMS estimated in adult participants with dyslexia (red lines) vs. controls (blue lines), depending on (A) metrical weight of a syllable, (B) individual sensitivity to acoustic cues d’ and (C) individual musicality scores. Table 7. Summary of the logistic mixed-effects model best ﬁtting the SMS probability data (weak syllables only). Factor AIC df χ2 p Nucleus type 3186.6 1 8.31 <0.01 Group × individual d’ 3182.4 1 4.17 <0.05 Group × musicality 3189.9 1 11.61 <0.001 Figure 3. Likelihood of SMS estimated in weak syllables, for (A) the main effect of syllable nucleus, (B) the interactions of group and d’-scores and (C) the interaction of group and musicality scores. Dyslexic data are plotted in red, control data in blue. Table 7. Summary of the logistic mixed-effects model best ﬁtting the SMS probability data (weak syllables only). Factor AIC df χ2 p Nucleus type 3186.6 1 8.31 <0.01 Group × individual d’ 3182.4 1 4.17 <0.05 Group × musicality 3189.9 1 11.61 <0.001 Figure 3. Likelihood of SMS estimated in weak syllables, for (A) the main effect of syllable nucleus, (B) the interactions of group and d’-scores and (C) the interaction of group and musicality scores. Dyslexic data are plotted in red, control data in blue. Figure 3. Likelihood of SMS estimated in weak syllables, for (A) the main effect of syllable nucleus, (B) the interactions of group and d’-scores and (C) the interaction of group and musicality scores. Dyslexic data are plotted in red, control data in blue. 17 of 31 Brain Sci. 2021, 11, 1303 17 of 31 3.3.3. Signed Asynchrony A linear mixed model (estimated using REML and the “nloptwrap” optimizer) was ﬁtted to signed asynchrony (scaled and centered around the group mean). Predictors were the metrical weight of the syllable (strong/weak), logarithmically transformed nucleus duration and rise-time of the amplitude envelope, vowel intensity (scaled and centered around the mean), participants’ musicality scores, their sensitivity d’ obtained in the phoneme monitoring task and their signed asynchronies collected in the paced tapping tasks (all individual values were scaled and centered around the group mean). Two-way interactions of each predictor with group (dyslexic/control) were tested. Random effects were participant and sentence. The ﬁrst model included a maximal random effect structure. The ﬁnal model retained only those random effects that produced no convergence or singular ﬁt issues. The total explanatory power of the best-ﬁt model was weak (conditional R2 = 0.03), with a smaller contribution of the ﬁxed effects alone (marginal R2 = 0.02). The estimated effects of the best-ﬁt model are shown in Figure 5. Accordingly, all participants (regardless of their group afﬁliation) tended to predict—that is, tap ahead of— longer syllable nuclei (β = −13.71, SE = 1.70, t = −8.39, p < 0.001, Figure 5A). A differential effect of the group came to the fore in participants with variable signed asynchronies measured in the paced tapping task (Figure 5B). More speciﬁcally, signed asynchronies produced in the task with simple linguistic stimuli playing repetitions of the syllable [bi:] at 600 ms IOI could predict signed asynchronies with the natural linguistic stimuli, but only in the control (β = 6.01, SE = 1.68, t = 3.57, p < 0.01) and not in the dyslexic (β = 0.91, SE = 1.39, t = 0.66, p = 0.52) group of participants. There was a positive linear relationship in the neurotypical participants’ performance, i.e., those predicting a vowel onset in simple, isochronous stimuli, also predicted nucleus onsets in complex, natural stimuli. The relevant results are summarized in Table 9. Table 9. Summary of the linear mixed-effects model best ﬁtting the signed asynchrony data. Table 9. Summary of the linear mixed-effects model best ﬁtting the signed asynchrony data. Factor Sum Sq. 3.3.2. Absolute Asynchrony 2021, 11, 1303 18 of 31 Table 8. Summary of the linear mixed-effects model best ﬁtting the absolute asynchrony data. Factor Sum Sq. df F p Metrical weight 27.15 1 25.25 <0.001 Group × metrical weight 0.01 1 0.01 0.92 Group × individual d’ 13.49 1 12.55 <0.01 Group × musicality 2.18 1 2.03 0.17 Group × rise-time 0.99 1 0.92 0.34 Group × duration 0.26 1 0.24 0.62 Group × intensity 1.29 1 1.20 0.27 Table 8. Summary of the linear mixed-effects model best ﬁtting the absolute asynchrony data. 3.3.2. Absolute Asynchrony We ﬁtted a linear mixed model (estimated using REML and “nloptwrap” optimizer) to predict logarithmically transformed absolute asynchronies with metrical weight of the syllable (strong/weak), nucleus duration, and rise-time of the amplitude envelope around the nucleus onset (both on a logarithmic scale), nucleus intensity (scaled and centered around the mean), participants’ sensitivity (d’), musicality scores and their individual abso- lute asynchronies measured in the paced tapping tasks (all individual values were scaled and centered around the group mean). Participant and sentence were deﬁned as random effects. We tested for two-way interactions of each predictor with group (dyslexic/control) and included a maximal random effect structure, successively reducing the complexity of the initial model by removing predictors that were not signiﬁcant and random effects that caused convergence or singular ﬁt issues. The total explanatory power of the best-ﬁt model was rather weak (conditional R2 = 0.02), equaling to the total contribution of the ﬁxed effects alone (marginal R2 = 0.02). g The magnitude of the effects from the best-ﬁt model is plotted in Figure 4. Accordingly, all participants (regardless of the group) showed longer asynchronies with metrically weak than with metrically strong syllables (β = 0.23, SE = 0.05, t = 5.03, p < 0.001, Figure 4A). A differential effect of the group membership was again found for participants with variable sensitivity (d’): while control participants with higher d’-scores tended to show smaller asynchronies (β = −0.06, SE = 0.03, z = −2.06, p = 0.058), dyslexic participants with higher d’-scores had larger asynchronies in their SMS with vowel onsets (β = 0.13, SE = 0.04, z = 3.08, p < 0.01, Figure 4B). In other words, the more phonemes the control participants were able to spot, the more accurate they were synchronizing with the nucleus onsets, while the opposite is true for the dyslexic participants: the more phonemes they were able to correctly identify in the phoneme monitoring task, the less accurate they tapped with nucleus onsets. A summary of the relevant results is given in Table 8. Figure 4. Predicted values of absolute asynchronies in (A) strong vs. weak syllables, and (B) dyslexic (red) vs. control (blue) participants with variable d’-scores (plotted on the x-axis). Figure 4. Predicted values of absolute asynchronies in (A) strong vs. weak syllables, and (B) dyslexic (red) vs. control (blue) participants with variable d’-scores (plotted on the x-axis). Brain Sci. 4. Discussion The present study set out to investigate rhythm perception and entrainment in dyslexia, and aimed at making a contribution toward a deeper understanding of prosodic deﬁcits and the underlying causes of dyslexia by studying adult dyslexic adults’ perfor- mance in an SMS task with natural speech. The following section discusses the results of the study with respect to the originally outlined open questions and hypotheses. 3.3.3. Signed Asynchrony df F p Nucleus duration 41,602 1 12.77 <0.01 Group × metrical weight 2645 1 0.82 0.36 Group × individual d’ 741 1 0.23 0.64 Group × musicality 1614 1 0.50 0.49 Group × signed asynchrony (600 ms IOI) 29,176 1 8.92 <0.01 Group × rise-time 713 1 0.22 0.64 Group × nucleus duration 102 1 0.03 0.86 Group × relative intensity 10,639 1 3.30 0.07 19 of 31 Brain Sci. 2021, 11, 1303 Figure 5. Predicted signed asynchronies in (A) syllable nuclei of variable duration, and (B) dyslexic (red) vs. control (blue) participants with variable signed asynchronies measured in the paced tapping task with the targets presented at IOI of 600 ms (plotted along the x-axis). On the y-axis, 0 ms refers to the nucleus onset. Figure 5. Predicted signed asynchronies in (A) syllable nuclei of variable duration, and (B) dyslexic (red) vs. control (blue) participants with variable signed asynchronies measured in the paced tapping task with the targets presented at IOI of 600 ms (plotted along the x-axis). On the y-axis, 0 ms refers to the nucleus onset. 4.1. Rhythm Perception and Motor Entrainment in Dyslexia Rhythm perception and motor entrainment were studied by means of a previously developed SMS paradigm that utilized ﬁnger tapping to the subjectively perceived beat of looped sentences [50]. To our knowledge, the present study is the ﬁrst to attest sensorimotor synchronization with natural, syntactically complex and metrically irregular sentences in dyslexic adults. Even though our materials lacked regular occurrences of strong and weak syllables, and contained syllables with non-vocalic nuclei (see Appendix A), they did not generally cause more difﬁculties to the dyslexic participants than to the age- matched controls during SMS. The performance observed in the dyslexic adults’ SMS was not indicative of a prevalent issue with the syllabic entrainment per se, as Leong and Goswami [22] suggested. Instead, we found that the dyslexic participants of the study synchronized with the nuclei of metrically weak syllables less frequently than the controls, without showing any differences in the synchronization accuracy, or prediction tendency for these syllables. At the same time, both groups generally produced fewer and less precise taps with weak syllable nuclei, particularly if those were occupied by sonorants. Both groups synchronized more frequently and more accurately with onsets of strong syllable nuclei. The latter ﬁnding is in line with the results discussed by Leong and Goswami [22], suggesting that adults with developmental dyslexia have no entrainment deﬁcits at larger timescales such as inter-stress intervals. Given the similarity of the overall performance across the two groups, these ﬁnd- ings cannot be easily reconciled with the idea that the hierarchical relationships be- tween stressed and unstressed syllables are inadequately encoded in dyslexic listeners (e.g., [22,110,111,115]). Overall, the metrical weight of syllables was equally well reﬂected Brain Sci. 2021, 11, 1303 20 of 31 20 of 31 in all participants’ SMS, with the only key difference that weak syllables were signiﬁcantly less at the center of rhythmic attention in the dyslexic participants than in the neurotypical controls (cf. [153]). As far as the ongoing discussions of neural entrainment deﬁcits in dyslexia are concerned, the results of the present study can be reconciled with the growing body of research reporting deviant patterns in the delta range (~0.5–4 Hz, [160,161]) which cover the timescale of plausible intervals spanning the distance between unstressed units of the prosodic hierarchy. 4.1. Rhythm Perception and Motor Entrainment in Dyslexia The reduced attention to weak syllables can hardly arise as a purely psychoacoustic effect of locally varied rise-time, duration or intensity, given that these measures had little inﬂuence on the participants’ performance in the SMS task. Even though the rise-time of the experimental materials measured signiﬁcant differences between strong vs. weak syllables and between weak syllables with vocalic vs. sonorant nuclei, such variability in signal acoustics did not have any differential effects on the participants from the two experimental groups. This ﬁnding suggests that rhythm processing in natural speech may not be inﬂuenced by signal rise-time as is sometimes assumed [22,127–129], and that it plays little role in the dyslexic perception of speech rhythm as previously discussed in light of correlational evidence (e.g., [110,115,125,126]). Importantly, the results of the present study stem from an experimental design, and provide innovative evidence in the extension of previous, exclusively correlational studies that showed some inconsistencies of the rise-time effect in dyslexia observed across different tasks (e.g., [110,162]). y g One acoustic-prosodic parameter, if any, might have indirectly shaped the group difference in SMS with weak syllables found in the present study. Since the relative intensity differed systematically between strong vs. weak syllables (but not between the two types of weak syllables), the reduced sensorimotor synchronization with weak syllables observed among dyslexic participants could be related to their rhythmic attention being more extensively reliant upon perceptual prominence than the rhythmic attention of the neurotypical participants. Acoustic intensity, corresponding to the psychoacoustic impression of loudness, is known to dominate the perception of auditory prominence, at least in English [121], and is the only acoustic parameter of the materials that displays a (distant) similarity with the group results found in the SMS experiment. The role of intensity in dyslexic speech processing would thus beneﬁt from a further experimental replication with new speech materials exhibiting prominence variability on a larger scale, and including emphatic and contrastive prominence. A further group-level ﬁnding of the SMS experiment demonstrates that the tendency to anticipate upcoming rhythmic events differed between the dyslexic and the neurotypical groups. In contrast to previous research by Thomson and Goswami [126], who found more anticipation among their dyslexic participants synchronizing with a metronome, there was no general anticipation tendency observed in the present group of dyslexic adults synchronizing either with simple one-word loops or with complex sentence loops. 4.2. Phonological Awareness and Prosody 4.2. Phonological Awareness and Prosody The ﬁndings of the present study contribute to the understanding of the role of prosody in phonological awareness. We considered several possibilities that could explain how the ability to perceive and encode rhythmic structure in speech might be related to the segmental phonological skills, and tested these relations by studying the cross-over effects of the participants’ performance in the two tasks: (1) phoneme monitoring that required segmentation and the processing of variable acoustic cues, and their mapping to the abstract representations of sonorant and obstruent phonemes; and (2) SMS that relied on the perception of a rhythmic beat and motor entrainment to the perceived beat in time. As far as phoneme monitoring is concerned, our prediction that dyslexic listeners would show more difﬁculties in identifying obstruents rather than sonorants (cf. [35,142]) was not borne out by the data, and there was also no effect of the participants’ SMS rates on their identiﬁcation of phonemes embedded in nonsense strings. However, participants’ sensitivity d’ derived from their performance in the phoneme monitoring task explained some individual and group-level variability found in SMS. Two aspects of participants’ SMS—frequency and accuracy (but not anticipation)— could be, at least partly, predicted by their performance during phoneme monitoring. Among the control participants, the individual ability to correctly segment and identify phonemes in nonsense strings was predictive of their tendency to attend to the rhythmic beat of each and every syllable. The more phonemic targets were correctly identiﬁed, the more syllabic nuclei were also synchronized to. This effect was absent in the dyslexic participants’ data, possibly due to a high level of individual variability observed across the two tasks (cf. [163]). However, participants with dyslexia differed signiﬁcantly from the control group in their synchronization accuracy, and showed opposite cross-over effects with regards to the sensitivity d’. While neurotypical participants tapped more accurately in time with the sentence beats if their sensitivity d’ in the phoneme monitoring task was high, dyslexic participants with the highest d’ produced the least accurate asynchronies in the SMS task. Overall, individual performance displayed a high level of cross-task congruence in the neurotypical but not in the dyslexic group of participants (cf. [163]). In a way, dyslexic re- sults can be viewed such that an increased accuracy in a segmental phonology task leads to a delayed response time in a rhythmic synchronization task. 4.1. Rhythm Perception and Motor Entrainment in Dyslexia Rather, our results concerning signed asynchronies indicate a difference in the group-level consistency of SMS behaviors. Among the control participants of the study, there was a high level of consistency in their SMS performance across rhythmically simple and complex tasks. The control participants’ synchronization with natural speech mirrored their performance with simple, isochronous sequences containing a looped monosyllabic word presented at 600 ms IOI. This high level of within-group consistency stands in a stark contrast to the results of the dyslexic participants whose individual tendency to predict an upcoming rhythmic event did not transfer from a simpler to a more complex rhythmic task. As noted in many previous studies, individuals diagnosed with developmental dyslexia frequently form no homogenous group and show high interpersonal variability in their behaviors [163]. Different “routes to failure” [132] might be responsible for the diverse behavioral proﬁles observed in individuals diagnosed with developmental dyslexia, including their non-clinical performance in certain tasks [164–167]. Brain Sci. 2021, 11, 1303 21 of 31 4.4. On the Role of Music Training in Dyslexia The role of music training in speech processing has been discussed in a number of stud- ies, demonstrating beneﬁts for aging adults, difﬁcult listening conditions and short-term memory capacity (e.g., [61–67,69,70,174]). Fueled by the auditory processing hypothe- sis of dyslexic deﬁcits, music-based therapies have been developed and implemented, though with mixed results, as a means of treating dyslexia in children (e.g., [80–83]). Assuming that music practice enhances general auditory skills and thus beneﬁts speech and language processing (e.g., [60,85–87]), we hypothesized that individually variable levels of previous music training would inﬂuence participants’ performance in the two experimental tasks of the present study—phoneme monitoring and SMS—and expected the effect of music training to potentially outweigh the dyslexic status of participants (cf. [88]). Unexpectedly, previous music training did not play a signiﬁcant role in both exper- imental tasks. Only the rhythmic-prosodic SMS task of the present study showed the hypothesized effect, with notable differences between the two groups of participants. This result concerned the likelihood, but not the asynchrony, of rhythmic synchronization with the syllabic targets. In the neurotypical group, we observed an increase in the ten- dency to synchronize with each syllable nucleus in participants with higher levels of music training which is in line with the predictions made by the general auditory skills account (e.g., [60,85–87]). The role of music training in speech processing has been discussed in a number of stud- ies, demonstrating beneﬁts for aging adults, difﬁcult listening conditions and short-term memory capacity (e.g., [61–67,69,70,174]). Fueled by the auditory processing hypothe- sis of dyslexic deﬁcits, music-based therapies have been developed and implemented, though with mixed results, as a means of treating dyslexia in children (e.g., [80–83]). Assuming that music practice enhances general auditory skills and thus beneﬁts speech and language processing (e.g., [60,85–87]), we hypothesized that individually variable levels of previous music training would inﬂuence participants’ performance in the two experimental tasks of the present study—phoneme monitoring and SMS—and expected the effect of music training to potentially outweigh the dyslexic status of participants (cf. [88]). Unexpectedly, previous music training did not play a signiﬁcant role in both exper- imental tasks. Only the rhythmic-prosodic SMS task of the present study showed the hypothesized effect, with notable differences between the two groups of participants. This result concerned the likelihood, but not the asynchrony, of rhythmic synchronization with the syllabic targets. 4.3. Lack of an Acoustic Inﬂuence on the Phonemic and Prosodic Processing 4.3. Lack of an Acoustic Inﬂuence on the Phonemic and Prosodic Processing The present study focused on the acoustic parameters such as rise-time, duration and intensity, and addressed their role in dyslexic processing of natural and nonsense speech. Instead of taking up the prevalent approach from previous research and employing purely psychoacoustic tests to estimate individual sensitivity to these aspects of acoustic signals (e.g., [83,110,115,129–131]), this study estimated the impact of variable signal rise- time, duration and intensity on the dyslexic and control participants’ performance in two experimental tasks, phoneme monitoring and SMS. If an auditory processing deﬁcit of (any of) the above-mentioned acoustic parameters was the main cause underlying dyslexic difﬁculties as sometimes suggested (see [117], for an overview), we would expect to ﬁnd an effect of (at least some of) these parameters on the dyslexic participants’ performance in the verbal tasks. However, the results showed that hardly any of these parameters accounted for the variance either in the phoneme monitoring task, or in the SMS task. Only the duration of a syllable nucleus had an effect on SMS and was predictive of the amount of anticipation. Longer syllable nuclei attracted larger signed asynchronies, i.e., were more anticipated, than shorter nuclei. No further effects of the acoustic properties of syllables were found. Moreover, the dyslexic and the neurotypical adult participants of the present study did not differ in this regard. These results speak to the proposal put forward by Protopapas [132] that the links between auditory difﬁculties observed in dyslexic participants’ non-verbal and verbal pro- cessing (e.g., [83,110,115,129–131,163]) might not be causational as is sometimes suggested, but concomitant, deriving from the same (multitude of) factors and affected by the same (or neighboring) cortical structures. Experimental research with non-correlational designs is needed to further elaborate on the nature of the relation between verbal and non-verbal auditory deﬁcits in individuals of different ages diagnosed with developmental dyslexia. Even though the idea that an auditory processing deﬁcit would cause inaccurate speech perception and therefore lead to inadequate phonological representations and subsequent reading difﬁculties appeals for its simplicity, conﬂicting evidence among existing studies (e.g., [171–173]; see [166] for a review) also points toward the need to revise the hypothesis of a general auditory origin of dyslexia. 4.2. Phonological Awareness and Prosody This might be reﬂective of in- dividually variable perception strategies fueled by speciﬁc speed–accuracy trade-offs in the context of limited cognitive resources [168], and adds to the previous research document- ing auditory short-term memory issues in dyslexia (e.g., [169]; see [29], for an overview). Upon review of a large body of experimental ﬁndings, Ramus and Szenkovits [29] suggest that phonological representations are likely to be intact in individuals diagnosed with developmental dyslexia, but their short-term memory often fails to retrieve them when nec- essary. More recently, Tierney et al. [170] demonstrated a link between reading ability and memory for rhythm. Our results support previous observations, and add further insights from individual variability among adults with dyslexia. Those dyslexic participants of our study who established better access to their segmental phonology, seem to have traded in the response time speed in a rhythmic-prosodic task. Taken together, these ﬁndings suggest that previously documented deﬁcits in the perception and encoding of rhythmic structures in speech [22,43,110–115] are unlikely to constitute the primary issue in dyslexia, but might rather be moderated by an individual severity of difﬁculties in the access to segmental phonological representations, arising as a consequence of a limited ability to attend to the relevant acoustic cues to segmental phono- logical contrasts. Dyslexic deﬁcits in phonological awareness may therefore be caused by insensitivity to sub-lexical units and consequently to acoustic cues encoding higher-level structures such as syllable boundaries, lexical stress and metrical hierarchies. Importantly, the present study outlines an innovative approach for understanding the sources of indi- vidual variability in the frequently heterogenous dyslexic group performance [164–167]. Given that no signiﬁcant group effects were observed during phoneme monitoring despite Brain Sci. 2021, 11, 1303 22 of 31 a high level of individual variability, this approach might in the future help to uncover hidden relationships between dyslexic participants’ non-clinical performance in certain tasks and their underlying deﬁcits. 4.4. On the Role of Music Training in Dyslexia In the neurotypical group, we observed an increase in the ten- dency to synchronize with each syllable nucleus in participants with higher levels of music training which is in line with the predictions made by the general auditory skills account (e.g., [60,85–87]). Brain Sci. 2021, 11, 1303 23 of 31 23 of 31 In contrast, dyslexic participants with more music training synchronized to fewer nuclei. Given that the main difference between the two groups lies in their synchronization with weak syllables, the effect may be interpreted as a shift of rhythmic attention to exclu- sively strong syllables in dyslexic participants with a high level of musical training. Such increased selective sensitivity to stressed syllables might be a compensatory mechanism that dyslexic listeners acquire while learning how to manage the complexities of speech and language processing in light of their limited cognitive resources. Discussing “the enigma of dyslexic musicians”, Weiss et al. [169] concluded that deﬁcits of the auditory short-term memory capacity remain the key issue even in very skilled musicians with developmental dyslexia, regardless of their extensive auditory training during music prac- tice. Since the perception of lexically stressed syllables is of the utmost importance for speech segmentation and lexical access [106,107], a decrease of rhythmic attention paid to unstressed syllables would save cognitive resources while preserving the key functionality in the perceptual language system. Using a prosodic skills battery [175], previous research demonstrated that the ability of dyslexic children to use linguistic prosody for communica- tively relevant purposes such as semantic or syntactic disambiguation was commensurate with their general linguistic abilities [176]. This means that some prosodic processing difﬁculties in dyslexia can indeed be well compensated in communicative interaction, even in young children with developmental dyslexia. Unlike its high relevance in the SMS task, music training did not play an important role in the phoneme monitoring task (either alone or in an interaction with the participant group). This ﬁnding suggests that the beneﬁts of music training may be speciﬁc to the per- ception of speech prosody and do not generalize to the perception of segmental phonology. Even though word-level segmentation skills improve in listeners with a higher level of musical skills [67,68], the auditory beneﬁts do not seem to transfer to the phoneme-level segmentation that was involved in the phoneme monitoring task of the present study. 4.4. On the Role of Music Training in Dyslexia Since language and music share primarily prosodic-acoustic features related to pitch, tim- ing and timbre [86], music training is likely to bring about an exclusive advantage for the processing of these aspects of auditory signals [177,178]. The exact mechanisms underpin- ning phonological awareness and the role of musical and prosodic skills in its development are yet to be fully established and require further research. 5. Conclusions The present study focused on dyslexic adults with and without music training, and investigated their rhythm perception in, and entrainment to, natural speech [50], along with their ability to perceive segmental phonological contrasts. Our results showed that both dyslexic and neurotypical participants attended less to metrically weak than to metrically strong syllables, particularly if the syllabic nuclei were occupied by sonorants; however, participants with dyslexia did so to a greater extent than the neurotypical, age-matched controls. Overall, participants’ performance during phoneme monitoring was predictive of their performance during SMS, but not vice versa, suggesting that prosodic processing is not the primary deﬁcit in dyslexia as previously suggested [22,43,110–115]. Against expectation, signal acoustics such as rise-time, duration and intensity played no role in the group performance during either the segmental or the prosodic task, calling for a revision of the general auditory deﬁcit hypothesis of dyslexia put forward on the basis of correlational evidence (e.g., [127–129]). This conclusion is further corroborated by the ﬁnding that higher levels of music training did not lead to an improved performance of our dyslexic participants across all tasks as the general auditory deﬁcit hypothesis would predict. Rather, the effect of increased music training was speciﬁc to the rhythmic task, and its direction was suggestive of a compensatory strategy in musically trained adults with dyslexia as a means of coping with limited short-term memory resources (cf. [29,166]). In their review of the controversial ﬁndings surrounding the nature of dyslexic deﬁcits, Ramus and Ahissar [166] concluded that prosody perception and/or production have only been found impaired in the tasks that involved metalinguistic judgements of prosody or Brain Sci. 2021, 11, 1303 24 of 31 24 of 31 other difﬁculty factors such as the presence of background noise, an increased short-term memory load or time pressure in a task at hand. The present results complement and corroborate this proposal by contributing innovative evidence to the understanding of individual pathways to compensation for language processing issues among dyslexic adults (cf. [132]). The study had not been designed to investigate the role of short-term memory in dyslexic processing of prosody, and its conclusions concerning the involvement of the cognitive resources require a follow-up investigation involving measurements of auditory short-term memory capacity, in order to corroborate the present ﬁndings. 5. Conclusions y y p y p g Of course, the results of the present study reﬂect, and are limited to, the performance of well-compensated adults who had been diagnosed with developmental dyslexia as children and reported merely mild dyslexic symptoms at the time of testing. It is possible that dyslexic children will show different experimental proﬁles indicative of more substantial deﬁcits, due to the lack of compensation strategies that adults may have developed over time (e.g., [179]). A longitudinal or cross-sectional study would be beneﬁcial in establishing individual trajectories in dyslexic language processing, and the role of music training in this development. p The conclusions above support the cognitive theories of dyslexia (cf. [165,180]), at least as far as auditory processing is concerned. Independent evidence exists on poor readers’ deﬁcits in senses other than audition, including vision (e.g., [181,182]), somatosen- sation [183], proprioception [184] and audiovisual integration ([185]; see [186], for a review). In order to advance the understanding of the underlying causes of developmental dyslexia, future research will beneﬁt from cross-disciplinary approaches and comprehensive, non- correlational designs to study the complexities involved in reading ability and skills. Author Contributions: Conceptualization, T.R.; Data curation, C.-Y.L.; Formal analysis, T.R. and C.-Y.L.; Funding acquisition, T.R.; Investigation, C.-Y.L.; Methodology, C.-Y.L.; Project administration, T.R.; Software, C.-Y.L.; Supervision, T.R. and C.-Y.L.; Writing—original draft, T.R.; Writing—review & editing, T.R. and C.-Y.L. All authors have read and agreed to the published version of the manuscript. Funding: This research (including APC) was funded by the Leverhulme Trust (RPG-2017-306 grant to the ﬁrst author). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Faculty of Humanities Research Ethics Advisory Group for Human Participants at the University of Kent (protocol code 0291920, received 6 January 2020). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The data collected for the present study can be made available upon request. Acknowledgments: The authors are extremely grateful to the study participants who were willing to attend the lab-based experiment despite a number of restrictions in place during the ongoing pandemic and would further like to thank Naomi Caplan who created the materials and collected part of the experimental data for the present study. Conﬂicts of Interest: The authors declare no conﬂict of interest. Appendix A The list of English sentences used in the study. 5. Conclusions Sentence Metrical Structure Total Number of Syllables Number of Non-Vocalic Nuclei Her cackle was not subtle. WSWWWSW 7 2 The cattle suddenly escaped. WSWSWWWS 8 2 The broken bottle made a rattle as it fell. WSWSWSWSWWWS 12 2 The milk carton is biodegradable. WSSWWSWSWW 11 1 The couple watched the stars twinkle. WSWSWSSW 8 2 Tom and his uncle explored the jungle. SWWSWWSWSW 10 2 Author Contributions: Conceptualization, T.R.; Data curation, C.-Y.L.; Formal analysis, T.R. and C.-Y.L.; Funding acquisition, T.R.; Investigation, C.-Y.L.; Methodology, C.-Y.L.; Project administration, T.R.; Software, C.-Y.L.; Supervision, T.R. and C.-Y.L.; Writing—original draft, T.R.; Writing—review & editing, T.R. and C.-Y.L. All authors have read and agreed to the published version of the manuscript. Funding: This research (including APC) was funded by the Leverhulme Trust (RPG-2017-306 grant to the ﬁrst author). Funding: This research (including APC) was funded by the Leverhulme Trust (RPG-2017-306 grant to the ﬁrst author). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Faculty of Humanities Research Ethics Advisory Group for Human Participants at the University of Kent (protocol code 0291920, received 6 January 2020). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The data collected for the present study can be made available upon request. Data Availability Statement: The data collected for the present study can be made available upon request Acknowledgments: The authors are extremely grateful to the study participants who were willing to attend the lab-based experiment despite a number of restrictions in place during the ongoing pandemic and would further like to thank Naomi Caplan who created the materials and collected part of the experimental data for the present study. Conﬂicts of Interest: The authors declare no conﬂict of interest. Appendix A The list of English sentences used in the study. Sentence Metrical Structure Total Number of Syllables Number of Non-Vocalic Nuclei Her cackle was not subtle. WSWWWSW 7 2 The cattle suddenly escaped. WSWSWWWS 8 2 The broken bottle made a rattle as it fell. WSWSWSWSWWWS 12 2 The milk carton is biodegradable. WSSWWSWSWW 11 1 The couple watched the stars twinkle. WSWSWSSW 8 2 Tom and his uncle explored the jungle. SWWSWWSWSW 10 2 Brain Sci. 5. Conclusions 2021, 11, 1303 25 of 31 Sentence Metrical Structure Total Number of Syllables Number of Non-Vocalic Nuclei Fresh apple juice is delicious. SSWSWWSW 8 1 The seamstress seldom lost her buttons. WSWSWSWSW 9 1 The only juice available was pineapple. WSWSWSWWWSWW 12 1 Passers-by had trodden down the autumnal leaves. SWSWSWSWWSWS 12 1 The café will certainly serve bacon on toast. WSWWSWWSSWWS 12 1 The student had written an exceptional essay. WSWWSWWWSWWSW 13 2 He felt sudden pain in his ankle. WSSWSWWSW 9 2 The wooden chair was not affordable. WSWSWWWSWW 10 2 Grandma’s rhubarb crumble is heaven. SWWSSWSW 9 1 In the autumn, she wears comfortable boots. WWSWWSSWWWS 11 1 There are lots of puddles in autumn. WWSWSWWSW 9 1 The purple bicycle was the fastest. WSWSWWWWSW 10 2 The door handle has broken. WSSWWSW 7 1 The people were woken by thunder. WSWWSWWSW 9 1 4. Nicolson, R.I.; Fawcett, A.J.; Dean, P. Developmental dyslexia: The cerebellar deﬁcit hypothesis. Trends Neurosci. 2001, 24, 508–511. [CrossRef] 4. Nicolson, R.I.; Fawcett, A.J.; Dean, P. Developmental dyslexia: The cerebellar deﬁcit hypothesis. Trends Neurosci. 2001, 24, 508–511. [CrossRef] [ ] 5. Pagliarini, E.; Guasti, M.T.; Toneatto, C.; Granocchio, E.; Riva, F.; Sarti, D.; Molteni, B.; Stucchi, N. Dyslexic children fail to comply with the rhythmic constraints of handwriting. Hum. Mov. Sci. 2015, 42, 161–182. [CrossRef] 6. Treiman, R. Spelling in Normal Children and Dyslexics. In Foundations of Reading Acquisition and Dyslexia, Implications for Early Intervention; Routledge: London, UK, 1997; pp. 191–218. g pp Seymour, P.H.K. Beyond the Phonological Deﬁcit Hypothesis. Child Psychol. Psychiatry Rev. 1998, 3, 22–23 F A Ni l R D l i Th l f h b ll El J R Ed P h l 2 35 58 8. Fawcett, A.; Nicolson, R. Dyslexia: The role of the cerebellum. Electron. J. Res. Educ. Psychol. 2004, 2, 35–58. tt, A.; Nicolson, R. Dyslexia: The role of the cerebellum g p y y 10. Richlan, F. Functional neuroanatomy of developmental dyslexia: The role of orthographic depth. Front. Hum. Neurosci. 2014, 8. [CrossRef] [PubMed] 11. Petkov, C.I.; O’Connor, K.N.; Benmoshe, G.; Baynes, K.; Sutter, M.L. Auditory perceptual grouping and attention in dyslexia. Cogn. Brain Res. 2005, 24, 343–354. [CrossRef] [PubMed] 12. Snowling, M. Dyslexia as a Phonological Deﬁcit: Evidence and Implications. Child Psychol. Psychiatry Rev. 1998, 3, 4–11. [CrossRef] 13. Stanovich, K.E.; Siegel, L.S. 20. Goswami, U.; Bryant, P. Phonological Skills and Learning to Read; Lawrence Erlbaum: Hove, UK, 1990. 5. Conclusions 2006, 29, 334–348. [CrossRef] N. The ability to tap to a beat relates to cognitive, linguistic, and perceptual skills. Brain Lang. 2013, 124 41. Tierney, A.; Kraus, N. The ability to tap to a beat relates to cognitive, linguistic, and perceptual skil 225–231. [CrossRef] 42. Rayner, K.; Pollatsek, A.; Bilsky, A.B. Can a temporal processing deﬁcit account for dyslexia? Psychon. Bull. Rev. 1995, 2, 501–507. [CrossRef] [ ] 43. Wood, C.; Terrell, C. Poor readers’ ability to detect speech rhythm and perceive rapid speech. Br. J. Dev. Psychol. 1998, 16, 397–413. [CrossRef] [ ] 44. Marcus, S.M. Acoustic determinants of perceptual center (P-center) location. Percept. Psychophys. 1981, 30, 247–256. [CrossRef] 45. Morton, J.; Marcus, S.; Frankish, C. Perceptual centers (P-centers). Psychol. Rev. 1976, 83, 405–408. [CrossRef] Acoustic determinants of perceptual center (P-center) location. Percept. Psychophys. 1981, 30, 247–256. [Cro 44. Marcus, S.M. Acoustic determinants of perceptual center (P-center) location. Percept. Psychophys. 1981, us, S.M. Acoustic determinants of perceptual center (P-center) location. Percept. Psychophys. 1981, 30, 247–25 on, J.; Marcus, S.; Frankish, C. Perceptual centers (P-centers). Psychol. Rev. 1976, 83, 405–408. [CrossRef] 45. Morton, J.; Marcus, S.; Frankish, C. Perceptual centers (P-centers). Psychol. Rev. 1976, 83, 405–408. [Cro 46. Leong, V. Prosodic Rhythm in the Speech Amplitude Envelope: Amplitude Modulation Phase Hierarchies University of Cambridge: Cambridge, UK, 2012. 46. Leong, V. Prosodic Rhythm in the Speech Amplitude Envelope: Amplitude Modulation Phase Hierarchies (AMPHs) and AMPH Models; University of Cambridge: Cambridge, UK, 2012. 47. Tilsen, S.; Arvaniti, A. Speech rhythm analysis with decomposition of the amplitude envelope: Characterizing rhythmic patterns within and across languages. J. Acoust. Soc. Am. 2013, 134, 628–639. [CrossRef] [PubMed] g g 48. Lin, C.-Y.; Rathcke, T. How to hit that beat: Testing acoustic anchors of rhythmic movement with s 10th International Conference on Speech Prosody, Tokyo, Japan, 24–28 May 2020; pp. 1–5. [CrossRe Lin, C.-Y.; Rathcke, T. How to hit that beat: Testing acoustic anchors of rhythmic movement with speech. 10th International Conference on Speech Prosody Tokyo Japan 24–28 May 2020; pp 1–5 [CrossRef] n, C.-Y.; Rathcke, T. How to hit that beat: Testing acoustic anchors of rhythmic movement with speech. In th International Conference on Speech Prosody, Tokyo, Japan, 24–28 May 2020; pp. 1–5. [CrossRef] 49. Rathcke, T.; Lin, C.-Y.; Falk, S.; Dalla Bella, S. When language hits the beat: Synchronising movement to simple tonal and verbal stimuli. 5. Conclusions 1991, 83, 346–360. [CrossRef] 26. Vellutino, F.R.; Fletcher, J.M.; Snowling, M.J.; Scanlon, D.M. Speciﬁc reading disability (dyslexia): What have we learned in the past four decades? J. Child. Psychol. Psychiatry 2004, 45, 2–40. [CrossRef] 27. Caravolas, M.; Lervåg, A.; Mousikou, P.; Efrim, C.; Litavský, M.; Onochie-Quintanilla, E.; Salas, N.; Schöffelová, M.; Deﬁor, S.; Mikulajová, M.; et al. Common Patterns of Prediction of Literacy Development in Different Alphabetic Orthographies. Psychol. Sci. 2012, 23, 678–686. [CrossRef] 29. Ramus, F.; Szenkovits, G. What Phonological Deﬁcit? Q. J. Exp. Psychol. 2008, 61, 129–141. [CrossRef] [PubMed] g p y .L. The Oxford Handbook of Cognitive Psychology. Reisberg, D., Ed.; Oxford University Press: Oxford, UK 30. Mattys, S.L. The Oxford Handbook of Cognitive Psychology. Reisberg, D., Ed.; Oxford University 391–412. 31. Serniclaes, W.; Sprenger-Charolles, L. Reading impairment: From behavior to brain. In Routledge Handbook of Communication Disorders; Bahr, R.H., Silliman, E.R., Eds.; Routledge: London, UK, 2015; pp. 34–45. [CrossRef] 32. Serniclaes, W.; Sprenger-Charolles, L.; Carré, R.; Demonet, J.-F. Perceptual Discrimination of Speech Sounds in Developmental Dyslexia. J. Speech Lang. Hear. Res. 2001, 44, 384–399. [CrossRef] 33. Vandermosten, M.; Boets, B.; Luts, H.; Poelmans, H.; Golestani, N.; Wouters, J.; Ghesquiere, P. Adults with dyslexia are impaired in categorizing speech and nonspeech sounds on the basis of temporal cues. Proc. Natl. Acad. Sci. USA 2010, 107, 10389–10394. [CrossRef] [PubMed] 34. Werker, J.F.; Tees, R.C. Speech perception in severely disabled and average reading children. Can. J. Psychol. Can. Psychol. 1987, 41, 48–61. [CrossRef] [PubMed] 35. Noordenbos, M.W.; Serniclaes, W. The Categorical Perception Deﬁcit in Dyslexia: A Meta-Analysis. Sci. Stud. Read. 2015, 19, 340–359. [CrossRef] 36. Noordenbos, M.; Segers, E.; Serniclaes, W.; Mitterer, H.; Verhoeven, L. Neural evidence of allophonic perception in children at risk for dyslexia. Neuropsychology 2012, 50, 2010–2017. [CrossRef] 37. Noordenbos, M.W.; Segers, E.; Serniclaes, W.; Verhoeven, L. Neural evidence of the allophonic mode of speech perception in adults with dyslexia. Clin. Neurophysiol. 2013, 124, 1151–1162. [CrossRef] [PubMed] p y precision and rhythm in developmental dyslexia. Read. Writ. 2002, 15, 179–206. [CrossRef] y p y 38. Wolff, P.H. Timing precision and rhythm in developmental dyslexia. Read. Writ. 2002, 15, 179–206. [Cr 39. Wolff, P.H.; Michel, G.F.; Ovrut, M.; Drake, C. Rate and timing precision of motor coordination in developmental dyslexia. Dev. Psychol. 1990, 26, 349–359. [CrossRef] 40. Thomson, J.; Fryer, B.; Maltby, J.; Goswami, U. Auditory and motor rhythm awareness in adults with dyslexia. J. Res. Read. 5. Conclusions Phenotypic performance proﬁle of children with reading disabilities: A regression-based test of the phonological-core variable-difference model. J. Educ. Psychol. 1994, 86, 24–53. [CrossRef] 13. Stanovich, K.E.; Siegel, L.S. Phenotypic performance proﬁle of children with reading disabilities: A regression-based test of the phonological-core variable-difference model. J. Educ. Psychol. 1994, 86, 24–53. [CrossRef] g 14. Wagner, R.K.; Torgesen, J.K. The nature of phonological processing and its causal role in the acquisition of reading skills. Psychol. Bull. 1987, 101, 192–212. [CrossRef] 15. Bradley, L.; Bryant, P.E. Categorizing sounds and learning to read—A causal connection. Nature 1983, 301, 419–421. [CrossRef] 16. Bruck, M.; Treiman, R. Phonological awareness and spelling in normal children and dyslexics: The case of initial consonant clusters. J. Exp. Child Psychol. 1990, 50, 156–178. [CrossRef] Å Bradley, L.; Bryant, P.E. Categorizing sounds and le 16. Bruck, M.; Treiman, R. Phonological awareness and spelling in normal children and dyslexics: The case of initial consonant clusters. J. Exp. Child Psychol. 1990, 50, 156–178. [CrossRef] p y 17. Lundberg, I.; Olofsson, Å.; Wall, S. Reading and spelling skills in the ﬁrst school years predicted from phonemic awareness skills in kindergarten. Scand. J. Psychol. 1980, 21, 159–173. [CrossRef] g y 18. Stahl, S.A.; Murray, B.A. Deﬁning phonological awareness and its relationship to early reading. J. Educ. Psychol. 1994, 86, 221–234. [CrossRef] 18. Stahl, S.A.; Murray, B.A. Deﬁning phonological awareness and its relationship to early reading. J. Educ. Psychol. 1994, 86, 221–234. [CrossRef] 20. Goswami, U.; Bryant, P. Phonological Skills and Learning to Read; Lawrence Erlbaum: Hove, UK, 1990. 20. Goswami, U.; Bryant, P. Phonological Skills and Learning to Read; Lawrence Erlbaum: Hove, UK, 1990. Brain Sci. 2021, 11, 1303 26 of 31 21. Goswami, U.; Leong, V. Speech rhythm and temporal structure: Converging perspectives? Lab. Phonol. J. Assoc. Lab. Phonol. 2013, 4, 67–92. [CrossRef] , [ ] 22. Leong, V.; Goswami, U. Assessment of rhythmic entrainment at multiple timescales in dyslexia: Evidence for disruption to syllable timing. Hear. Res. 2014, 308, 141–161. [CrossRef] y g 23. Snowling, M.J. From language to reading and dyslexia. Dyslexia 2001, 7, 37–46. [CrossRef] [PubMed] 24. Ziegler, J.C.; Goswami, U. Reading Acquisition, Developmental Dyslexia, and Skilled Reading Across guistic Grain Size Theory. Psychol. Bull. 2005, 131, 3–29. [CrossRef] g y y 25. Treiman, R. Children’s spelling errors on syllable-initial consonant clusters. J. Educ. Psychol. 1991, 83, 3 25. Treiman, R. Children’s spelling errors on syllable-initial consonant clusters. J. Educ. Psychol. 5. Conclusions In Proceedings of the 19th International Congress of Phonetic Sciences, Melbourne, Australia, 5–9 August 2019; pp. 1505–1509. 50. Rathcke, T.; Lin, C.-Y.; Falk, S.; Bella, S.D. Tapping into linguistic rhythm. Lab. Phonol. J. Assoc. Lab. Phono T.; Lin, C.-Y.; Falk, S.; Bella, S.D. Tapping into linguistic rhythm. Lab. Phonol. J. Assoc. Lab. Phonol. 2021, 12, 11. Metrical Stress Theory: Principles and Case Studies; Chicago University of Chicago Press: Chicago IL USA 1 51. Hayes, B. Metrical Stress Theory: Principles and Case Studies; Chicago University of Chicago Press: Chica 52. Liberman, M.; Prince, A. On Stress and Linguistic Rhythm. Linguist. Inq. 1977, 8, 249–336. [CrossRef] 53. Selkirk, E. On derived domains in sentence phonology. Phonol. Yearb. 1986, 3, 371–405. [CrossRef] 27 of 31 Brain Sci. 2021, 11, 1303 54. Lidji, P.; Palmer, C.C.; Peretz, I.I.; Morningstar, M. Listeners feel the beat: Entrainment to English and French speech rhythms. Psychon. Bull. Rev. 2011, 18, 1035–1041. [CrossRef] y 55. Repp, B.H. Sensorimotor synchronization: A review of the tapping literature. Psychon. Bull. Rev. 2005, 12, 969–992. [CrossRef] [PubMed] [ ] 56. Repp, B.H.; Su, Y.-H. Sensorimotor synchronization: A review of recent research (2006–2012). Psychon. Bull. Rev. 2013, 20, 403–452. [CrossRef] [ ] 57. Bella, S.D.; Farrugia, N.; Benoit, C.-E.; Begel, V.; Verga, L.; Harding, E.; Kotz, S.A. BAASTA: Battery for the Assessment of Auditory Sensorimotor and Timing Abilities. Behav. Res. Methods 2017, 49, 1128–1145. [CrossRef] 58. Baer, L.; Thibodeau, J.L.N.; Gralnick, T.M.; Li, K.; Penhune, V. The role of musical training in emergent and event-based timing. Front. Hum. Neurosci. 2013, 7, 191. [CrossRef] 59. Drake, C.; Jones, M.R.; Baruch, C. The development of rhythmic attending in auditory sequences: Attu focal attending. Cognition 2000, 77, 251–288. [CrossRef] R.; Baruch, C. The development of rhythmic attending in auditory sequences: Attunement, referent period nition 2000, 77, 251–288. [CrossRef] 60. Kraus, N.; Chandrasekaran, B. Music training for the development of auditory skills. Nat. Rev. Neurosci. 2010, 11, 599–605. [CrossRef] 61. Parbery-Clark, A.; Skoe, E.; Lam, C.; Kraus, N. Musician Enhancement for Speech-In-Noise. Ear Hear. 2009, 30, 653–661. [CrossRef] 62 Strait D ; Parbery Clark A ; Hittner E ; Kraus N Musical training during early childhood enhances the neural encoding of 61. Parbery-Clark, A.; Skoe, E.; Lam, C.; Kraus, N. Musician Enhancement for Speech-In-Noise. Ear Hear. 2009, 30, 653–661. [CrossRef] 62. Strait, D.; Parbery-Clark, A.; Hittner, E.; Kraus, N. 5. Conclusions Musical training during early childhood enhances the neural encoding of speech in noise. Brain Lang. 2012, 123, 191–201. [CrossRef] p 63. Zendel, B.R.; Alain, C. Musicians experience less age-related decline in central auditory processing. Psychol. Aging 2012, 27, 410–417. [CrossRef] 64. Chan, A.S.; Ho, Y.-C.; Cheung, M.C. Music training improves verbal memory. Nat. Cell Biol. 1998, 396, 128. [CrossRef] 65. Strait, D.L.; Kraus, N.; Parbery-Clark, A.; Ashley, R. Musical experience shapes top-down auditory me masking and auditory attention performance. Hear. Res. 2010, 261, 22–29. [CrossRef] g y p 66. Tierney, A.T.; Bergeson, T.R.; Pisoni, D.B. Effects of Early Musical Experience on Auditory Sequence Memory. Empir. Music. Rev. 2008, 3, 178–186. [CrossRef] [PubMed] 67. François, C.; Chobert, J.; Besson, M.; Schön, D. Music Training for the Development of Speech Segmentation. Cereb. Cortex 2013, 23, 2038–2043. [CrossRef] [PubMed] 68. François, C.; Jaillet, F.; Takerkart, S.; Schön, D. Faster Sound Stream Segmentation in Musicians than in Nonmusicians. PLoS ONE 2014, 9, e101340. [CrossRef] [PubMed] 69. Bidelman, G.M.; Weiss, M.W.; Moreno, S.; Alain, C. Coordinated plasticity in brainstem and auditory cortex contributes to enhanced categorical speech perception in musicians. Eur. J. Neurosci. 2014, 40, 2662–2673. [CrossRef] 70. Slater, J.; Skoe, E.; Strait, D.L.; O’Connell, S.; Thompson, E.; Kraus, N. Music training improves speech-in-noise perception: Longitudinal evidence from a community-based music program. Behav. Brain Res. 2015, 291, 244–252. [CrossRef] g y p g 71. Gérard, C.; Rosenfeld, M. Pratique musicale et régulations temporelles. L’Année Psychol. 1995, 95, 5 y p g osenfeld, M. Pratique musicale et régulations temporelles. L’Année Psychol. 1995, 95, 571–591. [CrossRef] 72. Pressing, J.; Jolley-Rogers, G. Spectral properties of human cognition and skill. Biol. Cybern. 1997, 76, 339–347. [CrossRef] [PubMed] 73. Repp, B.H.; Penel, A. Auditory dominance in temporal processing: New evidence from synchronization with simultaneous visual and auditory sequences. J. Exp. Psychol. Hum. Percept. Perform. 2002, 28, 1085–1099. [CrossRef] [PubMed] y q p y p f 74. Repp, B.H. Sensorimotor synchronization and perception of timing: Effects of music training and tas 2010, 29, 200–213. [CrossRef] 75. Cogo-Moreira, H.; Avila, C.R.B.; Ploubidis, G.B.; Mari, J.D.J. Effectiveness of Music Education for the Improvement of Reading Skills and Academic Achievement in Young Poor Readers: A Pragmatic Cluster-Randomized, Controlled Clinical Trial. PLoS ONE 2013, 3, e59984. [CrossRef] 76. Herrera, L.; Lorenzo, O.; Deﬁor, S.; Fernandez-Smith, G.; Costa-Giomi, E. Effects of phonological and reading readiness of native- and foreign-Spanish-speaking children. Psychol. Music. 2011, 39, 68–81. 5. Conclusions Language, music, and the brain: A resource-sharing framework. In Language and Music as Cognitive Systems; R b h t P R h i M H ki J A C I Ed O f d U i it P O f d UK 2012 204 223 86. Patel, A.D. Language, music, and the brain: A resource-sharing framework. In Language and Mu Rebuschat, P., Rohmeier, M., Haw-kins, J.A., Cross, I., Eds.; Oxford University Press: Oxford, UK, 2012 J y pp 87. Tierney, A.; Ekraus, N. Auditory-motor entrainment and phonological skills: Precise auditory timing hypothesis (PATH). Front. Hum. Neurosci. 2014, 8, 949. [CrossRef] [PubMed] 88. Boll-Avetisyan, N.; Bhatara, A.; Höhle, B. Processing of Rhythm in Speech and Music in Adult Dyslexia. Brain Sci. 2020, 10, 261. [CrossRef] [PubMed] 89. Chen, J.L.; Penhune, V.B.; Zatorre, R.J. Listening to Musical Rhythms Recruits Motor Regions of the Brain. Cereb. Cortex 2008, 18, 2844–2854. [CrossRef] 89. Chen, J.L.; Penhune, V.B.; Zatorre, R.J. Listening to Musical Rhythms Recruits Motor Regions of the Brain. Cereb. Cortex 2008, 18, 2844–2854. [CrossRef] 90. Grahn, J.A.; Rowe, J. Feeling the Beat: Premotor and Striatal Interactions in Musicians and Nonmusicians during Beat Perception. ] Feeling the Beat: Premotor and Striatal Interactions in Musicians and Nonmusicians during Beat Perception 7540–7548. [CrossRef] 90. Grahn, J.A.; Rowe, J. Feeling the Beat: Premotor and Striatal Interactions in Musicians and Nonmusi J. Neurosci. 2009, 29, 7540–7548. [CrossRef] 91. Iversen, J.R.; Repp, B.H.; Patel, A.D. Top-Down Control of Rhythm Perception Modulates Early Auditory Responses. Ann. N. Y. Acad. Sci. 2009, 1169, 58–73. [CrossRef] [PubMed] 92. Morillon, B.; Schroeder, C.E.; Wyart, V. Motor contributions to the temporal precision of auditory attention. Nat. Commun. 2014, 5, 5255. [CrossRef] 93. Stupacher, J.; Hove, M.J.; Novembre, G.; Schütz-Bosbach, S.; Keller, P. Musical groove modulates motor cortex excitability: A TMS investigation. Brain Cogn. 2013, 82, 127–136. [CrossRef] 94. Su, Y.-H.; Pöppel, E. Body movement enhances the extraction of temporal structures in auditory sequences. Psychol. Res. 2012, 76, 373–382. [CrossRef] 95. Zatorre, R.J.; Chen, J.L.; Penhune, V.B. When the brain plays music: Auditory–motor interactio production. Nat. Rev. Neurosci. 2007, 8, 547–558. [CrossRef] p 96. Ainsworth, W.A. Duration as a Cue in the Recognition of Synthetic Vowels. J. Acoust. Soc. Am. 1972, 51, 648–651. [CrossRef] p 96. Ainsworth, W.A. Duration as a Cue in the Recognition of Synthetic Vowels. J. Acoust. Soc. Am. 1972, 51, 648–651. [CrossRef] 97. Fischer, R.M.; Ohde, R.N. 5. Conclusions Spectral and duration properties of front vowels as cues to ﬁnal stop-consonant voicing. J. Acoust. Soc. Am. 1990, 88, 1250–1259. [CrossRef] , g y J , , [ ] 97. Fischer, R.M.; Ohde, R.N. Spectral and duration properties of front vowels as cues to ﬁnal stop-consonant voicing. J. Acoust. Soc. Am. 1990, 88, 1250–1259. [CrossRef] 98. Mermelstein, P. Difference limens for formant frequencies of steady-state and consonant-bound vowels. J. Acoust. Soc. Am. 1978, 63, 572–580. [CrossRef] 99 S W E l i h i f l i J A t S A 1989 85 2081 2087 [C R f] 63, 572–580. [CrossRef] 99. Strange, W. Evolving theories of vowel perception. J. Acoust. Soc. Am. 1989, 85, 2081–2087. [CrossRef] Strange, W. Evolving theories of vowel perception. J. Acoust. Soc. Am. 1989, 85, 2081–2087. [CrossRef] 100. Whalen, D.H. Vowel and consonant judgments are not independent when cued by the same information. Percept. Psychophys. 1989, 46, 284–292. [CrossRef] [PubMed] 101. Kemps, R.J.J.K.; Wurm, L.H.; Ernestus, M.; Schreuder, R.; Baayen, H. Prosodic cues for morphological complexity in Dutch and English. Lang. Cogn. Process. 2005, 20, 43–73. [CrossRef] 102. Cho, T.; McQueen, J.; Cox, E.A. Prosodically driven phonetic detail in speech processing: The case of in English. J. Phon. 2007, 35, 210–243. [CrossRef] ; Cox, E.A. Prosodically driven phonetic detail in speech processing: The case of domain-initial strengthening 2007, 35, 210–243. [CrossRef] 103. Davis, M.H.; Marslen-Wilson, W.D.; Gaskell, M.G. Leading up the lexical garden path: Segmentation and ambiguity in spoken word recognition. J. Exp. Psychol. Hum. Percept. Perform. 2002, 28, 218–244. [CrossRef] g p y p f 104. Salverda, A.P.; Dahan, D.; McQueen, J. The role of prosodic boundaries in the resolution of lexical embedding in speech comprehension. Cognition 2003, 90, 51–89. [CrossRef] 105. Smith, R.; Rathcke, T. Glasgow Gloom or Leeds Glue? Dialect-Speciﬁc Vowel Duration Constrains Lexical Segmentation and Access. Phonetica 2017, 74, 1–24. [CrossRef] [PubMed] 106. Cutler, A.; Norris, D. The role of strong syllables in segmentation for lexical access. J. Exp. Psychol. Hum. Percept. Perform. 1988, 14, 113–121. [CrossRef] 107. Klein, H.B. Learning to stress: A case study. J. Child Lang. 1984, 11, 375–390. [CrossRef] [PubMed] 107. Klein, H.B. Learning to stress: A case study. J. Child Lang. 1984, 11, 375–390. [CrossRef] [PubMed] 108. Cutler, A.; Carter, D.M. The predominance of strong initial syllables in the English vocabulary. Comput. Speech Lang. 1987, 2, 133–142. [CrossRef] 108. Cutler, A.; Carter, D.M. 5. Conclusions [C g g p p g y , , [ ] 77. Moreno, S.; Marques, C.; Santos, A.; Santos, M.; Castro, S.L.; Besson, M. Musical Training Inﬂuences Linguistic Abilities in 8-Year-Old Children: More Evidence for Brain Plasticity. Cereb. Cortex 2009, 19, 712–723. [CrossRef] 77. Moreno, S.; Marques, C.; Santos, A.; Santos, M.; Castro, S.L.; Besson, M. Musical Training Inﬂuenc 8-Year-Old Children: More Evidence for Brain Plasticity. Cereb. Cortex 2009, 19, 712–723. [CrossRef] 78. Moreno, S.; Friesen, D.; Bialystok, E. Effect of Music Training on Promoting Preliteracy Skills: Preliminary Causal Evidence. Music. Percept. Interdiscip. J. 2011, 29, 165–172. [CrossRef] p p 79. Myant, M.; Armstrong, W.; Healy, N. Can music make a difference? A small scale longitudinal study into the ef-fects of music instruction in nursery on later reading ability. Educ. Child Psychol. 2008, 25, 83–96. y g y 80. Bhide, A.; Power, A.; Goswami, U. A Rhythmic Musical Intervention for Poor Readers: A Comparison of Efﬁcacy with a Letter-Based Intervention. Mind Brain Educ. 2013, 7, 113–123. [CrossRef] 81. Overy, K. Dyslexia and Music: From Timing Deﬁcits to Musical Intervention. Ann. N. Y. Acad. Sci. 2003, 999, 497–505. [CrossRef] 82. Register, D.; Darrow, A.-A.; Swedberg, O.; Standley, J. The Use of Music to Enhance Reading Skills of Second Grade Students and Students with Reading Disabilities. J. Music. Ther. 2007, 44, 23–37. [CrossRef] [PubMed] 81. Overy, K. Dyslexia and Music: From Timing Deﬁcits to Musical Intervention. Ann. N. Y. Acad. Sci. 82. Register, D.; Darrow, A.-A.; Swedberg, O.; Standley, J. The Use of Music to Enhance Reading Skills o Students with Reading Disabilities. J. Music. Ther. 2007, 44, 23–37. [CrossRef] [PubMed] g 83. Thompson, J.M.; Nuerk, H.-C.; Moeller, K.; Kadosh, R.C. The link between mental rotation ability and basic numerical representa- tions. Acta Psychol. 2013, 144, 324–331. [CrossRef] [PubMed] y 84. Vidal, M.M.; Lousada, M.; Vigário, M. Music effects on phonological awareness development in 3-year-old children. Appl. Psycholinguist. 2020, 41, 299–318. [CrossRef] Brain Sci. 2021, 11, 1303 28 of 31 28 of 31 85. Gordon, R.L.; Fehd, H.M.; McCandliss, B.D. Does Music Training Enhance Literacy Skills? A Meta-Analysis. Front. Psychol. 2015, 6, 1777. [CrossRef] , [ ] 86. Patel, A.D. Language, music, and the brain: A resource-sharing framework. In Language and Music as Cognitive Systems; Rebuschat, P., Rohmeier, M., Haw-kins, J.A., Cross, I., Eds.; Oxford University Press: Oxford, UK, 2012; pp. 204–223. 86. Patel, A.D. 5. Conclusions The predominance of strong initial syllables in the English vocabulary. Comput. Speech Lang. 1987, 2, 133–142. [CrossRef] 109. Lindﬁeld, K.C.; Wingﬁeld, A.; Goodglass, H. The Role of Prosody in the Mental Lexicon. Brain Lang. 1999, 68, 312–317. [CrossRef] 110. Goswami, U.; Gerson, D.; Astruc, L. Amplitude envelope perception, phonology and prosodic sensitivity in children with developmental dyslexia. Read. Writ. 2010, 23, 995–1019. [CrossRef] 109. Lindﬁeld, K.C.; Wingﬁeld, A.; Goodglass, H. The Role of Prosody in the Mental Lexicon. Brain Lang. , ; g , ; g , y g , , [ ] 110. Goswami, U.; Gerson, D.; Astruc, L. Amplitude envelope perception, phonology and prosodic sensitivity in children with developmental dyslexia. Read. Writ. 2010, 23, 995–1019. [CrossRef] 111. Goswami, U.; Huss, M.; Mead, N.; Fosker, T.; Verney, J.P. Perception of patterns of musical beat distribution in phonological developmental dyslexia: Signiﬁcant longitudinal relations with word reading and reading comprehension. Cortex 2013, 49, 1363–1376. [CrossRef] 112. Holliman, A.J.; Wood, C.; Sheehy, K. Does speech rhythm sensitivity predict children’s reading ability 1 year later? J. Educ. Psychol. 2010, 102, 356–366. [CrossRef] 113. Holliman, A.J.; Wood, C.; Sheehy, K. A cross-sectional study of prosodic sensitivity and reading difﬁculties. J. Res. Read. 2012, 35, 32–48. [CrossRef] 114. Kitzen, K.R. Prosodic sensitivity, morphological ability, and reading ability in young adults with and without childhood histories of reading difﬁculty. In Dissertation Abstracts International Section A: Humanities and Social Sciences; ProQuest Dissertations Publishing: Ann Arbor, MI, USA, 2001. Brain Sci. 2021, 11, 1303 29 of 31 29 of 31 115. Leong, V.; Hamalainen, J.; Soltész, F.; Goswami, U. Rise time perception and detection of syllable stress in adults with develop- mental dyslexia. J. Mem. Lang. 2011, 64, 59–73. [CrossRef] 116. Whalley, K.M.; Hansen, J.A. The role of prosodic sensitivity in children’s reading development. J. Res. Read. 2006, 29, 288–303. [CrossRef] ] äläinen, J.A.; Salminen, H.K.; Leppänen, P. Basic Auditory Processing Deﬁcits in Dyslexia: Systematic 117. Hämäläinen, J.A.; Salminen, H.K.; Leppänen, P. Basic Auditory Processing Deﬁcits in Dyslexia: Systematic Review of the Behavioral and Event-Related Potential/Field Evidence. J. Learn. Disabil. 2013, 46, 413–427. [CrossRef] [PubMed] vioral and Event-Related Potential/Field Evidence. J. Learn. Disabil. 2013, 46, 413–427. [CrossRef] [PubMed] r, K.J. From Communicative Functions to Prosodic Forms. Phonetica 2013, 70, 24–65. [CrossRef] [PubMed] 118. Kohler, K.J. From Communicative Functions to Prosodic Forms. Phonetica 2013, 70, 24–65. [C 119. Gussenhoven, C.; Chen, A. (Eds.) Introduction. 5. Conclusions In The Oxford Handbook of Language Prosody; Oxford University Press: Oxford, UK, 2021; pp. 1–15. [CrossRef] 120. Gordon, M.; Roettger, T.B. Acoustic correlates of word stress: A cross-linguistic survey. Linguist. Vanguard 2017, 3. [CrossRef] 121. Kochanski, G.; Grabe, E.; Coleman, J.; Rosner, B. Loudness predicts prominence: Fundamental frequency lends little. J. Acoust. Soc. Am. 2005, 118, 1038–1054. [CrossRef] 120. Gordon, M.; Roettger, T.B. Acoustic correlates of word stress: A cross-linguistic survey. Linguist. Vangu 120. Gordon, M.; Roettger, T.B. Acoustic correlates of word stress: A cross-linguistic survey. Linguist. Vanguard 2017, 3. [CrossRef] 121 K h ki G G b E C l J R B L d di t i F d t l f l d littl J A t 120. Gordon, M.; Roettger, T.B. Acoustic correlates of word stress: A cross-linguistic survey. Linguist. Vanguard 2017, 3. [CrossRef] 121. Kochanski, G.; Grabe, E.; Coleman, J.; Rosner, B. Loudness predicts prominence: Fundamental frequency lends little. J. Acoust. 121. Kochanski, G.; Grabe, E.; Coleman, J.; Rosner, B. Loudness predicts prominence: Fundamental freque Soc. Am. 2005, 118, 1038–1054. [CrossRef] 122. Braun, B.; Lemhöfer, K.; Mani, N. Perceiving unstressed vowels in foreign-accented English. J. Acoust. Soc. Am. 2011, 129, 376. [CrossRef] 123. Yu, V.Y.; Andruski, J.E. A Cross-Language Study of Perception of Lexical Stress in English. J. Psycholinguist. Res. 2009, 39, 323–344. [CrossRef] 124. Abrams, D.A.; Nicol, T.; Zecker, S.; Kraus, N. Abnormal Cortical Processing of the Syllable Rate of Speech in Poor Readers. J. Neurosci. 2009, 29, 7686–7693. [CrossRef] 125. Goswami, U.; Fosker, T.; Huss, M.; Mead, N.; Szucs, D. Rise time and formant transition duration in the discrimination of speech sounds: The Ba-Wa distinction in developmental dyslexia. Dev. Sci. 2011, 14, 34–43. [CrossRef] 126. Thomson, J.M.; Goswami, U. Rhythmic processing in children with developmental dyslexia: Auditory a reading and spelling. J. Physiol. 2008, 102, 120–129. [CrossRef] reading and spelling. J. Physiol. 2008, 102, 120 129. [CrossRef] 127 Goswami U A temporal sampling framework for developmental dyslexia Trends Cogn Sci 2011 15 3–10 [CrossRef] g p g J y [ ] 127. Goswami, U. A temporal sampling framework for developmental dyslexia. Trends Cogn. Sci. 2011, 15, 3–10. [CrossRef] 127. Goswami, U. A temporal sampling framework for developmental dyslexia. Trends Cogn. Sci. 2011, 15, 3–10. [CrossRef] 128. Goswami, U. Speech rhythm and language acquisition: An amplitude modulation phase hierarchy perspective. Ann. N. Y. Acad. Sci. 2019, 1453, 67–78. [CrossRef] [PubMed] 128. Goswami, U. 5. Conclusions Speech rhythm and language acquisition: An amplitude modulation phase hierarchy perspective. Ann. N. Y. Acad. Sci. 2019, 1453, 67–78. [CrossRef] [PubMed] 129. Goswami, U.; Thomson, J.; Richardson, U.; Stainthorp, R.; Hughes, D.; Rosen, S.; Scott, S. Amplitu developmental dyslexia: A new hypothesis. Proc. Natl. Acad. Sci. USA 2002, 99, 10911–10916. [CrossRe 129. Goswami, U.; Thomson, J.; Richardson, U.; Stainthorp, R.; Hughes, D.; Rosen, S.; Scott, S. Amplitude envelope onsets and developmental dyslexia: A new hypothesis. Proc. Natl. Acad. Sci. USA 2002, 99, 10911–10916. [CrossRef] [PubMed] 130 H l i J L P T M M ll K L i H D i f d i i b d l i h d l i B i 129. Goswami, U.; Thomson, J.; Richardson, U.; Stainthorp, R.; Hughes, D.; Rosen, S.; Scott, S. Amplitude envelope onsets and developmental dyslexia: A new hypothesis. Proc. Natl. Acad. Sci. USA 2002, 99, 10911–10916. [CrossRef] [PubMed] 130. Hamalainen, J.; Leppänen, P.; Torppa, M.; Müller, K.; Lyytinen, H. Detection of sound rise time by adults with dyslexia. Brain y y 130. Hamalainen, J.; Leppänen, P.; Torppa, M.; Müller, K.; Lyytinen, H. Detection of sound rise time by Lang. 2005, 94, 32–42. [CrossRef] [PubMed] g 131. Muneaux, M.; Ziegler, J.; Truc, C.; Thomson, J.; Goswami, U. Deﬁcits in beat perception and dyslexia: Evidence from French. NeuroReport 2004, 15, 1255–1259. [CrossRef] [PubMed] p 132. Protopapas, A. From temporal processing to developmental language disorders: Mind the gap. Philos. Trans. R. Soc. B Biol. Sci. 2014, 369, 20130090. [CrossRef] 133. Howell, P.; Rosen, S. Production and perception of rise time in the voiceless affricate/fricative distinction. J. Acoust. Soc. Am. 1983, 73, 976–984. [CrossRef] 134. Walsh, M.A.; Diehl, R.L. Formant Transition Duration and Amplitude Rise Time as Cues to the Stop/Glide Distinction. Q. J. Exp. Psychol. Sect. A 1991, 43, 603–620. [CrossRef] [PubMed] y 135. Bella, S.D.; Biału´nska, A.; Sowinski, J. Why Movement Is Captured by Music, but Less by Speech: Rol PLoS ONE 2013, 8, e71945. [CrossRef] É a, A.; Sowinski, J. Why Movement Is Captured by Music, but Less by Speech: Role of Temporal Regularity 71945. [CrossRef] 136. Lagrois, M.-É.; Palmer, C.; Peretz, I. Poor Synchronization to Musical Beat Generalizes to Speech. Brain Sci. 2019, 9, 157. [CrossRef] [PubMed] [ ] 137. Wells, J. The Phonological Status of Syllabic Consonants in English R.P. Phonetica 1965, 13, 110–113. [CrossRef] 138. Ohala, J.J.; Ohala, M. 5. Conclusions SIMR: An R package for power analysis of generalized linear mixed models by Evol. 2016, 7, 493–498. [CrossRef] 152. Wickham, H. ggplot2: Elegant Graphics for Data Analysis; Springer: New York, NY, USA, 2016. [Cros 152. Wickham, H. ggplot2: Elegant Graphics for Data Analysis; Springer: New York, NY, USA, 2016. [CrossRef] 153. Large, E.W.; Jones, M.R. The dynamics of attending: How people track time-varying events. Psychol. Rev. 1999, 106, 119–159. [CrossRef] 153. Large, E.W.; Jones, M.R. The dynamics of attending: How people track time-varying events. Psy [CrossRef] 154. Borchers, H.W. Pracma: Practical Numerical Math Functions. 2018. Available online: https://cran.r- (accessed on 22 September 2021). 155. Bates, D.; Mächler, M.; Bolker, B.; Walker, S. Fitting Linear Mixed-Effects Models Using lme4. J. Stat. Softw. 2015, 67, 1–48. [CrossRef] [ ] 156. Lenth, R.V. Emmeans: Estimated Marginal Means, aka Least-Squares Means. R Package Version 1.6.0. 2021. Available online: https://CRAN.R-project.org/package=emmeans (accessed on 22 September 2021). 157. Lüdecke, D. sjPlot: Data Visualization for Statistics in Social Science. 2019. Available online: https://cran.r-project.org/web/ packages/sjPlot/sjPlot.pdf (accessed on 22 September 2021). [CrossRef] 158. Baayen, R.H. Analyzing Linguistic Data: A Practical Introduction to Statistics Using R; Cambridge University Press: Cambridge, UK, 2008. [CrossRef] g 159. Barr, D.J.; Levy, R.; Scheepers, C.; Tily, H.J. Random effects structure for conﬁrmatory hypothesis Mem. Lang. 2013, 68, 255–278. [CrossRef] 159. Barr, D.J.; Levy, R.; Scheepers, C.; Tily, H.J. Random effects structure for conﬁrmatory hypothesis testing: Keep it maximal. J. Mem. Lang. 2013, 68, 255–278. [CrossRef] 159. Barr, D.J.; Levy, R.; Scheepers, C.; Tily, H.J. Random effects structure for conﬁrmatory hypothesis testing: Keep it maximal. J. Mem. Lang. 2013, 68, 255–278. [CrossRef] 160. Molinaro, N.; Lizarazu, M.; Lallier, M.; Bourguignon, M.; Carreiras, M. Out-of-synchrony speech entrainment in developmental 160. Molinaro, N.; Lizarazu, M.; Lallier, M.; Bourguignon, M.; Carreiras, M. Out-of-synchrony speech en dyslexia. Hum. Brain Mapp. 2016, 37, 2767–2783. [CrossRef] azu, M.; Lallier, M.; Bourguignon, M.; Carreiras, M. Out-of-synchrony speech entrainment in developmental n Mapp. 2016, 37, 2767–2783. [CrossRef] y pp 161. Colling, L.J.; Noble, H.L.; Goswami, U. Neural Entrainment and Sensorimotor Synchronization to the Beat in Children with Developmental Dyslexia: An EEG Study. Front. Neurosci. 2017, 11, 360. [CrossRef] 162. Surányi, Z.; Csépe, V.; Richardson, U.; Thomson, J.M.; Honbolygó, F.; Goswami, U.; Thomson, J. Sensitivity to rhythmic parameters in dyslexic children: A comparison of Hungarian and English. Read. Writ. 2008, 22, 41–56. 5. Conclusions [CrossRef] 163 T ll l P A dit t l ti h i d di di biliti i hild B i L 1980 9 182 198 [C R f] y temporal perception, phonics, and reading disabilities in children. Brain Lang. 1980, 9, 182–198. [CrossRe 163. Tallal, P. Auditory temporal perception, phonics, and reading disabilities in children. Brain Lang. 1 y p p p p g g 164. Lallier, M.; Thierry, G.; Tainturier, M.-J. On the importance of considering individual proﬁles when investigating the role of auditory sequential deﬁcits in developmental dyslexia. Cognition 2013, 126, 121–127. [CrossRef] y y 165. Ramus, F. Developmental dyslexia: Speciﬁc phonological deﬁcit or general sensorimotor dysfunction? Curr. Opin. Neurobiol. 2003, 13, 212–218. [CrossRef] 166. Ramus, F.; Ahissar, M. Developmental dyslexia: The difﬁculties of interpreting poor performance, and the importance of normal performance. Cogn. Neuropsychol. 2012, 29, 104–122. [CrossRef] 167. Talcott, J.B.; Witton, C.; Stein, J.F. Probing the neurocognitive trajectories of children’s reading skills. Neuropsychology 2013, 51, 472–481. [CrossRef] 168. Schouten, J.F.; Bekker, J.A.M. Reactlon Tine and Accuracy. Acta Psychol. 1967, 27, 143–153. [CrossRef] y y H.; Granot, R.; Ahissar, M. The enigma of dyslexic musicians. Neuropsychology 2014, 54, 28–40. [CrossRef] 169. Weiss, A.H.; Granot, R.; Ahissar, M. The enigma of dyslexic musicians. Neuropsychology 2014, 54, 28–40 g y p y gy 170. Tierney, A.; Gomez, J.C.; Fedele, O.; Kirkham, N.Z. Reading ability in children relates to rhythm perception across modalities. J. Exp. Child Psychol. 2021, 210, 105196. [CrossRef] 171. Georgiou, G.K.; Protopapas, A.; Papadopoulos, T.C.; Skaloumbakas, C.; Parrila, R. Auditory temporal processing and dyslexia in an orthographically consistent language. Cortex 2010, 46, 1330–1344. [CrossRef] g p y g g 172. Georgiou, G.K.; Papadopoulos, T.C.; Zarouna, E.; Parrila, R. Are Auditory and Visual Processing Deﬁcits Related to Developmental Dyslexia? Dyslexia 2012, 18, 110–129. [CrossRef] y 173. Papadopoulos, T.C.; Georgiou, G.K.; Parrila, R. Low-level deﬁcits in beat perception: Neither necessary nor sufﬁcient for explaining developmental dyslexia in a consistent orthography. Res. Dev. Disabil. 2012, 33, 1841–1856. [CrossRef] [PubMed] 174. Parbery-Clark, A.; Strait, D.; Anderson, S.; Hittner, E.; Kraus, N. Musical Experience and the Aging Auditory System: Implications for Cognitive Abilities and Hearing Speech in Noise. PLoS ONE 2011, 6, e18082. [CrossRef] [PubMed] 175. McCann, J.; Peppé, S.J. Prosody in autism spectrum disorders: A critical review. Int. J. Lang. Commun. [CrossRef] [PubMed] 176. Marshall, C.; Harcourt-Brown, S.; Ramus, F.; Van Der Lely, H.K.J. 5. Conclusions The Phonetics of Nasal Phonology: Theorems and Data. In Nasals, Nasalization, a Amsterdam, The Netherlands, 1993; pp. 225–249. pp 139. Selkirk, E. On the major class features and syllable theory. In Language Sound Structure; Aronoff, M., Oehrle, R.T., Eds.; MIT Press: Cambridge, MA, USA, 1984. g 140. Van Son, R.J.J.H.; Pols, L.C.W. A comparison between the acoustics of vowel and consonant reduction. Proc. Inst. Phon. Sci. 1996, 20, 13–25. Available online: https://www.fon.humanities.uva.nl/archive/1996/Proc20SonPols.pdf (accessed on 22 September 2021). Septe be 0 ) 141. Lust, B.C. Child language: Acquisition and growth. In Child Language: Acquisition and Growth; Cornell University: New York, NY, USA, 2006. [CrossRef] p 141. Lust, B.C. Child language: Acquisition and growth. In Child Language: Acquisition and Growth; Cornell U USA, 2006. [CrossRef] 142. Mody, M.; Studdert-Kennedy, M.; Brady, S. Speech Perception Deﬁcits in Poor Readers: Auditory Processing or Phonological Coding? J. Exp. Child Psychol. 1997, 64, 199–231. [CrossRef] g p y 143. Huggins, A.W.F. Just Noticeable Differences for Segment Duration in Natural Speech. J. Acoust. Soc. Am. 1972, 51, 1270–1278. [CrossRef] 144. Stefanics, G.; Fosker, T.; Huss, M.; Mead, N.; Szucs, D.; Goswami, U. Auditory sensory deﬁcits in developmental dyslexia: A longitudinal ERP study. NeuroImage 2011, 57, 723–732. [CrossRef] [PubMed] 145. Todd, J.; Michie, P.T. Do perceived loudness cues contribute to duration mismatch negativity (MMN)? NeuroReport 2000, 11, 3771–3774. [CrossRef] Brain Sci. 2021, 11, 1303 30 of 31 146. Boersma, P.; Weenink, D. Praat: Doing Phonetics by Computer [Computer Program]. Version 6.1.52. 2021. Available online: http://www.praat.org/ (accessed on 22 September 2021). p p g p 147. Stuart-Smith, J.; Sonderegger, M.; Rathcke, T.; Macdonald, R. The private life of stops: VOT in a real-time corpus of spontaneous Glaswegian. Lab. Phonol. J. Assoc. Lab. Phonol. 2015, 6, 505–549. [CrossRef] Š g 148. Šturm, P.; Volin, J. P-centres in natural disyllabic Czech words in a large-scale speech-metronome synchronization experiment. J. Phon. 2016, 55, 38–52. [CrossRef] 149. Smythe, I.; Everatt, J. Dyslexia in the Digital Age; Bloomsbury Publishing: London, UK, 2001; Available online: https://media. bloomsbury.com/rep/ﬁles/ditda-worksheets-appendix-1.pdf (accessed on 22 September 2021). 150. Snowling, M.; Dawes, P.; Nash, H.; Hulme, C. Validity of a Protocol for Adult Self-Report of Dyslexia and Related Difﬁculties. Dyslexia 2012, 18, 1–15. [CrossRef] [PubMed] C. SIMR: An R package for power analysis of generalized linear mixed models by simulation. Methods Ecol 8. [CrossRef] 151. Green, P.; MacLeod, C. 5. Conclusions The link between prosody and language skills in children with speciﬁc language impairment (SLI) and/or dyslexia. Int. J. Lang. Commun. Disord. 2009, 44, 466–488. [CrossRef] 31 of 31 31 of 31 Brain Sci. 2021, 11, 1303 177. Fujioka, T.; Trainor, L.; Ross, B.; Kakigi, R.; Pantev, C. Musical Training Enhances Automatic Encoding of Melodic Contour and Interval Structure. J. Cogn. Neurosci. 2004, 16, 1010–1021. [CrossRef] g 178. Johnson, K.L.; Nicol, T.; Zecker, S.G.; Kraus, N. Developmental Plasticity in the Human Auditory Brainstem. J. Neurosci. 2008, 28, 4000–4007. [CrossRef] 179. Goswami, U. Why theories about developmental dyslexia require developmental designs. Trends Cogn. Sci. 2003, 7, 534–540. [CrossRef] [PubMed] 180. Goswami, U. Sensory theories of developmental dyslexia: Three challenges for research. Nat. Rev. Neurosci. 2015, 16, 43–54. [CrossRef] 181. Lovegrove, W.; Bowling, A.; Badcock, D.; Blackwood, M. Speciﬁc reading disability: Differences in contrast sensitivity as a function of spatial frequency. Science 1980, 210, 439–440. [CrossRef] Â 182. Vilhena, D.D.A.; Guimarães, M.R.; Guimarães, R.Q.; Vieira Pinheiro, Â.M. Magnocellular visual fu dyslexia: Deﬁcit in frequency-doubling perimetry and ocular motor skills. Arq. Bras. Oftalmol. 2021, 84 182. Vilhena, D.D.A.; Guimarães, M.R.; Guimarães, R.Q.; Vieira Pinheiro, Â.M. Magnocellular visual function in developmental dyslexia: Deﬁcit in frequency-doubling perimetry and ocular motor skills. Arq. Bras. Oftalmol. 2021, 84, 442–448. [CrossRef] 183. Stoodley, C.J.; Talcott, J.B.; Carter, E.L.; Witton, C.; Stein, J.F. Selective deﬁcits of vibrotactile sensitivity in dyslexic readers. Neurosci. Lett. 2000, 295, 13–16. [CrossRef] 183. Stoodley, C.J.; Talcott, J.B.; Carter, E.L.; Witton, C.; Stein, J.F. Selective deﬁcits of vibrotactile sensitivity in dyslexic readers. Neurosci. Lett. 2000, 295, 13–16. [CrossRef] 184. Laprevotte, J.; Papaxanthis, C.; Saltarelli, S.; Quercia, P.; Gaveau, J. Movement detection thresholds reveal proprioceptive impairments in developmental dyslexia. Sci. Rep. 2021, 11, 299. [CrossRef] [PubMed] 185. Harrar, V.; Tammam, J.; Perez-Bellido, A.; Pitt, A.; Stein, J.; Spence, C. Multisensory Integration and Attention in Developmental Dyslexia. Curr. Biol. 2014, 24, 531–535. [CrossRef] [PubMed] 186. Hahn, N.; Foxe, J.J.; Molholm, S. Impairments of multisensory integration and cross-sensory learning as pathways to dyslexia. Neurosci. Biobehav. Rev. 2014, 47, 384–392. [CrossRef] [PubMed]
https://openalex.org/W4394959356	https://www.frontiersin.org/articles/10.3389/fninf.2024.1323203/pdf?isPublishedV2=False	English	null	Building a realistic, scalable memory model with independent engrams using a homeostatic mechanism	Frontiers in neuroinformatics	2,024	cc-by	18,018	TYPE Original Research PUBLISHED 19 April 2024 DOI 10.3389/fninf.2024.1323203 TYPE Original Research PUBLISHED 19 April 2024 DOI 10.3389/fninf.2024.1323203 OPEN ACCESS OPEN ACCESS EDITED BY Dong Song, University of Southern California, United States REVIEWED BY Shuangming Yang, Tianjin University, China Georgios Detorakis, Independent Researcher, Irvine, United States CORRESPONDENCE Marvin Kaster marvin.kaster@tu-darmstadt.de RECEIVED 17 October 2023 ACCEPTED 27 March 2024 PUBLISHED 19 April 2024 CITATION Kaster M, Czappa F, Butz-Ostendorf M and Wolf F (2024) Building a realistic, scalable memory model with independent engrams using a homeostatic mechanism. Front. Neuroinform. 18:1323203. doi: 10.3389/fninf.2024.1323203 OPEN ACCESS EDITED BY Dong Song, University of Southern California, United States Marvin Kaster1, Fabian Czappa1, Markus Butz-Ostendorf1,2 and Felix Wolf1 Marvin Kaster1, Fabian Czappa1, Markus Butz-Ostendorf1,2 and 1 1Laboratory for Parallel Programming, Department of Computer Science, Technical University of Darmstadt, Darmstadt, Germany, 2Data Science, Translational Medicine and Clinical Pharmacology, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany Memory formation is usually associated with Hebbian learning and synaptic plasticity, which changes the synaptic strengths but omits structural changes. A recent study suggests that structural plasticity can also lead to silent memory engrams, reproducing a conditioned learning paradigm with neuron ensembles. However, this study is limited by its way of synapse formation, enabling the formation of only one memory engram. Overcoming this, our model allows the formation of many engrams simultaneously while retaining high neurophysiological accuracy, e.g., as found in cortical columns. We achieve this by substituting the random synapse formation with the Model of Structural Plasticity. As a homeostatic model, neurons regulate their activity by growing and pruning synaptic elements based on their current activity. Utilizing synapse formation based on the Euclidean distance between the neurons with a scalable algorithm allows us to easily simulate 4 million neurons with 343 memory engrams. These engrams do not interfere with one another by default, yet we can change the simulation parameters to form long-reaching associations. Our model’s analysis shows that homeostatic engram formation requires a certain spatiotemporal order of events. It predicts that synaptic pruning precedes and enables synaptic engram formation and that it does not occur as a mere compensatory response to enduring synapse potentiation as in Hebbian plasticity with synaptic scaling. Our model paves the way for simulations addressing further inquiries, ranging from memory chains and hierarchies to complex memory systems comprising areas with diferent learning mechanisms. CITATION Kaster M, Czappa F, Butz-Ostendorf M and Wolf F (2024) Building a realistic, scalable memory model with independent engrams using a homeostatic mechanism. Front. Neuroinform. 18:1323203. OPEN ACCESS doi: 10.3389/fninf.2024.1323203 COPYRIGHT © 2024 Kaster, Czappa, Butz-Ostendorf and Wolf. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. KEYWORDS learning, memory, homeostatic plasticity, structural plasticity, scalable COPYRIGHT © 2024 Kaster, Czappa, Butz-Ostendorf and Wolf. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. learning, memory, homeostatic plasticity, structural plasticity, scalable Frontiers in Neuroinformatics Building a realistic, scalable memory model with independent engrams using a homeostatic mechanism OPEN ACCESS EDITED BY Dong Song, University of Southern California, United States REVIEWED BY Shuangming Yang, Tianjin University, China Georgios Detorakis, Independent Researcher, Irvine, United States CORRESPONDENCE Marvin Kaster marvin.kaster@tu-darmstadt.de RECEIVED 17 October 2023 ACCEPTED 27 March 2024 PUBLISHED 19 April 2024 CITATION Kaster M, Czappa F, Butz-Ostendorf M and Wolf F (2024) Building a realistic, scalable memory model with independent engrams using a homeostatic mechanism. Front. Neuroinform. 18:1323203. doi: 10.3389/fninf.2024.1323203 1 Introduction Memory is a key ingredient in our thinking and learning process. However, the understanding of learning and memories is still very limited. In 1921, Richard Semon described the idea of a memory engram, the neurophysiological trace of a memory. Nowadays, working memory is usually characterized by persistent neuron activity (Barak and Tsodyks, 2014). Recent study (Fiebig and Lansner, 2017) modeled this persistent activity with a partly plastic network and synaptic plasticity. However, synaptic plasticity is limited to changing the weights of already existing connections and prohibits the formation of new synapses between neurons. A memory engram, e.g., in the form of a strongly interconnected group of neurons, could represent an abstract memory. If we now want to form an association with another memory in the form of a memory engram, we are limited to strengthening existing synapses with synaptic plasticity alone. We cannot build an association if there is no or only sparse Frontiers in Neuroinformatics 01 10.3389/fninf.2024.1323203 Kaster et al. memory engrams simulated with homeostatic structural plasticity. We showed that the engrams do not interfere with each other. connectivity between these two engrams. Moreover, ignoring structural plasticity limits the storage capacity of the brain drastically (Chklovskii et al., 2004), and structural plasticity can overcome both problems. An alternative for a model using only synaptic plasticity is to use all-to-all connectivity, but this is impractical for large networks. Additionally, recent study showed that structural plasticity also plays an essential role in the biology of memory formation (Butz et al., 2009; May, 2011; Holtmaat and Caroni, 2016) and especially long-term memory, on the other hand, is usually characterized by silent synapses (Gallinaro et al., 2022). The structure of the model underlying our simulation draws inspiration from the human cortex, which is organized in columns further subdivided into minicolumns (Mountcastle, 1997). Each of these minicolumns consists of excitatory pyramid cells that act as a group for a speciﬁc feature depending on the cortex area. For example, a minicolumn in the visual cortex represents the orientation of an object in a visual ﬁeld (Hubel and Wiesel, 1962). The diﬀerent minicolumns combined comprise a hypercolumn that enables encoding all orientations for a certain spot on the retina. Similar organizations can be found in the auditory cortex (Reale and Imig, 1980) and the somatosensory cortex (Ruben et al., 2001). 1 Introduction The columns of higher associative cortices represent more complex features such as colors (Hadjikhani et al., 1998), objects (López- Aranda et al., 2009), or persons (Downing and Peelen, 2011). In our study, we adopted the cortical organization of the associative cortices, where complex features can be learned and represented in a column. Hence, we split our network into boxes that represent a column. Each column represents a feature that acts independently from neighboring columns, and, therefore, all columns can be active simultaneously. In our example with the visual cortex, two neighboring columns encode an orientation for their ﬁeld of view, respectively, without interference. is usually characterized by silent synapses (Gallinaro et al., 2022). While most structures (Kalisman et al., 2005; Mizrahi, 2007) and synapses in the developed brain remain stable, there is evidence that learning (Holtmaat and Svoboda, 2009) and sensory input leads to an increased synapse turnover, growth of dendritic spines, and axonal remodeling (Barnes and Finnerty, 2010). For example, the alternate trimming of whisker hair of mice leads to a changed sensory input and an increased dendritic spine turnover (Trachtenberg et al., 2002), with 50% of the formed spines becoming stable. Another example is the change in the brain’s gray matter when adults learn juggling (Boyke et al., 2008). Recently, it was proposed that synaptic plasticity plays a vital role in memory consolidation (Butz et al., 2009; Caroni et al., 2012; Holtmaat and Caroni, 2016). This is the process of transmitting information from the short-term memory into the long-term memory. First, learning increases the synaptic turnover and the formation of vacant synaptic elements (Butz et al., 2009; Holtmaat and Svoboda, 2009; Caroni et al., 2012). Synaptic elements are dendritic spines and axonal boutons, which, if unoccupied, generate potential synapses (Stepanyants et al., 2002). Some potential synapses later materialize, forming actual new synapses (Butz et al., 2009; Caroni et al., 2012). The correlation between spine stabilization and the performance of animals in tests (Xu et al., 2009) supports this hypothesis further. Moreover, when newly formed synapses during training are impaired, the performance of the animal decreases (Hayashi-Takagi et al., 2015), indicating that the formation of new synapses is essential for learning. However, how the learning process interacts with the increased turnover and stabilization of speciﬁc spines, without overall modiﬁcation of the structures in the brain, remains unclear (Caroni et al., 2012). 1 Introduction Our contributions are: • We increased the neurophysiological plausibility of homeostatic memory models, which enables us to model cortical-like structures. • Our model maintains the introduced structure of the cortical columns without additional constraints on how the neurons can connect. • We formed multiple ensembles in parallel without unwanted interference between them. This is comparable to the brain with its many concurrent activities. Frontiers in Neuroinformatics 2.1.1 Growth model dCa dt = ( −Ca τ + β if neuron ﬁres −Ca τ otherwise (1) dCa dt = ( −Ca τ + β if neuron ﬁres −Ca τ otherwise (1) (1) The calcium concentration that acts as a proxy for the ﬁring rate is described in Equation (1). It decreases over time with the time constant τ and increases by β when the neuron ﬁres. Based on the calcium concentration, the number of synaptic elements of a neuron grows or shrinks as described in Equations (2–4). It depends on the growth rate ν, the current calcium concentration Ca, the minimum calcium concentration η required to grow synaptic elements, and the target calcium rate ǫ. Hence, if the calcium concentration is higher than the target, the neuron prunes synaptic elements; it grows new elements if the concentration is lower than the target. We follow Butz and van Ooyen (2013) for selecting our minimum calcium concentration ηaxon, ηden_ex, and ηden_inh, where they showed that a high ηaxon and a low ηden 2 Materials and methods In the experiment, we followed the study by Gallinaro et al. (2022), modeling a conditioned learning paradigm. In a conditioned learning paradigm, a subject learns the relationship between an unconditioned stimulus and a neutral stimulus. Before the experiment, the subject shows an unconditioned reaction to the unconditioned stimulus. During the experiment, both unconditioned (e.g., food) and neutral stimuli (e.g., bell) are presented simultaneously. After the experiment, the subject shows the same previously unconditioned (now conditioned) reaction (e.g., salivating) for the previously neutral (now conditioned) stimulus. At the level of neuronal networks, we modeled a stimulus by stimulating neurons and a reaction as the ﬁring of a downstream, so-called readout, neuron. We split the whole population into boxes of equal side lengths and divided the neurons within each box into four distinct neuron ensembles: Unconditioned stimulus (US), conditioned stimulus 1 (C1), conditioned stimulus 2 (C2), and the rest. We added a single readout neuron (R) to each box that Dammasch (1990) proposed the idea of forming memory engrams based on structural plasticity. Recently, Gallinaro et al. (2022) developed a model that can form silent memory engrams with only structural plasticity on a homeostatic basis. They showed that a homeostatic rule implicitly models a Hebbian learning rule and demonstrated this principle with a conditioned learning paradigm. However, their model connects neurons uniformly at random, making the simultaneous formation of clustered neurons as distinct memory engrams impossible. The stimulation of multiple memory engrams at once would lead to one single large memory engram instead of multiple small ones, which diﬀers from the brain, where diﬀerent regions are active simultaneously without interfering with one another. We extended their work with a more neurophysiological accurate approach by connecting neurons depending on their distance and forming memory engrams simultaneously. We simulated 4 million neurons with 343 engrams, enormously increasing the number of simulated neurons and Frontiers in Neuroinformatics 02 frontiersin.org Kaster et al. 10.3389/fninf.2024.1323203 FIGURE 1 Initial setup of our model network as a 2D simpliﬁcation with 2 × 2 boxes. We split the boxes into equally sized boxes and grouped a subset of neurons from each box in three equally sized ensembles representing the unconditional stimulus (US), the ﬁrst conditional stimulus (C1), and the second conditional stimulus (C2). The neurons of the ensemble US are fully connected with an additional readout neuron (R). The remaining neurons did not belong to any ensemble. Frontiers in Neuroinformatics 2 Materials and methods FIGURE 1 Initial setup of our model network as a 2D simpliﬁcation with 2 × 2 boxes. We split the boxes into equally sized boxes and grouped a subset of neurons from each box in three equally sized ensembles representing the unconditional stimulus (US), the ﬁrst conditional stimulus (C1), and the second conditional stimulus (C2). The neurons of the ensemble US are fully connected with an additional readout neuron (R). The remaining neurons did not belong to any ensemble. is fully connected to the neurons in the ensemble US with static connections (Figure 1) to monitor the unconditioned reaction of the network. With these neuron ensembles, we can model a conditioned learning paradigm by representing a neuron ensemble with a stimulus. Similar to the conditioned learning paradigm, where an unconditioned learning stimulus is presented at the same time as a conditioned stimulus and, therefore, learns a relationship between them, the network shall learn a relationship between US and C1 within each box. However, there should be no relationship associated with C2 or across diﬀerent boxes, as C2 acts as our control ensemble. To accomplish this, we stimulated the ensembles US and C1 together. We performed these stimulations simultaneously for every memory that we wanted to learn. with up to 109 neurons. Using current technology, human-scale simulations with 1011 neurons are possible in principle. We provide all model parameters in Table 1. frontiersin.org 2.1.3 Electrical activity The electrical input I is the sum of three inputs: synaptic, background, and stimulation, as described in Equation (7). The synaptic input Equation (8) is calculated over all ﬁred input neurons j. Each of these synapses has a weight that of 1 for excitatory input neurons or −1 for inhibitory ones and is multiplied by the ﬁxed synapse conductance k. The sum over those products for all input synapses of neuron j whose source neurons ﬁred is the synaptic input for j. Additionally, the neurons are driven by a random, normally distributed background activity (Equation 9). random, normally distributed background activity (Equation 9). I = Isynaptic + Ibackground + Istimulation (7 (7) I = Isynaptic + Ibackground + Istimulation matches the experimental data and enables functional remapping. (8) dz dt = ν 2 ∗exp − Ca −ξ ζ 2! −1 ! (2) ξ = η + ǫ 2 (3) ζ = η −ǫ 2√−ln(0.5) (4) 2.2 Izh We ch neuron m dz dt = ν 2 ∗exp − Ca −ξ ζ 2! −1 ! (2) ξ = η + ǫ 2 (3) ζ = η −ǫ 2√−ln(0.5) (4) Isynaptic = X j k ∗wj Ibackground ∼N(µ, σ 2) 2.2 Izhikevich model We chose the Izhikevich model (Izhik neuron model because it enables modelin dz dt = ν 2 ∗exp − Ca −ξ ζ 2! −1 ! (2) ξ = η + ǫ 2 (3) ζ = η −ǫ 2√−ln(0.5) (4) dz dt = ν 2 ∗exp − Ca −ξ ζ 2! −1 ! (2) (9) (3) (3) 2.2 Izhikevich model (4) We chose the Izhikevich model (Izhikevich, 2004) as our neuron model because it enables modeling diﬀerent spiking patterns and strikes a good compromise between biological accuracy and computational eﬃciency. We describe the membrane potential in Equation 10, where k0, k1, and k2 are constants and u is the membrane recovery variable as described in Equation 11. The membrane recovery depends on the ﬁxed parameters a, b, and c and accounts for the hyperpolarization period of a neuron. Finally, we assumed that a neuron spikes if the membrane potential reaches 30mV. Then, we reset the membrane potential and membrane recovery variables according to Equation 12. 2.1 Model of structural plasticity The Model of Structural Plasticity (MSP) (Butz and van Ooyen, 2013) enables structural plasticity based on a homeostatic mechanism. Each neuron controls its excitability and, therefore, the number of synapses based on its calcium concentration, which acts as a proxy for its ﬁring rate. An approximation of MSP (Rinke et al., 2018; Czappa et al., 2023) reduced the model’s computational complexity from O(n2) to O(n log n), which enabled simulations 03 frontiersin.org Kaster et al. 10.3389/fninf.2024.1323203 TABLE 1 Parameters for the experiments. on the distance between the positions xi of the neuron of the inititaiting axonal element and the candidate’s neuron position xj, as described in Equation (5). Note that the implementation uses an approximation (Rinke et al., 2018; Czappa et al., 2023) so that it does not need to calculate the probability for all candidates. on the distance between the positions xi of the neuron of the inititaiting axonal element and the candidate’s neuron position xj, as described in Equation (5). Note that the implementation uses an approximation (Rinke et al., 2018; Czappa et al., 2023) so that it does not need to calculate the probability for all candidates. Parameter Parameter description Value µbackground Mean of the background activity 5 σbackground Standard deviation of the background activity 2 θ Barnes–Hut acceptance criterion 0.3 τvac Retract ratio 100 ǫ Target calcium level 0.7 ηaxon Minimum calcium level to grow axons 0.4 ηden_ex Minimum calcium level to grow excitatory dendrites 0.1 ηden_inh Minimum calcium level to grow inhibitory dendrites 0 σ Gaussian scaling parameter 12 k Synapse conductance 3 τ Calcium decay constant 10,000 β Calcium intake 0.001 νaxon Growth rate of axons 0.0003 νden_ex Growth rate of excitatory dendrites 0.0006 νden_inh Growth rate of inhibitory dendrites 0.0006 aizh Izhikevich model parameter 0.1 bizh Izhikevich model parameter 0.2 cizh Izhikevich model parameter -65 dizh Izhikevich model parameter 2 ki,j = exp −\|\|xj −xi\|\|2 2 σ 2 ! (5) (5) Synaptic elements that are not connected are removed over time depending on the time constant τvac, as described in Equation (6). dvac dt = −vac τvac (6) (6) Frontiers in Neuroinformatics 2.4 Validity check To check the validity of our results, we needed to make an assumption about the distribution of the neurons’ ﬁring rate. We hypothesized that the ﬁring rates follow a normal distribution and showed this with the Kolmogorov–Smirnov test. It calculates the probability that the distribution of our data is not the same as the given distribution to which we compare (in our case: normal distribution). For this, we simulated the network that resulted from our main experiment for 100,000 steps and recorded the times the readout neurons ﬁre. Then, we split the steps into bins with the size of 1,000 steps and calculated the ﬁring rate of each readout neuron for each bin. As we hypothesized a normal distribution, we calculated the mean and standard deviation for the calculated ﬁring rates. The histogram of the ﬁring rates is shown in Supplementary Figure 1. We applied the Kolmogorov–Smirnov test (Massey Jr, 1951) to the calculated ﬁring rates and retrieved a p-value of 5.34 ∗10−6. As a value of p smaller than 0.05 can be interpreted as non-signiﬁcant, we can assume that our ﬁring rates are normally distributed. We started our network in a fully disconnected state and let it build up the connectivity from scratch for 1,000,000 steps. In more detail, the neurons received a normally distributed background activity such that they have a certain baseline activity. As this baseline is insuﬃcient to reach the neurons’ target calcium level, they grow synapses to connect to other neurons and receive more input. When the neurons reach the target calcium level, they stop growing synapses and they perform only small modiﬁcations to their synapses due to the ﬂuctuations in the input noise and the resulting ﬂuctuations in the neurons’ ﬁring rate. In this state, the network is in equilibrium, as every neuron is approximately at its target calcium level, and only small modiﬁcations are made to the network. We saved the newly formed network and used it as a starting point for our experiment. Hence, we continued the simulation with this network and waited for 150,000 steps until the network stabilized itself. Then, we continued the simulation with three stimulation phases similar to Gallinaro et al. (2022): baseline, encoding, and retrieval. We performed all stimulations for 2,000 steps with 20mV. Frontiers in Neuroinformatics 2.3 Network setup and stimulation Unless noted otherwise, our network consisted of 337,500 neurons, each with 20% inhibitory and 80% excitatory neurons. We distributed the neurons uniformly at random in a 3-dimensional cubic space. We split the network into 3 × 3 × 3 = 27 equally sized cubes with about 12,500 neurons each, following Gallinaro et al. (2022). In each box, we grouped a part of the neurons into disjoint ensembles of 333 excitatory neurons. We selected them randomly from all neurons in the box. Figure 1 visualizes the setup as a 2D example. 2.1.2 Forming and pruning of synapses Since we already determined how many synaptic elements a neuron forms, we must now actually form synapses between unbound synaptic elements or prune them if the number of grown synaptic elements of a neuron is smaller than its actual synapses. In the last case, we chose a synapse to prune uniformly at random from all synapses of the neuron. Consequently, the synapse will be removed from the source and target neuron, independently of the number of grown synaptic elements of the partner neuron. When we need to form a new synapse, we search for vacant dendritic elements for every vacant axonal element. We choose a target element randomly based on weighted probabilities. Each target dendritic candidate is weighted with a probability based dv dt = k2v2 + k1v + k0 −u (10) (10) du dt = a(bv −u) (11) (11) frontiersin.org 04 frontiersin.org 10.3389/fninf.2024.1323203 Kaster et al. 10.3389/fninf.2024.1323203 if v ≥30mV, then ( v ←c u ←u + d (12) consecutively in isolation, one after the other, with a pause of 18,000 steps between them. Note that this is diﬀerent from the previous two phases, where we stimulated the same ensemble type (e.g., US) from each box at the same time. In the end, we stimulated all C2 ensembles together at step 1,190,000 as a control. We expect that the readout neuron of a box ﬁres at an increased rate when we stimulate C1 of the same box due to the learned relationship and not if we stimulate the C2 ensembles. The protocol is visualized in Figure 2A. (12) • We stimulate its ensemble US during the baseline phase or, 2.4 Validity check In the baseline phase, we stimulated all US ensembles in step 150,000, followed by all C1 ensembles (250,000), and ﬁnally, all ensemble C2 (350,000). Note that US, C1, and C2 were stimulated after each other, but all ensembles of the same type across all boxes were stimulated at the same time. The goal of this phase is to strengthen the connectivity within each neuron ensemble. We selected the pause between the stimulation so that the neurons’ calcium levels return to their target level to ensure that there is no priming eﬀect. Moreover, the C2 ensemble acts as a control to counter a priming eﬀect as well as we would see an eﬀect not only in US and C1 but also in C2. Now, we can continue with our validity check by checking the ﬁring rate of the readout neuron during the retrieval phase to ensure that the network behaves as expected. We assumed that the ﬁring rates follow a normal distribution and detect abnormal behavior of the readout neuron when its ﬁring rate is signiﬁcantly outside of the normal distribution. For it, we used the 3-σ rule (Pukelsheim, 1994), as described in Equation (13) with f as the ﬁring rate of the readout neuron, µ as its mean, and σ its standard deviation without any stimulation. Approximately 99.7% of the normal ﬁring rate lies within the interval of three times the standard deviation around the mean of the normal distribution of the ﬁring rate. We considered a ﬁring rate outside of this interval for at least 500 steps to be signiﬁcantly diﬀerent from the normal ﬁring rate of the neuron when the network is in an uninﬂuenced state. Then, in the encoding phase, we wanted to learn the relationship between US and C1 and, therefore, stimulated all US and C1 ensembles together in step 450,000 and all C2 ensembles in step 550,000. The stimulation of the C2 ensembles alone acts as a control against a priming eﬀect. That would be the case when US or C1 form connections to C2 despite it being stimulated alone. Similar to the baseline phase, we stimulated the speciﬁed ensemble types from each box at the same time. • We stimulate its ensembles US and C1 during the encoding phase, or 2.4 Validity check If the model successfully learns the relationship between the ensembles US and C1 of the same box, neurons of ensemble US will also ﬁre at an increased rate. (A) Stimulation protocol of the experiment. The experiment is split into three stimulation phases: baseline, encoding, and retrieval. (B) Firing rates of all 27 readout neurons laid over each other. (C) Firing rate of a single readout neuron. (D) Scatter plot when readout neurons ﬁre at an increased rate. The 27 readout neurons are distributed along the y-axis. A red dot indicates that a readout neuron ﬁres at an increased rate. The stimulation of all US (blue), C1 (red), C2 (brown), and single C1 ensembles (gray) are marked with a vertical bar at the bottom. Frontiers in Neuroinformatics 2.4 Validity check f ∈[0, µ −3σ) Signiﬁcantly decreased ﬁring rate f ∈[µ −3σ, µ + 3σ] Normal ﬁring rate f ∈(µ + 3σ, 1000] Signiﬁcantly increased ﬁring rate (13) We expected a readout neuron to ﬁre at an increased rate if (13) Later, in the retrieval phase, we turned the plasticity oﬀ so that no further modiﬁcations were possible. We did this to see how the network behaves without any further modiﬁcations. Here, we stimulated, beginning in step 650,000, all C1 ensembles • We stimulate its ensemble US during the baseline phase or, • We stimulate its ensembles US and C1 during the encoding phase, or • We stimulate its ensembles US and C1 during the encoding phase, or frontiersin.org 05 Kaster et al. 10.3389/fninf.2024.1323203 FIGURE 2 Stimulation protocol and ﬁring rates (y-axis) of all readout neurons plotted over time (x-axis). The vertical dashed lines represent the stimulation of the ensembles US (blue), C1 (red), and C2 (brown). The gray lines and red line on the right represent single stimulations of C1. The readout neurons ﬁre at an increased rate when US is stimulated in the baseline and encoding phase. If the model successfully learns the relationship between the ensembles US and C1 of the same box, neurons of ensemble US will also ﬁre at an increased rate. (A) Stimulation protocol of the experiment. The experiment is split into three stimulation phases: baseline, encoding, and retrieval. (B) Firing rates of all 27 readout neurons laid over each other. (C) Firing rate of a single readout neuron. (D) Scatter plot when readout neurons ﬁre at an increased rate. The 27 readout neurons are distributed along the y-axis. A red dot indicates that a readout neuron ﬁres at an increased rate. The stimulation of all US (blue), C1 (red), C2 (brown), and single C1 ensembles (gray) are marked with a vertical bar at the bottom. • We stimulate its ensemble C1 during the retrieval phase. 2.5 Large-scale formation of memory engrams FIGURE 2 Stimulation protocol and ﬁring rates (y-axis) of all readout neurons plotted over time (x-axis). The vertical dashed lines represent the stimulation of the ensembles US (blue), C1 (red), and C2 (brown). The gray lines and red line on the right represent single stimulations of C1. The readout neurons ﬁre at an increased rate when US is stimulated in the baseline and encoding phase. 2.4 Validity check If the model successfully learns the relationship between the ensembles US and C1 of the same box, neurons of ensemble US will also ﬁre at an increased rate. (A) Stimulation protocol of the experiment. The experiment is split into three stimulation phases: baseline, encoding, and retrieval. (B) Firing rates of all 27 readout neurons laid over each other. (C) Firing rate of a single readout neuron. (D) Scatter plot when readout neurons ﬁre at an increased rate. The 27 readout neurons are distributed along the y-axis. A red dot indicates that a readout neuron ﬁres at an increased rate. The stimulation of all US (blue), C1 (red), C2 (brown), and single C1 ensembles (gray) are marked with a vertical bar at the bottom. FIGURE 2 Stimulation protocol and ﬁring rates (y-axis) of all readout neurons plotted over time (x-axis). The vertical dashed lines represent the stimulation of the ensembles US (blue), C1 (red), and C2 (brown). The gray lines and red line on the right represent single stimulations of C1. The readout neurons ﬁre at an increased rate when US is stimulated in the baseline and encoding phase. If the model successfully learns the relationship between the ensembles US and C1 of the same box, neurons of ensemble US will also ﬁre at an increased rate. (A) Stimulation protocol of the experiment. The experiment is split into three stimulation phases: baseline, encoding, and retrieval. (B) Firing rates of all 27 readout neurons laid over each other. (C) Firing rate of a single readout neuron. (D) Scatter plot when readout neurons ﬁre at an increased rate. The 27 readout neurons are distributed along the y-axis. A red dot indicates that a readout neuron ﬁres at an increased rate. The stimulation of all US (blue), C1 (red), C2 (brown), and single C1 ensembles (gray) are marked with a vertical bar at the bottom. FIGURE 2 Stimulation protocol and ﬁring rates (y-axis) of all readout neurons plotted over time (x-axis). The vertical dashed lines represent the stimulation of the ensembles US (blue), C1 (red), and C2 (brown). The gray lines and red line on the right represent single stimulations of C1. The readout neurons ﬁre at an increased rate when US is stimulated in the baseline and encoding phase. 3 Results Gallinaro et al. (2022) showed that the stimulation of 50% of the neurons of a single engram was necessary to increase the activity of the rest of the engram; we will improve upon that in both the necessary threshold and the technical contribution. While we stimulated all neurons of ensemble C1 during the retrieval in the previous experiment, we now show that it is suﬃcient to stimulate only a subgroup of neurons in ensemble C1 to trigger the readout neuron in the associated ensemble US. Hence, after the previous learning experiment with 27 boxes, we continued simulating the network, picked a single ensemble C1 from one box, and stimulated varying numbers of neurons. To this end, we continued the simulation and waited for 60,000 steps until the network stabilized. Then, we turned the plasticity oﬀ and stimulated 5% randomly chosen neurons of the ensemble C1 of the center box for 2,000 steps. After a pause of 98,000 steps, we added a further 5% randomly chosen neurons of the ensemble to the stimulated neurons and stimulated them again. We repeated this cycle until we stimulated 100% of the ensemble. This section is divided into multiple parts. We will start with the analysis of the process of the formation of a single memory engram. Then, we will discuss the simultaneous formation of multiple memory engrams. Finally, we provide the results of the large-scale and advanced simulations. • We stimulate its ensemble C1 during the retrieval phase. It should not ﬁre at an increased ﬁring rate otherwise. We successfully repeated the experiment with a larger network to show how well our approach scales. Instead of 3 × 3 × 3 = 27 boxes, we split a larger network into 7 × 7 × 7 = 343 We allowed the increased ﬁring rates to start (end) within 100ms of the start (end) of the associated stimulation to allow for some variation in the neurons’ behavior. frontiersin.org 06 Kaster et al. 10.3389/fninf.2024.1323203 2.7 Ablation studies boxes with a total of 343 · 12, 500 = 4, 287, 500 neurons and stimulated the network following the same pattern as before. We analyzed the ﬁring rates of the readout neurons to check whether they ﬁred only when US was stimulated during the baseline and encoding phases and only when C1 in the same box was stimulated during the retrieval phase as described in Section 2.4. boxes with a total of 343 · 12, 500 = 4, 287, 500 neurons and stimulated the network following the same pattern as before. We analyzed the ﬁring rates of the readout neurons to check whether they ﬁred only when US was stimulated during the baseline and encoding phases and only when C1 in the same box was stimulated during the retrieval phase as described in Section 2.4. We investigated how the network reacts to applied lesions. We are interested in two cases: The loss of synapses and the loss of neurons. For both cases, we continued the simulation from our main experiment and applied the lesion after 150,000 steps. Then, we waited 200,000 steps for the network to stabilize again so that each neuron reached its calcium level again. In step 350,000, we turned the plasticity oﬀand stimulated all C1 ensembles after each other and all C2 ensembles as described for the retrieval phase in Section 2.3. We randomly selected a center of the lesion in each box and selected a fraction of neurons that are closest to this center. In step 150,000, we removed all connections from and to the selected neurons. If we decided to lesion the neurons, we also removed them from the network so they were not available for reconnecting their synapses. 2.6 Advanced simulations The following two experiments started with the network that was stimulated as described in Section 2.3 because they analyze the newly created engrams. Frontiers in Neuroinformatics 3.1 Process of engram formation Our experiment consists of three phases: baseline, encoding, and retrieval. We started with the baseline phase. As described in Section 2.3, the network was in a stable state with only minor changes in the connectivity at the beginning of the baseline phase (Figure 3) . At this point, the network’s connectivity was random based on the neurons’ distances (Figure 4B). This phase aims to form neuron ensembles with strong connectivity within the ensemble. The additional stimulations resulted in an increased ﬁring rate (Figure 3A) of the neurons, which in consequence lead to an increase in their calcium level and therefore to a pruning of synapses (Figure 3B). After the stimulation ends, the ﬁring rate fell before its usual level due to the smaller synaptic input caused by the pruning of synapses. Consequently, the neurons within the ensemble started regrowing their synapses until they reached a stable ﬁring rate and calcium level as its target value. All neurons within the ensemble regrew their synapses simultaneously and are, therefore, looking for new synapse partners at the same time. This high ratio of potential partners within the same ensemble results in strong connectivity within this ensemble (Figure 3C). We stimulated a single ensemble, such as US, from each box at the same time. This and the property of forming new synapses based on the distance of neurons result in a strong within-connectivity of the ensemble in a box. After a short delay, we continued with the second C1 ensembles, after another delay with C2 ensembles. As a result, we had three strongly within-connected ensembles in each box representing an engram (Figure 4C). We refer to the study by Gallinaro et al. (2022) for details of this process. Figure 4 shows the process of reorganizing the connectivity during the entire experiment. 2.6.2 Forming long-distance connections d b ld (A) Mechanism of a single engram formation shown with a single ensemble group US. The stimulation in step 150,000 starts the engram formation. (A) Intracellular Calcium (right y-axis) as an indicator for the average ﬁring rate (left y-axis) of all neurons in a randomly selected example US ensemble. The stimulation is visible as a high spike in the ﬁring rate. This was followed by decreased activity caused by homeostatic network reorganization as depicted in (B, C). (B) Changes in intracellular calcium triggered growth of synaptic elements of the neurons in the exemplary ensemble. The synaptic elements decrease after the stimulation as calcium was higher than the homeostatic set-point and grew again afterwards when activities fell below the set-point as a consequence of compensatory pruning of synapses. (C) Average connectivity of an exemplary ensemble to itself and other neurons. As a consequence of the changing number of synaptic elements, we see a drop in the connectivity to neurons outside of the ensemble directly after the stimulation which was slowly restored afterwards. Connectivity within the ensemble increased likewise. Next, we continued with the encoding phase. In this phase, the model learned the association between the memory ensembles US and C1 within each box. For this, we stimulated the ensembles US and C1 together. Consequently, the ensembles US and C1 in a single box form many connections using the same mechanism as in the baseline phase (Figure 4D). ensemble C1 from the same box was stimulated and not if we stimulated our control ensemble C2 or any other ensemble from another box. Frontiers in Neuroinformatics 2.6.2 Forming long-distance connections d b ld 2.6.2 Forming long-distance connections During our main experiment, we wanted to build memory engrams only within a box. Now, we want to show that we can still form connections over long distances and therefore combine memory engrams from diﬀerent regions. For this, we continued the simulation with a larger Gaussian scaling parameter σdistant that makes connections over long distances more probable. We selected two edge boxes on the opposite side of the simulation area to maximize the distance. We waited for 150,000 steps so that the network could stabilize ﬁrst. Then, we stimulated the ensembles US and C1 from both boxes together for 2,000 steps. After a pause, we turned the plasticity oﬀ in step 250,000 and stimulated each ensemble C1 from the boxes after each other with a pause of 8,000 steps between them to check if the network learned the relationship between the ensembles in the two boxes without interfering with other boxes. As a last stimulation in the step 520,000, all of the ensembles C2 were stimulated to check the response to the control ensembles. Frontiers in Neuroinformatics frontiersin.org 07 Kaster et al. 10.3389/fninf.2024.1323203 GURE 3 echanism of a single engram formation shown with a single ensemble group US. The stimulation in step 150,000 starts the engram formation. (A) tracellular Calcium (right y-axis) as an indicator for the average ﬁring rate (left y-axis) of all neurons in a randomly selected example US ensemble. he stimulation is visible as a high spike in the ﬁring rate. This was followed by decreased activity caused by homeostatic network reorganization as epicted in (B, C). (B) Changes in intracellular calcium triggered growth of synaptic elements of the neurons in the exemplary ensemble. The synaptic ements decrease after the stimulation as calcium was higher than the homeostatic set-point and grew again afterwards when activities fell below e set-point as a consequence of compensatory pruning of synapses. (C) Average connectivity of an exemplary ensemble to itself and other eurons. As a consequence of the changing number of synaptic elements, we see a drop in the connectivity to neurons outside of the ensemble rectly after the stimulation which was slowly restored afterwards. Connectivity within the ensemble increased likewise. FIGURE 3 Mechanism of a single engram formation shown with a single ensemble group US. The stimulation in step 150,000 starts the engram formation. 3.2 Simultaneous formation of memory engrams (A) Visualization of the network at the beginning with no connectivity besides the static connections to the readout neuron. (B) Visualization of the network after the growth period with random connectivity. (C) Visualization of the network after the baseline phase with increased connectivity within each ensemble. (D) Visualization of the network after the encoding phase showing that the learned relationship between US and C1 is brought about by an additional increased connectivity between the ensembles C1 and US as marked in the heatmap with red dots. stimulation of our control ensembles C2 still did not inﬂuence the readout neurons. neurons in Figures 2B, D, we show that the network learns the relationships between US and C1 in each box and not between US and C2. Without stimulation, the readout neurons ﬁred at about 600Hz. All readout neurons ﬁred at steps 150,000 and 450,000 with the maximal frequency of 1000Hz. This was followed by a decrease of the ﬁring rate to about 450Hz until it recovers to its normal frequency of 600Hz. In step 150,000, we stimulated the US ensembles directly connected to their readout neurons during the baseline phase. During the stimulations of C1 in step 250,000 and C2 in step 350,000, the readout neurons did not ﬁre at an increased rate compared to their baseline frequency. This shows that the network works as desired because the unconditioned reaction is shown after the unconditioned stimulus. Later, during the encoding phase, the readout neurons ﬁred again, as expected when US and C1 were stimulated together in step 450,000 but not when C2 was stimulated in step 550,000. Finally, in the retrieval phase, we stimulated the ensemble C1 from each box after each other. The readout neuron of the same box ﬁred when its ensemble C1 was stimulated, indicating that the network learned the relationship between US and C1 only within the same box but not beyond box boundaries. The ﬁring rates were lower than during the stimulation in the encoding phase but still very distinguishable from the rest of the activity. We noticed small spikes of some readout neurons when another box was stimulated. However, the The learned relationship between US and C1 in each box can be explained with the newly formed connections, as visualized in Figure 5. At the beginning of the simulation, all ensembles were randomly connected (Figure 5A). 3.2 Simultaneous formation of memory engrams Finally, in the retrieval phase, we checked if the model learned the association between US and C1. The ﬁring rate of an exemplary single box’s readout neuron is visualized in Figure 2C. In addition to the two spikes during the earlier phases when US was stimulated, the readout neuron ﬁred only at an increased rate when the Our network consisted of 3 × 3 × 3 = 27 boxes, each with three ensembles: US, C1, and C2. By plotting the readout frontiersin.org 08 frontiersin.org frontiersin.org Kaster et al. 10.3389/fninf.2024.1323203 FIGURE 4 Left: Schematic illustration of the connectivity within a single box. Right: Heatmap of the neurons’ normalized number of input (row) and output (column) connections of each ensemble (O), US, C1, and C2. Normalized with the size of the source and target ensemble.. (A) Visualization of the network at the beginning with no connectivity besides the static connections to the readout neuron. (B) Visualization of the network after the growth period with random connectivity. (C) Visualization of the network after the baseline phase with increased connectivity within each ensemble. (D) Visualization of the network after the encoding phase showing that the learned relationship between US and C1 is brought about by an additional increased connectivity between the ensembles C1 and US as marked in the heatmap with red dots. FIGURE 4 Left: Schematic illustration of the connectivity within a single box. Right: Heatmap of the neurons’ normalized number of input (row) and output (column) connections of each ensemble (O), US, C1, and C2. Normalized with the size of the source and target ensemble.. (A) Visualization of the network at the beginning with no connectivity besides the static connections to the readout neuron. (B) Visualization of the network after the growth period with random connectivity. (C) Visualization of the network after the baseline phase with increased connectivity within each ensemble. (D) Visualization of the network after the encoding phase showing that the learned relationship between US and C1 is brought about by an additional increased connectivity between the ensembles C1 and US as marked in the heatmap with red dots. FIGURE 4 Left: Schematic illustration of the connectivity within a single box. Right: Heatmap of the neurons’ normalized number of input (row) and output (column) connections of each ensemble (O), US, C1, and C2. Normalized with the size of the source and target ensemble.. Frontiers in Neuroinformatics 3.2 Simultaneous formation of memory engrams After the baseline phase, the connectivity within the ensembles increased, and the connectivity between diﬀerent ensembles decreased (Figure 5B). This increased connectivity within ensembles indicates the formation of memory engrams similar to Gallinaro et al. (2022). However, in contrast to what they did, we stimulated all US and C1 ensembles together only once instead of thrice. After the encoding phase, the connectivity between the ensembles US and C1 in the same box increases signiﬁcantly, clarifying that the network learned the relationship between US and C1 (Figure 5C). However, we could observe a slightly increased connectivity between the ensembles of neighboring boxes due to the connectivity probability being based on the distance of neurons. This explains the slightly increased activity of the readout neurons sometimes when another box is stimulated. Furthermore, the C2 ensembles formed connections primarly within their ensembles and did not connect to neurons in other boxes or ensembles, conﬁrming that these control ensembles did not inﬂuence the learning process. Frontiers in Neuroinformatics 09 frontiersin.org Kaster et al. 10.3389/fninf.2024.1323203 FIGURE 5 Connectivity between chosen ensembles in the network. From the top left box of the ﬁrst layer (box 00) over the center box of the second layer (box 13) to the bottom right box of the last layer (box 26). (A) During baseline phase, before ﬁrst stimulation at step 150,000, connectivity is randomly distributed but predominantly remains within a box. (B) After baseline phase at step 450,000, connectivity has increased within each neuron ensembles. (C) After the encoding phase at step 650,000, connectivity also increased between ensembles US and C1 within the same box. Connectivity between chosen ensembles in the network. From the top left box of the ﬁrst layer (box 00) over the center box of the second layer (box 13) to the bottom right box of the last layer (box 26). (A) During baseline phase, before ﬁrst stimulation at step 150,000, connectivity is randomly distributed but predominantly remains within a box. (B) After baseline phase at step 450,000, connectivity has increased within each neuron ensembles. (C) After the encoding phase at step 650,000, connectivity also increased between ensembles US and C1 within the same box. Frontiers in Neuroinformatics 3.3 Spatiotemporal dynamics of homeostatic engram formation growth curves for this activity level were at around zero as the model is in an equilibrium state with only minor modiﬁcations to the network, which is also visible in the almost constant number of axons and dendrites, as illustrated in Figures 6C, D. The connectivity between US and C1 was almost zero, as there is no relationship between them yet. Instead, the connectivity from within the ensemble US to external neurons was only high for neurons outside of any ensemble just because of the higher number of neurons not belonging to an ensemble compared to the size of the ensembles C1 and C2. Next, we applied the stimulation to the US and C1 ensembles (Figure 6 (0)), causing their activity to increase and moving the calcium for these ensembles to the right in Figure 7A (center). The values of the ensembles’ growth curves were almost -1 for this calcium level, We observe the average calcium level, grown axons and excitatory dendrites, and the connectivity of the neurons over the entire reorganization process for the ensembles US, C1, and C2 for an exemplary single box in Figure 6 to analyze the mechanism behind the synaptic reorganization further. Moreover, we visualize the average calcium level and, therefore, indirectly, the activity of the neuron groups at diﬀerent times in the context of the growth curves for the diﬀerent types of synaptic elements in Figure 7A. Before the ﬁrst stimulation in the encoding phase of the ensembles US and C1, the calcium level of all groups was around their target level (Figure 7A left). The values of the 10 frontiersin.org Kaster et al. 10.3389/fninf.2024.1323203 FIGURE 6 Homeostatic reorganization of neuron ensembles within a single box during the encoding phase. All graphs are averaged over all neurons of the respective ensemble within a single box. We stimulated the ensembles US and C1 at step 450,000 (vertical red-blue line) and the ensemble C2 at step 550,000 (vertical brown line). Panels show the average intracellular calcium concentration (A), connectivity (B), amounts of excitatory dendritic elements (C) and axonal elements (D). Networks are in a homeostatic equilibrium before ﬁrst stimulation (0). After stimulation, activity of the ensembles US and C1 is increased, resulting in pruning of synapses until calcium levels fall below the target value (1). Then, synaptic elements are formed, and calcium level may rise again (2). 3.3 Spatiotemporal dynamics of homeostatic engram formation Axonal and dendritic elements are simultaneously formed by neurons of ensembles US and C1, which become available for synapse formation and explain the observed connectivity increase between these ensembles. The growth phase is followed by a transient overshoot (3) and subsequent minor pruning until a homeostatic equilibirum is reached again (4). The stimulation of C2 (5) follows the same trend [(5)-(9)] with the main diference that neurons from ensemble C2 are the only ones that grow axonal and dendritic elements at the same time. The consequence is a massive increase of connections within the ensemble but not between C2 and ensemble US. GU 6 Homeostatic reorganization of neuron ensembles within a single box during the encoding phase. All graphs are averaged over all neurons of the respective ensemble within a single box. We stimulated the ensembles US and C1 at step 450,000 (vertical red-blue line) and the ensemble C2 at step 550,000 (vertical brown line). Panels show the average intracellular calcium concentration (A), connectivity (B), amounts of excitatory dendritic elements (C) and axonal elements (D). Networks are in a homeostatic equilibrium before ﬁrst stimulation (0). After stimulation, activity of the ensembles US and C1 is increased, resulting in pruning of synapses until calcium levels fall below the target value (1). Then, synaptic elements are formed, and calcium level may rise again (2). Axonal and dendritic elements are simultaneously formed by neurons of ensembles US and C1, which become available for synapse formation and explain the observed connectivity increase between these ensembles. The growth phase is followed by a transient overshoot (3) and subsequent minor pruning until a homeostatic equilibirum is reached again (4). The stimulation of C2 (5) follows the same trend [(5)-(9)] with the main diference that neurons from ensemble C2 are the only ones that grow axonal and dendritic elements at the same time. The consequence is a massive increase of connections within the ensemble but not between C2 and ensemble US. reducing the synaptic elements at maximum speed. Consequently, the number of axons and dendrites and their connectivity started decreasing. In the end, the calcium levels of all groups returned to their set level (Figure 7A bottom, Figure 6 (4)) and the connectivity as well as the number of synaptic elements remained at a stable level. Frontiers in Neuroinformatics frontiersin.org 3.3 Spatiotemporal dynamics of homeostatic engram formation During the entire stimulation and reorganization of the ensembles US and C1, the calcium level, number of synaptic elements, and connectivity of the control ensemble C2 remained unchanged. The reaction of the ensemble C2 followed the same trend as the ensembles US and C1 during and after its stimulation (Figure 6 (5)-(9)). As we stimulate the ensemble C2 on its own, we see no eﬀect on the other ensembles. After the stimulation ended, the calcium levels fell below their target (Figure 6 (1)). The neurons started rebuilding their synapses as soon as the calcium level dropped below its target level. The decrease in the calcium level was slowed down until it reached its lowest level (Figure 7A right, Figure 6 (2)). At this calcium level, the ensembles US and C1 correspond to the spike of the growth curve of the axons. The growth curves of the dendrites are lower but still positive. Hence, the ensembles US and C1 build new synapses fast, as shown in Figures 6C, D. As the ensembles US and C1 built new synapses simultaneously, they formed their synapses to a large degree between themselves, increasing connectivity between them (Figure 6B). The fast formation of synapses caused the calcium level to exceed its target (Figure 6 (3)) due to the increased synaptic input and, therefore, led again to a slight reduction of synaptic elements. When we look at the course of the calcium levels before (Figure 7B left) and after (Figure 7B center) we removed all connectivity between a group of neurons (Figure 7B), we observe that the levels dropped from their target level for the lesioned group of neurons. The decreased calcium level corresponds almost to the peak of the growth curve for axons (Figure 7B center), meaning that axons were built at their maximum speed. Moreover, dendrites 11 frontiersin.org Kaster et al. 10.3389/fninf.2024.1323203 FIGURE 7 The average calcium level of groups of neurons of a single box (x-axis) with calcium-dependent growth curves (y-axis). (A) Calcium level before and after the ensembles US and C1 were stimulated at step 450,000. Subsequentially, their calcium levels increased and growth curves caused synaptic elements to decrease and synapses to prune. When stimulation stopped and synapses were pruned, activities and calcium levels, respectively, dropped below the homeostatic set-point which, in turn, triggered the growth of synaptic elements and potentially also the growth of new synapses. 3.3 Spatiotemporal dynamics of homeostatic engram formation Note, that calcium levels below the set-point were in an optimal regime for axonal element formation while dendritic elements grew slower. A surplus of axonal elements may result in more long-range connections while a prolongued growth of dendritic elements extended the phase in which new engrams could form because it may take longer until activities return to a homeostatic set-point. (B) Average calcium levels during ablation studies in which we removed connectivity of 50% of the neurons in a box. Directly after the stimulation, calcium levels of lesioned neurons dropped due to the lack of input. As a result, the neurons start regrowing synaptic elements until enough synapses were formed to restore activity homeostasis. The homeostatic reorganization is comparable to engram formation after stimulation in A. Note, that even for higher deletion rates neurons will return average ﬁring rates to the homeostatic set-point (data not shown) very much as in B, however without functional recovery of trained engrams. The average calcium level of groups of neurons of a single box (x-axis) with calcium-dependent growth curves (y-axis). (A) Calcium level before and after the ensembles US and C1 were stimulated at step 450,000. Subsequentially, their calcium levels increased and growth curves caused synaptic elements to decrease and synapses to prune. When stimulation stopped and synapses were pruned, activities and calcium levels, respectively, dropped below the homeostatic set-point which, in turn, triggered the growth of synaptic elements and potentially also the growth of new synapses. Note, that calcium levels below the set-point were in an optimal regime for axonal element formation while dendritic elements grew slower. A surplus of axonal elements may result in more long-range connections while a prolongued growth of dendritic elements extended the phase in which new engrams could form because it may take longer until activities return to a homeostatic set-point. (B) Average calcium levels during ablation studies in which we removed connectivity of 50% of the neurons in a box. Directly after the stimulation, calcium levels of lesioned neurons dropped due to the lack of input. As a result, the neurons start regrowing synaptic elements until enough synapses were formed to restore activity homeostasis. The homeostatic reorganization is comparable to engram formation after stimulation in A. Frontiers in Neuroinformatics 3.3 Spatiotemporal dynamics of homeostatic engram formation Note, that even for higher deletion rates neurons will return average ﬁring rates to the homeostatic set-point (data not shown) very much as in B, however without functional recovery of trained engrams. were also built fast but not at their maximum speed. As a result of the build-up of synaptic elements, the activity and, therefore, the calcium levels of the lesioned neurons started increasing until they reached their target level again (Figure 7B right). The calcium level of the non-lesioned neurons remained mostly unchanged. The rebuilding of the synaptic elements after completely removing them for the lesioned neurons is similar to rebuilding the synapses after the stimulation during the conditioned learning experiment. This explains why the network can only recover its learned relationships if all or a high ratio of the lesioned neurons are part of the learned relationship. Otherwise, the network expresses the same learning eﬀect as before but between all lesioned neurons regardless of the aﬃliation of the neurons. As we could observe, the synaptic reorganization in our model always follows the same pattern. First, there is a loss of connectivity either directly caused by a lesion or indirectly caused by stimulation and the following pruning of synaptic elements caused by the increased activity and the growth rule of our model itself (Figure 6 (0)-(1)). In consequence, the activity of the neurons decreases because of the smaller synaptic input. Then, neurons start rebuilding the synapses, connecting mostly among themselves (Figure 6 (1)-(4)). In the end, the activity of the neurons returns Frontiers in Neuroinformatics 12 frontiersin.org 10.3389/fninf.2024.1323203 Kaster et al. 10.3389/fninf.2024.1323203 to its initial level. This contrasts Hebbian plasticity, where we cannot observe such a sequence of events. Neurons with similar activity patterns, e.g., because of stimulation, directly strengthen their connections. Strengthening their connections makes it more likely that they ﬁre together and increase their activity. As they are now more likely to ﬁre together, Hebbian plasticity continues to strengthen their synapses. This can lead to continuous strengthening of synapses and increasing neuron activities with unbound synaptic weights. Multiple counter mechanisms have been discussed (Chen et al., 2013; Chistiakova et al., 2015; Fox and Stryker, 2017) to counter the runaway activity of Hebbian plasticity. Integrating our model of structural homeostatic plasticity with Hebbian plasticity could also counter this problem. 3.7 Ablation studies To see if our model can recover from lesions, we visualize the reaction of a single C1 ensemble to the deletion of all connections from 50% of the neurons of the ensemble in Figure 9A. Initially, the connectivity from neurons outside US and C1 was by far the strongest. The connectivity from C1 to itself and US to C1 was approximately equal, ranging at about one connection per neuron. We can immediately see the removed connections in step 150,000 as the connectivity from all ensembles decreased drastically. Then, every neuron received less input. Half of the neurons in the ensemble received no synaptic input because we had entirely disconnected them; the other half kept only connections to neurons outside of the ensemble and among each other. As a result, the calcium level started decreasing, and the neurons formed new synapses to rebuild their connections. 3.3 Spatiotemporal dynamics of homeostatic engram formation While most models are diﬃcult to observe in experiments, our model shows an observable sequence of events during learning that could be tested in experiments. engrams within a box. To demonstrate this, we increased σ and stimulated the ensembles US and C1 from two boxes together and checked in the retrieval phase whether they formed a single, interconnected engram. The ﬁring rates are visualized in Figure 8. We can see that the readout neurons of both boxes ﬁred once we stimulated them together in step 150,000 and during retrieval when the C1 ensembles of both boxes were stimulated at diﬀerent times. Thus, we can connect engrams that were not associated before and are distant from each other. 3.5 Pattern completion We want to see if our network can reactivate the learned pattern if we stimulate it only partly. As we see in Supplementary Figure 2, the ﬁring rate of the corresponding readout neuron of US increased with more stimulated neurons of C1 except for the change between 0%–5%, 45%–50%, and 80%–85%, where the ﬁring rate decreased slightly. We considered a ﬁring rate larger than three times higher than its mean signiﬁcantly diﬀerent from its baseline, as 99.7% of the baseline activity lies within this interval (3 σ rule; Pukelsheim, 1994). This was the case for 45% or more stimulated neurons of the ensemble, concluding that at least 150 stimulated neurons are necessary to increase the ﬁring rate of the readout neuron signiﬁcantly. Therefore, it is enough to stimulate 45% of the neurons of the ensemble C1 to signiﬁcantly reactivate the pattern so that the readout neurons ﬁre with a signiﬁcantly increased ﬁring rate. This shows that we can also perform pattern completion over the engram pairs C1 and US due to our high connectivity within a box. In Figure 9B, we illustrate the reaction of the network when we permanently apply a lesion to half of the neurons in C1 in step 150,000. A lesion in our model includes removing all connections from and to the lesioned neuron and removing them from the network so that no new connections can be formed. As we completely removed the lesioned neurons from the network, they were no longer considered when calculating the average connectivity after step 150,000. Hence, we see a large decrease in the total connectivity in average connectivity but not in the average connectivity per neuron simply because of the decreased size of the ensemble. 3.4 Large-scale formation of memory engrams To show how well our approach scales, we repeated the experiment with 343 boxes. We assessed that the network behaves correctly for 341 readout neurons ﬁring only with a signiﬁcantly increased rate when expected. Two readout neurons behaved not as expected. One of the readout neurons ﬁred only at an increased rate for 1,400 steps instead of the entire 2,000 steps of the stimulation of the associated ensemble C1 during the retrieval phase. In contrast, the other readout neuron ﬁred at an increased rate for 500 steps during the retrieval phase when another ensemble C1 was stimulated. However, the newly grown synapses connected diﬀerently than the recently removed ones. The connectivity from US to C1 was approximately only half as strong as before after the network returned to an equilibrium. In exchange, the connectivity from other neurons outside of US and C1 increased slightly. Interestingly, the connectivity within C1 increased to approximately 2, doubling the connectivity strength within the ensemble. We can explain this behavior with the same eﬀect observed for the learning mechanism with the homeostatic model in Section 3.1. All the neurons from which we removed the connections rebuild their synapses simultaneously so that they start looking for new partners simultaneously. This led to a huge ratio of potential synapse partners within the ensemble. During the retrieval phase at the end, all readout neurons ﬁred at an increased rate at the expected times when we stimulated the C1 of the corresponding box. Nevertheless, the rewiring caused three readout neurons to ﬁre at an increased rate when a C1 from another box was stimulated. Frontiers in Neuroinformatics frontiersin.org 3.6 Forming long-distance connections As described in Equation 5, we can control the probability with which neurons connect by adjusting the Gaussian scaling parameter σ. We can connect engrams of diﬀerent boxes when we increase this scaling parameter after we have already formed First, the average connectivity shifted immediately after the lesion in step 150,000 because the remaining neurons have slightly 13 frontiersin.org frontiersin.org Kaster et al. 10.3389/fninf.2024.1323203 FIGURE 8 Firing rates of the two readout neurons from the boxes boxes that we stimulated to form long-distance connections. Both neurons ﬁred at a rate of 1kHz when both ensembles were stimulated together. They also started ﬁring together when only a single ensemble C1 was stimulated, indicating that both boxes had formed a new engram. The blue (red) dashed line mark the stimulation of the ensemble US (C1) of the depending box. (A) Firing rate of a read-out neuron located in the upper left box on the top side of the cubic simulation model [see inset (A)]. (B) Firing rate of a read-out neuron in the right bottom box at the bottom side of the cubic simulation model [see inset (B)]. E 8 FIGURE 8 Firing rates of the two readout neurons from the boxes boxes that we stimulated to form long-distance connections. Both neurons ﬁred at a rate of 1kHz when both ensembles were stimulated together. They also started ﬁring together when only a single ensemble C1 was stimulated, indicating that both boxes had formed a new engram. The blue (red) dashed line mark the stimulation of the ensemble US (C1) of the depending box. (A) Firing rate of a read-out neuron located in the upper left box on the top side of the cubic simulation model [see inset (A)]. (B) Firing rate of a read-out neuron in the right bottom box at the bottom side of the cubic simulation model [see inset (B)]. neurons outside of US and C1. This increase continued until the neurons were satisﬁed with their input again. The connectivity from US and C1 remained low as the probability of connecting to these neurons is much smaller due to the small size of the ensembles compared to the rest of the network. However, the remaining connectivity was still suﬃcient to excite the readout neurons enough during the retrieval phase so that they ﬁred at an increased rate. diﬀerent average connectivity. Frontiers in Neuroinformatics 3.6 Forming long-distance connections Moreover, the average connectivity from C1 to itself decreased slightly because a fraction of these connections were to lesioned neurons and dropped out. The newly freed synapses can connect instantly with neurons outside US and C1. This leads to a small decrease in the ﬁring rate, which could be observed in the decrease in the average calcium level. Second, the neurons regrew synapses to return to their target activity level. In contrast to our network with only removed connections, mostly the connectivity from neurons outside of US and C1 increased. This is caused by the much smaller fraction of potential synapse partners from neurons within C1, as the lesioned neurons were not looking for new synapse partners. Note that the average connectivity of C1 after the lesions varied due to the randomness involved, as we can see in the shift in the connectivity from other neurons to C1. Other boxes may have diﬀerent shifts and, therefore, diﬀerent initial conditions for regrowing their synapses, but all boxes had in common that the connectivity within C1 was not or only slightly strengthened. However, the connectivity from outside of US and C1 was strengthened. Nevertheless, the remaining connectivity from C1 to US was still large enough to enable ﬁring all readout neurons at an increased rate during the retrieval phase. If we removed the connections of even more neurons, as visualized in Figure 9D, the trend was similar but to a larger degree. More connections were removed and restructured mostly to random neurons, leaving only a small number of connections between US and C1. This was visible in the retrieval, and three readout neurons did not ﬁre at an increased rate when we removed 50% of the connections. When we removed all connections from 70% of the neurons (Figure 9E), the number of connections between US and C1 was close to zero and, therefore, insuﬃcient to trigger an increased ﬁring rate of the readout neurons in 19 out of 27 boxes. frontiersin.org 3.8 Memory capacity We visualize the network’s connectivity if we remove all connections from 30% of all neurons within a box regardless of whether they are part of an ensemble in Figure 9C. The decrease in connectivity in step 150,000 is visible for the connection from US and C1. As the newly freed synapses reconnected immediately to random neurons, we see an increase in the connectivity from We will take a closer look at the memory capacity of our model. Fusi (2024) deﬁned the memory capacity of a neural network over the signal-to-noise ratio (SNR), with which memories can be recalled and the number of stored memories. Instead of 14 frontiersin.org frontiersin.org Kaster et al. 10.3389/fninf.2024.1323203 FIGURE 9 Network response to simulated lesions over time (x-axis). Left: Average input connectivity per neuron for all non-lesioned neurons within a randomly selected example ensemble C1. Input synapses originate from neurons of the same receiving ensemble (blue), from the ensemble US (yellow), or from other ensembles (green) within the same box. The dotted line visualizes the average calcium level of all non-lesioned neurons (right y-axis). A successful functional recovery would require re-formation of input connectivity from ensemble US. Right: Successful functional recovery indicated by red dots meaning increased ﬁring of readout neurons in the retrieval phase as in Figure 2D. Lack of increased ﬁring is marked with a gray cross. The stimulation of single C1 (all C2) ensembles is marked with a vertical gray (brown) bar at the bottom of each diagram. (A) Lesioned network loosing the entire connectivity from 50% of the input neurons of ensemble C1. (B) Lesioned network in which 50% of the input neurons from the ensemble C1 are completely removed from the network. (C) Lesioned network with input connectivity from 30% of randomly selected neurons from the entire box is deleted. (D) Lesioned network as in C but with a deletion rate of 50%. (E) Lesioned network as in C but with a deletion rate of 70%. FIGURE 9 Network response to simulated lesions over time (x-axis). Left: Average input connectivity per neuron for all non-lesioned neurons within a randomly selected example ensemble C1. Input synapses originate from neurons of the same receiving ensemble (blue), from the ensemble US (yellow), or from other ensembles (green) within the same box. The dotted line visualizes the average calcium level of all non-lesioned neurons (right y-axis). 3.8 Memory capacity A successful functional recovery would require re-formation of input connectivity from ensemble US. Right: Successful functional recovery indicated by red dots meaning increased ﬁring of readout neurons in the retrieval phase as in Figure 2D. Lack of increased ﬁring is marked with a gray cross. The stimulation of single C1 (all C2) ensembles is marked with a vertical gray (brown) bar at the bottom of each diagram. (A) Lesioned network loosing the entire connectivity from 50% of the input neurons of ensemble C1. (B) Lesioned network in which 50% of the input neurons from the ensemble C1 are completely removed from the network. (C) Lesioned network with input connectivity from 30% of randomly selected neurons from the entire box is deleted. (D) Lesioned network as in C but with a deletion rate of 50%. (E) Lesioned network as in C but with a deletion rate of 70%. Our model uses only structural plasticity, so we cannot apply the same calculations directly. In our model, each neuron has approximately the same number of synapses sneuron in the equilibrium state. We ﬁrst remove some of the existing synapses of each stimulated neuron and their partners due to the stimulation. Then, the synapses regrow in a structured way so that they encode the newly created memory. The number of retraced and regrown synapses per neuron is roughly the same for each stimulated neuron that we call sengram with sengram ≤sneuron. Similar to Fusi (2024), we ﬁrst calculate the memory signal that can be retrieved directly after creating the memory. Each memory consists of nengram number of neurons that we stimulate and recruit. This means that we have sinit = nengram ∗sengram synapses retracted and regrown that represent a single memory initially. As a result, we can express the measuring the neurons’ activity during the memory recall, they observe the changes in the network structure and assume that the changes happening in the network during learning represent a memory. Their model uses synaptic plasticity; therefore, the changed synaptic weights represent memory and the signal. When the network learns more memories, synaptic weights are changed again, reducing the ability to recognize the ﬁrst memory of the network if it overrides the same synaptic weights. In other words, adding more memories reduces the SNR. Frontiers in Neuroinformatics 4.1 Comparison between homeostatic engram formation and synaptic plasticity-driven learning models In the human brain, synapses are formed only sparsely. With synaptic plasticity alone, only the weights of these synapses can be changed. This can be a problem if learning patterns require strong connectivity between certain neurons, but their connectivity is low (Knoblauch, 2017). Structural plasticity can overcome those suboptimal connectivity patterns of sparsely connected networks by forming new synapses between the required neurons (and reducing cross-talk in neuronal assemblies by pruning synapses). Moreover, this is an important property when the network needs to learn an entirely new input or when the network needs to recover from a partial lesion. Computational models with synaptic plasticity avoid this problem either by all-to-all connectivity (Rolls et al., 2013; Huang and Wei, 2021)—which is computationally demanding on the one hand and not comparable to the brain where connectivity is only sparse on the other hand—or they require the creation of a ﬁxed number of synapses between random neurons (Szatmáry and Izhikevich, 2010; Savin and Triesch, 2014; Fiebig and Lansner, 2017) at the beginning, limiting the ability to form memories between neuron pairs drastically. Our approach overcomes this limitation, allowing the forming of connections between all neurons and inﬂuencing the range over which neurons connect by changing our Gaussian probability parameter σ. When we talk about large and small memories, we mean the relative size of a memory in ratio to the network size. More precisely, we measure the size of a memory as the total number of synapses a memory occupies. Hence, the larger a memory is, the higher the ratio of synapses out of the total number of synapses in the network. A memory with the same number of synapses is relatively smaller in a network with many neurons than in a small network. Hence, we can increase the SNR of a single memory either by increasing the number of total synapses per memory or by decreasing the number of synapses in the network. Again, the total number of synapses for either a single memory or the entire network can be modiﬁed by changing either the number of neurons (Figure 10A) or the number of synapses per neuron (Figure 10C). The number of neurons in the network we recruit for a single memory is a direct parameter we can modify in our experiment. 3.8 Memory capacity This SNR can then be interpreted as an upper bound of the memory capacity—an upper bound because it cannot tell whether the network can recall a memory, only whether its structure is still present in the network, which is necessary but not suﬃcient. Their study describes that memory is forgotten when its SNR falls below a threshold. measuring the neurons’ activity during the memory recall, they observe the changes in the network structure and assume that the changes happening in the network during learning represent a memory. Their model uses synaptic plasticity; therefore, the changed synaptic weights represent memory and the signal. When the network learns more memories, synaptic weights are changed again, reducing the ability to recognize the ﬁrst memory of the network if it overrides the same synaptic weights. In other words, adding more memories reduces the SNR. This SNR can then be interpreted as an upper bound of the memory capacity—an upper bound because it cannot tell whether the network can recall a memory, only whether its structure is still present in the network, which is necessary but not suﬃcient. Their study describes that memory is forgotten when its SNR falls below a threshold. Frontiers in Neuroinformatics 15 frontiersin.org Kaster et al. 10.3389/fninf.2024.1323203 SNR of the memory in comparison to the noise of the network as proportional to snrinit ∝sinit/(ntotal ∗sneuron). If we add additional memory to the network, we remove and replace sinit synapses in our network. A single synapse in our memory has the probability of q = nengram/ntotal ∗sengram/sneuron being removed. The ﬁrst quotient represents the probability that either the pre- or the postsynaptic neuron belongs to the stimulated neurons, and the second quotient is the probability of a single synapse from a single neuron belonging to the deleted synapses. As a result, the number of synapses still encoding our memory after t added memories is proportional to sinit ∗(1 −q)t and the SNR is proportional to snr(t) ∝snrinit ∗(1 −q)t. Moreover, the information storage capacity could be increased by recruiting neurons to more than one ensemble. This can explain how the human brain can learn many concepts and associations with a limited number of neurons. 4 Discussion We could show that the Model of Structural Plasticity (MSP) (Butz and van Ooyen, 2013) can form multiple and non-interfering memory engrams in a recurrent and sparsely connected network of spiking neurons. This is remarkable as MSP does not aim to form associative memories. With MSP engram formation solely results from the reciprocal interplay of altered activity levels (after stimulation of selected neuronal ensembles and caused by rewired network connectivity) with local homeostatic morphogenetic changes of neurons (forming and deleting of axonal and dendritic elements) and distance-dependent synapse formation and deletion. Hence, MSP emulates associative memory formation with distinct properties that distinguish it from pure Hebbian synaptic plasticity. We visualize the SNR for our model and diﬀerent parameters in Figure 10. A higher SNR indicates a better-recallable memory. Our initial SNR depends on the number of neurons in the engram, the number of synapses, and the total number of synapses in the network. We can say that a larger memory—either by a larger number of recruited neurons (Figure 10D) or a larger number of belonging synapses (Figure 10B)—increases the initial SNR as we have a lot of synapses (signal) compared to our noise. The larger memory has the disadvantage that we can store fewer memories in our network as synapses are more likely to be overwritten by other synapses. This is visible in the faster fall of the SNR with an increasing number of memories. In contrast, a smaller memory has the disadvantage of an initial small SNR but the advantage of a much slower decreasing SNR as we have more unrelated synapses in our network. This behavior is similar to the plasticity–stability tradeoﬀdescribed by Fusi (2024), where an increase in synaptic plasticity (e.g., by a large learning rate) allows faster learning of memories but also faster forgetting. In conclusion, our SNR depends on the ratio between the size of a memory compared to the size of the network. 3.8 Memory capacity In our simulations, neurons are mostly in a single memory engram, but forming multiple engrams in a single box with overlapping neurons could signiﬁcantly increase the number of engrams that can be simulated with the same number of neurons. frontiersin.org Frontiers in Neuroinformatics 4.1 Comparison between homeostatic engram formation and synaptic plasticity-driven learning models On the other hand, we can also indirectly inﬂuence the number of synapses as it depends on the calcium level of the neuron. Possible parameters that we can modify to increase the number of synapses per neuron are the target calcium level η, the parameters β and τ that inﬂuence the accumulation of the calcium level, or the parameters ν and µ to inﬂuence the growth curve of synaptic elements based on the calcium level. Additionally, if we want to modify the number of synapses retracted and regrown by the stimulation of a neuron, we can modify the stimulation intensity or duration. If we repeat our experiment with only synaptic plasticity with all-to-all connectivity instead of structural plasticity, we would need n ∗(n −1) synapses, where n is the number of neurons in our network. Our network of 27 boxes consists of 337,500 Frontiers in Neuroinformatics 16 frontiersin.org Kaster et al. 10.3389/fninf.2024.1323203 FIGURE 10 The signal-to-noise ratio (SNR) of our model with varying parameters for an increasing number of stored memories in the network. The orange line visualizes the SNR of the model used in our main experiment with 27 boxes. The blue and green lines visualize the change in the SNR by decreasing (blue) or increasing (green) a single parameter. (A) SNR with varying numbers of total neurons in the network. (B) SNR with varing number of synapses overwritten per neuron in the engram. (C) SNR with varying numbers of synapses per neuron. (D) SNR with varying numbers of neurons per engram. The signal-to-noise ratio (SNR) of our model with varying parameters for an increasing number of stored memories in the network. The orange line visualizes the SNR of the model used in our main experiment with 27 boxes. The blue and green lines visualize the change in the SNR by decreasing (blue) or increasing (green) a single parameter. (A) SNR with varying numbers of total neurons in the network. (B) SNR with varing number of synapses overwritten per neuron in the engram. (C) SNR with varying numbers of synapses per neuron. (D) SNR with varying numbers of neurons per engram. networks (Yang et al., 2021b). Synaptic plasticity is also supported by many platforms enabling fast learning (Yang et al., 2021a). 4.1 Comparison between homeostatic engram formation and synaptic plasticity-driven learning models These platforms are built biologically motivated, mimicking some aspects of the brain architectures, e.g., a cerebellum network for motor learning (Yang et al., 2021c), dendritic on-line learning (Yang et al., 2023b), and context-dependent learning (Yang et al., 2021a). Based on the platform for context- dependent learning, a real-world application for smart traﬃc systems was developed (Yang et al., 2023a). Moreover, recent studies showed that structural plasticity can be modeled on some of these platforms (Bogdan et al., 2018; Billaudelle et al., 2021). Our approach, which enormously decreases the required number of synapses, could beneﬁt from a neuromorphic platform implementation with an adaptable routing system to further increase its scalability. neurons and 3,009,795 synapses. With all-to-all connectivity, each neuron would need 337,499 synapses compared to our model’s average of 9 synapses, resulting in 113,905,912,500 synapses. If we compare the numbers of our large-scale experiment with 343 boxes and 4,287,500 neurons, we see that while our ﬁnal network had 38,296,669 synapses, all-to-all connectivity would require about 1013 synapses. Even if we connect only a tiny fraction of neuron pairs with synapses, simulating such a large number of synapses is impractical, and the quadratic dependency of the number of synapses on the number of neurons limits the size of the network enormously. In contrast, our network’s synapses grow linearly with the number of neurons; as in our homeostatic model, each neuron aims for the same number of synapses. An alternative approach could use neuromorphic computing, where specialized hardware circuits are used for the simulations with spiking neuronal networks instead of general-purpose CPUs. This enables optimizations for neuron simulations in the form of power usage, parallelism, and speed. The hardware architectures range from hybrid systems with FPGAs (Park et al., 2016), analog circuits, ASICs (Davies et al., 2018) to massively parallel supercomputers (Furber et al., 2014). A considerable advantage is the parallel simulation of neurons through direct hardware support, e.g., having a separate circuit for each neuron. The platforms diﬀer in the way that they transmit spikes between the neurons. They can be transmitted over buses (Mortara et al., 1995), grids (Merolla et al., 2007), or special routing Frontiers in Neuroinformatics 4.2 Biological implications Our ablation studies showed that the memory engrams are resistant. If a loss of connections aﬀected only neurons within the memory engram, it could recover from it and even strengthen its connections. Even a lesion of 50% of the neurons did not lead to forgetting a memory as the connectivity between the memory ensemble was still high enough. This implies some inherent reserve capacity in cortical connectivity (as currently being discussed for Alzheimer’s disease Teipel et al., 2016). If exceeded and connectivity dropped below a critical level, the network would not regrow the connections. As a result, memory engrams may not be recovered. This is similar to our model, where engrams are more likely to form locally, leading to a clustering of neurons forming an engram. In our model, one box represents a concept comparable to a place ﬁeld. With our simulation, we could show that it is possible to form these place cells by simultaneous stimulation comparable to long- term potentation (Teyler and DiScenna, 1987). When rats were placed in a new environment, within minutes, new place cells were activated in the hippocampus and recruited so that they could be recalled weeks later (Rotenberg et al., 1996; Lever et al., 2002). Our model explains this process through the increased activity of the neurons, which leads to a locally limited cluster formation. Only a small cue, such as a perceived environmental one or the recall of the location in the imagination, can reactivate the cluster of place cells (O’Keefe and Conway, 1978). This is similar to our model, where only a partial activation of the unconditioned stimulus (similar to an environmental cue) leads to the reactivation of the conditioned stimulus. For this reserve capacity, distant connections may also play a role. Moreover, forming synapses in a distant-dependent manner enables the simulation of ongoing neuron activity in diﬀerent brain areas without interference. Without this property, all of our ensembles, US and C1, would connect to each other, forming a single large memory engram instead of various smaller ones. Nevertheless, engrams can be largely distributed over the brain brought about by the Gaussian scaling parameter σ. An increment of σ would be comparable with a phase in which the subject learns a new concept and increases plasticity in the brain. 4.2 Biological implications We showed that the Model of Structural Plasticity enables the simultaneous formation of multiple memory engrams. We saw clear, distinct spikes during the retrieval phase, indicating that the network learned the relationship between the ensembles US and C1. However, the ﬁring rate of the readout neurons was lower than during the direct stimulation of US with 1kHz. Moreover, while conﬁrming the general neurophysiological conclusions by 17 frontiersin.org Kaster et al. 10.3389/fninf.2024.1323203 discarded this idea (Eichenbaum et al., 1999). Nevertheless, place cells have been found in the hippocampus of humans and rats that are active when the subject is at a certain location (O’Keefe and Dostrovsky, 1971; Hill and Best, 1981; Thompson and Best, 1989; Eichenbaum et al., 1999; Moser et al., 2008). These place cells allow reliable location detection but are not spatially distributed in the hippocampus. However, place cells of the same or nearby location tend to cluster (Eichenbaum et al., 1989), and the distance between these place cells correlates with the real-world locations they represent (O’Keefe and Burgess, 1996). Neurons representing similar concepts, such as similar locations, are often located close to each other. Other examples can be found in the retinotopic organization of the visual cortex (Hubel and Wiesel, 1962; Tusa et al., 1979) and the cerebral cortex (Penﬁeld and Rasmussen, 1950). discarded this idea (Eichenbaum et al., 1999). Nevertheless, place cells have been found in the hippocampus of humans and rats that are active when the subject is at a certain location (O’Keefe and Dostrovsky, 1971; Hill and Best, 1981; Thompson and Best, 1989; Eichenbaum et al., 1999; Moser et al., 2008). These place cells allow reliable location detection but are not spatially distributed in the hippocampus. However, place cells of the same or nearby location tend to cluster (Eichenbaum et al., 1989), and the distance between these place cells correlates with the real-world locations they represent (O’Keefe and Burgess, 1996). Neurons representing similar concepts, such as similar locations, are often located close to each other. Other examples can be found in the retinotopic organization of the visual cortex (Hubel and Wiesel, 1962; Tusa et al., 1979) and the cerebral cortex (Penﬁeld and Rasmussen, 1950). Gallinaro et al. (2022), we demonstrated them at a much greater scale—with a network of 4,287,500 neurons and 343 ensemble pairs. And given the favorable scaling properties of the underlying algorithm, we see no obvious limit to further increases. 4.2 Biological implications This could be comparable to the release of proteins during learning phases that allow higher plasticity in the brain (Poo, 2001; Lamprecht and LeDoux, 2004). Another possibility is to model diﬀerent types of neurons with diﬀerent σ to ensure that some neurons can project into other brain areas while others project only locally to their direct neighbors. Frontiers in Neuroinformatics Funding The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fninf.2024. 1323203/full#supplementary-material The author(s) declare ﬁnancial support was received for the research, authorship, and/or publication of this article. The authors 4.4 Predictions on the nature of homeostatic engram formation The organization of our 3D model in distinct boxes is comparable to the modular organization of the cortex in columns and hyper-columns (Hubel and Wiesel, 1962; Mountcastle, 1997). One concept is learned in one box very much within a single module of the associative cortices in the brain. However, our model can still learn relationships between more than one box. These memory engrams do not inﬂuence the local memory engrams within a box comparable to the human brain, where inactive memories remain unchanged while non-associated memories are active. Our modeling approach predicts that pruning of synapses is an integral part of homeostatic engram formation and does not occur as mere compensatory response to enduring synapse potentiation associated with memory formation (as in Hebbian plasticity with synaptic scaling Turrigiano, 2008 as synaptic homeostasis rule) but, in fact, precedes and ﬁrst enables associative synapse formation. Homeostatic engram formation requires a certain spatiotemporal order and availability of vacant axonal and dendritic elements for synapse formation. If vacant synaptic elements are not available in certain ensembles, learning or re- learning of engrams may fail. This is particularly seen in ablation studies and clearly distinguishes homeostatic engram formation from associative Hebbian learning, in which the formation of new engrams merely depends on the coincidence or temporal order of input signals. Homeostatic engram formation can further be boosted by inhibition opening up critical periods for ampliﬁed synapse formation (Rinke et al., 2017) and possibly also for engram formation (still to be shown computationally). With this, the present modeling approach provides a number of testable predictions on the nature of homeostatic engram formation. Another view on our model is its similarity to the hippocampus, which is an essential area in memory formation (Squire et al., 2004) and shows structural changes during learning (Leuner et al., 2003; Groussard et al., 2010; Leuner and Gould, 2010). Declarative and spatial memory are often associated with the hippocampus (Eichenbaum and Cohen, 2014; Deuker et al., 2016; Eichenbaum, 2017). Spatial memory enables us to navigate and capture the relation of locations. It was originally suspected that the brain consists of a spatial allocentric map that would allow a mapping of locations in an absolute coordinate system (Tolman, 1948; O’keefe and Nadel, 1979; O’Keefe, 1991), but later research 18 frontiersin.org frontiersin.org 10.3389/fninf.2024.1323203 Kaster et al. Kaster et al. Conﬂict of interest The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Acknowledgments The authors gratefully acknowledge having conducted a part of this study on the Lichtenberg high-performance computer of TU Darmstadt. The authors acknowledge the support of the Open Access Publishing Fund of Technical University of Darmstadt. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Author contributions MK: Investigation, Methodology, Software, Writing – original draft, Writing – review & editing. FC: Methodology, Software, Supervision, Writing – review & editing. MB-O: Methodology, Supervision, Writing – review & editing. FW: Funding acquisition, Methodology, Supervision, Writing – review & editing. Data availability statement The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the ﬁnal decision. The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: the simulator code can be found here: https://github.com/tuda-parallel/RELeARN. Our analysis scripts and data can be found here: https://doi.org/10.5281/ zenodo.8287234. 4.5 Conclusion would like to thank the NHR-Verein e.V. for supporting this work within the NHR Graduate School of National High Performance Computing (NHR). The authors gratefully acknowledge the German Federal Ministry of Education and Research (BMBF) and the state of Hesse (HMWK) for supporting this project as part of the NHR funding. This research has also received funding from the European Union’s Horizon 2020 Framework Programme for Research and Innovation under the Speciﬁc Grant Agreement No. 945539 (Human Brain Project SGA3). would like to thank the NHR-Verein e.V. for supporting this work within the NHR Graduate School of National High Performance Computing (NHR). The authors gratefully acknowledge the German Federal Ministry of Education and Research (BMBF) and the state of Hesse (HMWK) for supporting this project as part of the NHR funding. This research has also received funding from the European Union’s Horizon 2020 Framework Programme for Research and Innovation under the Speciﬁc Grant Agreement No. 945539 (Human Brain Project SGA3). We believe that our approach lays the foundation for the understanding of more complex memory systems. We could show that the homeostatic engram formation and functional recovery from lesions trigger a neuronal growth mechanism (implemented as homeostatic growth rules) and, hence, may recapitulate neural development. As we have further demonstrated, we can form engrams locally within a box as well as over longer distances by adapting the Gaussian scaling parameter σ. In addition to the already discussed recruiting of neurons in multiple engrams, we could build associations between multiple engrams locally or distribute them across the network. This would be an important extension because memories in the brain do not exist in a single one-to-one relationship but rather depend on many other memories. Another possibility is to build a memory hierarchy with directed associations where one memory engram activates another but not the other way around. In that case, an abstract concept represented as a memory engram is more strongly associated with another than vice versa. A combination of these extensions would enable the building of a more complex memory system. Butz, M., Wörgötter, F., and Ooyen, A. V. (2009). Activity-dependent structural plasticity. Brain Res. Rev. 60, 287–305. doi: 10.1016/j.brainresrev.2008. 12.023 Boyke, J., Driemeyer, J., Gaser, C., Büchel, C., and May, A. (2008). Training- induced brain structure changes in the elderly. J. Neurosci. 28, 7031–7035. doi: 10.1523/JNEUROSCI.0742-08.2008 References doi: 10.1523/JNEUROSCI.09-08-02764.1989 Moser, E. I., Kropﬀ, E., and Moser, M.-B. (2008). Place cells, grid cells, and the brain’s spatial representation system. Annu. Rev. Neurosci. 31, 69–89. doi: 10.1146/annurev.neuro.31.061307.090723 Fiebig, F., and Lansner, A. (2017). A spiking working memory model based on hebbian short-term potentiation. J. Neurosci. 37, 83–96. doi: 10.1523/JNEUROSCI.1989-16.2017 Mountcastle, V. B. (1997). The columnar organization of the neocortex. Brain 120, 701–722. doi: 10.1093/brain/120.4.701 O’Keefe, J. (1991). An allocentric spatial model for the hippocampal cognitive map. Hippocampus 1, 230–235. doi: 10.1002/hipo.450010303 Fox, K., and Stryker, M. (2017). Integrating Hebbian and homeostatic plasticity: introduction. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 372:20160413. doi: 10.1098/rstb.2016.0413 O’Keefe, J., and Burgess, N. (1996). Geometric determinants of the place ﬁelds of hippocampal neurons. Nature 381, 425–428. doi: 10.1038/381425a0 Furber, S. B., Galluppi, F., Temple, S., and Plana, L. A. (2014). The spinnaker project. Proc. IEEE 102, 652–665. doi: 10.1109/JPROC.2014.2304638 O’Keefe, J., and Conway, D. H. (1978). Hippocampal place units in the freely moving rat: why they ﬁre where they ﬁre. Experim. Brain Res. 31:, -590. doi: 10.1007/BF00239813 Fusi S. (2024). “Memory capacity of neural network models,” in Oxford Handbook of Human Memory, eds M. Kahana and W. Anthony (Oxford University Press). O’Keefe, J., and Dostrovsky, J. (1971). The hippocampus as a spatial map: preliminary evidence from unit activity in the freely-moving rat. Brain Res. 34, 171–5. doi: 10.1016/0006-8993(71)90358-1 Gallinaro, J. V., Gašparovi´c, N., and Rotter, S. (2022). Homeostatic control of synaptic rewiring in recurrent networks induces the formation of stable memory engrams. PLoS Comput. Biol. 18, 1009836. doi: 10.1371/journal.pcbi.1009836 O’keefe, J., and Nadel, L. (1979). Précis of o’keefe & nadel’s the hippocampus as a cognitive map. Behav. Brain Sci. 2, 487–494. doi: 10.1017/S0140525X000 63949 Groussard, M., La Joie, R., Rauchs, G., Landeau, B., Chetelat, G., Viader, F., et al. (2010). When music and long-term memory interact: eﬀects of musical expertise on functional and structural plasticity in the hippocampus. PLoS ONE 5, e13225. doi: 10.1371/journal.pone.0013225 Park, J., Yu, T., Joshi, S., Maier, C., and Cauwenberghs, G. (2016). Hierarchical address event routing for reconﬁgurable large-scale neuromorphic systems. IEEE trans. Neural netw. Learn. Syst. 28, 2408–2422. doi: 10.1109/TNNLS.2016.2572164 Hadjikhani, N., Liu, A. K., Dale, A. M., Cavanagh, P., and Tootell, R. B. (1998). Retinotopy and color sensitivity in human visual cortical area v8. Nat. Neurosci. 1, 235–241. doi: 10.1038/681 Penﬁeld, W., and Rasmussen, T. (1950). References doi: 10.1016/j.jpdc.2022.09.001 Leuner, B., and Gould, E. (2010). Structural plasticity and hippocampal function. Annu. Rev. Psychol. 61, 111–140. doi: 10.1146/annurev.psych.093008.100359 Dammasch, I. E. (1990). “Structural realization of a hebb-type learning rule,” in Models of Brain Function, ed. R. M. J. Cotterill (Cambridge University Press, Cambridge), 539–552. Lever, C., Wills, T., Cacucci, F., Burgess, N., and O’Keefe, J. (2002). Long-term plasticity in hippocampal place-cell representation of environmental geometry. Nature 416, 90–94. doi: 10.1038/416090a Davies, M., Srinivasa, N., Lin, T.-H., Chinya, G., Cao, Y., Choday, S. H., et al. (2018). Loihi: A neuromorphic manycore processor with on-chip learning. IEEE Micro 38, 82–99. doi: 10.1109/MM.2018.112130359 López-Aranda, M. F., López-Téllez, J. F., Navarro-Lobato, I., Masmudi-Martín, M., Gutiérrez, A., and Khan, Z. U. (2009). Role of layer 6 of v2 visual cortex in object-recognition memory. Science. 325, 87–89. doi: 10.1126/science.1170869 Deuker, L., Bellmund, J. L., Navarro Schröder, T., and Doeller, C. F. (2016). An event map of memory space in the hippocampus. Elife 5, e16534. doi: 10.7554/eLife.16534 Massey Jr, F. J. (1951). The kolmogorov-smirnov test for goodness of ﬁt. J. Am. Stat. Assoc. 46, 68–78. doi: 10.1080/01621459.1951.10500769 Downing, P. E., and Peelen, M. V. (2011). The role of occipitotemporal body-selective regions in person perception. Cogn. Neurosci. 2, 186–203. doi: 10.1080/17588928.2011.582945 May, A. (2011). Experience-dependent structural plasticity in the adult human brain. Trends Cogn. Sci. 15, 475–482. doi: 10.1016/j.tics.2011.08.002 Eichenbaum, H. (2017). On the integration of space, time, and memory. Neuron 95, 1007–1018. doi: 10.1016/j.neuron.2017.06.036 Merolla, P. A., Arthur, J. V., Shi, B. E., and Boahen, K. A. (2007). Expandable networks for neuromorphic chips. IEEE Trans. Circuits Syst. I: Regular Papers 54, 301–311. doi: 10.1109/TCSI.2006.887474 Eichenbaum, H., and Cohen, N. J. (2014). Can we reconcile the declarative memory and spatial navigation views on hippocampal function? Neuron 83, 764–770. doi: 10.1016/j.neuron.2014.07.032 Mizrahi, A. (2007). Dendritic development and plasticity of adult-born neurons in the mouse olfactory bulb. Nat. Neurosci. 10, 444–452. doi: 10.1038/nn1875 Eichenbaum, H., Dudchenko, P., Wood, E., Shapiro, M., and Tanila, H. (1999). The hippocampus, memory, and place cells: is it spatial memory or a memory space? Neuron 23, 209–226. doi: 10.1016/S0896-6273(00)80773-4 Mortara, A., Vittoz, E. A., and Venier, P. (1995). A communication scheme for analog vlsi perceptive systems. IEEE J. Solid-State Circuits 30, 660–669. doi: 10.1109/4.387069 Eichenbaum, H., Wiener, S. I., Shapiro, M., and Cohen, N. (1989). The organization of spatial coding in the hippocampus: a study of neural ensemble activity. J. Neurosci. 9, 2764–2775. References Barak, O., and Tsodyks, M. (2014). Working models of working memory. Curr. Opin. Neurobiol. 25, 20–24. doi: 10.1016/j.conb.2013.10.008 Boyke, J., Driemeyer, J., Gaser, C., Büchel, C., and May, A. (2008). Training- induced brain structure changes in the elderly. J. Neurosci. 28, 7031–7035. doi: 10.1523/JNEUROSCI.0742-08.2008 Barnes, S. J., and Finnerty, G. T. (2010). Sensory experience and cortical rewiring. The Neuroscientist 16, 186–198. doi: 10.1177/1073858409343961 Butz, M., and van Ooyen, A. (2013). A simple rule for dendritic spine and axonal bouton formation can account for cortical reorganization after focal retinal lesions. PLoS Comput. Biol. 9, 39–43. doi: 10.1371/annotation/e8b7df48-4639-4ac1-8a98-cb13dea3415b Billaudelle, S., Cramer, B., Petrovici, M. A., Schreiber, K., Kappel, D., Schemmel, J., et al. (2021). Structural plasticity on an accelerated analog neuromorphic hardware system. Neural Netw. 133, 11–20. doi: 10.1016/j.neunet.2020.09.024 Bogdan, P. A., Rowley, A. G., Rhodes, O., and Furber, S. B. (2018). Structural plasticity on the spinnaker many-core neuromorphic system. Front. Neurosci. 12, 434. doi: 10.3389/fnins.2018.00434 Frontiers in Neuroinformatics 19 frontiersin.org Kaster et al. 10.3389/fninf.2024.1323203 Izhikevich, E. (2004). Which model to use for cortical spiking neurons? IEEE Trans. Neural Netw. 15, 1063–1070. doi: 10.1109/TNN.2004.832719 Caroni, P., Donato, F., and Muller, D. (2012). Structural plasticity upon learning: regulation and functions. Nat, Revi, Neurosci, 13, 478–490. doi: 10.1038/nrn3258 Kalisman, N., Silberberg, G., and Markram, H. (2005). The neocortical microcircuit as a tabula rasa. Proc. Nat. Acad. Sci. 102, 880–885. doi: 10.1073/pnas.0407088102 Chen, J.-Y., Lonjers, P., Lee, C., Chistiakova, M., Volgushev, M., and Bazhenov, M. (2013). Heterosynaptic plasticity prevents runaway synaptic dynamics. J. Neurosci. 33, 15915–15929. doi: 10.1523/JNEUROSCI.5088-12.2013 Knoblauch, A. (2017). “Chapter 17 - impact of structural plasticity on memory formation and decline,” The Rewiring Brain, eds. A. van Ooyen, and M. Butz-Ostendorf (San Diego: Academic Press), 361–386. Chistiakova, M., Bannon, N. M., Chen, J.-Y., Bazhenov, M., and Volgushev, M. (2015). Homeostatic role of heterosynaptic plasticity: models and experiments. Front. Comput. Neurosci. 9, 89. doi: 10.3389/fncom.2015.00089 Lamprecht, R., and LeDoux, J. (2004). Structural plasticity and memory. Nat. Rev. Neurosci. 5, 45–54. doi: 10.1038/nrn1301 Chklovskii, D. B., Mel, B. W., and Svoboda, K. (2004). Cortical rewiring and information storage. Nature 431, 3012. doi: 10.1038/nature03012 Leuner, B., Falduto, J., and Shors, T. J. (2003). Associative memory formation increases the observation of dendritic spines in the hippocampus. J. Neurosci. 23, 659–665. doi: 10.1523/JNEUROSCI.23-02-00659.2003 Czappa, F., Geiß, A., and Wolf, F. (2023). Simulating structural plasticity of the brain more scalable than expected. J. Parallel Distrib. Comput. 171, 24–27. References The cerebral cortex of man; a clinical study of localization of function. J. Am. Med. Assoc. 144, 1412–1412. doi: 10.1001/jama.1950.02920160086033 Hayashi-Takagi, A., Yagishita, S., Nakamura, M., Shirai, F., Wu, Y. I., Loshbaugh, A. L., et al. (2015). Labelling and optical erasure of synaptic memory traces in the motor cortex. Nature 525, 333–338. doi: 10.1038/nature15257 Poo, M. (2001). Neurotrophins as synaptic modulators. Nat. Rev. Neurosci. 2, 24–32. doi: 10.1038/35049004 Hill, A. J., and Best, P. J. (1981). Eﬀects of deafness and blindness on the spatial correlates of hippocampal unit activity in the rat. Exp. Neurol. 74, 204–217. doi: 10.1016/0014-4886(81)90159-X Pukelsheim, F. (1994). The three sigma rule. Am. Stat. 48, 88–91. doi: 10.1080/00031305.1994.10476030 Holtmaat, A., and Caroni, P. (2016). Functional and structural underpinnings of neuronal assembly formation in learning. Nat. Neurosci. 19, 1553–1562. doi: 10.1038/nn.4418 Reale, R. A., and Imig, T. J. (1980). Tonotopic organization in auditory cortex of the cat. J. Comparat. Neurol. 192, 265–291. doi: 10.1002/cne.901920207 Rinke, S., Butz-Ostendorf, M., Hermanns, M. A., Naveau, M., and Wolf, F. (2018). A scalable algorithm for simulating the structural plasticity of the brain. J. Parallel Distrib. Comput. 120, 251–266. doi: 10.1016/j.jpdc.2017.11.019 Holtmaat, A., and Svoboda, K. (2009). Experience-dependent structural synaptic plasticity in the mammalian brain. Nat. Rev. Neurosci. 10, 647–658. doi: 10.1038/nrn2699 Rinke, S., Naveau, M., Wolf, F., and Butz-Ostendorf, M. (2017). “Chapter 8 - critical periods emerge from homeostatic structural plasticity in a full-scale model of the developing cortical column,” in The Rewiring Brain, eds. A. van Ooyen, and M. Butz-Ostendorf (San Diego: Academic Press), 177–201. Huang, Q.-S., and Wei, H. (2021). A computational model of working memory based on spike-timing-dependent plasticity. Front. Comput. Neurosci. 15, 630999. doi: 10.3389/fncom.2021.630999 Butz-Ostendorf (San Diego: Academic Press), 177–201. Hubel, D. H., and Wiesel, T. N. (1962). Receptive ﬁelds, binocular interaction and functional architecture in the cat’s visual cortex. J. Physiol. 160, 106. doi: 10.1113/jphysiol.1962.sp006837 Rolls, E. T., Dempere-Marco, L., and Deco, G. (2013). Holding multiple items in short term memory: a neural mechanism. PLoS ONE 8, e61078. doi: 10.1371/journal.pone.0061078 Frontiers in Neuroinformatics 20 frontiersin.org 10.3389/fninf.2024.1323203 Kaster et al. Rotenberg, A., Mayford, M., Hawkins, R. D., Kandel, E. R., and Muller, R. U. (1996). Mice expressing activated camkii lack low frequency ltp and do not form stable place cells in the ca1 region of the hippocampus. Cell 87, 1351–1361. doi: 10.1016/S0092-8674(00)81829-2 Trachtenberg, J. T., Chen, B. E., Knott, G. W., Feng, G., Sanes, J. References R., Welker, E., et al. (2002). Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex. Nature 420, 788–794. doi: 10.1038/nature01273 Turrigiano, G. G. (2008). The self-tuning neuron: synaptic scaling of excitatory synapses. Cell 135, 422–435. doi: 10.1016/j.cell.2008.10.008 Ruben, J., Schwiemann, J., Deuchert, M., Meyer, R., Krause, T., Curio, G., et al. (2001). Somatotopic organization of human secondary somatosensory cortex. Cereb. Cortex 11, 463–473. doi: 10.1093/cercor/11.5.463 Tusa, R., Rosenquist, A., and Palmer, L. (1979). Retinotopic organization of areas 18 and 19 in the cat. J. Comparat, Neurol, 185, 657–678. doi: 10.1002/cne.901850405 Savin, C., and Triesch, J. (2014). Emergence of task-dependent representations in working memory circuits. Front. Comput. Neurosci. 8, 57. doi: 10.3389/fncom.2014.00057 Xu, T., Yu, X., Perlik, A. J., Tobin, W. F., Zweig, J. A., Tennant, K., et al. (2009). Rapid formation and selective stabilization of synapses for enduring motor memories. Nature 462, 915–919. doi: 10.1038/nature08389 Squire, L. R., Stark, C. E., and Clark, R. E. (2004). The medial temporal lobe. Annu. Rev. Neurosci. 27, 279–306. doi: 10.1146/annurev.neuro.27.070203.144130 Yang, S., Tan, J., Lei, T., and Linares-Barranco, B. (2023a). Smart traﬃc navigation system for fault-tolerant edge computing of internet of vehicle in intelligent transportation gateway. IEEE Trans. Intellig. Transport. Syst. 24, 13011–13022. doi: 10.1109/TITS.2022.3232231 Stepanyants, A., Hof, P. R., and Chklovskii, D. B. (2002). Geometry and structural plasticity of synaptic connectivity. Neuron 34, 275–288. doi: 10.1016/S0896-6273(02)00652-9 Yang, S., Wang, H., Pang, Y., Azghadi, M. R., and Linares-Barranco, B. (2023b). Nadol: Neuromorphic architecture for spike-driven online learning by dendrites. IEEE Trans. Biomed. Circuits Syst. 18, 186–199. doi: 10.1109/TBCAS.2023.3316968 Szatmáry, B., and Izhikevich, E. M. (2010). Spike-timing theory of working memory. PLoS Comput. Biol. 6, e1000879. doi: 10.1371/journal.pcbi.1000879 Teipel, S., Grothe, M. J., Zhou, J., Sepulcre, J., Dyrba, M., Sorg, C., et al. (2016). Measuring cortical connectivity in alzheimer’s disease as a brain neural network pathology: toward clinical applications. J. Int. Neuropsychological Society 22, 138–163. doi: 10.1017/S1355617715000995 Yang, S., Wang, J., Deng, B., Azghadi, M. R., and Linares-Barranco, B. (2021a). Neuromorphic context-dependent learning framework with fault- tolerant spike routing. IEEE Trans. Neural Netw. Learn. Syst. 33, 7126–7140. doi: 10.1109/TNNLS.2021.3084250 Teyler, T. J., and DiScenna, P. (1987). Long-term potentiation. Annu. Rev. Neurosci. 10, 131–161. doi: 10.1146/annurev.ne.10.030187.001023 Yang, S., Wang, J., Hao, X., Li, H., Wei, X., Deng, B., et al. (2021b). Bicoss: toward large-scale cognition brain with multigranular neuromorphic architecture. IEEE Trans. Neural Netw. Learn. Syst. 33, 2801–2815. References doi: 10.1109/TNNLS.2020.3045492 Thompson, L., and Best, P. (1989). Place cells and silent cells in the hippocampus of freely-behaving rats. J. Neurosci. 9, 2382–2390. doi: 10.1523/JNEUROSCI.09-07-02382.1989 Yang, S., Wang, J., Zhang, N., Deng, B., Pang, Y., and Azghadi, M. R. (2021c). Cerebellumorphic: large-scale neuromorphic model and architecture for supervised motor learning. IEEE Trans. Neural Netw. Learn. Syst. 33, 4398–4412. doi: 10.1109/TNNLS.2021.3057070 Tolman, E. C. (1948). Cognitive maps in rats and men. Psychol. Rev. 55, 189. doi: 10.1037/h0061626 21 frontiersin.org Frontiers in Neuroinformatics Frontiers in Neuroinformatics
https://openalex.org/W2063521888	https://europepmc.org/articles/pmc4450062?pdf=render	English	null	A Membrane-bound eIF2 Alpha Kinase Located in Endosomes Is Regulated by Heme and Controls Differentiation and ROS Levels in Trypanosoma cruzi	PLOS pathogens	2,015	cc-by	16,954	Abstract Citation: da Silva Augusto L, Moretti NS, Ramos TCP, de Jesus TCL, Zhang M, Castilho BA, et al. (2015) A Membrane-bound eIF2 Alpha Kinase Located in Endosomes Is Regulated by Heme and Controls Differentiation and ROS Levels in Trypanosoma cruzi. PLoS Pathog 11(2): e1004618. doi:10.1371/journal.ppat.1004618 Translation initiation has been described as a key step for the control of growth and differen- tiation of several protozoan parasites in response to environmental changes. This occurs by the activation of protein kinases that phosphorylate the alpha subunit of the translation initia- tion factor 2 (eIF2α), which decreases translation, and in higher eukaryotes favors the ex- pression of stress remedial response genes. However, very little is known about the signals that activate eIF2α kinases in protozoan parasites. Here, we characterized an eIF2α kinase of Trypanosoma cruzi (TcK2), the agent of Chagas’ disease, as a transmembrane protein lo- cated in organelles that accumulate nutrients in proliferating parasite forms. We found that heme binds specifically to the catalytic domain of the kinase, inhibiting its activity. In the ab- sence of heme, TcK2 is activated, arresting cell growth and inducing differentiation of prolif- erative into infective and non-proliferative forms. Parasites lacking TcK2 lose this Translation initiation has been described as a key step for the control of growth and differen- tiation of several protozoan parasites in response to environmental changes. This occurs by the activation of protein kinases that phosphorylate the alpha subunit of the translation initia- tion factor 2 (eIF2α), which decreases translation, and in higher eukaryotes favors the ex- pression of stress remedial response genes. However, very little is known about the signals that activate eIF2α kinases in protozoan parasites. Here, we characterized an eIF2α kinase of Trypanosoma cruzi (TcK2), the agent of Chagas’ disease, as a transmembrane protein lo- cated in organelles that accumulate nutrients in proliferating parasite forms. We found that heme binds specifically to the catalytic domain of the kinase, inhibiting its activity. In the ab- sence of heme, TcK2 is activated, arresting cell growth and inducing differentiation of prolif- erative into infective and non-proliferative forms. Parasites lacking TcK2 lose this differentiation capacity and heme is not stored in reserve organelles, remaining in the cyto- sol. TcK2 null cells display growth deficiencies, accumulating hydrogen peroxide that drives the generation of reactive oxygen species. A Membrane-bound eIF2 Alpha Kinase Located in Endosomes Is Regulated by Heme and Controls Differentiation and ROS Levels in Trypanosoma cruzi Leonardo da Silva Augusto1, Nilmar Silvio Moretti1, Thiago Cesar Prata Ramos1, Teresa Cristina Leandro de Jesus1¤, Min Zhang2, Beatriz A. Castilho1, Sergio Schenkman1* Leonardo da Silva Augusto1, Nilmar Silvio Moretti1, Thiago Cesar Prata Ramos1, Teresa Cristina Leandro de Jesus1¤, Min Zhang2, Beatriz A. Castilho1, Sergio Schenkman1* 1 Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil, 2 Department of Pathology, New York University School of Medicine, New York, New York, United States of America ¤ Current address: Instituto de Física de São Carlos (IFSC), Universidade de São Paulo. São Carlos, São Paulo, Brazil * sschenkman@unifesp.br ¤ Current address: Instituto de Física de São Carlos (IFSC), Universidade de São Paulo. São Carlos, São Paulo, Brazil * sschenkman@unifesp br RESEARCH ARTICLE Introduction The phosphorylation of the alpha subunit of the trimeric eukaryotic initiation factor 2 (eIF2) com- plex is a key event in the cellular stress response [1]. This phosphorylation inhibits the conversion of GDP to GTP in eIF2 by the eIF2B, a guanosine exchange factor, thus decreasing the levels of the ternary complex eIF2-GTP-Met-tRNAi available for new rounds of translation initiation. At the same time that global translation is inhibited, there is preferential translation of specific mes- sages that promote the remediation from stresses in several eukaryotes [2]. The phosphorylation of eIF2α is catalyzed by serine-threonine kinases specifically activated by the different stress condi- tions [3]. Mammals have four kinases that phosphorylate eIF2α: a) The general control nondere- pressible 2 (GCN2) kinase (KEGG K16196, also known as eIF2AK4), which is activated by amino acid deprivation through binding to uncharged tRNAs [4]. b) The RNA-activated protein kinase (PKR) (KEGG 16195, also known as eIF2AK2), which is activated by double strand RNA in cells infected with viruses [5, 6, 7]. c) The PKR-like endoplasmic reticulum kinase (PERK) (KEGG K08860, also known as eIF2AK3), which is activated by unfolded proteins in the endoplasmic re- ticulum [8]. d) The heme-regulated eIF2α kinase (HRI) (KEGG K16194, also known as eIF2AK1), which is activated by heme deficiency [9]. Different stress conditions result in confor- mational changes within the catalytic domain of these kinases leading to autophosphorylation and activation, allowing for the binding and subsequent phosphorylation of the eIF2α substrate. Similar kinases of eIF2α are present in other organisms and are involved in stress sensing and remediation [10]. While GCN2, or a GCN2-like kinase, is present in virtually all eukaryotic organisms, other eIF2α kinases are more scattered through different organisms. For example, HRI is present in some fungi including Schizosaccharomyces pombe. PERK orthologues have been identified in worms, insects and vertebrates. Specific phosphorylation of eIF2α by unique kinases is also directly involved in responses to environmental changes throughout the lifecycle of protozoan parasites (reviewed in [11, 12]). For example, the eIF2α kinase eIK2 controls the latency of Plasmodium sporozoites in insect salivary glands through eIF2α phosphorylation [13]. The eIK1 is homologous to GCN2, as it is activated by amino acid starvation, regulating the erythrocyte cycle of Plasmodium [14]. The PK4 has a topology similar to PERK and con- trols the growth of schizonts and gametocytes of Plasmodium [15]. eIF2 Kinase Regulated by Heme 50586-6 to TCPR, 2003/12722-9 to TCLJ, 2012/ 50399-4 to MZ, 2009/52047-5 to BAC and by Conselho Nacional de Desenvolvimento Científico e tecnológico (http://www.cnpq.br) through grants 477143/2011-3 and 304359/2009-2 to SS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 50586-6 to TCPR, 2003/12722-9 to TCLJ, 2012/ 50399-4 to MZ, 2009/52047-5 to BAC and by Conselho Nacional de Desenvolvimento Científico e tecnológico (http://www.cnpq.br) through grants 477143/2011-3 and 304359/2009-2 to SS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. parasite moves towards the hindgut and when the blood is completely digested, these or- ganelles are consumed. At this moment, the insect is ready for a new feeding cycle that promotes the release of infective metacyclic-trypomastigote forms. We have previously found that such differentiation involves protein synthesis arrest through the phosphoryla- tion of the eukaryotic translation initiation factor 2α (eIF2α). Now, we show that one of the kinases (TCK2) that phosphorylate eIF2α is localized in these endosomes. TcK2 binds and is specifically inhibited by heme derived from blood hemoglobin. We also found that heme inhibits differentiation, suggesting that it is an important signal for differentiation. By generating knockouts of TcK2, we observed an increased accumulation of heme in the cytosol, which induced cellular damage by affecting the reactive oxygen metabolism in the parasite. We conclude that this eIF2α kinase senses cytosolic heme obtained from the blood meal, promoting its storage in the cytosolic organelles. When heme levels are de- creased in the cytosol, TcK2 activation can then arrest protein synthesis that is followed by the induction of the differentiation of proliferative epimastigote forms to infective meta- cyclic-trypomastigotes. Competing Interests: The authors have declared that no competing interests exist Competing Interests: The authors have declared that no competing interests exist Abstract The augmented level of hydrogen peroxide oc- curs as a consequence of increased superoxide dismutase activity and decreased peroxide activity. These phenotypes could be reverted by the re-expression of the wild type but not of a TcK2 dead mutant. These findings indicate that heme is a key factor for the growth control and differentiation through regulation of an unusual type of eIF2α kinase in T. cruzi. Editor: Martin Taylor, London School of Hygiene & Tropical Medicine, UNITED KINGDOM Received: September 28, 2014 Accepted: December 10, 2014 Published: February 6, 2015 Editor: Martin Taylor, London School of Hygiene & Tropical Medicine, UNITED KINGDOM Editor: Martin Taylor, London School of Hygiene & Tropical Medicine, UNITED KINGDOM Received: September 28, 2014 Accepted: December 10, 2014 Published: February 6, 2015 Copyright: © 2015 da Silva Augusto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. differentiation capacity and heme is not stored in reserve organelles, remaining in the cyto- sol. TcK2 null cells display growth deficiencies, accumulating hydrogen peroxide that drives the generation of reactive oxygen species. The augmented level of hydrogen peroxide oc- curs as a consequence of increased superoxide dismutase activity and decreased peroxide activity. These phenotypes could be reverted by the re-expression of the wild type but not of a TcK2 dead mutant. These findings indicate that heme is a key factor for the growth control and differentiation through regulation of an unusual type of eIF2α kinase in T. cruzi. Data Availability Statement: All relevant data are whitin the paper and its Supporting Information files except for the sequence TcCLB.506559.129, TcCLB.511071.190, ADWP02013222, available from Tritryp (http://tritrypdb.org) and gene identification 945,1 available from NCBI; and Q9BQI3.2 from UniProtKB/Swiss-Prot. Author Summary Trypanosoma cruzi proliferates as epimastigotes in the midgut of the insect vector filled with blood meal. There, it accumulates nutrients in specific endosomal organelles. The Funding: This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (http:// www.fapesp.br) through grants 2011/51973-3 to SS, 2009/54364-8 to LSA, 2012/09403-8 to NSM, 2011/ 1 / 27 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 eIF2 Kinase Regulated by Heme latent in the vertebrate host, or during parasite passage through the extracellular medium, when the parasite is subjected to lower amino acid levels [16, 17, 18]. latent in the vertebrate host, or during parasite passage through the extracellular medium, when the parasite is subjected to lower amino acid levels [16, 17, 18]. The phosphorylation of eIF2α is also involved in the control of trypanosome differentiation in response to variations in nutrients and temperature. In these organisms, there are three pu- tative eIF2α kinases: a) K1, which is similar to GCN2, b) K2 with a topology similar to PERK, and c) K3, with little similarity to the known eIF2 kinases [19]. The physiological significance of these kinases is mostly unknown. In Leishmania, the K2 ortholog (LinPERK) is localized in the endoplasmic reticulum and regulates the differentiation of promastigotes into amastigotes by unknown stimulus [20, 21]. Interestingly, in Trypanosoma brucei the homologous kinase (TbeIF2αK2) is localized in the flagellar pocket membrane, site of intense membrane internali- zation, and early endocytic vesicles [19]. TbeIF2αK3 has been recently shown to migrate from the endoplasmic reticulum to the nucleus upon persistent stress, abrogating transcription events [22]. In Trypanosoma cruzi, we have demonstrated that eIF2α phosphorylation is re- quired for the differentiation of the non-infective epimastigotes into infective metacyclic trypo- mastigotes (metacyclogenesis) stimulated by starvation [23], mimicking what happens in the insect hindgut [24, 25]. Metacyclogenesis takes about 72 h to be accomplished when the para- sites finally cease translation [23]. As nutrients stored in endosome-like organelles, also called reservosomes, are consumed during metacyclogenesis [26], we presumed that TcK2, the kinase homologous to the T. brucei flagellar pocket eIF2 kinase (TbeIF2αK2) might be involved in T. cruzi differentiation. Herein, we studied the expression and the localization of TcK2 kinase in different life stages of the parasite and generated knockout parasites of this kinase. We pro- vide evidence that in T. cruzi, TcK2 is located in the endosomal compartment and it is regulat- ed by heme, controlling the parasite’s growth and differentiation. Introduction In Toxoplasma gondii eIF2α phosphorylation takes place during the differentiation to bradyzoites, which remain PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 2 / 27 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 eIF2 Kinase Regulated by Heme Figure 1. T. cruzi K2 is a membrane bound protein kinase that undergoes autophosphorylation and phosphorylates the parasite eIF2α. (A) Comparative topology of T. cruzi (TcK2) and Mus musculus (Mm) PERK indicating the signal sequence (SS), the transmembrane (TMD) and the kinase (KD) domains. (B) Western blot of total extracts of T. cruzi epimastigotes incubated with an antiserum from a rabbit immunized with a recombinant protein corresponding to T. brucei K2 KD or with a mAb 5D10 prepared against a recombinant K2 fragment located as indicated in panel (A). (C) Western blot of the 100,000 g supernatant (Sol) (1 h centrifugation) from parasites extracts; of the supernatant from the previous pellet after extraction with 1% Triton X100 in the same buffer extraction (Memb) and of the insoluble fraction after detergent extraction (Pellet). T. cruzi epimastigotes were lysed in 0.1 M NaCl, 50 mM Tris-HCl (pH 7.4), 2 mM MgCl2, 10 mM ethylene-glycol tetra-acetic acid, containing the cOmplete cocktail by three freezing and thawing cycles. The blots were probed with anti-TcK2-KD and anti-Hsp70 antibodies. (D) Western blot of lysates of epimastigotes (Tot), or the supernatants of epimastigotes lysed in the presence of indicted amounts of digitonin as described [21] and probed with and anti-TcK2 and anti-BiP and anti-HsP70 antibodies. (E) SDS- PAGE of 6XHis-tagged TceIF2α incubated in the presence of indicated proteins for 15 min with [32P]-γ-ATP. The upper panel shows the autoradiogram and the bottom panel the same gel stained with Coomassie Blue R250. (F) and (G) are blots of, respectively, total extracts of T. cruzi epimastigotes treated with CIAP or of cells pre-incubated for 2 h without (−) or with (+) 10 nM of calyculin A. Anti-β-tubulin was used as a loading control. The histograms below the gel represent the relative amounts of the upper phosphorylated band of TcK2 (TcK2[Up]) and the fast migrating band of TcK2 (TcK2[Low]). The values are mean and standard deviations of triplicates. Asterisks indicate statistically significant differences (p < 0.05) based on t- Figure 1. T. cruzi K2 is a membrane bound protein kinase that undergoes autophosphorylation and phosphorylates the parasite eIF2α. (A) Comparative topology of T. cruzi (TcK2) and Mus musculus (Mm) PERK indicating the signal sequence (SS), the transmembrane (TMD) and the kinase (KD) domains. (B) Western blot of total extracts of T. cruzi epimastigotes incubated with an antiserum from a rabbit immunized with a recombinant protein corresponding to T. brucei K2 KD or with a mAb 5D10 prepared against a recombinant K2 fragment located as indicated in panel (A). (C) Western blot of the 100,000 g supernatant (Sol) (1 h centrifugation) from parasites extracts; of the supernatant from the previous pellet after extraction with 1% Triton X100 in the same buffer extraction (Memb) and of the insoluble fraction after detergent extraction (Pellet). T. cruzi epimastigotes were lysed in 0.1 M NaCl, 50 mM Tris-HCl (pH 7.4), 2 mM MgCl2, 10 mM ethylene-glycol tetra-acetic acid, containing the cOmplete cocktail by three freezing and thawing cycles. The blots were probed with anti-TcK2-KD and anti-Hsp70 antibodies. (D) Western blot of lysates of epimastigotes (Tot), or the supernatants of epimastigotes lysed in the presence of indicted amounts of digitonin as described [21] and probed with and anti-TcK2 and anti-BiP and anti-HsP70 antibodies. (E) SDS- PAGE of 6XHis-tagged TceIF2α incubated in the presence of indicated proteins for 15 min with [32P]-γ-ATP. The upper panel shows the autoradiogram and the bottom panel the same gel stained with Coomassie Blue R250. (F) and (G) are blots of, respectively, total extracts of T. cruzi epimastigotes treated with CIAP or of cells pre-incubated for 2 h without (−) or with (+) 10 nM of calyculin A. Anti-β-tubulin was used as a loading control. The histograms below the gel represent the relative amounts of the upper phosphorylated band of TcK2 (TcK2[Up]) and the fast migrating band of TcK2 (TcK2[Low]). The values are mean and standard deviations of triplicates. Asterisks indicate statistically significant differences (p < 0.05) based on t- Student test. doi:10.1371/journal.ppat.1004618.g001 TcK2 is a membrane bound eIF2α kinase that undergoes autophosphorylation and phosphorylates the parasite eIF2α The predicted topology of T. cruzi K2 was found similar to mammalian PERK. TcK2 contains a kinase domain at its C-terminal, a transmembrane domain in the middle, and a signal peptide at its N-terminal (Fig. 1A). Polyclonal antibodies generated against the kinase domain of TbeI- F2αK2 [19] and a monoclonal antibody (mAb) 5D10 obtained from mice immunized with the TcK2 N-terminal region recognized specifically two bands (140–160 kDa) in Western blots of total parasite lysates (Fig. 1B). The other bands seen in the Western blot were either degrada- tion products, or non-specific antibody labeling, as they were not seen in all experiments. The protein is associated with parasite membranes as it could be solubilized only in the presence of detergents, in contrast to the soluble cytosolic heat shock protein (Hsp) 70 (Fig. 1C). Moreover, TcK2 was only solubilized in high concentrations of digitonin, differently from BiP, present in the endoplasmic reticulum lumen or the Hsp70, located in the cell cytosol (Fig. 1D). The kinase domain of TcK2 fused with the glutathione S-transferase (GST) was expressed in E. coli. The recombinant GST-TcK2, but not GST, obtained from E. coli was able to phosphorylate the purified His6x-tagged-TceIF2α in vitro, as shown in Fig. 1E. In addition, it became phosphorylated, as indicated by the presence of the second labeled band in the autoradiogram with the expected size of GST-TcK2 (68 kDa). Phosphorylation of TcK2 was also suggested to occur in vivo, as treatment of cell lysates with calf intestine alkaline phospha- tase (CIAP) decreased the relative intensity of the slow migrating band (Fig. 1F). In addition, pre-treatment of live parasites with calyculin A, a general phosphatase inhibitor, increased the levels of the slow migrating band. These observations indicated that this slow migrating band might correspond to the autophosphorylated and active form of the TcK2 kinase. PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 3 / 27 Hemin starvation causes an increase in the slow migrating TcK2 form Epimastigotes proliferate in the insect gut, a compartment full of blood meal. The parasite stores different nutrients, including heme [27] and lipids, such as cholesterol [28], in organelles derived from endosomes called reservosomes [29]. As nutrients become scarce, differentiation of epimastigotes into metacyclic-trypomastigotes is triggered and concomitantly these endo- somes are consumed [26]. As heme is largely present in the blood meal and is required for epi- mastigote growth [30], we tested the effect of adding heme in the form of hemin into the culture medium and checked parasite growth, TcK2 localization and the enzyme migration by gel electrophoresis. As shown in Fig. 3A, parasites maintained in heme-rich medium (LIT with hemin) propagated faster than parasites cultivated in medium without heme supplementation, as shown previously [31]. In the heme-rich medium, TcK2 was detected mainly in endosomes, co-localizing with cruzipain (Fig. 3B) a protease present in these organelles [32]. Both proteins were found more diffuse in the parasites maintained in media with reduced amounts of hemin (Fig. 3B). This is compatible with the different solubilization of TcK2 by digitonin in trypomas- tigote forms when compared to epimastigotes (S1 Fig. and Fig. 1D), or when cells were main- tained in reduced amounts of hemin (Fig. 2A), suggesting a different localization of TcK2 in the various parasite stages. In parasites cultivated in medium supplemented with hemin, electron microscopy showed more organelles appearing as electron-dense vesicles, while in hemin poor medium electron- lucent structures predominate (Fig. 3C). These results are in agreement with the proposal that heme causes the elevated electron density of these T. cruzi structures, located in the posterior end of the cell, possibly reservosomes, based on incorporation of tracers added externally to parasites [32]. More importantly, in the absence of hemin, the upper band of TcK2 predominated in total parasite lysates (Fig. 3D). Similarly, when parasites were transferred to saline buffer (PBS), the upper band of TcK2 prevailed and this was inhibited by addition of hemin (Fig. 3E). These re- sults indicate that heme accumulation in the endosomes could be related to TcK2 inactivation. eIF2 Kinase Regulated by Heme (Fig. 2A and 2C). In epimastigotes and amastigotes, respectively the forms that proliferate in the insect gut and in the cytosol of mammalian cells, the enzyme showed poor colocalization with BiP, an ER-resident protein (Fig. 2A). Increased, but incomplete colocalization with BiP was observed in trypomastigote stages, which are the infective and non-proliferative forms. In- terestingly, in proliferating epimastigotes and amastigotes, the lower band, which may corre- spond to the non-phosphorylated form of TcK2, predominated while in the infective forms (metacyclic-trypomastigotes and cell-derived trypomastigotes) the upper phosphorylated band was predominant (Fig. 2C), suggesting that the kinase could be more active in the infective forms. T. cruzi K2 is localized in endosomes and the upper phosphorylated band predominates in non-proliferative stages Immunofluorescence showed that TcK2 is located in cellular organelles in the epimastigote form of the parasite (Fig. 2A). This form corresponds to the stage that proliferates in the gut of the insect vector. The same result was obtained with a C-terminal HA-tagged TcK2 expressed in epimastigotes (Fig. 2B). TcK2 was found expressed in both replicative and infective forms PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 4 / 27 Hemin binds to and specifically inhibits TcK2 Two putative heme binding sites in the catalytic domain of TcK2 could be predicted based on sequence analysis [33], as shown in Fig. 4A. These sites are represented in more detail as hori- zontal bars in the alignment of the kinase domain of HRI, PERK and the two kinases of T. cruzi (S2 Fig.). Indeed, a GST-TcK2 recombinant protein displayed a typical Soret spectral band, with a peak from 410 to 420 nm in the presence of heme, not seen in the GST-TcK2 alone, as detected by spectroscopy analysis (Fig. 4B). This spectral band is formed in proteins that bind to heme. Quantitative assays using different amounts of hemin revealed that the in- crease in absorbance at 410 nm was saturable, suggesting specific binding and allowing us to es- timate an apparent heme binding constant (Kdapp) of 2.8 ± 0.6 μM (Fig. 4C). 5 / 27 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 eIF2 Kinase Regulated by Heme Figure 2. TcK2 is located in the endosomal compartment and is phosphorylated in non-proliferating parasite stages of T. cruzi. (A) Wild type epimastigotes (Epi), metacyclics (Meta), intracellular amastigotes (Ama) and cell-released trypomastigotes (Trypo) were fixed, permeabilized, and incubated with anti-BiP antibodies (Bip, green) and mAb 5D10 (TcK2, red). The images show individual immunofluorescence and DAPI labeling, DIC images and the merged immunofluorescence labeling from an enlarged field (Bars = 5 μm). (B) Wild type (WT) or HA-tagged K2 (TcK2-HA) epimastigotes were adsorbed to glass slides, fixed with paraformaldehyde and permeabilized with 0.1% Triton X100. Adhered cells were then stained with mAb 5D10 (TcK2, green) or anti-HA (HA, red), and DAPI (blue). The images show individual labeling and the fluorescence signals merged with differential interference contrast (DIC). (C) Western blots of total extracts of parasites of the indicated stages revealed by anti-TcK2 (anti-KD) antibody. Anti-β-tubulin was used as a loading control. The graph shows the mean and standard deviation of the TcK2[Up]/TcK2[Low] ratio (n = 3). Figure 2. TcK2 is located in the endosomal compartment and is phosphorylated in non-proliferating parasite stages of T. cruzi. (A) Wild type epimastigotes (Epi), metacyclics (Meta), intracellular amastigotes (Ama) and cell-released trypomastigotes (Trypo) were fixed, permeabilized, and incubated with anti-BiP antibodies (Bip, green) and mAb 5D10 (TcK2, red). The images show individual immunofluorescence and DAPI labeling, DIC images and the merged immunofluorescence labeling from an enlarged field (Bars = 5 μm). doi:10.1371/journal.ppat.1004618.g002 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 Hemin binds to and specifically inhibits TcK2 (B) Wild type (WT) or HA-tagged K2 (TcK2-HA) epimastigotes were adsorbed to glass slides, fixed with paraformaldehyde and permeabilized with 0.1% Triton X100. Adhered cells were then stained with mAb 5D10 (TcK2, green) or anti-HA (HA, red), and DAPI (blue). The images show individual labeling and the fluorescence signals merged with differential interference contrast (DIC). (C) Western blots of total extracts of parasites of the indicated stages revealed by anti-TcK2 (anti-KD) antibody. Anti-β-tubulin was used as a loading control. The graph shows the mean and standard deviation of the TcK2[Up]/TcK2[Low] ratio (n = 3). doi:10.1371/journal.ppat.1004618.g002 6 / 27 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 eIF2 Kinase Regulated by Heme Figure 3. Heme promotes parasite multiplication with accumulation of electron dense endo and changes in TcK2 gel migration. (A) Epimastigotes were cultivated in LIT medium prepared w adding hemin (black circle), or with 10 μg (black square) or 30 μg (black triangle) of hemin per ml a along the indicated times. Values are mean and standard deviation (n = 3). (B) Epimastigotes were maintained for 96 h in the absence of added hemin (−Hemin), or in the presence of 30 μg/ml of hem processed for immunofluorescence with mAb 5D10, or with anti-cruzipain (Cz). The images also s DAPI staining; the merged immunofluorescences of the two antibodies, and the corresponding fiel with dotted squares in the respective DIC images (Bars = 5 μm). (C) Transmission electron microg parasites maintained in LIT prepared with 30 μg/ml of hemin (+Hemin) or without hemin suppleme (−Hemin) (Bars = 2 μm). Arrowheads indicate the reservosomes filled with electron dense materia Figure 3. Heme promotes parasite multiplication with accumulation of electron dense endosomes and changes in TcK2 gel migration. (A) Epimastigotes were cultivated in LIT medium prepared without adding hemin (black circle), or with 10 μg (black square) or 30 μg (black triangle) of hemin per ml and counted along the indicated times. Values are mean and standard deviation (n = 3). (B) Epimastigotes were maintained for 96 h in the absence of added hemin (−Hemin), or in the presence of 30 μg/ml of hemin, and processed for immunofluorescence with mAb 5D10, or with anti-cruzipain (Cz). The images also show the DAPI staining; the merged immunofluorescences of the two antibodies, and the corresponding field marked with dotted squares in the respective DIC images (Bars = 5 μm). TcK2 null cells show decreased differentiation and proliferation rates To better understand the TcK2 function in the parasite, we generated TcK2-null cell lines by a two-step gene replacement through homologous recombination and antibiotic selection as il- lustrated in Fig. 5A. The integration of resistance genes (Neo and Hygro, or Blast) in the TcK2 locus of the two homologous chromosomes was confirmed by PCR using specific primers (Fig. 5B). The TcK2 gene was not amplified from the double KOs (−/−) (Fig. 5C) and the cells did not express the kinase as shown by Western blot (Fig. 5D) and immunofluorescence assays (Fig. 5E). TcK2 null cells still expressed cruzipain in organelles (Fig. 5E), showing that the ki- nase is not necessary for cruzipain incorporation into endosomes. As expected, epimastigotes with deletion of one TcK2 allele (−/+) and the TcK2 null cell line (−/−) showed a progressive inhibition in differentiation to metacyclic-trypomastigotes (Fig. 5F), compatible with the heme starvation followed by eIF2α phosphorylation being a factor inducing differentiation. Strick- ling, in hemin-containing medium, only the TcK2 null cells showed a reduced growth rate (Fig. 5G). The same phenotypes were obtained in four independent clones, two of them using blasticidin instead of hygromycin resistance genes (see below). eIF2 Kinase Regulated by Heme arrows indicate the same structures with less electron dense material. Western blots using anti-TcK2-KD and anti-β-tubulin antibodies of samples prepared from parasites kept in the medium without () or with (+) hemin supplementation (30 μg/ml) (D), or from parasites kept 6 h in LIT medium containing hemin (10 μg/ml), PBS, or PBS with 30 μg/ml of hemin (E). The gel shows biological duplicates and below are the means and standard deviation of the TcK2 [Up]/TcK2[Low] (n = 3). The differences are statistically significant (p < 0.05) based on the t-Student test. arrows indicate the same structures with less electron dense material. Western blots using anti-TcK2-KD and anti-β-tubulin antibodies of samples prepared from parasites kept in the medium without () or with (+) hemin supplementation (30 μg/ml) (D), or from parasites kept 6 h in LIT medium containing hemin (10 μg/ml), PBS, or PBS with 30 μg/ml of hemin (E). The gel shows biological duplicates and below are the means and standard deviation of the TcK2 [Up]/TcK2[Low] (n = 3). The differences are statistically significant (p < 0.05) based on the t-Student test. doi:10.1371/journal.ppat.1004618.g003 To test the effect of heme in the regulation of TcK2 activity, we assessed the in vitro phosphorylation of TceIF2α by GST-TcK2 in the presence of heme and several analogs. Heme (Fe-Protoporphyrin IX) was able to inhibit the activity of GST-TcK2, while protoporphyrin IX, mesoporphyrin IX, and Sn protoporphyrin had no effect (Fig. 4D). Importantly, this inhibitory effect was specific, as the PK4 kinase from P. falciparum did not bind to heme and heme did not inhibit eIF2α phosphorylation by PK4 (S3B Fig.) [15]. When we analyzed the effect of heme and its analogs, we also found that only heme inhib- ited the differentiation of epimastigotes into metacyclic trypomastigotes (Fig. 4E). As previous- ly reported, the differentiation depends on eIF2α phosphorylation and protein synthesis attenuation [23]. Taken together, these data suggest that heme binding inactivates TcK2, thus inhibiting T. cruzi differentiation. Hemin binds to and specifically inhibits TcK2 (C) Transmission electron micrographs of parasites maintained in LIT prepared with 30 μg/ml of hemin (+Hemin) or without hemin supplementation (−Hemin) (Bars = 2 μm). Arrowheads indicate the reservosomes filled with electron dense material and PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 7 / 27 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 eIF2 Kinase Regulated by Heme Figure 4. Heme binds specifically to the catalytic domain of TcK2 and inhibits TceIF2α phosphorylation and metacyclogenesis. (A) Prediction of heme binding sites along the TcK2 sequence according to the Hemebind software [33]. The vertical dark bars are positive hits for each amino acid residue. (B) Absorption spectra of 10 μM hemin (dashed line), GST + 10 μM hemin (dotted line) and GST-TcK2 (dotted/dashed line), and GST-TcK2 incubated with 10 μM hemin (continuous line). (C) The figure shows the relative absorbance at the indicated concentrations of hemin incubated with GST-TcK2 at 25°C. (D) SDS- PAGE of His6x-TceIF2α incubated without (−) or with (+) GST-TcK2 for 15 min with [32P]-γ-ATP in the absence or presence of 10 μM of the indicated porphyrins. The upper gel shows the autoradiogram and at the bottom is the same gel stained with Coomassie Blue R250. Panel (E) shows the percentages of differentiated cells (metacyclics-trypomastigotes) after 96 h in the presence of the indicated porphyrins counted by direct observation of 250 cells per replicate in the fluorescence microscope after DAPI staining. The numbers are mean and standard deviation (n = 3). The asterisk indicates a significant difference (p < 0.05) based on the t- Student test. Figure 4. Heme binds specifically to the catalytic domain of TcK2 and inhibits TceIF2α h h l i d l i (A) P di i f h bi di i l h T K Figure 4. Heme binds specifically to the catalytic domain of TcK2 and inhibits TceIF2α phosphorylation and metacyclogenesis. (A) Prediction of heme binding sites along the TcK2 sequence according to the Hemebind software [33]. The vertical dark bars are positive hits for each amino acid residue. (B) Absorption spectra of 10 μM hemin (dashed line), GST + 10 μM hemin (dotted line) and GST-TcK2 (dotted/dashed line), and GST-TcK2 incubated with 10 μM hemin (continuous line). (C) The figure shows the relative absorbance at the indicated concentrations of hemin incubated with GST-TcK2 at 25°C. (D) SDS- PAGE of His6x-TceIF2α incubated without (−) or with (+) GST-TcK2 for 15 min with [32P]-γ-ATP in the absence or presence of 10 μM of the indicated porphyrins. The upper gel shows the autoradiogram and at the bottom is the same gel stained with Coomassie Blue R250. PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 Ascorbate restores the growth rate and cell viability but not the full differentiation capacity The slow growth phenotype of the TcK2 null cells could be due to oxidative damage generated by the hemin in parasites lacking this kinase. Therefore, we examined the effect of antioxidant agents in parasite growth. As shown in Fig. 6A, ascorbate restored normal growth when used at concentration of 10 μM; with 1 mM of ascorbate, growth was initially restored but the proliferation rate diminished after a few days. At the concentration of 10 mM, ascorbate was not effective. The effect of ascorbate was not because it restored the levels of sulfhydryl groups, as N-acetyl-cysteine, a sulfhydryl reducing agent, did not restore growth. Moreover, the effect of ascorbate disappeared after three days when cells were washed and maintained in medium without ascorbate (Fig. 6B), suggesting that a factor that prevents growth progressively accu- mulates in the medium of TcK2-KO cells. 8 / 27 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 Panel (E) shows the percentages of differentiated cells (metacyclics-trypomastigotes) after 96 h in the presence of the indicated porphyrins counted by direct observation of 250 cells per replicate in the fluorescence microscope after DAPI staining. The numbers are mean and standard deviation (n = 3). The asterisk indicates a significant difference (p < 0.05) based on the t- Student test. doi:10.1371/journal.ppat.1004618.g004 9 / 27 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 eIF2 Kinase Regulated by Heme Figure 5. TcK2-KO cells are impaired in cellular differentiation and display reduced growth. (A) Schematic diagram illustrating the homologous recombination strategy to generate knockouts of TcK2. (B) PCR products of specific resistance gene using as a template the DNA extracted from wild type (WT), partia KO (+/−) and the TcK2 null (−/−) parasites. (C) Same as in B, but using oligonucleotides to amplify part of the open reading frame of TcK2. The localization of oligonucleotides used in (B) and (C) is indicated in panel (A) (D) Western blot of extracts obtained from wild type, partial KO (+/−) and TcK2 null (−/−) parasites, using ant TcK2-KD (top panel) or anti-β-tubulin (bottom panel) antibodies. (E) Immunofluorescence of the wild type an TcK2 null epimastigotes using mAb 5D10 (green) and anti-cruzipain (red) (Bars = 2 μm). (F) Percentages of differentiated (metacyclics-trypomastigotes) obtained from wild type, partial KO (+/−) and TcK2 null (−/−) cells. (G) Growth curve of wild type (black circle), partial KO (white square), and TcK2 null parasites (black losangle). In F and G numbers are mean and standard deviation (n = 3) and asterisks indicate a significant difference (p < 0.05) based on the t-Student test. Figure 5. TcK2-KO cells are impaired in cellular differentiation and display reduced growth. (A) Figure 5. TcK2 KO cells are impaired in cellular differentiation and display reduced growth. (A) Schematic diagram illustrating the homologous recombination strategy to generate knockouts of TcK2. (B) PCR products of specific resistance gene using as a template the DNA extracted from wild type (WT), partial KO (+/−) and the TcK2 null (−/−) parasites. (C) Same as in B, but using oligonucleotides to amplify part of the open reading frame of TcK2. The localization of oligonucleotides used in (B) and (C) is indicated in panel (A). (D) Western blot of extracts obtained from wild type, partial KO (+/−) and TcK2 null (−/−) parasites, using anti- TcK2-KD (top panel) or anti-β-tubulin (bottom panel) antibodies. (E) Immunofluorescence of the wild type and TcK2 null epimastigotes using mAb 5D10 (green) and anti-cruzipain (red) (Bars = 2 μm). (F) Percentages of differentiated (metacyclics-trypomastigotes) obtained from wild type, partial KO (+/−) and TcK2 null (−/−) cells. (G) Growth curve of wild type (black circle), partial KO (white square), and TcK2 null parasites (black losangle). In F and G numbers are mean and standard deviation (n = 3) and asterisks indicate a significant difference (p < 0.05) based on the t-Student test. doi:10.1371/journal.ppat.1004618.g005 On the other hand, 10 μM ascorbate only partially reverted the poor differentiation capacity of TcK2 null epimastigotes to metacyclic-trypomastigotes (Fig. 6C). This partial reversion of differentiation is suggestive that TcK2 affects differently the growth and the metacyclogenesis. The generated TcK2 null metacyclics also showed a reduced capacity to invade mammalian cells (Fig. 6D). Moreover, after infection, the TcK2 null parasites were able to differentiate into PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 10 / 27 eIF2 Kinase Regulated by Heme Figure 6. Ascorbate restores TcK2 null parasite growth, differentiation and infectivity, but not intracellular proliferation. (A) Growth of wild type (WT, white losangle) and TcK2 null cells in the absence (black circle) or in the presence of 10 μM (black inverted triangle), 1 mM (black triangle) or 10 mM (black square) ascorbate, or 5 mM N-acetyl-cysteine (white circle). (B) Growth of WT (white losangel) and TcK2 null parasites in the absence of ascorbate (black circle), or after removal of 10 μM ascorbate on day 0 (white losangle). On day 6, the cells were diluted 10 fold (see arrow). (C) and (D) show, respectively, the percentage of cellular differentiation into metacyclics-trypomastigotes after 96 h and the invasion of mammalian cells by the same number of wild type (WT) and the TcK2 null parasites in the absence (empty bars) or presence of 10 μM ascorbate (filled bars). (E) Intracellular proliferation of amastigotes after infection by the wild type (WT, white losangle), TcK2 null cells previously differentiated in the absence (black circle) or in the presence of 10 μM ascorbate (black inverted triangle). In all cases, the values are mean and standard deviation of triplicates. Asterisks indicate statistically significant differences between the WT and null lines in each case based on t-Student test (* p < 0.01, ** p < 0.3). d i 10 1371/j l t 1004618 006 Figure 6. TcK2 null cells display more electron lucent structures The TcK2 null cells still showed organelles that resemble reservosomes, as revealed by the cru- zipain signal in the immunofluorescence analysis (Fig. 5E). Indeed, TcK2 null parasites exam- ined by transmission electron microscopy showed cytoplasmic organelles that were more electron-lucent than in wild type parasites (Fig. 7). The empty structures were also found in parasites cultivated in medium without hemin supplementation (Fig. 3C), suggesting that heme does not accumulate in these organelles in cells lacking TcK2. These structures were clearly distinguishable from the mitochondrion, which was identified by the double mem- branes. Remarkably, in TcK2 null parasites treated with 10 μM ascorbate, which restored epi- mastigotes growth, these organelles remained electron-lucent, probably not accumulating heme. This observation suggests that the reduced proliferation in the absence of ascorbate was not because of the lack of heme in the endosomal compartment. Heme levels and superoxide dismutase activity increase and peroxidase decreases in TcK2 null parasites Next, we quantified the total amount of heme in the parasites after gentle lysis, expecting to measure mainly molecules present in the cytosol. The TcK2 null parasites had larger amounts of heme than wild type parasites, and this increase was not reversed by ascorbate, as shown in Fig. 8A. We also observed a larger uptake of the fluorescent heme analogue [Zn(II) Mesopor- phyrin IX] by the TcK2 null cells when compared to the wild type cells. This uptake was strongly inhibited by heme in TcK2 null parasites as compared to wild type parasites, ruling out a non-specific binding to the cell (Fig. 8B). These results suggest that TcK2 could act in sensing and/or regulating the amount of heme that is present in the cells. Together with the electron-microscopy results, such data indicate that in parasites lacking TcK2, heme taken up by the cell is not directed to the lumen of endosomes, remaining in the cytosol. The absence of TcK2 also caused a decrease in the total peroxidase activity (Fig. 8C), and an increase in the Fe-superoxide dismutase (SOD) activity when compared to wild type parasites (Fig. 8D). Consequently, the total levels of hydrogen peroxide were largely increased (Fig. 8E), independently of the presence of ascorbate. However, there was an increase in the amount of reactive oxygen species (ROS), as measured using CellRox (Fig. 8F), which was reduced by ascorbate addition. An increase from 600 fluorescence units in the case of control and 1000 fluorescence units corresponding to ROS levels was also detected using 5CM-H2DCFDA. However, this reagent provided a much higher background, due to the presence of medium. Anyway, the reduced growth of the TcK2 null cells correlated with the increased levels of ROS, probably produced by the augmented levels of H2O2, which might react with free iron or cop- per to generate hydroxyl radicals, causing parasite damage. eIF2 Kinase Regulated by Heme Ascorbate restores TcK2 null parasite growth, differentiation and infectivity, but not intracellular proliferation. (A) Growth of wild type (WT, white losangle) and TcK2 null cells in the absence (black circle) or in the presence of 10 μM (black inverted triangle), 1 mM (black triangle) or 10 mM (black square) ascorbate, or 5 mM N-acetyl-cysteine (white circle). (B) Growth of WT (white losangel) and TcK2 null parasites in the absence of ascorbate (black circle), or after removal of 10 μM ascorbate on day 0 (white losangle). On day 6, the cells were diluted 10 fold (see arrow). (C) and (D) show, respectively, the percentage of cellular differentiation into metacyclics-trypomastigotes after 96 h and the invasion of mammalian cells by the same number of wild type (WT) and the TcK2 null parasites in the absence (empty bars) or presence of 10 μM ascorbate (filled bars). (E) Intracellular proliferation of amastigotes after infection by the wild type (WT, white losangle), TcK2 null cells previously differentiated in the absence (black circle) or in the presence of 10 μM ascorbate (black inverted triangle). In all cases, the values are mean and standard deviation of triplicates. Asterisks indicate statistically significant differences between the WT and null lines in each case based on t-Student test (* p < 0.01, ** p < 0.3). doi:10.1371/journal.ppat.1004618.g006 doi:10.1371/journal.ppat.1004618.g006 amastigotes but did not grow (Fig. 6E), or produce trypomastigotes, even after long incuba- tions. Also, when metacyclics-trypomastigotes from TcK2 null cells were produced in the pres- ence of ascorbate, the invasion was largely increased (Fig. 6D), without intracellular multiplication (Fig. 6E). 11 / 27 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 Only the active kinase restores the wild type phenotypes in TcK2 null parasites To determine whether the changes observed in the null parasites were due to an indirect effect related to the absence of TcK2 as a protein, or to the requirement of an enzymatically active ki- nase, we generated parasites expressing the wild type and an enzymatically inactive TcK2 mu- tant. For this, we used null parasites obtained by replacement of the two copies of the endogenous kinase with Neo and Blast genes. This was done because these parasites were trans- fected with pROCK, which is a plasmid that integrates in the tubulin locus, being selected by hygromycin resistance [34]. The resulting constructs were expected to express constitutively the full length TcK2 gene. In one set of transfections the full-length wild type kinase was PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 12 / 27 eIF2 Kinase Regulated by Heme Figure 7. TcK2 null parasites lose electron dense organelles and their contents are not restored by ascorbate. Transmission electron micrographs of wild type (WT), TcK2 null and TcK2 null parasites maintained in 10 μM ascorbate (Bars = 2 μm). Arrowheads indicate the endosomal organelles and thin arrows sections of the mitochondrion in all cases. Figure 7. TcK2 null parasites lose electron dense organelles and their contents are not restored by ascorbate. Transmission electron micrographs of wild type (WT), TcK2 null and TcK2 null parasites maintained in 10 μM ascorbate (Bars = 2 μm). Arrowheads indicate the endosomal organelles and thin arrows sections of the mitochondrion in all cases. doi:10.1371/journal.ppat.1004618.g007 inserted, while in the other, a kinase with the lysine 695 replaced by alanine was used. The posi- tion of this mutation is indicated by an asterisk in S2 Fig., which corresponds to the lysine 622 of human PERK. This residue is located in the ATP binding site and inactivates the enzyme [35]. As shown in Fig. 9A, both forms of TcK2 were localized in similar vesicular structures in the posterior end of the parasite at the same position shown by cruzipain. The reinsertion of the wild type gene restored the growth defect, while the slow growth was maintained in the case of the mutated and probably inactive kinase. In addition, the differentiation capacity and ROS levels were restored by the wild type but not by the mutant kinase (Fig. 9C and D). West- ern blotting showed the presence of similar levels of the TcK2 protein in both cases, which were similar also to TcK2 expression levels in untransfected parasites, whereas the Neo/Blast null line that was used for these constructions has no TcK2 signal (Fig. 9E). The upper band was also seen in the kinase dead add-back. Nevertheless, upon incubation in PBS without hemin, the intensity of the upper phosphorylated band increased only in the original cells and in the wild type add-back of TcK2, not in the parasite line containing the kinase dead construct. These results indicate that the absence of the TcK2 activity is responsible for the growth defect and alterations in the oxidative metabolism observed in the null cells. inserted, while in the other, a kinase with the lysine 695 replaced by alanine was used. PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 (C) and (D) respectively indicate the total peroxidase and FeSOD activities in WT and TcK2 null parasites. (E) and (F) respectively show the H2O2 and ROS levels in WT and TcK2 null cells cultivated in the absence (empty bars) or presence (filled bars) of 10 μM ascorbate. In all cases, the values are mean and standard deviation of triplicates. Asterisks indicate statistically significant differences based on t-Student test (p < 0.01). doi:10.1371/journal.ppat.1004618.g008 eIF2 Kinase Regulated by Heme Figure 8. TcK2 null parasites incorporate more heme than WT cells, resulting in increased H2O2 and ROS levels. (A) Intracellular heme levels in wild type (WT) and TcK2 null parasites growing in the absence (empty bars) or in the presence (filled bars) of 10 μM ascorbate. (B) Zn (II) mesoporphyrin IX incorporation measured in WT and TcK2 null cells in the absence (empty bars) or in the presence (filled bars) of 10 μM heme. (C) and (D) respectively indicate the total peroxidase and FeSOD activities in WT and TcK2 null parasites. (E) and (F) respectively show the H2O2 and ROS levels in WT and TcK2 null cells cultivated in the absence (empty bars) or presence (filled bars) of 10 μM ascorbate. In all cases, the values are mean and standard deviation of triplicates. Asterisks indicate statistically significant differences based on t-Student test (p < 0.01). doi:10.1371/journal.ppat.1004618.g008 Figure 8. TcK2 null parasites incorporate more heme than WT cells, resulting in increased H2O2 and Figure 8. TcK2 null parasites incorporate more heme than WT cells, resulting in increased H2O2 and ROS levels. (A) Intracellular heme levels in wild type (WT) and TcK2 null parasites growing in the absence (empty bars) or in the presence (filled bars) of 10 μM ascorbate. (B) Zn (II) mesoporphyrin IX incorporation measured in WT and TcK2 null cells in the absence (empty bars) or in the presence (filled bars) of 10 μM heme. (C) and (D) respectively indicate the total peroxidase and FeSOD activities in WT and TcK2 null parasites. (E) and (F) respectively show the H2O2 and ROS levels in WT and TcK2 null cells cultivated in the absence (empty bars) or presence (filled bars) of 10 μM ascorbate. In all cases, the values are mean and standard deviation of triplicates. Asterisks indicate statistically significant differences based on t-Student test (p < 0.01). Figure 8. TcK2 null parasites incorporate more heme than WT cells, resulting in increased H2O2 and ROS levels. (A) Intracellular heme levels in wild type (WT) and TcK2 null parasites growing in the absence (empty bars) or in the presence (filled bars) of 10 μM ascorbate. (B) Zn (II) mesoporphyrin IX incorporation measured in WT and TcK2 null cells in the absence (empty bars) or in the presence (filled bars) of 10 μM heme. The posi- tion of this mutation is indicated by an asterisk in S2 Fig., which corresponds to the lysine 622 of human PERK. This residue is located in the ATP binding site and inactivates the enzyme [35]. As shown in Fig. 9A, both forms of TcK2 were localized in similar vesicular structures in the posterior end of the parasite at the same position shown by cruzipain. The reinsertion of the wild type gene restored the growth defect, while the slow growth was maintained in the case of the mutated and probably inactive kinase. In addition, the differentiation capacity and ROS levels were restored by the wild type but not by the mutant kinase (Fig. 9C and D). West- ern blotting showed the presence of similar levels of the TcK2 protein in both cases, which were similar also to TcK2 expression levels in untransfected parasites, whereas the Neo/Blast null line that was used for these constructions has no TcK2 signal (Fig. 9E). The upper band was also seen in the kinase dead add-back. Nevertheless, upon incubation in PBS without hemin, the intensity of the upper phosphorylated band increased only in the original cells and in the wild type add-back of TcK2, not in the parasite line containing the kinase dead construct. These results indicate that the absence of the TcK2 activity is responsible for the growth defect and alterations in the oxidative metabolism observed in the null cells. Importantly, TcK2 immunoprecipitated from epimastigotes previously incubated for 6 h in PBS had more eIF2α phosphorylation activity, as compared to the immunoprecipitates ob- tained from epimastigotes maintained in LIT medium (Fig. 9F). Control immunoprecipitations with null epimastigotes did not show any activity, confirming that the ability to phosphorylate the eIF2α in vitro was due to TcK2. Altogether, these data support the notion that the enzyme, migrating mainly as the top band is active in phosphorylating eIF2α. 13 / 27 doi:10.1371/journal.ppat.1004618.g008 eIF2 Kinase Regulated by Heme Figure 9. Wild type, but not the kinase dead TcK2 restores the knockout phenotypes. (A) Immunofluorescence of the indicated lines of epimasti using anti-TcK2 (green) and anti-cruzipain (red) antibodies. The images also show the DAPI and DIC merged images. Bars = 5 μm. The figure also sh growth curve (B) and the percentage of metacyclics after 96 h (C) of the indicated strains. (D) ROS levels were quantified by using the CellRox in non- modified parasites (+/+), in the knockout (Neo/Blas replacements, −/−) and in the add backs with the full TcK2 genes (Wild type, WT and kinase dead, The quantification was also measured in the parasites maintained for 5 days in the presence of 10 μM ascorbate (Asc). The numbers in C-D are mean standard deviation (n = 3) and asterisks indicate the relevant significant difference (p < 0.05) based on the t-Student test. (E) Western blot of the indica lines of parasites maintained in regular medium (Control) or incubated 6 h in PBS, probed with anti-TcK2 or anti-Tubulin (Tub) antibodies. (F) Autorad of a 10% SDS-PAGE containing immunoprecipitates of non-transfected epimastigotes (+/+) or the null epimastigote line (Neo/Blast, −/−) pre-incubate h at 28°C in complete LIT medium (LIT) or in PBS. The epimastigotes were lysed and the cleared extracts incubated with the rabbit anti-TcK2 and Pro Sepharose. The beads were washed and incubated with 2 μg/ml of 6xHis-TceIF2α in the presence of [32P]-γ-ATP for 1 h at 37°C. The top panel show autoradiogram and the bottom the gel stained with Coomassie Blue R Figure 9. Wild type, but not the kinase dead TcK2 restores the knockout phenotypes. (A) Immunofluorescence of the indicated lines of epimastigotes using anti-TcK2 (green) and anti-cruzipain (red) antibodies. The images also show the DAPI and DIC merged images. Bars = 5 μm. The figure also shows the growth curve (B) and the percentage of metacyclics after 96 h (C) of the indicated strains. (D) ROS levels were quantified by using the CellRox in non- modified parasites (+/+), in the knockout (Neo/Blas replacements, −/−) and in the add backs with the full TcK2 genes (Wild type, WT and kinase dead, KD). The quantification was also measured in the parasites maintained for 5 days in the presence of 10 μM ascorbate (Asc). Discussion We have demonstrated that T. cruzi expresses a membrane bound protein kinase in the endo- somes that is able to phosphorylate eIF2α in response to heme deprivation. In the absence of heme, the parasite undergoes differentiation in a process that is dependent on the active form of the kinase. In parasites lacking the kinase, heme accumulates in the cell, most likely in the 14 / 27 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 doi:10.1371/journal.ppat.1004618.g009 eIF2 Kinase Regulated by Heme cytosol, causing an increase in hydrogen peroxide levels, which leads to the generation of reac- tive oxygen species and parasite damage, as proposed in the model depicted in the Fig. 10. Therefore, this unusual eIF2α kinase is involved in the control of both cell differentiation and heme homeostasis in T. cruzi, a parasite that deals with environments presenting variable amounts of heme and other oxidant products [36]. The topology of TcK2 is very similar to PERK. Indeed, we have demonstrated that TcK2 is linked to the membrane, as shown for its orthologues in T. brucei [19] and L. infantum [21]. However, it is not predominantly in the ER, which agrees with our previous observation that T. brucei K2 is located in the flagellar pocket, a compartment related to the endocytic/exocytic pathway, and early endocytic vesicles [19]. In contrast to T. brucei, in T. cruzi the K2 is found in organelles more spread throughout the cytoplasm, which might reflect differences in protein trafficking in these organisms. Our immunofluorescence data strongly suggest that TcK2 is lo- cated in the endosomal membranes. Detection of the enzyme in purified reservosomes or other organelle, as well as their immunolocalization by electron microscopy could strengthen this conclusion. These findings do not exclude the possibility that some TcK2 is localized in ER mainly in the case of trypomastigotes, as there is some degree of co-localization with BiP. Fur- ther studies on endosome formation, its relationship to the ER and its remodeling during dif- ferentiation are required for a more detailed understanding of TcK2 trafficking in the cells. Our data also suggest that TcK2 may be involved in the control of protein synthesis in the different parasite stages. We have previously shown that metacyclogenesis requires the inhibi- tion of translation by phosphorylation of eIF2α [23]. In contrast to a cell line overexpressing a wild type form of eIF2α, a cell line overexpressing non-phosphorylatable form of eIF2α (Thr169Ala mutation) does not undergo metacyclogenesis under conditions that lack heme and other nutrients. Those previous data give strong support for the findings described here on TcK2 role in phosphorylating eIF2α in vivo as a requirement for differentiation. The numbers in C-D are mean and standard deviation (n = 3) and asterisks indicate the relevant significant difference (p < 0.05) based on the t-Student test. (E) Western blot of the indicated lines of parasites maintained in regular medium (Control) or incubated 6 h in PBS, probed with anti-TcK2 or anti-Tubulin (Tub) antibodies. (F) Autoradiogram of a 10% SDS-PAGE containing immunoprecipitates of non-transfected epimastigotes (+/+) or the null epimastigote line (Neo/Blast, −/−) pre-incubated for 6 h at 28°C in complete LIT medium (LIT) or in PBS. The epimastigotes were lysed and the cleared extracts incubated with the rabbit anti-TcK2 and Protein G- Sepharose. The beads were washed and incubated with 2 μg/ml of 6xHis-TceIF2α in the presence of [32P]-γ-ATP for 1 h at 37°C. The top panel shows the autoradiogram and the bottom the gel stained with Coomassie Blue R. PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 15 / 27 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 The relatively smaller amounts of the upper band, which partially corresponds to the phos- phorylated enzyme form in epimastigotes and amastigotes implies that TcK2 could be in a less active state in proliferating cells, which in fact present high rates of protein synthesis [37]. In contrast, protein synthesis is decreased in metacyclic-trypomastigotes and trypomastigotes, which is compatible with the predominant presence of relatively larger amounts of the upper phosphorylated band in the Western blotting. While epimastigotes proliferate and synthesize most of the housekeeping genes, trypomastigotes predominantly produce proteins involved in cell invasion and virulence, belonging to the trans-sialidase large family of genes and surface glycoproteins as mucins and mucin associated proteins [38]. Thus, it is possible that when hemin levels are low, the kinase is activated resulting in protein synthesis inhibition through TceIF2α phosphorylation, a requirement for T. cruzi differentiation, perhaps by allowing pref- erential translation of trypomastigote specific mRNAs, in agreement with recent findings that a subpopulation of mRNAs is translated in trypomastigotes [39]. Several other factors have been shown to promote T. cruzi differentiation. Examples are nu- trient starvation [24], hemoglobin derived peptides [40], proteasome inhibition [41]. These fac- tors could directly act on the differentiation, or could be part of a stress response generated by critical changes in environment, perhaps affecting heme levels or the activation of other eIF2α kinase. For example, amino acid starvation or proteasome inhibition may activate TcK1, the homolog of GCN2. We observed that heme, but not other porphyrins, prevents parasite differentiation to meta- cyclic-trypomastigotes forms. In a similar way, it specifically binds and inhibits TcK2 activity in vitro. Specific binding was inferred by the saturable interaction as observed for HRI, which interacts more strongly to hemin (Fe+3-protoporphyrin) when compared to other porphyrins [42]. Further work would be necessary to precisely identify the heme binding site in TcK2. The PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 16 / 27 eIF2 Kinase Regulated by Heme Figure 10. Model of the proposed actions of TcK2 in response to heme during growth and differentiation of T. cruzi. TcK2 is located in the membrane of endosomes and is involved in heme accumulation inside these organelles through an unknown transporter. Extracellular heme enters the cell through ABC transporters and inhibits TcK2, allowing general translation. In heme deprivation, heme is released in the cytosol, and when cytosolic levels reach low levels, TcK2 is activated leading to TceIF2α phosphorylation, decreasing general translation and allowing expression of trypomastigote specific proteins. When excess heme accumulates in the cytosol, for example in the TcK2 null parasites, FeSODs are activated and peroxidases are inhibited resulting in the production and accumulation of H2O2, which outcomes in the increase of ROS levels by Fenton Reaction. We propose that the Fenton Reaction occurs extracellularly as ascorbate is not internalized by T. cruzi and washed parasites sustains better growth. doi:10.1371/journal.ppat.1004618.g010 Figure 10. Model of the proposed actions of TcK2 in response to heme during growth and differentiation of T. cruzi. TcK2 is located in the membrane of endosomes and is involved in heme accumulation inside these organelles through an unknown transporter. Extracellular heme enters the cell through ABC transporters and inhibits TcK2, allowing general translation. In heme deprivation, heme is released in the cytosol, and when cytosolic levels reach low levels, TcK2 is activated leading to TceIF2α phosphorylation, decreasing general translation and allowing expression of trypomastigote specific proteins. When excess heme accumulates in the cytosol, for example in the TcK2 null parasites, FeSODs are activated and peroxidases are inhibited resulting in the production and accumulation of H2O2, which outcomes in the increase of ROS levels by Fenton Reaction. We propose that the Fenton Reaction occurs extracellularly as ascorbate is not internalized by T. cruzi and washed parasites sustains better growth. doi:10.1371/journal.ppat.1004618.g010 doi:10.1371/journal.ppat.1004618.g010 presence of heme, but not other analogs, also promotes parasite growth by activating CaM kinase II [30]. This activation was shown to occur because of a non-deleterious increase in ROS [43]. As TcK2 is probably in the membrane of organelles, which are consumed when epimasti- gotes move across the insect gut, we presume that the kinase activation by heme depletion could be one key factor regulating differentiation. In fact, when parasites migrate to the midgut the amount of nutrients decrease and the electron dense structure gradually disappear [24]. As differentiation is accomplished, the content of these organelles that contain heme are con- sumed and cruzipain is secreted, participating in cell invasion [28, 44]. These findings are in agreement with our observations that electron lucent structures predominated in the parasites, as seen by EM in medium lacking hemin. PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 eIF2 Kinase Regulated by Heme T. cruzi has lost the capacity to synthesize heme, which is an essential growth factor for the parasite [47]. Heme appears to be internalized directly into the cytosol by ABC-transporters [27]. Therefore, heme accumulation in the endosomal compartment could occur through an unknown heme transporter in their membranes as a mechanism to store and prevent excessive levels in the cytosol. Alternatively, heme could be scavenged from the medium and stored in specific organelles through the parasite cytostome, as shown for other macromolecules [32]. T. brucei acquires heme through the haptoglobin-transferrin receptor localized in the flagellar pocket [48], where TbK2 is also localized [19]. However, the haptoglobin-transferrin receptor is found only in African Trypanosomes, which proliferate in the vertebrate blood and utilizes heme from blood. In Leishmania amazonensis, a parasite that develops in the insect gut and in- side intracellular vacuoles, heme is taken up by the Leishmania heme response 1 (LHR1) pro- tein, located in the parasite membrane and on acidic intracellular organelles [49]. A paralog of LHR1 is present in T. cruzi (TcCLB.511071.190, http://tritrypdb.org) and we are investigating its possible association and role in heme transport. The generation of TcK2 null parasites confirms the kinase role in cellular differentiation, probably through the phosphorylation of eIF2α, as shown earlier [23]. We also inferred that the decrease in metacyclic-trypomastigote formation was not due to a less efficient cell growth, because the single knockout of K2 partially inhibited differentiation without affecting cellular proliferation. In addition, ascorbate that fully restored the growth rate did not fully restore dif- ferentiation. This was not the case for the cell invasion by metacyclic-trypomastigotes, which was largely affected by the absence of TcK2, and was even increased by adding ascorbate during the differentiation period. Most likely, it was due to the presence of high levels of ROS, causing parasite damage, or other modifications in the parasites that affected invasion. Our results also showed that the lack of active TcK2 is directly related to the observed phe- notypes as only the reintroduction of the wild type kinase, not of the protein mutated in the ATP binding site, abrogated the effects of its gene depletion. We also found that the incapacity to restore the normal growth phenotype and ROS was not due to protein mislocalization or poor expression. PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 Thus, heme levels seems to decrease initially in these organelles, then in the cytosol, most likely causing TcK2 activation on the later stages of differ- entiation. In fact, an increased phosphorylation of TcK2 was detected during metacyclogenesis by phosphoproteome analysis [45], supporting the notion that the enzyme is activated in try- pomastigotes. Likewise, more active TcK2 was found in stressed cells compared to non- stressed epimastigotes. presence of heme, but not other analogs, also promotes parasite growth by activating CaM kinase II [30]. This activation was shown to occur because of a non-deleterious increase in ROS [43]. [ ] [ ] As TcK2 is probably in the membrane of organelles, which are consumed when epimasti- gotes move across the insect gut, we presume that the kinase activation by heme depletion could be one key factor regulating differentiation. In fact, when parasites migrate to the midgut the amount of nutrients decrease and the electron dense structure gradually disappear [24]. As differentiation is accomplished, the content of these organelles that contain heme are con- sumed and cruzipain is secreted, participating in cell invasion [28, 44]. These findings are in agreement with our observations that electron lucent structures predominated in the parasites, as seen by EM in medium lacking hemin. Thus, heme levels seems to decrease initially in these organelles, then in the cytosol, most likely causing TcK2 activation on the later stages of differ- entiation. In fact, an increased phosphorylation of TcK2 was detected during metacyclogenesis by phosphoproteome analysis [45], supporting the notion that the enzyme is activated in try- pomastigotes. Likewise, more active TcK2 was found in stressed cells compared to non- stressed epimastigotes. The mechanism by which the organelles that accumulate heme are consumed and by which TcK2 and cruzipain disperse is unknown. It might involve events such as proteolysis, or other post-translational modifications. Interestingly, there is evidence that eIF2α phosphorylation and translation arrest are linked to membrane trafficking in mammalian cells [46], which could explain the differences in the localization and distribution of the kinase and the different endosomes. PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 17 / 27 Ethics statement This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Brazilian National Council of Animal Experimen- tation (http://www.cobea.org.br/). The protocol was approved by the Committee on the Ethics of Animal Experiments of the Institutional Animal Care and Use at the Federal University of São Paulo (Id # CEP 1982/11). Moreover, when submitted to stress, only the endogenous and the wild type add-back TcK2 showed an increase in the upper band, which supports the notion that this band corresponds to an active and autophosphorylated kinase. Also, the presence of low levels of the upper band in the kinase dead mutant suggest that it could be phosphorylated by other kinases, in agreement with the presence of several phosphor-sites in the C-terminus of the pro- tein [50], causing eIF2α phosphorylation. This modulation is also observed in mammalian PERK. Alternatively, it could be due to other modifications such as glycosylation, as shown in the T. brucei enzyme [19]. Our data also indicated that in the TcK2 null cells, the SOD dismutase activity increased and the peroxidase activity decreased, which would generate high levels of intracellular H2O2. This might occur as direct effect of the increased heme levels in the cytosol due to the failure to transport heme into endosomes. Otherwise, it could be a consequence of the differential trans- lation mediated by increased eIF2α phosphorylation, or by phosphorylation of another sub- strates, as in the case of phosphorylation of the transcription factor Nrf2 in mammalian cells [51]. A possibility remains that heme is also acting as a source of iron, which can directly bind and activate superoxide dismutase, a strictly iron-dependent enzyme in trypanosomatids [52]. In addition, the reason why in the absence of TcK2 there was an increase in the heme detected in the soluble fraction after a gentle lysis is unclear at this moment. One possibility is that the kinase affects directly the heme intake into endosomes by interacting, or phosphorylating an unknown carrier. In fact, there is a link between the levels of heme and several stress elements [53], but this mechanism remains obscure. In mammalian cells, for example, PERK mediates apoptosis in response to ROS by facilitating the interaction of the ER with the mitochondria, PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 18 / 27 eIF2 Kinase Regulated by Heme independently of the kinase activity [54]. PERK knockouts were shown to affect largely the un- folded protein responses causing high levels of ER stress and its fragmentation [55]. The observation that ascorbate was effective at low concentrations suggests that it could protect the parasite as an antioxidant towards reactive species, donating one electron to oxidiz- ing radicals such as hydroxyl radical [56]. This agrees with our observation that ROS levels in- creased in the TcK2 null parasites being restored to normal levels in cells growing in the presence of ascorbate. Therefore, it is possible that hydroxyl radicals would be generated by the Fenton reaction, which would occur with the increased levels of H2O2 produced by the parasite and minimal amounts of free Fe2+ present in the medium. This is in agreement with the results showing that inhibition of growth occurs a few days after ascorbate removal. At concentrations higher than 1 mM, ascorbate would act as a pro-oxidant agent, reducing Fe3+ to Fe2+, which would increase the production of more hydroxyl radicals in the presence of H2O2 by the Fenton reaction. Heme as regulatory molecule controlling TcK2 activity also appears to occur in the mam- malian stages of the parasite, as the kinase is active in trypomastigotes released from infected cells and it is mainly inactive in proliferating amastigotes. In addition, our observations indi- cate that intracellular amastigotes growth is largely diminished in the TcK2 null cells. The fact that growth is not restored by ascorbate could be due to its inability to decrease intracellular H2O2. These observations agree with recent findings indicating that iron and heme availability influences T. cruzi infection and disease progression [57]. Moreover, the intake of heme and/or iron has been found critical for Leishmania survival and infection [58] and several other hemo- parasites [59]. In conclusion, our results indicate that heme affects the eIF2α kinase 2 of T. cruzi regulating development in insect and most likely in mammalian hosts. Future understanding of how this kinase regulates heme levels across the different parasite compartments and the development of specific inhibitors for this kinase could be useful in the control and in the understanding of the Chagas’ disease. PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 Immunoblotting Extracts were prepared from parasites collected by centrifugation at 2000 g for 5 min at 4°C and washed once with PBS before lysis in SDS-polyacrylamide gel electrophoresis (SDS- PAGE) sample buffer. Alternatively, extracts were prepared by lysis of 1 × 107 cells in 50 μl of ice-cold 50 mM NaCl, 20 mM Tris-HCl (pH 7.4) containing 1% Triton X100, 1 mM phenyl- methanesulfonyl fluoride (PMSF), and the cOmplete protease inhibitor cocktail, EDTA-free (Roche). The lysates were centrifuged at 6000 g for 10 min at 4°C and the supernatants were in- cubated with 5 U of calf intestinal alkaline phosphatase (Promega) for 10 min and the reaction was stopped by adding sample buffer and further incubation at 95°C for 5 min. Immunoblots were performed with extracts of cells submitted to electrophoresis on SDS- PAGE (7.5%) and transferred to nitrocellulose membranes. The membranes were stained with 0.3% Ponceau S in 10% acetic acid and then treated with 5% nonfat milk in PBS containing 0.05% Tween-20 for 60 min. The membranes were incubated 1 h with the primary antibodies in PBS containing 0.1% Tween-20. Anti-TcK2 rabbit polyclonal antiserum, which was obtained by immunization with a recombinant protein corresponding to the kinase domain (KD) of the T. brucei K2 expressed in Escherichia coli BL21 [19]. It was used at 1:1500 dilution. A monoclo- nal antibody (mAb) 5D10, obtained as described in [62] by immunization with a recombinant protein corresponding to the residue 346 to 511 of T. cruzi K2, and expressed from pET28a (Novagen) in E. coli BL21. It was used at 1:500 dilution. Anti-Hsp70 [63] was used at 1:20,000, anti-T. cruzi β-tubulin [64] at 1:20,000 and anti-BiP [65] at 1:20,000 dilutions. The membranes were washed three times with PBS containing 0.5% Tween-20 (10 min each) followed by 1 h in- cubation with peroxidase labeled goat anti-mouse or rabbit IgG antibody (Invitrogen) at 1:10,000 dilutions. Bound antibodies were detected by ECL (Millipore) by using a LI-COR Od- yssey imaging apparatus. eIF2 Kinase Regulated by Heme 120 to 144 h after infection) as described previously [61]. Cell invasion assays were carried out by seeding the parasites onto each well of 24-well plates containing 13-mm diameter round glass coverslips coated with 2 × 104 cells. After 2 h incubation with parasites (2 × 105 parasites), the medium was removed, and the wells were washed with PBS and incubated with 1 ml of 4% paraformaldehyde in PBS at room temperature for 20 min. The coverslips were then washed with PBS and mounted in Prolong Gold Antifade Reagent (Invitrogen) in the presence of 10 μg/ml of 4-6-diamidino-2-phenylindole (DAPI). Images were analyzed in an Olympus (BX-61) fluorescence microscope. PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 Parasite cultures and differentiation T. cruzi epimastigotes (Y strain) were cultivated in liver infusion tryptose (LIT) medium sup- plemented with 10% fetal bovine serum at 28°C [60]. For the differentiation into metacyclic- trypomastigotes, exponentially growing epimastigotes (1 × 107 cells per ml) were collected by centrifugation (2000 g for 10 min) and resuspended at a density of 5 × 108 cells per ml in Tria- toma artificial urine (TAU) medium [190 mM NaCl, 17 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 8 mM phosphate buffer (pH 6.0)]. After 2 h culture at 28°C the cells were diluted to 5 × 106 in TAU supplemented with 2.5% sodium bicarbonate, 500 U/ml penicillin, 10 mM L-proline, 50 mM L-glutamic acid, 2 mM L-aspartic acid, 10 mM glucose and incubated at 28°C for 96 h [37]. Intracellular amastigotes were obtained from LLCMK2 (ATCC CCL-7) monkey kidney cells through lysis 72 h after infection. Trypomastigotes were collected from the cell medium (from PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 19 / 27 Immunoprecipitation Parasites at exponential growth phase were collected by centrifugation at 2000 g for 5 min at 4° C and resuspended at 4°C in 137 mM NaCl, 2 mM EDTA and 20 mM Tris-HCl (pH 8.0) con- taining protease inhibitor cocktail 1% Nonidet P40, for lysis. The lysates were centrifuged at 12000 g for 20 min and the supernatants transferred to new tubes, which were incubated at 4°C with the anti-TcK2 rabbit polyclonal antiserum for 16 h. Immune complexes were collected after 2 h incubations with Protein G Sepharose (GE). The immunoprecipitates were centri- fuged at 500 g for 2 min, washed once in 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM Tris-HCl (pH 8.0) containing 1% Triton-X100. Electron microscopy Parasites were fixed at room temperature with 2% formaldehyde/2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2). The fixed parasites were then decanted overnight on ACLAR (Electron Microscopy Sciences) film precoated with 0.01% poly-L-lysine. The ACLAR films were washed three times with 0.1 M sodium cacodylate (pH 7.2) and then fixed with 1% osmium tetroxide in the same buffer at room temperature. The preparation was then washed with cacodylate buffer followed by three washes with water. Subsequently, the ACLARfilms were treated with 0.4% uranyl acetate, gradually dehydrated in a series of ethanol solutions, and embedded in Epon resin (Electron Microscopy Sciences). After sectioning to produce con- secutive sections, the material was stained with uranyl acetate and lead citrate, mounted on Formvar grids, and observed in a JEOL 1200 EX II transmission electron microscope at 80 kV. eIF2 Kinase Regulated by Heme Apo-oil objective (NA 1.4). Acquisitions were at every 0.2 μm for each set of excitation/ emission filters. Blind deconvolution was performed by employing the AutoQuant X2.2 software (Media Cybernetics). PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 Immunofluorescence Parasites were washed with PBS and attached to glass slides (Tekdon Incorporated) pretreated with 0.01% poly-L-lysine for 5 min. The excess of parasites was removed and the slides incubat- ed with 4% paraformaldehyde in PBS at room temperature for 20 min, washed in PBS and per- meabilized 5 min in PBS containing 0.1% Triton X100. Fixed and permeabilized cells were washed with PBS, incubated for 20 min with PBS containing 2% BSA and for 1 h at room tem- perature with primary antibodies diluted in the same buffer. Anti-human influenza hemagglu- tinin (HA) (Covance) was used at 1:1,000, the mAb of anti-5D10 at 1:500, anti-Binding immunoglobulin protein (BiP) at 1:5,000, and anti-T. cruzi cruzipain mAb (a gift of Prof. Dr. Julio Scharfstein [66], at 1:2,000 dilutions. The slides were washed with PBS, incubated 1 h at room temperature with Alexa Fluor 594, or 488, goat anti-rabbit or anti-mouse IgG (Invitro- gen) diluted 1:1000, washed once more, and mounted in Prolong Gold Antifade Reagent (Invi- trogen) in the presence of 10 μg of DAPI per ml. Images were acquired by using a Hamamatsu Orca R2 CCD camera coupled to an Olympus (BX-61) microscope equipped with a ×100 plan PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 20 / 27 eIF2 Kinase Regulated by Heme (Lonza), program U-033. Cells were diluted and selected by growing in 0.2 to 0.5 mg/ml G418, 0.5 mg/ml Hygro, or 0.05 mg/ml Blast depending on the plasmid used for transfection. Limiting dilution was further used to clone the parasites. The presence of resistance marker insertions was detected by PCR amplification of DNA ex- tracted from parasite clones using oligonucleotides 5K2fow at the 50UTR of K2 and G418Rev for G418, HigroR for Hygro, and BlastRev for Blast. The removal of K2 was confirmed by PCR amplification using oligonucleotides TcK2-KD-pGEX5X-1(F) and TcK2-KD-pGEX5X-1(R). T. cruzi containing the original and the kinase death were obtaining by cloning both con- structs in the XbaI and XhoI sites of the pROCK-GFP-HYG plasmid [34]. The full-length gene was amplified from the genomic DNA of T. cruzi Y strain by PCR using the primers Tck2Xba- fow and TcK2XhoI-rev. The mutated protein was generated by amplification of two fragments encompassing the full-length gene with two sets of primers. One of the fragments was generat- ed with Pfx50 polymerase (Life Technologies) by using the primers Tck2XhoIrev and Tck2mut-fow and the other with the primers TceiF2-K2mut-rev and the TcK2Xbafow to re- place the lysine 695 by an alanine. The two fragments were gel purified and assembled by a sec- ond PCR using the primers Tck2Xba-fow and TcK2XhoI-rev. The final fragment was inserted into pCR2.1 Topo (Life Technologies) and subcloned into the XbaI and XhoI sites of pROCK- GFP-HYG. The obtained plasmids were confirmed by restriction analysis and sequencing. Recombinant proteins, kinase and binding assays Soluble recombinant T. cruzi eIF2α containing a histidine-tag was expressed from pDEST17 [23] in E. coli BL21. Expression was induced for 16 h at 30°C with 0.5 mM isopropyl β-D-1- thiogalactopyranoside (IPTG); the cells collected and resuspended in 0.25 M NaCl, 50 mM Tris-HCl (pH 8.0) and the cOmplete protease inhibitor cocktail and lysed by using a French press. The recombinant protein was purified by affinity chromatography on a nickel agarose column (Qiagen) by elution with the lysis solution containing 0.5 M imidazole. The kinase do- main of TcK2 was amplified from total T. cruzi DNA with the oligonucleotides TcK2-KD- pGEX5X-1 and TcK2-KD-pGEX5X-1 and cloned into the EcoRI and XhoI sites of the plasmid pGEX5X-1 (GE) to generate a protein fused with GST. The TcK2 recombinant was expressed in E. coli BL21 after induction with 0.5 mM IPTG for 16 h at 30°C. The protein was purified from bacteria lysed by the French press by using affinity chromatography on GST-Sepharose (GE) equilibrated in PBS containing cOmplete cocktail, by elution in the same buffer contain- ing 0.1 M glutathione. Both proteins were dialyzed in the kinase buffer (50 mM NaCl, 20 mM Tris-HCl (pH 7.5) at 4°C for 18 h. The kinase assays were performed in the presence of 5 μg/ml eIF2α, 1 μg/ml TcK2, 10 mM MgCl2, 10 μCi of [32P] γATP (3000 Ci/mmol, Perkin Elmer) and 0.5 mM ATP at 37°C for 1 h. When indicated the heme derivatives (Frontiers Scientific) were added 15 min before addition of ATP. Reactions were stopped by the addition of SDS-PAGE sample buffer. The labeling was quantified by using 9200 Typhon imaging apparatus (GE) after electrophoresis and Coomassie R-250 staining. For the binding assays hemin at indicated concentrations were incubated 30 min with the indicated with 5 μg/ml of recombinant proteins in 0.1 M Tris-HCl (pH 7.4) and 50 mM NaCl and the absorbance read in a SpectraMax M3 reader (Molecular Devices), at 25°C. Dissociation constant determination was estimated by using the Prisma 6 software (Graphpad). Generation of recombinant parasites TcK2 knockouts were generated by gene replacement using antibiotic resistance markers flanked by untranslated regions (UTR) of the K2 gene (http://tritrypdb.org flanked by untranslated regions (UTR) of the K2 gene (http://tritrypdb.org TcCLB.506559.129). The 50-UTR region was amplified from T. cruzi (Y-strain) genomic DNA [67] using oligonucleotides 5k2fowApaI and 5revXbaI. The 30-UTR was amplified using oligo- nucleotides 30K2fowSal and 3K2RevSac. Primer sequences used in this study are presented in S1 Table. The amplified fragments were cloned into plasmids containing the neomycin phos- photransferase (Neo), hygromycin phosphotransferase (Hygro), or blasticidin (Blast) genes in- serted into the XbaI and SalI sites of pGEM-T (a gift from Dr. Carlos Renato Machado from the Universidade Federal de Minas Gerais, Brazil). The blasticidin gene was amplified from pNAT6Myc [68] with oligonucleotides BlastFowXbaI and BlastRevSalI. Restriction mapping and DNA sequencing confirmed all constructs. HA-epitope tagged TcK2 was prepared in a modified pTEX plasmid [69] containing one HA encoding sequence 5-TACCCTTACGACGTTCCTGATTACGCTAGCTGA between the BamH1 and XhoI sites. Two segments corresponding to the C-terminus sequence of TcK2 were amplified from T. cruzi genomic DNA (Y strain) with oligonucleotides K2TcXbaFor / K2TcNotRev and K2TcNotFor / K2TcBam and cloned sequentially into pTEX-HA to intro- duce a Not I site in the sequence and create a 900 base sequence followed by the HA tag. Thirty micrograms of each plasmid linearized with NotI for the HA-tag, and SacI for the gene replacements, were incubated with T. cruzi epimastigotes previously washed and resuspended to 5 × 106 parasites in 100 μl of 137 mM NaCl, 5 mM KCl, 5.5 mM Na2HPO4, 0.77 mM glucose and 21 mM Hepes (pH 7.0) and pulsed with the Amaxa Nucleofactor PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 21 / 27 eIF2 Kinase Regulated by Heme flow cytometry using a FACS Accuri C6 (BD Biosciences) as recommended by the manufactur- er. Hydrogen peroxide levels were measured by incubating 2 × 107 parasites per ml of PBS with 1 U/ml horseradish peroxidase in the presence of 2 μM Amplex Red (Invitrogen) for 30 min. The parasites were then centrifuged and the resulting fluorescence measured in the superna- tants (excitation 530 nm and emission 590 nm) by using a SpectraMax M3 Reader. Total per- oxidase activity was measured in the supernatant of 2 × 107 parasites lysed in 50 μl of ice-cold 50 mM NaCl, 20 mM Tris-HCl (pH 7.4) containing 1% Triton X100. The activity was quanti- fied after 30 min incubation at 25°C by adding 2 μM Amplex Red (Invitrogen) and 2 mM H2O2. SOD activity was measured in the supernatant of 1 × 107 parasites previously lysed by three sonication cycles (20 sec each) in 200 μl of 50 mM potassium phosphate buffer (pH 7.4) and 0.1 mM EDTA (SOD buffer). Reactions were done at 25°C by the addition of 25 μl of the lysate into 200 μl of SOD buffer containing 10 mg/ml β-nicotinamide adenine dinucleotide and 28 mg/ml nitrotetrazolium blue chloride (both from Sigma) followed by 25 μl of 3.3 mM phen- azine methosulfate (Sigma). The SOD activity was determined by the kinetics of nitrotetrazo- lium blue chloride reduction followed by absorption at 560 nm [70]. Intracellular heme levels and incorporation of heme analogues We employed the method described in [71]. Briefly, 2 × 107 cells were centrifuged, washed with PBS, and resuspended in 1 ml of 10 mM Tris-HCl (pH 7.4) containing the cOmplete cock- tail. The cells were lysed by freezing and thawing (three cycles), the insoluble material removed by centrifugation at 10,000 g for 10 min and 840 μl transferred to a new tube containing 100 μl of 1 M NaOH and 200 μl of pyridine. Heme levels were then measured in a 1 ml cuvette after addition of 2 mg of solid sodium dithionite at 450 nm by using the Spectra Max M3 Reader. The incorporation of the fluorescent Zn(II) Mesoporphyrin IX (Frontier Scientific) was per- formed in 2 × 107 parasites resuspended in 100 μl of PBS. Ten μM of the porphyrin was added and the mixture incubated for 5 min followed by the addition of the indicated concentrations of hemin as a competitor. After more 30 min, the cells were chilled by adding 800 μl of cold PBS, centrifuged and washed with cold PBS and lysed in PBS containing 0.5% Triton X-100. The fluorescent porphyrin was measured in the supernatant (excitation 405 nm and emission at 578 nm). Reactive species of oxygen, H2O2 and peroxidases Parasites at 2 × 107 cells per ml were pre-loaded with 5 μM of CellRox (Invitrogen) in PBS for 30 min, or with 5 μM CM-H2DCFDA (Invitrogen). In the case of this later reagent, cells were further incubated for 30 min in LIT medium. Reactive species of oxygen were then detected by PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 22 / 27 Acknowledgments We would like to thank Victor Nussenzweig for helpful suggestions and comments; Dr. Thom- as Dever (NIH-NICHD) for reading the manuscript; Dr. Marcia Paes (UERJ) for providing us the heme analogs and for helpful suggestions; Dr. Carlos Renato Machado, for giving us the pGEM vectors and for valuable comments; Dr. Julio Scharfstein (UFRJ) for the anti-cruzipain antibodies; Vânia d’Almeida and Lisandro Lungato for helping with the SOD assays. We are also grateful to Claudeci Medeiros and Claudio Rogério for the constant technical help during this work. Author Contributions Conceived and designed the experiments: LSA NSM TCLJ MZ BC SS. Performed the experi- ments: LSA NSM TCPR TCLJ. Analyzed the data: LSA NSM TCLJ MZ BAC SS. Contributed reagents/materials/analysis tools: MZ BAC SS. Wrote the paper: LSA NSM BAC MZ SS. eIF2 Kinase Regulated by Heme S3 Fig. Heme binding is specific for TcK2. (A) Absorption spectra of heme (yellow line), GST + hemin (blue line) and GST-TcK2 (green line), GST-TcK2 incubated with hemin (black line) and GST-PfK4 (red line). (B) SDS-PAGE of His6x-TceIF2α incubated without (−) or with (+) 10 μM hemin in the presence of GST-TcK2, or GST-PK4 for 30 min with [32P]-γ-ATP. The upper gel shows the autoradiogram and at the bottom is the same gel stained with Coomassie Blue R250. (TIF) Supporting Information S1 Fig. TcK2 is more soluble in trypomastigotes. Parasites were collected by centrifugation, resuspended once in PBS containing the cocktail of protease inhibitors. Increased concentra- tions of digitonin were added and the lysates incubated for 5 min at 37°C then with 0.3 M su- crose. Each fraction corresponds to the supernatant of 10000 g (5 min) that was mixed with SDS-PAGE sample buffer, submitted to Western Blotting and probed with the indicated antibodies. S2 Fig. Alignment of the kinase domains of TcK2, TcK3, PERK and HRI. The alignment was generated using the amino acid sequence from the kinases domain of TcK2 from CL- Brener sequence from residues 638–984 (TcCLB.506559.129, http://tritrypdb.org), TcK3 based on the sequence between residues 547 and 1062 of the putative kinase of T. cruzi Sylvio X10/1 strain (ADWP02013222, http://tritrypdb.org), the kinase domain of human PERK containing residues 599 to1073 of the NCBI gene identification 945,1 and the kinase domain of human HRI from residues 167–582 (UniProtKB/Swiss-Prot, Q9BQI3.2). The asterisk indicates the ly- sine 695 required for ATP binding in the catalytic site. The underlined residues are the 23 / 27 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 eIF2 Kinase Regulated by Heme 10. Wek RC, Jiang HY, Anthony TG (2006) Coping with stress: eIF2 kinases and translational control. Bio- chem Soc Trans 34: 7–11. PMID: 16246168 11. Zhang M, Joyce BR, Sullivan WJ Jr., Nussenzweig V (2013) Translational control in Plasmodium and Toxoplasma parasites. Eukariot Cell 12: 161–167. doi: 10.1128/EC.00296-12 PMID: 23243065 12. Vonlaufen N, Kanzok SM, Wek RC, Sullivan WJ Jr. (2008) Stress response pathways in protozoan par- asites. Cell Microbiol 10: 2387–2399. doi: 10.1111/j.1462-5822.2008.01210.x PMID: 18647172 13. Zhang M, Fennell C, Ranford-Cartwright L, Sakthivel R, Gueirard P, et al. (2010) The Plasmodium eu- karyotic initiation factor-2alpha kinase IK2 controls the latency of sporozoites in the mosquito salivary glands. J Exp Med 207: 1465–1474. doi: 10.1084/jem.20091975 PMID: 20584882 14. Fennell C, Babbitt S, Russo I, Wilkes J, Ranford-Cartwright L, et al. (2009) PfeIK1, a eukaryotic initia- tion factor 2alpha kinase of the human malaria parasite Plasmodium falciparum, regulates stress-re- sponse to amino-acid starvation. Malar J 8: 99. doi: 10.1186/1475-2875-8-99 PMID: 19435497 15. Zhang M, Mishra S, Sakthivel R, Rojas M, Ranjan R, et al. (2012) PK4, a eukaryotic initiation factor 2alpha(eIF2alpha) kinase, is essential for the development of the erythrocytic cycle of Plasmodium. Proc Natl Acad Sci USA 109: 3956–3961. doi: 10.1073/pnas.1121567109 PMID: 22355110 16. Konrad C, Wek RC, Sullivan WJ Jr. (2011) A GCN2-like eukaryotic initiation factor 2 kinase increases the viability of extracellular Toxoplasma gondii parasites. Eukariot Cell 10: 1403–1412. doi: 10.1128/ EC.05117-11 PMID: 21908594 17. Joyce BR, Queener SF, Wek RC, Sullivan WJ Jr. (2010) Phosphorylation of eukaryotic initiation factor- 2{alpha} promotes the extracellular survival of obligate intracellular parasite Toxoplasma gondii. Proc Natl Acad Sci USA 107: 17200–17205. doi: 10.1073/pnas.1007610107 PMID: 20855600 18. Narasimhan J, Joyce BR, Naguleswaran A, Smith AT, Livingston MR, et al. (2008) Translation regula- tion by eukaryotic initiation factor-2 kinases in the development of latent cysts in Toxoplasma gondii. J Biol Chem 283: 16591–16601. doi: 10.1074/jbc.M800681200 PMID: 18420584 19. Moraes MCS, Jesus TCL, Hashimoto NN, Dey M, Schwartz KJ, et al. (2007) Novel membrane-bound eIF2a kinase in the flagellar pocket of Trypanosoma brucei. Eukariot Cell 6: 1979–1991. PMID: 17873083 20. Cloutier S, Laverdiere M, Chou M-N, Boilard N, Chow C, et al. (2012) Translational control through eIF2alpha phosphorylation during the Leishmania differentiation process. PLoS One 7: e35085. doi: 10.1371/journal.pone.0035085 PMID: 22693545 21. PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 References 1. Sonenberg N, Dever TE (2003) Eukaryotic translation initiation factors and regulators. Curr Opin Struct Biol 13: 56–63. PMID: 12581660 1. Sonenberg N, Dever TE (2003) Eukaryotic translation initiation factors and regulators. Curr Opin Struct Biol 13: 56–63. PMID: 12581660 1. Sonenberg N, Dever TE (2003) Eukaryotic translation initiation factors and regulators. Curr Opin Struct Biol 13: 56–63. PMID: 12581660 2. Sonenberg N, Hinnebusch A (2009) Regulation of translation initiation in eukaryotes: mechanisms and biological targets. Cell 136: 731–745. doi: 10.1016/j.cell.2009.01.042 PMID: 19239892 3. Donnelly N, Gorman AM, Gupta S, Samali A (2013) The eIF2alpha kinases: their structures and func- tions. Cell Mol Life Sci 70: 3493–3511. doi: 10.1007/s00018-012-1252-6 PMID: 23354059 4. Castilho BA, Shanmugam R, Silva RC, Ramesh R, Himme BM, et al. (2014) Keeping the eIF2 alpha ki- nase Gcn2 in check. Biochim Biophys Acta. 1843: 1848–1968. doi: 10.1016/j.bbamcr.2014.04.006 PMID: 24732012 5. Rothenburg S, Deigendesch N, Dey M, Dever TE, Tazi L (2008) Double-stranded RNA-activated pro- tein kinase PKR of fishes and amphibians: varying the number of double-stranded RNA binding do- mains and lineage-specific duplications. BMC Biology 6: 12. doi: 10.1186/1741-7007-6-12 PMID: 18312693 6. Garcia MA, Meurs EF, Esteban M (2007) The dsRNA protein kinase PKR: virus and cell control. Biochi- mie 89: 799–811. PMID: 17451862 7. Dey M, Cao C, Dar AC, Tamura T, Ozato K, et al. (2005) Mechanistic link between PKR dimerization, autophosphorylation, and eIF2alpha substrate recognition. Cell 122: 901–913. PMID: 16179259 8. Ron D, Harding HP (2012) Protein-folding homeostasis in the endoplasmic reticulum and nutritional regulation. Cold Spring Harb Perspect Biol 4: 1–13. PMID: 23209157 9. Chen JJ, London IM (1995) Regulation of protein synthesis by heme-regulated eIF-2 alpha kinase. Trends Bioch Sci 20: 105–108. PMID: 7709427 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 24 / 27 eIF2 Kinase Regulated by Heme 30. Souza CF, Carneiro AB, Silveira AB, Laranja GA, Silva-Neto MA, et al. (2009) Heme-induced Trypano- soma cruzi proliferation is mediated by CaM kinase II. Biochem Biophys Res Commun 390: 541–546. doi: 10.1016/j.bbrc.2009.09.135 PMID: 19818332 31. Lara Fa, Sant’anna C, Lemos D, Laranja GaT, Coelho MGP, et al. (2007) Heme requirement and intra- cellular trafficking in Trypanosoma cruzi epimastigotes. Biochem Biophys Res Commun 355: 16–22. PMID: 17292866 32. Sant’anna C, de Souza W, Cunha e S (2004) Biogenesis of the reservosomes of Trypanosoma cruzi. Micros Microanal 10: 637–646. PMID: 15525436 33. Liu R, Hu J (2011) HemeBIND: a novel method for heme binding residue prediction by combining struc- tural and sequence information. BMC bioinformatics 12: 207. doi: 10.1186/1471-2105-12-207 PMID: 21612668 34. DaRocha WD, Silva RA, Bartholomeu DC, Pires SF, Freitas JM, et al. (2004) Expression of exogenous genes in Trypanosoma cruzi: improving vectors and electroporation protocols. Parasitol Res 92: 113– 120. PMID: 14634799 35. Harding HP, Zhang Y, Ron D (1999) Protein translation and folding are coupled by an endoplasmic-re- ticulum-resident kinase. Nature 397: 271–274. PMID: 9930704 36. Piacenza L, Alvarez MN, Peluffo G, Radi R (2009) Fighting the oxidative assault: the Trypanosoma cruzi journey to infection. Curr Opin Microbiol 12: 415–421. doi: 10.1016/j.mib.2009.06.011 PMID: 19616990 37. Contreras VT, Salles JM, Thomas N, Morel CM, Goldenberg S (1985) In vitro differentiation of Trypano- soma cruzi under chemically defined conditions. Mol Biochem Parasitol 16: 315–327. PMID: 3903496 38. Goldenberg S, Avila AR (2011) Aspects of Trypanosoma cruzi stage differentiation. Adv Parasit 75: 285–305. doi: 10.1016/B978-0-12-385863-4.00013-7 PMID: 21820561 39. De Gaudenzi JG, Carmona SJ, Aguero F, Frasch AC (2013) Genome-wide analysis of 30-untranslated regions supports the existence of post-transcriptional regulons controlling gene expression in trypano- somes. Peer Journal 1: e118. doi: 10.7717/peerj.118 PMID: 23904995 40. Garcia ES, Gonzalez MS, De Azambuja P, Baralle FE, Fraidenraich D, et al. (1995) Induction of Trypa- nosoma cruzi metacyclogenesis in the gut of the hematophagous insect vector, Rhodnius prolixus, by hemoglobin and peptides carrying alpha D-globin sequences. Exp Parasitol 81: 255–261. PMID: 7498422 41. Cardoso J, Soares MJ, Menna-Barreto RF, Le Bloas R, Sotomaior V, et al. (2008) Inhibition of protea- some activity blocks Trypanosoma cruzi growth and metacyclogenesis. Parasitol Res 103: 941–951. doi: 10.1007/s00436-008-1081-6 PMID: 18581141 42. Igarashi J, Murase M, Iizuka A, Pichierri F, Martinkova M, et al. PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 Chow C, Cloutier S, Dumas C, Chou MN, Papadopoulou B (2011) Promastigote to amastigote differen- tiation of Leishmania is markedly delayed in the absence of PERK eIF2alpha kinase-dependent eIF2al- pha phosphorylation. Cell Microbiol 13: 1059–1077. doi: 10.1111/j.1462-5822.2011.01602.x PMID: 21624030 22. Hope R, Ben-Mayor E, Friedman N, Voloshin K, Biswas D, et al. (2014) Phosphorylation of the TATA- binding protein activates the spliced leader silencing pathway in Trypanosoma brucei. Sci Signal 7: ra85. doi: 10.1126/scisignal.2005234 PMID: 25185157 23. Tonelli RR, Augusto LdS, Castilho BA, Schenkman S (2011) Protein Synthesis Attenuation by Phos- phorylation of eIF2alpha Is Required for the Differentiation of Trypanosoma cruzi into Infective Forms. PLoS One 6: e27094. doi: 10.1371/journal.pone.0027904 PMID: 22114724 24. Figueiredo RC, Rosa DS, Soares MJ (2000) Differentiation of Trypanosoma cruzi epimastigotes: meta- cyclogenesis and adhesion to substrate are triggered by nutritional stress. J Parasitol 86: 1213–1218. PMID: 11191893 25. Zeledon R, Bolanos R, Rojas M (1984) Scanning electron microscopy of the final phase of the life cycle of Trypanosoma cruzi in the insect vector. Acta Trop 41: 39–43. PMID: 6143481 26. Figueiredo RC, Rosa DS, Gomes YM, Nakasawa M, Soares MJ (2004) Reservosome: an endocytic compartment in epimastigote forms of the protozoan Trypanosoma cruzi (Kinetoplastida: Trypanoso- matidae). Correlation between endocytosis of nutrients and cell differentiation. Parasitol 129: 431–438. PMID: 15521631 27. Cupello MP, Souza CFD, Buchensky C, Soares JBRC, Laranja GAT, et al. (2011) The heme uptake process in Trypanosoma cruzi epimastigotes is inhibited by heme analogues and by inhibitors of ABC transporters. Acta Trop 120: 211–218. doi: 10.1016/j.actatropica.2011.08.011 PMID: 21903090 28. Pereira MG, Nakayasu ES, Sant’Anna C, De Cicco NN, Atella GC, et al. (2011) Trypanosoma cruzi epi- mastigotes are able to store and mobilize high amounts of cholesterol in reservosome lipid inclusions. PLoS One 6: e22359. doi: 10.1371/journal.pone.0022359 PMID: 21818313 29. Porto-Carreiro I, Attias M, Miranda K, de Souza W, Cunha e S (2000) Trypanosoma cruzi epimastigote endocytic pathway: cargo enters the cytostome and passes through an early endosomal network be- fore storage in reservosomes. Eur J Cell Biol 79: 858–869. PMID: 11139150 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 25 / 27 eIF2 Kinase Regulated by Heme 50. de Godoy LM, Marchini FK, Pavoni DP, Rampazzo Rde C, Probst CM, et al. (2012) Quantitative proteo- mics of Trypanosoma cruzi during metacyclogenesis. Proteomics 12: 2694–2703. doi: 10.1002/pmic. 201200078 PMID: 22761176 51. Cullinan SB, Zhang D, Hannink M, Arvisais E, Kaufman RJ, et al. (2003) Nrf2 is a direct PERK substrate and effector of PERK-dependent cell survival. Mol Cell Biol 23: 7198–7209. PMID: 14517290 52. Dufernez F, Yernaux C, Gerbod D, Noel C, Chauvenet M, et al. (2006) The presence of four iron-con- taining superoxide dismutase isozymes in trypanosomatidae: characterization, subcellular localization, and phylogenetic origin in Trypanosoma brucei. Free Radic Biol Med 40: 210–225. PMID: 16413404 53. Koskenkorva-Frank TS, Weiss G, Koppenol WH, Burckhardt S (2013) The complex interplay of iron metabolism, reactive oxygen species, and reactive nitrogen species: insights into the potential of vari- ous iron therapies to induce oxidative and nitrosative stress. Free Radic Biol Med 65: 1174–1194. doi: 10.1016/j.freeradbiomed.2013.09.001 PMID: 24036104 54. Verfaillie T, Rubio N, Garg AD, Bultynck G, Rizzuto R, et al. (2012) PERK is required at the ER-mito- chondrial contact sites to convey apoptosis after ROS-based ER stress. Cell Death Differ 19: 1880– 1891. doi: 10.1038/cdd.2012.74 PMID: 22705852 55. Huang G, Yao J, Zeng W, Mizuno Y, Kamm KE, et al. (2006) ER stress disrupts Ca2+-signaling com- plexes and Ca2+ regulation in secretory and muscle cells from PERK-knockout mice. J Cell Sci 119: 153–161. PMID: 16352659 56. Du J, Cullen JJ, Buettner GR (2012) Ascorbic acid: chemistry, biology and the treatment of cancer. Bio- chim Biophys Acta 1826: 443–457. doi: 10.1016/j.bbcan.2012.06.003 PMID: 22728050 57. Paiva CN, Feijo DF, Dutra FF, Carneiro VC, Freitas GB, et al. (2012) Oxidative stress fuels Trypano- soma cruzi infection in mice. J Clin Invest 122: 2531–2542. doi: 10.1172/JCI58525 PMID: 22728935 58. Andrews NW (2012) Oxidative stress and intracellular infections: more iron to the fire. J Clin Invest 122: 2352–2354. doi: 10.1172/JCI64239 PMID: 22728929 59. Toh SQ, Glanfield A, Gobert GN, Jones MK (2010) Heme and blood-feeding parasites: friends or foes? Parasit Vectors 3: 108–108. doi: 10.1186/1756-3305-3-108 PMID: 21087517 60. Camargo EP (1964) Growth and differentiation in Trypanosoma cruzi: Origin of metacyclic trypomasti- gotes in liquid media. Rev Inst Med Tropical S Paulo 6: 93–100. 61. (2008) Elucidation of the heme binding site of heme-regulated eukaryotic initiation factor 2alpha kinase and the role of the regulatory motif in heme sensing by spectroscopic and catalytic studies of mutant proteins. J Biol Chem 283: 18782– 18791. doi: 10.1074/jbc.M801400200 PMID: 18450746 43. Nogueira NP, de Souza CF, Saraiva FM, Sultano PE, Dalmau SR, et al. (2011) Heme-induced ROS in Trypanosoma cruzi activates CaMKII-like that triggers epimastigote proliferation. One helpful effect of ROS. PLoS One 6: e25935. doi: 10.1371/journal.pone.0025935 PMID: 22022475 44. Sant’Anna C, Pereira MG, Lemgruber L, de Souza W, Cunha e Silva NL (2008) New insights into the morphology of Trypanosoma cruzi reservosome. Microsc Res Techniq 71: 599–605. doi: 10.1002/ jemt.20592 PMID: 18452191 45. Marchini FK, de Godoy LM, Rampazzo RC, Pavoni DP, Probst CM, et al. (2011) Profiling the Trypano- soma cruzi phosphoproteome. PLoS One 6: e25381. doi: 10.1371/journal.pone.0025381 PMID: 21966514 46. Kloft N, Neukirch C, von Hoven G, Bobkiewicz W, Weis S, et al. (2012) A subunit of eukaryotic transla- tion initiation factor 2alpha-phosphatase (CreP/PPP1R15B) regulates membrane traffic. J Biol Chem 287: 35299–35317. doi: 10.1074/jbc.M112.379883 PMID: 22915583 47. Salzman TA, Stella AM, Wider de Xifra EA, Batlle AM, Docampo R, et al. (1982) Porphyrin biosynthesis in parasitic hemoflagellates: functional and defective enzymes in Trypanosoma cruzi. Comparative Biochmestry and Physiology B 72: 663–667. PMID: 6751683 48. Vanhollebeke B, De Muylder G, Nielsen MJ, Pays A, Tebabi P, et al. (2008) A haptoglobin-hemoglobin receptor conveys innate immunity to Trypanosoma brucei in humans. Science 320: 677–681. doi: 10. 1126/science.1156296 PMID: 18451305 49. Huynh C, Yuan X, Miguel DC, Renberg RL, Protchenko O, et al. (2012) Heme uptake by Leishmania amazonensis is mediated by the transmembrane protein LHR1. PloS Pathog 8: e1002795. doi: 10. 1371/journal.ppat.1002795 PMID: 22807677 26 / 27 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015 Abuin G, Freitas-Junior LHG, Colli W, Alves MJ, Schenkman S (1999) Expression of trans-sialidase and 85 kDa glycoprotein genes in Trypanosoma cruzi is differentially regulated at the post-transcription- al level by labile protein factors. J Biol Chem 274: 13041–13047. PMID: 10224055 62. Cornejo A, Oliveira CR, Wurtele M, Chung J, Hilpert K, et al. (2012) A novel monoclonal antibody against the C-terminus of beta-tubulin recognizes endocytic organelles in Trypanosoma cruzi. Protein Pept Lett 19: 636–643. PMID: 22519535 63. McDowell MA, Ransom DM, Bangs JD (1998) Glycosylphosphatidylinositol-dependent secretory trans- port in Trypanosoma brucei. Biochem J 335: 681–689. PMID: 9794811 64. Chung J, Rocha AA, Tonelli RR, Castilho BA, Schenkman S (2013) Eukaryotic initiation factor 5A de- phosphorylation is required for translational arrest in stationary phase cells. Biochem J 451: 257–267. doi: 10.1042/BJ20121553 PMID: 23368777 65. Bangs JD, Uyetake L, Brickman MJ, Balber AE, Boothroyd JC (1993) Molecular cloning and cellular lo- calization of a BiP homologue in Trypanosoma brucei. Divergent ER retention signals in a lower eukary- ote. J Cell Sci 105: 1101–1113. PMID: 8227199 66. Monteiro AC, Abrahamson M, Lima AP, Vannier-Santos MA, Scharfstein J (2001) Identification, char- acterization and localization of chagasin, a tight-binding cysteine protease inhibitor in Trypanosoma cruzi. J Cell Sci 114: 3933–3942. PMID: 11719560 67. Medina-Acosta E, Cross GAM (1993) Rapid isolation of DNA from trypanosomatid protozoa using a simple ‘mini-prep’ procedure. Mol Biochem Parasitol 59: 327–330. PMID: 8341329 68. Alsford S, Horn D (2008) Single-locus targeting constructs for reliable regulated RNAi and transgene expression in Trypanosoma brucei. Mol Biochem Parasitol 161: 76–79. doi: 10.1016/j.molbiopara. 2008.05.006 PMID: 18588918 69. Kelly JM, Ward HM, Miles MA, Kendall G (1992) A shuttle vector which facilitates the expression of transfected genes in Trypanosoma cruzi and Leishmania. Nucleic Acids Res 20: 3963–3969. PMID: 1324472 70. Ewing JF, Janero DR (1995) Microplate superoxide dismutase assay employing a nonenzymatic super- oxide generator. Anal Biochem 232: 243–248. PMID: 8747482 71. Miguel DC, Flannery AR, Mittra B, Andrews NW (2013) Heme uptake mediated by LHR1 is essential for Leishmania amazonensis virulence. Infect Immun 81: 3620–3626. doi: 10.1128/IAI.00687-13 PMID: 23876801 27 / 27 PLOS Pathogens \| DOI:10.1371/journal.ppat.1004618 February 6, 2015
https://openalex.org/W4376600241	https://zenodo.org/records/7937975/files/Paper-5-V2I3.pdf	English	null	Enhancing the Capabilities of Remotely PilotedAerial Systems Through Object Detection, FaceTracking, Digital Mapping and Gesture Control	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	4,564	Lavanya N L . Anish Bhat . Bhanuranjan S B . K G Lakshmi Narayan . Manjunath Bharadwaj V Lavanya N L . Anish Bhat . Bhanuranjan S B . K G Lakshmi Narayan . Manjunath Bharadwaj V Lavanya N L . Anish Bhat . Bhanuranjan S B . K G Lakshmi Narayan . Manjunath Bharadwaj V Department of Computer Science Sapthagiri College of Engineering, Bangalore, Karnataka, India. Received: 23 April 2023 / Revised: 30 April 2023 / Accepted: 11 May 2023 ©Milestone Research Publications, Part of CLOCKSS archiving DOI: 10.5281/zenodo.7937975 Abstract – Remotely Piloted Aerial Systems (RPAS) for remote sensing, a significant way of obtaining geographic data, has benefits like real-time, adaptability, high-resolution, cost-effectiveness, etc., and it can acquire data in risky environments without jeopardizing flight crews. It has great potential and a promising future since RPAS remote sensing is a powerful companion to airborne and spaceborne remote sensing. This work provides a comprehensive view of recent advancements in the field of Remotely Piloted Aerial Systems (RPAS) with machine learning features. The focus is on some specific areas: Face tracking, Object Detection, Surveillance. The paper describes the methods and algorithms used for these applications, discusses their performance and accuracy, and highlights the challenges faced in the implementation of such systems. The paper also provides an overview of the various platforms and tools used for the development of these systems, including hardware and software components. The review concludes by highlighting the future directions for research and development in this field. Index Terms – Remotely Piloted Aerial Systems; Remote sensing application; Object detection; Face tracking; Gesture control; Digital mapping 147 147 RESEARCH ARTICLE I. INTRODUCTION A remotely piloted aerial system (RPAS) is a type of aerial vehicle which requires minimum human control for its operation. We propose a RPAS which is capable of video-based surveillance, object detection, face tracking, lane following and a digitally mapped system which takes coordinates from the user instantaneously and follows the path given out by the user on a screen. The RPAS is usually called an Unmanned Aerial Vehicle (UAV) which employs aerodynamic forces to lift the vehicle, is scalable or 148 recoverable, can fly autonomously, and can carry a lethal or nonlethal payload. Its usage is currently limited by difficulties such as satellite communication and cost, but the advent of programmable drones has enabled engineers to implement various technologies in an unmanned aerial vehicle which has applications in numerous fields. RPAS were originally developed for the aerospace and defence sectors, but they have now been made available to the civilian population due to the higher levels of effectiveness as well as the security they provide. Fig 1: Architecture of a Remotely Piloted Aerial System. Fig 1: Architecture of a Remotely Piloted Aerial System. These autonomous robotic drones have variable degrees of autonomy as they fly. An RPAS's autonomy can range from remotely piloted, wherein an individual commands its movements, to advanced autonomy, where it makes decisions about its movement using a collection of sensors and LiDAR detectors. Drones are ideal for some of the world's most difficult undertakings since they can be flown at different altitudes and distances while being controlled remotely. They can be found assisting in the hunt for survivors after a hurricane, supporting the military and law enforcement with an aerial surveillance capability during terrorist incidents, and promoting scientific study in some of the world's most hostile climates. Even in our homes, drones provide amusement for enthusiasts and are an indispensable tool for photographers. II. BACKGROUND The popularity of unmanned aerial vehicles (UAVs), also known as drones or remotely piloted aircraft systems (RPAS), has increased recently as a result of their capacity to fly independently and collect detailed aerial data and pictures. The use of RPAS for surveillance, object detection, face tracking, lane following, and digital mapping has an array of range of applications, from military and law enforcement to commercial and recreational use. RPAS can be equipped with cameras, sensors, and other technologies that enable them to capture detailed images and data from the air. This data can be used for a variety of purposes, such as monitoring traffic, detecting objects and people, tracking movements, and creating detailed maps of terrain and structures. For example, in the military and law enforcement sectors, 149 RPAS are used for reconnaissance, surveillance, and intelligence gathering. They can be used to monitor and track potential threats, survey areas that are difficult to access, and provide real-time situational awareness to ground troops. Agricultural operations, infrastructural inspection, search and rescue missions, and environmental monitoring are just a few of the business uses for RPAS. For example, drones equipped with thermal imaging cameras can be used to detect heat signatures and identify individuals who may be lost or injured in a wilderness area. In the field of transportation, RPAS are being used for lane following and object detection in autonomous vehicles. They can help vehicles navigate complex road networks and avoid obstacles in real-time. Digital mapping is another important application of RPAS. RPAS can produce incredibly accurate maps of the landscape and built environments through the collection of aerial photography and data. These maps may be utilized for a range of tasks, including urban planning, disaster response, and environmental preservation. The usage of RPAS for monitoring, recognizing objects, face tracking methods lane following, and mapping purposes has a wide range of applications in many different industries, and it is anticipated that their use will increase over the next several years as technology develops and new uses are found. III. PROBLEM STATEMENT In this paper, we attempt to find out whether we can integrate multiple functionalities like surveillance, object detection, face tracking, lane following and digital mapping into a single remotely piloted aerial system therefore making it a versatile agent for use in various sectors like security, scientific research, defense, urban planning, law enforcement etc. We try to integrate all these features so that they can work seamlessly and complement each other's functionalities to form a complete product of high value using minimal computing resources and having less power requirements. Furthermore, we make use of a wide variety of learning algorithms to implement the aforementioned features. This makes the RPAS more intelligent, and its working can improve over time. V. SYSTEM DESIGN A quadcopter has to be able to move in three separate directions—vertically, laterally, and rotateally—in order to be able to fly. Each of them may be accomplished utilising the quadcopter's four propellers in accordance with Newton's third law. The spinning propellers force air down. Newton's third rule of motion involves pushing the air downward, just like a helicopter does. The quadcopter is propelled upward by the response, which is a force known as lift. Each propeller will provide lift, and the quadcopter's overall lift will be equal to the sum of the lifts produced by each of the four propellers. For the quadcopter to take off, the overall force of the lift must be greater than the force of gravity. When the forces of lift and gravity are equal, the quadcopter may hover in the air without moving vertically. The quadcopter travels vertically when the lift force acts straight up. But a lift can also move laterally if it operates at an angle. This is due to the lift's uneven distribution of forces, which causes lateral movement that can be both forward and backward as well as side to side. By changing the propellers' speed, lateral movement is achieved. Uneven levels of lift are produced on the two sides of the quadcopter by altering the speed of the quadcopter's two propellers on one side and/or the other. Fig 2 :Direction Of Propellor Rotation. Fig 2 :Direction Of Propellor Rotation. In comparison to the lift produced on the opposite side, the lift produced on the side with the faster spinning propellers is greater. As a result, the quadcopter moves towards the side where less lift is produced. A quadcopter's propellers also experience torque when they revolve since each propeller generates torque on its own. As a result, two propellers produce torque that operates in a clockwise direction, while two produce torque that acts in an anticlockwise direction. The forces thus cancel one another out. The quadcopter may also be made to rotate by using this torque. The anticlockwise force operating on the quadcopter will be stronger than the clockwise torque acting on it if these propellers revolve more quickly than propellers 2 and 4. The quadcopter turns anticlockwise as a consequence. Naturally, the quadcopter will rotate in a clockwise direction if propellers 2 and 4 spin more quickly than propellers 1 and 3. IV. OBJECTIVES The objectives for a project titled RPAS (Remotely Piloted Aircraft System) with the following features could include - Surveillance: Develop a system that enables the RPAS to conduct surveillance activities such as monitoring borders, traffic, wildlife, and other activities. Face Tracking: Design a system that enables the RPAS to track human faces and recognize them using machine learning algorithms. Object Detection: Develop a system that allows the RPAS to detect objects and identify them with high accuracy, using computer vision techniques and deep learning models. Lane Following: Create a system that enables the RPAS to follow designated lanes autonomously, using advanced navigation systems and sensors. Digital Mapping: Develop a system that enables the RPAS to create accurate and detailed digital maps of the surrounding environment, using LiDAR or other mapping technologies. Overall, the objective of the project is to create an RPAS that can perform a range of tasks with high precision and reliability, by integrating advanced technologies such as machine learning, computer vision, navigation systems, and mapping technologies. 150 Surveillance Module We can perform surveillance on a given area by controlling the movement of the RPAS by using either a joystick or a keyboard. The live feed from the RGB-D sensors can be viewed on a screen which is connected to the same network as the drone. Still images can be captured at any moment using a pre- programmed key on the controller device. One of the benefits of using drones for surveillance is their ability to cover large areas quickly and efficiently. Drones can be equipped with high-resolution cameras, thermal sensors, and other advanced technologies that provide real-time data and imagery. This information can be used to monitor borders, search for missing persons, monitor wildlife, and track criminal activities. This has many uses in home security, search and rescue, military operations, exploration, videography. V. SYSTEM DESIGN Two propellers push air upward while two push air downward as they rotate in opposing directions. When these pressures are 151 combined, the overall lift is zero, preventing the quadcopter from taking off. Engineers employ two distinct propeller blades to get around this. Figure shows that the propeller on the left has a higher front left edge than the other, which has a higher front right edge. Due to this design, even though the two propellers rotate in different directions, the air is really propelled in the same direction by both of them. Fig 3: Movement of RPAS. Fig 3: Movement of RPAS. Object Detection Module Object detection for RPAS is implemented using the YOLO algorithm. YOLO stands for “You Only Look Once” which recognizes and detects several objects in a picture. The class probabilities of the discovered photos are provided by the object identification process in YOLO, which is carried out as a regression problem. Convolutional neural networks (CNN) are used by the YOLO method to recognize items instantly. The approach just needs one forward propagation through a neural network to identify objects, as the name would imply. This indicates that a single algorithm run is used to do prediction throughout the full picture. Multiple class probabilities and bounding boxes are simultaneously predicted using the CNN. The YOLO algorithm works by dividing the image into N grids, each having an equal dimensional region of SxS. Each of these N grids takes part in the detection and localization of the object it contains. Correspondingly, these grids predict bounding box B coordinates relative to their cell 152 152 152 coordinates, along with the object label along with confidence value of the object being present in the cell. The following reasons make YOLO Algorithm significant: Speed, High Accuracy, Learning capabilities. coordinates, along with the object label along with confidence value of the object being present in the cell. The following reasons make YOLO Algorithm significant: Speed, High Accuracy, Learning capabilities. Fig 4: Object Detection Fig 4: Object Detection Face Tracking Module The implementation for face tracking is done by first setting up a frame and identifying a face. The identification of a face is done using the Viola-Jones method. Upon finding a face, the RPAS follows the person or the closest person among a group of people based on distance from starting point. Developed in 2001 by Paul Viola and Michael Jones, the Viola-Jones algorithm is an object-recognition framework that allows the detection of image features in real-time. Before detecting a face, the image is converted into grayscale, since it is easier to work with and there’s lesser data to process. The Viola-Jones algorithm first detects the face on the grayscale image and then finds the location on the coloured image. Viola-Jones outlines a box (as you can see on the right) and searches for a face within the box. It is essentially searching for these haar-like features. The features below show a box with a light side and a dark side, which is how the machine determines what the feature is. Sometimes one side will be lighter than the other, as in an edge of an eyebrow. Sometimes the middle portion may be shinier than the surrounding boxes, which can be interpreted as a nose. The speed of tracking depends upon the distance between the person and the RPAS. Fig 5: Face detection using Haar-like features Fig 5: Face detection using Haar-like features Fig 5: Face detection using Haar-like features Digital Mapping 153 Digital Mapping is performed using visual odometry. The use of digital mapping in remotely piloted aerial systems (RPAS) has brought about significant advancements in various fields such as urban planning, environmental management, and agriculture. 2-D digital mapping, in particular, has proven to be a valuable tool for capturing and analyzing geographic information in real-time. 2-D digital mapping involves the creation of two-dimensional maps that provide a bird's eye view of the earth's surface. This mapping technology allows for the identification and visualization of objects, terrain features, and geographic information such as streets, buildings, and land cover. RPAS equipped with high-resolution cameras and sensors can capture detailed imagery and data, which can then be processed into 2-D maps. The velocity of the drone and its location from the bounding boxes is used to determine the current location of the drone relative to the image which is used as the reference. This is used to create a digital map of the path of the drone. This map is visible on a display device and the location is outputted as relative coordinates. Used for topographic surveys, photogrammetric solutions, progress monitoring, disaster management, and agriculture and forestry. Gesture Control Gesture control involves recognizing hand gestures in real time using machine learning techniques and computer vision and utilizing them to control the movement of the RPAS. A machine learning-based software library called MediaPipe Gesture Recognizer has been developed by Google that can be used with a webcam or other camera to recognize different hand gestures in real-time. The MediaPipe Gesture Recognizer analyses video data using a neural network to recognize various hand gestures based on the user's finger and palm movements. Convolutional neural networks (CNNs) and recurrent neural networks (RNNs) are used in combination by MediaPipe Gesture Recognizer to analyze video data and identify hand motions. Fig 6: Recognition of hand gesture and display of hand landmarks Fig 6: Recognition of hand gesture and display of hand landmarks Fig 6: Recognition of hand gesture and display of hand landmarks 154 Fig 7: Various hand gestures for RPAS control Fig 7: Various hand gestures for RPAS control The RNN is used to recognize a particular hand gesture by looking at the temporal sequence of the hand motions, while the CNN is used to extract information from the video frames and locate the hand. Real-time hand gesture recognition is possible with the MediaPipe Gesture Recognizer task, which also displays the results of the recognition as well as the hand landmarks of the hands that were found. This task receives either static data or a continuous stream and uses an ML model to act on image data. The task yields hand landmarks in world coordinates, hand landmarks in picture coordinates, and types of hand gestures. After recognizing a hand gesture, it can be used to control the movements of the RPAS. For instance, pointing up with index finger to increase altitude, pointing down to decrease altitude, closed fist to stop movement, pointing right or left to move right or left respectively and “OK” hand gesture to land the RPAS. It has several use cases. Robotics is one area where the MediaPipe library is put to use for gesture control of drones. Building hand gesture recognition models for real-time drone control, researchers and developers employ MediaPipe. Applications for this technology include surveillance, inspection, and search and rescue operations. VIII. DISCUSSION RPAS can cover a large area in a short span of time, making them more efficient than traditional methods of data collection. This can be particularly useful for tasks such as crop monitoring, land surveying, and infrastructure inspections. By using RPAS, data can be collected more quickly and accurately, allowing for faster decision-making and better resource allocation. RPAS can provide highly accurate and precise data, making them useful for tasks that require a high degree of accuracy, such as surveying and mapping. The data collected by RPAS can be used to create detailed 3D models, which can be used for planning and analysis purposes. Additionally, RPAS can be equipped with high- resolution cameras and sensors, allowing for detailed data collection and analysis. RPAS can be used to perform tasks in hazardous or hard-to- reach areas, such as inspecting power lines or pipelines, without putting human operators at risk. This can reduce the risk of accidents and injuries, while also increasing the safety and efficiency of operations. RPAS can be a cost-effective alternative to traditional methods of data collection, such as manned aircraft or ground-based surveys. RPAS are generally less expensive to operate than manned aircraft, and they can be used for a wider range of tasks. Additionally, they require less manpower to operate, reducing the overall cost of operations. The use of RPAS is subject to regulations and restrictions that vary by country and can be complex and time-consuming to navigate. Operators must obtain appropriate licenses and permits, and adhere to strict guidelines on where and how RPAS can be flown. Failure to comply with these regulations can result in fines and legal consequences. RPAS can be used to collect data on individuals without their knowledge or consent, raising privacy concerns. For example, RPAS equipped with cameras could be used for surveillance purposes, raising questions about the appropriate use of the technology. To address these concerns, regulations have been introduced in many countries to restrict the use of RPAS for privacy-invasive purposes. RPAS operations can be impacted by weather conditions, such as high winds or precipitation, which can limit their usefulness. For instance, high winds can cause RPAS to lose control, while precipitation can damage sensitive electronics. This means that RPAS may not be suitable for certain tasks in certain weather conditions. VIII. DISCUSSION RPAS technology is still evolving and can have technical limitations, such as limited battery life, which can limit their capabilities and potential applications. VII. RESULTS Fig 8: Surveillance (with movement parameters) Fig 9: Object detection(person) Fig 9: Object detection(person) Fig 9: Object detection(person) Fig 8: Surveillance (with movement parameters) 155 Fig 10: Object detection(Laptop and mouse) Fig 11: Face tracking Fig 12: Digital mapping Fig 13: Gesture control(Stop) Fig 10: Object detection(Laptop and mouse) Fig 11: Face tracking Fig 10: Object detection(Laptop and mouse) Fig 11: Face tracking Fig 10: Object detection(Laptop and mouse) Fig 11: Face tracking Fig 11: Face tracking Fig 11: Face tracking Fig 10: Object detection(Laptop and mouse) Fig 12: Digital mapping Fig 13: Gesture control(Stop) Fig 12: Digital mapping Fig 13: Gesture control(Stop) Fig 12: Digital mapping Fig 13: Gesture control(Stop) Fig 13: Gesture control(Stop) Fig 14: Gesture control(up) Fig 14: Gesture control(up) 156 REFERENCES 1. Anish Bhat, Bhanuranjan S B, K G Lakshmi Narayan, Manjunath Bharadwaj V, & Lavanya N L. (2023). Survey On Object Detection, Face Tracking, Digital Mapping and Lane Following For Remotely Piloted Aerial Systems (RPAS) . International Journal of Human Computations & Intelligence, 2(2), 94–105. https://doi.org/10.5281/zenodo.7900522 1. Anish Bhat, Bhanuranjan S B, K G Lakshmi Narayan, Manjunath Bharadwaj V, & Lavanya N L. (2023). Survey On Object Detection, Face Tracking, Digital Mapping and Lane Following For Remotely Piloted Aerial Systems (RPAS) . International Journal of Human Computations & Intelligence, 2(2), 94–105. https://doi.org/10.5281/zenodo.7900522 1. Anish Bhat, Bhanuranjan S B, K G Lakshmi Narayan, Manjunath Bharadwaj V, & Lavanya N L. (2023). Survey On Object Detection, Face Tracking, Digital Mapping and Lane Following For Remotely Piloted Aerial Systems (RPAS) . International Journal of Human Computations & Intelligence, 2(2), 94–105. https://doi.org/10.5281/zenodo.7900522 2. Kim, T. Y., & Kim, J. N. (2020). Implementation of unmanned aerial system for surveillance mode. International Conference on Information and Communication Technology Convergence. https://doi.org/10.1109/ictc49870.2020.9289181 3. Tayara, H., Soo, K. G., & Lu, N. (2018). Vehicle Detection and Counting in High-Resolution Aerial Images Using Convolutional Regression Neural Network. IEEE Access, 6, 2220–2230. htt //d i /10 1109/ 2017 2782260 2. Kim, T. Y., & Kim, J. N. (2020). Implementation of unmanned aerial system for surveillance mode. International Conference on Information and Communication Technology Convergence. https://doi.org/10.1109/ictc49870.2020.9289181 2. Kim, T. Y., & Kim, J. N. (2020). Implementation of unmanned aerial system for surveillance mode. International Conference on Information and Communication Technology Convergence. https://doi.org/10.1109/ictc49870.2020.9289181 3. Tayara, H., Soo, K. G., & Lu, N. (2018). Vehicle Detection and Counting in High-Resolution Aerial Images Using Convolutional Regression Neural Network. IEEE Access, 6, 2220–2230. https://doi.org/10.1109/access.2017.2782260 3. Tayara, H., Soo, K. G., & Lu, N. (2018). Vehicle Detection and Counting in High-Resolution Aerial Images Using Convolutional Regression Neural Network. IEEE Access, 6, 2220–2230. https://doi.org/10.1109/access.2017.2782260 4. Sambolek, S., & Ivašić-Kos, M. (2021). Automatic Person Detection in Search and Rescue Operations Using Deep CNN Detectors. IEEE Access, 9, 37905–37922. https://doi.org/10.1109/access.2021.3063681 4. Sambolek, S., & Ivašić-Kos, M. (2021). Automatic Person Detection in Search and Rescue Operations Using Deep CNN Detectors. IEEE Access, 9, 37905–37922. https://doi.org/10.1109/access.2021.3063681 5. Saetchnikov, I., Tcherniavskaia, E. A., & Skakun, V. S. (2021b). Object Detection for Unmanned Aerial Vehicle Camera via Convolutional Neural Networks. IEEE Journal on Miniaturization for Air and Space Systems, 2(2), 98– 103. https://doi.org/10.1109/jmass.2020.3040976 5. Saetchnikov, I., Tcherniavskaia, E. A., & Skakun, V. S. (2021b). REFERENCES Object Detection for Unmanned Aerial Vehicle Camera via Convolutional Neural Networks. IEEE Journal on Miniaturization for Air and Space Systems, 2(2), 98– 103. https://doi.org/10.1109/jmass.2020.3040976 6. Karahan, M., Kurt, H., & Kasnakoglu, C. (2020). Autonomous Face Detection and Tracking Using Quadrotor UAV. 2020 4th International Symposium on Multidisciplinary Studies and Innovative Technologies (ISMSIT). https://doi.org/10.1109/ismsit50672.2020.9254469 7. Çetinkaya, O., Sandal, S., Bostanci, E., Güzel, M. S., Osmanoglu, M., & Kanwal, N. (2019). A Fuzzy Rule Based Visual Human Tracking System for Drones. 2019 4th International Conference on Computer Science and Engineering (UBMK) https://doi org/10 1109/ubmk 2019 8907104 (UBMK). https://doi.org/10.1109/ubmk.2019.8907104 8. Surinta, O., & Khruahong, S. (2019). Tracking People and Objects with an Autonomous Unmanned Aerial Vehicle using Face and Color Detection. ECTI DAMT-NCON. https://doi.org/10.1109/ecti-ncon.2019.8692269 9. Rabah, M., Rohan, A., Mohamed, S. a. M., & Kim, S. (2019). Autonomous Moving Target-Tracking for a UAV Quadcopter Based on Fuzzy-PI. IEEE Access, 7, 38407–38419. https://doi.org/10.1109/access.2019.2906345 10. Morbale, Naregalkar, Singh, A., Dwivedi, P., & Mathur, R. (2022). “Quadcopter Drone with Face Recognition.” IJSERM. 11. Kumar, S. S., Ahmed, S. T., Vigneshwaran, P., Sandeep, H., & Singh, H. M. (2021). Two phase cluster validation approach towards measuring cluster quality in unstructured and structured numerical datasets. Journal of Ambient Intelligence and Humanized Computing, 12, 7581-7594. 12. Ahmed, S. T., Kumar, V. V., Singh, K. K., Singh, A., Muthukumaran, V., & Gupta, D. (2022). 6G enabled federated learning for secure IoMT resource recommendation and propagation analysis. Computers and Electrical Engineering, 102, 108210. 13. Shen, Q., Jiang, L., & Xiong, H. (2018). Person Tracking and Frontal Face Capture with UAV. International Conference on Communication Technology. https://doi.org/10.1109/icct.2018.8600068 14. Feng, Y., Yu, S., Peng, H., Li, Y., & Zhang, J. (2021). Detect Faces Efficiently: A Survey and Evaluations. IEEE Transactions on Biometrics, Behavior, and Identity Science, 4(1), 1–18. https://doi.org/10.1109/tbiom.2021.3120412 15. Nguyen, T. T., Mann, G. K. I., & Gosine, R. G. (2014). Vision-based qualitative path-following control of quadrotor i l hi l I i l C f U d Ai f S h //d i /10 1109/i 2014 6842281 14. Feng, Y., Yu, S., Peng, H., Li, Y., & Zhang, J. (2021). Detect Faces Efficiently: A Survey and Evaluations. IEEE Transactions on Biometrics, Behavior, and Identity Science, 4(1), 1–18. https://doi.org/10.1109/tbiom.2021.3120412 15. Nguyen, T. T., Mann, G. K. I., & Gosine, R. G. (2014). Vision-based qualitative path-following control of quadrotor aerial vehicle. International Conference on Unmanned Aircraft Systems. https://doi.org/10.1109/icuas.2014.6842281 16. IX. CONCLUSION In this paper, we try to zero in on an effective way to integrate multiple features into an aerial system which is either remotely controlled or partially automated. The features are namely surveillance, face tracking, object detection, lane following and digital mapping. We first look at multiple methods to perform surveillance using RPAS. Then we explore the various algorithms to perform object detection and their dependencies. This is an ambitious and challenging endeavour that requires a multidisciplinary approach. By integrating advanced technologies such as machine learning, computer vision, navigation systems, and mapping technologies, the RPAS can perform a range of tasks with high precision and 157 reliability. The developed system can provide valuable support to various fields, such as security, transportation, wildlife conservation, and urban planning, by enhancing the efficiency and accuracy of data collection and analysis. Overall, the project has the potential to contribute to the development of innovative RPAS systems that can meet the increasing demands for advanced aerial platforms in various applications. REFERENCES Zhou, H., Kong, H., Wei, L., Creighton, D., & Nahavandi, S. (2015). Efficient Road Detection and Tracking for Unmanned Aerial Vehicle. IEEE Transactions on Intelligent Transportation Systems, 16(1), 297–309. https://doi.org/10.1109/tits.2014.2331353 158 17. Oliveira, T., Aguiar, A. P., & Encarnação, P. (2016). Moving Path Following for Unmanned Aerial Vehicles With Applications to Single and Multiple Target Tracking Problems. IEEE Transactions on Robotics, 32(5), 1062–1078. https://doi.org/10.1109/tro.2016.2593044 18. Ahmed, S., Guptha, N., Fathima, A., & Ashwini, S. (2021, January). Multi-View Feature Clustering Technique for Detection and Classification of Human Actions. In Proceedings of the First International Conference on Advanced Scientific Innovation in Science, Engineering and Technology, ICASISET 2020, 16-17 May 2020, Chennai, India. 19. Ahmed, S. T., Guptha, N. S., Lavanya, N. L., Basha, S. M., & Fathima, A. S. (2022). IMPROVING MEDICAL IMAGE PIXEL QUALITY USING MICQ UNSUPERVISED MACHINE LEARNING TECHNIQUE. Malaysian Journal of Computer Science, 53-64. f p 20. Muthukumaran, V., Vasudevan, S., & Siddiqha, S. A. (2023). Secure Public Key Cryptosystem for in Smart City using Algebraic Structure. International Journal of Human Computations & Intelligence, 2(1), 20-25.
https://openalex.org/W2087780959	https://zenodo.org/records/1258401/files/article.pdf	English	null	Computer simulation studies of high-energy collision cascades	Nuclear instruments and methods in physics research. Section B, Beam interactions with materials and atoms/Nuclear instruments & methods in physics research. Section B, Beam interactions with materials and atoms	1,992	public-domain	4,239	1. Introduction varied as E 2 and dominated the computation above a few keV. A better procedure uses the fact that a nearby lattice site is associated with each atom in a MARLOWE cascade. A so-called "hashing" algorithm is used to find such sites in the same way used for vacant ones: the lattice vector to a site defines a location in a table. If the site is vacant or associated with a stopped nonlattice atom, a pointer to the site or atom occupies the place indicated; otherwise, it is marked as empty. Separate tables are maintained for vacancies and atoms. With large enough tables and with provisions for conflicts between different sites for places in them, most searching is eliminated, the pro- gram execution time is proportional to E 0, and high- energy cascade calculations are not inhibited. Computer simulation in the binary collision approxi- mation has long been used to study the development of collision cascades in crystals. Previous work with the MARLOWE program [1] showed how such collisions may be ordered properly in time and how nonlinear effects in cascade development may be approximated by letting .'~lready-stopped cascade atoms become tar- gets in later collisions, but the work was limited to initial kinetic energies in the low kilovolt region by a need for very frequent searches of the entire list of atoms in the developing cascades. Because of this limitation, the effects of nonlinearities, though clearly evident, were modest and there were incentives for further study. This paper sketches recent changes in the MARLOWE model, including a new algorithm for locating pcltential targets and other refinements, allow- ing calcula~tions to be extended to initial kinetic ener- gies of 100 keV or more in both Cu and Au. Projectiles in MARLOWE are followed as long as their kinetic energies exceed a value E c. To be dis- placed, lattice targets must surmount a binding energy E b if the projectile continues moving, or E~, if it stops, and still have kinetic energy > E c. Originally, a nonlat- tice target receiving kinetic energy in a collision was always followed and no binding energy was used. To be redisplaced in the revised model, a nonlattice target must surmount an energy E~ and still have kinetic energy > E c. If redisplacements do not occur, encoun- ters with nonlattice targets are classed as subthreshold. * Research sponsored by the Division of Materials Science, U.S. Department of Energy, under contract no. DE-AC05- 84OR21,100 with Martin Marietta Energy Systems, Inc. Computer simulation studies of high-energy collision cascades * Mark T. Robinson Solid State Dicision, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, TN 37831-6032, USA Mark T. Robinson A modified binary-collision-approximation algorithm allowing the proper order of the collisions in time was used to study cascades in Cu and Au at primary kinetic energies up to 100 keV. Nonlinearities were approximated by letting already-stopped cascade atoms become targets in later collisions, using an improved method of locating potential targets to extend the calculations to energies much higher than heretofore. Beside the effect of the approximate nonlinearity, the effect of thermal disorder in the targets was examined. Target redisplacemen!s reduce the damage in Cu by 3% at most, but in Au they reduce it by amounts up to 20% at 1O0 keV. Thermal disorder is also important: by disrupting crystal effects, the damage is reduced significantly. 1. Introduction events: some atoms once regarded as redisplaced [1] are now in this group. The residual kinetic energy of a nonlattice target when it stops is ignored in determin- ing its new kinetic energy. Nuclear Instruments & Methods in Physics Research sechon B Nuclear Instruments & Methods in Physics Research sechon B Nuclear Instruments and Methods in Physics Research B67 (1992) 396-400 North-Holland Nuclear Instruments and Methods in Physics Research B67 (1992) 396-400 North-Holland 0168-583X/92/$05.00 © 1992 - Elsevier Science Publishers B.V. All rights reserved 2. The computational model Most a~spects of the MARLOWE model are de- scribed in detail elsewhere [1-4]. Potential nonlattice (stopped cascade atom) targets used to be found by scanning a table of cascade particles exhaustively at every collision: since the table length and the number of times it was scanned were each roughly proportional to the initial kinetic energy E0, the time for this task Records are now kept of the times at which redis- placed atoms initially stop as well as of the times at which they are redisplaced: this alters the counting of atoms in motion in a cascade. Records are kept of the energy transferred in redisplacement events and in subthreshold encounters with nonlattice atoms. For the 0168-583X/92/$05.00 © 1992 - Elsevier Science Publishers B.V. All rights reserved M.T. Robinson / Computer simulation of high-energy collision cascades 397 producing new or redisplaced recoils. Fig. la shows {Ek(t)) for 20 keV cascades in Au, where Ek(t) is the kinetic energy of the moving recoils at time t. When redisplacements are allowed in a static target, (Ek(t)) is unaffected below about 0.18 ps, but then falls faster than otherwise. In both static calculations, there is a pronounced tail extending to long times, which repre- sents the slow decrease of kinetic energy along linear collision sequences (LCSs). If thermal displacements are added, long LCSs do not occur, eliminating the tail. Moreover, energy dissipation from the cascade is increased at all times. Only the 1000 K results are shown in fig. la; those for other temperatures fall between this line and the static case. producing new or redisplaced recoils. Fig. la shows {Ek(t)) for 20 keV cascades in Au, where Ek(t) is the kinetic energy of the moving recoils at time t. When redisplacements are allowed in a static target, (Ek(t)) is unaffected below about 0.18 ps, but then falls faster than otherwise. In both static calculations, there is a pronounced tail extending to long times, which repre- sents the slow decrease of kinetic energy along linear collision sequences (LCSs). If thermal displacements are added, long LCSs do not occur, eliminating the tail. Moreover, energy dissipation from the cascade is increased at all times. Only the 1000 K results are shown in fig. la; those for other temperatures fall between this line and the static case. present work, a special analysis evaluated projectile kinetic energy spectra at intervals during cascade de- velopment. 2. The computational model Cascades were generated in Cu and Au for E o from 0.1 to 100 keV. Up to 20 keV, samples of 1000 cas- cades were generated; 500 were evaluated at 50 keV and 250 at 100 keV. Quantities averaged over sets of cascades are enclosed in { ~. In each case, E~ = E~ = 0.1E b, E b = E c, and Ec = 3.50 eV in Cu and 3.81 eV in Au. The fcc lattice constant a 0 was 0.36]5 nm in Cu and 0.4078 nm in Au. The MoliEre potential was used with screening lengths of 7.38 pm in Cu and 7.50 pm in Au. Collisions with impact parameters < 0.62a 0 were evaluated. "Local" inelastic energy losses [3] were scaled to the LSS model [5]. Thermal disorder in the targets was described by the Debye model [6] with 0 D = 315 K in Cu and 175 K in Au. Calculations with redisplacements were made for static targets and for temperatures of 0, 300, and 1000 K. Static calculations were also made without redisplacements. The colli- sions were time-ordered with a time interval of 1 fs. Calculations were carried out on a Data General AViiON TM 300 workstation and required up to several hours per case. Figs. lb-ld show recoil energy spectra in static calculations without redisplacements at three times. The spectra for other cases are similar. It is convenient to plot f( E, t)= E 2 p( E, t)against l/E, where p( E, t) dE is the number of recoils at time t with kinetic energy between E and E + dE. In the hard-core ap- proximation in structureless media, f(E, t) is a con- stant [7]. Early on, most recoils have high kinetic ener- gies and there is a peak in the spectrum which moves to lower energies and spreads out as time advances. At long times, the spectrum approaches hard-core behav- ior at low recoil energies. The spectrum in fig. ld has two noteworthy features. One is a small peak at the high-energy end of the spectrum which persists for a rather long time even with thermal disorder: it repre- sents channeled atoms which lag behind most other recoils in slowing down. The other is a small peak near 7 eV, marked by an arrow, which also persists for a 3. The temporal development of cascades At 7 eV, Au atoms require about 0.11 ps to move a nearestmeighbor distance, accounting for the slow dissipation. long period: it eventually dissipates slowly, but is ab- sent when thermal disorder is included. It represents long LCSs, along which energy dissipation is rather slow. At 7 eV, Au atoms require about 0.11 ps to move a nearestmeighbor distance, accounting for the slow dissipation. p Fig. 2a shows (N(t)), the mean number of atoms in motion in the same 20 keV cascades in Au. With redisplacements allowed, (N(t)) is reduced and falls more rapidly at longer times than otherwise. This be- havior reflects the added loss of energy from the cas- cade because of the encounters of recoils with nonlat- rice target: atoms. In the static calculations, a persistent tail due Ito LCSs is evident. If thermal disorder is added, (N(t)) drops further at long times, removing the tail almost entirely, but the number of moving atoms increases at short times. This is a result of the reduction of blocking effects by thermal disorder: many encounters in which moderate amounts of energy are transferred replace a smaller number of very hard encounters. Fig. 2b shows histograms of the times at which recoils stop which are later redisplaced and of the times at which they are redisplaced. The centroids of these distributions are about 70 fs apart, but distri- butions ot! the delay times show that half the atoms are redisplaeed < 60 fs after they stop, many of them almost immediately, while a few are delayed for > 300 fs. Distributions of the delay times are independent of initial kinetic energy and are unaffected by thermal disorder, it should be noted that the bulk of the redisplacements occur while the largest numbers of recoils are in motion. 4. Overall cascade properties At the end of a MARLOWE computation, the defects produced are arranged in Frenkel pairs and sorted into classes [1,2]. Only so-cal.:d distant pairs are regarded as permanent displacements: pairs of other classes are unstable and annihilate promptly. The num- ber of pairs may be discussed in terms of the familiar "damage energy" model of radiation damage [9]: the damage energy E is E o less the energy lost in electron excitation. The number of Frenkel pairs produced in a cascade is u = E/Q, E > Q, a displacement energy. In the well-known model of Kinchin and Pease [10], for example, Q = 2E u, with E u the displacement thresh- old, about 29 and 43 eV for Cu and Au, respectively [11]. Fig. 3 shows the effective displacement energy Q = (E)/(u), as a function of E 0, derived from calcu- lations in both Cu and Au. The MARLOWE results for both metals are roughly what is expected from the Kinchin-Pease model, namely ~ 58 eV for Cu and ~ 86 eV for Au. It must be emphasized that the only explicit displacement threshold MARLOWE is E¢ + E h, 7.02 eV for Cu and 7.62 eV for Au. Z o 700 _z 600 ~ 200 ~ 100 g o 3" ~ T "l T-T- (b) 20 keY CASCADES IN STATIC Au ~ ,2 t ,r=,~--.EO,SP~.C ....... ME _.m -: ~ ..... TIME OF STOPPING ! \ ; '.°t- : i BEFORE t ~ g-I- r ~ .Eo,sP..CE .... i ~;-\ ~ o.41- .i r j L h o. I \".. \ o o.1 0.2 0.3 0.4 o.s 0.6 t "~ ''- X t. TIME (pS) / ~ ",. ~. (a) 20keVCASCADES IN Au / ~".. ~ ~ STATIC. NO REDISPLACEMENTS ] ~ "... ~ ........ STATIC. REDISPLACEMENTS 0.1 0.2 0.3 0.4 0.5 0.6 0.7 t, TIME (ps) Fig. 2. (a) The mean number of atoms in motion as a function of time for 20 keV cascades in Au. (b) 'The times of occurrence of redisplacements in 20 keV cascades in static Au and the times at which recoils stop which are to be redisplaced later. Z o 700 _z 600 ~ 200 ~ 100 g o 3" ~ T "l T-T- (b) 20 keY CASCADES IN STATIC Au ~ ,2 t ,r=,~--.EO,SP~.C ....... ME _.m -: ~ ..... TIME OF STOPPING ! 3. The temporal development of cascades The initial kinetic energy of the primary recoil is dissipated partly in exciting target electrons, partly in subthreshold encounters in which the target atoms receive kinetic energy < E c, and partly in encounters ~ 25 ,,z, 20 O_ v 15 ,~ 5 I i i ~ E (OV~ i i..,,..... E (oV) i ~3t<o). ;1~' ~"'1°"' e ? ;~t [/'~"~"~' ~...10....s ? 'i] tV.2 ~ , 0 S0 100 150 200 250 41 ............... I \\ (a) 20 ksV CASCADES IN Ag '~ I / ~ STATIC, '~ NO REDISPLACEMENTS ~ 2 t ~ _ _ J ~ ........ STATIC, =" / . . ~ ,"-- "~ REDISPLACEMENTS u; 11" ,,,,r- t 1 "~% -- looo~ = [ / ' I "~ REDISPLACEMENTS 0 I~" , I I I I I , 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 t, TIME (lOS) Fig. 1. (a) The mean cascade kinetic energy, {Eg(t)), as a function of time for 20 keV cascades in Au. (b)-(d) The kinetic energy spectra of recoils in static Au without redisplacements at 0.04, 0.16, and 0.32 ps. VJ. CASCADES, D AMAGE, RANGES ~ 25 ,,z, 20 O_ v 15 ,~ 5 I i i ~ E (OV~ i i..,,..... E (oV) i ~3t<o). ;1~' ~"'1°"' e ? ;~t [/'~"~"~' ~...10....s ? 'i] tV.2 ~ , 0 S0 100 150 200 250 41 ............... I \\ (a) 20 ksV CASCADES IN Ag '~ I / ~ STATIC, '~ NO REDISPLACEMENTS ~ 2 t ~ _ _ J ~ ........ STATIC, =" / . . ~ ,"-- "~ REDISPLACEMENTS u; 11" ,,,,r- t 1 "~% -- looo~ = [ / ' I "~ REDISPLACEMENTS 0 I~" , I I I I I , 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 t TIME (lOS) M.T. Robhzson / Computersimulation of high-energy coUision cascades M.T. Robhzson / Computersimulation of high-energy coUision cascades 398 Cascades in Au show similar behavior at other energies, with the effects of redisplacements becoming greater at higher energies. In Cu, on the other hand, the effects are much smaller. Both results are consis- tent with theoretical expectations [8] that cascade non- linearities are more important at high energies and in high-Z targets. long period: it eventually dissipates slowly, but is ab- sent when thermal disorder is included. It represents long LCSs, along which energy dissipation is rather slow. 4. Overall cascade properties \ ; '.°t- : i BEFORE t ~ g-I- r ~ .Eo,sP..CE .... i ~;-\ ~ o.41- .i r j L h o. I \".. \ o o.1 0.2 0.3 0.4 o.s 0.6 t "~ ''- X t. TIME (pS) / ~ ",. ~. (a) 20keVCASCADES IN Au / ~".. ~ ~ STATIC. NO REDISPLACEMENTS ] ~ "... ~ ........ STATIC. REDISPLACEMENTS 0.1 0.2 0.3 0.4 0.5 0.6 0.7 t, TIME (ps) Fig. 2. (a) The mean number of atoms in motion as a function of time for 20 keV cascades in Au. (b) 'The times of occurrence of redisplacements in 20 keV cascades in static Au and the times at which recoils stop which are to be redisplaced later. M. T. Robinson / Computer simulation of high-energy collision cascades 39 A 70 A V ~ 5O 2, <WV 40 0.1 Fig. 3. The effective displacement energy, (E)/(v), as a 100 I-'.~"" ." t> ~. ~.3.n-- .-o- -- -~ ---w- . ...... o'"'":~-~ STATIC, NO DISPLACEMENTS J ---0--, STATIC. REDISPLACEMENTS J ---B-- 0 K. REDISPLACEMENTS -J ---D-- 1000 K, FIEDISPLACEMENTS / 43 ~ ~ ~ O. ~----'C2-- ------.O -- ---- .~_.o.~_o-~---~-:: ....... ~---~---} ~ , o ~ - . - - = - - - ~ . . . . ~ - ..... Q ........ : .......... : ........ - :~lr" - ~ cu j I I ! 1 10 100 E 0, INITIAL PRIMARY RECOIL KINETIC ENERGY (keV) function of the initial kinetic energy for cascades in Cu and Au. 399 Fig. 3. The effective displacement energy, (E)/(v), as a E 0, INITIAL PRIMARY RECOIL KINETIC ENERGY (keV) function of the initial kinetic energy for cascades in Cu and Au. In both metals, static calculations without redis- placements show rather constant values of Q except in regions near 0.35 keV in Cu and 1.25 keV in Au, where Q is significantly higher (that is, (v) is significantly lower) than at other energies. When redisplacements arc added to the static model, Q increases slightly in Cu and substantially in Au, but the regions of maxi- mum Q persist. When thermal disorder is introduced, however, the maxima are removed, showing them to he associated with lattice effects, mainly the formation of long LCSs. 4. Overall cascade properties The smaller cascade volume increases the concentra- tion of nonlattice targets and, therefore, the number of redisplacements show some evidence of the proximity of the threshold near 0.1 keV; similar behavior may be developing in the 0 K data in the same region. No such behavior is seen in Cu, where Q is well below 100 eV. Another way of looking at the importance of redisplacemcnts in cascade development is given in fig. 4a, which shows the ratio of (p), the mean number of redisplacements per cascade, to (v). In Cu, the ratio is small (~ 0.1) and essentially independent of E o. In Au, on the other hand, it is ~arger and increases almost linearly with E 0. Above about 30 keV, there are more redisplacements than displacements in Au. The picture which emerges from these calculations is the following. As a collision cascade develops, stopped atoms accumulate until there are enough of them to supply targets for still-moving recoils. During the time when the number of recoils is the largest, collisions with nonlattice targets lead to redisplace- ments. The smallness of the delay between the stop- ping of a recoil and its redisplacement indicates that there is a very high density of recoils during this phase, but further study is required to analyze this situation in more detail. As E0 rises, the number of potential nonlattice targets rises also, as does the number of moving recoils in each energy interval. The rate of such encounters depends on the product of these densities. Most encounters with nonlattice targets involve only low-energy recoils, but the highest energy at which such events occur rises slowly with E0. Fig. 4b shows the mean maximum kinetic energy at which redisplacements occur in cascades in both met- als. In all cases, this quantity rises with energy, varying roughly as E ~/2. The spectrum of initial redisplace- ment energies is strongly peaked near E c. Although there are occasional redisplaced atoms which carry 100 eV or more, they are quite rare, especially in Cu, and do not influence overall cascade properties signifi- cantly. Finally, there seem not to be any "local" struc- tural diffelrences in cascades caused by the redisplace- ment process. That is, the distribution of Frenkel-pair separation distances was not altered when redisplace- ments were included. 4. Overall cascade properties In fact, defining "long" LCSs to be those with three or more members, only above about 0.25 keV in Cu and about 0.7 keV in Au does essentially every cascade have at least one such sequence. In the regions ,'-" "he maxima, the LCSs are more efficient in carrying kinetic energy out of the cascade than they are at other energies. At lower energies, the number of LCSs is reduced; at higher energies, their lengths are limited by defocusing. This behavior is not affected by the redisplacement of cascade atoms. The much greater importance of redisplacements in Au is evident in fig. 3: Q increases by ~ 16% at 10 keV and ~ 19% at 100 keV, while in Cu it increases by only ~ 3% at each energy. Thermal disorder further increases Q in both metals; again the effects are much smaller in Cu than in Au. The 1000 K data from Au CASCADES WITH REDISPLACEMENTS: 1.0 1 10 100 STATIC ~ ~ _ 1000K I (b) r/j'c. ° ,o~J" , 1 10 100 E 0 INITIAL PRIMARY RECOIL KINETIC ENERGY (keY) CASCADES WITH REDISPLACEMENTS: 1.0 1 10 100 STATIC ~ ~ _ 1000K I (b) r/j'c. ° ,o~J" , 1 10 100 E 0 INITIAL PRIMARY RECOIL KINETIC ENERGY (keY) Fig. 4. (a) The number of redisplacements per Frenkel pair as a function of the initial kinetic energy for cascades in Cu and Au. (b) The maximum rcdisplacement energies in the same cascades. VI. CASCADES, DAMAGE, RANGES E 0 INITIAL PRIMARY RECOIL KINETIC ENERGY (keY) ber of redisplacements per Frenkel pair as a function of the initial kinetic energy for cascades in Cu and Au. (b) The maximum rcdisplacement energies in the same cascades. Fig. 4. (a) The number of redisplacements per Frenkel pair as a function of the initial kinetic energy for cascades in Cu and Au. (b) The maximum rcdisplacement energies in the same cascades. Fig. 4. (a) The number of redisplacements per Frenkel pair as a function of the initial kinetic energy for cascades in Cu and Au. (b) The maximum rcdisplacement energies in the same cascades. VI. CASCADES, DAMAGE, RANGES VI. CASCADES, DAMAGE, RANGES M.T. Robinson / Computer simulation of high-energy collision cascades 400 hand, the volume decreases by about 13% at 100 keV. References [1] M.T. Robinson, Nucl. Instr. and Meth. B48 (1990) 408. [2] M.T. Robinson, Phys. Rev. B40 (1989) 10717. [3] O.S. Pen and M.T. Robinson, Nucl. Instr. and Meth. 132 (1976) 647. [4] M.T. Robinson, User's Guide to MARLOWE (Version 13), unpublished; this version of the program will be available from the Radiation Shielding Information Cen- ter, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, TN 37831-6362, USA, in early 1992. 4. Overall cascade properties Finally, it must be noted that at the highest energies in Au, the number of encounters between recoils and nonlattice targets is great enough to suggest that fur- ther nonlinearities, involving collisions between low-en- ergy moving recoils really need to be included in the model to obtain a more complete picture. A few calculations were made to test the sensitivity of the resuiits to the value of E¢. It is easily deduced in the hard-core approximation that the number of stopped cascade atoms and the number of low-energy recoils are both inversely proportional to E c, so the frequency of encounters between them may be ex- pected to vary as E~ -2 and this rule proves to describe high-energy cascades in Au fairly well. Both the num- ber of redisplacements and the number of encounters with nonlattice targets show this behavior. 5. Discussion Encounters between recoil atoms and nonlattice target atoms influence cascade development partly through tl~eir role in energy dissipation. The differ- ences between the static calculations with and without redisplacements can be analyzed in this manner: the damage energy is modified by excluding energy trans- ferred to nonlattice targets and a modified effective threshold is obtained. This agrees well with the value obtained without the nonlinearity in Cn. The agree- ment for Au is less good, suggesting that other factors are also significant. One of these factors is the cascade volume, of which one measure is the total number of lattice sites visited by the recoils. The cascade volume decreases when redisplacements are allowed, but in Cu by only about 5% at 100 keV. In Au, on the other Ridge, TN 37831-6362, USA, in early 1992. [5] J. Lindhard, M. Scharff and H.E. Schi¢tt, [5] J. Lindhard, M. Scharff and H.E. Schi¢tt, K. Dan. Vi- densk. Selsk. Mat. Fys. Medd. 33 (14) (1963). densk. Selsk. Mat. Fys. Medd. 33 (14) (1963). [6] M. Blackman, in: Handbuch der Physik, vol. 7, part 1 (Springer, Berlin, 1955) p. 192. [7] M.T. Robinson, Philos. Mag. 12 (1965) 145. [8] See, e.g.: H.H. Andersen, Nucl. Instr. and Meth. BI8 (1987) 321. [9] See, e.g.: M.J. Norgen, M.T. Robinson and I.M. Torrens 5 5 [9] See, e.g.: M.J. Norgen, M.T. Robinson and I.M. Torrens, Nucl. Eng. Des. 3~ (1975) 50. [ ] , g g , , Nucl. Eng. Des. 3~ (1975) 50. [10] G.H. Kinchin and R.S. Pease, Rep. Prog. Phys. 18 (1955) 1. Nucl. Eng. Des. 3~ (1975) 50. Nucl. Eng. Des. 3 (1975) 50. [10] G.H. Kinchin and R.S. Pease, Rep. Prog. Phys. 18 (1955) 1 g ( ) [10] G.H. Kinchin and R.S. Pease, Rep. Prog. Phys. 18 (1955) 1. [11] P. Lucasson, in: Fundamental Aspects of Radiation Damage in Metals, vol. 1, eds. M.T. Robinson and F.W. Young Jr. (U.S. Energy Research and Development Ad- ministration Report CONF-751006, 1975) p. 42.
https://openalex.org/W2996570483	https://www.macrothink.org/journal/index.php/ijafr/article/download/15319/12515	English	null	Fourth Quarter Earnings Volatility: Case of Firms Listed in DFM	International journal of accounting and financial reporting	2,019	cc-by	7,871	International Journal of Accounting and Financial Reporting ISSN 2162-3082 2019, Vol. 9, No. 4 Fourth Quarter Earnings Volatility: Case of Firms Listed in DFM Nadia Sbei Trabelsi American University in Dubai, Dubai, UAE E-mail: nsbeitrabelsi@aud.edu 1. Introduction In 2010, the International Accounting Standards Board (IASB) and the Financial Accounting Standards Board (FASB) developed jointly the Conceptual Framework for Financial Reporting (the conceptual framework). This document has replaced the primary characteristic of “reliability” with “faithful representation”. Faithful representation of information is attained when the representation of an economic event is complete, neutral, and free from material error. Timeliness and verifiability aspects of information have been considered as enhancing characteristics. International Accounting Standard 34 (IAS34) “Interim Financial Reporting” does not require the publication of interim reports. It encourages disclosing interim reports for listed entities. Mandatory disclosure of quarterly financial reports for publicly-traded companies, in the majority of jurisdictions around the world, is the direct consequence of applying “timeliness”. Having relevant information available sooner would improve its capacity to influence decisions. However, the interim reports are not required to be audited. In some jurisdictions, they need only be reviewed. Financial statements audits are expensive and time-consuming. Unaudited interim reports would affect the verifiability of financial information. Boritz (2007) proposed “more frequent reporting of relevant and reliable information through an integrated audit focusing on interim reporting rather than focusing excessively on the annual financial statements”. Disclosing more information is preferred to less (Gigler et al., 2014). (Tsao et al., 2018) consider that switching from semiannual to quarterly reporting regime contributes in accrual mispricing. Research that has analyzed the quality of accounting information disclosed in interim reports did not lead to sharp results. Rácz and Huszár (2019) and Chakraborty and Chetan (2018) consider that quarterly earnings are relevant for investors: the market reacts when they are announced. Others consider quarterly reports as irrelevant because they are misstated (Saidin et al., 2016) and lead to managerial short-termism (Ernstberger et al., 2017). Reviewing interim financial statements by external auditors is expected to improve their quality (Manry et al., 2003, Malek et al., 2016). Manry et al. (2003) performed a research in U. S where the auditor review is mandatory. Results indicate that the review increases the quality of accounting disclosures. Research carried out in Canada where the auditor review of interim financial reports is allowed but not required (Lightstone et al., 2012), indicated that the volatility of net income in each of the first three quarters is significantly lower than in the final quarter. The fourth-quarter adjustments are considerably different from the previous three. Abstract Mandatory disclosure of quarterly financial reports for publicly traded companies, in the majority of jurisdictions around the world, is the direct consequence of applying “timeliness” as presented in the Conceptual Framework for Financial Reporting (the conceptual framework) developed jointly in 2010 by the International Accounting Standards Board (IASB) and the Financial Accounting Standards Board (FASB). Having relevant information available sooner would improve its capacity to influence decisions. However, the interim reports are not required to be audited. In UAE, companies whose securities are listed on a securities and commodities market licensed by the Securities and Commodities Authority (SCA) are required to notify and provide interim financial reports, which are reviewed by the external auditor of the company. The objective of this paper is to analyze, in UAE, the volatility of the fourth-quarter earnings compared with the previous three. This study includes four years (2012-2015) of quarterly financial statements of firms listed in Dubai Financial Market (DFM). In order to determine if interim results are suspect, the paper analyzes the magnitude of differences in fourth quarter earnings and revenues relative to the first three quarters by using the Kiger‟s 1974 methodology. Overall, results indicate that the volatility of earnings and revenue in the fourth quarter is significantly higher than those of the first three quarters. This main finding would be explained by the necessary adjustments to the fourth quarter earnings and revenues in order to correct the estimation. In fact, the quarterly financial statements require the use of more estimates than those prepared at the end of the fiscal year. This research would contribute to better understanding the quality of interim reports in an emerging market context. Keywords: Earnings quality, Interim reports, Auditing, Volatility http://ijafr.macrothink.org 351 International Journal of Accounting and Financial Reporting ISSN 2162-3082 2019, Vol. 9, No. 4 International Journal of Accounting and Financial Reporting ISSN 2162-3082 2019, Vol. 9, No. 4 International Journal of Accounting and Financial Reporting ISSN 2162-3082 2019, Vol. 9, No. 4 and revenues relative to the first three quarters by using the methodology of Kiger (1974). Overall, results indicate that the volatility of earnings and revenue in the fourth quarter is significantly higher than those of the first three quarters. This main finding would be explained by the necessary adjustments to the fourth quarter earnings and revenues in order to correct the estimation. In fact, the quarterly financial statements require the use of more estimates than those prepared at the end of the fiscal year. The findings documented in this paper contribute to the debate on quarterly earnings‟ quality. It allows shedding light on this issue in an emerging market where reviewing interim accounting reports prepared using IFRS is mandatory. The reminder of this paper is organized as follows. Continuance of this section contains literature review and hypothesis development. The second section related to methodology subjects and selecting data and sample. The third section discusses research findings. Summary and discussions are presented in the final section. IAS 34 encourages publicly traded entities to disclose interim accounting reports without specifying the frequency of publication. This position is the consequence of timeliness: enhancing qualitative characteristic of accounting information according to the conceptual framework. Several researchers analyzed the quality of quarterly accounting numbers (Kerstein and Rai, 2007, Das et al., 2009, Chakraborty and Chetan, 2018, Kajüter et al., 2018, Rácz and Huszár, 2019). Results found by Das et al. (2009) indicate that 22% of firms demonstrate a reversal in fourth quarter earnings. Likewise Kerstein and Rai (2007) noted a significant increase in earnings management during the fourth quarter. Empirical research suggested that a mandatory auditor review should be performed in order to improve the faithful representation of accounting numbers reported quarterly (Boritz, 2007, Lightstone et al., 2012). In Germany, where security regulations allow listed entities to decide whether to have quarterly reports reviewed by an external auditor, Kajüter et al. (2016) found that reviewed interim financial statements show higher earnings quality and that they yield more decision-useful information for investors compared to un-reviewed reports. In United States, where the auditor review is compulsory, empirical studies support the thought that the review increases the quality of reported accounting information. This paper would contribute to this debate by analyzing the case of firms listed in an emerging market: DFM. 1. Introduction Therefore, faithful representation of information would be distorted which prevents providing relevant information for decision making. In UAE, companies whose securities are listed on a securities and commodities market licensed by the Securities and Commodities Authority (SCA) are required to notify and provide interim financial reports which are reviewed by the external auditor of the company within forty-five days from the end of the specified time period (SCA, 2000). The objective of this paper is to analyze, in UAE, the volatility of the fourth-quarter earnings compared with the previous three. This study includes four years (2012-2015) of quarterly financial statements of firms listed in Dubai Financial Market (DFM). In order to determine if interim results are suspect, the paper analyzes the magnitude of differences in fourth quarter earnings http://ijafr.macrothink.org 352 http://ijafr.macrothink.org International Journal of Accounting and Financial Reporting ISSN 2162-3082 2019, Vol. 9, No. 4 Several papers such as Das et al. (2009), Kerstein and Rai (2007), and Kiger (1975) have analyzed the distribution of quarterly earnings in order to explain the adjustments performed during fourth quarter. Examining a sample of US firms, Kerstein and Rai (2007) explain that earnings management during quarter four is more noticeable for firms having small accumulated losses or those with small accumulated profits during the first three quarters. It allows the formers to disclose small annual profit and the latters to avoid disclosing small annual loss. Kiger (1975) explains that fourth quarter adjustments could be a result of the correction of errors or the adjustment of estimated inventories in previous quarters, interim allocation procedures used, real diﬀerences in proﬁtability in the quarter, as well as diﬀerences caused by the multiplier eﬀect on income as sales move away from the break-even point. He indicates that fourth quarter earnings volatility could also be the result of either lack of care in the preparation of quarterly reports or earnings management from the shifting of income from quarter to quarter or from one year to the next. In U.S. Gu et al. (2005) found that regulated ﬁrms are less likely to engage in significant earnings management than unregulated ﬁrms. Studies in Singapore, where quarterly reporting is voluntary, found that when interim reports are prepared, the reports play a monitoring role. Rahman et al. (2007) concluded that focusing attention on the quarterly ﬁnancial statements resulted in lower discretionary accruals and that fewer major adjustments were found in the fourth quarter. Other papers suggested that interim ﬁnancial statements provide an opportunity for management to defer discretionary income-reducing cost estimates to the fourth quarter (Dempsey, 1994, Mendenhall and Nichols, 1988). Accordingly, the ﬁrst three quarters‟ income statements may report better earnings than are justiﬁed by the annual results. This was supported by an earlier ﬁnding of unusually lar ge adjustments in the fourth quarter of U.S. quarterly ﬁnancial statements in companies without timely quarterly reviews by auditors (Ettredge et al., 2000). Results from accounting literature examining the impact of reviewing interim reports by auditors on the quality of quarterly financial information are contradictory to certain extent. Malek et al. (2016) examined the earnings response coefficient of quarterly accounts for a matched pair sample of 60 Malaysian firms. The objective of this paper is to measure and analyze the degree of differences in fourth quarter earnings and revenue compared to the first three quarters. If fourth quarter adjustments are greater than those in the previous three quarters, this may indicate that interim ﬁnancial statements are not complete and therefore are not faithful representations of each quarter‟s results (Bédard and Courteau, 2015, Porumb et al., 2018). User reliance on these reports may not be warranted. In order to determine whether quarterly results are suspect, this paper examines the variability associated with each quarter‟s reported net income and revenue over a period of four years. When earnings and revenue changes in the ﬁrst three quarters are relatively small and the fourth quarter varies by a signiﬁcant margin, a conclusion could be drawn that adjustments that relate to each quarter are, in fact, being postponed and included in the fourth quarter‟s results. http://ijafr.macrothink.org 353 providing higher quality information. Yet this did not prevent some firms to voluntarily reviewing their quarterly reports while it‟s not mandatory by law. It allows to reduce the cost of capital (Porumb et al., 2018). Several empirical research provide extensive evidence of the use of discretionary accruals by managers for earnings management (Graham et al., 2005, Brown and Caylor, 2005, Dechow et al., 2003, Dechow and Skinner, 2000, Healy and Wahlen, 1999, Degeorge et al., 1999, Burgstahler and Dichev, 1997, Dempsey, 1994). The use of judgment allowed by accounting standards gives managers discretion in their choices of accounting policies. They may have an opportunistic behavior by selecting methods and estimates in favor of certain parties. Research provides explanations specifically related to quarterly earnings management. In Brazil Rodrigues et al. (2019) analyze the behavior of discretionary accruals throughout quarters. Results indicate that the level of discretionary accruals is significantly higher during the fourth quarter. They are used by managers in order to attain target profit. Lin and Lai (2019) demonstrate, for a sample of listed companies in Taiwan, that managers may manage earnings in quarter four in order to avoid small losses or decrease in earnings. Quarterly earnings management may result from capital investment decisions taken by managers. Managers differ investment decisions in order to achieve quarterly earnings thresholds (Canace and Salzsieder, 2015). This how certain researchers have wondered about the quality of quarterly earnings guidance. Lin (2017) considers that poor quality of quarterly earnings guidance misleads investors and may cause earnings management. Cessation of quarterly earnings guidance reduces pressure on managers and therefore contributes in reducing earnings management (Kim et al., 2017). The literature presented above arouses interest on the degree of relevance and reliability of quarterly accounts. The “accounting community”, including IASB and FASB, agrees on the importance of providing financial information to users on a timely basis. However, there is no clear cut answer on the “optimal” way of disclosing interim reports. Critics have been addressed on several levels. Publication of quarterly financial reports may lead to short-termism from managers (Gigler et al., 2014, Kim et al., 2017): managers may have a short term perspective in taking investment decisions as they are under market pressure. Other studies have analyzed the quality of quarterly reports. Finding support that explanatory power of the model is sensitive to auditors‟ involvement. Earnings response coefficient is higher for companies publishing reviewed interim reports. Similar results were found by (Manry et al., 2003) for a sample of US firms. The association between quarterly returns and quarterly earnings is significantly stronger for earnings audited timely compared to those audited retrospectively at the end of the year. Timely reviews are those done by auditors at the end of each quarter and are different from retrospective quarterly reviews performed at year end. In the same way Ettredge et al. (2000) found that “the frequency of adjustments during the ﬁrst three quarters is greater for companies with timely reviews, while the frequency and proportion of fourth quarter adjustments is smaller. However other studies are not sure about the positive impact of reviewing interim reports on earnings quality. The study of Bédard and Courteau (2015) conducted in Canada, failed to demonstrate a significant improvement in the quality of quarterly financial statement if they are audited. In addition to this, authors explain that the increase in audit fees -associated with the review- by 18% cannot be justified by 354 http://ijafr.macrothink.org Although results of several studies confirm the relevance of interim earnings such as recent works of Chakraborty and Chetan (2018) and Rácz and Huszár (2019), others had reservations. Thus, quarterly accounts may disclose poor quality information because of earnings volatility (Kerstein and Rai, 2007, Das et al., 2009, Ernstberger et al., 2017). Findings of Lee et al. (2016) indicate that in Singapore investors rely more on reports disclosed at year end than previous interim reports. It is in this context that comes the motivation of this paper. The main objective is to analyze the distribution and the degree of volatility of quarterly financial information for a sample of firms listed in DFM. In UAE, listed companies have to communicate quarterly financial statements reviewed by independent auditor. Accounting reports should be prepared using IFRS. Results would contribute to this debate in the context of an emerging market. 355 http://ijafr.macrothink.org 2. Methodology The sample was drawn from the total population of companies (equities) listed in Dubai Financial market (DFM) during the period 2012-2015, excluding investment and financial services (45 companies). Companies in the investment and financial services group as classified by DFM as the presentation of their financial statements doesn‟t allow getting the required data for analysis: revenue and expenses are not reported separately, they report in the income statement the net profit or loss from main activities. After eliminating three additional firms because of unavailable data (2) and fiscal year ending on March 31 (1), the final population consists of 42 firms. Financial data were collected manually from each company‟s financial statements posted on DFM or from their websites. The net income (loss) and revenue reported for the first three quarters and for the year were collected for the sample companies from 2012 to 2015. As companies do not publish separate fourth quarter financial statements, the fourth quarter‟s results were interfered from the annual amounts. In total the analysis is conducted for a sample of 164 firm-quarter observations. Following the approach used by Kiger (1974) and Lightstone et al. (2012), a measure of volatility was determined for each company‟s quarterly results for both earnings and revenue. Therefore the volatility measure was determined by using a three-step approach. Step 1: Quarterly and annual net income (loss) for each company in each of the four years from 2012 to 2015 was recorded. Amounts in AED Company Year Quarter 1 Quarter 2 Quarter 3 Quarter 4 Total Aramex 2015 97049000 102983000 78574000 66805000 345411000 Step 2: A measure of quarterly net income volatility was calculated for each entity. For each year, each quarter‟s net income was expressed as percentage of annual results. Amounts in AED Company Year Quarter 1 Quarter 2 Quarter 3 Quarter 4 Total Aramex 2015 97049000 102983000 78574000 66805000 345411000 Step 2: A measure of quarterly net income volatility was calculated for each entity. For each year, each quarter‟s net income was expressed as percentage of annual results. Company Year Quarter 1 Quarter 2 Quarter 3 Quarter 4 Total Aramex 2015 28.10 29.81 22.75 19.34 100.00 Step 3: The volatility measure for each company was determined for each quarter by subtracting the lowest percentage over the four years from the highest percentage. International Journal of Accounting and Financial Reporting ISSN 2162-3082 2019, Vol. 9, No. 4 2. Methodology Company 2012 Q 1 % 2013 Q 1 % 2014 Q 1 % 2015 Q 1 % High Low Difference Aramex 25.52 24.99 24.90 28.10 28.10 24.90 3.20 The earnings volatility measure for Aramex for quarter 1over the 2012-2015 period is 3.20. he earnings volatility measure for Aramex for quarter 1over the 2012-2015 period is 3.2 A quarterly revenue volatility measure was calculated for reported revenue by using the same procedures: the three-step approach. In addition an annual measure of quarterly volatility was determined for each company for each year by subtracting the minimum percentage of 356 http://ijafr.macrothink.org 2.1 Additional Variable: Size Effect In order to provide supplementary perceptiveness into findings and to determine whether the viability of quarterly results is related to company size, the sample was segmented by size. Companies were classified into two groups based on average total assets over the 2012-2015 period. Therefore group 1 consists on firms with average total assets between $0 and $100,000,000 (n=21) and group 2 with average total assets more than $100,000,000 (n=21). In accordance with findings of previous research and the discussion above, the following hypotheses were developed: In accordance with findings of previous research and the discussion above, the following hypotheses were developed:  Hypothesis 1: Quarter four net income volatility is signiﬁcantly greater than the volatility of net income in each of the ﬁrst three quarters.  Hypothesis 2: Quarter four net income volatility is signiﬁcantly greater than the volatility of net income in each of the ﬁrst three quarters for companies with at least one quarterly loss reported compared with firms with no quarterly losses.  Hypothesis 3: There is no signiﬁcant diﬀerence between any of quarters one, two, and three with respect to net income volatility. Following Kiger (1974) who included revenue volatility in order “to measure the clarity and consistency of patterns in the data”, this paper is investigating revenue in relation to earnings management. It includes volatility in revenue as a measure of stabilization. A conclusion of earnings management might be supported in situations where companies have either stable revenue patterns and volatile net incomes or volatile revenue patterns and stable net incomes. Accordingly, the following hypotheses were added:  Hypothesis 4: Quarter four revenue volatility is signiﬁcantly greater than the volatility of revenue in each of the ﬁrst three quarters.  Hypothesis 4: Quarter four revenue volatility is signiﬁcantly greater than the volatility of revenue in each of the ﬁrst three quarters.  Hypothesis 5: There is no signiﬁcant diﬀerence between any of quarters one, two, and three with respect to revenue volatility.  Hypothesis 5: There is no signiﬁcant diﬀerence between any of quarters one, two, and three with respect to revenue volatility. International Journal of Accounting and Financial Reporting ISSN 2162-3082 2019, Vol. 9, No. 4 contribution to the annual result from the maximum percentage contribution. For example, the annual volatility measure for 2015 for the firm represented in step 2 illustrations above is (29.81-19.34). This procedure was carried out for both net income and revenue reported. contribution to the annual result from the maximum percentage contribution. For example, the annual volatility measure for 2015 for the firm represented in step 2 illustrations above is (29.81-19.34). This procedure was carried out for both net income and revenue reported. 3.1 Descriptive Statistics Table 1 represents descriptive statistics of firms. It shows results for net income, revenue, assets, and stockholders „equity. Calculation of descriptive statistics indicators was performed separately for all companies then for firms with no quarterly losses reported and finally for companies with at least one quarterly loss reported. From the group of companies with at least one quarterly loss reported, 48% (11 firms) disclosed quarterly losses only during the fourth quarter. 357 357 http://ijafr.macrothink.org International Journal of Accounting and Financial Reporting ISSN 2162-3082 2019, Vol. 9, No. 4 Results related to all companies show that firms in the sample are very heterogeneous in size. The range in total assets is very high ($110m). Tests that will be performed later in order to detect the size effect allow to analyze the degree of income and revenue volatility related to size. Results related to all companies show that firms in the sample are very heterogeneous in size. The range in total assets is very high ($110m). Tests that will be performed later in order to detect the size effect allow to analyze the degree of income and revenue volatility related to size. size. Table 1 All companies (n=42) Net income Revenue Assets Stockholders „equity Mean (USD 000) 121322 696253 5850350 1285242 Median (USD 000) 23900 199533 911363 360078 Standard deviation 290609 1099996 15961850 2446400 Min (USD 000) -756603 2577 14390 4215 Max (USD 000) 1939527 4657653 110690666 13816867 Kurtosis 12.91 3.96 25.11 11.26 Skewness 2.98 2.15 4.80 3.30 Companies with no quarterly losses reported (n=19) Net income Revenue Assets Stockholders „equity Mean (USD 000) 265512 1200203 11036078 2375612 Median (USD 000) 91696 529663 2215585 1197903 Standard deviation 371908 1401710 22569378 3293755 Min (USD 000) 3922 34902 57287 44342 Max (USD 000) 1939527 4657653 110690666 13816867 Kurtosis 5.64 0.25 9.89 3.43 Skewness 2.20 1.23 3.13 2.04 Table 1 http://ijafr.macrothink.org http://ijafr.macrothink.org 358 International Journal of Accounting and Financial Reporting ISSN 2162-3082 2019, Vol. 9, No. 4 Companies with at least one quarterly loss reported (n=23) Net income Revenue Assets Stockholders „equity Mean (USD 000) 2209 285865 1566488 384501 Median (USD 000) 8096 96160 416966 175305 Standard deviation 96347 463574 2595432 478237 Min (USD 000) -756604 2577.223 14390.34 4215 Max (USD 000) 174432 2257873 14484903 1928773 Kurtosis 43.47 6.57 10.33 2.21 Skewness -5.77 2.54 3.02 1.69 Table 2 represents descriptive statistics of net income and revenue percentage (step 2) for each quarter across the four years related to the sample. Calculation of descriptive statistics indicators was performed separately for all companies then for firms with no quarterly losses reported and finally for companies with at least one quarterly loss reported. T bl 2 4 Skew 7.93 -10.09 -12.68 11.29 0.49 1.02 0.54 0.00 Kurt 70.52 119.94 163.19 141.53 1.99 9.38 3.34 6.56 Quarterly net income and revenue percentage: Companies with no quarterly losses reported (n=19) Net income (loss) Revenue Quarter 1 Quarter 2 Quarter 3 Quarter 4 Quarter 1 Quarter 2 Quarter 3 Quarter 4 Mean 26.31 26.61 23.05 24.04 25.16 26.08 23.80 24.96 Median 23.74 25.77 23.88 24.71 24.34 25.18 24.79 24.95 SD 11.04 8.66 8.96 11.89 5.59 6.84 3.99 6.98 Min 1.39 2.69 1.54 4.50 6.61 2.12 11.55 1.21 Max 65.21 60.97 53.28 80.56 41.00 62.84 30.98 58.18 Skew 1.10 0.88 0.41 1.86 0.87 1.82 -0.90 1.25 Kurt 1.94 4.49 2.20 6.95 2.86 13.63 0.79 8.87 Quarterly net income and revenue percentage: Companies with at least one quarterly loss reported (n=23) Net income (loss) Revenue Quarter 1 Quarter 2 Quarter 3 Quarter 4 Quarter 1 Quarter 2 Quarter 3 Quarter 4 Mean 40.82 11.11 -7.54 55.61 24.76 26.11 24.53 24.60 Median 23.72 24.97 25.05 28.97 23.68 26.02 24.10 24.50 SD 138.49 159.26 373.29 393.37 5.64 6.64 5.46 7.46 Min -116.90 -1378.96 -3525.06 -680.23 7.84 2.40 10.33 -11.22 360 International Journal of Accounting and Financial Reporting ISSN 2162-3082 2019, Vol. 9, No. 4 Max 1040.46 312.25 310.63 3633.39 42.41 56.80 46.58 42.38 Skew 5.86 -7.47 -9.39 8.35 0.20 0.31 0.91 -0.82 Kurt 37.89 65.51 89.45 77.41 1.41 6.14 3.17 5.28 3.2 Volatility of Quarterly Net Income Tables 3, 4, and 5 provide the resulting descriptive statistics for each quarter across the four years related to the sample. Table 3 reports the analysis when all companies are included in the database, Table 4 excludes companies that reported at least one quarterly loss, and Table 5 excludes firms with no quarterly losses reported. Table 3 Net income volatility measure: all companies (n=42) Quarter 1 Quarter 2 Quarter 3 Quarter 4 Mean 80.09 85.82 124.69 180.24 Median 25.13 25.24 19.67 33.26 Standard deviation 194.73 262.80 543.86 662.85 Min 2.55 2.12 1.57 2.18 Max 1029.54 1691.21 3543.57 4313.62 Kurtosis 17.37 36.14 40.87 39.47 Skewness 4.14 5.86 6.36 6.20 Table 4 Net income volatility measure: Companies with no quarterly losses reported (n=19) International Journal of Accounting and Financial Reporting ISSN 2162-3082 2019, Vol. 9, No. Table 2 represents descriptive statistics of net income and revenue percentage (step 2) for each quarter across the four years related to the sample. Calculation of descriptive statistics indicators was performed separately for all companies then for firms with no quarterly losses reported and finally for companies with at least one quarterly loss reported. Table 2 represents descriptive statistics of net income and revenue percentage (step 2) for each quarter across the four years related to the sample. Calculation of descriptive statistics indicators was performed separately for all companies then for firms with no quarterly losses reported and finally for companies with at least one quarterly loss reported. Table 2 Quarterly net income and revenue percentage: All companies (n=43) Net income (loss) Revenue Quarter 1 Quarter 2 Quarter 3 Quarter 4 Quarter 1 Quarter 2 Quarter 3 Quarter 4 Mean 34.26 18.12 6.30 41.33 24.94 26.10 24.20 24.76 Median 23.72 25.49 24.26 25.35 24.20 25.37 24.66 24.84 SD 102.75 117.96 276.05 290.91 5.60 6.71 4.85 7.23 Min -116.90 -1378.96 -3525.06 -680.23 6.61 2.12 10.33 -11.22 Max 1040.46 312.25 310.63 3633.39 42.41 62.84 46.58 58.18 Quarterly net income and revenue percentage: All companies (n=43) 359 International Journal of Accounting and Financial Reporting ISSN 2162-3082 2019, Vol. 9, No. 4 Max 1040.46 312.25 310.63 3633.39 42.41 56.80 46.58 42.38 Skew 5.86 -7.47 -9.39 8.35 0.20 0.31 0.91 -0.82 Kurt 37.89 65.51 89.45 77.41 1.41 6.14 3.17 5.28 3.2 Volatility of Quarterly Net Income Tables 3, 4, and 5 provide the resulting descriptive statistics for each quarter across the four years related to the sample. Table 3 reports the analysis when all companies are included in the database, Table 4 excludes companies that reported at least one quarterly loss, and Table 5 excludes firms with no quarterly losses reported. Table 3 Net income volatility measure: all companies (n=42) Quarter 1 Quarter 2 Quarter 3 Quarter 4 Mean 80.09 85.82 124.69 180.24 Median 25.13 25.24 19.67 33.26 Standard deviation 194.73 262.80 543.86 662.85 Min 2.55 2.12 1.57 2.18 Max 1029.54 1691.21 3543.57 4313.62 Kurtosis 17.37 36.14 40.87 39.47 Skewness 4.14 5.86 6.36 6.20 Table 4 Net income volatility measure: Companies with no quarterly losses reported (n=19) Quarter 1 Quarter 2 Quarter 3 Quarter 4 International Journal of Accounting and Financial Reporting ISSN 2162-3082 2019, Vol. 9, No. 4 Max 1040.46 312.25 310.63 3633.39 42.41 56.80 46.58 42.38 Skew 5.86 -7.47 -9.39 8.35 0.20 0.31 0.91 -0.82 Kurt 37.89 65.51 89.45 77.41 1.41 6.14 3.17 5.28 3 2 Volatility of Quarterly Net Income Tables 3, 4, and 5 provide the resulting descriptive statistics for each quarter across the four years related to the sample. Table 3 reports the analysis when all companies are included in the database, Table 4 excludes companies that reported at least one quarterly loss, and Table 5 excludes firms with no quarterly losses reported. Table 3 Net income volatility measure: all companies (n=42) Quarter 1 Quarter 2 Quarter 3 Quarter 4 Mean 80.09 85.82 124.69 180.24 Median 25.13 25.24 19.67 33.26 Standard deviation 194.73 262.80 543.86 662.85 Min 2.55 2.12 1.57 2.18 Max 1029.54 1691.21 3543.57 4313.62 Kurtosis 17.37 36.14 40.87 39.47 Skewness 4.14 5.86 6.36 6.20 Table 4 Net income volatility measure: Companies with no quarterly losses reported (n=19) Quarter 1 Quarter 2 Quarter 3 Quarter 4 Mean 9.49 12.65 8.07 16.17 Median 5.56 6.80 6.93 10.60 361 International Journal of Accounting and Financial Reporting ISSN 2162-3082 2019, Vol. 9, No. 4 Standard deviation 7.66 13.14 5.58 16.79 Min 2.55 2.12 1.57 2.18 Max 27.93 56.51 18.55 75.11 Kurtosis 0.18 6.52 -0.97 8.40 Skewness 1.17 2.39 0.57 2.62 Table 5 Net income volatility measure: Companies with at least one quarterly loss reported (n=23) Quarter 1 Quarter 2 Quarter 3 Quarter 4 Mean 138.41 146.26 221.03 315.77 Median 53.73 53.13 53.99 73.18 Standard deviation 250.52 346.59 727.84 880.98 Min 9.80 10.66 7.17 20.43 Max 1029.54 1691.21 3543.57 4313.62 Kurtosis 8.64 20.15 22.49 21.88 Skewness 3.02 4.40 4.72 4.63 Ki (1974) l d i l ili b i l di i h l l i i h Net income volatility measure: Companies with at least one quarterly loss reported (n=23) Quarter 1 Quarter 2 Quarter 3 Quarter 4 Mean 138.41 146.26 221.03 315.77 Median 53.73 53.13 53.99 73.18 Standard deviation 250.52 346.59 727.84 880.98 Min 9.80 10.66 7.17 20.43 Max 1029.54 1691.21 3543.57 4313.62 Kurtosis 8.64 20.15 22.49 21.88 Skewness 3.02 4.40 4.72 4.63 come volatility measure: Companies with at least one quarterly loss reported (n=23) Kiger (1974) analyzed net income volatility by including in the sample only companies with no quarterly losses reported. It is considered that including firms with quarterly losses in the sample might bias results by reporting less volatility than currently exists. Following this earlier study and in order to eliminate any possible noise generated by quarterly losses in the data, this paper analyzed separately the data for those companies with no quarterly losses. There were 19 companies that met this requirement, and Table 4 shows the descriptive statistics for the 19 remaining. Consistent with Kiger‟s expectation, restricting the population to entities with no quarterly losses reported does dampen the measures of volatility. Analysis of the fourth quarter‟s volatility for all companies supports Hypothesis 1. The fourth quarter‟s earnings volatility (M = 180.24, SD = 662.85) was signiﬁcantly higher than that of the ﬁrst quarter (M = 80.09, SD = 194.73), the second quarter (M = 85.82, SD = 262.80), and the third quarter (M = 124.69, SD = 543.86). Similar results were found when the same tests 362 http://ijafr.macrothink.org http://ijafr.macrothink.org were conducted for firms that did not report any quarterly losses in the four-year period. Providing further support for Hypothesis 1, the fourth quarter‟s earnings volatility (M = 16.17, SD = 16.79) was signiﬁcantly higher than that of the ﬁrst quarter (M = 9.49, SD = 7.66), the second quarter (M = 12.65, SD = 13.14), and the third quarter (M = 8.07, SD = 5.58). Comparison between results provided in tables 3 and 4 supports Hypothesis 2. Quarter four net income volatility is signiﬁcantly greater than the volatility of net income in each of the ﬁrst three quarters for companies with at least one quarterly loss reported compared with firms with no quarterly losses. In fact the fourth quarter‟s volatility for all companies (M = 180.24, SD = 662.85) is significantly higher than that of firms with no quarterly losses (M = 16.17, SD = 16.79). No signiﬁcant diﬀerences in quarterly earnings volatility were found between any of quarters one, two, and three, either for the complete population or for the non–loss reporting group. These results support Hypothesis 3. 3.3 Variability of Quarterly Revenue The results of descriptive statistical tests related to revenue for all companies are presented in Table 6. It included all firms in the sample as all quarterly revenues are positive. Companies not reporting revenue where excluded when the sample was formed at the beginning. Quarterly revenue volatility measure: All companies (n=42) Quarter 1 Quarter 2 Quarter 3 Quarter 4 Mean 6.15 9.25 6.22 10.53 Median 4.06 4.90 4.36 5.50 Standard deviation 5.02 10.99 5.61 12.17 Min 0.72 0.30 0.84 0.46 Max 20.61 49.88 26.63 53.59 Kurtosis 0.72 4.85 3.21 4.21 Skewness 1.17 2.18 1.67 2.04 Results of the repeated measures test were found to be signiﬁcant only for quarters one and three. The volatility of fourth quarter revenue reported (M = 10.53, SD = 5.5) was positive and signiﬁcantly diﬀerent from that of the ﬁrst quarter (M = 6.15, SD = 5.02), and the third quarter (M = 6.22, SD = 5.61). Accordingly, Hypothesis 4 is not completely supported. Quarterly revenue volatility measure: All companies (n=42) Results of the repeated measures test were found to be signiﬁcant only for quarters one and three. The volatility of fourth quarter revenue reported (M = 10.53, SD = 5.5) was positive and signiﬁcantly diﬀerent from that of the ﬁrst quarter (M = 6.15, SD = 5.02), and the third quarter (M = 6.22, SD = 5.61). Accordingly, Hypothesis 4 is not completely supported. Results of the repeated measures test were found to be signiﬁcant only for quarters one and three. The volatility of fourth quarter revenue reported (M = 10.53, SD = 5.5) was positive and signiﬁcantly diﬀerent from that of the ﬁrst quarter (M = 6.15, SD = 5.02), and the third quarter (M = 6.22, SD = 5.61). Accordingly, Hypothesis 4 is not completely supported. http://ijafr.macrothink.org 363 http://ijafr.macrothink.org 363 ISSN 2162-3082 2019, Vol. 9, No. 4 Moreover, and contrary to the earnings volatility results, the second quarter‟s revenue volatility was also positive and signiﬁcantly diﬀerent from each of the first and third quarters. Moreover, and contrary to the earnings volatility results, the second quarter‟s revenue volatility was also positive and signiﬁcantly diﬀerent from each of the first and third quarters. 3.4 Quarterly Net Income and Revenue Variability Table 7 summarizes results from analyzing relationship between quarterly earnings and quarterly revenue volatility. Tests were performed only for companies with no quarterly losses reported. Table 7 Quarterly Net Income and Revenue Variability: Companies with no quarterly losses reported (n=19) Net income Revenue Mean 20.20 10.08 Median 12.95 6.40 SD 17.02 11.63 Min 0.86 0.79 Max 79.17 61.63 Kurtosis 0.85 6.55 Skewness 1.14 2.38 Results indicate that the volatility in quarterly revenue is, in fact, signiﬁcantly diﬀerent than that for quarterly earnings, and is so for each quarter: Net income volatility (M = 20.20, SD = 17.02) is significantly higher than revenue volatility 9m = 10.08, SD = 11.63). 3 5 Addi i l V i bl Si Eff Quarterly Net Income and Revenue Variability: Companies with no quarterly losses re (n=19) Results indicate that the volatility in quarterly revenue is, in fact, signiﬁcantly diﬀerent than that for quarterly earnings, and is so for each quarter: Net income volatility (M = 20.20, SD = 17.02) is significantly higher than revenue volatility 9m = 10.08, SD = 11.63). Results indicate that the volatility in quarterly revenue is, in fact, signiﬁcantly diﬀerent than that for quarterly earnings, and is so for each quarter: Net income volatility (M = 20.20, SD = 17.02) is significantly higher than revenue volatility 9m = 10.08, SD = 11.63). 3.5 Additional Variable: Size Effect In order to investigate whether the extent of quarterly earnings volatility is dependent on of the size of a company, the same tests conducted previously were performed separately for the two sub-samples: firms with total assets below $100,000,000 (firms A) and those with total assets above $100,000,000 (firms B). Table 8 represents descriptive statistics for companies classified by size. Results indicate that for sub-samples A and B, the volatility of the fourth quarter‟s reported earnings are signiﬁcantly higher than that of each of the other three quarters. For sub-sample A, fourth quarter‟s earnings volatility (M = 45.33, SD = 43.54) is significantly higher than that of the first three quarters. Similar results were detected for sub-sample B: fourth 364 http://ijafr.macrothink.org International Journal of Accounting and Financial Reporting ISSN 2162-3082 2019, Vol. 9, No. 4 quarter‟s earnings volatility (M = 315.15, SD = 927.67) is significantly higher than those of the first three quarters. The above results consolidate tests run for Hypothesis 1 and 2. 4. Summary and Discussions This paper has measured and analyzed the degree of difference in fourth quarter earnings and revenue compared to the first three quarters. If the adjustments in the fourth quarter earnings and revenue are higher than those in the previous three quarters, this may support that interim financial statements are not compete and therefore are not representing faithfully each quarter‟s results. Moreover in order to determine whether quarterly results are dubious, this study examines the variability associated with each quarter‟s reported net income and revenue over a period of four years. When earnings and revenue changes in the ﬁrst three quarters are relatively small and the fourth quarter varies by a signiﬁcant margin, a conclusion could be drawn that adjustments that relate to each quarter are, in fact, being postponed and included in the fourth quarter‟s results. Tests were carried out on a sample of companies listed in DFM during the period 2012-2015. Data was collected manually and directly from interim reports and annual reports as reported by companies. Additional tests were done in order to better analyze results. Therefore supplementary analysis was carried out for firms with no quarterly losses reported. In addition to this the size effect was tested. Results indicate that the volatility of earnings and revenue in the fourth quarter is significantly higher than those of the first three quarters. This main finding would be explained by the necessary adjustments to the fourth quarter earnings and revenues in order to correct the estimation. In fact, the quarterly financial statements require the use of more estimates than those prepared at the end of the fiscal year. Tests related to the analysis of size effect, show similarities in results to certain extent. Overall results indicate higher volatility for the subsample of big firms. Findings from this study are relevant to standard setters and regulators for future directions in developing accounting standards. The results may be helpful to investors for understanding the information content of interim reports, and may also provide insights for accounting standard setters and regulators. However, results do not allow detecting the effect of reviewing quarterly reports by independent auditors on earnings volatility. This limitation is due to the fact that reviewing interim accounting reports is mandatory to all listed companies. Table 8 Quarterly net income and revenue volatility measure: Total assets>$100,000,000 (n=21) Net income Revenue Quarter 1 Quarter 2 Quarter 3 Quarter 4 Quarter 1 Quarter 2 Quarter 3 Quarter 4 Mean 23.45 28.02 25.95 45.33 5.40 8.92 5.50 10.37 Median 9.80 20.34 9.97 29.48 3.04 4.55 2.95 4.59 SD 26.25 29.31 33.28 43.54 5.35 12.49 4.76 12.38 Min 2.73 2.12 1.60 2.18 0.72 0.42 1.13 0.64 Max 102.56 118.17 115.24 151.62 20.61 49.88 15.71 43.21 Kurt 2.88 3.36 2.34 0.52 2.98 5.51 -0.67 2.94 Skew 1.70 1.75 1.81 1.08 1.85 2.34 0.92 1.86 Quarterly net income and revenue volatility measure: Total assets<$100,000,000 (n=21) Net income Revenue Quarter 1 Quarter 2 Quarter 3 Quarter 4 Quarter 1 Quarter 2 Quarter 3 Quarter 4 Mean 136.73 143.63 223.44 315.15 6.90 9.58 6.95 10.69 Median 46.92 34.17 49.98 43.96 6.56 5.86 4.71 7.99 SD 265.16 365.65 764.70 927.67 4.67 9.57 6.38 12.26 Min 2.55 3.85 1.57 4.91 0.89 0.30 0.84 0.46 Max 1029.54 1691.21 3543.57 4313.62 16.04 40.23 26.63 53.59 365 International Journal of Accounting and Financial Reporting ISSN 2162-3082 2019, Vol. 9, No. 4 Kurt 7.49 18.19 20.50 19.81 -0.79 4.53 3.82 7.04 Skew 2.83 4.18 4.51 4.40 0.51 2.03 1.90 2.39 4. Summary and Discussions Future research may be conducted in order to analyze, in the context of an emerging market, other aspects of quarterly earnings quality such as earnings management and earnings response coefficient. Moreover it would be interesting to examine the effect of the level compliance with IFRS on quarterly earnings quality. Indeed studies of Bédard, J., & Courteau, L. (2015). Benefits and Costs of Auditor's Assurance: Evidence From the Review of Quarterly Financial Statements. Contemporary Accounting Research, 32, 308-335. References Bédard, J., & Courteau, L. (2015). Benefits and Costs of Auditor's Assurance: Evidence From the Review of Quarterly Financial Statements. Contemporary Accounting Research, 32, 308-335. http://ijafr.macrothink.org 366 ISSN 2162-3082 2019, Vol. 9, No. 4 Gu, Z., Lee, C.-W. J., & Rosett, J. G. (2005). What Determines the Variability of Accounting Accruals?. Review of Quantitative Finance and Accounting, 24, 313-334. Gu, Z., Lee, C.-W. J., & Rosett, J. G. (2005). What Determines the Variability of Accounting Accruals?. Review of Quantitative Finance and Accounting, 24, 313-334. Healy, P. M., & Wahlen, J. M. (1999). A Review of the Earnings Management Literature and Its Implications for Standard Setting. Accounting Horizons, 13, 365-383. Kajüter, P., Klassmann, F., & Nienhaus, M. (2016). Do Reviews by External Auditors Improve the Information Content of Interim Financial Statements?. The International Journal of Accounting, 51, 23-50. Kajüter, P., Klassmann, F., & Nienhaus, M. (2018). The Effect of Mandatory Quarterly Reporting on Firm Value. The Accounting Review, 94, 251-277. Kerstein, J., & Rai, A. (2007). Intra-Year Shifts in the Earnings Distribution and Their Implications for Earnings Management. Journal of Accounting and Economics, 44, 399-419. Kiger, J. E. (1974). Volatility in Quarterly Accounting Data. The Accounting Review, 4 Kiger, J. E. (1975). Implications of Volatility in Quarterly Accounting Data: A Reply. The Accounting Review, 50, 130. Kim, Y., Su, L., & Zhu, X. (2017). Does the Cessation of Quarterly Earnings Guidance Reduce Investors' Short-Termism?. Review of Accounting Studies, 22, 715-752. Lee, A., Lim, C. Y., & Zhang, T. C. (2016). Cross-Quarter Differential Market Reactions: An Investigation of the Audit Effect Hypothesis. Pacific Accounting Review, 28, 219-235. Lightstone, K., Young, N. M., & Mcfadden, T. (2012). Information Quality of Interim Financial Statements. Accounting Perspectives, 11, 297-313. Lin, K. C. (2017). Quality Concerns Over Managers' Quarterly Earnings Guidance. Advances in Accounting, 38, 113-125. Lin, S.-H., & Lai, H.-H. (2019). Earnings Management During the Fourth Quarter: Evidence From Taiwan. Emerging Markets Finance and Trade, 1-20. Malek, M., Saidin, S. F., Osman, H., & Noor, M. (2016). Audited Quarterly Accounts and Earnings Response Coefficients. DLSU Business & Economics Review, 25, 53-62. Manry, D., Tiras, S. L., & Wheatley, C. M. (2003). The Influence of Interim Auditor Reviews on the Association of Returns With Earnings. The Accounting Review, 78, 251-274. Mendenhall, R. R., & Nichols, W. D. (1988). Bad News and Differential Market Reactions to Announcements of Earlier-Quarters Versus Fourth-Quarter Earnings. Journal of Accounting Research, 63-86. Porumb, V.-A., Karaibrahimoglu, Y. Z., & Wang, S. (2018, December). Spend Money to Make Money?: Voluntary Audit Reviews and Firms‟ Cost of Debt. International Journal of Accounting and Financial Reporting ISSN 2162-3082 2019, Vol. 9, No. 4 Boritz, J. E. (2007). Forum on Maintaining Quality Capital Markets Through Quality Information. Accounting Perspectives, 6, 263-268. Brown, L. D., & Caylor, M. L. (2005). A Temporal Analysis of Quarterly Earnings Thresholds: Propensities and Valuation Consequences. The Accounting Review, 80, 423-440. Burgstahler, D., & Dichev, I. (1997). Earnings Management to Avoid Earnings Decreases and Losses. Journal of Accounting and Economics, 24, 99-126. Canace, T. G., & Salzsieder, L. (2015). The Timing of Asset Purchases to Achieve Earnings Thresholds. Journal of Management Accounting Research, 28, 81-106. Chakraborty, S., & Chetan, G. K. (2018). A Study of Quarterly Earnings Announcement and Stock Price Reactions - With Reference to Nifty Midcap 150. Journal of Commerce & Accounting Research, 7, 1-12. Das, S., Shroff, P. K., & Zhang, H. (2009). Quarterly Earnings Patterns and Earnings Management. Contemporary Accounting Research, 26, 797-831. Dayanandan, A., Donker, H., Ivanof, M., & Karahan, G. (2016). IFRS and Accounting Quality: Legal Origin, Regional, and Disclosure Impacts. International Journal of Accounting and Information Management, 24, 296-316. Dechow, P. M., & Skinner, D. J. (2000). Earnings Management: Reconciling the Views of Accounting Academics, Practitioners, and Regulators. Accounting Horizons, 14, 235-250. Dechow, P. M., Richardson, S. A., & Tuna, I. (2003). Why Are Earnings Kinky? An Examination of the Earnings Management Explanation. Review of Accounting Studies, 8, 355-384. Degeorge, F., Patel, J., & Zeckhauser, R. (1999). Earnings Management to Exceed Thresholds. The Journal of Business, 72, 1-33. Dempsey, S. J. (1994). Interim Earnings Management and the Fourth Quarter Good News Effect. Journal of Business Finance & Accounting, 21, 889-908. Ernstberger, J., Link, B., Stich, M., & Vogler, O. (2017). The Real Effects of Mandatory Quarterly Reporting. The Accounting Review, 92, 33-60. Ettredge, M. L., Simon, D. T., Smith, D. B., & Stone, M. S. (2000). The Effect of the External Accountant's Review on the Timing of Adjustments to Quarterly Earnings. Journal of Accounting Research, 38, 195-207. Gigler, F., Kanodia, C., Sapra, H., & Venugopalan, R. (2014). How Frequent Financial Reporting Can Cause Managerial Short-Termism: An Analysis of the Costs and Benefits of Increasing Reporting Frequency. Journal of Accounting Research, 52, 357-387. Graham, J. R., Harvey, C. R., & Rajgopal, S. (2005). The Economic Implications of Corporate Financial Reporting. Journal of Accounting and Economics, 40, 3-73. http://ijafr.macrothink.org 367 Voluntary Audit Reviews and Firms’ Cost of Debt. 368 http://ijafr.macrothink.org http://ijafr.macrothink.org Rácz, D. A., & Huszár, G. (2019). The Effects of Earnings Surprises in Quarterly Reports on S&P 500 Components. Public Finance Quarterly (0031-496x), 64, 239-259. Rahman, A. R., Tay, T. M., Ong, B. T., & Cai, S. (2007). Quarterly Reporting in A Voluntary Disclosure Environment: Its Benefits, Drawbacks and Determinants. The International Journal of Accounting, 42, 416-442. Rodrigues, R. M. R. C., Melo, C. L. L. D., & Paulo, E. (2019). Earnings Management and Quarterly Discretionary Accruals Level in the Brazilian Stock Market. Bbr. Brazilian Business Review, 16, 297-314. Saidin, S. F., Malek, M., Ibrahim, D. N., & Kee, P. L. (2016). Events After Reporting Period and Misstatements in Quarterly Accounts. International Journal of Economics and Financial Issues, 6, 42-46. SCA (Ed.). (2000). Decision No. (3/R) of 2000 Concerning Regulations as to Disclosures and Transparency. UAE. Tawiah, V., & Boolaky, P. (2019). Determinants of IFRS Compliance in Africa: Analysis of Stakeholder Attributes. International Journal of Accounting & Information Management. Tsao, S.-M., Lu, H.-T., & Keung, E. C. (2018). Interim Reporting Frequency and the Mispricing of Accruals. Accounting Horizons, 32, 29-47. Watts, R. L., & Zimmerman, J. L. (1986). Positive Accounting Theory. Copyright Disclaimer Copyright for this article is retained by the author(s), with first publication rights granted to the journal. This is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/) 369 http://ijafr.macrothink.org
https://openalex.org/W2951563520	https://www.frontiersin.org/articles/10.3389/fpls.2017.02185/pdf	English	null	Morphometrics Reveals Complex and Heritable Apple Leaf Shapes	Frontiers in plant science	2,018	cc-by	10,847	Morphometrics Reveals Complex and Heritable Apple Leaf Shapes Zoë Migicovsky 1, Mao Li 2, Daniel H. Chitwood 3, 4 and Sean Myles 1 1 Department of Plant, Food and Environmental Sciences, Faculty of Agriculture, Dalhousie University, Truro, NS, Canada, 2 Donald Danforth Plant Science Center, St. Louis, MO, United States, 3 Department of Horticulture, Michigan State University, East Lansing, MI, United States, 4 Computational Mathematics, Science and Engineering, Michigan State University, East Lansing, MI, United States Apple (Malus spp.) is a widely grown and valuable fruit crop. Leaf shape is important for ﬂowering in apple and may also be an early indicator for other agriculturally valuable traits. We examined 9,000 leaves from 869 unique apple accessions using linear measurements and comprehensive morphometric techniques. We identiﬁed allometric variation as the result of differing length-to-width aspect ratios between accessions and species of apple. The allometric variation was due to variation in the width of the leaf blade, not the length. Aspect ratio was highly correlated with the ﬁrst principal component (PC1) of morphometric variation quantiﬁed using elliptical Fourier descriptors (EFDs) and persistent homology (PH). While the primary source of variation was aspect ratio, subsequent PCs corresponded to complex shape variation not captured by linear measurements. After linking the morphometric information with over 122,000 genome- wide single nucleotide polymorphisms (SNPs), we found high SNP heritability values even at later PCs, indicating that comprehensive morphometrics can capture complex, heritable phenotypes. Thus, techniques such as EFDs and PH are capturing heritable biological variation that would be missed using linear measurements alone. Reviewed by: y Fabrizio Costa, Fondazione Edmund Mach, Italy Marie Monniaux, UMR5667 Reproduction et Developpement des Plantes (RDP), France Fabrizio Costa, Fondazione Edmund Mach, Italy Marie Monniaux, UMR5667 Reproduction et Developpement des Plantes (RDP), France Keywords: apple, leaf shape, morphometrics, elliptical Fourier descriptors, persistent homology, Malus domestica Edited by: Catherine Anne Kidner, University of Edinburgh, United Kingdom INTRODUCTION Correspondence: Zoë Migicovsky zoe.migicovsky@dal.ca Apples (Malus spp.) are one of the world’s most widely grown fruit crops, with the third highest global production quantity of over 84 million tons in 2014 (Food Agriculture Organization of the United Nations, 2017). The shape and size of apple leaves can impact access to light, ultimately inﬂuencing ﬂower number and fruit quality (Wünsche and Lakso, 2000; Dennis, 2003). Apple leaves are generally simple, with an elliptical-to-ovate shape. Previous studies in apple used linear measurements, such as length and width, to quantify leaf shape (Liebhard et al., 2003; Bassett et al., 2011). The length-to-width aspect ratio is a major source of variation in leaf shape. Diﬀering aspect ratios lead to a disproportionate increase or decrease in length relative to width, resulting in allometric variation, in leaves (Gurevitch, 1992; Chitwood et al., 2013). While linear measurements such as leaf length and width are useful, they fail to capture the full extent of leaf shape diversity. Failing to measure leaf shape comprehensively also limits our ability to discern the total underlying genetic contributions. Specialty section: This article was submitted to Plant Evolution and Development, a section of the journal Frontiers in Plant Science Received: 06 October 2017 Accepted: 12 December 2017 Published: 04 January 2018 ORIGINAL RESEARCH published: 04 January 2018 doi: 10.3389/fpls.2017.02185 Citation: Migicovsky Z, Li M, Chitwood DH and Myles S (2018) Morphometrics Reveals Complex and Heritable Apple Leaf Shapes. Front. Plant Sci. 8:2185. doi: 10.3389/fpls.2017.02185 Eigenshape analysis is a traditional morphometric technique for comprehensively quantifying the outline of a shape, using a number of semilandmarks placed at equal distances around the shape (Lohmann, 1983). In comparison, Elliptical Fourier descriptors (EFD) analysis ﬁrst converts the outline to a chaincode, a lossless data compression method that encodes shape by January 2018 \| Volume 8 \| Article 2185 Frontiers in Plant Science \| www.frontiersin.org 1 Complex and Heritable Apple Leaf Shapes Migicovsky et al. a chain of numbers, in which each number indicates step-by- step movements to reconstruct the pixels comprising the shape. A Fourier decomposition is subsequently applied to the chaincode, quantifying the shape as a harmonic series. EFDs have been used extensively to quantify leaf shape in diverse species, such as grape (Chitwood et al., 2014), tomato (Chitwood et al., 2012), and Passiﬂora (Chitwood and Otoni, 2017). Previous work used EFDs to assess apple fruit shape (Currie et al., 2000), but this technique has not yet been applied to apple leaves. A newly developed morphometric technique, persistent homology (PH), provides another method for estimating leaf shape. PH, like EFDs, is normalized to diﬀerences in size, but it is also orientation invariant. PH treats the pixels of a contour as a 2D point cloud before applying a neighbor density estimator to each pixel. A series of annulus kernels of increasing radii is used to isolate and smooth the contour densities. The number of connected components is recorded as a function of density for each annulus, resulting in a curve (a reduced version of persistent barcode) that quantiﬁes shape as topology. The topology-based PH approach can also be applied to serrations and root architecture, allowing a similar framework to be used across disparate plant shapes and structures (Li et al., 2017a,b,c). examples of genomic analyses of leaf shape in apple. However, a previous bi-parental linkage mapping study found two suggestive quantitative trait loci for leaf size (Liebhard et al., 2003). Work by Khan et al. (2014) measured several leaf traits such as area, perimeter and circularity, in 158 apple accessions. The study linked these measurements with 901 single nucleotide polymorphisms (SNPs), but found no signiﬁcant genotype- phenotype relationships. Citation: Thus far, eﬀorts have not been made to estimate the genetic heritability of comprehensive morphometric leaf phenotypes, such as those described using EFDs and PH. It therefore remains unclear to what extent these methods are capturing biologically meaningful, heritable variation. To fully understand the genetic basis of leaf shape, it is essential to include both linear and morphometric estimates of shape. Decreasing sequencing costs and access to a large and diverse germplasm collection allowed us to analyze approximately 9,000 leaves from over 800 unique accessions which we linked to over 122,000 genome-wide SNPs. We present the ﬁrst comprehensive analysis of leaf shape in apple, revealing that both accessions and species show allometric variation due to diﬀerences in the width of the leaf blade. While the primary axis of variation in apple using EFDs and PH is due to this allometric variation, we ﬁnd high SNP heritability values even at later principal components, indicating that comprehensive estimates of shape capture heritable variation which would be missed by linear estimates alone. Comprehensively measuring leaf shape, using approaches such as EFDs and PH, may reveal shape features associated with fruit shape or quality traits. For example, recent work examining persimmon (Diospyros kaki) found weak but signiﬁcant correlations between EFD measurements for leaf and fruit shape (Maeda et al., 2018). Similarly, in previous work on apple, several leaf traits such as area and perimeter were correlated with fruit size (Khan et al., 2014). There are also several cases of unique leaf characteristics providing an early marker for other genetic diﬀerences in apple. For example, the gene underlying red fruit ﬂesh color may lead to anthocyanin accumulation in the leaves, causing red foliage (Chagné et al., 2007; Espley et al., 2009) while columnar tree architecture may be accompanied by an increase in leaf number, area, weight per unit area and length-to-width ratio (Talwara et al., 2013). Leaf pH has also been proposed as an early indicator of low acid fruit (Visser and Verhaegh, 1978). Frontiers in Plant Science \| www.frontiersin.org Sample Collection p Apple trees in Kentville, Nova Scotia, Canada were budded onto M.9 rootstocks in spring 2012. In the fall, the trees were uprooted and kept in cold storage until spring 2013, when trees were planted in an incomplete block design (see “REstricted Maximum Likelihood (REML)” below). Leaves from over 900 trees were collected from August 24th to September 16th 2015. Ten mature leaves were collected from each tree and eﬀorts were made to collect leaves from portions of the branch representing third year growth. No leaves were collected from trees which had been heavily pruned due to the presence of disease. Leaves were ﬂattened and placed to avoid touching, then scanned using Canon CanoScan (LiDE 220) Colour Image Scanners. Leaves were then dried for 48 h at 65◦C and weighed to estimate the total dry weight (g) for each tree. In addition to serving as early markers for other traits, leaf shape and size may inﬂuence the amount of light a tree receives, and light exposure is crucial for ﬂowering in apple. Light penetration results in higher levels of ﬂowering, while leaf injury or defoliation can reduce ﬂowering (Dennis, 2003). Thinning apple trees to a particular leaf-to-fruit ratio is a common practice to attain optimal fruit color and size. Contrastingly, trees with fewer fruit may increase vegetative growth and thus leaf area (Wünsche et al., 2000). Clearly, there is an important relationship between the leaves and the fruit of an apple tree, and comprehensively quantifying the variation in leaf shape is a crucial component to understanding this relationship. Morphometric Analyses p y Leaf scans were converted into a separate binary image for each leaf using custom ImageJ macros, which included the “make binary” function (Abràmoﬀet al., 2004). Images were converted to RGB .bmp ﬁles and a chain code analysis was performed using SHAPE (Iwata and Ukai, 2002). The chain code was used to calculate normalized elliptical Fourier descriptors (EFDs) in SHAPE. The normalized EFDs were read into Momocs v1.1.5 (Bonhomme et al., 2014) in R (R Core Team, 2016) where Leaves are the main photosynthetic organs of apple, but the genetic basis underlying their shape and size remains unknown. Although there are examples of a single locus controlling major variation in leaf shape (Kimura et al., 2008; Cartolano et al., 2015; Andres et al., 2017), in most instances leaf shape appears to be controlled by numerous small-eﬀect loci (Langlade et al., 2005; Tian et al., 2011; Chitwood et al., 2013). There are limited January 2018 \| Volume 8 \| Article 2185 2 Complex and Heritable Apple Leaf Shapes Migicovsky et al. harmonics B and C were removed to eliminate asymmetrical variation in leaf shape. aspect ratio, length, width, major and minor axis, and surface area. Variance was calculated as the coeﬃcient of variation using the “cv” function in the raster package (Hijmans, 2016) in R to estimate within-tree variability in leaf size, which is indicated as “var” throughout this manuscript. p The binary leaf images were also analyzed using PH (Li et al., 2017c). To numerically estimate the shape of the leaves using PH, we extracted the leaf contour using a 2D point cloud (Figure 1A). After centering and normalizing the contour to its centroid size, we used a Gaussian density estimator (Figure 1B), which assigns high values (red) to pixels with many neighboring pixels, and low values (blue) to pixels with fewer neighboring pixels. We multiplied the density estimator by an annulus kernel, or ring (Figure 1C), which emphasizes the shape in an annulus at the centroid and is thus invariant to orientation (Figure 1D). The resulting function can also be visualized from the side view (Figures 1E,F). As we moved a plane from the top to the bottom, we recorded the number of connected components above the plane, forming a curve. With each new component this value increased, and each time components were merged, it decreased (Figure 1G). REstricted Maximum Likelihood (REML) Adjustment of Phenotype Data The orchard sampled in this study is an incomplete block design with 1 of 3 standards per grid. The standards, or “control trees”—‘Honeycrisp,’ ‘SweeTango,’ and ‘Ambrosia’—are replicated across the grid. Leaves from these trees were sampled multiple times across the orchard which allowed us to correct for positional eﬀects. Each phenotype was adjusted using a REstricted Maximum Likelihood (REML) model which resulted in one adjusted value per accession, even when multiple trees were measured. The impact of row grid (rGrid), column grid (cGrid), and rGrid × cGrid eﬀects were adjusted for using the following REML model: phenotype ∼accession + ( 1 \| rGrid) + ( 1 \| cGrid) + ( 1 \| cGrid : rGrid) Only leaves for which both EFDs and PH shape estimations were successfully calculated were included in subsequent analyses. Additionally, only trees with 8–10 leaves were included, as leaves were sometimes removed due to tears, folding, or the absence of a petiole, which did not allow for accurate quantiﬁcation of shape. The ﬁnal dataset consisted of 915 trees with 8–10 leaves, which included 869 unique accessions and 8,995 leaves. We ﬁt a linear mixed-eﬀects model via REML using the “lmer” function in the lme4 package in R (Bates et al., 2015) and then calculated the least squares means using the “lsmeans” function in the lsmeans R package (Lenth, 2016). Thus, while the initial phenotype data were collected for 915 trees, following REML adjustment, one value remained per unique accession, resulting in 869 accessions. REML-adjustment was applied directly to all size, weight and variance estimates. For PH and EFDs, we applied the REML following PCA and thus the percent contribution for each PC was calculated using unadjusted values. The adjusted data for all 24 phenotypes are included in Table S1. EFDs and PH values were averaged across leaves from an individual tree. The contribution of EFD harmonics 1–15 to the mean leaf shape across all trees was visualized using the “hcontrib” function in the Momocs R package (Figure 2). To allow for discrimination between accessions based on leaf shape, principal component analysis (PCA) was performed using the Momocs “PCA” function (Bonhomme et al., 2014) for EFDs, and the “prcomp” function in R for PH values, which center but do not scale the data. The resulting principal component (PC) values were adjusted using REstricted Maximum Likelihood (see below). Morphometric Analyses For each leaf we computed 16 curves corresponding to 16 expanding rings. For computational purposes, each curve was divided into 500 numbers, ultimately resulting in the shape of each leaf being represented by 8,000 (16∗500) values. Frontiers in Plant Science \| www.frontiersin.org REstricted Maximum Likelihood (REML) Adjustment of Phenotype Data Subsequently, we identiﬁed the accession with the minimum and maximum value along each of the ﬁrst 5 PCs. Phenomic Analyses The correlation between leaf phenotypes was calculated using Pearson’s correlation and p-values were Bonferroni-corrected for multiple comparisons. The resulting heatmap was visualized using the “geom_tile” function in ggplot2 in R (Wickham, 2009). Next, we examined the leaves for allometry using the “SMA” function in the smartr R package (Warton et al., 2012) to estimate if the slope between the log-transformed minor and major axis diﬀered from 1. In addition to estimating the contour of the leaf using EFDs and PH, we used several more metrics to describe the leaves. Using ImageJ, we automated the measurement of leaf surface area (cm2), length (cm) of the leaf and width (cm) of the leaf as well as major (blade length) and minor (blade width) axes of the best ﬁtting ellipse—which excluded the petiole—through batch processes (Abràmoﬀet al., 2004). Throughout the manuscript, we use “major” when referring to the length of the leaf blade, and “minor” when referencing the width of the leaf blade. We also calculated the aspect ratio of the leaf, by dividing the major axis by the minor axis. Additionally, leaf mass per area was calculated for 780 trees where we possessed surface area data for all 10 leaves, by calculating the ratio of dry weight to surface area (g/cm2). Accessions were labeled as either Malus × domestica Borkh. or Malus sieversii Lebed. based on information provided by the United States Department of Agriculture (USDA) Germplasm Resources Information Network website (https://npgsweb.ars- grin.gov) (Table S2). We used a Mann-Whitney U-test to test if any phenotypes diﬀered between species and Bonferroni- corrected all p-values for multiple comparisons. Genomic Analyses DNA was extracted using commercial extraction kits. Genotyping-by-sequencing (GBS) libraries were prepared using ApeKI and PstI-EcoT221I restriction enzymes according While linear phenotypes were calculated as an average value for a particular tree, we also estimated variance within a tree for January 2018 \| Volume 8 \| Article 2185 3 Complex and Heritable Apple Leaf Shapes Migicovsky et al. FIGURE 1 \| Visualization of persistent homology technique for annulus kernel 7. Binary images were converted into a 2D point cloud (A) which was then normalized using a Gaussian density estimator (B). For each leaf, 16 annulus kernels were used. Annulus kernel 7, indicated in purple (C) is used as an example for this visualization. The density estimator is multiplied by ring 7 (D). The function can also be visualized from the side view (E,F). As a plane moves from top to bottom, the number of connected components is recorded along the curve (G). Below (G) are ﬁve visualizations of curves that are represented as red vertical dotted lines in (G). FIGURE 1 \| Visualization of persistent homology technique for annulus kernel 7. Binary images were converted into a 2D point cloud (A) which was then normalized using a Gaussian density estimator (B). For each leaf, 16 annulus kernels were used. Annulus kernel 7, indicated in purple (C) is used as an example for this visualization. The density estimator is multiplied by ring 7 (D). The function can also be visualized from the side view (E,F). As a plane moves from top to bottom, the number of connected components is recorded along the curve (G). Below (G) are ﬁve visualizations of curves that are represented as red vertical dotted lines in (G). FIGURE 1 \| Visualization of persistent homology technique for annulus kernel 7. Binary images were converted into a 2D point cloud (A) which was then normalized using a Gaussian density estimator (B). For each leaf, 16 annulus kernels were used. Annulus kernel 7, indicated in purple (C) is used as an example for this visualization. The density estimator is multiplied by ring 7 (D). The function can also be visualized from the side view (E,F). As a plane moves from top to bottom, the number of connected components is recorded along the curve (G). Below (G) are ﬁve visualizations of curves that are represented as red vertical dotted lines in (G). Genomic Analyses January 2018 \| Volume 8 \| Article 2185 Frontiers in Plant Science \| www.frontiersin.org Frontiers in Plant Science \| www.frontiersin.org Frontiers in Plant Science \| www.frontiersin.org 4 Migicovsky et al. Complex and Heritable Apple Leaf Shapes FIGURE 2 \| Contribution of elliptical Fourier descriptor harmonics to leaf shape. The leaf shapes depicted are the mean leaf shapes based on all 915 trees. Harmonics 1–15 are represented on the x-axis and each harmonic is multiplied by the ampliﬁcation factor on the y-axis to visualize their contribution to mean leaf shape. An ampliﬁcation factor of 0 indicates the removal of the harmonic; a factor of 1 results in the normal shape; and values above 1 exaggerate effects to better visualize the harmonic’s contribution to the ﬁnal shape. FIGURE 2 \| Contribution of elliptical Fourier descriptor harmonics to leaf shape. The leaf shapes depicted are the mean leaf shapes based on all 915 trees. Harmonics 1–15 are represented on the x-axis and each harmonic is multiplied by the ampliﬁcation factor on the y-axis to visualize their contribution to mean leaf shape. An ampliﬁcation factor of 0 indicates the removal of the harmonic; a factor of 1 results in the normal shape; and values above 1 exaggerate effects to better visualize the harmonic’s contribution to the ﬁnal shape. FIGURE 2 \| Contribution of elliptical Fourier descriptor harmonics to leaf shape. The leaf shapes depicted are the mean leaf shapes based on all 915 trees. Harmonics 1–15 are represented on the x-axis and each harmonic is multiplied by the ampliﬁcation factor on the y-axis to visualize their contribution to mean leaf shape. An ampliﬁcation factor of 0 indicates the removal of the harmonic; a factor of 1 results in the normal shape; and values above 1 exaggerate effects to better visualize the harmonic’s contribution to the ﬁnal shape. FIGURE 2 \| Contribution of elliptical Fourier descriptor harmonics to leaf shape. The leaf shapes depicted are the mean leaf shapes based on all 915 trees. Harmonics 1–15 are represented on the x-axis and each harmonic is multiplied by the ampliﬁcation factor on the y-axis to visualize their contribution to mean leaf shape. An ampliﬁcation factor of 0 indicates the removal of the harmonic; a factor of 1 results in the normal shape; and values above 1 exaggerate effects to better visualize the harmonic’s contribution to the ﬁnal shape. Genomic Analyses Prior to performing PCA, SNPs were pruned for linkage disequilibrium (LD) using PLINK. We considered a window of 10 SNPs, removing one SNP from a pair if R2 > 0.5, then shifting the window by 3 SNPs and repeating (PLINK command: indep- pairwise 10 3 0.5). This resulted in a set of 75,973 SNPs for 816 accessions. PCA was performed on the LD-pruned genome- wide SNPs using “prcomp” in R. The ﬁrst 2 genomic PCs were visualized using ggplot2 in R (Wickham, 2009). to Elshire et al. (2011). GBS libraries were sequenced using Illumina Hi-Seq 2000 technology. Reads which failed Illumina’s “chastity ﬁlter” were removed from raw fastq ﬁles. Remaining reads were aligned to the Malus × domestica v1.0 pseudo haplotype reference sequence (Velasco et al., 2010) using the Burrows-Wheeler aligner tool v0.7.12 (Li and Durbin, 2009) and the Tassel version 5 pipeline (Glaubitz et al., 2014). Tassel parameters included a minKmerL of 30, mnQS of 20, mxKmerNum of 50000000 and batchSize of 20. The kmerlength was set to 82 for ApeKI and 89 for PstI-EcoT22I based on the max barcode size. The minMAF for the DiscoverySNPCallerPluginV2 was set to 0.01. All other default parameters were used. Non-biallelic sites and indels were removed using VCFtools v.0.1.14 (Danecek et al., 2011). Variant Call Format (VCF) ﬁles for both enzymes were then merged using a custom perl script, preferentially keeping SNPs called by PstI-EcoT22I at overlapping sites, since those sites tended to have higher coverage. We performed a genome-wide association study (GWAS) using the mixed linear model in Tassel (version 5) for each phenotype, adjusting for relatedness among individuals using a kinship matrix as well as the ﬁrst 3 PCs for population structure (Bradbury et al., 2007; Zhang et al., 2010). The threshold for signiﬁcance was calculated using simpleM (Gao et al., 2008, 2010) which estimates the number of PCs needed to explain 0.995 of the variance, or the number of independent SNPs. The inferred Meﬀ used to calculate the signiﬁcance threshold was 91,667 SNPs. We searched the regions surrounding any signiﬁcant GWAS SNPs using the Genome Database for Rosaceae GBrowse tool for Malus × domestica v1.0 p genome (Jung et al., 2014). Variation in Apple Leaf Shape We examined 24 phenotypes related to apple leaf shape and size including length, width, surface area, dry weight, leaf mass per area, within-tree variance, and overall shape estimated using PCs derived from EFD and PH data (see Materials and Methods and Figures 1–2). The distribution and raw values for each phenotype are provided (Figure S1, Table S1). To visualize the primary axes of morphometric variation, we chose a representative leaf from accessions with the minimum and maximum values along the ﬁrst 5 PCs for EFDs and PH (Figure 3A). The accessions with extreme values along PC1 for both methods are similar. In fact, ‘Binet Rouge’ has the lowest value along PC1 for EFD and PH, with the axis clearly representing a decrease in the length-to-width (aspect) ratio. The annulus kernels most strongly contributing to PH PC1 (Figure S2) provide further evidence that this PC captures variation in aspect ratio. Variation in leaf shape captured by higher-order PCs is more complex and cryptic, and is thus not captured using linear measurements alone. In addition, while the primary axis of variation (PC1) using EFDs and PH may explain similar aspects of leaf morphology, the morphospaces resulting from the two techniques diﬀer (Figure 3B). In addition to variation between accessions, we investigated diﬀerences in leaf shape and size between species by comparing M. domestica, the domesticated apple, with its primary progenitor species, M. sieversii (Table S4). PCA of the genome- wide SNP data reveals a primary axis of genetic variation that separates M. domestica and M. sieversii, although separation is incomplete (Figure 6A). The major axis (p = 0.975) of the leaves does not diﬀer between species (Figure 6B). However, the minor axis (p = 4 × 10−4) of M. domestica leaves are signiﬁcantly larger than M. sieversii (Figure 6C) and the aspect ratio (p = 0.023) is signiﬁcantly less (Figure 6D). Thus, there is allometric variation both within (Figure 5) and between (Figure 6) Malus species. Next, we examined the correlation between all measured traits (Table S3). By assessing the correlation of PCs resulting from a classical morphometric technique such as EFDs with a novel, topology-based morphometric approach like PH, we reveal how complementary the methods are (Figure 4, Figure S3). Genomic Analyses We used a window of ±5,000 bp (10 kb) surrounding the signiﬁcant SNP to check for genes, and when identiﬁed, we used the basic local alignment search tool (BLAST) from NCBI to search for the mRNA sequence and reported the result with the max score (Altschul et al., 1990). Missing genotypes were imputed using LinkImputeR v0.9 (Money et al., 2017) with global thresholds of 0.01 for minor allele frequency (MAF) and 0.70 for missingness. We examined depths of 3–8 and selected a case for imputation with a max position/sample missingness of 0.70, a minimum depth of 5, and an imputation accuracy of 94.9%. The VCF was converted to a genotype table using PLINK v1.07 (Purcell et al., 2007; Purcell, 2009). Genomic prediction was performed using the “x.val” function in the R package PopVar (Mohammadi et al., 2015). The rrBLUP model was selected and 5-fold (nFold = 5) cross-validation was repeated 3 times (nFold.reps = 3) with no further ﬁltering (min.maf = 0) from the set of 122,596 SNPs used for GWAS. All other default parameters were used. In addition to performing genomic prediction on the main 24 phenotypes examined in this study, we performed genomic prediction on all 40 PCs for EFDs and on the ﬁrst 40 PCs for PH values. We also used the “rnorm” Of the 869 accessions assessed in this study, 816 had genomic data following imputation and ﬁltering and were included in downstream analyses. The resulting genotype table consisted of 816 accessions and 197,565 SNPs. Subsequently, a 0.05 MAF ﬁlter was applied using PLINK, after which 128,132 SNPs remained. SNPs with more than 90% heterozygous genotypes were removed. The ﬁnal genotype table consisted of 816 samples and 122,596 SNPs. January 2018 \| Volume 8 \| Article 2185 Frontiers in Plant Science \| www.frontiersin.org 5 Complex and Heritable Apple Leaf Shapes Migicovsky et al. function in R to generate 1,000 random phenotypes with a mean of 0 and a standard deviation of 1, and performed genomic prediction using these random phenotypes to obtain the range of genomic prediction accuracies one can expect at random. Lastly, we used genome-wide complex trait analysis (GCTA) v.1.26.0 which estimates the genetic relationships between individuals based on genome-wide SNPs and uses this information to calculate the variance explained by these SNPs. Genomic Analyses The ratio of additive genetic variation to phenotypic variance is used to calculate SNP heritability (h2) of a trait (Yang et al., 2011). We used GCTA to estimate heritability for each phenotype, including the ﬁrst 40 PCs for EFD and PH. We also estimated the correlation between genomic prediction accuracy (r) and SNP heritability (h2) using a Pearson’s correlation. (r = −0.734, p < 1 × 10−15). The correlation between the minor axis of a leaf and surface area (r = 0.939, p < 1 × 10−15) is higher than the correlation between the major axis (blade length) and surface area (r = 0.810, p < 1 × 10−15). As expected, leaf surface area is also highly correlated with average leaf dry weight (r = 0.934, p < 1 × 10−15), indicating that larger leaves are heavier. Allometry in Apple Leaves The high correlation between aspect ratio and PC1 for both EFD and PH methods indicates that length-to-width ratio is the primary source of variation in apple leaf shape. If there is an allometric relationship between the minor and major axis, and thus, the length and width of a leaf do not increase at equal rates, a slope signiﬁcantly diﬀering from 1 is expected. We ﬁnd that the slope between the two measurements is signiﬁcantly >1 (95% CI = 1.506–1.678, R2 = 0.343, p < 1 × 10−15), indicating that the minor axis increases at a greater rate than the major axis. While there is no signiﬁcant correlation between the major axis (blade length) and EFD PC1 (R2 = 0.001, p = 1) or PH PC1 (R2 = 0.002, p =1), there is a signiﬁcant correlation for the minor axis (blade width) and EFD PC1 (R2 = 0.541, p < 1 × 10−15) and PH PC1 (R2 = 0.573, p < 1 × 10−15) (Figure 5). As PC1 explains 80.23% of the variation in the leaf shape for EFDs, and 62.20% for PH, it is apparent that the width of the leaf blade, and not length, is the major source of leaf shape variation in apple. In fact, the aspect ratio, calculated as the ratio of the major axis to minor axis, is even more strongly correlated with EFD and PH PC1, with an R2 of 0.732 for EFD PC1 (p < 1 × 10−15) and an R2 of 0.771 for PH PC1 (p < 1 × 10−15). Given the signiﬁcant correlation between EFD PC1 and PH PC1 (Table S3, Figure S3), it is not surprising that aspect ratio is highly correlated with both. Variation in Apple Leaf Shape While there is a highly signiﬁcant correlation between PC1 for both methods (R2 = 0.949, p < 1 × 10−15), later PCs are often not signiﬁcantly correlated, with the most notable exception being EFD PC2 and PH PC3 (R2 = 0.432, p < 1 × 10−15), although several other PCs also show weak correlations. Thus, while the primary axis of variation (PC1) is consistent and highly correlated between methods, each method captures distinct aspects of leaf morphology in subsequent PCs. The Genetic Basis of Leaf Shape in Apple GWAS of the 24 leaf phenotypes examined in this study yielded few signiﬁcant results (Figure S4). We identiﬁed 70 signiﬁcant SNPs representing 5 phenotypes which are reported in Table S5. Manhattan plots for 4 phenotypes which include 69 of the 70 signiﬁcant SNPs are shown in Figure 7. We examined the regions surrounding signiﬁcant SNPs for candidate genes using the GBrowse tool (Table S6; Jung et al., 2014). We searched within a ±5,000 bp window, which should capture any linked causal variation given the rapid LD decay observed in a diverse collection of apples that is largely replicated in the germplasm Many of the leaf phenotypes show a strong correlation with each other (Figure 4). In particular, aspect ratio is highly correlated with PH PC1 (r = −0.878, p < 1 × 10−15), EFD PC1 (r = −0.855, p < 1 × 10−15) and minor axis (leaf blade width) January 2018 \| Volume 8 \| Article 2185 Frontiers in Plant Science \| www.frontiersin.org 6 Migicovsky et al. Complex and Heritable Apple Leaf Shapes GURE 3 \| Examples of leaf shape across PCs derived from EFDs and PH. Binary images of leaves from accessions with minimum and maximum values along PCs –5 for EFD and PH estimates. PCs were calculated using averages estimated across 8–10 leaves but only a single representative leaf is displayed. PCs were EML-adjusted based on tree position in the orchard. The accession name is also listed (A). Visualization of PC1 vs. PC2 for EFD and PH data. Accessions with inimum and maximum values along PC1 and PC2 are indicated (B). FIGURE 3 \| Examples of leaf shape across PCs derived from EFDs and PH. Binary images of leaves from accessions with minimum and maximum values along PCs 1–5 for EFD and PH estimates. Variation in Apple Leaf Shape PCs were calculated using averages estimated across 8–10 leaves but only a single representative leaf is displayed. PCs were REML-adjusted based on tree position in the orchard. The accession name is also listed (A). Visualization of PC1 vs. PC2 for EFD and PH data. Accessions with minimum and maximum values along PC1 and PC2 are indicated (B). FIGURE 3 \| Examples of leaf shape across PCs derived from EFDs and PH. Binary images of leaves from accessions with minimum and maximum values along PCs 1–5 for EFD and PH estimates. PCs were calculated using averages estimated across 8–10 leaves but only a single representative leaf is displayed. PCs were REML-adjusted based on tree position in the orchard. The accession name is also listed (A). Visualization of PC1 vs. PC2 for EFD and PH data. Accessions with minimum and maximum values along PC1 and PC2 are indicated (B). January 2018 \| Volume 8 \| Article 2185 7 Frontiers in Plant Science \| www.frontiersin.org Complex and Heritable Apple Leaf Shapes Migicovsky et al. FIGURE 4 \| Correlations among leaf phenotypes. Values above the diagonal are colored according to the Pearson’s correlation coefﬁcient, and those below the diagonal indicate Bonferroni-corrected p-values. The box enclosed by the dotted lines indicates comparisons between phenotypes captured by comprehensive morphometric analyses. FIGURE 4 \| Correlations among leaf phenotypes. Values above the diagonal are colored according to the Pearson’s correlation coefﬁcient, and those below the diagonal indicate Bonferroni-corrected p-values. The box enclosed by the dotted lines indicates comparisons between phenotypes captured by comprehensive morphometric analyses. studied here (Migicovsky et al., 2016). However, no strong candidate genes were identiﬁed. correlated with genomic prediction accuracies (Figure S5B, R2 = 0.936, p < 1 × 10−15), which is not surprising given that both techniques involve predicting a phenotype from genome-wide SNP data. None of the phenotypes measuring variance within the 8–10 leaves sampled had heritability estimates signiﬁcantly diﬀerent from 0. While GWAS examines the genome for single, large-eﬀect loci, genomic prediction estimates our ability to predict a phenotype using genome-wide marker data. We complimented our GWAS with genomic prediction and observed prediction accuracies (r) ranging from −0.10 to 0.52 (Table S7, Figure S5A). Aspect ratio is the primary source of variation in leaf shape (Figure 5C) and it was also the leaf measurement that had the highest genomic prediction accuracy (0.52). January 2018 \| Volume 8 \| Article 2185 Variation in Apple Leaf Shape Other phenotypes highly correlated with aspect ratio, such as leaf width (0.51), minor axis (0.49), EFD PC1 (0.48), and PH PC1 (0.47), all had relatively high prediction accuracies. PH PC3 (0.51) was also among the most well-predicted using genetic data. While the primary axis of variation in leaf shape detected by EFDs and PH is aspect ratio, we were also interested in determining if higher-order PCs, which capture variation not readily visible to the eye, are extracting information that is biologically meaningful. Using genomic prediction and heritability estimates, we found evidence of a genetic basis for these complex “hidden phenotypes,” which are unmeasurable using linear techniques. For example, the heritability of phenotypes such as PH PC6 (0.48), PH PC9 (0.35), PH PC10 (0.33), and EFD PC9 (0.33) is similar to traditionally measured phenotypes such as leaf length (0.44) and leaf mass per area (0.40). While higher PCs may have relatively high heritability values, after a certain point the values (± standard error) Similarly, estimates of SNP heritability (h2) calculated using GCTA (Yang et al., 2011) ranged from 0 to 0.75, with the highest heritability observed for aspect ratio (0.75) followed by leaf width (0.71), EFD PC1 (0.71), minor axis (0.69), and PH PC1 (0.65) (Figure 8, Table S8). Heritability estimates were highly January 2018 \| Volume 8 \| Article 2185 Frontiers in Plant Science \| www.frontiersin.org 8 Complex and Heritable Apple Leaf Shapes Migicovsky et al. FIGURE 5 \| Correlation between the primary axis of variation (PC1) captured using EFD and PH values and leaf shape measures. EFD PC1 is plotted against the major axis (length of leaf blade) (A), minor axis (width of leaf blade) (B) and aspect ratio (ratio of length-to-width of blade) (C). PH PC1 is plotted against the same measures in (D–F). The percent variances explained by PC1, prior to REML-adjustment, is shown in parentheses. All p-values are Bonferonni-corrected based on the number of comparisons in Figure 4. A regression line from a linear model with a shaded 95% conﬁdence interval is also shown. FIGURE 5 \| Correlation between the primary axis of variation (PC1) captured using EFD and PH values and leaf shape measures. EFD PC1 is plotted against the major axis (length of leaf blade) (A), minor axis (width of leaf blade) (B) and aspect ratio (ratio of length-to-width of blade) (C). PH PC1 is plotted against the same measures in (D–F). Variation in Apple Leaf Shape The percent variances explained by PC1, prior to REML-adjustment, is shown in parentheses. All p-values are Bonferonni-corrected based on the number of comparisons in Figure 4. A regression line from a linear model with a shaded 95% conﬁdence interval is also shown. FIGURE 6 \| Genetic and phenotypic comparison of the domesticated apple and its wild ancestor. PCs 1 and 2 were derived from 75,973 genome-wide SNPs and samples are labeled as M. domestica (purple), M. sieversii (green) or unknown (gray) (A). M. domestica leaves do not differ from M. sieversii leaves along the major axis (B), but they have a larger minor axis (C) and aspect ratio (D). P-values reported are Bonferroni-corrected based on multiple comparisons (Table S4). Species labels are based on USDA classiﬁcation. FIGURE 6 \| Genetic and phenotypic comparison of the domesticated apple and its wild ancestor. PCs 1 and 2 were derived from 75,973 genome-wide SNPs and samples are labeled as M. domestica (purple), M. sieversii (green) or unknown (gray) (A). M. domestica leaves do not differ from M. sieversii leaves along the major axis (B), but they have a larger minor axis (C) and aspect ratio (D). P-values reported are Bonferroni-corrected based on multiple comparisons (Table S4). Species labels are based on USDA classiﬁcation. FIGURE 6 \| Genetic and phenotypic comparison of the domesticated apple and its wild ancestor. PCs 1 and 2 were derived from 75,973 genome-wide SNPs and samples are labeled as M. domestica (purple), M. sieversii (green) or unknown (gray) (A). M. domestica leaves do not differ from M. sieversii leaves along the major axis (B), but they have a larger minor axis (C) and aspect ratio (D). P-values reported are Bonferroni-corrected based on multiple comparisons (Table S4). Species labels are based on USDA classiﬁcation. January 2018 \| Volume 8 \| Article 2185 Frontiers in Plant Science \| www.frontiersin.org 9 Migicovsky et al. Complex and Heritable Apple Leaf Shapes FIGURE 7 \| Manhattan plots of GWAS results for traits of interest. Results are shown for EFD PC3, PH PC4, leaf mass speciﬁc area, and width variance. P-values are log-transformed. The horizontal dotted line represents the simpleM-corrected signiﬁcance threshold. Chromosome R indicates SNPs found on contigs unanchored to the reference genome. All remaining Manhattan plots are included in Figure S4. FIGURE 7 \| Manhattan plots of GWAS results for traits of interest. Variation in Apple Leaf Shape Results are shown for EFD PC3, PH PC4, leaf mass speciﬁc area, and width variance. P-values are log-transformed. The horizontal dotted line represents the simpleM-corrected signiﬁcance threshold. Chromosome R indicates SNPs found on contigs unanchored to the reference genome. All remaining Manhattan plots are included in Figure S4. not length. Leaf surface area was also more highly correlated with the minor axis than the major axis. Variation in leaf width is therefore essential to both the size and shape of apple leaves, similar to previous work in tomato (Schwarz and Kläring, 2001). overlap with 0, indicating that they are not heritable. The cutoﬀfor morphometric PCs with a heritable genetic basis is approximately PC17. These results suggest that by making use of morphometric techniques that measure shape comprehensively, we are describing biologically meaningful, heritable phenotypes which would be missed by simple measurements such as leaf length, width and surface area. The width of the leaf blade is not only the source of variation between apple accessions, but also between M. domestica and M. sieversii. The presence of the same allometric relationship within and between species suggests that the genetic loci controlling intra-speciﬁc leaf shape variation within M. domestica may be the same as those controlling the divergence in leaf shape observed between the domesticated apple and its wild ancestor. For example, in birds, while PC1 and PC2 of bill shape explain the majority of variation across 2,000 species, they are also consistently associated with the variation between higher taxa (possessing >20 species) (Cooney et al., 2017). Our results suggest that the increase in leaf size since domestication has not been an overall increase in leaf size but speciﬁcally an increase in blade width leading to larger leaves with a reduced length-to- width ratio. Frontiers in Plant Science \| www.frontiersin.org DISCUSSION The proportion of the total phenotypic variance explained by each PC is indicated in parentheses. a simple genetic basis of leaf shape, such as in Arabidopsis thaliana, where the ANGUSTIFOLIA and ROTUNDIFOLIA3 independently control leaf width and length (Tsuge et al., 1996). In barley, transcript levels of BFL1 limit leaf width, with overexpression resulting in narrower leaves and loss of BFL1 function resulting in a reduced length-to-width ratio (Jöst et al., 2016). Using GWAS, we found no robust associations with shape phenotypes, observed a low ratio of signiﬁcant SNPs to the number of phenotypes examined, and found that signiﬁcant SNPs were sparsely distributed across multiple chromosomes. In addition, the small number of signiﬁcant SNPs are likely spurious associations due to poor correction for cryptic relatedness, as evidenced by the QQ plots (Figure S4). These observations suggest that leaf shape is likely polygenic and controlled by a large number of small eﬀect loci, such as in tomato and maize (Tian et al., 2011; Chitwood et al., 2013). In comparison, GWAS on apple fruit phenotypes, such as color and ﬁrmness, have revealed strong associations resulting from a small number of large eﬀect loci (Migicovsky et al., 2016). However, it is possible that large eﬀect loci were missed in the present study, either because of poor reference genome assembly or inadequate marker density due to rapid LD decay. Improvements where the length-to-width ratio was the major source of shape variation (>40%) (Chitwood et al., 2013). Similarly, work in Passiﬂora and Vitis species performed using two independent morphometric techniques identiﬁed allometric variation as the primary source of variation in PC1, which explained at least 40% of the variation in leaf shape (Chitwood and Otoni, 2017; Klein et al., 2017). Thus, linear measurements—in particular aspect ratio—are an important source of information when describing leaf shape. However, linear measurements are not suﬃcient for capturing the full spectrum of diversity. In our study, PC1 accounts for 62.20% or 80.23% of the variation, depending on the technique used. By simply quantifying apple leaves using linear measurements, we would miss nearly 40% of the variation in some cases. While PC1 is highly correlated with aspect ratio, later PCs represent orthogonal variation that can likely only be captured through morphometric techniques such as EFDs and PH. Therefore, to fully quantify variation in leaf shape, comprehensive morphometric techniques are essential. DISCUSSION Leaves play a crucial role in the growth and development of apple trees. To elucidate the genetic basis of variation in apple leaves, we quantiﬁed leaf shape using traditional linear measurements and comprehensive morphometric techniques. Our work oﬀers the ﬁrst comparison between the novel topology-based technique, PH, and EFDs, which we ﬁnd are complementary but distinct methods. For both methods, PC1 was highly correlated with the aspect ratio, providing evidence that the primary axis of variation in apple leaf shape can be captured using linear measurements. The minor axis, or width of the leaf blade, was also highly correlated with PC1, while the major axis was not. Thus, variation in the aspect ratio is due to variation in the leaf blade width, Our work provides evidence that allometry is the primary source of morphometric variation in apple leaves. These ﬁndings are consistent with work reported in other species such as tomato, Frontiers in Plant Science \| www.frontiersin.org January 2018 \| Volume 8 \| Article 2185 10 Complex and Heritable Apple Leaf Shapes Migicovsky et al. FIGURE 8 \| SNP heritability (h2) of leaf phenotypes. Values represent the additive genetic variance (Vg) divided b 2 FIGURE 8 \| SNP heritability (h2) of leaf phenotypes. Values represent the additive genetic variance (Vg) divided by the phenotypic variance (Vp) with a standard error as calculated using GCTA (Yang et al., 2011). The dotted lines are found at h2 = 0, indicating that none of the phenotypic variance is explained by the genetic data. The proportion of the total phenotypic variance explained by each PC is indicated in parentheses FIGURE 8 \| SNP heritability (h2) of leaf phenotypes. Values represent the additive genetic variance (Vg) divided by the phenotypic variance (Vp) with a standard error as calculated using GCTA (Yang et al., 2011). The dotted lines are found at h2 = 0, indicating that none of the phenotypic variance is explained by the genetic data. The proportion of the total phenotypic variance explained by each PC is indicated in parentheses. FIGURE 8 \| SNP heritability (h2) of leaf phenotypes. Values represent the additive genetic variance (Vg) divided by the phenotypic variance (Vp) with a standard error as calculated using GCTA (Yang et al., 2011). The dotted lines are found at h2 = 0, indicating that none of the phenotypic variance is explained by the genetic data. Frontiers in Plant Science \| www.frontiersin.org CONCLUSIONS It is clear from our work that variation in apple leaf shape and size are under genetic control. Further, high genomic prediction and heritability estimates for higher morphometric PCs indicate that techniques such as EFDs and PH are capturing heritable biological variation that will be missed if researchers restrict leaf shape estimates to linear measurements. Additionally, a better understanding of the variation in leaf shape and size in apple could have important implications for canopy management, where light exposure is crucial to ﬂowering (Dennis, 2003). Ultimately, through the ﬁrst in-depth study of leaf shape in apple, we uncover allometry between accessions and species, as well as evidence that complex and heritable phenotypes can be captured using comprehensive morphometric techniques. Figure S5 \| Genomic prediction accuracy (r) (A) and correlation between genomic prediction results and SNP heritability estimates (h2) for all leaf phenotypes (B). Genomic prediction accuracies represent the average correlation (± standard deviation) between observed and predicted phenotype scores, based on 5-fold cross-validation with 3 iterations. Dotted red lines indicate the minimum and maximum prediction average accuracy (r) achieved using 1,000 randomly generated phenotypes. The percent variance explained by each PC was calculated prior to REML-adjustment and is indicated in parentheses. Table S1 \| All leaf phenotypes assessed in apple, following REML-adjustment. Accessions are identiﬁed by their unique “apple id.” Species information for these accessions is available in Table S2. Table S2 \| Species (M. domestica, M. sieversii, or unknown) for all accessions assessed in this study. Table S3 \| Correlation between leaf phenotypes as well as Bonferroni-adjusted p-values. Pearson’s product moment correlation coefﬁcients are reported. These results are visualized in Figure 2. AUTHOR CONTRIBUTIONS Table S4 \| Comparison of leaf phenotypes between accessions based on species. Bonferroni-adjusted p-values resulting from a Mann-Whitney U-test estimating the difference between accessions based on species (M. domestica/M. sieversii) for the leaf phenotypes examined. ZM, DC, and SM designed the research. ZM and ML performed the research. ZM analyzed the data. DC and SM supervised the experiments. ZM, ML, DC, and SM wrote the article. ZM, DC, and SM designed the research. ZM and ML performed the research. ZM analyzed the data. DC and SM supervised the experiments. ZM, ML, DC, and SM wrote the article. Table S5 \| Positional information for signiﬁcant GWAS results. Additional information about signiﬁcant SNPs are included such as p-value, marker R2, minor and major allele, minor and major effect and MAF. DISCUSSION To discern the genetic contributions to leaf shape, we paired both linear and comprehensive morphometric estimates of shape with genome-wide SNP data. There are examples of January 2018 \| Volume 8 \| Article 2185 11 Complex and Heritable Apple Leaf Shapes Migicovsky et al. in genome assembly and increases in marker number will aid to further reveal the genetic architecture of apple leaf shape variation. present members of the Myles Lab for their work in maintaining the apple orchard. This research was supported in part, thanks to funding from the Canada Research Chairs program (SM), the National Sciences and Engineering Research Council of Canada (SM, ZM), Genome Canada (SM) and a Killam Predoctoral Scholarship from Dalhousie University (ZM). This research was published originally as part of the doctoral thesis of ZM (Migicovsky, 2017). Lastly, we investigated the degree to which leaf shape is heritable and can be predicted using genome-wide SNP data. We ﬁnd that the genomic prediction accuracies of the primary axes of leaf shape variation are similar to previously reported estimates for fruit width (0.48) and length (0.47), indicating that leaf shape is as heritable as fruit shape (Migicovsky et al., 2016). In combination with few signiﬁcant GWAS results, high SNP heritability estimates support a polygenic basis for leaf shape. Aspect ratio was identiﬁed as the primary source of variation in leaf shape in apple and had the highest genomic prediction and heritability estimates, indicating that there is a genetic, heritable basis for allometric variation in apple. Further, although the ﬁrst 5 PCs for both EFDs and PH explain the majority of the variation in apple leaf shape, most PCs from 1 to 14 have heritability estimates above 0.20 and may still represent crucial diﬀerences in leaf shape from an ecological, evolutionary, or agricultural perspective. Thus, while our ability to detect the primary axes of variation in leaf shape using genome-wide data is expected, our observation that higher level PCs are also heritable conﬁrms that these comprehensive morphometric methods capture biologically meaningful variation that would be missed by linear measurements alone. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2017. 02185/full#supplementary-material Figure S1 \| Distribution of leaf phenotypes following REML-adjustment. N is equal to the total number of unique samples. Figure S2 \| Visualization of contributions of each annulus kernel to PH PC1. Annulus kernels 6, 7 and 16 contribute the most to leaf shape according to PH PC1. The placement of each annulus kernel is visualized on a leaf representing the minimum and maximum value along PC1 (A). The contribution to PC1 of each of the 16 annulus kernels is also shown (B). Figure S2 \| Visualization of contributions of each annulus kernel to PH PC1. Annulus kernels 6, 7 and 16 contribute the most to leaf shape according to PH PC1. The placement of each annulus kernel is visualized on a leaf representing the minimum and maximum value along PC1 (A). The contribution to PC1 of each of the 16 annulus kernels is also shown (B). Figure S3 \| Comparison of morphometric EFD and PH PCs 1 to 5. Correlation between ﬁrst 5 PCs, estimated using Pearson’s correlation, including R2 and Bonferroni corrected p-values based on Figure 4, Table S3. Figure S4 \| GWAS results for all 24 leaf phenotypes examined. Manhattan and QQ plots are included for each phenotype. The QQ-plot shows both the results of a naive GWAS (Pearson correlation) and the results from applying the mixed model. P-values are log-transformed and the threshold for signiﬁcance is simpleM-corrected and indicated by a horizontal dotted line. Chromosome R indicates SNPs found on contigs unanchored to the reference genome. REFERENCES Sources of variation in leaf shape among two populations of achillea lanulosa. Genetics 130, 385–394. Hijmans, R. J. (2016). raster: Geographic Data Analysis and Modeling. R package version 2.5–8. Cartolano, M., Pieper, B., Lempe, J., Tattersall, A., Huijser, P., Tresch, A., et al. (2015). Heterochrony underpins natural variation in Cardamine hirsuta leaf form. Proc. Natl. Acad. Sci. U.S.A. 112, 10539–10544. doi: 10.1073/pnas.1419791112 Iwata, H., and Ukai, Y. (2002). SHAPE: a computer program package for quantitative evaluation of biological shapes based on elliptic Fourier descriptors. J. Hered. 93, 384–385. doi: 10.1093/jhered/93.5.384 Chagné, D., Carlisle, C. M., Blond, C., Volz, R. K., Whitworth, C. J., Oraguzie, N. C., et al. (2007). Mapping a candidate gene (MdMYB10) for red ﬂesh and foliage colour in apple. BMC Genomics 8:212. doi: 10.1186/1471-2164-8-212 Jöst, M., Hensel, G., Kappel, C., Druka, A., Sicard, A., Hohmann, U., et al. (2016). The INDETERMINATE DOMAIN protein BROAD LEAF1 limits barley leaf width by restricting lateral proliferation. Curr. Biol. 26, 903–909. doi: 10.1016/j.cub.2016.01.047 Chitwood, D. H., Headland, L. R., Kumar, R., Peng, J., Maloof, J. N., and Sinha, N. R. (2012). The developmental trajectory of leaﬂet morphology in wild tomato species. Plant Physiol. 158, 1230–1240. doi: 10.1104/pp.111.192518 Jung, S., Ficklin, S. P., Lee, T., Cheng, C. H., Blenda, A., Zheng, P., et al. (2014). The Genome Database for Rosaceae (GDR): year 10 update. Nucleic Acids Res. 42, D1237–D1244. doi: 10.1093/nar/gkt1012 Chitwood, D. H., Kumar, R., Headland, L. R., Ranjan, A., Covington, M. F., Ichihashi, Y., et al. (2013). A quantitative genetic basis for leaf morphology in a set of precisely deﬁned tomato introgression lines. Plant Cell 25, 2465–2481. doi: 10.1105/tpc.113.112391 Khan, M. A., Olsen, K. M., Sovero, V., Kushad, M. M., and Korban, S. S. (2014). Fruit quality traits have played critical roles in domestication of the apple. Plant Genome 7, 1–18. doi: 10.3835/plantgenome2014.04.0018 Kimura, S., Koenig, D., Kang, J., Yoong, F. Y., and Sinha, N. (2008). Natural variation in leaf morphology results from mutation of a novel KNOX gene. Curr. Biol. 18, 672–677. doi: 10.1016/j.cub.2008.04.008 Chitwood, D. H., and Otoni, W. C. (2017). Morphometric analysis of Passiﬂora leaves: the relationship between landmarks of the vasculature and elliptical Fourier descriptors of the blade. Gigascience 6, 1–13. doi: 10.1093/gigascience/gix070 Klein, L. L., Caito, M., Chapnick, C., Kitchen, C., O’hanlon, R., Chitwood, D. H., et al. (2017). REFERENCES Elshire, R. J., Glaubitz, J. C., Sun, Q., Poland, J. A., Kawamoto, K., Buckler, E. S., et al. (2011). A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species. PLoS ONE 6:e19379. doi: 10.1371/journal.pone.00 19379 Abràmoﬀ, M. D., Magalhães, P. J., and Ram, S. J. (2004). Image processing with ImageJ. Biophoton. Int. 11, 36–42. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990). Basic local alignment search tool. J. Mol. Biol. 215, 403–410. doi: 10.1016/S0022-2836(05)80360-2 Espley, R. V., Brendolise, C., Chagné, D., Kutty-Amma, S., Green, S., Volz, R., et al. (2009). Multiple repeats of a promoter segment causes transcription factor autoregulation in red apples. Plant Cell 21, 168–183. doi: 10.1105/tpc.108.059329 Andres, R. J., Coneva, V., Frank, M. H., Tuttle, J. R., Samayoa, L. F., Han, S. W., et al. (2017). Modiﬁcations to a LATE MERISTEM IDENTITY1 gene are responsible for the major leaf shapes of Upland cotton (Gossypium hirsutum L.). Proc. Natl. Acad. Sci. U.S.A. 114, E57–E66. doi: 10.1073/pnas.16135 93114 Food and Agriculture Organization of the United Nations (2017). FAOSTAT. Available online at: http://www.fao.org/faostat/en/#data (Accessed Mar 9, 2017). Gao, X., Becker, L. C., Becker, D. M., Starmer, J. D., and Province, M. A. (2010). Avoiding the high Bonferroni penalty in genome-wide association studies. Genet. Epidemiol. 34, 100–105. doi: 10.1002/gepi.20430 Bassett, C. L., Glenn, D. M., Forsline, P. L., Wisniewski, M. E., and Farrell, R. E. (2011). Characterizing water use eﬃciency and water deﬁcit responses in apple (Malus × domestica Borkh. and Malus sieversii Ledeb.) M. Roem. HortScience 46, 1079–1084. Gao, X., Starmer, J., and Martin, E. R. (2008). A multiple testing correction method for genetic association studies using correlated single nucleotide polymorphisms. Genet. Epidemiol. 32, 361–369. doi: 10.1002/gepi. 20310 Bates, D., Mächler, M., Bolker, B., and Walker, S. (2015). Fitting linear mixed- eﬀects models using lme4. J. Stat. Softw. 67, 1–48. doi: 10.18637/jss.v067.i01 Glaubitz, J. C., Casstevens, T. M., Lu, F., Harriman, J., Elshire, R. J., Sun, Q., et al. (2014). TASSEL-GBS: a high capacity genotyping by sequencing analysis pipeline. PLoS ONE 9:e90346. doi: 10.1371/journal.pone.0090346 Bonhomme, V., Picq, S., Gaucherel, C., and Claude, J. (2014). Momocs: outline analysis using R. J. Stat. Softw. 56, 1–24. doi: 10.18637/jss.v056.i13 Bradbury, P. J., Zhang, Z., Kroon, D. E., Casstevens, T. M., Ramdoss, Y., and Buckler, E. S. (2007). TASSEL: software for association mapping of complex traits in diverse samples. Bioinformatics 23, 2633–2635. doi: 10.1093/bioinformatics/btm308 Gurevitch, J. (1992). ACKNOWLEDGMENTS Table S6 \| Genes found within ±5 kb of SNPs with signiﬁcant GWAS associations. Results are listed according to the Genome Database for Rosaceae GBrowse (accessed January 27, 2017). Overlapping mRNA, length, contig, GO category, GO term accession, GO term name, InterPro Term, InterPro Description, and NCBI sequence with Max Score when BLASTed using NCBI are reported. Table S6 \| Genes found within ±5 kb of SNPs with signiﬁcant GWAS associations. Results are listed according to the Genome Database for Rosaceae GBrowse (accessed January 27, 2017). Overlapping mRNA, length, contig, GO category, GO term accession, GO term name, InterPro Term, InterPro Description, and NCBI sequence with Max Score when BLASTed using NCBI are reported. We would like to acknowledge Gavin Douglas and Sherry Fillmore for their help setting up the statistical design of the orchard and SNP-calling pipeline. We also thank all past and Frontiers in Plant Science \| www.frontiersin.org January 2018 \| Volume 8 \| Article 2185 12 Complex and Heritable Apple Leaf Shapes Migicovsky et al. Table S8 \| SNP heritability (h2) for leaf phenotypes. h2 represents the genetic variance (Vg) divided by the phenotypic variance (Vp). The standard error (SE) is also reported. These results are visualized in Figure 8. Table S8 \| SNP heritability (h2) for leaf phenotypes. h2 represents the genetic variance (Vg) divided by the phenotypic variance (Vp). The standard error (SE) is also reported. These results are visualized in Figure 8. Table S7 \| Genomic prediction accuracies (r) for leaf phenotypes. r_avg represents the average correlation between observed and predicted phenotype scores, based on 5-fold cross-validation with 3 iterations. The standard deviation (r_sd) is also reported. These results are visualized in Figure S5A. Figure 8. REFERENCES Digital morphometrics of two North American grapevines (Vitis: Vitaceae) quantiﬁes leaf variation between species, within species, and among individuals. Front. Plant Sci. 8:373. doi: 10.3389/fpls.2017.00373 Chitwood, D. H., Ranjan, A., Martinez, C. C., Headland, L. R., Thiem, T., Kumar, R., et al. (2014). A modern ampelography: a genetic basis for leaf shape and venation patterning in grape. Plant Physiol. 164, 259–272. doi: 10.1104/pp.113.229708 Langlade, N. B., Feng, X., Dransﬁeld, T., Copsey, L., Hanna, A. I., Thébaud, C., et al. (2005). Evolution through genetically controlled allometry space. Proc. Natl. Acad. Sci. U.S.A. 102, 10221–10226. doi: 10.1073/pnas.0504210102 Cooney, C. R., Bright, J. A., Capp, E. J. R., Chira, A. M., Hughes, E. C., Moody, C. J. A., et al. (2017). Mega-evolutionary dynamics of the adaptive radiation of birds. Nature 542, 344–347. doi: 10.1038/nature21074 Lenth, R. V. (2016). Least-squares means: the R package lsmeans. J. Stat. Softw. 69, 1–33. doi: 10.18637/jss.v069.i01 Currie, A. J., Ganeshanandam, S., Noiton, D. A., Garrick, D., Shelbourne, C. J. A., and Oraguzie, N. (2000). Quantitative evaluation of apple (Malus × domestica Borkh.) fruit shape by principal component analysis of Fourier descriptors. Euphytica 111, 219–227. doi: 10.1023/A:1003862525814 Li, H., and Durbin, R. (2009). Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760. doi: 10.1093/bioinformatics/btp324 Danecek, P., Auton, A., Abecasis, G., Albers, C. A., Banks, E., Depristo, M. A., et al. (2011). The variant call format and VCFtools. Bioinformatics 27, 2156–2158. doi: 10.1093/bioinformatics/btr330 Li, M., An, H., Angelovici, R., Bagaza, C., Batushansky, A., Clark, L., et al. (2017a). Persistent homology demarcates a leaf morphospace. bioRxiv. 1–23. doi: 10.1101/151712 Dennis, F. Jr. (2003). “Flowering, pollination and fruit set and development,” in Apples: Botany, Production and Uses, eds D. C. Ferree and I. J. Warrington (Wallingford, UK: CABI Publishing), 153–166. Li, M., Duncan, K., Topp, C. N., and Chitwood, D. H. (2017b). Persistent homology and the branching topologies of plants. Am. J. Bot. 104, 349–353. doi: 10.3732/ajb.1700046 January 2018 \| Volume 8 \| Article 2185 Frontiers in Plant Science \| www.frontiersin.org 13 Complex and Heritable Apple Leaf Shapes Migicovsky et al. Li, M., Frank, M. H., Coneva, V., Mio, W., Topp, C. N., and Chitwood, D. H. (2017c). Persistent homology: a tool to universally measure plant morphologies across organs and scales. bioRxiv. 1–25. doi: 10.1101/ 104141 Tian, F., Bradbury, P. J., Brown, P. J., Hung, H., Sun, Q., Flint-Garcia, S., et al. (2011). REFERENCES Genome-wide association study of leaf architecture in the maize nested association mapping population. Nat. Genet. 43, 159–162. doi: 10.1038/ng.746 Tsuge, T., Tsukaya, H., and Uchimiya, H. (1996). Two independent and polarized processes of cell elongation regulate leaf blade expansion in Arabidopsis thaliana (L.) Heynh. Development 122, 1589–1600. Liebhard, R., Kellerhals, M., Pfammatter, W., Jertmini, M., and Gessler, C. (2003). Mapping quantitative physiological traits in apple (Malus × domestica Borkh.). Plant Mol. Biol. 53, 511–526. doi: 10.1023/A:10248865 00979 Velasco, R., Zharkikh, A., Aﬀourtit, J., Dhingra, A., Cestaro, A., Kalyanaraman, A., et al. (2010). The genome of the domesticated apple (Malus × domestica Borkh.). Nat. Genet. 42, 833–839. doi: 10.1038/ng.654 Lohmann, G. (1983). Eigenshape analysis of microfossils: a general morphometric procedure for describing changes in shape. Math. Geol. 15, 659–672. doi: 10.1007/BF01033230 Visser, T., and Verhaegh, J. (1978). Inheritance and selection of some fruit characters of apple. II. The relation between leaf and fruit pH as a basis for preselection. Euphytica 27, 761–765. doi: 10.1007/BF00023712 Maeda, H., Akagi, T., and Tao, R. (2018). Quantitative characterization of fruit shape and its diﬀerentiation pattern in diverse persimmon (Diospyros kaki) cultivars. Sci. Hortic. 228, 41–48. doi: 10.1016/j.scienta.2017.10.006 Warton, D. I., Duursma, R. A., Falster, D. S., and Taskinen, S. (2012). smatr 3- an R package for estimation and inference about allometric lines. Methods Ecol. Evol. 3, 257–259. doi: 10.1111/j.2041-210X.2011.00153.x Migicovsky, Z. (2017). Patterns of Genomic and Phenomic Diversity in Apple and Grape. PhD thesis, Dalhousie University. Wickham, H. (2009). ggplot2: Elegant Graphics for Data Analysis. New York, NY: Springer-Verlag. Migicovsky, Z., Gardner, K. M., Money, D., Sawler, J., Bloom, J. S., Moﬀett, P., et al. (2016). Genome to phenome mapping in apple using historical data. Plant Genome 9, 1–15. doi: 10.3835/plantgenome2015.11.0113 Wünsche, J. N., and Lakso, A. N. (2000). The relationship between leaf area and light interception by spur and extension shoot leaves and apple orchard productivity. HortScience 35, 1202–1206. Mohammadi, M., Tiede, T., and Smith, K. P. (2015). PopVar: a genome- wide procedure for predicting genetic variance and correlated response in biparental breeding populations. Crop Sci. 55, 2068–2077. doi: 10.2135/cropsci2015.01.0030 Wünsche, J. N., Palmer, J. W., and Greer, D. H. (2000). Eﬀects of crop load on fruiting and gas-exchange characteristics of “Braeburn”/M. 26 apple trees at full canopy. J. Am. Soc. Hortic. Sci. 125, 93–99. Yang, J., Lee, S. H., Goddard, M. E., and Visscher, P. M. (2011). Frontiers in Plant Science \| www.frontiersin.org January 2018 \| Volume 8 \| Article 2185 REFERENCES GCTA: a tool for genome-wide complex trait analysis. Am. J. Hum. Genet. 88, 76–82. doi: 10.1016/j.ajhg.2010.11.011 Money, D., Migicovsky, Z., Gardner, K., and Myles, S. (2017). LinkImputeR: user-guided genotype calling and imputation for non-model organisms. BMC Genomics 18:523. doi: 10.1186/s12864-017-3873-5 Purcell, S. (2009). PLINK v.1.07. Available online at: http://zzz.bwh.harvard.edu/ plink/ Zhang, Z., Ersoz, E., Lai, C.-Q., Todhunter, R. J., Tiwari, H. K., Gore, M. A., et al. (2010). Mixed linear model approach adapted for genome-wide association studies. Nat. Genet. 42, 355–360. doi: 10.1038/ng.546 Purcell, S., Neale, B., Todd-Brown, K., Thomas, L., Ferreira, M. A., Bender, D., et al. (2007). PLINK: a tool set for whole-genome association and population- based linkage analyses. Am. J. Hum. Genet. 81, 559–575. doi: 10.1086/ 519795 Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. R Core Team (2016). R: A Language and Environment for Statistical Computing. Vienna: R Foundation for Statistical Computing. Copyright © 2018 Migicovsky, Li, Chitwood and Myles. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Schwarz, D., and Kläring, H.-P. (2001). Allometry to estimate J. Plant Nutr. 24, 1291–1309. doi: 10.1081/PLN-100106982 Talwara, S., Grout, B. W. W., and Toldam-Andersen, T. B. (2013). Modiﬁcation of leaf morphology and anatomy as a consequence of columnar architecture in domestic apple (Malus × domestica Borkh.) trees. Sci. Hortic. 164, 310–315. doi: 10.1016/j.scienta.2013.08.025 January 2018 \| Volume 8 \| Article 2185 Frontiers in Plant Science \| www.frontiersin.org 14
https://openalex.org/W4285793373	https://portal.findresearcher.sdu.dk/files/205613263/s00702_022_02530_4.pdf	English	null	Neuromelanin granules of the substantia nigra: proteomic profile provides links to tyrosine hydroxylase, stress granules and lysosomes	Journal of neural transmission	2,022	cc-by	11,259	Citation for pulished version (APA): Wulf, M., Barkovits, K., Schork, K., Eisenacher, M., Riederer, P., Gerlach, M., Eggers, B., & Marcus, K. (2022). Neuromelanin granules of the substantia nigra: proteomic profile provides links to tyrosine hydroxylase, stress granules and lysosomes. Journal of Neural Transmission, 129(10), 1257-1270. https://doi.org/10.1007/s00702- 022-02530-4 Download date: 24. Oct. 2024 University of Southern Denmark Neuromelanin granules of the substantia nigra proteomic profile provides links to tyrosine hydroxylase, stress granules and lysosomes Wulf, Maximilian; Barkovits, Katalin; Schork, Karin; Eisenacher, Martin; Riederer, Peter; Gerlach Manfred; Eggers Britta; Marcus Katrin proteomic profile provides links to tyrosine hydroxylase, stress granules and lysosomes Wulf, Maximilian; Barkovits, Katalin; Schork, Karin; Eisenacher, Martin; Riederer, Peter; Gerlach, Manfred; Eggers, Britta; Marcus, Katrin y Wulf, Maximilian; Barkovits, Katalin; Schork, Karin; Eisenacher, Martin; Riederer, Peter; Gerlach, Manfred; Eggers, Britta; Marcus, Katrin Go to publication entry in University of Southern Denmark's Research Portal Terms of use This work is brought to you by the University of Southern Denmark. Unless otherwise specified it has been shared according to the terms for self-archiving. If no other license is stated, these terms apply: Abstract Neuromelanin is a black-brownish pigment, present in so-called neuromelanin granules (NMGs) in the cell bodies of dopa- minergic neurons in the substantia nigra (SN) pars compacta. These neurons are lost in neurodegenerative diseases, such as Parkinson’s disease and dementia with Lewy bodies. Although it is known that lipids, proteins, and environmental toxins accumulate in NMGs, the function of NMGs has not yet been finally clarified as well as their origin and the synthesis of neuromelanin. We, therefore, isolated NMGs and surrounding SN tissue from control patients by laser microdissection and analyzed the proteomic profile by tandem mass spectrometry. With our improved workflow, we were able to (1) strengthen the regularly reported link between NMGs and lysosomes, (2) detect tyrosine hydroxylase to be highly abundant in NMGs, which may be related to neuromelanin synthesis and (3) indicate a yet undescribed link between stress granules (SGs) and NMGs. Based on our findings, we cautiously hypothesize, that SGs may be the origin of NMGs or form in close proximity to them, potentially due to the oxidative stress caused by neuromelanin-bound metals. Keywords Neuromelanin granules · Substantia nigra pars compacta · Laser microdissection · Mass spectrometry · Stress granules · Neuromelanin synthesis Neuromelanin granules of the substantia nigra: proteomic profile provides links to tyrosine hydroxylase, stress granules and lysosomes Maximilian Wulf1,2 · Katalin Barkovits1,2 · Karin Schork1,2 · Martin Eisenacher1,2 · Peter Ried Manfred Gerlach5 · Britta Eggers1,2 · Katrin Marcus1,2 Received: 28 April 2022 / Accepted: 5 July 2022 © The Author(s) 2022 Received: 28 April 2022 / Accepted: 5 July 2022 © The Author(s) 2022 Terms of use • You may download this work for personal use only. y p y • You may not further distribute the material or use it y y p • You may freely distribute the URL identifying this open access version at this document breaches copyright please contact us providing details and we will investigate your claim. enquiries to puresupport@bib.sdu.dk If you believe that this document breaches copyright please contact us providing details and we will in Please direct all enquiries to puresupport@bib.sdu.dk Journal of Neural Transmission https://doi.org/10.1007/s00702-022-02530-4 NEUROLOGY AND PRECLINICAL NEUROLOGICAL STUDIES - ORIGINAL ARTICLE NEUROLOGY AND PRECLINICAL NEUROLOGICAL STUDIES - ORIGINAL ARTICLE 5 Center of Mental Health, Department of Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy, University Hospital of Wuerzburg, University of Wuerzburg, Wuerzburg, Germany Introduction Therefore, the origin and role of NMGs are still widely discussed in the scientific community and need further clarification, as well as the synthesis of NM. 1. Due to similarities between NM and melanin present in human skin, one hypothesis believes the enzyme tyrosi- nase, responsible for melanin synthesis (Raper 1928; Mason 1965; Sánchez-Ferrer et al. 1995), to play an essential role in NM synthesis (Carballo-Carbajal et al. 2019). However, so far, only tyrosinase–mRNA could be detected and no evidence was found for the presence of tyrosinase protein in human SN tissue (Miranda et al. 1984; Xu et al. 1997; Ikemoto et al. 1998; Tief et al. 1998; Tribl et al. 2007). To gain insights into these important aspects of NMG biology, an in-depth characterization of NMGs and SN tis- sue surrounding NMGs (hereafter called SNSurr.) is indis- pensable. Therefore, we established a revised and optimized version (Wulf et al. 2021) of our previously published work- flow (Plum et al. 2016), optimizing NMG collection and in-depth proteomic characterization, leading to the identifi- cation of 170% more proteins and an improved quantification of protein abundances. In total, we were able to identify 2693 proteins in all of our measured samples of SNSurr. tis- sue and NMGs. 2. A second enzyme believed to potentially play a role in NM synthesis is tyrosine hydroxylase (TH), the rate-lim- iting enzyme in the catecholamine–synthesis pathway, as NM is mainly restricted to brain regions containing catecholaminergic neurons (Saper and Petito 1982; Gas- par et al. 1983; Halliday et al. 1988; Hirsch et al. 1988). Indeed, the presence of TH protein in human brain tis- sue was reported in several publications (Mogi et al. 1988; Nagatsu et al. 2019), as TH is commonly used as a marker for dopaminergic neurons in immunological stainings (Robert and Meghan 2012). Nevertheless, TH is also present in the SN of rodents, such as mice and rats, in which no production of NM can be observed (Marsden 1961; Aumann et al. 2011). Even TH-overex- pression did not lead to the induction of NM synthesis in mice (Mor et al. 2017). 2. A second enzyme believed to potentially play a role in NM synthesis is tyrosine hydroxylase (TH), the rate-lim- iting enzyme in the catecholamine–synthesis pathway, as NM is mainly restricted to brain regions containing catecholaminergic neurons (Saper and Petito 1982; Gas- par et al. 1983; Halliday et al. 1988; Hirsch et al. Introduction Neuromelanin (NM) is a black-brownish pigment, mainly observable in the cell bodies of dopaminergic neurons in the substantia nigra (SN) pars compacta. In the human brain, NM is stored in membrane-surrounded neuromelanin gran- ules (NMGs) in association with proteins and lipids. These NMGs are first observable in infants while increasing in number and density of pigmentation during aging (Halli- day et al. 2006). Investigating the molecular composition of NMGs is of high relevance, since the selective loss of NMG- containing dopaminergic neurons in the SN leads to cardinal symptoms in neurodegenerative diseases, such as Parkin- son’s disease (PD) and dementia with Lewy bodies (DLB). Therefore, robust and reproducible OMICs techniques are of immense value, enabling the study of genes, transcripts, proteins and lipids. Nevertheless, as NMGs are absent in common laboratory animals (Marsden 1961), researchers rely on human post mortem brain tissue for their studies, which are limited as only a static view of NMG composition can be observed. Thus, the mechanism of NM synthesis as well as the origin of NMGs have not been finally clarified Britta Eggers and Katrin Marcus have contributed equally to this work. * Katrin Marcus katrin.marcus@rub.de 1 Medizinisches Proteom‑Center, Medical Faculty, Ruhr- University Bochum, Bochum, Germany 2 Medical Proteome Analysis, Center for Proteindiagnostics (PRODI), Ruhr-University Bochum, Bochum, Germany 3 Center of Mental Health, Clinic and Policlinic for Psychiatry, Psychosomatics and Psychotherapy, University Hospital Wuerzburg, Margarete‑Hoeppel‑Platz 1, 97080 Wuerzburg, Germany 4 Psychiatry Department of Clinical Research, University of Southern Denmark Odense University Hospital, Odense C, Denmark 5 Center of Mental Health, Department of Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy, University Hospital of Wuerzburg, University of Wuerzburg, Wuerzburg, Germany 1 Medizinisches Proteom‑Center, Medical Faculty, Ruhr- University Bochum, Bochum, Germany 2 Medical Proteome Analysis, Center for Proteindiagnostics (PRODI), Ruhr-University Bochum, Bochum, Germany 3 Center of Mental Health, Clinic and Policlinic for Psychiatry, Psychosomatics and Psychotherapy, University Hospital Wuerzburg, Margarete‑Hoeppel‑Platz 1, 97080 Wuerzburg, Germany 4 Psychiatry Department of Clinical Research, University of Southern Denmark Odense University Hospital, Odense C, Denmark 5 Center of Mental Health, Department of Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy, University Hospital of Wuerzburg, University of Wuerzburg, Wuerzburg, Germany (0121 3456789) 3 M. Wulf et al. (Zecca et al. 2001; Vila 2019). Concerning the NM synthe- sis, several hypotheses have been raised: lead to neuroinflammation when released (Oberländer et al. 2011; Zhang et al. 2011; Cebrián et al. 2014). Ethical statement Flash frozen, human post mortem SN tissue slices of 5 µm thickness were provided on 1.0 PEN-membrane glass slides (Carl Zeiss Microscopy GmbH, Göttingen, Germany) by the Navarrabiomed Biobank (Pamplona, Spain). The use of human brain tissue was approved by the ethics committee of the Ruhr-University Bochum, Germany (file number 4760- 13), according to German regulations and guidelines. Furthermore, first studies elucidating the molecular composition of NMGs on the level of proteins resulted in hypotheses on their potential origin. In-depth proteomic characterization, utilizing either density gradient purifica- tion via ultracentrifugation (Tribl et al. 2006; Zucca et al. 2018) or selective enrichment via laser microdissection (Plum et al. 2016), revealed an increased abundance of lysosomal proteins. These findings led to the assumption that NMGs may play a protective role in the human brain shielding toxic compounds, such as metals and environ- mental toxins from the cytoplasm (D'Amato et al. 1986; Lindquist et al. 1988; Aime et al. 1994; Zecca et al. 1996; Bridelli et al. 1999; Double et al. 2003; Tribl et al. 2006; Bohic et al. 2008; Biesemeier et al. 2016; Plum et al. 2016; Zucca et al. 2018). Other studies state that this neuroprotec- tive role may transition ultimately resulting in neurotoxic- ity, as the NMG composition of highly toxic products can Introduction 1988). Indeed, the presence of TH protein in human brain tis- sue was reported in several publications (Mogi et al. 1988; Nagatsu et al. 2019), as TH is commonly used as a marker for dopaminergic neurons in immunological stainings (Robert and Meghan 2012). Nevertheless, TH is also present in the SN of rodents, such as mice and rats, in which no production of NM can be observed (Marsden 1961; Aumann et al. 2011). Even TH-overex- pression did not lead to the induction of NM synthesis in mice (Mor et al. 2017). Thus, the present study provides the largest proteomic data set focusing on NMG biology. Concerning NM syn- thesis, we provide evidence for TH to be of high abundance in NMGs, while we were not able to detect tyrosinase in any of our analyzed samples. In addition, we strengthen the assumed link between NMGs and lysosomes based on their proteomic profile. For the first time, we revealed an enrichment of ribosomal and translation-associated proteins in NMGs, pointing towards a potential new aspect of their biology. With our results, we contribute to the knowledge of NMG origin and function, opening the possibility to reconsider and extend current hypotheses. 3. A third hypothesis assumes an enzyme-free mechanism in which autooxidation of dopamine is expected to be the main process in NM synthesis (Fornstedt et al. 1986; Sulzer and Zecca 2000; Zucca et al. 2017). However, based on this assumption, a formation of NM should be observable in the SN of rats and mice as well. Laser microdissection and sample preparation samples were measured over the course of all LC–MS/MS measurements to check for technical variances and total ion chromatogram as well as basepeak intensities were checked to ensure identical sample load. The LC–MS/MS measure- ments were performed on an Ultimate 3000 RSLC nano LC system (Dionex, Idstein, Germany) coupled to an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific). At first, peptides were loaded on a pre-column (Acclaim PepMap nanoViper, Thermo Fisher Scientific; 100 µm × 2 cm, 5 µm particle size) and washed for 7 min with 0.1% TFA at a flow rate of 30 µl/min. Subsequently, the pre-column was connected to an analytical C18 column (Acclaim PepMap nanoViper, Thermo Fisher Scientific; 75 µm × 50 cm, 2 µm particle size). Separation of peptides was performed at a flow rate of 400 nL/min with a gradient starting with 95% solution A (0.1% FA) and 5% solution B (84% acetonitrile, 0.1% FA). The concentration of solution B was increased up to 30% after 105 min, then within 2 min to 95% and maintained at that level for 3 further minutes. Subsequently, the column was adjusted back to 5% solu- tion B. These gradient settings were used for data dependent acquisition (DDA)-experiments, as well as for parallel reac- tion monitoring (PRM)-experiments. The nano LC-system was directly coupled to the electrospray ionization source (Thermo Fisher Scientific) of the Orbitrap Fusion Lumos Tribrid mass spectrometer. The SN tissue slices were stored at − 80 °C until further use. The isolation of NMGs and SNSurr. tissue was performed as previously reported (Wulf et al. 2021). In brief, NMGs covering a tissue area of 500,000 µm2 were isolated at 400 x-magnification and collected in water-filled caps of non- adhesive microtubes (MicroTube 500, Carl Zeiss Micros- copy GmbH) using a PALM Micro Beam (P.A.L.M.-Sys- tem, Carl Zeiss Microscopy GmbH). Subsequently, SNSurr. tissue was selected at 50 x-magnification and an area of 1,000,000 µm2 was collected in a fresh tube. All samples were stored at -80 °C until further use. Sample preparation The sample preparation was performed as previously reported (Wulf et al. 2021). Briefly, samples were treated with formic acid (FA) and sonicated. After a drying step in a vacuum-centrifuge (Concentrator plus, Eppendorf AG, Hamburg, Germany), samples were refilled with ammonium bicarbonate, reduced with dithiothreitol and acetylated with iodoacetamide. Trypsin was added to the samples according to the collected tissue area and digestion was performed at 37 °C overnight. The digestion was stopped by adding trif- luoroacetic acid (TFA). At last, samples were vacuum-dried and peptides were stored in 20 µl of 0.1% TFA at -80 °C. Subjects SN tissue slices of five control (CTRL) patients, which are identified as controls based on a combination of clinical and pathological measures by the Navarrabiomed Brain Bank, were used. Additional information can be found in Table 1. SN tissue of two patients of the study cohort as well as SN tissue of two additional independent CTRL patients was used for verification experiments (Table 1, highlighted in italic). 1 3 3 Neuromelanin granules of the substantia nigra: proteomic profile provides links to tyrosine… Neuromelanin granules of the substantia nigra: proteomic profile provides links to tyrosine… Table 1 Composition of study group Ø Age ± SD average age and standard deviation; PMI post mortem interval, Ø PMI (h) ± SD average post mortem interval and standard deviation. Samples from patients highlighted in italic were used for verifica- tion experiments Group Sex Age (in years) Ø Age ± SD PMI (h) Ø PMI (h) ± SD Control (n = 5) Male 91 78.8 ± 12.2 4:00 4:23 ± 2:01 Male 66 8:00 Female 66 3:20 Female 82 3:20 Female 89 3:20 Control Verifica- tion (n = 2) Male 79 75.5 ± 4.9 11:00 10:00 ± 1:25 Male 72 9:00 Ø Age ± SD average age and standard deviation; PMI post mortem interval, Ø PMI (h) ± SD average post mortem interval and standard deviation. Samples from patients highlighted in italic were used for verifica- tion experiments Targeted mass spectrometry decoy database. Peptide identification was carried out with an initial allowed precursor mass deviation up to 5 parts per million (ppm) and an allowed fragment mass deviation of 20 ppm. Match between runs was enabled to enhance and optimize peptide identification. Label free quantifica- tion (LFQ) was performed using the MaxQuant LFQ nor- malization, whereby the Top10 unique and razor peptides were assessed for protein quantification (Cox et al. 2014). In addition, intensity based absolute quantification (iBAQ) value calculation was enabled. IBAQ values were normal- ized for each sample and multiplied by 1000 to obtain per mille values. Of these normalized iBAQ values, mean values were created for each sample group (NMG, SNSurr.). Protein groups (hereafter referred to as proteins) with normalized mean iBAQ values > 0 were considered as identified. For PRM-experiments, the system operated within a scan range of 350 to 1400 m/z with a resolution of 120,000 and a maximum injection time of 50 ms. An inclusion list contain- ing 7 precursor ions, representing unique peptides of tyros- ine hydroxylase, glycerol-3-phosphate dehydrogenase and cytoplasmic dynein 1 heavy chain 1 was applied (Table 2). For each peptide precursor, a retention time (RT) window of 3 min was defined in the inclusion list. During the RT win- dow, the peptide precursor was selected by the Quadrupole for fragmentation at a collision energy of 28% by HCD. The fragment ion scan was carried out in the Orbitrap at a resolu- tion of 60,000 with a maximum injection time of 200 ms. The mass spectrometry proteomics data have been depos- ited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al. 2022) partner repository with the data set identifier PXD033533. i The resulting protein group output was analyzed using the Perseus software (version 1.6.15.0), as part of the MaxQuant environment (Tyanova et al. 2016b), whereby master mixes were excluded for the subsequent analysis. Contaminants and decoys were filtered out and LFQ values were subse- quently log2 transformed for further statistical analysis. A minimum of 70% valid values per group was set as an additional filtering criterion prior to relative quantification of proteins. Prior to statistical analysis, the remaining miss- ing values were imputed from a normal distribution using a width of 0.3 and a downshift of 1.8. Mass spectrometric analysis For global DDA-experiments, the system operated within a scan range from 350 to 1400 m/z with a resolution of 120,000 and a maximum injection time of 80 ms. In a fixed cycle time of 2 s, all precursor ions with an intensity above 1 × 104 were selected for fragmentation at a fixed collision energy of 28% by higher energy collisional dissociation (HCD). Precursor ions selected for fragmentation were maintained on a dynamic exclusion list for 30 s. Fragment ion scans were performed at a resolution of 30,000 with a maximum injection time of 80 ms. For liquid-chromatography tandem mass spectrometry (LC–MS/MS) analysis, peptide samples were diluted in 0.1% TFA. To ensure an identical peptide load, the volume of pep- tide solution was adapted according to the amount of col- lected tissue. As for each NMG sample 500,000 µm2, and for each SNSurr. sample 1,000,000 µm2 of tissue were collected, 5 µl of peptide solution were used for mass spectrometric measurements of NMG samples, while 2.5 µl were used for the measurements of the SNSurr. samples. Mixtures of all 1 3 3 M. Wulf et al. Targeted mass spectrometry Paired Student’s t test as well as p value correction by Benjamini–Hochberg were applied to the data set (leading to q values) to determine proteins with significantly differential abundances. Proteins with a p value < 0.05 were defined as being significantly differential, while q values were applied as more stringent statistical criteria. Fold changes were determined by calcu- lating the ratio of means between the two assessed groups (NMG, SNSurr.). Global proteomic characterization of NMGs and surrounding tissue We first set out to characterize the individual protein profiles of NMGs and SNSurr. tissue (Fig. 1, Supplemen- tary Table 1). As the main components of brain tissue are neurons (in case of SN tissue dopaminergic neurons), oligodendrocytes and astrocytes, we confirmed the high abundance of selected marker proteins for each cell type in NMGs and SNSurr. tissue (Fig. 1, neurons: alpha-synuclein (SNCA), neurofilament light (NEFL), medium (NEFM) and heavy chain (NEFH), amyloid beta precursor protein (APP), microtubule associated protein tau (MAPT); dopa- minergic neurons: tyrosine hydroxylase (TH), aromatic- L-amino-acid decarboxylase (DDC), solute carrier family 6 member 3 (SLC6A3); astrocytes: glial fibrillary acidic protein (GFAP), S100 calcium binding protein B (S100B); oligodendrocytes: myelin basic protein (MBP), proteolipid protein 1 (PLP1)). For principal component analysis (PCA), analysis was conducted and graphics were created in R using the same software versions as for the Volcano plot. LFQ values from MaxQuant were taken and log2-transformed, zero values were treated as missing values. Proteins with more than 50% missing values were excluded and the remaining missing values were imputed by the mean of the corresponding pro- tein intensities. A PCA was calculated using the setting that scales the data beforehand. The first two principal compo- nents (PC) were plotted.fi Differentially expressed proteins as well as identified proteins in SNSurr. and NMGs based on iBAQ were further analyzed by GO (gene ontology)-term and pathway enrich- ment analysis. In brief, proteins were used for annotation enrichment using the web version of DAVID Bioinformatics Resources 6.8 (Da Huang et al. 2009a, 2009b) to gain a first overview of differences in the proteomic profile between NMGs and SNSurr. tissue hinting towards characteristic functions. The homo sapiens proteome served as a refer- ence and GO-terms from the categories Biological Process (BP), Cellular Compartment (CC), Molecular Function (MF) and KEGG Pathways were taken for in-depth analysis. A p value < 0.05 was used as a significance threshold and fold enrichment scores were taken to assess the enrichment of resulting GO-terms and pathways. To search for information on specific proteins, the UniProt database was utilized (The UniProt Consortium 2021). Especially neuronal and astrocytic markers such as SNCA, NEFL, S100B, NEFH and GFAP were found to be highly abundant in SNSurr. tissue, indicating the pres- ence of astrocytes and neurons in the tissue surrounding NMGs, whereas the dopaminergic marker proteins DDC and TH were of lower abundance. Targeted mass spectrometry Verification of differentially expressed proteins with signifi- cant p values was carried out by PRM experiments for tyros- ine hydroxylase, glycerol-3-phosphate dehydrogenase and cytoplasmic dynein 1 heavy chain 1. For that, the resulting peptide output of MaxQuant was processed in an identical manner as our protein group output and peptides assigned to TH were examined for significantly differential expres- sion (p value < 0.05), fold change, retention time and charge state. As housekeeping proteins, glycerol-3-phosphate dehy- drogenase and cytoplasmic dynein 1 heavy chain 1 were chosen, as they were found to be neither significant nor dif- ferential between NMGs and SNSurr. tissue and additionally displayed a low standard deviation on the level of intensi- ties. Evaluation of PRM data was performed using Skyline software (MacLean et al. 2010). Group comparisons were performed on peak areas for each protein and an adjusted p value < 0.05, according to Benjamini–Hochberg, was used as a significance threshold on MS2-level. Results (uncorrected) are plotted against the log2-transformed fold changes. Proteins that reach a corrected p value < 0.05 are marked in red. On each side, the ten proteins with the largest Euclidean distance from the origin in the Volcano plot are labeled with the corresponding gene name. Global proteomic characterization of NMGs and surrounding tissue DDC and TH were instead found to be of higher abun- dance in NMGs, while both astrocytic markers, GFAP and S100B, were found to be lower abundant in NMGs. Neu- ronal markers SNCA, NEHL and NEFH showed a similar abundance pattern as in SNSurr. tissue. Marker proteins for oligodendrocytes showed low abundance levels in both sam- ple types, PLP1 was not even identified in NMGs. These findings support the localization of NMGs in cell bodies of dopaminergic neurons and allow the characterization of the SNSurr. tissue as mainly composed of neurons and astrocytes, while oligodendrocytes play only a minor role. Global mass spectrometry The MaxQuant software (version 1.6.17.0) was used for the analysis of raw files. The resulting peak lists were searched against the human UniProt FASTA reference proteome (version 20th January 2021; 75,796 entries) and a common contaminants database provided in the Andromeda search engine (Cox and Mann 2008; Cox et al. 2011; Tyanova et al. 2016a). Due to sample preparation, carbamidomethlyation of methionine was set as fixed modification and carbamido- methylation of any N-terminus, oxidation of methionine as well as deamidation of asparagine and glutamine were set as variable modifications. Trypsin was set as digestion enzyme and a maximum number of 2 missed cleavages were toler- ated in the search. The false discovery rate (FDR) was set to 1% for peptides (minimum length of seven amino acids) and proteins and was determined by searching against a reverse The results of this analysis are presented as a Volcano plot, created in R version 4.0.3 (R Foundation for Statis- tical Computing 2020) using the packages ggplot2 3.3.3 and ggrepel 0.9.1. Negative log10-transformed p values Table 2 Peptides selected for parallel reaction monitoring-experiments m/z mass-to-charge ratio, RT retention time Protein Peptide m/z Charge state RT-window mean [± 1.5 min] Tyrosine hydroxylase AGGPHLEYFVR 415.884 3 + 43.4 FDPDLDLDHPGFSDQVYR 712.6605 3 + 75.6 TGFQLRPVAGLLSAR 529.3106 3 + 69.8 Glycerol-3-phosphate-dehydrogenase NHVVDISESGLITIAGGK 603.995 3 + 67.3 LAFLNVQAAEEALPR 821.4516 2 + 89.5 Cytoplasmic dynein 1 heavy chain 1 ESPEVLLTLDILK 735.4267 2 + 105.6 EFGPVVIDYGK 612.319 2 + 66.1 Table 2 Peptides selected for parallel reaction monitoring-experiments 1 Neuromelanin granules of the substantia nigra: proteomic profile provides links to tyrosine… NMGs and surrounding tissue can be separated based on their proteomic profile As the majority of proteins present in the human brain are not restricted to a single cell type or cell compart- ment, a proteomic study based only on the identification of proteins may miss important information about NMGs. Therefore, we also included the abundances of proteins in our analysis to gain information about proteins being enriched in either NMGs or SNSurr.. Our data set included 1088 proteins, which suited the criteria for our quantitative comparison (Supplementary 1 3 Fig. 1 Ranking plots of protein abundances in NMGs and surround- ing tissue. Plots show ranked normalized mean iBAQ-values of all identified proteins in both sample groups (NMG, SNSurr.). A Neuronal and dopaminergic markers rank highest in NMGs, while markers for astrocytes and oligodendrocytes are relatively low in abundance. PLP1 was not identified in NMG samples. B Compared to NMGs, astrocytic markers are higher abundant in SNSurr. tissue, while dopa- minergic markers are lower abundant. Oligodendrocyte markers are higher abundant compared to NMGs. SNCA alpha-synuclein, NEFL neurofilament light chain, NEFM neurofilament medium chain, NEFH: neurofilament heavy chain, APP: amyloid beta precursor pro- tein, MAPT microtubule associated protein tau, TH tyrosine hydroxy- lase, DDC aromatic-L-amino-acid decarboxylase, SLC6A3 solute car- rier family 6 member 3, GFAP glial fibrillary acidic protein, S100B S100 calcium binding protein B, MBP myelin basic protein, PLP1 proteolipid protein 1 M. Wulf et al. Fig. 2 Principal component analysis (PCA). The measured samples form two clusters based on the sample groups (NMG, SNSurr.) on Principal Component 1 (PC1) Fig. 2 Principal component analysis (PCA). The measured samples form two clusters based on the sample groups (NMG, SNSurr.) on Principal Component 1 (PC1) Differential proteins hint towards an involvement of NMGs in protein synthesis Since especially proteins with high and significant fold changes may provide insights into characteristics of NMGs and SNSurr. tissue, we focused especially on these proteins when evaluating the results of our quantitative compari- son. Indeed, 345 proteins showed a statistically significant differential abundance (adjusted p value < 0.05), displayed in the Volcano Plot (Fig. 3). For further in-depth charac- terization, we focused on the Top 10 proteins (Fig. 3B, C) regarding high and significant fold changes. Fig. 1 Ranking plots of protein abundances in NMGs and surround- ing tissue. Plots show ranked normalized mean iBAQ-values of all identified proteins in both sample groups (NMG, SNSurr.). A Neuronal and dopaminergic markers rank highest in NMGs, while markers for astrocytes and oligodendrocytes are relatively low in abundance. PLP1 was not identified in NMG samples. B Compared to NMGs, astrocytic markers are higher abundant in SNSurr. tissue, while dopa- minergic markers are lower abundant. Oligodendrocyte markers are higher abundant compared to NMGs. SNCA alpha-synuclein, NEFL neurofilament light chain, NEFM neurofilament medium chain, NEFH: neurofilament heavy chain, APP: amyloid beta precursor pro- tein, MAPT microtubule associated protein tau, TH tyrosine hydroxy- lase, DDC aromatic-L-amino-acid decarboxylase, SLC6A3 solute car- rier family 6 member 3, GFAP glial fibrillary acidic protein, S100B S100 calcium binding protein B, MBP myelin basic protein, PLP1 proteolipid protein 1 i Among these proteins, lysosomal sialate O-acetylester- ase (SIAE) showed the highest enrichment in NMGs (Fig. 3C), strengthening the relationship between lys- osomes and NMGs. Furthermore, a link to protein synthe- sis was found, since three tRNA ligases (isoleucine–tRNA ligase (IARS), phenylalanine–tRNA ligase beta subunit (FARSB), arginine–tRNA ligase (RARS)) and three ribosomal proteins (40S ribosomal protein S4 X isoform (RPS4X), 60S ribosomal protein L7a (RPL7A), 40S ribo- somal protein S9 (RPS9)) showed higher levels in NMGs. In contrast, the top ten proteins enriched in SNSurr. tis- sue (Fig. 3B) were found to be characteristic for neuron- surrounding tissue, such as the myelin-associated glyco- protein (MAG) as a part of the neuronal myelin sheath. Proteins exclusively identified in SNSurr. tissue stressed the neuronal character of the tissue, as for example myelin Table 2). As the principal component analysis (PCA) in Fig. 2 shows, NMGs and SNSurr. can be distinguished already with a high percentage on Principal Component 1, exceptionally stressing their high individuality on the proteome level. Table 2). As the principal component analysis (PCA) in Fig. 2 shows, NMGs and SNSurr. Differential proteins hint towards an involvement of NMGs in protein synthesis can be distinguished already with a high percentage on Principal Component 1, exceptionally stressing their high individuality on the proteome level. 1 3 3 Neuromelanin granules of the substantia nigra: proteomic profile provides links to tyrosine… proteolipid protein, dynein light chain 1 and peripherin were included in this list (Supplementary Table 1). In summary, our quantitative comparison strengthens the previously reported relation between lysosomes/endosomes and NMGs (Plum et al. 2016; Zucca et al. 2018). Neverthe- less, as proteins associated with protein synthesis were found i This finding is supported by the results of a GO-term analysis of proteins exclusively identified in NMGs, which revealed a variety of proteins to be related to ribosomes, mRNA-binding and translation (Supplementary Table 1). Stress granule‑related proteins are significantly Fig. 3 Volcano plot show- ing the relationship between fold change (FC) and p value for the comparison of protein abundance in NMGs and SNSurr. tissue. A Red dots indicate pro- teins with a significant q value (< 0.05). The 10 proteins with the highest Euclidean distances in both FC directions are anno- tated with their gene names and FC, p and q values are displayed in the tables shown in B and C. SIRT2 NAD-dependent protein deacetylase sirtuin-2, MAG myelin-associated glycoprotein, CNTNAP1 contactin-associated protein 1, PAK1 serine/threo- nine–protein kinase PAK 1, SH3GLB2 endophilin-B2, PHGDH D-3-phosphoglyc- erate dehydrogenase, GNB2 guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2, AKR1A1: alcohol dehydrogenase [NADP( +)], SEPTIN7 Septin-7, BLVRB flavin reductase (NADPH), SIAE sialate O-acetylesterase, IARS isoleucine–tRNA ligase, RPS9 40S ribosomal protein S9, FARSB phenylalanine–tRNA ligase beta subunit, DPP7 dipeptidyl peptidase 2, RARS arginine–tRNA ligase, RPL7A 60S ribosomal protein L7a, RPS4X 40S ribosomal protein S4 X isoform, DDOST dolichyl- diphosphooligosaccharide–pro- tein glycosyltransferase 48 kDa subunit, RCN2 reticulocalbin-2 proteolipid protein, dynein light chain 1 and peripherin were included in this list (Supplementary Table 1). This finding is supported by the results of a GO-term analysis of proteins exclusively identified in NMGs, which revealed a variety of proteins to be related to ribosomes, mRNA-binding and translation (Supplementary Table 1). In summary, our quantitative comparison strengthens the previously reported relation between lysosomes/endosomes and NMGs (Plum et al. 2016; Zucca et al. 2018). Neverthe- less, as proteins associated with protein synthesis were found to be significantly higher abundant in NMGs, our analysis may open a new perspective of NMG biology as, to our knowledge, enhanced abundances of ribosomal, mRNA-binding and trans- lation-related proteins in NMGs were never reported before. Stress granule‑related proteins are significantly higher abundant in NMGs Especially, the high abundances of proteins of the 40S ribosomal subunit in combination with several eukaryotic 1 M. Wulf et al. used, tyrosinase was not detected in any of our measure- ments, neither in SNSurr. tissue, nor in NMGs. For proteins involved in dopamine metabolism, we focused on tyrosine hydroxylase (TH) as the rate limiting enzyme in catechola- mine biosynthesis and, therefore, essential in the synthesis of dopamine, as well as aromatic-L-amino-acid decarboxy- lase (DDC), monoamine oxidase A and B (MAOA, MAOB), vesicular monoamine transporter 2 (VMAT2/SLC18A2) and dopamine transporter (DAT/SLC6A3). translation initiation factors (eIF) and RNA-binding proteins raised the question of a potential link between NMGs and stress granules (SGs). SGs are transient structures, which form after the inhibition or interruption of translation and, therefore, contain the aforementioned protein species (Ked- ersha et al. 2016). To further characterize this potential link, we analyzed the results of our quantitative comparison with a special focus on SG-specific proteins based on a previously published list of 37 proteins (Wolozin and Ivanov 2019). Nine well-known SG proteins, amongst them polyadenylate-binding protein 1 (PABPC1), which is frequently used in immunohistochemi- cal stainings as a marker protein for SGs, were significantly higher abundant in NMGs with fold changes between 2.1 and 4.9-fold (Fig. 4).i In our quantitative comparison, we found both proteins required for dopamine synthesis, TH (fold change: 2.5, q value: 0.03) and DDC (fold change: 3.3; q value: 0.01), to be significantly higher abundant in NMGs. We could further verify the significantly higher abundance of TH in NMGs in independent targeted MS-experiments (Fig. 5), in which only peptides unique for TH were quantified. To ensure iden- tical sample load we used two reference proteins (Glycerol- 3-phosphate dehydrogenase, mitochondrial (GPDM) and cytoplasmic dynein 1 heavy chain 1 (DYHC1)), thus verifi- ying the increased abundance of TH in NMG tissue. Due to the strict filter criteria for our quantitative com- parison, we also checked our data set for the global pro- teome analysis. In this, 23 additional SG-specific proteins were included, all of them showing higher abundances in NMGs compared to SNSurr. tissue. In addition, three SG- proteins (Caprin 1, Caprin 2 and probable ATP-dependent RNA helicase DDX5) were exclusively identified in NMGs (Supplementary Table 1). Further proteins associated with dopamine metabolism did not display such a cohesive picture. Conversely, MAOA showed a comparable abundance in SNSurr. Stress granule‑related proteins are significantly higher abundant in NMGs tissue and NMGs (fold change: 0.83; q value: 0.18), while MAOB was signifi- cantly higher abundant in SNSurr. tissue (fold change: 0.59; q value: 0.04). In summary, our complete data set contains data for 32 of the previously published 37 SG-proteins, of which all are higher abundant in NMGs. Through our quantita- tive comparison, we provide clear evidence for elemental components of SGs (Kedersha et al. 2005) such as poly- adenylate-binding protein 1 (PABPC1), subunits of eukary- otic translation initiation factor 3 (eIF3A, eIF3B) and eIF4 gamma 1 (eIF4G1) being significantly higher abundant in NMGs than in surrounding tissue. VMAT2 was not identified at all in any of our samples. DAT instead did not match the criteria of our quantitative comparison, but protein levels showed a higher abundance in NMGs. Summarizing our findings, we provide evidence for enzymes required for dopamine synthesis, TH and DDC, being highly abundant in NMGs. Discussion Since the findings presented up to this point were primar- ily the result of a systematic comparison, we wanted to take another look at those aspects of NMG biology that have already been intensively studied in the past, namely, the involvement in the iron metabolism of the SN and the mechanism of NM synthesis. Our here presented study represents the largest data set concerning the global proteome of NMGs, including 2693 identified proteins, of which 1088 were examined using a relative quantification approach allowing an in-depth charac- terization of protein abundances in NMGs and surrounding SN tissue for the first time. Both, identified and quantified proteins, give further insights into open questions of NMG biology concerning origin and function of NMGs as well as NM synthesis. Stressing the importance of iron metabolism in the SN, ferritin heavy chain was amongst the highest abundant proteins in both NMGs and SNSurr. tissue, with a signifi- cantly higher abundance in SNSurr. tissue (1.8-fold, Supple- mentary Tables 1, 2), while ferritin light chain was com- parably abundant in both of our sample groups as well as serotransferrin. 1 3 Origin and function of neuromelanin granules 2016; Zucca et al. 2018). In a first approach on characterizing NMGs, we (Plum et al. 2016) further hypothesized that NM may be first generated in the cytosol and then taken up by lysosomes, resulting in NMG formation. In the present study, we can confirm the link between NMGs and lysosomes based on higher abundances of lyso- somal proteins such as SIAE, Lysosome-associated mem- brane glycoprotein 2 (LAMP2) and several cathepsines in Summary of the LFQ- quantitative comparison e stress granule marker ns. A LFQ-values for nine granule marker proteins gher in NMGs (left) n SNSurr. tissue (right) five patients included in udy showing the higher n abundances. B Fold es between NMGs and are rather mild, but the f nces are significant after mini–Hochberg correc- DDX1 ATP-dependent helicase DDX1, DDX3x ependent RNA helicase X, DDX6 probable ATP- dent RNA helicase DDX6, 7 probable ATP-depend- NA helicase DDX17, eukaryotic translation on factor 3 subunit A, eukaryotic translation on factor 3 subunit B, 1 eukaryotic translation on factor 4 gamma 1, PA2B1 heterogeneous r ribonucleoproteins A2/ ABPC1 polyadenylate- g protein 1 Fig. 4 Summary of the LFQ- based quantitative comparison for nine stress granule marker proteins. A LFQ-values for nine stress granule marker proteins are higher in NMGs (left) than in SNSurr. tissue (right) for all five patients included in this study showing the higher protein abundances. B Fold changes between NMGs and SNSurr. are rather mild, but the differences are significant after Benjamini–Hochberg correc- tion. DDX1 ATP-dependent RNA helicase DDX1, DDX3x ATP-dependent RNA helicase DDX3X, DDX6 probable ATP- dependent RNA helicase DDX6, DDX17 probable ATP-depend- ent RNA helicase DDX17, eIF3A eukaryotic translation initiation factor 3 subunit A, eIF3B eukaryotic translation initiation factor 3 subunit B, eIF4G1 eukaryotic translation initiation factor 4 gamma 1, hnRNPA2B1 heterogeneous nuclear ribonucleoproteins A2/ B1, PABPC1 polyadenylate- binding protein 1 approach on characterizing NMGs, we (Plum et al. 2016) further hypothesized that NM may be first generated in the cytosol and then taken up by lysosomes, resulting in NMG formation.i approach on characterizing NMGs, we (Plum et al. 2016) further hypothesized that NM may be first generated in the cytosol and then taken up by lysosomes, resulting in NMG formation.i SN. Until now, the most widely accepted theory regarding NMG origin claims a close relation between NMGs and lys- osomes or endosomes (Tribl et al. 2006; Plum et al. 2016; Zucca et al. 2018). Origin and function of neuromelanin granules The origin and function of NMGs are widely discussed. With our proteomic characterization we were able to high- light several key characteristics of the proteomic compound of NMGs, potentially revealing distinct functions in the Concerning NM synthesis, we focused on proteins involved in dopamine metabolism as well as tyrosinase, since it is often assumed to be involved in NM synthesis. Although a highly sensitive state-of-the-art mass spectrometer was 1 3 Neuromelanin granules of the substantia nigra: proteomic profile provides links to tyrosine… SN. Until now, the most widely accepted theory regarding NMG origin claims a close relation between NMGs and lys- osomes or endosomes (Tribl et al. 2006; Plum et al. 2016; Zucca et al. 2018). This theory is based on the results of several proteomic studies and the observation that NMGs are surrounded by a double membrane, a distinct feature of organelle structures (D'Agostino and Luse 1964; Tribl i approach on characterizing NMGs, we (Plum et al. 2016) further hypothesized that NM may be first generated in the cytosol and then taken up by lysosomes, resulting in NMG formation. In the present study, we can confirm the link between NMGs and lysosomes based on higher abundances of lyso- somal proteins such as SIAE, Lysosome-associated mem- Fig. 4 Summary of the LFQ- based quantitative comparison for nine stress granule marker proteins. A LFQ-values for nine stress granule marker proteins are higher in NMGs (left) than in SNSurr. tissue (right) for all five patients included in this study showing the higher protein abundances. B Fold changes between NMGs and SNSurr. are rather mild, but the differences are significant after Benjamini–Hochberg correc- tion. DDX1 ATP-dependent RNA helicase DDX1, DDX3x ATP-dependent RNA helicase DDX3X, DDX6 probable ATP- dependent RNA helicase DDX6, DDX17 probable ATP-depend- ent RNA helicase DDX17, eIF3A eukaryotic translation initiation factor 3 subunit A, eIF3B eukaryotic translation initiation factor 3 subunit B, eIF4G1 eukaryotic translation initiation factor 4 gamma 1, hnRNPA2B1 heterogeneous nuclear ribonucleoproteins A2/ B1, PABPC1 polyadenylate- binding protein 1 1 3 Until now, the most widely accepted theory regarding G origin claims a close relation between NMGs and lys- es or endosomes (Tribl et al. 2006; Plum et al. 2016; a et al. 2018). This theory is based on the results of al proteomic studies and the observation that NMGs urrounded by a double membrane, a distinct feature ganelle structures (D'Agostino and Luse 1964; Tribl 2006; Plum et al. Origin and function of neuromelanin granules This theory is based on the results of several proteomic studies and the observation that NMGs are surrounded by a double membrane, a distinct feature of organelle structures (D'Agostino and Luse 1964; Tribl et al. 2006; Plum et al. 2016; Zucca et al. 2018). In a first In the present study, we can confirm the link between NMGs and lysosomes based on higher abundances of lyso- somal proteins such as SIAE, Lysosome-associated mem- brane glycoprotein 2 (LAMP2) and several cathepsines in 1 3 M. Wulf et al. Fig. 5 PRM results for proteins chosen for verification. A–C Graphs show intensities of selected peptides for GPDM (A), DYHC1 B and TH C as mean values for NMGs and SNSurr. tissue. Mean values for peptides of GPDM and DYHC1 are comparable, only the mean value of peptides for TH is significantly higher in NMGs compared to SNSurr. tissue. D Summary of the results of our PRM-experiments, showing an even higher fold change (~ 6) for TH than the DDA- experiments. *: q value < 0.001. GPDM glycerol-3-phosphate dehydrogenase, mitochondrial, DYHC1 cytoplasmic dynein 1 heavy chain 1 Fig. 5 PRM results for proteins chosen for verification. A–C Graphs show intensities of selected peptides for GPDM (A), DYHC1 B and TH C as mean values for NMGs and SNSurr. tissue. Mean values for peptides of GPDM and DYHC1 are comparable, only the mean value of peptides for TH is significantly higher in NMGs compared to SNSurr. tissue. D Summary of the results of our PRM-experiments, showing an even higher fold change (~ 6) for TH than the DDA- experiments. : q value < 0.001. GPDM glycerol-3-phosphate dehydrogenase, mitochondrial, DYHC1 cytoplasmic dynein 1 heavy chain 1 to SNSurr. tissue. D Summary of the results of our PRM-experiments, showing an even higher fold change (~ 6) for TH than the DDA- experiments. **: q value < 0.001. GPDM glycerol-3-phosphate dehydrogenase, mitochondrial, DYHC1 cytoplasmic dynein 1 heavy chain 1 Fig. 5 PRM results for proteins chosen for verification. A–C Graphs show intensities of selected peptides for GPDM (A), DYHC1 B and TH C as mean values for NMGs and SNSurr. tissue. Mean values for peptides of GPDM and DYHC1 are comparable, only the mean value of peptides for TH is significantly higher in NMGs compared 2013; Kedersha et al. 2013; Wheeler et al. 2016). 1 3 Origin and function of neuromelanin granules However, recent studies suggest that the frequently observed aggre- gates in various neurodegenerative disorders may originate from SGs, which develop into persistent protein aggre- gates under constant cell stress (Wolozin 2012; Dobra et al. 2018). Indeed, we could find TAR DNA-binding protein 43 (TDP43), which is the main component of protein aggre- gates in amyotrophic lateral sclerosis (Neumann et al. 2009), to be higher abundant in NMGs than in SNSurr. tissue. NMGs compared to SNSurr. tissue (Supplementary Tables 1, 2). In addition, the identification of typically non-neuronal proteins such as GFAP in NMGs may be another hint towards their lysosomal role. To ensure, that the presence of these proteins in our samples is not based on contamina- tions due to our methodological approach, we compared our data set with previously published studies focusing on the proteomic content of NMGs (Plum et al. 2016; Zucca et al. 2018). Since GFAP was identified in NMGs in both studies, although the latter one (Zucca et al. 2018) used an independ- ent methodological approach for the purification of NMGs in density gradient purification via ultracentrifugation, this seems to be a reliable finding. Our data thus indicate that NMGs, similar to protein aggregates in neurodegenerative diseases, contain several SG marker proteins. We, therefore, cautiously raise two pos- sible hypotheses, potentially explaining the relation between SGs and NMGs: i While these observations would assume that NMGs are a degradative organelle, we are the first to provide evidence for a link between SGs and NMGs, as we detected several well-known SG-associated proteins in the NMGs. From 37 SG proteins (Wolozin and Ivanov 2019), we were able to identify 32 in our data set, all of them being higher abundant in NMGs. 1. SGs may be the origin of NMGs as a result of chronic cell stress. 2. SGs form in close proximity to NMGs. A major stress factor contributing to transient SG for- mation and potentially their transformation into persistent NMG structures could be oxidative stress caused by NM- bound metals (Sian-Hülsmann et al. 2011; Riederer et al. In general, SGs are known to form under external stress and influence mRNA function, localization as well as sign- aling pathways (Buchan and Parker 2009; Buchan et al. 1 3 Neuromelanin granules of the substantia nigra: proteomic profile provides links to tyrosine… 2021). Neuromelanin synthesis In catecholaminergic neurons, it is widely accepted that TH is localized in the neuronal cell body in the substantia nigra, as well as in the presynaptic axon terminals in the striatum (Muñoz et al. 2012). As we used substantia nigra pars compacta tissue for our analysis, cell bodies of dopa- minergic neurons should be present, while presynaptic ter- minals of dopaminergic neurons should be absent. TH is essential for the production of dopamine, further such as DDC and vesicular VMAT2 are also involved in dopamine synthesis and packaging. DDC catalyzes the conversion of L-dihydroxyphenylalanine (L-DOPA) into dopamine, while the packaging into synaptic vesicles is achieved by the activity of VMAT2 (Cartier et al. 2010). VMAT2 was not identified in any of our MS-measurements, but for DDC, a significantly higher abundance in NMGs compared to SNSurr. tissue was revealed in our quantitative compari- son. MAOA and MAOB, proteins involved in dopamine degradation (Finberg 2019), were found to be comparably abundant in SNSurr. tissue and NMGs instead (MAOA) or significantly higher abundant in SNSurr. tissue (MAOB), while immunohistochemical stainings only revealed the Origin and function of neuromelanin granules It was reported that metal concentrations in SN are high and that metal ions are associated with neuromelanin within NMGs (Zecca and Swartz 1993). In addition, we were able to show that iron-binding proteins such as ferritin light and heavy chain were highly abundant in NMGs, but also in the surrounding tissue, assuming that they are not NMG specific (Supplementary Table 1). presence of MAOA and MAOB in glial cells surrounding NMG-containing neurons. (Konradi et al. 1988).i presence of MAOA and MAOB in glial cells surrounding NMG-containing neurons. (Konradi et al. 1988). These findings suggest that production of dopamine may take place in NMGs of the SN, although our data cannot prove the production of dopamine by functional TH and DDC. Thus, this hypothesis needs to be further investigated in the future. Nevertheless, it is known that dopamine not imported into vesicles by VMAT2 either oxidizes into aminochrome, a precursor of neuromelanin, or can be degraded by MAOA (Smythies 1996; Muñoz et al. 2012; Goldstein 2021).i i Still, we have to point out that NMGs, unlike SGs, are membrane-surrounded and thus may be closer related to lysosomes, since both inherit misfolded protein species and lyososomal/endosomal proteins (Tribl et al. 2005, 2006; Plum et al. 2016; Zucca et al. 2018). Unfortunately, we are not able to verify our hypotheses with the methods applied in this manuscript. To study the potential link between SGs and NMGs and to commit to one of our hypotheses, functional studies in suitable neuromelanin-like pigments producing models (Hasegawa et al. 2003; Jo et al. 2016; Carballo- Carbajal et al. 2019) will be of immense importance, for example based on immunological stainings of SG marker proteins. Another interesting finding in this context is the higher abundance of DAT/SLC6A3 in NMGs compared to SNSurr. tissue. Although the mediation of the reuptake of dopa- mine from the synaptic cleft is the main task of DAT, it is also reported that DAT is able to release dopamine in the extracellular space (Khoshbouei et al. 2003). In addi- tion to its localization in the plasma membrane, DAT was observed to be localized in the soma of dopaminergic neurons at the endoplasmic reticulum, the Golgi appara- tus and in “multivesicular body vesicles” (Hersch et al. 1997). Therefore, the localization of DAT in NMGs could indicate a functional role, although the direction of dopa- mine transport mediated by DAT in NMGs needs further examination. Neuromelanin synthesis Besides origin and function of NMGs, the synthesis of NM is highly debated as well. The three most common hypoth- eses focus either on TH or on tyrosinase to be involved in an enzymatic synthesis or assume an autooxidative generation with dopamine being a potential precursor for NM. With our proteomics approach, we are able to provide evidence for TH to be of high abundance in NMGs, while not a single peptide belonging to tyrosinase was detected in any of our samples. In catecholaminergic neurons, it is widely accepted that TH is localized in the neuronal cell body in the substantia nigra, as well as in the presynaptic axon terminals in the striatum (Muñoz et al. 2012). As we used substantia nigra pars compacta tissue for our analysis, cell bodies of dopa- minergic neurons should be present, while presynaptic ter- minals of dopaminergic neurons should be absent. TH is essential for the production of dopamine, further such as DDC and vesicular VMAT2 are also involved in dopamine synthesis and packaging. DDC catalyzes the conversion of L-dihydroxyphenylalanine (L-DOPA) into dopamine, while the packaging into synaptic vesicles is achieved by the activity of VMAT2 (Cartier et al. 2010). VMAT2 was not identified in any of our MS-measurements, but for DDC, a significantly higher abundance in NMGs compared to SNSurr. tissue was revealed in our quantitative compari- son. MAOA and MAOB, proteins involved in dopamine degradation (Finberg 2019), were found to be comparably abundant in SNSurr. tissue and NMGs instead (MAOA) or i ifi tl hi h b d t i SN ti (MAOB) Besides origin and function of NMGs, the synthesis of NM is highly debated as well. The three most common hypoth- eses focus either on TH or on tyrosinase to be involved in an enzymatic synthesis or assume an autooxidative generation with dopamine being a potential precursor for NM. With our proteomics approach, we are able to provide evidence for TH to be of high abundance in NMGs, while not a single peptide belonging to tyrosinase was detected in any of our samples. However, it is important to mention that not all TH-pos- itive neurons in the human SN contain NMGs (Hirsch et al. 1989; Saper and Petito 1982) and that no NM synthesis can be observed in mice or rats, although dopamine is synthe- sized in their brains. belonging to tyrosinase was detected in any of our samples. Conclusions Concluding, our data provides us with further evidence on NM synthesis and NMG origin. Based on our analysis of the proteomic profile of NMGs and surrounding SN tissue, we hypothesize that SGs resulting from cellular stress may be the origin of NMGs or that they form in close proximity to NMGs. In addition, we provide further evidence for the link of NMGs and lysosomes. Concerning NM synthesis, we hypothesize that NM may be synthesized in a pathway involving TH and autooxidative processes. Still, to verify our findings, functional analyses in appropriate cell culture or animal models are inevitable to further elucidate NMG genesis and function in disease progression. Supplementary Information The online version contains supplemen- tary material available at https://doi.org/10.1007/s00702-022-02530-4. Acknowledgements The authors would like to thank the team of the Navarrabiomed Brain Bank in Pamplona for providing human post- mortem tissue. 1 3 M. Wulf et al. Author contributions Conceptualization: KM, BE; methodology: MW, BE, KB; software: KS, ME; verification: MW; formal analysis: MW, BE, KS; resources: KM, ME, PR, M.G.; writing—original draft preparation: M.W., K.S., B.E.; writing—review and editing: KB, KM, PR, MG; supervision: KM, BE; project administration: KM; funding acquisition: ME, KM. All authors have read and agreed to the pub- lished version of the manuscript. Carballo-Carbajal I, Laguna A, Romero-Giménez J, Cuadros T, Bové J, Martinez-Vicente M, Parent A, Gonzalez-Sepulveda M, Peñuelas N, Torra A, Rodríguez-Galván B, Ballabio A, Hasegawa T, Bor- tolozzi A, Gelpi E, Vila M (2019) Brain tyrosinase overexpression implicates age-dependent neuromelanin production in Parkinson’s disease pathogenesis. Nat Commun 10(1):973 Cartier EA, Parra LA, Baust TB, Quiroz M, Salazar G, Faundez V, Egaña L, Torres GE (2010) A biochemical and functional protein complex involving dopamine synthesis and transport into synaptic vesicles. J Biol Chem 285(3):1957–1966 Funding Open Access funding enabled and organized by Projekt DEAL. This work was supported by de.NBI, a project of the German Federal Ministry of Education and Research (BMBF) (grant number FKZ 031 A 534A) and P.U.R.E. (Protein Research Unit Ruhr within Europe) and Center for Protein Diagnostics (ProDi) grants, both from the Ministry of Innovation, Science and Research of North-Rhine West- phalia, Germany. Cebrián C, Zucca FA, Mauri P, Steinbeck JA, Studer L, Scherzer CR, Kanter E, Budhu S, Mandelbaum J, Vonsattel JP, Zecca L, Loike JD, Sulzer D (2014) MHC-I expression renders catecholaminergic neurons susceptible to T-cell-mediated degeneration. References Dobra I, Pankivskyi S, Samsonova A, Pastre D, Hamon L (2018) Relation between stress granules and cytoplasmic protein aggre- gates linked to neurodegenerative diseases. Curr Neurol Neu- rosci Rep 18(12):107 Aime S, Fasano M, Bergamasco B, Lopiano L, Valente G (1994) Evidence for a glycidic-lipidic matrix in human neuromelanin, potentially responsible for the enhanced iron sequestering ability of substantia nigra. J Neurochem 62(1):369–371 Double KL, Gerlach M, Schünemann V, Trautwein AX, Zecca L, Gallorini M, Youdim MB, Riederer P, Ben-Shachar D (2003) Iron-binding characteristics of neuromelanin of the human sub- stantia nigra. Biochem Pharmacol 66(3):489–494f Aumann TD, Egan K, Lim J, Boon WC, Bye CR, Chua HK, Baban N, Parish CL, Bobrovskaya L, Dickson P, Horne MK (2011) Neuronal activity regulates expression of tyrosine hydroxylase in adult mouse substantia nigra pars compacta neurons. J Neurochem 116(4):646–658 stantia nigra. Biochem Pharmacol 66(3):489–494 Finberg JPM (2019) Inhibitors of MAO-B and COMT: their effects on brain dopamine levels and uses in Parkinson’s disease. J Neural Transm 126(4):433–448 Biesemeier A, Eibl O, Eswara S, Audinot J-N, Wirtz T, Pezzoli G, Zucca FA, Zecca L, Schraermeyer U (2016) Elemental mapping of Neuromelanin organelles of human Substantia Nigra: correlative ultrastructural and chemical analysis by analytical transmission electron microscopy and nano-secondary ion mass spectrometry. J Neurochem 138(2):339–353 Fornstedt B, Rosengren E, Carlsson A (1986) Occurrence and distri- bution of 5-S-cysteinyl derivatives of dopamine, dopa and dopac in the brains of eight mammalian species. Neuropharmacology 25(4):451–454 Gaspar P, Berger B, Gay M, Hamon M, Cesselin F, Vigny A, Javoy- Agid F, Agid Y (1983) Tyrosine hydroxylase and methio- nine-enkephalin in the human mesencephalon. J Neurol Sci 58(2):247–267 Bohic S, Murphy K, Paulus W, Cloetens P, Salomé M, Susini J, Double K (2008) Intracellular chemical imaging of the developmental phases of human neuromelanin using synchrotron X-ray micro- spectroscopy. Anal Chem 80(24):9557–9566 Goldstein DS (2021) The catecholaldehyde hypothesis for the patho- genesis of catecholaminergic neurodegeneration: what we know and what we do not know. Int J Mol Sci. https://doi.org/10.3390/ ijms22115999f Bridelli MG, Tampellini D, Zecca L (1999) The structure of neu- romelanin and its iron binding site studied by infrared spectros- copy. FEBS Lett 457(1):18–22 Halliday GM, Li YW, Joh TH, Cotton RG, Howe PR, Geffen LB, Blessing WW (1988) Distribution of monoamine-synthesiz- ing neurons in the human medulla oblongata. J Comp Neurol 273(3):301–317 Buchan JR, Parker R (2009) Eukaryotic stress granules: the ins and outs of translation. Conflicts of interest The authors declare no conflict of interest. Cox J, Michalski A, Mann M (2011) Software lock mass by two- dimensional minimization of peptide mass errors. J Am Soc Mass Spectrom 22(8):1373–1380 Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Cox J, Hein MY, Luber CA, Paron I, Nagaraj N, Mann M (2014) Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. MCP 13(9):2513–2526 Da Huang W, Sherman BT, Lempicki RA (2009a) Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res 37(1):1–13 Da Huang W, Sherman BT, Lempicki RA (2009b) Systematic and integrative analysis of large gene lists using DAVID bioinfor- matics resources. Nat Protoc 4(1):44–57 D’AgostinoLuse AJSA (1964) Electron microscopic observations on the human substantia nigra. Neurology 14:529–536 D’Amato RJ, Lipman ZP, Snyder SH (1986) Selectivity of the par- kinsonian neurotoxin MPTP: toxic metabolite MPP+ binds to neuromelanin. Science 231(4741):987–989 Conclusions Nat Com- mun 5:3633i Cox J, Mann M (2008) MaxQuant enables high peptide identifi- cation rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26(12):1367–1372 Declarations Conflicts of interest The authors declare no conflict of interest. References Mol Cell 36(6):932–941 Buchan JR, Kolaitis R-M, Taylor JP, Parker R (2013) Eukaryotic stress granules are cleared by autophagy and Cdc48/VCP function. Cell 153(7):1461–1474 1 3 Neuromelanin granules of the substantia nigra: proteomic profile provides links to tyrosine… KA, Caldwell GA, Wolfe JH, Ischiropoulos H (2017) Dopamine induces soluble α-synuclein oligomers and nigrostriatal degenera- tion. Nat Neurosci 20(11):1560–1568 Halliday GM, Fedorow H, Rickert CH, Gerlach M, Riederer P, Double KL (2006) Evidence for specific phases in the development of human neuromelanin. J Neural Transm 113(6):721–728 Muñoz P, Huenchuguala S, Paris I, Segura-Aguilar J (2012) Dopamine oxidation and autophagy. Parkinson’s Dis 2012:920953 Hasegawa T, Matsuzaki M, Takeda A, Kikuchi A, Furukawa K, Shiba- hara S, Itoyama Y (2003) Increased dopamine and its metabolites in SH-SY5Y neuroblastoma cells that express tyrosinase. J Neu- rochem 87(2):470–475 Nagatsu T, Nakashima A, Ichinose H, Kobayashi K (2019) Human tyrosine hydroxylase in Parkinson’s disease and in related disor- ders. J Neural Transm 126(4):397–409 Hersch SM, Yi H, Heilman CJ, Edwards RH, Levey AI (1997) Sub- cellular localization and molecular topology of the dopamine transporter in the striatum and substantia nigra. J Comp Neurol 388(2):211–227 Neumann M, Kwong LK, Lee EB, Kremmer E, Flatley A, Xu Y, For- man MS, Troost D, Kretzschmar HA, Trojanowski JQ, Lee VM-Y (2009) Phosphorylation of S409/410 of TDP-43 is a consistent feature in all sporadic and familial forms of TDP-43 proteinopa- thies. Acta Neuropathol 117(2):137–149 Hirsch E, Graybiel AM, Agid YA (1988) Melanized dopaminergic neu- rons are differentially susceptible to degeneration in Parkinson’s disease. Nature 334(6180):345–348 Oberländer U, Pletinckx K, Döhler A, Müller N, Lutz MB, Arzberger T, Riederer P, Gerlach M, Koutsilieri E, Scheller C (2011) Neu- romelanin is an immune stimulator for dendritic cells in vitro. BMC Neurosci 12:116 Hirsch EC, Graybiel AM, Agid Y (1989) Selective vulnerability of pigmented dopaminergic neurons in Parkinson’s disease. Acta Neurol Scand Suppl 126:19–22 Ikemoto K, Nagatsu I, Ito S, King RA, Nishimura A, Nagatsu T (1998) Does tyrosinase exist in neuromelanin-pigmented neurons in the human substantia nigra? Neurosci Lett 253(3):198–200 Perez-Riverol Y, Bai J, Bandla C, García-Seisdedos D, Hewapathi- rana S, Kamatchinathan S, Kundu DJ, Prakash A, Frericks-Zip- per A, Eisenacher M, Walzer M, Wang S, Brazma A, Vizcaíno JA (2022) The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences. References Nucleic Acids Res 50(D1):D543–D552 Jo J, Xiao Y, Sun AX, Cukuroglu E, Tran H-D, Göke J, Tan ZY, Saw TY, Tan C-P, Lokman H, Lee Y, Kim D, Ko HS, Kim S-O, Park JH, Cho N-J, Hyde TM, Kleinman JE, Shin JH, Weinberger DR, Tan EK, Je HS, Ng H-H (2016) Midbrain-like organoids from human pluripotent stem cells contain functional dopaminergic and neuromelanin-producing neurons. Cell Stem Cell 19(2):248–257 Plum S, Steinbach S, Attems J, Keers S, Riederer P, Gerlach M, May C, Marcus K (2016) Proteomic characterization of neu- romelanin granules isolated from human substantia nigra by laser-microdissection. Sci Rep 6:37139 Kedersha N, Stoecklin G, Ayodele M, Yacono P, Lykke-Andersen J, Fritzler MJ, Scheuner D, Kaufman RJ, Golan DE, Anderson P (2005) Stress granules and processing bodies are dynamically linked sites of mRNP remodeling. J Cell Biol 169(6):871–884 R Foundation for Statistical Computing (2020) R. A language and environment for statistical computing. R Core Team, Vienna Raper HS (1928) The aerobic oxidases. Physiol Rev 8(2):245–282 Kedersha N, Ivanov P, Anderson P (2013) Stress granules and cell signaling: more than just a passing phase? Trends Biochem Sci 38(10):494–506 Riederer P, Monoranu C, Strobel S, Iordache T, Sian-Hülsmann J (2021) Iron as the concert master in the pathogenic orches- tra playing in sporadic Parkinson’s disease. J Neural Transm 128(10):1577–1598 Kedersha N, Panas MD, Achorn CA, Lyons S, Tisdale S, Hickman T, Thomas M, Lieberman J, McInerney GM, Ivanov P, Anderson P (2016) G3BP-Caprin1-USP10 complexes mediate stress gran- ule condensation and associate with 40S subunits. J Cell Biol 212(7):845–860 Robert BW, Meghan GT (2012) Moving beyond tyrosine hydroxy- lase to define dopaminergic neurons for use in cell replacement therapies for Parkinson’s disease. CNSNDDT 11(4):340–349 Á Sánchez-Ferrer Á, Neptuno Rodríguez-López J, García-Cánovas F, García-Carmona F (1995) Tyrosinase: a comprehensive review of its mechanism. Biochim Biophys Acta 1247(1):1–11 Khoshbouei H, Wang H, Lechleiter JD, Javitch JA, Galli A (2003) Amphetamine-induced dopamine efflux. A voltage-sensitive and intracellular Na+-dependent mechanism. J Biol Chem 278(14):12070–12077 Saper CB, Petito CK (1982) Correspondence of melanin-pigmented neurons in human brain with A1–A14 catecholamine cell groups. Brain : a Journal of Neurology 105(Pt 1):87–101 Konradi C, Svoma E, Jellinger K, Riederer P, Denney R, Thibault J (1988) Topographic immunocytochemical mapping of monoam- ine oxidase-A, monoamine oxidase-B and tyrosine hydroxylase in human post mortem brain stem. References Neuroscience 26(3):791–802 Sian-Hülsmann J, Mandel S, Youdim MBH, Riederer P (2011) The relevance of iron in the pathogenesis of Parkinson’s disease. J Neurochem 118(6):939–957 Smythies J (1996) On the functional of neuromelanin. Proc R Soc Lond B 263(1369):487–489 Lindquist NG, Larsson BS, Lydén-Sokolowski A (1988) Autoradiogra- phy of 14Cparaquat or 14Cdiquat in frogs and mice: accumulation in neuromelanin. Neurosci Lett 93(1):1–6 Sulzer D, Zecca L (2000) Intraneuronal dopamine-quinone synthesis: a review. Neurotox Res 1(3):181–195 MacLean B, Tomazela DM, Shulman N, Chambers M, Finney GL, Frewen B, Kern R, Tabb DL, Liebler DC, MacCoss MJ (2010) Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 26(7):966–968 The UniProt Consortium (2021) UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res 49(D1):D480–D489 Tief K, Schmidt A, Beermann F (1998) New evidence for presence of tyrosinase in substantia nigra, forebrain and midbrain. Mol Brain Res 53(1–2):307–310 Marsden CD (1961) Pigmentation in the nucleus substantiae nigrae of mammals. J Anat 95:256–261 Tribl F, Gerlach M, Marcus K, Asan E, Tatschner T, Arzberger T, Meyer HE, Bringmann G, Riederer P (2005) “Subcellular pro- teomics” of neuromelanin granules isolated from the human brain. MCP 4(7):945–957 Mason HS (1965) Oxidases. Annu Rev Biochem 34:595–6 Miranda M, Botti D, Bonfigli A, Ventura T, Arcadi A (1984) Tyrosi- nase-like activity in normal human substantia nigra. Gen Phar- macol 15(6):541–544 Tribl F, Marcus K, Meyer HE, Bringmann G, Gerlach M, Riederer P (2006) Subcellular proteomics reveals neuromelanin gran- ules to be a lysosome-related organelle. J Neural Transm 113(6):741–749 Mogi M, Harada M, Kiuchi K, Kojima K, Kondo T, Narabayashi H, Rausch D, Riederer P, Jellinger K, Nagatsu T (1988) Homospe- cific activity (activity per enzyme protein) of tyrosine hydroxylase increases in parkinsonian brain. J Neural Transm 72(1):77–82 Tribl F, Arzberger T, Riederer P, Gerlach M (2007) Tyrosi- nase is not detected in human catecholaminergic neurons by Mor DE, Tsika E, Mazzulli JR, Gould NS, Kim H, Daniels MJ, Doshi S, Gupta P, Grossman JL, Tan VX, Kalb RG, Caldwell 1 3 M. Wulf et al. immunohistochemistry and Western blot analysis. J Neural Transm. https://doi.org/10.1007/978-3-211-73574-9_8 Zecca L, Shima T, Stroppolo A, Goj C, Battiston GA, Gerbasi R, Sarna T, Swartz HM (1996) Interaction of neuromelanin and iron in substantia nigra and other areas of human brain. Neuroscience 73(2):407–415 Tyanova S, Temu T, Cox J (2016a) The MaxQuant computational plat- form for mass spectrometry-based shotgun proteomics. References Nat Protoc 11(12):2301–2319 Zecca L, Tampellini D, Gerlach M, Riederer P, Fariello RG, Sulzer D (2001) Substantia nigra neuromelanin: structure, synthesis, and molecular behaviour. Mol Pathol 54(6):414–418 Tyanova S, Temu T, Sinitcyn P, Carlson A, Hein MY, Geiger T, Mann M, Cox J (2016b) The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat Methods 13(9):731–740 Zhang W, Phillips K, Wielgus AR, Liu J, Albertini A, Zucca FA, Faust R, Qian SY, Miller DS, Chignell CF, Wilson B, Jackson-Lewis V, Przedborski S, Joset D, Loike J, Hong J-S, Sulzer D, Zecca L (2011) Neuromelanin activates microglia and induces degen- eration of dopaminergic neurons: implications for progression of Parkinson’s disease. Neurotox Res 19(1):63–72 Vila M (2019) Neuromelanin, aging, and neuronal vulnerability in Par- kinson’s disease. Mov Disord 34(10):1440–1451 Wheeler JR, Matheny T, Jain S, Abrisch R, Parker R (2016) Distinct stages in stress granule assembly and disassembly. Elife. https:// doi.org/10.7554/eLife.18413 Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. g g doi.org/10.7554/eLife.18413 Zucca FA, Segura-Aguilar J, Ferrari E, Muñoz P, Paris I, Sulzer D, Sarna T, Casella L, Zecca L (2017) Interactions of iron, dopa- mine and neuromelanin pathways in brain aging and Parkinson’s disease. Prog Neurobiol 155:96–119 g Wolozin B (2012) Regulated protein aggregation: stress granules and neurodegeneration. Mol Neurodegener 7:56 g g Wolozin B, Ivanov P (2019) Stress granules and neurodegeneration. Nat Rev Neurosci 20(11):649–666 Zucca FA, Vanna R, Cupaioli FA, Bellei C, de Palma A, Di Silvestre D, Mauri P, Grassi S, Prinetti A, Casella L, Sulzer D, Zecca L (2018) Neuromelanin organelles are specialized autolysosomes that accumulate undegraded proteins and lipids in aging human brain and are likely involved in Parkinson’s disease. NPJ Parkin- son’s Dis 4:17 Wulf M, Barkovits-Boeddinghaus K, Sommer P, Schork K, Eisenacher M, Riederer P, Gerlach M, Kösters S, Eggers B, Marcus K (2021) Laser microdissection-based protocol for the LC-MS/MS analysis of the proteomic profile of neuromelanin granules. JoVE. https:// doi.org/10.3791/63289 Xu Y, Stokes AH, Freeman WM, Kumer SC, Vogt BA, Vrana KE (1997) Tyrosinase mRNA is expressed in human substantia nigra. Brain Res Mol Brain Res 45(1):159–162 Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Zecca L, Swartz HM (1993) Total and paramagnetic metals in human substantia nigra and its neuromelanin. J Neural Transm Gen Sect 5(3):203–213 1 3
https://openalex.org/W3039924735	http://umu.diva-portal.org/smash/get/diva2:1429025/FULLTEXT01	English	null	Polymer Featuring Thermally Activated Delayed Fluorescence as Emitter in Light-Emitting Electrochemical Cells	The journal of physical chemistry letters	2,020	cc-by	8,531	http://www.diva-portal.org http://www.diva-portal.org http://www.diva-portal.org This is the published version of a paper published in Journal of Physical Chemistry Letters. pubs.acs.org/JPCL Polymer Featuring Thermally Activated Delayed Fluorescence as Emitter in Light-Emitting Electrochemical Cells Polymer Featuring Thermally Activated Delayed Fluorescence as Emitter in Light-Emitting Electrochemical Cells ABSTRACT: Semiconducting polymers that feature thermally activated delayed ﬂuorescence (TADF) can deliver a much desired combination of high-eﬃciency and metal-free electroluminescence and cost-eﬃcient solution-based fabrication. A TADF polymer is thus a very good ﬁt for the emitting compound in light-emitting electrochemical cells (LECs) because the commonly employed air-stabile and few- layer LEC architecture is well suited for such solution-based fabrication. Herein we report on the ﬁrst LEC device based on a TADF polymer as the emitting species, which delivers a luminance of 96 cd m−2 at 4 V and a current eﬃcacy of 1.4 cd A−1 and >600 cd m−2 at 6 V, which is competitive with the performance of multilayer organic light-emitting diodes based on the same TADF polymer. We further utilize the established sensitivity of the emission of the TADF polymer to its environment to draw conclusions on the exciton populations in host-guest and host-free TADF LEC devices. A recently invented class of heavy-metal free organic compounds addresses the above issue through the emission process of thermally activated delayed ﬂuorescence (TADF).28−32 Eﬃcient TADF emitters are designed so that the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) are spatially separated, which render the lowest excited singlet state (S1) and the lowest excited triplet state (T1) positioned close in energy; with such a design, a thermally promoted reverse intersystem crossing (RISC) from the nonemissive T1 to the emissive S1 can take place.33 Accordingly, all of the electrically generated singlet and triplet excitons can in a TADF emitter be utilized for light emission via prompt and delayed ﬂuorescence, respectively. T he emergence of thin, ﬂexible, and area-emissive technologies is projected to open up for new and functionalized applications of light in, for example, medicine1,2 and security.3 The frontrunner in the area-emissive ﬁeld is the organic light-emitting diode (OLED). At industrial grade, OLEDs widely adopt a multitude of diﬀerent layers, which are deposited with nanometer precision under high vacuum. As such, the OLED is, and will probably remain, expensive, which prohibits its use in several of the emerging applications. Citation for the original published paper (version of record): Citation for the original published paper (version of record): Citation for the original published paper (version of record): Lundberg, P., Wei, Q., Ge, Z., Voit, B., Reineke, S. et al. (2020) Polymer Featuring Thermally Activated Delayed Fluorescence as Emitter in Light- Emitting Electrochemical Cells Journal of Physical Chemistry Letters, 11(15): 6227-6234 https://doi.org/10.1021/acs.jpclett.0c01506 Access to the published version may require subscription. N.B. When citing this work, cite the original published paper. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-170537 http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-170537 This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. © 2020 American Chemical Society Letter pubs.acs.org/JPCL The Journal of Physical Chemistry Letters The operation of LEC devices is intrinsically dependent on the existence of mobile ions in the active material, and we further establish that the emissive state of the polymeric TADF emitter, because of its intramolecular charge-transfer character, is highly sensitive to the proximity of the polar ions. With this information in hand, we are able to draw conclusions regarding the exciton proﬁles that are present in diﬀerent LEC devices during operation, and we notably ﬁnd that the excitons can be self-trapped at ion-induced low-energy sites positioned at the edges of the emission zone. for enhanced solubility in common organic solvents, such as toluene, tetrahydrofuran, and chloroform. It is a common practice to disperse TADF (and triplet) emitters as a minority guest in a larger energy-gap host matrix because this inhibits the diﬀusion of the long-lived triplet excitons and thereby suppresses nondesired triplet−triplet and triplet−polaron quenching interactions. The host-guest design is also beneﬁcial because it lowers the self-absorption of the guest-generated light within the active material. The drawback is that the guest emitter can function as a trap for electron and hole transport, especially in thick host-guest layers. Herein we have, after careful screening (following the criteria detailed later), selected to employ 2,5-di(9H-carbazol-9-yl)pyridine (PYD-2Cz) as the host compound for the P1 guest emitter. The active material in LEC devices must, by deﬁnition, comprise an electrolyte, and the ionic liquid tetrahexylammo- nium tetraﬂuoroborate (THABF4) was employed because of its broad electrochemical stability window43 and because it has demonstrated good LEC performance in previous studies. A small amount of poly(styrene) (PS) was ﬁnally included in the LEC active material because it was found to render the device performance more repeatable in that it eﬀectively eliminated problems with electrical short circuits. The chemical structures of the active-material constituents are displayed in Figure 1a. ( ) Figure 1a presents the chemical structure of the TADF- active macrocycle conjugated polymer P1, which was synthesized by a Yamamoto-type polymerization of the non- TADF monomer (4-(3,6-dibromo-carbazol-9-yl)phenyl)(4- (dodecyloxy)phenyl)methanone.42 P1 is designed to feature a twist between the electron-donating carbazole unit (marked with a cyan background) and the electron-accepting benzophenone unit (marked with a purple background), which results in the HOMO positioned on the carbazole being spatially well separated from the LUMO localized on the benzophenone. The Journal of Physical Chemistry Letters Letter pubs.acs.org/JPCL Figure 1. (a) Chemical structure of the active-material constituents: the P1 polymeric TADF emitter, the PYD-2Cz host compound, the THABF4 electrolyte, and the PS additive. The electron-donor unit of P1 is marked with a cyan background color, and the electron-acceptor unit is marked with a purple background color. (b) Cyclic voltammetry traces recorded on thin ﬁlms of P1 (solid cyan line) and PYD-2Cz (dashed purple line). The vertical dotted lines indicate the onset potentials for oxidation and reduction. The scan speed was 0.05 V s−1. (c) Normalized absorbance of a thin ﬁlm comprising P1 dispersed in PS (open black circles; note that the contribution of PS is eﬀectively eliminated by the measurement procedure) and the normalized photoluminescence (PL) of neat ﬁlms of PYD-2Cz (solid purple squares) and P1 (solid cyan circles). (d) Normalized PL of diﬀerent active-material ﬁlms, with the mass ratio of the constituents deﬁned in the inset. The Journal of Physical Chemistry Letters pubs.acs.org/JPCL Letter Figure 1. (a) Chemical structure of the active-material constituents: the P1 polymeric TADF emitter, the PYD-2Cz host compound, the THABF4 electrolyte, and the PS additive. The electron-donor unit of P1 is marked with a cyan background color, and the electron-acceptor unit is marked with a purple background color. (b) Cyclic voltammetry traces recorded on thin ﬁlms of P1 (solid cyan line) and PYD-2Cz (dashed purple line). The vertical dotted lines indicate the onset potentials for oxidation and reduction. The scan speed was 0.05 V s−1. (c) Normalized absorbance of a thin ﬁlm comprising P1 dispersed in PS (open black circles; note that the contribution of PS is eﬀectively eliminated by the measurement procedure) and the normalized photoluminescence (PL) of neat ﬁlms of PYD-2Cz (solid purple squares) and P1 (solid cyan circles). (d) Normalized PL of diﬀerent active-material ﬁlms, with the mass ratio of the constituents deﬁned in the inset. Herein we address this issue and report on the pioneering realization of functional three-layer LEC devices based on a polymeric TADF emitter, which turn on and emit light at a low voltage below 4 V and which emit with a bright luminance of >600 cd m−2 at 6 V. pubs.acs.org/JPCL In this context, the light-emitting electrochemical cell (LEC) is an interesting alternative.4−9 The LEC can also feature con- formable areal emission, but, by the virtue of its in situ doping operational mechanism,10−14 it can be fabricated with low-cost solution-based printing and coating methods under ambient air.5,15−17 T p y A vast majority of today’s TADF emitters are small molecules, which commonly are best ﬁt for deposition by (expensive) physical vapor deposition under high vacuum. In contrast, polymers and oligomers allow for facile solution- based processing, but the number of such materials exhibiting TADF emission is very limited, in part due to a complicated synthesis protocol,34−41 and, so far, no LEC device based on an oligomeric or polymeric TADF emitter has been reported However, a current drawback is that today’s most eﬃcient LECs (and OLEDs) comprise phosphorescent organic semi- conducting emitters, which are based on expensive and rare heavy metals from the platinum group in the periodic table.18−24 This is because excitons (bound electron−hole pairs) form in a ∼3:1 triplet/singlet ratio in organic semiconductors during electric driving,25,26 and it is only such heavy-metal-based phosphorescent emitters that exhibit eﬃcient and high-brightness electroluminescence (EL) from the triplet state with a reasonable excited-state lifetime in the range of a few microseconds due to strong spin−orbit coupling. In addition, the singlet excitons in phosphorescent emitters can be eﬃciently transferred to the triplet state by intersystem crossing (ISC), so that, in principle, all electrically generated excitons can be harvested for light emission.27 oligomeric or polymeric TADF emitter has been reported. Received: May 15, 2020 Accepted: July 6, 2020 Published: July 6, 2020 7 https://dx.doi.org/10.1021/acs.jpclett.0c01506 J. Phys. Chem. Lett. 2020, 11, 6227−6234 Received: May 15, 2020 Accepted: July 6, 2020 Published: July 6, 2020 Received: May 15, 2020 Accepted: July 6, 2020 Published: July 6, 2020 © 2020 American Chemical Society © 2020 American Chemical Society https://dx.doi.org/10.1021/acs.jpclett.0c01506 J. Phys. Chem. Lett. 2020, 11, 6227−6234 6227 https://dx.doi.org/10.1021/acs.jpclett.0c01506 J. Phys. Chem. Lett. 2020, 11, 6227−6234 The Journal of Physical Chemistry Letters We observe that the PL emission of the PYD-2Cz host (solid purple squares) signiﬁcantly overlaps the absorption of the P1 guest (open black circles), which demonstrates that a principle requirement for Förster resonance energy transfer (FRET) from PYD-2Cz to P1 is fulﬁlled.47 The substantial separation, and the lack of overlap, between the PL spectrum of the emitter P1 (solid cyan circles) and the absorption spectrum of the host PYD-2Cz with an onset at 350 nm46 signals that the self-absorption within a thin layer of active material should be low. 6 y g The most dramatic spectral eﬀect is, however, observed following the addition of a small amount (8 mass %) of THABF4 electrolyte (and 9 mass % inert PS); see Figure 1d, solid cyan circles. The PL peak is red-shifted by 33 nm with respect to the neat P1 ﬁlm, whereas the PLQY is 47% under N2 and 41% under ambient air. Figure S2 shows that the inclusion of the THABF4 electrolyte has a negligible eﬀect on the absorption spectrum. Combined, these results therefore imply that the polar THABF4 electrolyte can interact strongly with the P1 emitter, but the eﬀect of this interaction is primarily manifested in the excited state of P1 (as detected with PL) and not in its ground state (as probed with absorption). We note that previous studies on TADF emitters report on a similar increasing red shift of the PL spectrum with increasing polar (solution) environment, which is in line with the fact that the excited state of TADF emitters exhibits an intramolecular charge-transfer character.50−52 y The triplet energy of PYD-2Cz is reported to be 2.93 eV.46 Because the singlet energy of P1 is 2.6 eV and because the singlet and triplet energies are essentially identical in TADF emitting materials by design, the conclusion is that Dexter energy transfer from PYD-2Cz to P1 should also be an eﬀective population pathway.48 The combined addition of both the PYD-2Cz host and the THABF4 electrolyte (and the PS polymer) to the P1 ﬁlm resulted in the highest PLQY values and a PL spectrum located in between that of the neat host-guest and the neat guest- electrolyte PL spectra. Speciﬁcally for the active-material ﬁlm optimized for LEC operation (Figure 1d, open red squares), featuring a {PYD-2Cz:P1:THABF4:PS} mass ratio of {66:17:8:9}, the PLQY is 71% under N2 and 63% under ambient air, and the PL peak is located at 505 nm. The Journal of Physical Chemistry Letters p g The HOMO and LUMO energy levels can be calculated from the CV-derived onset potentials for oxidation and reduction, respectively, using the equation: EHOMO/LUMO (eV) = −(4.8 eV + eVFc/Fc+ ox/red).45 Table S1 shows that the derived HOMO and LUMO levels for P1 are −5.2 and −2.4 eV, respectively, whereas the corresponding values for PYD- 2Cz are −5.5 and −2.0 eV. This results in the electrochemical energy gap being 2.8 eV for P1 and 3.5 eV for PYD-2Cz. The optical energy gap, as derived from the onset of absorption, is 2.6 eV for P1 (see Figure 1c) and 3.5 eV for PYD-2Cz.46 y g y We further ﬁnd that the PYD-2Cz host PL, with its peak located at 380 nm (Figure 1c, solid purple squares), is completely absent in the host-guest blend, which demonstrates that the host-to-guest transfer, as desired, is complete. We also note that signiﬁcant changes to the P1 emission capacity have been reported following the inclusion of other small-molecule host compounds, although the exact changes appear to be intimately linked to the detailed chemical and energy structure of the host compound.42,49 Our conclusion is that the PYD- 2Cz host and the P1 guest interact strongly in the host-guest blend, as manifested in the fact that the host-to-guest transfer is complete and the PLQY of P1 increases strongly, and we suggest that the majority PYD-2Cz host acts as a solid-state solvent for the minority P1 guest.50 g With the energy-level data in hand, the conclusion is that an intermolecular charge-transfer (or exciplex) state between the host and guest will not form in the PYD-2Cz:P1 host−guest system. Speciﬁcally, we ﬁnd that the LUMO oﬀset (or electron trap depth) for the PYD-2Cz:P1 host-guest active material is 0.4 eV, whereas the HOMO oﬀset (or hole trap depth) is 0.3 eV. It is noted that such balanced values for the electron and hole trap depths have been reported to facilitate the eﬃcient operation of host-guest LECs.43 p g Figure 1c presents the absorption spectrum of a thin ﬁlm comprising P1 diluted in PS at 10 mass % (left y-axis) and the photoluminescence (PL) spectra of neat PYD-2Cz and P1 thin ﬁlms (right y-axis). The Journal of Physical Chemistry Letters Letter voltammetry (CV). Figure 1b shows CV traces of thin ﬁlms of the TADF emitter P1 (solid cyan line) and the host PYD-2Cz (dashed purple line). We observe that both P1 and PYD-2Cz feature signiﬁcant and partially reversible oxidation reactions with onsets (as indicated by the vertical dotted lines in Figure 1b) at +0.4 and +0.7 V vs Fc/Fc+, respectively. The reduction reactions were of smaller magnitude and less reversible, and their onsets were located at −2.4 and −2.8 V vs Fc/Fc+ for P1 and PYD-2Cz, respectively. We mention that at least two prescans were required before the reduction of P1 could be observed. These results imply that the facile electrochemical p- type doping of both the P1 emitter and the PYD-2Cz host can be expected during LEC operation, but they raise some concerns regarding the eﬃciency and the extent of the n-type doping reaction.44 PL emission. Speciﬁcally, the PL peak remained at 485 nm, although a minor shoulder appeared at lower energies (Figure S1, solid black line), and the PLQY featured a very minor decrease by 3 to 4%. We rationalize that the retained emission performance of P1 following the addition of PS is due to the fact that the two polymers were poorly mixed and well- separated within the blend because of the characteristic low entropic gain that results from the mixing of polymers. p g g p y In contrast, the inclusion of a majority amount of a semiconducting small-molecule host provoked a much stronger eﬀect on the PLQY. Although the PL spectrum is essentially retained following the addition of 79 mass% PYD-2Cz (Figure 1d, open purple triangles), the PLQY almost doubled to 62% under N2 and increased to 51% under ambient air. The signiﬁcant diﬀerence between the PLQY under inert N2 and ambient air is in line with the fact that the P1 polymer is a TADF emitter. Speciﬁcally, a signiﬁcant fraction of the initially UV-excited singlets on the P1 polymer are transferred to the long-lived triplet state by ISC, where they are quenched by O2 molecules in ambient air. Under inert N2, in contrast, the triplets survive for a suﬃciently long time so that they can be recycled back to the emissive singlet state by RISC. https://dx.doi.org/10.1021/acs.jpclett.0c01506 J. Phys. Chem. Lett. 2020, 11, 6227−6234 The Journal of Physical Chemistry Letters The appearance of TADF emission from the P1 polymer, but not the constituent monomer, originates in a conjugation-induced reduction of the energy diﬀerence between the S1 and T1 states while keeping the radiative S1− S0 decay rate fast.42 The dodecyloxy end group was included The active material (or rather the organic semiconductor) in a functional LEC is electrochemically p-type-doped (oxidized) at the anode and n-type-doped (reduced) at the cathode during operation, so that a light-emitting p−n junction doping structure can form at the steady state. We investigated whether this basic doping requirement was fulﬁlled with cyclic 6228 pubs.acs.org/JPCL The Journal of Physical Chemistry Letters The Journal of Physical Chemistry Letters (c) Normalized EL spectrum measured at peak luminance for the host free LEC (solid cyan circles) and the host guest LEC (open orange squares) For reference emporal evolution of the luminance (left y-axis) and the current density (right y-axis) of ITO/active material/Al hos Figure 2. (a) Temporal evolution of the luminance (left y-axis) and the current density (right y-axis) of ITO/active material/Al host-free LECs during constant-voltage driving at 4 and 6 V. The host-free LECs featured an active-material mass ratio of {P1:THABF4:PS} = {83:8:9}, and the active-material thickness was 70 nm. (b) Temporal evolution of the luminance (left y-axis, solid cyan line) and the voltage (right y-axis, dashed black line) for ITO/PEDOT:PSS/active material/Al host-guest LEC during constant current-density driving at 100 A m−2. The host−guest LECs featured an active-material mass ratio of {PYD-2Cz:P1:THABF4:PS} = {66:17:8:9}, and the active-material thickness was 120 nm. (c) Normalized EL spectrum measured at peak luminance for the host-free LEC (solid cyan circles) and the host-guest LEC (open orange squares). For reference, the PL spectra of the electrolyte-free host−guest ﬁlm (dashed black line) and the host-free active-material ﬁlm (dotted black line) are included. The PL excitation wavelength was 300 nm. Figure 2. (a) Temporal evolution of the luminance (left y axis) and the current density (right y axis) of ITO/active material/Al host free LECs during constant-voltage driving at 4 and 6 V. The host-free LECs featured an active-material mass ratio of {P1:THABF4:PS} = {83:8:9}, and the active-material thickness was 70 nm. (b) Temporal evolution of the luminance (left y-axis, solid cyan line) and the voltage (right y-axis, dashed black line) for ITO/PEDOT:PSS/active material/Al host-guest LEC during constant current-density driving at 100 A m−2. The host−guest LECs featured an active-material mass ratio of {PYD-2Cz:P1:THABF4:PS} = {66:17:8:9}, and the active-material thickness was 120 nm. (c) Normalized EL spectrum measured at peak luminance for the host-free LEC (solid cyan circles) and the host-guest LEC (open orange squares). For reference, the PL spectra of the electrolyte-free host−guest ﬁlm (dashed black line) and the host-free active-material ﬁlm (dotted black line) are included. The PL excitation wavelength was 300 nm. lowered guest concentration) is an increase in the PLQY and a blue shift of the PL envelope. The Journal of Physical Chemistry Letters (See Figure S1.) We mention that the ﬂuctuating current without light emission during the ﬁrst few minutes of operation at 4 V was a relatively common observation for the host-free LECs, which we attribute to the existence and burnoﬀof minor microshorts through the 70 nm thin active-material ﬁlm. An eﬀective means to improve the quality and thickness of the active material was to include the high-solubility PYD-2Cz majority host compound in the active material in the host-guest LECs. We found that the best performance for the host-guest LEC was attained with an active-material mass ratio of {PYD- 2Cz:P1:THABF4:PS} = {66:17:8:9} and an active-material thickness of 120 nm. We have systematically evaluated the performance of two distinct types of P1-emitter-based LEC devices: “a host-free LEC” and “a host-guest LEC”. During the course of the project, we have tested >18 devices in each category, and the device yield was ∼50%. We found that the best performance for the host-free LECs was obtained at constant-voltage driving, whereas the host-guest LECs excelled at constant- current driving and with a thin layer of poly(3,4- ethylenedioxythiophene):poly(styrene-sulfonate) (PE- DOT:PSS) included between the indium-tin-oxide (ITO) anode and the active material. The presented results are for the champion devices, but a similar performance was recorded on at least three additional devices in each category. Figure 2b displays the temporal evolution of the luminance (left y-axis, solid cyan line) and the voltage (right y-axis, dashed black line) of such an optimized pristine host-guest ITO/ PEDOT:PSS/active material/Al LEC during driving by a constant current density of 100 A m−2. The luminance increased and reached 194 cd m−2 after 5 min of operation, which corresponds to a peak current eﬃcacy of 1.9 cd A−1 and a power eﬃcacy of 0.36 lm W−1. The voltage, following a short initial increase, consistently decreased during almost 1 h of operation, which is a direct consequence of the electrochemical doping rendering the active material more conductive. The consistently higher drive voltage for the host-guest LECs compared with the host-free LECs at the same current density is due to the fact that the undoped p−n junction in the former comprises transport-limiting nonﬁlled trap sites with a trap depth of 0.3 to 0.4 eV (see Table S1) in the form of P1 guest molecules dispersed in the PYD-2Cz host matrix. https://dx.doi.org/10.1021/acs.jpclett.0c01506 J. Phys. Chem. Lett. 2020, 11, 6227−6234 The Journal of Physical Chemistry Letters Figure 2a displays the temporal evolution of the luminance (left y-axis) and the current density (right y-axis) of pristine host-free LECs when driven by a constant voltage of 4 and 6 V. These devices featured a simple three-layer ITO/active material/Al structure, where the optimized 70 nm thick host- free {P1:THABF4:PS} active material exhibited a mass ratio of {83:8:9}. A simultaneous increase in current and luminance was observed during the initial operation, which originates in the mobile-ion-facilitated characteristic in situ electrochemical doping operation of LEC devices that improves the charge injection and renders the active material more conductive. As expected, identical devices void of mobile ions in the active material feature a constant (and much lower) current and no measurable luminance. The peak luminance at 4 V is 96 cd m−2 at a current density of 66 A m−2, corresponding to a current eﬃcacy exceeding 1.4 cd A−1. With the assumption of Lambertian emission, this performance translates to a power eﬃcacy of 1.0 lm W−1 at the peak luminance. By increasing the drive voltage to 6 V, the peak luminance increased to >600 cd m−2 but at a lowered current eﬃcacy of 0.9 cd A−1. We note that the luminance peaks before the current density, which implies that exciton− polaron quenching interactions start to play a signiﬁcant role when the fronts of the two doping regions approach each other and the eﬀective width of the p−n junction decreases.53 The corresponding data for the host-free LEC during driving with a constant current density of 100 A m−2 are presented in Figure S3 We also note that the same TADF-active P1 emitter has been implemented and evaluated in OLEDs, comprising a multitude (n = 3−5) of vacuum-evaporated and solution- processed functional layers in between the two electrodes.49 The structurally much simpler LEC devices in this study were found to be competitive in several performance metrics, and we call particular attention to the lower turn-on voltage and the higher power eﬃcacy at signiﬁcant luminance (>100 cd m−2) for the host-free LECs. Figure 2c presents the normalized EL spectra of the host- free LEC (solid cyan circles) and the host-guest LEC (open orange squares) recorded at peak luminance. For the host- guest LEC, no emission from the PYD-2Cz host (PL peak = 380 nm, solid purple squares in Figure 1c) can be detected, S3. 6230 https://dx.doi.org/10.1021/acs.jpclett.0c01506 J. Phys. Chem. Lett. 2020, 11, 6227−6234 https://dx.doi.org/10.1021/acs.jpclett.0c01506 J. The Journal of Physical Chemistry Letters The general trend with increasing host concentration (at the expense of a Figure 1d and Figure S1 present PL spectra of a number of diﬀerent thin ﬁlms comprising the P1 polymer. The neat P1 ﬁlm exhibits a featureless broad PL spectrum, with its emission peak at 485 nm (Figure 1d, dashed black line). The corresponding values for the PL quantum yield (PLQY) of the neat P1 ﬁlm are 33% in inert N2 and 32% under ambient air. We note that these results are essentially identical to those reported in a previous study on the P1 polymer.49 The addition of an insulating polymer to the P1 polymer in the form of 90 mass % PS resulted in marginal changes to the 6229 https://dx.doi.org/10.1021/acs.jpclett.0c01506 J. Phys. Chem. Lett. 2020, 11, 6227−6234 Figure 2. (a) Temporal evolution of the luminance (left y-axis) and the current density (right y-axis) of ITO/active material/Al host-free LECs during constant-voltage driving at 4 and 6 V. The host-free LECs featured an active-material mass ratio of {P1:THABF4:PS} = {83:8:9}, and the active-material thickness was 70 nm. (b) Temporal evolution of the luminance (left y-axis, solid cyan line) and the voltage (right y-axis, dashed black line) for ITO/PEDOT:PSS/active material/Al host-guest LEC during constant current-density driving at 100 A m−2. The host−guest LECs featured an active-material mass ratio of {PYD-2Cz:P1:THABF4:PS} = {66:17:8:9}, and the active-material thickness was 120 nm. (c) Normalized EL spectrum measured at peak luminance for the host-free LEC (solid cyan circles) and the host-guest LEC (open orange squares). For reference, the PL spectra of the electrolyte-free host−guest ﬁlm (dashed black line) and the host-free active-material ﬁlm (dotted black line) are included. The PL excitation wavelength was 300 nm. The Journal of Physical Chemistry Letters pubs.acs.org/JPCL Letter The Journal of Physical Chemistry Letters pubs.acs.org/JPCL Letter y y Figure 2. (a) Temporal evolution of the luminance (left y-axis) and the current density (right y-axis) of ITO/active material/Al host-free LECs during constant-voltage driving at 4 and 6 V. The host-free LECs featured an active-material mass ratio of {P1:THABF4:PS} = {83:8:9}, and the active-material thickness was 70 nm. (b) Temporal evolution of the luminance (left y-axis, solid cyan line) and the voltage (right y-axis, dashed black line) for ITO/PEDOT:PSS/active material/Al host-guest LEC during constant current-density driving at 100 A m−2. The host−guest LECs featured an active-material mass ratio of {PYD-2Cz:P1:THABF4:PS} = {66:17:8:9}, and the active-material thickness was 120 nm. ■EXPERIMENTAL METHODS j Accordingly, it seems plausible that the red-shifted EL from the host-free LEC at steady state is solely taking place from P1 polymers in close proximity to either the THA cations or the BF4 anions located within the doping regions, whereas the two- component EL from the host-guest LEC originates from a combination of P1 polymers solely surrounded by host molecules (the majority component at ∼500 nm) and from P1 polymers in proximity to the polar ions (the minority component at ∼550 nm). CV was carried out with a three-electrode setup equipped with a computer-controlled potentiostat (Autolab PGSTAT302, software: GPES). The working electrode comprised the material under study drop-cast on a Au-covered glass substrate, a Pt rod as the counter electrode, a Ag wire as the quasi-reference electrode, and 0.1 mol L−1 tetrabutylam- monium hexaﬂuorophosphate (TBAPF6, Sigma-Aldrich) in acetonitrile as the electrolyte solution. The scan rate was 0.05 V s−1. Directly after each CV scan, a calibration scan was run with a small amount of ferrocene added to the electrolyte so that the CV potentials could be reported versus the ferrocene/ ferrocenium ion (Fc/Fc+) reference potential. The reduction/ oxidation onset potentials were deﬁned as the intersection of the baseline with the tangent of the current at the half-peak height. The CV preparation and characterization were executed within a N2-ﬁlled glovebox ([O2] < 1 ppm, [H2O] < 0.5 ppm). Our qualitative explanation for these ﬁndings is as follows: In the host-free LEC, the excitons are free to diﬀuse around within the p−n junction until they are “trapped” on the lower energy P1 sites that exist close to the ions in the neighboring doping regions, from which they will emit (thus the red-shifted single-emission species). In the host−guest LEC, the exciton diﬀusion is greatly suppressed because the host compound is the majority component. In this scenario, the excitons will be quickly trapped on the P1 guest sites distributed over the entire p−n junction so that the emission will take place from both P1 sites in the central part of the p−n junction that are unaﬀected by the ions in the doping regions (the majority of the excitons) and from P1 sites close to the doping boundaries that will be aﬀected by the presence of the ions (the minority of the excitons). The Journal of Physical Chemistry Letters Letter LEC devices, which deliver a luminance of 96 cd m−2 at 4 V and a current eﬃcacy of 1.4 cd A−1 and >600 cd m−2 at 6 V. We note that this single-layer TADF LEC performance is competitive with that of multilayer OLEDs based on the same TADF polymer. We further show that the emission of the TADF polymer is highly sensitive to the proximity of the mobile ions, which are a fundamental constituent in LEC devices. This observation was utilized to shed light on the operation of TADF LEC devices, and we ﬁnd that in host− guest TADF LECs, the excitons are distributed over the entire p−n junction region, whereas in host-free TADF LECs the excitons appear to be strongly localized in close proximity to the doping regions. Our study thus identiﬁes and clariﬁes an interesting interplay between the excitons and the mobile ions in TADF LECs and also provides a contribution to the development of sustainable emissive devices that can be entirely fabricated with low-energy and cost-eﬃcient solution methods. which demonstrates that the host-to-guest energy transfer is complete. We further ﬁnd that the spectral diﬀerences between the two LEC systems are very large, with the host-free LEC emitting with a relatively narrow EL spectrum with its peak positioned at 547 nm (Commission Internationale de l’Éclairage (CIE) 1931 color space x,y coordinates = {0.39, 0.54}), whereas the host-guest LEC features a broader and distinctly blue-shifted EL spectrum with its peak emission at 500 nm (CIE x,y coordinates = {0.31, 0.44}). A closer inspection reveals that the EL spectrum of the host-free LEC appears to originate in one emitting species centered at 547 nm, whereas the EL spectrum of the host-guest LEC appears to be a superposition of two emitting components: a majority species with a peak wavelength of 500 nm and a minority species with a center wavelength of ∼550 nm. g To facilitate the discussion of the origin of these distinct spectral diﬀerences, Figure 2c also presents the PL spectra of the host-guest ﬁlm void of the THABF4 electrolyte (dashed black line) and the host-free active-material ﬁlm comprising the same electrolyte (dotted black line). ■EXPERIMENTAL METHODS The TADF polymer P1 (Mw = 20 500 g mol−1) was synthesized by Yamamoto-type polymerization of the mono- mer (4-(3,6-dibromo-carbazol-9-yl)phenyl)(4-(dodecyloxy)- phenyl)methanone using the procedure detailed in ref 42. The host PYD-2Cz (409.48 g mol−1, Lumtec), the electrolyte THABF4 (217.07 g mol−1, Sigma-Aldrich), and the polymer PS (Mw = 900 000 g mol−1 Sigma-Aldrich) were used as received. The master inks were prepared by separately dissolving the solutes in toluene at a concentration of 10 (P1) or 20 g L−1 (PYD-2Cz, THABF4 and PS) by stirring on a magnetic hot plate at 323 K for >3 h. The blend and active-material inks were prepared by blending the master inks in a desired mass ratio, followed by stirring on a magnetic hot plate at 323 K for >3 h. The ink-under-study was spin-coated (2000 rpm, 1000 rpm s−1, 60 s) on a carefully cleaned substrate and thereafter dried at 343 K for >2 h. The thickness of the dry active- material ﬁlm was measured with a proﬁlometer (DekTak XT, Bruker). The TADF polymer P1 (Mw = 20 500 g mol−1) was synthesized by Yamamoto-type polymerization of the mono- mer (4-(3,6-dibromo-carbazol-9-yl)phenyl)(4-(dodecyloxy)- phenyl)methanone using the procedure detailed in ref 42. The host PYD-2Cz (409.48 g mol−1, Lumtec), the electrolyte THABF4 (217.07 g mol−1, Sigma-Aldrich), and the polymer PS (Mw = 900 000 g mol−1 Sigma-Aldrich) were used as received. g g The master inks were prepared by separately dissolving the solutes in toluene at a concentration of 10 (P1) or 20 g L−1 (PYD-2Cz, THABF4 and PS) by stirring on a magnetic hot plate at 323 K for >3 h. The blend and active-material inks were prepared by blending the master inks in a desired mass ratio, followed by stirring on a magnetic hot plate at 323 K for >3 h. The ink-under-study was spin-coated (2000 rpm, 1000 rpm s−1, 60 s) on a carefully cleaned substrate and thereafter dried at 343 K for >2 h. The thickness of the dry active- material ﬁlm was measured with a proﬁlometer (DekTak XT, Bruker). https://dx.doi.org/10.1021/acs.jpclett.0c01506 J. Phys. Chem. Lett. 2020, 11, 6227−6234 The Journal of Physical Chemistry Letters These data remind us that the existence of polar ions in the neighborhood of the P1 emitter is concomitant with a distinct red shift of the emission spectrum, whereas the replacement of the polar electrolyte by the host molecules results in a blue shift of the P1 emission. We also remember that the steady-state operation of an LEC device dictates that all of the mobile ions are locked up in the doping regions as electrostatically compensating counterions.54 More speciﬁcally, at the steady state, the positive tetrahex- ylammonium (THA) cations are locked up in the n-type region in between the negative cathode and the p−n junction, whereas the negative BF4 anions are positioned in the p-type region between the positive anode and the emissive p−n junction. ■EXPERIMENTAL METHODS The Journal of Physical Chemistry Letters Phys. Chem. Lett. 2020, 11, 6227−6234 pubs.acs.org/JPCL The Journal of Physical Chemistry Letters * sı Supporting Information * sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jpclett.0c01506. Energy levels of the neat host and guest ﬁlms, optical properties of the neat host and guest ﬁlms and the host- free and host−guest active-material ﬁlms, and additional LEC device performance data (PDF) (5) Sandström, A.; Edman, L. Towards High-Throughput Coating and Printing of Light-Emitting Electrochemical Cells: A Review and Cost Analysis of Current and Future Methods. Energy Technology 2015, 3, 329−339. (6) Hu, S. Y.; Gao, J. Shaping Electroluminescence with a Large, Printed Bipolar Electrode Array: Solid Polymer Electrochemical Cells with over a Thousand Light-Emitting P-N Junctions. ChemElec- troChem 2020, 7, 1748−1751. Corresponding Authors (7) Yi, R. H.; Lo, C. L.; Luo, D.; Lin, C. H.; Weng, S. W.; Lu, C. W.; Liu, S. W.; Chang, C. H.; Su, H. C. Combinational Approach to Realize Highly Efficient Light-Emitting Electrochemical Cells. ACS Appl. Mater. Interfaces 2020, 12, 14254−14264. Qiang Wei −Ningbo Institute of Materials Technology & Engineering, Chinese Academy Sciences, Ningbo 315201, P. R. China; Email: weiqiang@nimte.ac.cn q g Ludvig Edman −The Organic Photonics and Electronics Group, Department of Physics, Umeå University, SE-901 87 Umeå, Sweden; LunaLEC AB, SE-901 87 Umeå, Sweden; pp f (8) Youssef, K.; Li, Y.; O’Keeffe, S.; Li, L.; Pei, Q. B. Fundamentals of Materials Selection for Light-Emitting Electrochemical Cells. Adv. Funct. Mater. 2020, 1909102. (9) Diethelm, M.; Grossmann, Q.; Schiller, A.; Knapp, E.; Jenatsch, S.; Kawecki, M.; Nüesch, F.; Hany, R. Optimized Electrolyte Loading and Active Film Thickness for Sandwich Polymer Light-Emitting Electrochemical Cells. Adv. Opt. Mater. 2019, 7, 1801278. orcid.org/0000-0003-2495-7037; Email: ludvig.edman@ umu.se The Journal of Physical Chemistry Letters pubs.acs.org/JPCL The Journal of Physical Chemistry Letters Sebastian Reineke −Dresden Integrated Center for Applied Physics and Photonic Materials (IAPP), Institute for Applied Physics, Technische Universität Dresden, DE-01069 Dresden, Germany; orcid.org/0000-0002-4112-6991 Sebastian Reineke −Dresden Integrated Center for Applied Physics and Photonic Materials (IAPP), Institute for Applied Physics, Technische Universität Dresden, DE-01069 Dresden, Germany; orcid.org/0000-0002-4112-6991 either an inert N2 atmosphere or an ambient-air atmosphere. The excitation wavelength in the PL and PLQY experiments was 300 nm. The ITO-coated glass substrates (ITO thickness = 160 nm, 20 Ω sq−1, Thin Film Devices) were cleaned by a sequential 20 min ultrasonic treatment in detergent (Extran MA 01, Merck), distilled water, acetone (VWR), and isopropanol (VWR), drying at 393 K for >4 h, and UV-ozone exposure for 20 min. PEDOT:PSS (, Clevios PVP AI 4083, Heraeus) was spin- coated (4000 rpm, 1000 rpm s−1, 60 s) on top of the ITO and thereafter dried at 393 K for >2 h. The thickness of the dry PEDOT:PSS ﬁlm was measured to be 35 nm. Complete contact information is available at: https://pubs.acs.org/10.1021/acs.jpclett.0c01506 ■REFERENCES The LEC devices were driven by and measured with a computer-controlled current−voltage−luminance system (OLED Lifetime Tester M6000, McScience), with the luminance measured in the forward direction. All LEC devices were characterized within 24 h of fabrication. The EL spectrum and intensity were measured with a carefully calibrated ﬁber- optic CCD-array spectrometer (Flame-S, Ocean Optics). (1) Yamanaka, T.; Nakanotani, H.; Hara, S.; Hirohata, T.; Adachi, C. Near-Infrared Organic Light-Emitting Diodes for Biosensing with High Operating Stability. Appl. Phys. Express 2017, 10, 074101. g p g y pp y p (2) Chaves, M. E. D.; de Araujo, A. R.; Piancastelli, A. C. C.; Pinotti, M. Effects of Low-Power Light Therapy on Wound Healing: Laser X Led. An. Bras. Dermatol. 2014, 89, 616−623. (3) Fischer, B.; Kreissl, S.; Boeffel, C.; Wedel, A. Multi-Layer Printing of Oleds as a Tool for the Creation of Security Features. Opt. Express 2012, 20, A219−A223. The authors declare no competing ﬁnancial interest. The authors declare no competing ﬁnancial interest. ■ACKNOWLEDGMENTS The active-material ink was spin-coated either on top of the ITO or on top of the ITO/PEDOT:PSS and dried at 343 K for >3 h. The thickness of the active material was controlled by the spin-coating parameters (800 rpm, 800 rpm s−1, 60 s). A 100 nm thick Al cathode was deposited on top of the active material by thermal vacuum evaporation at a base pressure below 2 × 10−6 mbar. The emission area 2 × 2 mm2 was deﬁned by the overlap between the Al top cathode and the ITO bottom anode. All of the above procedures, with the exception of the PEDOT:PSS deposition, were carried out in two interconnected N2-ﬁlled glove boxes ([O2] < 1 ppm, [H2O] < 0.5 ppm). We acknowledge the ﬁnancial support from the Swedish Research Council, the Swedish Energy Agency, Carl Tryggers stiftelse, Kempestiftelserna, Stiftelsen Olle Engkvist Byggmäs- tare, Bertil & Britt Svenssons stiftelse för belysningsteknik, the National Natural Science Foundation of China (21805296, 21876158, 51773212, 21574144, and 21674123), Zhejiang Provincial Natural Science Foundation of China (LR16b040002), Natural Science Foundation of Ningbo City (2018A610134), and Zhejiang Provincial Natural Science Foundation of China (grant no. LY20B040002). ■ASSOCIATED CONTENT (4) Choi, J.; Kanagaraj, S.; Choe, Y. Utilization of Novel Phenanthrene-Imidazole-Based Ionic Small Molecules for Blue Light-Emitting Electrochemical Cells. J. Mater. Chem. C 2020, 8, 4580−4587. ■EXPERIMENTAL METHODS Accordingly, the observed ion-induced shift of the TADF emission, as exempliﬁed in Figure 1d, can also shed light on the exciton distributions in host-free and host-guest TADF-emitting LEC devices. The optical transmission of 100 nm thick spin-coated thin ﬁlms on quartz substrates (thickness = 1 mm, Ted Pella) was measured with a spectrometer (C9920-02G, Hamamatsu Photonics), with a 100 nm thick PS ﬁlm on a quartz substrate as a reference. The absorbance was estimated as [1 − transmission], thus neglecting the eﬀects of reﬂectance. The PL spectrum and the PLQY of the thin ﬁlms were measured with an integrated sphere connected to the spectrometer under To summarize, we report that a novel TADF-emitting polymer can be utilized as the metal-free eﬃcient emitter in 6231 https://dx.doi.org/10.1021/acs.jpclett.0c01506 J. Phys. Chem. Lett. 2020, 11, 6227−6234 Letter The Journal of Physical Chemistry Letters High-Efficiency Blue Organic Light-Emitting Diodes Based on Thermally Activated Delayed Fluorescence from Phenoxaphosphine and Phenoxathiin Derivatives. Adv. Mater. 2016, 28, 4626−4631. (22) Costa, R. D.; Orti, E.; Bolink, H. J.; Monti, F.; Accorsi, G.; Armaroli, N. Luminescent Ionic Transition-Metal Complexes for Light-Emitting Electrochemical Cells. Angew. Chem., Int. Ed. 2012, 51, 8178−8211. (40) Yang, Y.; Wang, S.; Zhu, Y.; Wang, Y.; Zhan, H.; Cheng, Y. Thermally Activated Delayed Fluorescence Conjugated Polymers with Backbone-Donor/Pendant-Acceptor Architecture for Nondoped Oleds with High External Quantum Efficiency and Low Roll-Off. Adv. Funct. Mater. 2018, 28, 1706916. (23) Slinker, J. D.; Rivnay, J.; Moskowitz, J. S.; Parker, J. B.; Bernhard, S.; Abruna, H. D.; Malliaras, G. G. Electroluminescent Devices from Ionic Transition Metal Complexes. J. Mater. Chem. 2007, 17, 2976−2988. (41) Wei, Q.; Ge, Z.; Voit, B. Thermally Activated Delayed Fluorescent Polymers: Structures, Properties, and Applications in Oled Devices. Macromol. Rapid Commun. 2019, 40, 1800570. (24) Fresta, E.; Monclus, M. A.; Bertz, M.; Ezquerro, C.; Molina- Aldareguia, J. M.; Berenguer, J. R.; Kunimoto, M.; Homma, T.; Costa, R. D. Key Ionic Electrolytes for Highly Self-Stable Light-Emitting Electrochemical Cells Based on Ir(Iii) Complexes. Adv. Opt. Mater. 2020, 8, 2000295. (42) Wei, Q.; et al. Conjugation-Induced Thermally Activated Delayed Fluorescence (Tadf): From Conventional Non-Tadf Units to Tadf-Active Polymers. Adv. Funct. Mater. 2017, 27, 1605051. df-Active Polymers. Adv. Funct. Mater. 2017, 27, 1605051. (25) Segal, M.; Baldo, M. A.; Holmes, R. J.; Forrest, S. R.; Soos, Z. G. Excitonic Singlet-Triplet Ratios in Molecular and Polymeric Organic Materials. Phys. Rev. B: Condens. Matter Mater. Phys. 2003, 68, 075211. (43) Tang, S.; Sandström, A.; Lundberg, P.; Lanz, T.; Larsen, C.; van Reenen, S.; Kemerink, M.; Edman, L. Design Rules for Light-Emitting Electrochemical Cells Delivering Bright Luminance at 27.5% External Quantum Efficiency. Nat. Commun. 2017, 8, 1190. Quantum Efficiency. Nat. Commun. 2017, 8, 1190 (26) Reufer, M.; Walter, M. J.; Lagoudakis, P. G.; Hummel, A. B.; Kolb, J. S.; Roskos, H. G.; Scherf, U.; Lupton, J. M. Spin-Conserving Carrier Recombination in Conjugated Polymers. Nat. Mater. 2005, 4, 340−346. (44) Tang, S.; Buchholz, H. A.; Edman, L. On the Selection of a Host Compound for Efficient Host-Guest Light-Emitting Electro- chemical Cells. J. Mater. Chem. C 2015, 3, 8114−8120. (45) Pommerehne, J.; Vestweber, H.; Guss, W.; Mahrt, R. F.; Bassler, H.; Porsch, M.; Daub, J. Efficient Two Layer Leds on a Polymer Blend Basis. Adv. Mater. 1995, 7, 551−554. (27) Köhler, A.; Bässler, H. The Journal of Physical Chemistry Letters Materials Chemistry Frontiers 2019, 3, 1699−1721. (36) Nikolaenko, A. E.; Cass, M.; Bourcet, F.; Mohamad, D.; Roberts, M. Thermally Activated Delayed Fluorescence in Polymers: A New Route toward Highly Efficient Solution Processable Oleds. Adv. Mater. 2015, 27, 7236−7240. ( ) (36) Nikolaenko, A. E.; Cass, M.; Bourcet, F.; Mohamad, D.; Roberts, M. Thermally Activated Delayed Fluorescence in Polymers: , , (18) Nuss, P.; Eckelman, M. J. Life Cycle Assessment of Metals: A Scientific Synthesis. PLoS One 2014, 9, No. e101298. A New Route toward Highly Efficient Solution Processable Oleds. Adv. Mater. 2015, 27, 7236−7240. y (19) Namanga, J. E.; Gerlitzki, N.; Mudring, A.-V. Scrutinizing Design Principles toward Efficient, Long-Term Stable Green Light- Emitting Electrochemical Cells. Adv. Funct. Mater. 2017, 27, 1605588. (20) Henwood, A. F.; et al. Blue-Emitting Cationic Iridium(Iii) Complexes Featuring Pyridylpyrimidine Ligands and Their Use in Sky-Blue Electroluminescent Devices. J. Mater. Chem. C 2017, 5, 9638−9650. (37) Luo, J.; Xie, G.; Gong, S.; Chen, T.; Yang, C. Creating a Thermally Activated Delayed Fluorescence Channel in a Single Polymer System to Enhance Exciton Utilization Efficiency for Bluish- Green Electroluminescence. Chem. Commun. 2016, 52, 2292−2295. Green Electroluminescence. Chem. Commun. 2016, 52, 2292−2295. (38) Nobuyasu, R. S.; Ren, Z.; Griffiths, G. C.; Batsanov, A. S.; Data, P.; Yan, S.; Monkman, A. P.; Bryce, M. R.; Dias, F. B. Rational Design of Tadf Polymers Using a Donor-Acceptor Monomer with Enhanced Tadf Efficiency Induced by the Energy Alignment of Charge Transfer and Local Triplet Excited States. Adv. Opt. Mater. 2016, 4, 653−653. (39) Lee, S. Y.; Adachi, C.; Yasuda, T. High-Efficiency Blue Organic Light-Emitting Diodes Based on Thermally Activated Delayed Fluorescence from Phenoxaphosphine and Phenoxathiin Derivatives. Adv. Mater. 2016, 28, 4626−4631. (38) Nobuyasu, R. S.; Ren, Z.; Griffiths, G. C.; Batsanov, A. S.; Data, P.; Yan, S.; Monkman, A. P.; Bryce, M. R.; Dias, F. B. Rational Design of Tadf Polymers Using a Donor-Acceptor Monomer with Enhanced Tadf Efficiency Induced by the Energy Alignment of Charge Transfer and Local Triplet Excited States. Adv. Opt. Mater. 2016, 4, 653−653. (39) Lee S Y Adachi C Yasuda T High Efficiency Blue Organic (21) Bolink, H. J.; Cappelli, L.; Coronado, E.; Gratzel, M.; Orti, E.; Costa, R. D.; Viruela, P. M.; Nazeeruddin, M. K. Stable Single-Layer Light-Emitting Electrochemical Cell Using 4,7-Diphenyl-1,10-Phe- nanthroline-Bis(2-Phenylpyridine) Iridium(Iii) Hexafluorophosphate. J. Am. Chem. Soc. 2006, 128, 14786−14787. (39) Lee, S. Y.; Adachi, C.; Yasuda, T. Authors p (10) Zhang, Z. T.; et al. A Colour-Tunable, Weavable Fibre-Shaped Polymer Light-Emitting Electrochemical Cell. Nat. Photonics 2015, 9, 233−238. Petter Lundberg −The Organic Photonics and Electronics Group, Department of Physics, Umeå University, SE-901 87 Umeå, Sweden (11) Lanz, T.; Sandström, A.; Tang, S.; Chabrecek, P.; Sonderegger, U.; Edman, L. A Light-Emission Textile Device: Conformal Spray- Sintering of a Woven Fabric Electrode. Flexible and Printed Electronics 2016, 1, 025004. Ziyi Ge −Ningbo Institute of Materials Technology & Engineering, Chinese Academy Sciences, Ningbo 315201, P. R. China; orcid.org/0000-0003-3656-6017 g Brigitte Voit −Leibniz-Institut für Polymerforschung Dresden e.V., DE-01069 Dresden, Germany; orcid.org/0000-0002- 4531-691X (12) Liang, J. J.; Li, L.; Niu, X. F.; Yu, Z. B.; Pei, Q. B. Elastomeric Polymer Light-Emitting Devices and Displays. Nat. Photonics 2013, 7, 817−824. 6232 https://dx.doi.org/10.1021/acs.jpclett.0c01506 J. Phys. Chem. Lett. 2020, 11, 6227−6234 y , , (30) Wong, M. Y.; Zysman-Colman, E. Purely Organic Thermally Activated Delayed Fluorescence Materials for Organic Light-Emitting Diodes. Adv. Mater. 2017, 29, 1605444. The Journal of Physical Chemistry Letters The Journal of Physical Chemistry Letters pubs.acs.org/JPCL Letter (31) Uoyama, H.; Goushi, K.; Shizu, K.; Nomura, H.; Adachi, C. Highly Efficient Organic Light-Emitting Diodes from Delayed Fluorescence. Nature 2012, 492, 234−238. (13) Asadpoordarvish, A.; Sandström, A.; Larsen, C.; Bollström, R.; Toivakka, M.; Österbacka, R.; Edman, L. Light-Emitting Paper. Adv. Funct. Mater. 2015, 25, 3238−3245. (14) Li, X. Y.; Gao, J.; Liu, G. J. Thickness Dependent Device Characteristics of Sandwich Polymer Light-Emitting Electrochemical Cell. Org. Electron. 2013, 14, 1441−1446. (32) Wong, M. Y.; La-Placa, M.-G.; Pertegas, A.; Bolink, H. J.; Zysman-Colman, E. Deep-Blue Thermally Activated Delayed Fluorescence (Tadf) Emitters for Light-Emitting Electrochemical Cells (Leecs). J. Mater. Chem. C 2017, 5, 1699−1705. g (15) Hernandez-Sosa, G.; Tekoglu, S.; Stolz, S.; Eckstein, R.; Teusch, C.; Trapp, J.; Lemmer, U.; Hamburger, M.; Mechau, N. The (15) Hernandez-Sosa, G.; Tekoglu, S.; Stolz, S.; Eckstein, R.; Teusch, C.; Trapp, J.; Lemmer, U.; Hamburger, M.; Mechau, N. The Compromises of Printing Organic Electronics: A Case Study of Gravure-Printed Light-Emitting Electrochemical Cells. Adv. Mater. 2014, 26, 3235−3240. ( ) J , , (33) Penfold, T. J.; Dias, F. B.; Monkman, A. P. The Theory of Thermally Activated Delayed Fluorescence for Organic Light Emitting Diodes. Chem. Commun. 2018, 54, 3926−3935. (33) Penfold, T. J.; Dias, F. B.; Monkman, A. P. The Theory of g Compromises of Printing Organic Electronics: A Case Study of Gravure-Printed Light-Emitting Electrochemical Cells. Adv. Mater. 2014, 26, 3235−3240. Thermally Activated Delayed Fluorescence for Organic Light Emitting Diodes. Chem. Commun. 2018, 54, 3926−3935. (34) Liu, Y.; Li, C.; Ren, Z.; Yan, S.; Bryce, M. R. All-Organic Thermally Activated Delayed Fluorescence Materials for Organic Light-Emitting Diodes. Nature Reviews Materials 2018, 3, 18020. (16) Sandström, A.; Dam, H. F.; Krebs, F. C.; Edman, L. Ambient Fabrication of Flexible and Large-Area Organic Light-Emitting Devices Using Slot-Die Coating. Nat. Commun. 2012, 3, 1002. g g , , (35) Jhulki, S.; Cooper, M. W.; Barlow, S.; Marder, S. R. Phosphorescent and Tadf Polymers and Dendrimers in Solution- Processed Self-Host Organic Light-Emitting Diodes: Structure Analysis and Design Perspectives. Materials Chemistry Frontiers 2019, 3, 1699−1721. (17) Zimmermann, J.; Schlisske, S.; Held, M.; Tisserant, J.-N.; Porcarelli, L.; Sanchez-Sanchez, A.; Mecerreyes, D.; Hernandez-Sosa, G. Ultrathin Fully Printed Light-Emitting Electrochemical Cells with Arbitrary Designs on Biocompatible Substrates. Adv. Mater. Technol. 2019, 4, 1800641. Phosphorescent and Tadf Polymers and Dendrimers in Solution- Processed Self-Host Organic Light-Emitting Diodes: Structure Analysis and Design Perspectives. https://dx.doi.org/10.1021/acs.jpclett.0c01506 J. Phys. Chem. Lett. 2020, 11, 6227−6234 The Journal of Physical Chemistry Letters Triplet States in Organic Semi- conductors. Mater. Sci. Eng., R 2009, 66, 71−109. (28) Lundberg, P.; Tsuchiya, Y.; Lindh, E. M.; Tang, S.; Adachi, C.; Edman, L. Thermally Activated Delayed Fluorescence with 7% External Quantum Efficiency from a Light-Emitting Electrochemical Cell. Nat. Commun. 2019, 10, 5307. y (46) Son, K. S.; Yahiro, M.; Imai, T.; Yoshizaki, H.; Adachi, C. Analyzing Bipolar Carrier Transport Characteristics of Diarylamino- Substituted Heterocyclic Compounds in Organic Light-Emitting Diodes by Probing Electroluminescence Spectra. Chem. Mater. 2008, 20, 4439−4446. (29) Zeng, W.; Lai, H.-Y.; Lee, W.-K.; Jiao, M.; Shiu, Y.-J.; Zhong, C.; Gong, S.; Zhou, T.; Xie, G.; Sarma, M.; Wong, K.-T.; Wu, C.-C.; Yang, C. Achieving Nearly 30% External Quantum Efficiency for Orange-Red Organic Light Emitting Diodes by Employing Thermally Activated Delayed Fluorescence Emitters Composed of 1,8-Naph- thalimide-Acridine Hybrids. Adv. Mater. 2018, 30, 1870033. (47) Scholes, G. D. Long-Range Resonance Energy Transfer in Molecular Systems. Annu. Rev. Phys. Chem. 2003, 54, 57−87. (47) Scholes, G. D. Long-Range Resonance Energy Transfer in Molecular Systems. Annu. Rev. Phys. Chem. 2003, 54, 57−87. (48) Dexter, D. L. A Theory of Sensitized Luminescence in Solids. J. Chem. Phys. 1953, 21, 836−850. (48) Dexter, D. L. A Theory of Sensitized Luminescence in Solids. J. Chem. Phys. 1953, 21, 836−850. y (30) Wong, M. Y.; Zysman-Colman, E. Purely Organic Thermally Activated Delayed Fluorescence Materials for Organic Light-Emitting Diodes. Adv. Mater. 2017, 29, 1605444. y (49) Li, Y.; Wei, Q.; Cao, L.; Fries, F.; Cucchi, M.; Wu, Z.; Scholz, R.; Lenk, S.; Voit, B.; Ge, Z.; Reineke, S. Organic Light-Emitting Diodes Based on Conjugation-Induced Thermally Activated Delayed 6233 https://dx.doi.org/10.1021/acs.jpclett.0c01506 J. Phys. Chem. Lett. 2020, 11, 6227−6234 https://dx.doi.org/10.1021/acs.jpclett.0c01506 J. Phys. Chem. Lett. 2020, 11, 6227−6234 Letter The Journal of Physical Chemistry Letters The Journal of Physical Chemistry Letters pubs.acs.org/JPCL Fluorescence Polymers: Interplay between Intra- and Intermolecular Charge Transfer States. Front. Chem. 2019, 7, 1−12. Fluorescence Polymers: Interplay between Intra- and Intermolecular Charge Transfer States. Front. Chem. 2019, 7, 1−12. g (50) Northey, T.; Stacey, J.; Penfold, T. J. The Role of Solid State Solvation on the Charge Transfer State of a Thermally Activated Delayed Fluorescence Emitter. J. Mater. Chem. C 2017, 5, 11001− 11009. (51) Zhang, D. D.; Cai, M. H.; Zhang, Y. G.; Zhang, D. Q.; Duan, L. Sterically Shielded Blue Thermally Activated Delayed Fluorescence Emitters with Improved Efficiency and Stability. Mater. Horiz. 2016, 3, 145−151. (52) Hosokai, T.; Noda, H.; Nakanotani, H.; Nawata, T.; Nakayama, Y.; Matsuzaki, H.; Adachi, C. Solvent-Dependent Investigation of Carbazole Benzonitrile Derivatives: Does the 3 le - 1 ct Energy Gap Facilitate Thermally Activated Delayed Fluorescence? J. Photonics Energy 2018, 8, 1. (53) van Reenen, S.; Akatsuka, T.; Tordera, D.; Kemerink, M.; Bolink, H. J. Universal Transients in Polymer and Ionic Transition Metal Complex Light-Emitting Electrochemical Cells. J. Am. Chem. Soc. 2013, 135, 886−891. (54) Fang, J.; Matyba, P.; Edman, L. The Design and Realization of Flexible, Long-Lived Light-Emitting Electrochemical Cells. Adv. Funct. Mater. 2009, 19, 2671−2676. 6234
W4247566585.txt	https://www.qeios.com/read/1CGHRT/pdf	fr		Bacteroides fragilis	Definitions	2,020	cc-by	106	Qeios · Definition, February 7, 2020 Ope n Pe e r Re v ie w on Qe ios Bacteroides fragilis National Cancer Institute Source National Cancer Institute. Bacteroides fragilis. NCI T hesaurus. Code C86172. A species of obligately anaerobic, Gram-negative, rod shaped bacteria assigned to the phylum Bacteroidetes. T his species is nonmotile, non-spore forming, indole positive, produces hydrogen sulfide, does not reduce nitrate, hydrolyzes esculin and starch, does not ferment trehalose, melezitose or arabinose, and is penicillin resistant. B. fragilis is commensal and part of the normal human gastrointestinal tract flora, but may become pathogenic in other body regions. Qeios ID: 1CGHRT · https://doi.org/10.32388/1CGHRT 1/1
https://openalex.org/W1900136306	https://repository.ubn.ru.nl//bitstream/handle/2066/152239/152239.pdf	English	null	A randomized controlled trial of web-based cognitive behavioral therapy for severely fatigued breast cancer survivors (CHANGE-study): study protocol	BMC cancer	2,015	cc-by	7,700	Version of the following full text: Publisher’s version Downloaded from: http://hdl.handle.net/2066/152239 Download date: 2024-10-24 Version of the following full text: Publisher’s version Downloaded from: http://hdl.handle.net/2066/152239 Download date: 2024-10-24 A randomized controlled trial of web-based cognitive behavioral therapy for severely fatigued breast cancer survivors (CHANGE-study): study protocol Abrahams, H.J.G.; Gielissen, M.F.M.; Goedendorp, M.M.; Berends, T.B.M.; Peters, M.E.W.J.; Poort, H.; Verhagen, C.A.H.H.V.M.; Knoop, H. 2015, Article / Letter to editor (BMC Cancer, 15, (2015), article 765) Doi link to publisher: https://doi.org/10.1186/s12885-015-1787-7 STUDY PROTOCOL Open Access © 2015 Abrahams et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Note: Note: To cite this publication please use the final published version (if applicable). To cite this publication please use the final published version (if applicable). Abrahams et al. BMC Cancer (2015) 15:765 DOI 10.1186/s12885-015-1787-7 A randomized controlled trial of web-based cognitive behavioral therapy for severely fatigued breast cancer survivors (CHANGE-study): study protocol H. J. G. Abrahams1, M. F. M. Gielissen1, M. M. Goedendorp2, T. Berends1, M. E. W. J. Peters3, H. Poort1, C. A. H. H. V. M. Verhagen3 and H. Knoop1 H. J. G. Abrahams1, M. F. M. Gielissen1, M. M. Goedendorp2, T. Berends1, M. E. W. J. Peters3, H. Poort1, C. A. H. H. V. M. Verhagen3 and H. Knoop1 Abstract Background: About one third of breast cancer survivors suffer from persistent severe fatigue after completion of curative cancer treatment. Face-to-face cognitive behavioral therapy (F2F CBT), especially designed for fatigue in cancer survivors, was found effective in reducing fatigue. However, this intervention is intensive and treatment capacity is limited. To extend treatment options, a web-based version of CBT requiring less therapist time was developed. This intervention is aimed at changing fatigue-perpetuating cognitions and behaviors. The efficacy of web-based CBT will be examined in a multicenter randomized controlled trial. Methods: In total, 132 severely fatigued breast cancer survivors will be recruited and randomized to either an intervention condition or care as usual (ratio 1:1). Participants will be assessed at baseline and 6 months thereafter. The intervention group will receive web-based CBT, consisting of three F2F sessions and maximally eight web-based modules over a period of 6 months. The care as usual group will be on a waiting list for regular F2F CBT. The total duration of the waiting list is 6 months. The primary outcome of the study is fatigue severity. Secondary outcomes are functional impairments, psychological distress and quality of life. Discussion: If web-based CBT is effective, it will provide an additional treatment option for fatigue in breast cancer survivors. Web-based CBT is expected to be less time-consuming for therapists than regular F2F CBT, which would result in an increased treatment capacity. Moreover, the intervention would become more easily accessible for a larger number of patients, and patients can save travel time and costs. Keywords: Breast cancer, Survivor, Fatigue, Cognitive behavioral therapy, Web-based CBT, E-health, Randomized controlled trial, Study protocol * Correspondence: Harriet.Abrahams@radboudumc.nl 1Expert Center for Chronic Fatigue (ECCF), Radboud University Medical Center, PO Box 9101, 9166500 HB Nijmegen, The Netherlands Full list of author information is available at the end of the article © 2015 Abrahams et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to Background concerns are raised about their long-term well-being. After completion of curative cancer treatment, side-effects can become chronic. One of these persistent side-effects is cancer-related fatigue [3]. The National Comprehensive Cancer Network defined cancer-related fatigue as “a dis- tressing, persistent, subjective sense of physical, emotional and/or cognitive tiredness, related to cancer or cancer treatment, that is not proportional to recent activity and interferes with usual functioning” [4]. Once the malig- nancy is successfully treated, the fatigue is expected to de- crease. Nevertheless, severe fatigue becomes a chronic Worldwide, breast cancer is the most common malig- nancy in women. About 1.7 million new cases were diag- nosed in 2012 [1]. In the last decades, survival rates have been improved due to early detection by screening pro- grams and advances in oncological treatments [2, 3]. Since the number of breast cancer survivors increases, * Correspondence: Harriet.Abrahams@radboudumc.nl 1Expert Center for Chronic Fatigue (ECCF), Radboud University Medical Center, PO Box 9101, 9166500 HB Nijmegen, The Netherlands Full list of author information is available at the end of the article Page 2 of 10 Abrahams et al. BMC Cancer (2015) 15:765 condition in approximately one-third of breast cancer sur- vivors [5–8]. face (F2F) therapy. However, this F2F CBT is intensive for both therapists and patients, since it consists of 12 to 14 F2F sessions over a period of 6 months. Therapists need to invest considerable time to deliver these sessions and a limited number of trained cognitive behavioral therapists provide this F2F therapy. Besides, patients need to travel to a treatment center to attend the sessions. The develop- ment of web-based CBT would reduce the therapist time needed to deliver the intervention and increase treatment accessibility for severely fatigued breast cancer survivors. Interventions for fatigue in cancer survivors Since persisting severe fatigue interferes with daily func- tioning and has profound effects on quality of life, it should not be left untreated [5, 9]. The evidence of avail- able interventions was recently evaluated in a practice guideline of the American Society of Clinical Oncology [10]. It was concluded that there is evidence for the efficacy of physical and psychosocial interventions. Initiating or maintaining adequate levels of physical activity [11–19], (cognitive) behavioral therapy [20–25], and (psycho) educational interventions [20, 25, 26] can reduce fatigue. In addition, there is some evidence for the efficacy of mindfulness-based approaches [21, 27, 28], yoga [29, 30], and acupuncture [31, 32]. Web-based CBT The fast-growing field of e-health has created new possi- bilities in the development of web-based interventions. Web-based CBT has been developed and examined for a wide range of mental health problems, and so far, results are promising. Multiple studies have shown that web- based CBT can be effective in reducing mental health problems [35]. To extend treatment options for severely fatigued breast cancer survivors, we have developed a web-based version of our F2F CBT protocol for severely fatigued cancer survivors, named “On the road to recovery”. The efficacy of this intervention will be examined in a RCT, named “the CHANGE-study”. The current study focuses on one of these evidence- based interventions: cognitive behavioral therapy (CBT). A CBT protocol for fatigue in cancer survivors with vari- ous tumor types was developed and tested in a random- ized controlled trial (RCT) at our treatment center, the Expert Center for Chronic Fatigue of the Radboud uni- versity medical center (Radboudumc) [22]. This RCT showed that patients reported a clinically significant re- duction in fatigue and functional impairments following CBT [22]. These effects were maintained at a 2-year follow-up [33]. The efficacy of the CBT protocol was re- cently replicated in a RCT of Prinsen et al. [34]. The CBT protocol is based on a model of precipitating and perpetuating factors of fatigue [22]. According to this model, the malignancy and its treatment are the precipi- tating factors that induced fatigue. However, other fac- tors are responsible for the persistence of severe fatigue after cancer treatment [22]. These fatigue-perpetuating factors and the overall explanatory model are captured in Fig. 1. Each fatigue-perpetuating factor is addressed in a module of the CBT protocol, offered as regular face-to- The right time to intervene In our previous RCT’s examining regular F2F CBT for severely fatigued cancer survivors, the intervention was offered at least 1 year after completion of cancer treat- ment [22, 34]. However, recent research has shown that the level of fatigue does not decrease further after 3 months following curative cancer treatment [36], and fatigue-perpetuating factors can already be identi- fied at 3 months following cancer treatment [37]. Therefore, it might be possible to treat fatigue in can- cer survivors at an earlier stage. To examine if this is the case, the web-based CBT will be offered at least 3 months after completion of cancer treatment. Fig. 1 Explanatory model of the CBT protocol Page 3 of 10 Abrahams et al. BMC Cancer (2015) 15:765 Table 1 In- and exclusion criteria Inclusion criteria 1) Women who are 18 years or older. 2) Treated for breast cancer with curative intent. 3) Breast cancer treatment (surgery, chemo- and/or radiotherapy) must be finished at least 3 months previously. There is no upper limit for the time since completion of cancer treatment. Patients who currently receive hormone and/or targeted therapy are eligible. 4) Disease-free at entry of the study, defined by the absence of somatic disease activity parameters. 5) Able to speak, read, and write Dutch. As a second recruitment strategy, nurse practitioners from selected participating hospitals will identify cohorts of eligible patients through medical records. They will inform these cohorts about the CHANGE-study by mail. Patients will receive an information leaflet with an accompanying letter. In this letter, patients are asked to contact the re- searcher if they want to participate in the study. 6) Severely fatigued, defined by a score of ≥35 on the fatigue severity subscale of the Checklist Individual Strength. 7) Having access to a computer with internet. Exclusion criteria 8) Presence of a co-morbidity that explains the presence of severe fatigue. 9) A depressive disorder, assessed with the BDI-PC and the M.I.N.I. Referrals by medical professionals Patients will be recruited by medical professionals (physicians and nurses) at the outpatient clinic of the departments of surgery and/or oncology of eight hospitals in the Netherlands (Radboudumc, Nijmegen; Canisius Wilhelmina hospital, Nijmegen; hospital Gelderse Vallei, Ede; hospital Bernhoven, Uden; hospital Pantein, Boxmeer; VieCuri medical center, Venlo; Elkerliek hospital, Hel- mond; Slingeland hospital, Doetinchem). Physicians and nurses will inform eligible patients about the study during regular medical follow-up consults and give them an infor- mation leaflet. If a patient agrees to be informed about the study by the researcher, the nurse practitioner will fill out a participation form and send it to the researcher (HA). Subsequently, the researcher will call the patient to give a detailed explanation about the study and to address questions. Participants All i All patients who want to participate in the study will first be screened for eligibility. The in- and exclusion cri- teria are shown in Table 1. To verify the medical criteria (criterion 2, 3, and 4) of self-referrals, patients will send a copy of the most recent report of their medical follow- up examination to the researcher. The researcher will administer an online screening questionnaire to verify the other criteria. All patients will sign informed consent before filling out this online screening. The Checklist In- dividual Strength [40] will be used to screen for severe fatigue (criterion 6). The Beck Depression Inventory for Primary Care (BDI-PC) [41, 42] will be used to screen for a depressive disorder (criterion 9). If the score on the BDI-PC is ≥4, the researcher will administer the Depres- sion module of the Mini-International Neuropsychiatric Interview (M.I.N.I.) [43] by phone to assess the presence of a major depression. If patients meet the criteria for major depression, they will be advised to contact their general practitioner for an appropriate referral. 3) To examine if time since completion of cancer treatment moderates the efficacy of web-based CBT with respect to fatigue severity. Methods The method section of this study protocol is written in accordance with the CONSORT statement for reporting parallel group randomized trials [38] and the CONSORT e-health criteria for reporting web-based interventions [39]. Design A non-blinded multicenter RCT (the CHANGE-study) will be conducted to evaluate the efficacy of web-based CBT compared to care as usual for severely fatigued breast cancer survivors. Aims of the CHANGE study and the Radboudumc (e.g. Facebook and Twitter). Patients can complete a participation form, integrated in an in- formative website. Subsequently, the researcher will contact patients by phone to inform them about the study and to address questions. 1) To examine the efficacy of web-based CBT for severely fatigued breast cancer survivors on fatigue severity compared to care as usual. 2) To examine the efficacy of web-based CBT for severely fatigued breast cancer survivors on functional impairments, psychological distress, and quality of life compared to care as usual. Procedure If patients are eligible and have signed written informed consent, they will start with a baseline assessment (T0). Following T0, participants will be randomized to either the intervention condition (web-based CBT) or the control Table 1 In- and exclusion criteria Inclusion criteria Randomization Stratified randomization will be based on time since completion of cancer treatment (3 months up to 1 year versus ≥1 year) and type of referral (referrals by medical professionals versus self-referrals). After T0, randomization will be performed by a test assistant in the presence of the patient. A computerized randomization tool, built by an in- dependent statistical expert, will be used to randomly allo- cate patients to either intervention or control condition. The allocation ratio will be 1:1 and block-randomization will be used with a block size of six. The test assistant, the researcher and the participants will be blinded to the alloca- tion sequence. They will not be blinded for the randomization outcome, because this is not possible in psychological treatments. Self-referrals 10) Current psychological treatment for a psychiatric disorder. Patients will also be informed about the study by leaflets and notifications on social media of patients’ associations 11) Current CBT for fatigue. 11) Current CBT for fatigue. Abrahams et al. BMC Cancer (2015) 15:765 Page 4 of 10 Experts in the field of fatigue in cancer survivors devel- oped the content of this web portal. Trained, experienced cognitive behavioral therapists (HK, TB) and researchers (MGI, HA) wrote the texts and assignments. In total, the web portal consists of 13 texts and 26 assignments. A graphic designer developed the lay-out of On the road to recovery, and a videographer made 13 videos together with a therapist (HK) and the researcher (HA). These videos are integrated in the web portal. In the first video, a med- ical oncologist (SV) explains the rationale of the CBT. The other 12 videos are interviews of three cancer survivors. These patients are recovered from fatigue after receiving F2F CBT, and tell about their experiences with the CBT modules. A screenshot of the web portal is provided in Fig. 3. For this occasion, the text is translated into English. condition (care as usual). After 6 months, all participants will be assessed again (T1). For participants assigned to the web-based CBT, this will be the post-intervention assess- ment. The overall study design is shown in Fig. 2. A test assistant will perform T0, T1 and the randomization. Development On the road to recovery is built in a web portal, designed with technical guidance from the Psychological and Psy- chiatric Care Innovation (Utrecht, The Netherlands) [44]. Fig. 2 Overall study design. T0 = baseline assessment; T1 = second assessment Usability testing l f Five severely fatigued breast cancer survivors, who were following F2F CBT, participated in a test pilot. The us- ability of the web portal was tested by using a “think aloud procedure” [45]. Participants were asked to think aloud while independently completing the modules. In the meanwhile, the researcher (HA) noted obstacles they encountered (i.e. usability problems and problems with text readability). Afterwards, all participants filled out a feedback form. They were asked about the sufficiency of information provided, text readability, and the lay-out and usability of the web portal. The findings of the us- ability testing were used to optimize the final version of the web portal. Fig. 3 Screenshot of On the road to recovery in working with the F2F CBT protocol for severe fatigue in cancer survivors. They will participate in a weekly supervision, in which cases are discussed in the presence of senior clinical psychologists (HK, TB). Changes in in- dividual treatment plans will be made according to the study protocol and to the CBT principles for severely fa- tigued cancer survivors. Assessment tools are used to assess which fatigue-per- petuating factors are present and to determine which treat- ment modules patients need to follow (Table 2). All treatment modules consist of three parts: psycho-education (“READING”), assignments in which participants work on fatigue-perpetuating factors (“DOING”) and a final as- signment, in which participants evaluate their progress (“REVIEW”). The content of the eight treatment modules is described in more detail in Additional file 1. Assessment tools are used to assess which fatigue-per- petuating factors are present and to determine which treat- ment modules patients need to follow (Table 2). All treatment modules consist of three parts: psycho-education (“READING”), assignments in which participants work on fatigue-perpetuating factors (“DOING”) and a final as- signment, in which participants evaluate their progress (“REVIEW”). The content of the eight treatment modules is described in more detail in Additional file 1. At the end of the study, a random 5 % of the e-mail messages send to the patients will be evaluated. An ex- perienced clinician (HK) and researcher (HA) will deter- mine whether the web-based CBT was delivered according to the predefined treatment protocol. To determine if web- based CBT is less time consuming than F2F CBT, therapists will register the invested time for each patient. Therapists will contact patients two-weekly by e-mail to give feedback on their progress and to answer questions. Therapists can also initiate video sessions with a secured video consultation system (Facetalk) [46]. These video ses- sions are in particular recommended for the modules “Fear of cancer recurrence” and “Coping with cancer and cancer treatment”. The guideline is to plan maximally two video sessions. The maximum duration of On the road to recovery is 6 months. Therapists will be blinded for the level of fatigue severity (primary outcome measure). Only after the post-treatment assessment (T1), they will be informed about the levels of fatigue severity on T0 and T1. Control condition: care as usual Participants in the control condition will be on a waiting list for regular F2F CBT for fatigue in cancer survivors. The total duration of the waiting list is 6 months. In this period, patients will receive care as usual. The usual care for breast cancer survivors in the Netherlands consists of follow-up examinations conform the Dutch guidelines for oncology care [47]. The frequency of these follow-up examinations depends on age, time since diagnosis and a possible BRCA1/2 mutation. In general, there will be a 3-month follow-up in the first year, a biannual follow-up in the second year, and an annual follow-up in the fol- lowing years up to 5 years after diagnosis. Recently, a guideline for the management of psy- chosocial distress in breast cancer survivors is imple- mented [48]. According to this guideline, psychosocial problems are identified and patients should be referred to The outcomes with respect to fatigue severity and other disabilities will be discussed with the participant in a final F2F session. In this session, the therapist and patient will determine if the patient is recovered from severe fatigue. If patients are not recovered from se- vere fatigue, F2F therapy will be offered outside the study context. Intervention condition: on the road to recovery y All participants in the intervention condition will follow On the road to recovery, a web-based version of the regular F2F CBT for severely fatigued cancer survivors. Participants will start with two F2F sessions with their therapist. In these sessions, the CBT model for fatigue in cancer survivors (Fig. 1) will be explained and a treat- ment plan will be made. Thereafter, participants will fol- low On the road to recovery online. The web-based CBT consists of eight treatment modules. All participants will start with setting their treatment goals (module 1). Then, they will work on the fatigue-perpetuating factors that are applicable to them: (1) poor coping with breast can- cer and breast cancer treatment; (2) high fear of cancer recurrence; (3) dysfunctional fatigue-related cognitions; (4) a deregulated sleep-wake rhythm; (5) a deregulated activity pattern; and/or (6) negative social interactions and low social support. Each of these six fatigue-perpetuating factors coincides with a treatment module (module 2–7). At baseline assessment, it is decided which modules are relevant for each participant. Finally, all participants will complete the therapy by realizing their treatment goals (module 8). On the road to recovery is tailor-made. Fig. 2 Overall study design. T0 = baseline assessment; T1 = second assessment Fig. 2 Overall study design. T0 = baseline assessment; T1 = second assessment Abrahams et al. BMC Cancer (2015) 15:765 Page 5 of 10 Fig. 3 Screenshot of On the road to recovery Intended statistical analyses The primary objective of the study is to examine the ef- fects of web-based CBT on reducing fatigue severity compared to care as usual. Therefore, an analysis of co- variance (ANCOVA) will be used with the CIS-fatigue score at T1 as dependent variable, the CIS-fatigue score at T0 as covariate and condition as fixed factor [39]. The clinical importance of the treatment effect will be deter- mined. Differences between the intervention and control condition on the amount of change in fatigue severity will be calculated on T0 and T1. Clinically meaningful change will be defined as a reliable change index of more than 1.96 and a decrease of the fatigue level to a normal range (i.e. a score of <35 on the fatigue severity subscale of the Checklist Individual Strength). The ef- fects of web-based CBT on the secondary outcomes of the study (functional impairments, psychological distress and quality of life) compared to care as usual will be de- termined with ANCOVA’s. For each secondary outcome measure, an ANCOVA will be performed with the score of the outcome measure at T1 as dependent variable, the score at T0 as covariate and condition as fixed Quality of life, measured by the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 (EORTC-QLQ-C30) [60]. This questionnaire consists of 30 items that cover five function scales (physical, role, cognitive, emotional and social func- tioning), three symptom scales (fatigue, pain, and nausea and vomiting), and a global health and quality of life scale. All scales are scored on a 4-point Likert scale. The EORTC-QLQ-C30 has been established as a valid and reliable measure [61]. Treatment integrity On the road to recovery will be given by licensed cogni- tive behavioral therapists. All therapists are experienced able 2 Tools to assess which treatment modules are indicated reatment module Instrument [REF] Outcome Response format Psychometric properties Cut-off value Coping with cancer and cancer treatment Impact of Event Scale [62] Intrusion and avoidance 4-point Likert scale, range 0–60 Cronbach’s α ranges between 0.87 and 0.96; adequate convergent validity [63] Score ≥10 for each separate scale Fear of cancer ecurrence Modified cancer acceptance scale [53] Fear of disease recurrence 4-point Likert scale, range 3–12 N/A Score ≥7 Cancer worry scale [64] Worries about the risk of developing cancer (again) 4-point Likert scale, range 8–32 Cronbach’s α = 0.87; good convergent and divergent validity [64] Score ≥14 [64] Helpful thinking Modified causal attribution list [52, 65] Somatic and non-somatic attributions 4-point Likert scale Cronbach’s α ranges between 0.71 and 0.77 [65] N/A Illness management questionnaire [65–67] Focusing on symptoms 6-point Likert-scale, range 9–54 Cronbach’s α ranges between 0.85 and 0.93 [66] Score ≥30 Fatigue catastrophizing scale [68] Catastrophizing in response to fatigue 5-point Likert scale, range 1–5 Cronbach’s α = 0.85 [68] Score ≥2 (magnifying); score ≥7 (ruminating). Self-efficacy scale [33, 65] Self-efficacy with respect to fatigue 4 point Likert scale, range 7–28 N/A Score ≤19 Sleep-wake rhythm Sleep-wake diary Sleep-wake rhythm Bedtimes and wake-up times of 12 consecutive days and nights N/A N/A Activity regulation An actometer, a motion-sensing device, worn to the ankle for 12 consecutive days and nights Activity pattern (relatively active versus low active) Average physical activity level (number of accelerations per 5 min period) [69] Adequate reliability and validity [73] N/A Social support Van Sonderen Social Support Inventory, subscales Interactions (SSLI) and Discrepancies (SSLD) [70] Discrepancy between actual and desired social support 4-point Likert Scale, range 34–136 Cronbach’s α = 0.93 (SSLI); α = 0.95 (SSLD); good content validity [71] Score ≥14 (SSLI); score ≥50 (SSLD) Table 2 Tools to assess which treatment modules are indicated Table 2 Tools to assess which treatment modules are indicated Treatment module Instrument [REF] Outcom Abrahams et al. BMC Cancer (2015) 15:765 Page 7 of 10 Page 7 of 10 Secondary outcomes l Functional impairments, measured by the Sickness Impact Profile 8 (SIP) [54, 55]. This questionnaire addresses the level of disability in eight domains: alertness behavior, sleep/ rest, homemaking, leisure activities, mobility, social interac- tions, ambulation, and work. The weighted total score on these eight domains will be used to assess functional disability, with higher scores indicating more disabil- ities. The SIP is a reliable measure with sufficient content validity [56]. Psychological distress, measured by the total score on the Brief Symptom Inventory 18 (BSI-18) [57]. This multidimensional questionnaire consists of 18 items, scored on a 5-point Likert scale. The range of scores is 0 to 72, with a higher score indicating more psychological distress. The BSI-18 is a shortened version of the Symp- tom Checklist 90 (SCL-90) [58]. The BSI-18 has high levels of sensitivity and specificity [59]. Primary outcome Fatigue severity, measured by the subscale Fatigue Severity (8 items, 7-point Likert Scale) of the Checklist Individual Strength (CIS) [49]. This subscale consists of eight items, scored on a 7-point Likert scale. The range of scores is 8 to 56, with a higher score indicating a higher level of fatigue. The cut-off score for severe fatigue is ≥35 [49]. The CIS has been established as a valid and reliable measure [50, 51], which showed sensitivity to detect change in previous stud- ies investigating fatigue in cancer survivors [22, 33, 52, 53]. Power specialized care providers. Participants may therefore be referred to other fatigue-oriented interventions during the study (e.g. psychosocial interventions, a rehabilitation tra- jectory, or physical therapy). At T1, all participants will be asked if they have received any treatment for fatigue dur- ing the study, and if so, they are asked to describe this treatment. The sample size calculation is based on the guidelines of Borm et al. for analysis of covariance (ANCOVA) in RCT’s [72]. A clinically relevant difference of six points is ex- pected for the primary outcome (fatigue severity subscale of the CIS) between the intervention and control condi- tion. This difference is based on a study of Knoop et al. [73], in which the efficacy of a minimal intervention for patients with chronic fatigue syndrome was examined [73]. A minimum number of 60 patients per condition would be needed for a t-test with an alpha of 0.05, a two- sided significance level and a power of 0.85. According to Borm et al. [72], this number of patients needs to be multiplied by a “design factor” to calculate the needed sample size for an ANCOVA [76]. This factor is one minus the squared correlation coefficient between the baseline and outcome measure of fatigue severity. In our previous study examining the efficacy of F2F CBT for fa- tigue in cancer survivors, the correlation of the baseline and outcome measure was 0.36 [22, 33]. This leads to a factor of 0.87 (1 - .362 = 0.87). Thus, the minimal number of patients in each condition is 53 (600.87 = 52.2) . The drop-out rate in our first RCT examining F2F CBT for fa- tigue in cancer survivors was 13 % [22, 33]. In the current study, patients might experience less support from their therapist in the web-based CBT. Therefore, the drop-out in the current RCT is estimated to be 50 % higher than in the first RCT (1,513 = 19,5 %) Therefore, a margin of 19,5 % for drop-out is added to the minimal number of 53 patients per condition. This results in a sample size of 132 severely fatigued breast cancer survivors. Other variables Demographic variables will be assessed by using a self- report questionnaire at T0. The instruments used to de- termine the relevant fatigue-perpetuating factors are shown in Table 2. Abrahams et al. BMC Cancer (2015) 15:765 Abrahams et al. BMC Cancer (2015) 15:765 Page 8 of 10 factor. The third objective of the study is to examine if time since completion of cancer treatment moderates the effects of web-based CBT. This will be analyzed with an ANCOVA with time since completion of cancer treatment (3 months-1 year versus ≥1 year) as covariate. The CIS-fatigue score at T1 will be the dependent vari- able, and the fatigue score at T0 will be the second co- variate. All data analyses will be based on intention to treat. Missing values on primary and secondary outcome measures will be replaced with multiple imputation using fully conditional specification with at least five im- putations. In case of statistically significant differences, a sensitivity analysis will be performed, based on different assumptions about the values of missing data. In conclusion, if web-based CBT is effective, it would provide an additional treatment option that is easily ac- cessible for breast cancer survivors suffering from severe fatigue. Acknowledgments f This study is financed by Pink Ribbon (project number 2012.WO26.C139). The participating hospitals are: Radboudumc (Nijmegen), Canisius Wilhelmina hospital (Nijmegen), hospital Gelderse Vallei (Ede), hospital Bernhoven (Uden), hospital Pantein (Boxmeer), VieCuri medical center (Venlo), Elkerliek hospital (Helmond) and Slingeland hospital (Doetinchem). Discussion The CHANGE study will examine the efficacy of a web- based version of an evidence-based CBT protocol for se- vere fatigue in breast cancer survivors. The efficacy of the intervention on fatigue, functional impairments, psycho- logical distress and quality of life will be examined as well. Web-based CBT has several advantages over F2F CBT; (i) e-mail contacts are expected to be less time consuming for therapists than F2F contacts, which would result in an increased treatment capacity; (ii) the intervention becomes more easily accessible for a larger number of patients, and (iii) the burden for patients can be reduced, because they can save travel time and costs to the treatment center. Be- sides, patients can work on the intervention at their own pace, at any preferred time. Abbreviations ANCOVA: Analysis of covariance; CBT: Cognitive behavioral therapy; CHANGE study: Cognitive behavioral therapy, an online intervention for fatigued breast cancer survivors; CIS: Checklist individual strength; ECCF: Expert Center for Chronic Fatigue; F2F: Face-to-face; RCT: Randomized controlled trial. Competing interests Competing interests The authors declare that they have no competing interests. This study has been reviewed and approved by the Med- ical Ethical Committee of the Radboudumc (reference no. 2013/167). The study has also been approved by the local ethical committees of each participating hospital (Radbou- dumc, Canisius Wilhelmina hospital, hospital Gelderse Vallei, hospital Bernhoven, hospital Pantein, VieCuri med- ical center, Elkerliek hospital and Slingeland hospital). The study is registered in the Dutch Trial Registry (reference no. NTR4309, date registered: December 6, 2013). p g The authors declare that they have no competing interests. Authors’ contributions HA is responsible for data acquisition and drafting the manuscript. HK, SV and MGI supervise the RCT. HK, TB, MGI and HA have written the content of the web-based CBT. HK, MGI, SV, HP, MGO, MP and HA have contributed to the study conception and design. All authors have critically revised the final manuscript. All authors have read and approved the final manuscript. Additional file Additional file 1: Overview of the treatment modules of On the road to recovery. (DOCX 64.3 kb) Author details 1 1Expert Center for Chronic Fatigue (ECCF), Radboud University Medical Center, PO Box 9101, 9166500 HB Nijmegen, The Netherlands. 2Department of Health Sciences, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands. 3Department of Medical Oncology, Radboud University Medical Center, Nijmegen, The Netherlands. Received: 24 July 2015 Accepted: 13 October 2015 Received: 24 July 2015 Accepted: 13 October 2015 References 1. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet‐Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin. 2015;65(2):87–108. 1. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet‐Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin. 2015;65(2):87–108. 2. Van Schoor G, Moss S, Otten J, Donders R, Paap E, den Heeten G, et al. Increasingly strong reduction in breast cancer mortality due to screening. Br J Cancer. 2011;104(6):910–4. After completion of the patient inclusion, the CHANGE study will be extended to form a non-inferiority trial. In this trial, stepped care will be compared to F2F CBT for severely fatigued breast cancer survivors. The first step in the stepped care condition will be web-based CBT. If patients are not recovered from severe fatigue after completion of web-based CBT, additional F2F CBT sessions will be of- fered. We will examine whether the effects of stepped care on fatigue severity are noninferior to regular F2F CBT after a waiting period. We will also determine whether stepped care requires less therapist time than regular F2F CBT. The non-inferiority trial is registered in the Dutch Trial Registry (reference no. NTR5179). 3. DeSantis CE, Lin CC, Mariotto AB, Siegel RL, Stein KD, Kramer JL, et al. Cancer treatment and survivorship statistics, 2014. CA Cancer J Clin. 2014;64(4):252–71. 4. Mock V, Atkinson A, Barsevick A, Cella D, Cimprich B, Cleeland C, et al. NCCN practice guidelines for cancer-related fatigue. Oncology. 2000;14(11A):151–61. 5. Bower JE, Ganz PA, Desmond KA, Rowland JH, Meyerowitz BE, Belin TR. Fatigue in breast cancer survivors: occurrence, correlates, and impact on quality of life. J Clin Oncol. 2000;18(4):743–53. 6. Servaes P, Gielissen M, Verhagen S, Bleijenberg G. The course of severe fatigue in disease‐free breast cancer patients: a longitudinal study. Psycho‐Oncol. 2007;16(9):787–95. y 7. Reinertsen KV, Cvancarova M, Loge JH, Edvardsen H, Wist E, Fosså SD. Predictors and course of chronic fatigue in long-term breast cancer survivors. J Cancer Surviv. 2010;4(4):405–14. 8. Minton O, Stone P. How common is fatigue in disease-free breast cancer survivors? A systematic review of the literature. Breast Cancer Res Treat. 2008;112(1):5–13. Page 9 of 10 Page 9 of 10 Abrahams et al. BMC Cancer (2015) 15:765 31. Johnston MF, Hays RD, Subramanian SK, Elashoff RM, Axe EK, Li J-J, et al. Patient education integrated with acupuncture for relief of cancer-related fatigue randomized controlled feasibility study. BMC Complement Altern Med. 2011;11(1):49. 9. References Minton O, Berger A, Barsevick A, Cramp F, Goedendorp M, Mitchell SA, et al. Cancer‐related fatigue and its impact on functioning. Cancer. 2013;119(S11):2124–30. 10. Bower JE, Bak K, Berger A, Breitbart W, Escalante CP, Ganz PA, et al. Screening, assessment, and management of fatigue in adult survivors of cancer: an American Society of Clinical oncology clinical practice guideline adaptation. J Clin Oncol. 2014;32(17):1840–50. 32. Molassiotis A, Sylt P, Diggins H. The management of cancer-related fatigue after chemotherapy with acupuncture and acupressure: a randomised controlled trial. Complement Ther Med. 2007;15(4):228–37. 33. Gielissen M, Verhagen C, Bleijenberg G. Cognitive behaviour therapy for fatigued cancer survivors: long-term follow-up. Br J Cancer. 2007;97(5):612–8. 11. Puetz TW, Herring MP. Differential effects of exercise on cancer-related fatigue during and following treatment: a meta-analysis. Am J Prev Med. 2012;43(2):E1–24. 34. Prinsen H, Bleijenberg G, Heijmen L, Zwarts M, Leer J, Heerschap A, et al. The role of physical activity and physical fitness in postcancer fatigue: a randomized controlled trial. Support Care Cancer. 2013;21(8):2279–88. 12. McMillan EM, Newhouse IJ. Exercise is an effective treatment modality for reducing cancer-related fatigue and improving physical capacity in cancer patients and survivors: a meta-analysis. Appl Physiol Nutr Metab. 2011;36(6):892–903. 35. Cuijpers P, van Straten A, Andersson G. Internet-administered cognitive behavior therapy for health problems: a systematic review. J Behav Med. 2008;31(2):169–77. 13. Cramp F, Daniel J. Exercise for the management of cancer‐related fatigue in adults. Cochrane Database Syst Rev. 2012;11:CD006145. 36. Goedendorp MM, Gielissen MF, Peters ME, Verhagen CA, Bleijenberg G. Moderators and long-term effectiveness of cognitive behaviour therapy for fatigue during cancer treatment. Psycho‐Oncol. 2012;21(8):877–85. 14. Schmitz KH, Courneya KS, Matthews C, Demark-Wahnefried W, Galvao DA, Pinto BM, et al. American College of Sports Medicine roundtable on exercise guidelines for cancer survivors. Med Sci Sports Exerc. 2010;42:1409–26. 37. Goedendorp MM, Gielissen MF, Verhagen CA, Bleijenberg G. Development of fatigue in cancer survivors: a prospective follow-up study from diagnosis into the year after treatment. J Pain Symptom Manage. 2013;45(2):213–22. 15. Brown JC, Huedo-Medina TB, Pescatello LS, Pescatello SM, Ferrer RA, Johnson BT. Efficacy of exercise interventions in modulating cancer-related fatigue among adult cancer survivors: a meta-analysis. Cancer Epidemiol Biomarkers Prev. 2011;20(1):123–33. 38. Schulz KF, Altman DG, Moher D. CONSORT 2010 statement: updated guidelines for reporting parallel group randomised trials. BMC Med. 2010;8(1):18. 16. Saarto T, Penttinen HM, Sievänen H, Kellokumpu-Lehtinen P-L, Hakamies-Blomqvist L, Nikander R, et al. References Effectiveness of a 12-month exercise program on physical performance and quality of life of breast cancer survivors. Anticancer Res. 2012;32(9):3875–84. 39. Eysenbach G, Group C-E. CONSORT-EHEALTH: improving and standardizing evaluation reports of Web-based and mobile health interventions. J Med Internet Res. 2011;13(4):126. 40. Vercoulen J, Alberts M, Bleijenberg G. De Checklist Individuele Spankracht (CIS). Gedragstherapie. 1999;32:131–6. 17. Winters-Stone KM, Dobek J, Bennett JA, Nail LM, Leo MC, Schwartz A. The effect of resistance training on muscle strength and physical function in older, postmenopausal breast cancer survivors: a randomized controlled trial. J Cancer Surviv. 2012;6(2):189–99. 41. Beck AT, Guth D, Steer RA, Ball R. Screening for major depression disorders in medical inpatients with the Beck Depression Inventory for Primary Care. Behav Res Ther. 1997;35:785–91. 18. Thorsen L, Courneya KS, Stevinson C, Fosså SD. A systematic review of physical activity in prostate cancer survivors: outcomes, prevalence, and determinants. Support Care Cancer. 2008;16(9):987–97. 42. Steer RA, Cavalieri TA, Leonard DM, Beck AT. Use of the Beck Depression Inventory for Primary Care to screen for major depression disorders. Gen Hosp Psychiatry. 1999;21(2):106–11. 19. Speck RM, Courneya KS, Mâsse LC, Duval S, Schmitz KH. An update of controlled physical activity trials in cancer survivors: a systematic review and meta-analysis. J Cancer Surviv. 2010;4(2):87–100. 43. Sheehan D, Lecrubier Y, Sheenhan K, Amorim P, Janavs J, Weiller E, et al. The Mini-International Neuropsychiatric Interview (MINI): the development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD-10. J Clin Psychiatry. 1998;59 Suppl 20:22–33. 20. Duijts SF, Faber MM, Oldenburg HS, van Beurden M, Aaronson NK. Effectiveness of behavioral techniques and physical exercise on psychosocial functioning and health‐related quality of life in breast cancer patients and survivors—a meta‐analysis. Psycho‐Oncol. 2011;20(2):115–26. y y 44. Psychological and Psychiatric Care Innovation. http://www.ippz.nl. Accessed 12 May 2015. 45. Cotton D, Gresty K. Reflecting on the think-aloud method for evaluating e-learning. Br J Educ Technol. 2006;37(1):45–54. 21. van der Lee ML, Garssen B. Mindfulness-based cognitive therapy reduces chronic cancer-related fatigue: a treatment study. Psycho‐Oncol. 2012;21(3):264–72. 46. Facetalk. http://www.facetalk.nl. Accessed 12 May 2015. 47. Dutch guideline for breast cancer. http://www.oncoline.nl/breastcancer. Accessed 24 March 2015. 22. Gielissen MF, Verhagen S, Witjes F, Bleijenberg G. Effects of cognitive behavior therapy in severely fatigued disease-free cancer patients compared with patients waiting for cognitive behavior therapy: a randomized controlled trial. J Clin Oncol. 2006;24(30):4882–7. 48. Guideline “Screening for Psychosocial Distress”. http://www.oncoline.nl/ screening-for-psychosocial-distress. Accessed 24 March 2015. References 49. Vercoulen JH, Swanink CM, Fennis JF, Galama JM, van der Meer JW, Bleijenberg G. Dimensional assessment of chronic fatigue syndrome. J Psychosom Res. 1994;38(5):383–92. 23. Dirksen SR, Epstein DR. Efficacy of an insomnia intervention on fatigue, mood and quality of life in breast cancer survivors. J Adv Nurs. 2008;61(6):664–75. 24. Jacobsen PB, Donovan KA, Vadaparampil ST, Small BJ. Systematic review and meta-analysis of psychological and activity-based interventions for cancer-related fatigue. Health Psychol. 2007;26(6):660–7. 50. Beurskens AJ, Bultmann U, Kant I, Vercoulen JH, Bleijenberg G, Swaen GM. Fatigue among working people: validity of a questionnaire measure. Occup Environ Med. 2000;57(5):353–7. 25. Kangas M, Bovbjerg DH, Montgomery GH. Cancer-related fatigue: a systematic and meta-analytic review of non-pharmacological therapies for cancer patients. Psychol Bull. 2008;134(5):700. 51. Dittner AJ, Wessely SC, Brown RG. The assessment of fatigue: a practical guide for clinicians and researchers. J Psychosom Res. 2004;56(2):157–70. 52. Servaes P, Verhagen S, Bleijenberg G. Determinants of chronic fatigue in disease-free breast cancer patients: a cross-sectional study. Ann Oncol. 2002;13(4):589–98. 26. Mitchell SA. Cancer-related fatigue: state of the science. PM R. 2010;2(5):364–83. 27. Lengacher CA, Johnson‐Mallard V, Post‐White J, Moscoso MS, Jacobsen PB, Klein TW, et al. Randomized controlled trial of Mindfulness-Based Stress Reduction (MBSR) for survivors of breast cancer. Psycho‐Oncol. 2009;18(12):1261–72. 53. Servaes P, Verhagen S, Schreuder HW, Veth RP, Bleijenberg G. Fatigue after treatment for malignant and benign bone and soft tissue tumors. J Pain Symptom Manage. 2003;26(6):1113–22. 54. Bergner M, Bobbitt RA, Carter WB, Gilson BS. The Sickness Impact Profile: development and final revision of a health status measure. Med Care. 1981;19(8):787–805. 28. Hoffman CJ, Ersser SJ, Hopkinson JB, Nicholls PG, Harrington JE, Thomas PW. Effectiveness of mindfulness-based stress reduction in mood, breast-and endocrine-related quality of life, and well-being in stage 0 to III breast cancer: a randomized, controlled trial. J Clin Oncol. 2012;30(12):1335–42. 55. Jacobs HM, Luttik A, Touw-Otten FW, de Melker RA. The sickness impact profile; results of an evaluation study of the Dutch version. Ned Tijdschr Geneeskd. 1990;134(40):1950–4. 29. Bower JE, Garet D, Sternlieb B, Ganz PA, Irwin MR, Olmstead R, et al. Yoga for persistent fatigue in breast cancer survivors. Cancer. 2012;118(15):3766–75. 56. De Bruin A, De Witte L, Stevens F, Diederiks J. Sickness Impact Profile: the state of the art of a generic functional status measure. Soc Sci Med. 1992;35(8):1003–14. 30. Banasik J, Williams H, Haberman M, Blank SE, Bendel R. References Effect of Iyengar yoga practice on fatigue and diurnal salivary cortisol concentration in breast cancer survivors. J Am Acad Nurse Pract. 2011;23(3):135–42. 57. Derogatis L. Brief Symptom Inventory (BSI) 18: administration, scoring and procedures manual. Minneapolis, MN: NCS Pearson Inc.; 2000. Page 10 of 10 Page 10 of 10 Abrahams et al. BMC Cancer (2015) 15:765 58. Arindell WA, Ettema JHM. Handleiding bij een multidimensionele psychopathologie-indicator. Symptom Checklist SCL-90. (Herziene uitgave). Lisse: Swets & Zeitlinger; 2003. 59. Zabora J, Brintzenhofeszoc K, Jacobsen P, Curbow B, Piantadosi S, Hooker C, et al. A new psychosocial screening instrument for use with cancer patients. Psychosomatics. 2001;42(3):241–6. 60. Aaronson NK, Ahmedzai S, Bergman B, Bullinger M, Cull A, Duez NJ, et al. The European Organization for Research and Treatment of Cancer QLQ-C30: a quality-of-life instrument for use in international clinical trials in oncology. J Natl Cancer Inst. 1993;85(5):365–76. 61. Albert US, Koller M, Lorenz W, Kopp I, Heitmann C, Stinner B, et al. Quality of life profile: from measurement to clinical application. Breast. 2002;11(4):324–34. 62. Brom D, Kleber RJ. De Schok Verwerkings Lijst. Ned Tijdschr Psychol. 1985;40:164–8. 63. van der Ploeg E, Mooren T, Kleber RJ, van der Velden PG, Brom D. Construct validation of the Dutch version of the impact of event scale. Psychol Assess. 2004;16(1):16–26. 64. Custers JA, van den Berg SW, van Laarhoven HW, Bleiker EM, Gielissen MF, Prins JB. The cancer worry scale: detecting fear of recurrence in breast cancer survivors. Cancer Nurs. 2014;37(1):E44–50. 65. Prins JB, Bleijenberg G, Bazelmans E, Elving LD, de Boo TM, Severens JL, et al. Cognitive behaviour therapy for chronic fatigue syndrome: a multicentre randomised controlled trial. Lancet. 2001;357(9259):841–7. 66. Ray C, Weir W, Stewart D, Miller P, Hyde G. Ways of coping with chronic fatigue syndrome: development of an illness management questionnaire. Soc Sci Med. 1993;37(3):385–91. 67. Andrykowski MA, Donovan KA, Laronga C, Jacobsen PB. Prevalence, predictors, and characteristics of off‐treatment fatigue in breast cancer survivors. Cancer. 2010;116(24):5740–8. 68. Jacobsen PB, Andrykowski MA, Thors CL. Relationship of catastrophizing to fatigue among women receiving treatment for breast cancer. J Consult Clin Psychol. 2004;72(2):355–61. 69. van der Werf SP, Prins JB, Vercoulen JH, van der Meer JW, Bleijenberg G. Identifying physical activity patterns in chronic fatigue syndrome using actigraphic assessment. J Psychosom Res. 2000;49(5):373–9. actigraphic assessment. J Psychosom Res. 2000;49(5):373–9. 70. Van Sonderen E. 71. Van Sonderen E, Ormel J. Het meten van aspecten van sociale steun en hun relatie met welbevinden. Gedrag Gezond. 1997;25:190–200. References Het meten van sociale steun met de Sociale Steun Lijst-Interacties (SLL-I) en Sociale Steun Lijst–Discrepanties (SSL-D): een handleiding. Groningen: Noordelijk Centrum voor Gezondheidsvraagstukken, Rijksuniversiteit Groningen; 1993. 71. Van Sonderen E, Ormel J. Het meten van aspecten van sociale steun en hun relatie met welbevinden. Gedrag Gezond. 1997;25:190–200. 72. Borm GF, Fransen J, Lemmens WA. A simple sample size formula for analysis of covariance in randomized clinical trials. J Clin Epidemiol. 2007;60(12):1234–8. 73. Knoop H, van der Meer JW, Bleijenberg G. Guided self-instructions for people with chronic fatigue syndrome: randomised controlled trial. Br J Psychiatry. 2008;193(4):340–1. 73. Knoop H, van der Meer JW, Bleijenberg G. Guided self-instructions for people with chronic fatigue syndrome: randomised controlled trial. Br J Psychiatry. 2008;193(4):340–1. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review
https://openalex.org/W2338531079	https://www.redalyc.org/pdf/4006/400644838002.pdf	Portuguese	null	A experiência estética nas práticas culinárias de uma organização gastronômica	Organizações & Sociedade	2,016	cc-by	13,395	Doutora em Psicologia do Trabalho e da Organização pela Università degli Studi di Bologna (Itália). Professora-adjunta do PPG em Administração da Universidade Estadual do Ceará (UECE). E-mail: ana.silvia@pq.cnpq.br Mestre em Administração pela Universidade Estadual do Ceará (UECE). Professora na Faculdade de Tecnologia do Nordeste (Fatene). E-mail: lumalouise@gmail.com *Mestre em Administração pela Universidade Estadual do Ceará (UECE). Professor da Universida- de Estadual do Ceará (UECE) e do Centro Universitário Christus (Unichristus). E-mail: elnivan@hotmail.com DOI: 10.1590/1984-9230771 DOI: 10.1590/1984-9230771 Palavras-chave: Experiência estética. Prática culinária. Gastronomia. Palavras-chave: Experiência estética. Prática culinária. Gastronomia. Palavras-chave: Experiência estética. Prática culinária. Gastronomia. Organizações & Sociedade ISSN: 1413-585X revistaoes@ufba.br Universidade Federal da Bahia Brasil Rocha Ipiranga, Ana Sílvia; Sousa Lopes, Luma Louise; Moreira de Souza, Elnivan A experiência estética nas práticas culinárias de uma organização gastronômica Organizações & Sociedade, vol. 23, núm. 77, abril-junio, 2016, pp. 191-210 Universidade Federal da Bahia Salvador, Brasil Disponível em: http://www.redalyc.org/articulo.oa?id=400644838002 Organizações & Sociedade ISSN: 1413-585X revistaoes@ufba.br Universidade Federal da Bahia Brasil Rocha Ipiranga, Ana Sílvia; Sousa Lopes, Luma Louise; Moreira de Souza, Elnivan A experiência estética nas práticas culinárias de uma organização gastronômica Organizações & Sociedade, vol. 23, núm. 77, abril-junio, 2016, pp. 191-210 Universidade Federal da Bahia Salvador, Brasil Disponível em: http://www.redalyc.org/articulo.oa?id=400644838002 Organizações & Sociedade ISSN: 1413-585X revistaoes@ufba.br Universidade Federal da Bahia Brasil Rocha Ipiranga, Ana Sílvia; Sousa Lopes, Luma Louise; Moreira de Souza, Elnivan A experiência estética nas práticas culinárias de uma organização gastronômica Organizações & Sociedade, vol. 23, núm. 77, abril-junio, 2016, pp. 191-210 Universidade Federal da Bahia Salvador, Brasil Disponível em: http://www.redalyc.org/articulo.oa?id=400644838002 Como citar este artigo Número completo Mais artigos Home da revista no Redalyc Sistema de Informação Científica Rede de Revistas Científicas da América Latina, Caribe , Espanha e Portugal Projeto acadêmico sem fins lucrativos desenvolvido no âmbito da iniciativa Acesso Aberto Como citar este artigo Número completo Mais artigos Home da revista no Redalyc Sistema de Informação Científica Sistema de Informação Científica Rede de Revistas Científicas da América Latina, Caribe , Espanha e Portugal cadêmico sem fins lucrativos desenvolvido no âmbito da iniciativa Acesso Aberto Rede de Revistas Científicas da América Latina, Caribe , Espanha e Portugal Projeto acadêmico sem fins lucrativos desenvolvido no âmbito da iniciativa Acesso Aberto A experiência estética nas práticas culinárias de uma organização gastronômica Ana Sílvia Rocha Ipiranga Luma Louise Sousa Lopes Elnivan Moreira de Souza* Resumo A i ti A prática culinária é um ato cultural, no qual o mundo sensível torna-se locus do conhecimento por meio da experiência estética. Nesse contexto de dis- cussão, este estudo teve como objetivo compreender como a experiência estética imbuída nas práticas culinárias influencia a organização gastronômica investigada. O delineamento metodológico seguido foi o proposto pela compreensão empática para compor o corpus empírico. Desse modo, os pesquisadores lançaram mão das técnicas da entrevista, da imersão e das anotações no diário de campo. Esse caminho revelou uma intensa experiência estética a partir da ativação das faculda- des sensoriais do paladar, da visão, do olfato, da audição, do tato e de juízo estético dos atores participantes da vida organizacional. Com isso, delineou-se uma teia de categorias estéticas, entre elas o belo e o sublime, o kitsch, o feio, o grandioso e o grotesco, o pitoresco, o gracioso e o sagrado, propiciando diferentes interpretações sobre a construção desse social. The aesthetic experience in culinary practices of a gastronomic organization Abstract Abstract T methodo T he culinary practice is a cultural act, in which the sensible world becomes locus of knowledge through aesthetic experience. In this context of discussion, this study aimed to understand how the aesthetic experience imbued in the culinary practices reflected in a gastronomic organization investigated. The methodological procedures were based on the principle of empathic understanding to compose the empirical corpus. Thus, the researchers resorted to the techniques of interview, immersion and notes in a field diary. This path revealed an intense aesthetic experience from activation of sensitive faculties of taste, vision, smell, hearing, touch and aesthetic judgment of the participating actors of organizational life. With this, was outlined a web of aesthetic categories among them the beautiful and the sublime, the kitsch, the ugly, the grand and the grotesque, the picturesque, the beautiful and the sacred, providing different interpretations of this social construction. Keywords: Aesthetic experience. Culinary practice. Gastronomy. Licença Creative Commons Attribution 3.0. 191 Ana Sílvia Rocha Ipiranga, Luma Louise Sousa Lopes & Elnivan Moreira de Souza Introdução A A prática de alimentar-se, além da natureza biológica, é um ato cultural, que envolve imaginários, símbolos, representações, escolhas e classificações que organizam as diversas visões de mundo no tempo e no espaço. Focadas na interpretação da coletividade, as investigações sobre alimentação argumen- tam que o ato de comer faz parte de um conjunto de práticas capaz de expressar a cultura de um grupo (GOMES; RIBEIRO, 2011; MACIEL, 2004; 2005). O simbolismo presente no ato de se alimentar também é encontrado no ato de preparar o alimento. Lévi-Strauss (1991) já alegava que o ato de cozinhar é universal, mas as cozinhas são diversas. A leitura da cozinha é uma viagem na consciência que as sociedades têm delas mesmas. Nesse sentido, a forma de preparar a comida, ou seja, a prática culinária de um dado grupo envolve um determinado estilo de vida, que produz uma mudança não só de estado, mas de sentido (MACIEL, 2004). A prática culinária pode ser entendida como um conjunto de aromas e sabo- res específicos de uma determinada cultura, enquanto resultado da interação do ser humano com o seu contexto, envolvendo diferentes dimensões, como a cultura, as sensações, as emoções e as lembranças, despertadas tanto pelos atos de preparar como de consumir alimento (DE PAULA, 2002; MACIEL, 2001; MINTZ, 2001). The aesthetic experience in culinary practices of a gastronomic organization Certeau, Giard e Mayol (1996) realçaram que os hábitos alimentares constituem um domínio em que a tradição e a inovação têm a mesma importância, em que o presente e o passado se entrelaçam, satisfazendo as necessidades do momento. Os autores também enfatizam o alto grau de ritualização e o considerável investimento afetivo, sendo as atividades culinárias consideradas por quem as pratica como um lugar de felicidade, de prazer e de invenção. g , p ç Nesse sentido, as percepções sensoriais e emotivas proporcionadas pelas prá- ticas de alimentação e da culinária imprimem no indivíduo conhecimentos sobre as ações que estão sendo realizadas. Trata-se de um processo em que o mundo sensível e corpóreo torna-se instrumento e locus do conhecimento (GHERARDI, 2001). Dessa maneira, compreender os aspectos subjetivos concernentes à experiência estética por meio da qual os sujeitos adquirem o conhecimento sensível se faz necessário, como uma forma de ampliar a compreensão das ações dos sujeitos e seus reflexos no contexto em que estão inseridos, entre estes as organizações (EWENSTEIN; WHYTE, 2007). Fine (1996) enfatizou a conjunção entre as práticas do preparo e da alimen- tação ao estudar as cozinhas e o mundo da culinária, evidenciando que as práticas dos restaurantes são julgadas esteticamente, sobretudo pela faculdade sensorial do paladar, assim como pelas faculdades da visão, do olfato e do tato. Para o autor, essa intensa diversidade estética possibilita uma ampla liberdade de escolhas nas práticas culinárias, envolvendo a sociabilidade e a organização dos restaurantes, assim como na composição dos cardápios e na preparação dos alimentos. p ç p p p ç Alexander Gottlieb Baumgartem (1993), que cunhou o termo “estética” em 1735, enfatiza a estética como a teoria do saber sensível, sinônimo do conhecimento obtido por meio dos sentidos. Em 1790, a estética foi tratada por Kant (1987) no âmbito filosófico e artístico como uma experiência aprendida enquanto intuição, sen- timentos, emoções, produzindo um efeito de prazer, de caráter desinteressado, que o autor caracteriza como experiência estética. p Para Strati (1992), a estética é entendida como a sensibilidade do ser humano ampliada para melhor observar os elementos da vida cotidiana. Isso implica em um esforço de ativação das faculdades sensoriais, no intuito de produzir uma experiência sensível na qual a estética diz respeito a toda complexidade de experiências humanas. The aesthetic experience in culinary practices of a gastronomic organization Na vida organizacional, a estética é compreendida por Strati (2007) como uma forma de conhecimento originado pelas faculdades perceptivas da audição, da visão, do tato, do olfato e do paladar, e pela capacidade de fazer um juízo estético. Nesse sentido, a estética está impregnada no dia a dia organizacional, sendo composta por nuances de histórias recontadas pelos atores organizacionais e suas interpretações. A dimensão estética contribui, portanto, para a análise organizacional em uma abordagem que lida com a complexidade, a ambiguidade e a sutileza presente nas organizações Para Strati (1992), a estética é entendida como a sensibilidade do ser humano ampliada para melhor observar os elementos da vida cotidiana. Isso implica em um esforço de ativação das faculdades sensoriais, no intuito de produzir uma experiência sensível na qual a estética diz respeito a toda complexidade de experiências humanas. Na vida organizacional, a estética é compreendida por Strati (2007) como uma forma de conhecimento originado pelas faculdades perceptivas da audição, da visão, do tato, do olfato e do paladar, e pela capacidade de fazer um juízo estético. Nesse sentido, a estética está impregnada no dia a dia organizacional, sendo composta por nuances de histórias recontadas pelos atores organizacionais e suas interpretações. A dimensão estética contribui, portanto, para a análise organizacional em uma abordagem que lida com a complexidade, a ambiguidade e a sutileza presente nas organizações o&s - Salvador, v. 23, n. 77, p. 191-210, Abr./Jun. 2016 www.revistaoes.ufba.br 192 A experiência estética nas práticas culinárias de uma organização gastronômica (STRATI, 1996; 2007). Igualmente para Ottensmeyer (1996), o entendimento da estética enquanto dimensão da vida organizacional é pertinente por constituir-se em uma parte inegável da produção de experiências organizacionais. (STRATI, 1996; 2007). Igualmente para Ottensmeyer (1996), o entendimento da estética enquanto dimensão da vida organizacional é pertinente por constituir-se em uma parte inegável da produção de experiências organizacionais. Os primeiros debates sobre a estética nas organizações estabeleceram diferentes articulações, como, por exemplo, as discussões sobre o processo de desconstrução do discurso organizacional oficial (GRAFTON-SMALL; LINSTEAD, 1985); a questão da relação entre a estética e a ética (BRADY, 1986); o estudo da identidade organizacional repassada por meios gráficos, levando em conta as imagens que transitam no inte- rior (BOLOGNINI, 1986); e no meio externo às organizações (SCHNEIDER; POWLEY, 1985); e, ainda, a experiência estética e os sentimentos no trabalho (SANDELANDS; BUCKNER, 1989). The aesthetic experience in culinary practices of a gastronomic organization Outros estudos, como o de Taylor e Hansen (2005), sugerem que as experiências estéticas não só transformam as organizações, mas as lentes que utilizamos para visualizá-las e, por fim, Witz, Warhurst e Nickson (2003) analisaram a estética no trabalho, discernindo formas nas quais a estética pode ser reconhecida e imbuída na organização. No Brasil, ainda são poucas as pesquisas que focalizaram a discussão da ex- periência estética nas organizações. Entre essas se relevam os estudos ensaísticos desenvolvidos por Leal (2000; 2002; 2007) e Wood e Csillag (2001). Por outro lado, Tavares e Kilimnik (2007), Oliveira (2012) e Schiavo (2010) desenvolveram inves- tigações focalizando de forma específica algumas dimensões organizacionais e sua relação com a experiência estética que, conforme Ottensmeyer (1996), permeia a organização como um todo. Recentemente, as investigações de Ipiranga et al. (2013), Lopes (2014) e Lopes, Souza e Ipiranga (2014) têm se dedicado aos estudos da es- tética em diferentes restaurantes, sejam de gastronomia popular ou especializados na culinária contemporânea. p No intuito de contribuir para o avanço dessa agenda de pesquisas e considerando, segundo Fine (1996), que a comida envolve mais dimensões sensoriais do que qualquer outra forma de atividade, este estudo focaliza a compreensão da experiência estética nas práticas culinárias de um pequeno restaurante de cultura italiana, localizado em Fortaleza, Ceará. Além disso, a escolha dessa organização gastronômica se deu pela observação do crescente número de restaurantes na cidade que, segundo pesquisas do setor, atrelado ao turismo regional, tem se mostrado um setor economicamente importante para o Estado. Tendo como base essas discussões, este estudo tem como objetivo compreender como a experiência estética imbuída nas práticas culinárias influencia a organização gastronômica investigada. O estudo de natureza qualitativa teve como base os três princípios metodo- lógicos para o desenvolvimento de pesquisa empírica sobre o tema da estética nas organizações, entre estes, o princípio da compreensão empática, que consiste em se imaginar no lugar do outro, de connoisseurship de suas faculdades sensoriais, e o princípio de observação das categorias estéticas, que são expressas pelos sujeitos organizacionais (STRATI, 2007). Com base nesses princípios, a pesquisa percorreu o itinerário proposto pela compreensão empática para compor o corpus empírico (BAUER; GASKELL, 2008; STRATI, 2007). A experiência estética e a estética nas organizaçõe A experiência estética e a estética nas organizações As origens da discussão sobre a estética a partir da categoria do belo se encon- tram no pensamento pré-socrático da Grécia Antiga e da Magna Grécia. Seu berço se deu nas polêmicas travadas por sofistas e céticos acerca da transformação da beleza, equivalente da estética, em uma das numerosas categorias da estética na filosofia europeia no final do século XVIII e início do século XIX, destacando-se: o Livro III de A república, de Platão (século IV a.C.), sobre as modalidades do estilo narrativo, gra- ciosidade, ritmos, melodias e harmonias que acompanham o discurso; os capítulos I a XII da Poética, de Aristóteles (registrada entre os anos 323 a.C. e 335 a.C.), que trata da poesia e da arte em sua época. Com Platão e Aristóteles a estética era estudada fundida com a lógica e a ética. O belo, o bom e o verdadeiro formavam uma unidade. Sobressai-se ainda a obra Do padrão do gosto, de Humes (1737-1740). Ainda nessa linha de discussão, relevam-se as pesquisas que discutem uma crítica do conceito de estética a partir da história do pensamento ocidental moderno, centrado nas complexas relações entre estética, ética e política. Destacam-se os trabalhos de Eagleton (1993), que trata a estética no contexto gramsciano da “hegemonia”, como um instrumento para sustentação do poder por meio do “consentimento”. A tradição estética “de esquerda” de Schiller (Sobre a educação estética do homem em uma se- quência de cartas, 1795), passando por Marx (os trechos de Manuscritos econômico- -filosóficos, de 1844, e na introdução de Para a crítica da economia política, de 1859) até Adorno (Teoria estética, 1982), tem muito a dizer da arte como crítica da alienação ao considerar o modernismo como um dos herdeiros dessa estetização radical. Etimologicamente, a palavra estética origina-se do grego aisthésis. A raiz grega aisth, no verbo aisthanomai, significa sentir com o coração ou com os sentimentos, evocando sensações, percepções sensíveis, conhecimento sensível ou dos fatos e objetos sensíveis, opondo-se à noética, traduzida por conhecimento intelectual ou teórico (PLATÃO, 1993). ii O uso corrente da estética, enquanto ciência filosófica, foi introduzido pelo filósofo alemão Alexander Gottlieb Baumgarten em 1735, na sua tese denominada Medifationes philosophicae de nonnullis ad poema pertinenfíbus (Reflexões filosóficas sobre algumas questões pertencentes à poesia). Nesse trabalho, a estética foi defini- da pelo autor enquanto a ciência da percepção em geral. The aesthetic experience in culinary practices of a gastronomic organization Dessa forma, durante visitas realizadas à organização gastronômica no período entre outubro de 2012 e março de 2013, procedeu-se à imersão dos pesquisadores no papel do outro, a partir da ativação de suas faculdades sensoriais e de seu juízo estéti- co, utilizando-se dos “sinais distintivos” (STRATI, 2007, p. 272) que manifestavam os humores, as impressões e as avaliações dos atores organizacionais. De forma a reviver a experiência na imaginação, lançou-se mão dos métodos da auto-observação e do exercício das faculdades intuitivas e da analogia (STRATI, 2007). Além das anotações no caderno de campo, foram entrevistados a partir de um roteiro aberto e relacional o chef e a gestora do restaurante. O processo de interpretação e escrita foi constituído na forma de “texto aber- to” (STRATI, 2007, p. 116), como um produto constante do reexame da experiência vivida. Nesse sentido, a descrição baseou-se no processo heurístico da evocação por meio da participação imaginada dos pesquisadores na situação organizacional descrita, o&s - Salvador, v. 23, n. 77, p. 191-210, Abr./Jun. 2016 www.revistaoes.ufba.br 193 Ana Sílvia Rocha Ipiranga, Luma Louise Sousa Lopes & Elnivan Moreira de Souza estabelecendo uma relação entre os pesquisadores (leitores), a descrição que se faz e seus autores (STRATI, 2007). Este trabalho está estruturado de maneira que as duas primeiras seções abordam a fundamentação teórica na qual se embasa a pesquisa; na sequência, versa-se sobre o desenho metodológico utilizado; a seção posterior traz a descrição e a discussão, seguidas das considerações finais. A experiência estética e a estética nas organizaçõe A intuição, ao contrário, é imediata e não discursiva e dá conta daquilo que há na realidade de único e original, assim como a sua continuidade, sua mobili- dade, sua atividade e a contingência do seu futuro. A intuição recorre a imagens, a comparações, a metáforas e a símbolos para veicular seus conteúdos. Há uma ana- logia entre a imagem ou metáfora presente e o objeto da intuição. A analogia define a compreensão abdutiva ou intuitiva (PARRET, 1997). Nesse sentido, enquanto Baumgarten enfatiza a dimensão da sensibilidade do conhecimento estético, Kant concentra-se em sua subjetividade. Esse retorno à sensi- bilidade é um retorno a Aristóteles (sensação aristotélica – aisthèsis), em combinação com o sentimento (Gefuhl kantiano) de Kant. Esse entrelaçamento possibilita a apre- ensão abdutiva e/ou intuitiva da realidade. Sobressai-se ainda a ênfase colocada por Croce (1990) para a experiência estética, enquanto resultante da expressão de uma intuição não formulada anteriormente (PARRET, 1997; STRATI, 2007). i ç ( , ; , ) A estética foi inserida no campo dos estudos organizacionais no final dos anos 1980, por meio da publicação de diversos estudos que traziam em seu bojo uma ins- piração estética. Dentre esses trabalhos, destacam-se os estudos da gestão enquanto prática artística (DEGÒT, 1987); da discussão sobre a elegância das organizações (RAMIREZ, 1987a) e da divergência entre o que as organizações de fato são e a ima- gem que projetam de si mesmas (RAMIREZ, 1987b). Todavia, foi no trabalho seminal de Strati (1992) que a estética foi compreendida como uma metáfora epistemológica apropriada para compreender a beleza como um elemento intrínseco às organizações. Para o autor, a discussão sobre a experiência estética constitui a corrente principal na qual a abordagem estética contemporânea das organizações se desenvolveu (STRATI, 2007). A estética na vida organizacional diz respeito a uma forma de conhecimento humano e, especificamente, ao conhecimento fornecido pelas faculdades perceptivas da audição, da visão, do tato, do olfato e do paladar, e pela capacidade de fazer um juízo estético (STRATI, 1992; 1996; 2007; TAYLOR; HANSEN, 2005). A compreensão da dimensão estética da organização exige formas de entendi- mento intuitivas. Strati (2007, p. 72) enfatiza que a “estética na vida organizacional é o motor da organização e não uma maquilagem dela [...]”, seja qual for a informa- ção dada aos sujeitos pela estética organizacional, isso é feito por meio de processos contínuos de construção, desconstrução e reconstrução simbólica. A experiência estética e a estética nas organizaçõe Por volta de 1750, no livro intitulado Aesthetica, Baumgarten (1993) constrói uma teoria estética sistemática, a que chama também, pela primeira vez, com nome de Estética, definindo-a enquanto uma teoria do saber sensível tomada como sinônimo de conhecimento pelos sentidos. p Em 1790, Kant atribuiu uma nova conotação à forma e ao conteúdo da estética na sua obra Crítica do julgamento (1987), na qual a noção de existência de uma beleza absoluta e paradigmática será substituída pela prioridade do juízo do gosto, e o belo (kalós) é considerado uma finalidade sem fim. A estética para Kant trata do estudo da experiência aprendida enquanto intuição, sentimentos, emoções, produzindo um efeito de prazer, de caráter desinteressado, que o autor denomina como experiência estética. O belo (kalós) é a condição de objeto da experiência estética. i ( ) ç j p A estética transcendental é definida como a ciência de todos os princípios da sensibilidade a priori, parte integrante no conjunto que é denominado por Kant (1987) de teoria do conhecimento. Nesse sentido, enfatiza o autor que na compreensão estética da realidade há, toda vez que nós transmitimos nossos pensamentos, dois modos (modi) de compô-los, um dos quais se chama maneira (modus aestheticus) e o outro método (modus logicus). Esses modos se distinguem entre si pelo fato de o&s - Salvador, v. 23, n. 77, p. 191-210, Abr./Jun. 2016 www.revistaoes.ufba.br 194 A experiência estética nas práticas culinárias de uma organização gastronômica que o primeiro modo não possui nenhum outro padrão que não o sentimento de que há unidade na apresentação (dos pensamentos), ao passo que o segundo segue em tudo princípio indeterminado (KANT, 1987, p. 181-88). O modus aestheticus de compreender a realidade é uma tendência de ultrapassar a impossibilidade de insights teóricos (logos) que só pode ser recuperada pelo racio- cínio imaginativo (ou pela compreensão abdutiva ou intuitiva). A imaginação é uma faculdade humana impregnada de sensibilidade (pathos) e é responsável por várias funções, entre elas: o reconhecimento dos conceitos; a produção de representações e suas sínteses; a identificação da consciência como lugar das figuras; a esquematização das figuras e os processos criativos e simbólicos (PARRET, 1997). A intuição (pathos do sensível) vem oposta ao conhecimento conceitual (logos – racional), que abstrai, generaliza e argumenta, uniformizando e imobilizando as realidades. Ana Sílvia Rocha Ipiranga, Luma Louise Sousa Lopes & Elnivan Moreira de Souza Ainda para Gagliardi (1990), a análise da estética nas organizações problematiza a fisicalidade dos elementos não humanos que marcam a vida organizacional. Nesse sentido, os artefatos são fenômenos culturais primários, algo resultante da ação hu- mana e, dessa forma, percebidos por nossos sentidos. p p Igualmente, Hatch e Jones (1997) e Ottensmeyer (1996) destacaram em seus trabalhos a influência da estética na compreensão da vida organizacional. Taylor e Hansen (2005) acrescentam que, além da relação da estética com o conhecimento que é criado a partir de nossas experiências sensoriais, se inclui também como nossos pen- samentos, sentimentos e a razão em torno deles comunicam-se às nossas cognições. Alguns recentes estudos focalizando a estética nas organizações relacionam-se com as questões da criação de estratégia (FILIPPI; TANNERY, 2009); das pessoas e do estilo de trabalho nas organizações (STRATI, 2010); do controle das ações por meio dos artefatos (SORENSEN, 2010); sobre o papel da estética na regulação da identidade e no rompimento com padrões culturais (WASSERMAN; FRENKEL, 2011). Outros trabalhos abordaram a discussão do conhecimento estético como elemento de ligação entre a gestão e o design (STEPHENS; BOLAND, 2011); o debate da beleza que perpassa as práticas de gestão, tornando possível a investigação da estética organizacional (TAYLOR, 2012) e a relação entre a ética e a estética, por meio da análise da noção de beau geste no âmbito da liderança (BOUILLOUD; DESLANDES, 2013). ) No Brasil, após as publicações da literatura ensaística que ratificou a contri- buição da estética para o estudo das organizações (p. ex. LEAL, 2000; 2002; 2007; WOOD; CSILLAG, 2001), as pesquisas empíricas começaram a despontar no sentido de formar um corpo coeso de estudos. É possível destacar as articulações envolven- do temáticas entre o conhecimento tácito e a estética (TAVARES; KILIMNIK, 2007); as discussões sobre a estética no contexto da construção civil (SCHIAVO, 2010) e no setor de móveis projetados (OLIVEIRA, 2012); as questões da estética e corpo- reidade (BERTOLIN; BRITO, 2012; FIGUEIREDO, 2012). E, por fim, destacam-se os estudos que iniciaram a agenda de pesquisas e têm como foco a estética e as prá- ticas culinárias em diferentes tipos de cozinhas contemporâneas (IPIRANGA et al., 2013; LOPES; SOUZA; IPIRANGA, 2014) e em restaurantes de um mercado popular (LOPES, 2014). Ensaios recentes (p. ex. BASSO; PAULI; BRESSAN, 2014) se propõem a associar a discussão da estética com as relações de gênero, com ênfase na cultura e no desempenho organizacional. A experiência estética e a estética nas organizaçõe Nesse sentido, a estética apresenta-se como uma parte irrefutável da produção de experiências e da realidade organizacional, dado que as experiências estéticas perpassam a organização em sua totalidade. Além disso, a estética não está encerrada em um ou outro de- partamento, mas é algo intrínseco a qualquer sujeito organizacional (OTTENSMEYER, 1996; STRATI, 1996). l ; , ) Gagliardi (2009) discutiu sobre a influência que a dimensão estética exerce sobre a organização, enfatizando três dimensões da experiência estética nos estudos organizacionais: (i) enquanto forma de conhecimento sensível diferente e em con- traposição ao conhecimento intelectual; (ii) enquanto forma de expressão da ação desinteressada, sem uma finalidade instrumental explicitada; e (iii) enquanto forma de comunicação, diferente da conversa ou diálogo que pode expressar sentimentos que não são explicitados ou codificados nas bases até então conhecidas. o&s - Salvador, v. 23, n. 77, p. 191-210, Abr./Jun. 2016 www.revistaoes.ufba.br 195 Ana Sílvia Rocha Ipiranga, Luma Louise Sousa Lopes & Elnivan Moreira de Souza A experiência estética nas práticas culinárias de uma organização gastronômica A experiência estética nas práticas culinárias de uma organização gastronômica A experiência estética nas práticas culinárias de uma organização gastronômica Com efeito, a imagem predominante transmitida pela literatura organizacional até meados da década de 1970 era a de que as organizações eram baseadas em ideias que se encontram e se fundem no plano racional; ideias, portanto, desprovidas de erotismo, de sensações belas ou feias, de perfumes e odores, de atração e repulsão. A teoria da organização e os estudos de gestão representavam as organizações de forma idealizada, privando-as de suas características terrestres de materialidade e corporeidade. Essa discussão sobre a “organização idealizada” (STRATI, 2007, p. 14) sofreu uma série de mudanças a partir dos anos 1970, quando a atenção dos estudiosos da organização começou a se voltar para a dimensão estética da rotina cotidiana nas organizações. Essas mudanças foram igualmente estimuladas pelo debate entre modernos e pós-modernos, que também envolveu estudos sobre a organização. Esse debate, mesmo quando se argumentou que a modernidade não havia sido substituída pela pós-modernidade, mas por uma modernidade reflexiva, considerou o recurso à dimensão estética. Para Strati (2007), tais discussões no contexto organizacional elevaram o juízo estético à categoria de sexto sentido que seria integrado aos demais, informando-nos sobre como a pessoa que expressa o juízo representa e constrói a organização para si mesma. O juízo estético tem como característica particular representar para o pes- quisador uma relação em que tanto o sujeito que julga quanto a organização julgada estão sempre presentes e, para tanto, utilizam as categorias estéticas, envolvendo as faculdades sensoriais. Desde Aristóteles as categorias estéticas são os principais instrumentos do juízo classificatório. As categorias do belo (kalós) e do sublime foram pensadas por Hume e Kant na segunda metade do século XVIII. Após isso, uma imensa diversidade de categorias estéticas expandiu-se rapidamente (PARRET, 1997). i Nesse contexto, o filósofo francês Souriau (1892-1979) propôs o famoso diagra- ma das categorias estéticas, incluindo 24 ramos classificatórios, no qual a categoria do sublime, ao estar ligada ao sentimento de transcendência e de excesso, é uma categoria que converge variadas emoções estéticas, podendo modificar qualquer valor estético, inclusive o belo (PARRET, 1997). Além das categorias do belo e do sublime, comumente citadas nos clássicos estudos filosóficos sobre estética, Strati (2007) destaca a multicategorialidade da estética para a compreensão estética da vida organizacional. A experiência estética nas práticas culinárias de uma organização gastronômica As categorias estéticas podem ser compreendidas como dimensões que compõem uma teia de interpretações expressa por um repertório de linguagens que manifesta o juízo estético elaborado pelos sujeitos a respeito da vida na organização. Autores como Taylor e Hansen (2005) e Strati (2007) evidenciaram as categorias estéticas do grandioso, do heroico e monstruoso, do trágico, do pitoresco e do cômico, do gracioso e do sagrado, entre outras, as quais refletem diferentes formas de experiências vivenciadas pelos sujeitos nas organizações. g ç Enfatiza-se ainda que as categorias estéticas sugerem e demarcam caminhos específicos para a análise da vida organizacional. Esses caminhos não dependem da estética filosófica e/ou da teoria da arte, mas de uma hibridação da estética com a teoria da organização na compreensão da vida organizacional. Nessa linha de discus- são, se enfatiza que há um entrelaçamento entre a vida organizacional, a faculdade de julgamento como sexto sentido do sujeito e as categorias estéticas. Essa articulação revela a compreensão subjetiva da vida organizacional cotidiana ao pesquisador que utiliza a abordagem estética, colocando diferentes questões no debate acerca do mé- todo nas ciências sociais ao iluminar o “pathos do sensível” (STRATI, 2007, p. 187). A partir da apreensão da experiência vivida pelas pessoas quando agem, a com- preensão estética da vida organizacional é considerada uma metáfora epistemológica, baseada no fato de que o conhecimento do sensível distingue de modo mais nítido as ciências sociais das ciências naturais e seus métodos de investigação. A forma de conhecimento aqui envolvida não se baseia no indício ou na prova, mas na imaginação ou na intuição. Essa experiência ocorre na imaginação do sujeito, e mesmo assim é uma experiência vivida (STRATI, 1992; 2007). Enfatiza-se ainda que as categorias estéticas sugerem e demarcam caminhos específicos para a análise da vida organizacional. Esses caminhos não dependem da estética filosófica e/ou da teoria da arte, mas de uma hibridação da estética com a teoria da organização na compreensão da vida organizacional. Nessa linha de discus- são, se enfatiza que há um entrelaçamento entre a vida organizacional, a faculdade de julgamento como sexto sentido do sujeito e as categorias estéticas. Essa articulação revela a compreensão subjetiva da vida organizacional cotidiana ao pesquisador que utiliza a abordagem estética, colocando diferentes questões no debate acerca do mé- todo nas ciências sociais ao iluminar o “pathos do sensível” (STRATI, 2007, p. 187). Ana Sílvia Rocha Ipiranga, Luma Louise Sousa Lopes & Elnivan Moreira de Souza Observa-se que a recente discussão da estética nos estudos organizacionais vem crescendo amplamente em razão da busca por métodos alternativos de construção de conhecimento e, mais especificamente, por conta da crise de representação no contexto da pesquisa nos estudos organizacionais (STRATI, 1992; 2007). Strati (2007) cita Croce (1990) para iluminar o papel exercido por Vico (1725) na ênfase anticartesianista radical realizada por este último autor, ao propor o con- ceito de estética no século XVIII. Vico opunha-se à explicação racional cartesiana e enfatizava o pensamento mítico, enquanto fantasia, metáfora e imagem, e a conexão direta entre esse pensamento e os sentimentos baseados nas faculdades sensoriais dos indivíduos e de seus corpos. O pensamento mítico é, portanto, uma maneira de ver e conhecer o mundo que não tem relação direta com análise, explicação ou razão, operando com base em uma ciência empírica que não é exata e nem verdadeira, mas baseada na participação dos indivíduos na construção do social (STRATI, 2007). Na discussão sobre a estética se reconhece, por exemplo, que a utilização da categoria do belo privilegia a faculdade perceptiva da visão. Essa característica tem origem na Grécia Antiga, na qual se dava prioridade aos sentidos da visão e da audição. Esses eram os sentidos “públicos” (STRATI, 2007, p. 286), ao passo que os outros – olfato, paladar e tato – não o eram. Primeiro por que produziam percepções nas quais o princípio da comensurabilidade não podia ser aplicado, ao contrário da visão e da audição; segundo, porque eram sentidos muito mais pessoais ou mesmo íntimos, como o paladar, por exemplo, que provoca percepções sensoriais no interior do corpo (STRATI, 2007). É essa característica idiossincrática da estética que possibilita o desabrochar do conhecimento a partir de dimensões racionalmente desconhecidas. o&s - Salvador, v. 23, n. 77, p. 191-210, Abr./Jun. 2016 www.revistaoes.ufba.br 196 A experiência estética nas práticas culinárias de uma organização gastronômica ã ê A partir da apreensão da experiência vivida pelas pessoas quando agem, a com- preensão estética da vida organizacional é considerada uma metáfora epistemológica, baseada no fato de que o conhecimento do sensível distingue de modo mais nítido as ciências sociais das ciências naturais e seus métodos de investigação. A forma de conhecimento aqui envolvida não se baseia no indício ou na prova, mas na imaginação ou na intuição. Essa experiência ocorre na imaginação do sujeito, e mesmo assim é uma experiência vivida (STRATI, 1992; 2007). o&s - Salvador, v. 23, n. 77, p. 191-210, Abr./Jun. 2016 www.revistaoes.ufba.br 197 Ana Sílvia Rocha Ipiranga, Luma Louise Sousa Lopes & Elnivan Moreira de Souza Ana Sílvia Rocha Ipiranga, Luma Louise Sousa Lopes & Elnivan Moreira de Souza O fio condutor desta pesquisa mostra que é possível adquirir conhecimento sensível, em vez de lógico-racional, da vida na organização, e que essa compreensão diz respeito às culturas, aos artefatos e aos símbolos organizacionais, bem como à estética criada, reconstruída ou destruída nas negociações organizacionais cotidianas (STRATI, 2007). A estética e as práticas culinárias Lévi-Strauss (1991) já alegava que o ato de cozinhar é universal, mas as co- zinhas são diversas. Para o autor, a cozinha é uma linguagem a partir da qual cada sociedade codifica mensagens que lhe permitem significar. Certeau, Giard e Mayol (1996) coadunam ao evidenciar que ao considerarmos a história das práticas culinárias nos surpreende a sua abundante diversidade, de uma sociedade a outra, estando os hábitos alimentares de uma dada sociedade ligados por coerências internas invisíveis, mas reais. Para os autores, a língua falada no decorrer da produção da prática culinária abrange quatro domínios distintos de objetos e ações: (i) dos ingredientes que são a matéria-prima; (ii) dos utensílios e recipientes; (iii) das operações, verbos de ação e descrições do hábil movimento das mãos; (iv) dos produtos finais e da nomeação dos pratos obtidos. Releva-se ainda a fase do consumo do produto final, envolvendo um léxico “saboroso”, expressando o gosto dos pratos, o prazer da degustação, as etapas da beatitude do comer e beber bem (CERTEAU; GIARD; MAYOL, 1996, p. 288). A experiência estética nas práticas culinárias de uma organização gastronômica Os autores se referem não apenas ao prazer de saborear bons pratos, mas ao prazer secreto e tenaz de cozinhar envolvido na manipulação e associação do inven- tário dos ingredientes, de modificar, inventar, organizar, combinar, aquela sequência de gestos entrelaçados que constituem a teia indefinida das práticas culinárias na intimidade das cozinhas: “na cozinha é preciso saber organizar-se, ter memória e bom gosto” (CERTEAU; GIARD; MAYOL, 1996, p. 269). g ( p ) Os hábitos alimentares são, portanto, veículos de profunda emoção, e as práticas culinárias, enquanto conjunção dos atos de preparo e de alimentar-se, expressam relações pessoais, sociais e culturais envolvidas nessas ações, abrangendo o sentido do prazer ao satisfazer as dimensões emocionais, psicológicas e estéticas do ser hu- mano (FIGUEIREDO et al., 2010; FINE, 1996; GARCIA; CASTRO, 2011; LEONARDO, 2009; MACIEL, 2004). ) DaMatta (1986) compara o alimento a uma grande moldura e a comida ao quadro, representando aquilo que foi devidamente escolhido dentre os alimentos e que deve ser visto e saboreado com os sentidos. A comida, segundo o autor, é tudo aquilo que se come com prazer, observando-se as regras da comunhão e comensa- lidade. É tudo aquilo que se refere a uma prática cotidiana e que auxilia a construir identidades (CANESQUI, 2005). Tanto o ato de alimentar-se como a prática culinária envolvem experiências sensoriais que geram algum tipo de conhecimento. A comida, conforme coloca Maciel (1996, p. 8), não é apenas “boa para comer, mas também boa para pensar”, pois ao se pensar em comida pensa-se, também, em simbolismos, pois a prática culinária compreende, além do preparo e da ingestão de nutrientes, a absorção de símbolos, ideias, imagens e sonhos que permitem uma vivência em coletividade. A alimentação está transida pela cultura e pela sociedade e compreendê-la exige a análise de várias dimensões, como as históricas, culturais, sociais e estéticas. As sensações despertadas tanto pelo ato de preparar como de consumir o alimento são tão intensas que alguns autores enfatizam que o gosto e o prazer se sobrepõem às questões nutricionais. O conjunto da cor, aroma, textura e sabor de um alimento faz alusão às mais longínquas lembranças de experiências e emoções vivenciadas em sucessivas épocas. Nesse contexto da alimentação, essas lembranças assumem valores simbólicos, intangíveis e de difícil interpretação, uma vez que cada ser humano apresenta experiências de vida singulares e não quantificáveis (DE PAULA, 2002; LEONARDO, 2009; MACIEL, 2001; MINTZ, 2001). o&s - Salvador, v. 23, n. 77, p. 191-210, Abr./Jun. 2016 www.revistaoes.ufba.br A experiência estética nas práticas culinárias de uma organização gastronômica A experiência estética nas práticas culinárias de uma organização gastronômica Dada a concepção de sistema alimentar como um conjunto de práticas culturais que vão desde o preparo do alimento até a composição de um cardápio, observa-se a presença dos sentidos como meio pelo qual acontece a experiência sensorial da ação de alimentar-se. Os sentidos são como códigos que transmitem mensagens, e no caso da prática culinária o registro dessa experiência sensorial é o resultado de um conjunto de sensações geradas pelo gosto, aroma, temperatura e textura que são julgados esteticamente (SUTTON, 2010). Destarte, a escolha dos alimentos pelas pessoas está ligada a um complexo estético e cultural, e por ser uma decisão a ser apreciada com os sentidos, logo se relaciona a comida ao paladar, por ser o sentido mais óbvio de ser ativado no ato de alimentar-se. Segundo Cascudo (1983), o paladar é tido como um elemento com- plementar à alimentação, sendo culturalmente construído. Pelo paladar são definidas as preferências alimentares humanas, além de estar profundamente arraigado nas normas culturais, consistindo em uma identidade por meio da qual os indivíduos e os grupos se assemelham ou se diferenciam (GONÇALVES, 2004). O gosto, a sensação experimentada pelas papilas gustativas, está relacionado de forma íntima ao olfato. A percepção a partir desses sentidos desempenha um papel essencial na prática culinária de escolha e preparo das comidas. O sabor experimen- tado, seja pelo gosto ou pelo aroma, constrói um conhecimento, remetendo o sujeito a diversas e diferentes memórias (MACIEL, 2001). i O paladar, segundo Brillat Savarin, em sua obra A fisiologia do gosto, editada em 1848, promove três diferentes sensações: (i) no momento de contato da comida com a boca ocorre o que o autor chama de sensação direta; (ii) a sensação completa se dá quando o alimento passa para o fundo da boca, liberando seu gosto e perfume; (iii) por último se dá a sensação refletida, que consiste no juízo interior a respeito das impressões geradas ao ingerir a comida. Aqui se enfatiza o reviver de experiências no momento em que se degusta a comida. This (2007) considera o gosto uma sensação muito mais ampla do que aquela proporcionada pelos órgãos envolvidos. Para o autor, os demais sentidos auxiliam na percepção e interpretação do gosto, associando a essa sensação atributos baseados nas experiências relacionadas às lembranças, emoções e aprendizados memorizados pelos indivíduos. o&s - Salvador, v. 23, n. 77, p. 191-210, Abr./Jun. 2016 www.revistaoes.ufba.br A experiência estética nas práticas culinárias de uma organização gastronômica o&s - Salvador, v. 23, n. 77, p. 191-210, Abr./Jun. 2016 www.revistaoes.ufba.br 198 Procedimentos metodológicos Procedimentos metodológicos Esta pesquisa trilhou o percurso metodológico proposto por Strati (1992; 2007) para o desenvolvimento da pesquisa empírica sobre estética organizacional. Tal caminho se mostrou alicerçado em três princípios que nortearam o fazer empírico. O primeiro princípio da compreensão empática consiste em se imaginar no lugar do outro, cujos propósitos, motivos e sentidos se pretende explicar (STRATI, 2007). Isso possibilita ao pesquisador o reconhecimento de humores, pensamentos e sentimentos estéticos intimamente ligados à ação dos atores organizacionais. Nesse sentido, evidencia-se o pathos com que um evento ou uma ação é enfatizada pelos informantes, consistindo “No que diz e como diz, este não só fornece ao pesquisador informações sobre processos, como também comunica uma emoção, uma sensação, um sentimento” (STRATI, 2007, p. 272). Assim, a ênfase recai sobre o compartilhamento entre sujeitos e pesquisadores da sensação de prazer ou desprazer causada por algum evento organizacional, pro- porcionando o “sinal distintivo” (STRATI, 2007, p. 272) para orientar a investigação. O sentimento caracteriza, portanto, o estudo estético da vida organizacional cotidia- na por ser a qualidade expressiva intrínseca aos fenômenos estéticos, este é “um modo de as coisas, as situações e as formas se oferecerem” (STRATI, 2007, p. 272). Buscou-se, portanto, apreender o sentimento em seu “ser-em-uso” (STRATI, 2007, p. 145), enquanto manifestação de estilo e de uma atitude intencional tanto no interior dos eventos como na relação entre os membros da organização e os pesquisadores. O segundo princípio refere-se à ideia de connoisseurship – de suas faculdades sensoriais e de juízo estético –, que vem demonstrado por todos os participantes da vida organizacional, incluindo aqui aqueles que a estudam, em relação aos elementos não humanos e também às relações interpessoais: É o connoisseurship – com seu conhecimento tácito e, não explícito, com sua com- preensão antes empática-estética do que analítico-racional – que possibilita que as pessoas afirmem a legitimidade organizacional de suas diferentes interpretações da construção social que participam (STRATI, 2007, p. 280). É o connoisseurship – com seu conhecimento tácito e, não explícito, com sua com- preensão antes empática-estética do que analítico-racional – que possibilita que as pessoas afirmem a legitimidade organizacional de suas diferentes interpretações da construção social que participam (STRATI, 2007, p. 280). o&s - Salvador, v. 23, n. 77, p. 191-210, Abr./Jun. 2016 www.revistaoes.ufba.br A experiência estética nas práticas culinárias de uma organização gastronômica O gosto pela culinária, tanto para quem prepara como para quem degusta, é uma forma de obter um prazer estético e sensorial (GARCIA; CASTRO, 2011). Fischler (1995) assinala que não são apenas o paladar e o olfato que colaboram para a apreciação dos alimentos. Ainda que com diferentes graus, os cinco sentidos humanos contribuem para tal, construindo a emissão de um juízo a respeito das práticas culinárias envolvidas. A audição, ainda que seja o sentido menos utilizado, auxilia na preparação dos alimentos por meio da sonoridade e de ruídos percebidos pela cocção ou na atividade de comer. Com o tato é possível reconhecer a densidade dos alimentos, sua temperatura e textura. Esse conhecimento obtido com o tato não se coloca somente pelo contato manual com os alimentos, mas com a própria boca, ao se mastigar um alimento o tato também está atuando, sendo imprescindível para uma boa degustação. g ç Para Tempass (2007), a visão é um sentido particularmente importante, a ponto de ser utilizada a expressão “comer com os olhos”. Além disso, e já se associando ao prazer gustativo do saborear, os produtos da prática culinária devem ser bem apresentados aos olhos. Conforme anteriormente colocado por Lévi-Strauss (1991) e Certeau, Giard e Mayol (1996), as lógicas das linguagens associadas à prática culiná- ria se baseiam, entre outras experiências, naquelas relacionadas aos produtos finais, como, por exemplo: a nomeação dos pratos obtidos e o consumo do produto final, que se estende à apreciação visual da forma como a comida está esteticamente apre- sentada, e aos arranjos dos utensílios, pratos, talheres e recipientes utilizados para tal. O sentido da visão também está associado à disposição, maneiras e sociabilidade das pessoas de estarem na mesa (TEMPASS, 2007). o&s - Salvador, v. 23, n. 77, p. 191-210, Abr./Jun. 2016 www.revistaoes.ufba.br 199 Ana Sílvia Rocha Ipiranga, Luma Louise Sousa Lopes & Elnivan Moreira de Souza Procedimentos metodológicos Procedimentos metodológicos Procedimentos metodológicos Procedimentos metodológicos E, por fim, para a compreensão estética da vida organizacional, enfatiza-se o princípio de observação das categorias estéticas que emergem e são ditas, bem como os termos de uso corrente na vida organizacional cotidiana, inerentes à lingua- gem habitual dos atores organizacionais. Para Strati (2007), são os próprios sujeitos selecionados para a pesquisa que expressam e nos chamam a atenção para essas categorias e que, por outro lado, os pesquisadores podem igualmente convidar os sujeitos a utilizarem categorias estéticas ao descreverem suas rotinas. Tendo como base esses princípios, esta pesquisa percorreu o itinerário proposto pela compreensão empática para compor o corpus empírico (BAUER; GASKELL, 2008; STRATI, 2007). Dessa forma, durante visitas realizadas à organização gastronômica sob estudo no período entre outubro de 2012 e março de 2013, procedeu-se à imersão dos pesquisadores no papel do outro, a partir da ativação de suas faculdades sensoriais e de seu juízo estético, utilizando-se dos “sinais distintivos” (STRATI, 2007, p. 272) que manifestavam os humores, as impressões e as avaliações dos atores organizacionais. Para isso, e visando reviver a experiência na imaginação, foram combinados os métodos da auto-observação (o pesquisador se observa enquanto se põe no lugar de outrem); do exercício das faculdades intuitivas (a fim de assumir o papel do ator organizacional); e da analogia (possibilitando que o pesquisador estabeleça uma re- lação dinâmica com o que pensa e sente o ator organizacional e, assim, com a ação intencional em exame) (STRATI, 2007). Os sinais observados e constatados pelos pesquisadores nesse processo de coleta de conhecimentos foram descritos no caderno de campo a partir dos humores provocados, das emoções sentidas, dos pensamentos secundários que surgiram e das categorias estéticas formuladas nas ações e na sua organização, revelando como o pesquisador se interpreta nos detalhes e no decorrer dos estágios da ação intencional sob estudo. o&s - Salvador, v. 23, n. 77, p. 191-210, Abr./Jun. 2016 www.revistaoes.ufba.br 200 A experiência estética nas práticas culinárias de uma organização gastronômica Além disso, e de forma auxiliar, utilizou-se de um roteiro de entrevista relacional com questões abertas formuladas a partir da leitura crítica da base teórica, sendo este aplicado ao chef e à gestora da organização gastronômica. Esse tipo de entrevista, segundo Spink (2004), é compreendida como fruto da interação entre sujeitos ativos, incluindo nessa interação os próprios pesquisadores. Procedimentos metodológicos O uso da entrevista somado aos métodos anteriormente mencionados possibilitou a triangulação a favor do enrique- cimento da interpretação (SPINK, 2004). p ç ( , ) Tendo como base os princípios da compreensão empática, o processo de in- terpretação foi constituído como um produto constante do reexame da experiência vivida, construindo dessa forma uma compreensão da experiência estética inerente às práticas culinárias cotidianas da organização gastronômica sob estudo. Nesse sentido, a descrição baseou-se no processo heurístico da evocação por meio da participação imaginada dos pesquisadores na situação organizacional, estabelecendo uma relação entre os pesquisadores (leitores), a descrição que se faz e seus autores (STRATI, 2007). Em relação ao estilo da descrição e considerando essas diferentes vozes, utilizou- -se do “texto aberto” (STRATI, 2007, p. 116), enfatizando o mesmo que se aplicou à maneira de ver no como escrever sobre os fenômenos, sem lhes retirar a principal dimensão, qual seja, a da estética. Diante da grande quantidade de anotações nos cadernos de campo, relatos e narrativas gravadas em horas e horas de observações e entrevistas que compuseram o “texto aberto” (STRATI, 2007, p. 116), para a apre- sentação da descrição optou-se pela organização em forma de “fragmentos da vida organizacional” (STRATI, 2007, p. 274). Fragmentos da experiência estética imbuídos nas práticas culinárias da organização gastronômica sob estudo Fragmentos da experiência estética imbuídos nas práticas culinárias da organização gastronômica sob estudo Considerando as práticas culinárias um artefato capaz de nos contar alguma coisa sobre a organização gastronômica, e com o intuito de lançar luzes sobre a ação intencional dos atores organizacionais envolvidos nesta pesquisa, foram recortados do “texto aberto” (STRATI, 2007, p. 116) alguns fragmentos da vida organizacional que iluminaram as experiências estéticas durante o processo interpretativo deste estudo. p p p Durante os primeiros contatos com o chef do restaurante, este iniciou sua nar- rativa revivendo as lembranças de sua infância ao lado da mãe na cozinha da família (MACIEL, 2001; MINTZ, 2001). Os fragmentos referentes a esses episódios possibilita- ram aos pesquisadores o compartilhamento empático dos pensamentos e sentimentos ligados a esses eventos, proporcionando o “sinal distintivo” (STRATI, 2007, p. 272) para orientar a investigação propriamente dita. Ao revivermos de forma empática essas lembranças, emergiram fragmentos que iluminaram a “estética dominante” (STRATI, 2007, p. 121), articulada das experiências de vida do chef, sendo essa posteriormente ativada no organizar das práticas culinárias do pequeno restaurante. Ana Sílvia Rocha Ipiranga, Luma Louise Sousa Lopes & Elnivan Moreira de Souza Além disso, o trecho anterior evidencia a relação entre experiência sensível e conhecimento tácito, qual seja, o conhecimento que o chef é capaz de pôr em prática, por que ele sabe por si mesmo o que fazer (STRATI, 2007; TAVARES; KILIMNIK, 2007). Autores como Strati (1992), Gagliardi (1990) e Sutton (2010) ressaltaram que a estética na vida organizacional diz respeito a uma forma de conhecimento originado pelas faculdades perceptivas da audição, da visão, do tato, do olfato e do paladar, e pela capacidade de fazer um juízo estético. Por outro lado, Fine (1996) ressaltou que existe uma intensa diversidade estética nos restaurantes que possibilita uma ampla liberdade de escolhas nas práticas culinárias, envolvendo a sociabilidade e a organi- zação dos restaurantes, assim como a composição de cardápios e a preparação dos alimentos. Essa diversidade estética revela nos fragmentos a seguir a categoria do sublime, ao convergir variadas emoções estéticas, transitando entre sentimentos transcendentes e de excessos (PARRET, 1997): “[...] no dia a dia a criação e montagem dos cardápios e a preparação dos pratos, a estética dos arranjos de como a comida ficará no prato interfere 100%. Os nossos clientes comem com os olhos, então o belo é importante. Eu uso pratos que favoreçam a visão do que está sendo servido. Tem uma sobremesa de sorvete com morango flam- bado que não serve para colocar em prato fundo, pois, esconde o visual da sobremesa que é muito bela. Outro dia a cozinheira me fez uma abobrinha, não refogou muito, aí ficou com uma cor branquela muito feia. Eu a joguei no lixo. Tem que refogar e deixa dourar lentamente, tem os tempos de cozimento, assim, deixa um colorido natural e bonito. [...] O olfato na cozinha? Não preciso nem enfatizar a importância. Se usa o olfato antes, durante e depois de todo o processo culinário para julgar se está bom ou ruim, juntamente ao paladar. O bom gustare é aquele que chega até lá, naquela perfeição ali. [...] O tato é a mão do cozinheiro, na textura, na diferença do cozido da massa al dente, por exemplo, se pega na massa, usa o tato, o tino para julgar o ponto certo.” (fragmentos dos relatos do chef). Ana Sílvia Rocha Ipiranga, Luma Louise Sousa Lopes & Elnivan Moreira de Souza Os sentidos são como códigos que transmitem mensagens, e no caso da prática culinária o registro dessa experiência sensível é o resultado de um conjunto de sen- sações geradas pelo olhar, gosto, aroma, temperatura, textura, cores, sonoridades, tempos e ritmos que são julgados esteticamente a partir da relevância da categoria do belo, modificando-se e envolvendo, conforme constatado, a diferença complementar do feio (FISCHLER, 1995; GONÇALVES, 2004; KANT, 1987; SUTTON, 2010). ( Ç ) Segundo Parret (1997) e Strati (2007), na pesquisa empírica as categorias do sublime e do belo modificam os valores estéticos ao se ligarem às outras categorias e, nesse sentido, alguns fragmentos que transitaram entre a tradição e a inovação das práticas culinárias sob estudo se entrelaçaram com elementos da cultura italiana de origem do chef, dando conta do que há nessa realidade de único e original. Esses fragmentos revelaram as experiências estéticas entremeadas pela invenção, criati- vidade, simbolismos, ritualização, prazer e desprazer e pelas categorias agógicas1. Nas práticas culinárias os ritmos e os movimentos são essenciais para a coordenação dos fluxos de trabalho, trazendo à luz os desdobramentos acerca das decisões orga- nizacionais (CERTEAU; GIARD; MAYOL, 1996; STRATI, 2007). “[...] Temos que simplificar no sentido de não lotar o restaurante, estou fazendo o primeiro prato, já inicia o segundo prato. No fogo tem uma lagosta grelhando e um camarão flambando, se o cliente me solicita cinco pedidos diferentes não tem como eu fazer por que as bocas de gás estão ocupadas, então é preciso pensar e organizar os fluxos e movimentos da cozinha. [...] Por outro lado, em Roma, na minha terra, com o clima do verão a comida é leve com peixes, a massa, refoga e já está pronto, os ritmos são rápidos. Você organiza os pedidos de acordo com grupos de proximida- des. É uma forma de organização e isso vem da experiência e da prática. [...] Muitas receitas nós inventamos, nunca foram feitas, combinamos os ingredientes. Nesses experimentos se renovam os pratos e a lógica é a da experiência do paladar. Eu te- nho que gostar do resultado. Quando eu gosto tudo acontece com um sentimento diferente.” (fragmentos dos relatos do chef). 1 Etimologicamente, o termo agógica tem as suas raízes no verbo grego ágo, que significa conduzir, andar, levar. 1 Etimologicamente, o termo agógica tem as suas raízes no verbo grego ágo, que significa conduzir, andar, levar. O significado do termo provém da cinética musical (do greco kine = movimento), também chamada de agógica (ou agoge), é a parte da música que estuda a velocidade ou o andamento com que cada peça musical deve ser executada. o&s - Salvador, v. 23, n. 77, p. 191-210, Abr./Jun. 2016 www.revistaoes.ufba.br Procedimentos metodológicos “[...] Antigamente se era acostumado a fazer tudo dentro de casa, tudo se passava na cozinha, eu via a minha mãe cozinhando e a ajudava. [...] Pegar, tocar o tomate, a cebola, cortando alguma coisa, saboreando. [...] Então a gente começa a ter esse amor pela cozinha, essa paixão pela comida, a desenvolver o tino.” (fragmentos dos relatos do chef). O fragmento descrito revela o modus aestheticus de compreender a realidade, ao evidenciar o conhecimento obtido por meio do sentimento de ser capaz de fazer determinada coisa a partir das faculdades sensoriais que foram vivenciadas. Há uma analogia entre a imagem metafórica presente e o objeto de intuição: os sentimentos de amor pela cozinha e a paixão pela comida foram aflorados pela imaginação ao reviver a imagem da mãe na cozinha; e pelo uso dos sentidos da visão, do tato e do paladar ao tocar e provar os alimentos, ao desenvolver o tino; e ainda do juízo es- tético ao informar sobre como essas lembranças imaginativas foram representadas, sintetizadas e reconstruídas (KANT, 1987; STRATI, 2007; THIS, 2007; PARRET, 1997). o&s - Salvador, v. 23, n. 77, p. 191-210, Abr./Jun. 2016 www.revistaoes.ufba.br 201 Ana Sílvia Rocha Ipiranga, Luma Louise Sousa Lopes & Elnivan Moreira de Souza Ana Sílvia Rocha Ipiranga, Luma Louise Sousa Lopes & Elnivan Moreira de Souza Ana Sílvia Rocha Ipiranga, Luma Louise Sousa Lopes & Elnivan Moreira de Souza O significado do termo provém da cinética musical (do greco kine = movimento), também chamada de agógica (ou agoge), é a parte da música que estuda a velocidade ou o andamento com que cada peça musical deve ser executada. o&s - Salvador, v. 23, n. 77, p. 191-210, Abr./Jun. 2016 www.revistaoes.ufba.br 202 A experiência estética nas práticas culinárias de uma organização gastronômica A experiência estética nas práticas culinárias de uma organização gastronômica Considerando o princípio do connoisseurship e no intuito de apreender o sen- timento em seu “ser-em-uso” (STRATI, 2007, p. 145), foram recortados do “texto aberto” alguns fragmentos que emergiram do reexame dessa experiência estética (Quadro 1), vivida e revivida na memória pelos pesquisadores ao recorreram a ima- gens, comparações, metáforas e símbolos, veiculando uma compreensão intuitiva, conotando diferentes categorias estéticas imbuídas na organização gastronômica sob estudo (CROCE, 1990). ( , ) Esses procedimentos interpretativos indicaram os sinais distintivos referentes à arquitetura e ao ambiente do restaurante, que transitaram entre os significados das categorias estéticas que evocaram a elegância do gracioso, a originalidade do pito- resco e a artificialidade embelezadora do kitsch, ao ressaltarem o que a arte evoca em código, causando um efeito imediato, um gosto específico, uma sensibilidade sobre o observador (STRATI, 2007; TAYLOR; HANSEN, 2005). ( , ; ; , ) Conforme Strati (1996), não é apenas a decoração e/ou o embelezamento da vida organizacional que constituem a esfera privilegiada da abordagem estética das organizações. Para o autor, o foco principal seriam os elementos que ligam as pessoas às organizações e aos seus trabalhos. Nesse sentido, releva-se que os fragmentos ar- ticulados, envolvendo a materialidade e não materialidade da estética organizacional, se referem também ao espaço como metáfora do tempo organizacional. Enfatizou-se, ainda, a influência na experiência estética de artefatos humanos e não humanos que representam eventos organizacionais em termos da cultura, dos simbolismos e da identidade dessa organização, assim como da identificação dos atores, dos pesqui- sadores e dos clientes com essa organização (CASCUDO, 1983; GAGLIARDI, 1990). Fonte: Diários de campo. o&s - Salvador, v. 23, n. 77, p. 191-210, Abr./Jun. 2016 www.revistaoes.ufba.br Ana Sílvia Rocha Ipiranga, Luma Louise Sousa Lopes & Elnivan Moreira de Souza A partir do exercício da evocação do processo heurístico realizado pelos pes- quisadores, outros trechos recortados do “texto aberto” referiram-se ao exame da prática da fotografia exercida anteriormente pelo chef, antes de ingressar na atividade gastronômica. Esses relatos foram conotados por uma forte emoção de um sentimento de orgulho, evocando a categoria do grandioso e do heroico (PARRET, 1997). g , g g ( , ) Considerando, segundo Strati (2007), que o sentimento é a qualidade expressiva intrínseca aos fenômenos estéticos, o modo de as coisas, as situações e as formas se oferecerem, esses “sinais distintivos” orientaram a investigação, possibilitando inferências relacionadas à conjunção entre artes – fotografia e gastronomia. Essas conjunções terminaram por influenciar a “organização enquanto arte” (STRATI, 2007, p. 265) colocada em ato criativamente por um chef – gestor-artista. Esses fragmentos relevam o conhecimento (modus aestheticus) obtido pelos sentidos como ponto de partida para: (i) a criatividade do chef no desenvolver das práticas culinárias; (ii) para a gestão dessa organização gastronômica praticada pelo chef e pela gestora, que se engajam em atividades relacionadas com a arte; e (iii) para as práticas organizacionais cotidianas, como aquelas referentes à culinária, que tem a ver com a arte (JONES; MOORE; SNYDER, 1988). Diversos fragmentos recortados do “texto aberto” relacionam-se com o evi- denciado por Certeau, Giard e Mayol (1996), Lévi-Strauss (1991) e Tempass (2007) referentes aos domínios das maneiras na mesa e da fase do consumo do produto final. O chef e a gestora do restaurante sublinharam as orientações recebidas pelo pessoal do atendimento às mesas, no sentido de oferecer um serviço com elegância, assim como do conhecimento necessário dos itens do cardápio. Contudo e apesar do ágil balé dos atendentes no salão, alguns deles não conheciam o significado de algumas nomeações do cardápio do restaurante, sobretudo as informações técnicas referentes ao cardápio dos vinhos. Essas observações inferem, segundo Strati (2007), que os estágios das negociações organizacionais cotidianas referentes à estética criada, re- construída ou destruída ainda é incipiente. Por outro lado, essa evidência da pesquisa, ao iluminar as categorias do feio, do trágico e do grotesco no contexto organiza- cional, ressalta a necessidade de se refletir criticamente sobre o conceito de estética, segundo Eagleton (1993), enquanto um instrumento para sustentação do poder por meio do “consentimento” e/ou da “resistência” a esse poder. Quadro 1 – Fragmentos acerca do reexame da experiência vivida pelos pesquisadores. A fachada, o paisagismo, o mobiliário, a iluminação, o tratamento do teto e das paredes, os revestimentos dos pisos, o uso das cores e das formas, as exibições de quadros, o grande arco de tijolo aparente construído e que divide, por cima, o salão ao meio, o fundo musical, enfim, a decoração do restaurante esforça-se para se aproximar da cultura e do estilo arquitetônico italiano. [...] A cozinha muito limpa, bem equipada com diferentes tipos de fogão, congeladores e refrigerado- res, bancadas e suportes para acomodação dos ingredientes, matéria-prima para as operações da cozinha: óleos, vinagres, manteigas, azeites, variados tipos de temperos e ervas, etc., utensílios como as tábuas e colheres de madeira de várias formas e tamanhos, talheres, conjunto de facas, recipientes, artefatos, elementos não humanos e quando não em uso bem arrumados nas diversas prateleiras, como por exemplo: frigideiras, panelas e caçarolas de diferentes formas e tamanhos e cinco visíveis escorredores de macarrão de formatos de plástico colorido e/ou inox, utensílio típico para a operação culinária de escorrer a água do spaghetti após cozido al dente e que bem caracteriza as massas enquanto prato típico da cultura gastronômica italiana. [...] As cores brancas dos aventais acompanhados pela simbologia da altura do chapéu do cozinheiro e seus auxiliares, os hábeis e ligeiros movimentos coordenados e em sequência das mãos e do olhar do chef na manipulação, combinação e associação dos diferentes ingredientes, utensílios e recipientes. A espera pelos tempos, da inclinação da cabeça para sentir o aroma transportado pelo vapor das panelas, da rotação dos corpos nos espaços da cozi- nha ao observarem, por exemplo, o prazer auditivo do vinho flambando o peixe quase cozido na frigideira tomada pelo fogo alto. [...] Os pratos saboreados, por nós pesquisadores, despertaram os nossos sentidos do olhar, através das cores, o olfato pelo aroma suculento, a visão, pelo conjunto harmonizado de comidas no menu degustação, e por fim, o paladar. [...] No salão, observou-se que entre os murmúrios das conversações cruzadas, os clientes primeiro saboreiam a comida com os olhos, questionam e falam entre si, expressando ao fim sorrisos e excla- mações de prazer e bem-estar à mesa. Fonte: Diários de campo. 203 Ana Sílvia Rocha Ipiranga, Luma Louise Sousa Lopes & Elnivan Moreira de Souza Ana Sílvia Rocha Ipiranga, Luma Louise Sousa Lopes & Elnivan Moreira de Souza Contudo, essa incipiência negocial era frequentemente compensada pela intensa sociabilidade do chef ao transitar da cozinha ao salão com seu alto e branquíssimo chapéu de cozinheiro, descrevendo minuciosamente aos seus clientes os ingredientes utilizados no menu degustation de mais de 15 porções mínimas e experimentais, re- fletindo sobre o ato de cozinhar e justificando de forma efusiva, e para a alegria dos seus convivas, o vinho adequado para melhor degustar e saborear o prato desejado. Nesse sentido, infere-se que a categoria do belo, enquanto a condição de objeto da experiência estética (KANT, 1987), se manifesta na sociabilidade do chef, ligando o indivíduo à organização gastronômica, significando o prazer que ele sente de pertencer àquela organização em particular. Ao revivermos essa experiência de participação na imaginação, o sentimento e as imagens que nós, pesquisadores, vislumbramos enquanto tradução do pensamento sensível foram da experiência comunal e da categoria estética do sagrado (TAYLOR; HANSEN, 2005; STRATI, 2007), da beatitude do comer e beber bem e da celebração da organização como festa (CERTEAU; GIARD; MAYOL, 1996; STRATI, 2007). Considerações finais Considerações finais Considerações finais No desenvolvimento desta pesquisa, considerou-se que o modus aestheticus de compreender a realidade delineou a compreensão estética da vida organizacional, indo além de insights teóricos (logos), caracterizando a qualidade expressiva intrínseca do fenômeno estético (pathos). Nesses termos, o percurso de análise aqui apresentado procurou articular, por meio do princípio da compreensão empática, os sentimentos em seu “ser-em-uso” o&s - Salvador, v. 23, n. 77, p. 191-210, Abr./Jun. 2016 www.revistaoes.ufba.br 204 A experiência estética nas práticas culinárias de uma organização gastronômica subjacentes aos eventos, aos estilos e à ação intencional dos atores organizacionais, e entre estes e os pesquisadores participantes do estudo. Esse caminho possibilitou a manifestação das faculdades sensoriais e de juízo estético (connoisseurship) dos atores participantes acerca da vida organizacional sob estudo, delineando diferentes representações, imagens, comparações, metáforas e símbolos na construção e interpretação desse social. Para isso, as práticas culinárias cotidianas da organização gastronômica, en- quanto unidade de análise da presente pesquisa, foram consideradas um artefato, no sentido de que elas nos dizem alguma coisa sobre essa organização. Ana Sílvia Rocha Ipiranga, Luma Louise Sousa Lopes & Elnivan Moreira de Souza Como resultados, releva-se que, ao descrevermos esteticamente as práticas culinárias, algumas categorias referentes ao trabalho e à própria organização gas- tronômica foram observadas e expressas pelos atores participantes, significando um conjunto de sensações e experiências geradas pelo: gosto, aroma, tempera- tura, textura, formas, movimentos, cores, conteúdos, arquiteturas, sonoridades e julgadas esteticamente. Embasando essa evidência, percebeu-se que ocorreu um entrelaçamento entre a vida organizacional cotidiana, a faculdade de julgamento e as categorias estéticas, iluminando o pathos do sensível na compreensão do organizar das práticas culinárias. Subsequentemente, relevou-se da pesquisa que as categorias do sublime e do belo, ao modificarem e desvelarem outras categorias que se manifestaram entrelaçadas com elementos da cultura culinária, delinearam a experiência estética dos sujeitos, entremeada pela invenção, criatividade, simbolismos, memórias, ritualização e ritmos, prazer e desprazer, trazendo à luz implicações acerca da coordenação dos fluxos e movimentos do trabalho e das decisões organizacionais. A consciência do modus aestheticus para a compreensão da vida organizacional busca dirigir a atenção do pesquisador para os aspectos sensíveis da organização, para a construção e redefinição da experiência sensível e para a experiência revivida na memória. Essa postura metodológica indicou os “sinais distintivos” referentes às formas e aos conteúdos das práticas do restaurante estudado, que transitaram entre os significados das categorias estéticas do gracioso e do pitoresco, causando uma sensibilidade sobre o observador; da artificialidade embelezadora do kitsch, da cate- goria do sagrado atribuído à beatitude do estar bem à mesa e da experiência comunal emersa com a sociabilidade do chef e seus convivas. Em termos de implicações desses resultados, entende-se que as percepções sensoriais e emotivas emersas das práticas culinárias, a partir da observação parti- cipante imaginada, imprimiram nos atores organizacionais e em nós pesquisadores conhecimento sensível sobre os eventos, ações e decisões organizacionais, ampliando, em consequência, a compreensão dos reflexos dessas dimensões do modus aestheticus para além de uma compreensão lógico racional do contexto organizacional. Nesses termos, inferiu-se da pesquisa que sentir, compreender e saber estão entremeados em seu ser-em-uso na organização gastronômica, constituídos pela prática das habilidades culinárias e pela prática da ação intencional dos atores organizacionais. l d d õ d õ d d d Conclui-se que, a partir das descrições das sensações dos entrevistados e das observações dos pesquisadores, foi possível iluminar, imbuído nas práticas culinárias, o modus aestheticus como forma de se obter conhecimentos a respeito da organiza- ção gastronômica. o&s - Salvador, v. 23, n. 77, p. 191-210, Abr./Jun. 2016 www.revistaoes.ufba.br Ana Sílvia Rocha Ipiranga, Luma Louise Sousa Lopes & Elnivan Moreira de Souza A perspectiva subjacente com a qual a organização foi analisada revela que há muito mais a ser conhecido do que a racionalidade instrumental pode revelar. O processo conjunto do ato de alimentar-se e do ato culinário em si é perme- ado por sensações e emoções despertadas pelos sentidos e que, consequentemente, promovem um juízo estético nos sujeitos, construindo conhecimento sensível sobre a organização. Quase como em uma prática artística, propriamente dita, é impossível dissociar a dimensão estética da prática culinária. Como limitação do estudo, releva-se que cada categoria estética emersa em si mesma não exauriu o conhecimento sensível da organização. E, nesse sentido, o “texto aberto” observado, imaginado, vivido, revivido, lido e reconstruído, por meio do pro- cesso evocativo para a aquisição de conhecimentos, não é o único possível que possa servir de base para a compreensão estética da organização gastronômica estudada. o&s - Salvador, v. 23, n. 77, p. 191-210, Abr./Jun. 2016 www.revistaoes.ufba.br 205 Ana Sílvia Rocha Ipiranga, Luma Louise Sousa Lopes & Elnivan Moreira de Souza Conforme Strati (2007, p. 35), essas considerações baseiam-se na natureza complexa e paradoxal do conhecimento organizacional enquanto processo “agente em vez de existente, enquanto concebido como uma construção social coletiva”. Por outro lado, a abordagem estética inspira-se nessa rede de interpretações intertextuais quando explora as categorias do sublime e do belo, do gracioso e do pitoresco, do kitsch, do feio e do trágico, do grandioso e do heroico, do agógico, do sagrado, e de como elas se manifestaram no decorrer da pesquisa. Enfatizamos, jun- tamente com Strati (2007), que essas categorias, apesar de se originarem da filosofia estética e da teoria da arte, não são meramente reaplicadas à vida organizacional, elas fazem parte da linguagem usual dos atores e dos pesquisadores e, assim, emergiram da análise e são inerentes à vida organizacional específica que foi estudada. Em termos de estudos futuros, sugere-se melhor aprofundar a questão inferida nesta pesquisa, relacionada aos estágios incipientes das negociações cotidianas entre os diferentes atores e fazeres organizacionais referentes à estética criada, reconstruída ou destruída. Outra sugestão de estudos diz respeito às discussões relativas à questão do feio, do trágico e do grotesco, do embaraçoso e do gosto execrável existentes na rotina e na vida cotidiana das pessoas nas organizações. Esses pontos sugerem a necessidade de aprofundar a reflexão crítica do conceito de estética aplicado às organizações. Ana Sílvia Rocha Ipiranga, Luma Louise Sousa Lopes & Elnivan Moreira de Souza i Por fim, releva-se que esses caminhos de pesquisa baseiam-se na hibridação que funde as diferentes interpretações filosóficas e/ou artísticas nas interpretações fornecidas pelo conhecimento do sensível de cada sujeito cognitivo – atores e pes- quisadores – na organização sob estudo. A arte não coincide com a estética, a observação dos pesquisadores do trabalho do chef e da gestora na arte de organizar se distingue por um de que a liga a uma atividade em particular, colocando em evi- dência o componente sensível da compreensão da ação intencional nessa organização (STRATI, 2007). Referências Referências ADORNO, T. W. Teoria estética. Lisboa: Edições 70, 1982. p. 11-24. ADORNO, T. W. Teoria estética. Lisboa: Edições 70, 1982. p. 11-24. Ó ARISTÓTELES. Poética. São Paulo: Abril, 1975. cap. I a XII, p. 241-252. (Os Pensadores). BASSO, K.; PAULI, J.; BRESSAN, V. P. Relações de gênero e estética organizacional: sugestões para estudos sobre relações, cultura e desempenho. Cadernos EBAPE.BR, v. 12, n. 3, p. 688-705, jul./set. 2014. BAUER, M; GASKELL, G. (Ed.). Pesquisa qualitativa com imagem texto e som: um manual prático. Tradução de Pedrinho A. Guarechi. 7. ed. Petrópolis: Vozes, 2008. p. 39-63. BAUMGARTEN, A. G. Meditationes philosophicae de nonnullis ad poema pertinentibus. Halle: Grunert, 1735. [Em português: Estética: a lógica da arte e do poema. Tradução de M. S. Medeiros. Petrópolis: Vozes, 1993. 191 p.]. BERTOLIN, R. V.; BRITO, M. J. de. Corporeidade e estética na aprendizagem organizacional: insights emergentes. In: ENCONTRO DE ESTUDOS ORGANIZACIONAIS, 7., 2012, Curitiba. Anais... Curitiba: ANPAD, 2012. 1 CD-ROM. BOLOGNINI, B. Images as identifying objects and as organizational integrators in two firms. Dragon, v. 1, n. 3, p. 61-75, fev. 1986. BOUILLOUD, J. P.; DESLANDES, G. From negativity to creativity: about the aesthetics of “Beau Geste” in leadership. In: EUROPEAN GROUP FOR ORGANIZATIONAL STUDIES COLLOQUIUM, 29., Montreal. Proceedings… Montreal: [s. n.], 2013. BRADY, F. N. Aesthetic components of management ethics. Academy of Management Review, v. 11, n. 2, p. 337-344, 1986. o&s - Salvador, v. 23, n. 77, p. 191-210, Abr./Jun. 2016 www.revistaoes.ufba.br 206 A experiência estética nas práticas culinárias de uma organização gastronômica CANESQUI, A. M. Comentários sobre os estudos antropológicos da alimentação. In: CANESQUI, A. M.; GARCIA, R. W. D. (Org.). Antropologia e nutrição: um diálogo possível. Rio de Janeiro: Fiocruz, 2005. p. 23-42. CASCUDO, L. da C. História da alimentação no Brasil. v. 2. São Paulo: Itatiaia, 1983. CERTEAU, M. de; GIARD, L.; MAYOL, P. Ana Sílvia Rocha Ipiranga, Luma Louise Sousa Lopes & Elnivan Moreira de Souza A invenção do cotidiano: 2. Morar, cozinhar. Petrópolis: Vozes, 1996. 372 p. CROCE, B. Estetica come scienza dell’ espressione e linguística generale. Milano: Adelphi, 1990. DAMATTA, R. O que faz o brasil, Brasil? Rio de Janeiro: Rocco, 1986. 128 p. DEGÒT, V. Portrait of the manager as an artist. Dragon, v. 2, n. 3, p. 13-50, dez. 1987. DE PAULA, N. M. Introdução ao conceito de hospitalidade em serviços de alimentação. In: DIAS, C. M. et al. (Org.). Hospitalidade: reflexões e perspectivas. São Paulo: Manole, 2002. EAGLETON, T. A ideologia da estética. Rio de Janeiro: Zahar, 1993. 327 p. EWENSTEIN, B.; WHYTE, J. Beyond words: aesthetics knowledge and knowing in organizations. Organization Studies, v. 28, n. 5, p. 689-708, 2007. FIGUEIREDO, S. M. de et al. Oficina de culinária: saberes e sabores dos alimentos. E-Scientia, v. 3, n. 1, p. 39-48, ago. 2010. FIGUEIREDO, M. D. de. O artesanato enquanto prática e materialidade: argumento para pensar a dimensão estética e a perspectiva do embodiment nos estudos organizacionais. In: ENCONTRO DA ASSOCIAÇÃO DE PÓS-GRADUAÇÃO E PESQUISA EM ADMINISTRAÇÃO, 36., Rio de Janeiro. Anais... Rio de Janeiro: ANPAD, 2012. 1 CD-ROM. FILIPPI, M.; TANNERY, F. Aesthetics drama and strategy creation. In: EUROPEAN GROUP FOR ORGANIZATIONAL STUDIES COLLOQUIUM, 25., Barcelona. Proceedings… Barcelona: [s. n.], 2009. FINE, G. A. Kitchens: the culture of restaurant work. Berkeley: University of California Press, 1996. 320 p. FISCHLER, C. El (h)omnívoro: el gusto, la cocina y el cuerpo. Barcelona: Anagrama, 1995. GAGLIARDI, P. (Ed.). Symbols and artifacts: views from the corporate landscape. Berlin; New York: W. de Gruyter, 1990. 428 p. GAGLIARDI, P. Explorando o lado estético da vida organizacional. In: CLEGG, S. R.; HARDY, C.; NORD, W. R. Handbook de Estudos Organizacionais. 1. ed.. 2. reimpr. São Paulo: Atlas, 2009. v. 2. p. 127-146. GARCIA, R. W. D.; CASTRO, I. R. R. de. A culinária como objeto de estudo e de intervenção no campo da alimentação e nutrição. Ciência e Saúde Coletiva, v. 16, n. 1, p. 91-98, jan. 2011. GHERARDI, S. From organizational learning to practice-based knowing. Human Relations, v. 54, n. 1, p. 131-139, jan. 2001. GOMES, P. C. da C.; RIBEIRO, L. P. Cozinha geográfica: o propósito da transformação de natureza em cultura. Espaço e Cultura, n. 29, p. 69-81, jan./jun. 2011. GONÇALVES, J. R. S. A fome e o paladar: a antropologia nativa de Luís da Câmara Cascudo. Estudos Históricos, v. 1, n. Ana Sílvia Rocha Ipiranga, Luma Louise Sousa Lopes & Elnivan Moreira de Souza 33, p. 40-55, 2004. o&s - Salvador, v. 23, n. 77, p. 191-210, Abr./Jun. 2016 www.revistaoes.ufba.br 207 Ana Sílvia Rocha Ipiranga, Luma Louise Sousa Lopes & Elnivan Moreira de Souza GRAFTON-SMALL, B.; LINSTEAD, S. Bricks and bricolages: deconstructing corporate images in stone and story. Dragon, v. 1, n. 1, p. 8-27, 1985. HATCH, M. J.; JONES, M. O. Photococylore at work: aesthics, collective criativity and the social construction of the organizations. Studies in Cults, Orgs. and Socs., v. 3, p. 263-287, 1997. HUME, D. Do padrão do gosto. São Paulo: Abril, 1980. 329 p. (Os Pensadores). IPIRANGA, A. S. R. et al. A experiência estética em uma organização gastronômica. In: ENCONTRO DA ASSOCIAÇÃO NACIONAL DE PESQUISA E PÓS-GRADUAÇÃO EM ADMINISTRAÇÃO, 1., 2013, Rio de Janeiro. Anais... Rio de Janeiro: ANPAD, 2013. 1 CD-ROM. JONES, M. O.; MOORE, M.; SNYDER, R. (Ed.). Inside organizations: understanding the human dimension. Newbury Park: Sage, 1988. KANT, I. Crítica da faculdade do juízo. 3. ed. Rio de Janeiro: Forense Universitária, 1987. 410 p. LEAL, R. S. A estética como elemento para compreensão da criatividade organizacional. Organizações & Sociedade, v. 14, n. 2, p. 67-82, jul./set. 2007. LEAL, R. S. Contribuições da estética para a análise organizacional: a abordagem de uma dimensão humana esquecida. In: ENCONTRO DE ESTUDOS ORGANIZACIONAIS, 1., 2000, Curitiba. Anais... Curitiba: ANPAD, 2000. 1 CD-ROM. LEAL, R. S. Razão, práxis e estética: integração de diferentes dimensões de análise organizacional. In: ENCONTRO DA ASSOCIAÇÃO NACIONAL DE PÓS-GRADUAÇÃO E PESQUISA EM ADMINISTRAÇÃO, 26., Salvador. Anais... Salvador: ANPAD, 2002. 1 CD-ROM. LEONARDO, M. Antropologia da alimentação. Revista Antropos, v. 3, n. 2, p. 1-6, dez. 2009. LÉVI-STRAUSS, C. O cru e o cozido. São Paulo: Brasiliense, 1991. LOPES, L. L. S. Sabores do cotidiano: a experiência estética nas práticas culinárias de uma organização gastronômica. 2014. Dissertação (Mestrado em Administração)–Universidade Estadual do Ceará, 2014. LOPES, L. L. S.; SOUZA, E. M.; IPIRANGA, A. S. R. Desvelando as categorias estéticas na organização de um pequeno restaurante. Revista Interdisciplinar de Gestão Social, v. 3, n. 1, p. 207-222, jan./abr. 2014. MACIEL, M. E. Churrasco à gaúcha. Horizontes Antropológicos, v. 29, n. 4, p. 34-48, jan./jun. 1996. MACIEL, M. E. Cultura e alimentação ou o que têm a ver os macaquinhos de Koshima com Brillat-Savarin? Horizontes Antropológicos, v. 7, n. 16, p. 145-156, dez. 2001. MACIEL, M. E. Identidade cultural e alimentação. In: CANESQUI, A. M.; GARCIA, R. W. D. o&s - Salvador, v. 23, n. 77, p. 191-210, Abr./Jun. 2016 www.revistaoes.ufba.br Ana Sílvia Rocha Ipiranga, Luma Louise Sousa Lopes & Elnivan Moreira de Souza (Org.). Antropologia e nutrição: um diálogo possível. Rio de Janeiro: Fiocruz, 2005. p. 49-55. MACIEL, M. E. Uma cozinha à brasileira. Estudos Históricos, v. 1, n. 33, p. 25-39, jan./jun. 2004. MARX, K. Manuscritos econômico-filosóficos. São Paulo: Abril, 1975. p. 17-21. (Os Pensadores). o&s - Salvador, v. 23, n. 77, p. 191-210, Abr./Jun. 2016 www.revistaoes.ufba.br 208 A experiência estética nas práticas culinárias de uma organização gastronômica A experiência estética nas práticas culinárias de uma organização gastronômica MARX, K. Para a crítica da economia política. São Paulo: Abril, 1975. p. 130-131. (Os Pensadores). MINTZ, S. W. Comida e antropologia: uma breve revisão. RBCS, v. 16, n. 47, p. 31- 41, out. 2001. OLIVEIRA, L. Y. M. de. A arquitetura dos processos de aprendizagem à luz da teoria da estética organizacional. 2012. Dissertação (Mestrado em Administração)–Escola de Administração, Universidade Federal do Rio Grande do Sul, 2012. OTTENSMEYER, E. J. Too strong to stop, too sweet to lose: aesthetics as a way to know organizations. Organization, v. 3, n. 2, p. 189-194, maio 1996. PARRET, H. A estética da comunicação. Além da pragmática. Campinas: Editora da Unicamp, 1997. 204 p. PLATÃO. A república. Tradução de M. H. R. Pereira. 7. ed. Lisboa: Fundação Calouste Gulbenkian, 1993. 518 p. RAMIREZ, R. An aesthetics theory of social organization. Dragon, v. 2, n. 3, p. 51- 64, dez. 1987a. RAMIREZ, R. The relationship between the aesthetics theory of social organization and some theories of organizational symbolism. Dragon, v. 2, n. 3, p. 65-84, dez. 1987b. SANDELANDS, L. E.; BUCKNER, G. C. Of art and work: aesthetic experience and the psychology of work feelings. Research in Organizational Behavior, v. 11, p. 105- 131, 1989. SCHIAVO, S. R. As práticas de trabalho e o processo de aprendizagem de trabalhadores da construção civil à luz da estética organizacional. 2010. 107 f. Dissertação (Mestrado em Administração)–Escola de Administração, Universidade Federal do Rio Grande do Sul, Porto Alegre, 2010. SCHILLER, J. C. F. Sobre a educação estética do homem em uma sequência de cartas – n. XXII a XXIV. In: DUARTE, R. O belo autônomo. Textos clássicos de filosofia. Belo Horizonte: Ed. UFMG, 1997. p. 123-134. SCHNEIDER, S. C.; POWLEY, E. The role of images in changing corporate culture: the case of A.T. & T. Dragon, v. 1, n. 2 p. 5-44, jan. 1985. SORENSEN, B. M. St Paul’s conversion: the aesthetic organization of labour. Organization Studies, v. 31, n. o&s - Salvador, v. 23, n. 77, p. 191-210, Abr./Jun. 2016 www.revistaoes.ufba.br o&s - Salvador, v. 23, n. 77, p. 191-210, Abr./Jun. 2016 www.revistaoes.ufba.br Ana Sílvia Rocha Ipiranga, Luma Louise Sousa Lopes & Elnivan Moreira de Souza 3, p. 307-326, mar. 2010. SPINK, M. J. (Org.). Práticas discursivas e produção de sentidos no cotidiano: aproximações teóricas e metodológicas. 3. ed. São Paulo: Cortez, 2004. 296 p. STEPHENS, J. P.; BOLAND, B. Legitimizing convergence: aesthetics as a bridge between managing and designing. In: EUROPEAN GROUP FOR ORGANIZATIONAL STUDIES COLLOQUIUM, 27., Gothenburg. Proceedings... Gothenburg: [s.n.], 2011. STRATI, A. Aesthetics understanding of organizational life. Academy of Management Review, v. 17, n. 3, p. 568-581, jul. 1992. STRATI, A. Aesthetics understanding of work and organizational life: approaches and research developments. Sociology Compass, v. 10, n. 4, p. 880-893, 2010. STRATI, A. Organização e estética. Tradução de Pedro Maia Soares. Rio de Janeiro: FGV, 2007. 320 p. STRATI, A. Organizations viewed through the lens of aesthetics. Organization, v. 3, n. 2, p. 209-218, maio 1996. 209 Ana Sílvia Rocha Ipiranga, Luma Louise Sousa Lopes & Elnivan Moreira de Souza SUTTON, D. E. Food and the senses. Annual Review of Anthropology, v. 39, p. 209- 223, 2010. TAVARES, M. das G. P.; KILIMNIK, Z. M. O conhecimento estético pode ser uma forma de explicação do conhecimento tácito? Reflexões a partir de dados empíricos. In: ENCONTRO DE ADMINISTRAÇÃO DA INFORMAÇÃO, 1., 2007, Florianópolis. Anais... Florianópolis: ANPAD, 2007. 1 CD-ROM. TAYLOR, S. S.; HANSEN, H. Finding form: looking at the field of organizational aesthetics. Journal of Management Studies, v. 42, n. 6, p. 1211-1231, set. 2005. TAYLOR, S. S. Little beauties: aesthetics, craft, skill, and the experience of beautiful action. Journal of Management Inquiry, v. 20, n. 2, p. 1-13, 2012. TEMPASS, M. C. O belo discreto: a estética alimentar Mbyá-Guarani. Espaço Ameríndio, v. 1, n. 1, p. 170-194, jul./dez. 2007. THIS, H. A ciência na cozinha, 2: Hervé This e os fundamentos da gastronomia molecular: corpo, máquina de comer. Scientific American. São Paulo: Duetto, 2007. WASSERMAN, V.; FRENKEL, M. Organizational aesthetics: caught between identity regulation and culture jamming. Organizational Science, v. 22, n. 2, p. 503-521, mar./abr. 2011. WITZ, A.; WARHURST, C.; NICKSON, D. The labour of aesthetics and the aesthetics of organization. Organization, v. 10, n. 1, p. 33-54, 2003. WOOD, T.; CSILLAG, P. Estética organizacional. Organização & Sociedade, v. 8, n. 21, p. 35-44, maio/ago. 2001. Submissão: 02/04/2014 Aprovação: 12/12/2014 210
https://openalex.org/W4236226139	https://periodicos.ufsc.br/index.php/textodigital/article/download/1807-9288.2012v8n2p393/23646	Portuguese	null	Burrocracia	Texto digital	2,012	cc-by	818	Franklin Valverde. O autor retoma uma forma de fazer poesia e a caracteriza de maneira especial. Há muito a poesia se libertou da folha de papel e procurou se expressar em outros suportes e com outros meios. Franklin Valverde. O autor retoma uma forma de fazer poesia e a caracteriza de maneira especial. Há muito a poesia se libertou da folha de papel e procurou se expressar em outros suportes e com outros meios. Franklin Valverde. O autor retoma uma forma de fazer poesia e a caracteriza de maneira especial. Há muito a poesia se libertou da folha de papel e procurou se expressar em outros suportes e com outros meios. Burrocracia. eucalipto folhas aplaudem ao vento amanhã guardarão os versos São Paulo: Littera, 2003. O trabalho repetitivo e monótono de que se fala tanto, e que tem sido tema de muitas poesias, acabou produzindo ele próprio a reprodução de repetições em graus cada vez mais elevados de automação. A produção em série é um dos exemplos do homem que procura imitar a máquina. Nas repartições públicas, escritórios e instituições financeiras, o processo de registro de dados foi tornando-se cada vez mais sofisticado, o que levou a produzir máquinas e mecanismos de repetição. O carimbo é uma das tecnologias da repetição e do enfado. Para deixar de escrever a mesma coisa, usa-se o carimbo. Ele repete, de maneira mais eficiente e com rapidez, o que precisamos dizer durante a rotina do trabalho. Ainda não esquecemos a imagem do homem sisudo e vazio, de óculos, atrás de um balcão, a procurar espaços em brancos em documentos para carimbar. E quantas vezes também nos tornamos carimbadores, no sentido real e figurado do tema, em guardarão os versos São Paulo: Littera, 2003. O trabalho repetitivo e monótono de que se fala tanto, e que tem sido tema de muitas poesias, acabou produzindo ele próprio a reprodução de repetições em graus cada vez mais elevados de automação. A produção em série é um dos exemplos do homem que procura imitar a máquina. Nas repartições públicas, escritórios e instituições financeiras, o processo de registro de dados foi tornando-se cada vez mais sofisticado, o que levou a produzir máquinas e mecanismos de repetição. O carimbo é uma das tecnologias da repetição e do enfado. Para deixar de escrever a mesma coisa, usa-se o carimbo. Ele repete, de maneira mais eficiente e com rapidez, o que precisamos dizer durante a rotina do trabalho. Ainda não esquecemos a imagem do homem sisudo e vazio, de óculos, atrás de um balcão, a procurar espaços em brancos em documentos para carimbar. DOI: 10.5007/1807-9288.2012v8n2p393 DOI: 10.5007/1807-9288.2012v8n2p393 Texto Digital, Florianópolis, v. 8, n. 2, p. 393-395, jul./dez. 2012. ISSNe: 1807-9288 Jorge Luiz Antonio Brasileiro, poeta, pesquisador, autor de Brazilian Digital Art and Poetry on the Web (2000), Cores, forma, luz, movimento: a poesia de Cesário Verde (SP, Musa, 2002), e Ciência, arte e metáfora na poesia de Augusto dos Anjos (SP, Navegar, 2004) e Poesia digital: teoria, historia, antologias (SP: Navegar; Columbus, Ohio, EUA: Luna Bisontes, 2010, livro e dvd). Texto recebido em 31/10/2012. Jorge Luiz Antonio Brasileiro, poeta, pesquisador, autor de Brazilian Digital Art and Poetry on the Web (2000), Cores, forma, luz, movimento: a poesia de Cesário Verde (SP, Musa, 2002), e Ciência, arte e metáfora na poesia de Augusto dos Anjos (SP, Navegar, 2004) e Poesia digital: teoria, historia, antologias (SP: Navegar; Columbus, Ohio, EUA: Luna Bisontes, 2010, livro e dvd). Texto Digital, Florianópolis, v. 8, n. 2, p. 393-395, jul./dez. 2012. ISSNe: 1807-9288 Franklin Valverde. O autor retoma uma forma de fazer poesia e a caracteriza de maneira especial. Há muito a poesia se libertou da folha de papel e procurou se expressar em outros suportes e com outros meios. E quantas vezes também nos tornamos carimbadores, no sentido real e figurado do tema, em Texto Digital, Florianópolis, v. 8, n. 2, p. 393-395, jul./dez. 2012. ISSNe: 1807-9288 393 nosso trabalho, família e vida. Assim como bancário é considerado popularmente como um louco atrás de papéis (e ai daquele que perder um papelzinho!), o carimbo é um dos instrumentos característicos de uma das primeiras automatizações. nosso trabalho, família e vida. Assim como bancário é considerado popularmente como um louco atrás de papéis (e ai daquele que perder um papelzinho!), o carimbo é um dos instrumentos característicos de uma das primeiras automatizações. Não há um papel-documento que vá de um lado para outro e que não leve um carimbo. Mas felizmente passou a existir o poema- carimbo ou rubber poetry, que já conta com seguidores como E. M. de Melo e Castro, o pioneiro Oswald de Andrade, Marcelo Dolabela e muitos outros. Autor do poema-objeto Bizcocho Z. Editor de Onda Latina. Texto Digital, Florianópolis, v. 8, n. 2, p. 393-395, jul./dez. 2012. ISSNe: 1807-9288 Texto Digital, Florianópolis, v. 8, n. 2, p. 393-395, jul./dez. 2012. ISSNe: 1807-9288 394 Texto recebido em 31/10/2012. Texto recebido em 31/10/2012. 395
https://openalex.org/W4220708172	https://birmingham.elsevierpure.com/files/196837920/s12874_021_01469_6.pdf	English	null	Completeness of reporting of clinical prediction models developed using supervised machine learning: a systematic review	BMC Medical research methodology	2,022	cc-by	8,147	University of Birmingham University of Birmingham Completeness of reporting of clinical prediction models developed using supervised machine learning Navarro, Constanza L. Andaur; Damen, Johanna A. A.; Takada, Toshihiko; Nijman, Steven W. J.; Dhiman, Paula; Ma, Jie; Collins, Gary S.; Bajpai, Ram; Riley, Richard D.; Moons, Karel G. M.; Hooft, Lotty DOI Document Version Publisher's PDF, also known as Version of record Citation for published version (Harvard): Navarro, CLA, Damen, JAA, Takada, T, Nijman, SWJ, Dhiman, P, Ma, J, Collins, GS, Bajpai, R, Riley, RD, Moons, KGM & Hooft, L 2022, 'Completeness of reporting of clinical prediction models developed using supervised machine learning: a systematic review', BMC Medical Research Methodology, vol. 22, no. 1, 12. https://doi.org/10.1186/s12874-021-01469-6 Citation for published version (Harvard): Navarro, CLA, Damen, JAA, Takada, T, Nijman, SWJ, Dhiman, P, Ma, J, Collins, GS, Bajpai, R, Riley, RD, Moons, KGM & Hooft, L 2022, 'Completeness of reporting of clinical prediction models developed using supervised machine learning: a systematic review', BMC Medical Research Methodology, vol. 22, no. 1, 12. https://doi.org/10.1186/s12874-021-01469-6 General rights U l li General rights Unless a licence is specified above, all rights (including copyright and moral rights) in this document are retained by the authors and/or the copyright holders. The express permission of the copyright holder must be obtained for any use of this material other than for purposes permitted by law. y y y p •Users may download and/or print one copy of the publication from the University of Birmingham research portal for the purpose of private study or non-commercial research. mercial research. racts from the document in line with the concept of ‘fair dealing’ under the Copyright, Designs and Patents Act 1988 (?) rther distribute the material nor use it for the purposes of commercial gain. y •User may use extracts from the document in line with the concept of ‘fair dealing’ under the Copyright, Designs and P •Users may not further distribute the material nor use it for the purposes of commercial gain. Where a licence is displayed above, please note the terms and conditions of the licence govern your use of this document. here a licence is displayed above, please note the terms and conditions of the licence govern your use of this document When citing, please reference the published version. Completeness of reporting of clinical prediction models developed using supervised machine learning: a systematic review C L A d N 1 2* J h A A D 1 2 T hihik T k d 1 S W J Nij 1 Constanza L. Andaur Navarro1,2* , Johanna A. A. Damen1,2 , Toshihiko Takada1 , Steven W. J. Nijman1 , Paula Dhiman3,4 , Jie Ma3 , Gary S. Collins3,4 , Ram Bajpai5 , Richard D. Riley5 , Karel G. M. Moons1,2 and Lotty Hooft1,2 Constanza L. Andaur Navarro1,2* , Johanna A. A. Damen1,2 , Toshihiko Takada1 , Steven W. J. Nijman1 , Paula Dhiman3,4 , Jie Ma3 , Gary S. Collins3,4 , Ram Bajpai5 , Richard D. Riley5 , Karel G. M. Moons1,2 and Lotty Hooft1,2 Abstract Background: While many studies have consistently found incomplete reporting of regression-based prediction model studies, evidence is lacking for machine learning-based prediction model studies. We aim to systematically review the adherence of Machine Learning (ML)-based prediction model studies to the Transparent Reporting of a multivariable prediction model for Individual Prognosis Or Diagnosis (TRIPOD) Statement. Methods: We included articles reporting on development or external validation of a multivariable prediction model (either diagnostic or prognostic) developed using supervised ML for individualized predictions across all medical fields. We searched PubMed from 1 January 2018 to 31 December 2019. Data extraction was performed using the 22-item checklist for reporting of prediction model studies (www.TRIPOD-statement.org). We measured the overall adherence per article and per TRIPOD item. Results: Our search identified 24,814 articles, of which 152 articles were included: 94 (61.8%) prognostic and 58 (38.2%) diagnostic prediction model studies. Overall, articles adhered to a median of 38.7% (IQR 31.0–46.4%) of TRIPOD items. No article fully adhered to complete reporting of the abstract and very few reported the flow of participants (3.9%, 95% CI 1.8 to 8.3), appropriate title (4.6%, 95% CI 2.2 to 9.2), blinding of predictors (4.6%, 95% CI 2.2 to 9.2), model specification (5.2%, 95% CI 2.4 to 10.8), and model’s predictive performance (5.9%, 95% CI 3.1 to 10.9). There was often complete reporting of source of data (98.0%, 95% CI 94.4 to 99.3) and interpretation of the results (94.7%, 95% CI 90.0 to 97.3). Conclusion: Similar to prediction model studies developed using conventional regression-based techniques, the completeness of reporting is poor. Essential information to decide to use the model (i.e. model specification and its performance) is rarely reported. However, some items and sub-items of TRIPOD might be less suitable for ML-based prediction model studies and thus, TRIPOD requires extensions. Overall, there is an urgent need to improve the reporting quality and usability of research to avoid research waste. Systematic review registration: PROSPERO, CRD42019161764. Correspondence: c.l.andaurnavarro@umcutrecht.nl 1 Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Full list of author information is available at the end of the article Correspondence: c.l.andaurnavarro@umcutrecht.nl 1 Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Full list of author information is available at the end of the article Open Access Open Access Take down policy hil h i i Take down policy While the University of Birmingham exercises care and attention in making items available there are rare occasions when an item has been uploaded in error or has been deemed to be commercially or otherwise sensitive. Take down policy While the University of Birmingham exercises care and attention in making items available there are rare occasions wh uploaded in error or has been deemed to be commercially or otherwise sensitive. If you believe that this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we will remove access to the work immediately and investigate. this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we will remove access ely and investigate. If you believe that this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we the work immediately and investigate. Download date: 24. Oct. 2024 Andaur Navarro et al. BMC Medical Research Methodology (2022) 22:12 https://doi.org/10.1186/s12874-021-01469-6 Andaur Navarro et al. BMC Medical Research Methodology (2022) 22:12 https://doi.org/10.1186/s12874-021-01469-6 Correspondence: c.l.andaurnavarro@umcutrecht.nl 1 Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Full list of author information is available at the end of the article Methods Our systematic review protocol was registered (PROS- PERO, CRD42019161764) and published [19]. We reported this systematic review following the PRISMA statement [20]. Background prognostic prediction model studies in recent literature using the TRIPOD Statement [15, 16]. Our results will highlight specific reporting areas that can inform report- ing guidelines for ML, such as TRIPOD-AI [17, 18]. Clinical prediction models are used extensively in health- care to aid patient diagnosis and prognosis of disease and health status. A diagnostic model combines multi- ple predictors or test results to predict the presence or absence of a certain disorder, whereas a prognostic model estimates the probability of future occurrence of an out- come [1–3]. Studies developing, validating, and updating prediction models are abundant in most clinical fields and their number will continue to increase as prediction models developed using artificial intelligence (AI) and machine learning (ML) are receiving substantial interest in the healthcare community [4]. Data source and search We searched PubMed on 19 December 2019 to identify primary articles describing prediction models (diagnostic or prognostic) using any supervised ML technique across all clinical domains published between 1 January 2018 and 31 December 2019. The search strategy is provided in the supplemental material. ML, a subset of AI, offers a class of models that can iteratively learn from data, identify complex data pat- terns, automate model building, and predict outcomes based on what has been learned using computer-based algorithms [5, 6]. ML is often described as more efficient and accurate than conventional regression-based tech- niques. ML-based prediction models, correctly devel- oped, validated, and implemented, can improve patient benefit and reduce disease and health system burden. There is increasing concern of the methodological and reporting quality of studies developing prediction mod- els, with research till date focusing on models developed with conventional statistical techniques such as logistic and Cox regression [7–11]. Recent studies have found limited application of ML-based prediction models because of poor study design and reporting [12, 13]. © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 13 Andaur Navarro et al. BMC Medical Research Methodology (2022) 22:12 Keywords: Prediction model, Diagnosis, Prognosis, Development, Validation, Reporting adherence, Reporting guideline, TRIPOD Study selection We included articles that described the development or validation of one or more multivariable prediction mod- els using any supervised ML technique aiming for indi- vidualized prediction of risk or outcomes. As there is still no consensus on a definition of ML, we defined a ‘study using ML’ as a study that describes the use of a non-gen- eralized linear models to develop or validate a prediction model (e.g. tree-based models, ensembles, deep learn- ing). Extensions to traditional statistical techniques such as generalized additive models and multivariable adap- tive regression splines were considered as non-machine learning for this study. Hence, studies that claimed to have used ML, but they reported only regression-based statistical techniques were excluded from this systematic review (e.g. logistic regression, lasso regression, ridge regression and elastic net). Specifically, we focused on supervised ML, a subdomain of ML, that is character- ized by the development of an algorithm that can predict (the risk of) outcomes for new observations (individuals) after learning from existing individuals and their labelled outcomes. For example, random forests, support vec- tor machine, neural network, naïve bayes, and gradient boosting machines. p y g p g Incomplete (or unclear) reporting makes ML-based prediction models difficult to interpret and impedes vali- dation by independent researchers, thus creating barriers to their use in daily clinical practice. Complete and accu- rate reporting of ML-based prediction model studies will improve its interpretability, reproducibility, risk of bias assessment, and applicability in daily medical practice and is, therefore, essential for high-quality research [14]. To improve transparency and reporting of prediction model studies, the Transparent Reporting of a multivari- able prediction model for Individual Prognosis Or Diag- nosis (TRIPOD) Statement, a checklist of 22 items, was designed (www.tripod-statement.org) [15, 16]. Specific guidance for ML-based prediction model studies is cur- rently lacking and has initiated the extension of TRIPOD for prediction models developed using ML or AI (TRI- POD-AI) [17, 18]. Articles reporting on the incremental value or model extension were also included. We included all articles regardless of study design, data source, or patient-related health outcome. Articles that investigated a single predic- tor, test or biomarker, or its causality with an outcome We conducted a systematic review to assess the com- pleteness of reporting of ML-based diagnostic and Page 3 of 13 Andaur Navarro et al. Results We identified 24,814 unique articles, of which we sampled ten random sets of 249 articles each with sampling replacement for screening. We screened title and abstracts of 2482 articles, assessed the full-text of 312 articles to finally included 152 eligible articles (Fig. 1). We performed a double data extraction for included articles. Two reviewers independently extracted data from each article using the standardized form which was available in REDCap, a data capture tool [23]. To accom- plish consistent data extraction, the form was piloted by all reviewers on five articles. One researcher (CLAN) extracted data from all articles and six researchers (TT, SWJN, PD, JM, RB, JAAD) collectively extracted data from the same articles. Discrepancies in data extrac- tion were discussed and resolved between each pair of reviewers. We included 94 (61.8%) prognostic and 58 (38.2%) diagnostic prediction model studies. 132 (86.8%) arti- cles described development with internal validation and 19 (12.5%) development with external validation (same model). One (0.6%) article was development with exter- nal validation (different model) and was included as a development with internal validation study in the pre- sent analysis. Prediction models were developed most often in oncology (21/152 [13.8%]). Detailed descrip- tion of the included studies is provided in supplemental material. Data extractionh The data extraction form was based on the TRIPOD adherence assessment form (www.tripod-statement.org) [22]. This form contains several adherence statements (hereafter called sub-items) per TRIPOD item. Some items and sub-items are applicable to all types of stud- ies, while others are only applicable to model develop- ment only or external validation only (Table 1). To judge reporting of the requested information, sub-items were formulated to be answered with ‘yes’, ‘no’, ‘not applicable’. We amended the published adherence form by omitting the ‘referenced’ option because we checked the informa- tion in the references, supplemental material, or appen- dix. Sub-items 10b and 16 were extracted per model rather than at study-level, as they refer to model perfor- mance. We limited our extraction and assessment to the first model reported in the Methods section so we could achieve a consistent evaluation of the items related to the Result section as well (item 13–17). Analyses were performed using R version 3.6.2 (R Core Team, Vienna, Austria). Results were summarized as per- centages with confidence intervals calculated using the Wilson score interval. In addition, we also used medians, IQR ranges, and visual plots. Study selection BMC Medical Research Methodology (2022) 22:12 development with external validation (same model), development with external validation (different model), and external validation only. Detailed definition of research aims can be found in the supplemental mate- rial. When articles described the development and/or validation of more than one prediction model, we chose the first ML model reported in the methods section for analysis. were excluded. Articles using ML to enhance reading of images or signals, or articles where ML models only used genetic traits or molecular markers as predictors, were also excluded. We also excluded systematic reviews, con- ference abstracts, tutorials, and articles for which full- text was unavailable via our institution. We restricted the search to human subjects and English-language articles. Further details are stated in our protocol [19]. We scored each TRIPOD item as ‘reported’ and ‘not reported’ based on answers to corresponding sub-items. If the answer to all sub-items of a TRIPOD item was scored ‘yes’ or ‘not applicable’, the corresponding item was considered ‘reported’. Two analyses were conducted: adherence per item and overall adherence per article. We calculated the adherence per TRIPOD item by dividing the number of studies that adhered to a specific item by the number of studies in which the item was applicable. The total number of TRIPOD items varies by the type of prediction model study (Table 1). We calculated the over- all adherence to TRIPOD per article by dividing the sum of reported TRIPOD items by the total number of appli- cable TRIPOD items for each study. If an item was ‘not applicable’ for a particular study, it was excluded when calculating the overall adherence, both in the numerator and denominator [22]. Two researchers, from a group of seven (CLAN, TT, SWJN, PD, JM, RB, JAAD), independently screened titles and abstracts to identify potentially eligible studies. Full- text articles were then retrieved, and two independent researchers reviewed them for eligibility using Rayyan [21]. One researcher (CLAN) screened all articles and six researchers (TT, SWJN, PD, JM, RB, JAAD) collec- tively screened the same articles. Disagreements between reviewers were resolved by a third researcher (JAAD). Data synthesis and analysis Across the 152 studies, 1429 models were developed and 219 were validated, with a range of 1 to 156 for both types of studies. The most commonly used ML tech- niques for the first reported model were Classification We categorized prediction model studies as progno- sis or diagnosis. We also classified studies by research aim: development (with or without internal validation), Andaur Navarro et al. BMC Medical Research Methodology (2022) 22:12 Page 4 of 13 Andaur Navarro et al. BMC Medical Research Methodology (2022) 22:12 Table 1 TRIPOD adherence reporting items Reporting Items Study design If applicable to studies Reporting items for TRIPOD adherence Development only Development and validation 1. Title D, V ✓ ✓ 2. Abstract D, V ✓ ✓ Introduction 3. Background and objectives a. Context and rationale D, V ✓ ✓ b. Objectives D, V ✓ ✓ Methods 4. Source of data a. Source of data D, V ✓ ✓ b. Key dates D, V ✓ ✓ 5. Participants a. Study setting D, V ✓ ✓ b. Eligibility criteria D, V ✓ ✓ c. Details of treatment D, V ✓ ✓ ✓ 6. Outcome a. Outcome definition D, V ✓ ✓ b. Blinding of outcome assessment D,V ✓ ✓ 7. Predictors a. Predictors definition D, V ✓ ✓ b. Blinding of predictor assessment D, V ✓ ✓ 8. Sample size Arrival at study size D,V ✓ ✓ 9. Missing Data Handling of missing data D, V ✓ ✓ 10. Statistical analysis a. Handling of predictors in the analysis D ✓ ✓ b. Specification of the model, all model building procedures, and internal valida- tion methods D ✓ ✓ c. For validation, description of how predictions were calculated V ✓ n.a. d. Specification of all measures used to assess model performance D, V ✓ ✓ e. Description of model updating V ✓ ✓ n.a. 11. Risk groups Details of how risk groups were created D, V ✓ ✓ ✓ 12. Development vs. validation For validation, description of differences between development and validation data V ✓ ✓ Results 13. Participants a. Flow of participants through the study D, V ✓ ✓ b. Description of characteristics of participants D, V ✓ ✓ c. For validation, comparison with development data V ✓ ✓ 14. Model development a. Number of participants and outcome in each analysis D ✓ ✓ b. 10. Statistical analysis a. Handling of predictors in the analysis Details of how risk groups were created Data synthesis and analysis Unadjusted association between each candidate predictor and outcome D ✓ ✓ ✓ 15. Model specification a. Presentation of full prediction model D ✓ ✓ ✓ b. Explanation of how to use the prediction model D ✓ ✓ Table 1 TRIPOD adherence reporting items Overall adherence per TRIPOD item Overall adherence per TRIPOD item Five TRIPOD items reached at least 75% adherence (background, objectives, source of data, limitations, and interpretation), whilst 12 TRIPOD items were below 25% adherence (Fig. 2). Results for the overall adherence per TRIPOD item stratified by study type, diagnosis and prognosis, and publication year are shown in Table 2. Introduction (item 3) and Regression Tree (CART [10.1%]), Support Vector Machine (SVM [9.4%]) and Random Forest (RF [9.4%]). Alongside ML techniques, 19.5% of studies reported also the development of a model using conventional sta- tistical techniques, such as logistic regression. Five out of 152 studies (3.3, 95% CI 1.4 to 7.5) stated following the recommendations of the TRIPOD Statement. Background and objectives were often reported TRIPOD items. Out of 152 studies, Background was provided in 123 studies (80.9, 95% CI 73.9 to 86.4), and the objectives were reported in 124 studies (81.6%, CI 74.6 to 86.9). 13. Participants Andaur Navarro et al. BMC Medical Research Methodology (2022) 22:12 Page 5 of 13 Table 1 (continued) Reporting Items Study design If applicable to studies Reporting items for TRIPOD adherence Development only Development and validation 16. Model performance Report of model performance measures D,V ✓ ✓ 17. Model updating Results from any model updating V ✓ ✓ n.a. Discussion 18. Limitations Limitations D, V ✓ ✓ 19. Interpretation a. For validation, interpretation of performance measure results V ✓ b. Overall interpretation of results D, V ✓ ✓ 20. Implications Potential clinical use of the model and implications for future research D, V ✓ ✓ Other information ✓ ✓ 21. Supplementary information Availability of supplementary resources D, V ✓ ✓ 22. Funding Source of funding and role of funders D, V ✓ ✓ Total number of applicable items for TRIPOD adherence score 31 37 (n.a) No included studies reported external validation only or model updating (Item 10c, 10e, and 17) Methods (item 4–12) Source of data was the most often reported item in the methods section, and across all TRIPOD items (98.0, 95% CI 94.4 to 99.3). Study setting was reported in 107/152 studies (70.4%, CI 62.7 to 77.1), eligibility criteria in 105/152 (69.1%, CI 61.3 to 75.9), and handling of pre- dictors in 105/ 152 studies (69.1%, CI 61.3 to 75.9). Ten studies assessed risk groups and five reported complete information (50.0%, CI 23.7 to 76.3). Differences between development and validation set were reported in 10 out of 19 studies were this item was applicable (52.6%, CI 31.7 to 72.7). For 72 studies, definition of outcome was reported (47.4%, CI 39.6% to 55.3). Key study dates such as start and end date of accrual, and length of follow-up were completely reported in 56 studies (36.8%, CI 29.6 to 44.7). Details of treatment were reported in 36 out of 116 studies were this item was applicable (31.0%, CI 23.3 to 39.9). Blinding of outcome and predictors were reported in 49/152 (32.2%, CI 25.3 to 40.0) and 7/152 studies (4.6%, CI 2.2 to 9.2), respectively. Title and abstract (item 1 and 2) Seven out of 152 studies (4.6, 95% CI 2.2 to 9.2) com- pletely adhered to title recommendations. Description of type of prediction model study (sub-item 1.i) was poorly reported (11.2%, CI 7.0 to 17.2), but outcome to be pre- dicted (sub-item 1.iv) was well reported (91.4%, CI 85.9 to 94.9). No study fully reported item 2, abstract (0%, CI 0 to 2.5). Andaur Navarro et al. BMC Medical Research Methodology (2022) 22:12 Page 6 of 13 Forty-four studies reported how missing data were handled (28.9%, 95% CI 22.3 to 36.6). The missing data item consists of four sub-items of which three were rarely addressed in included studies. Within 28 stud- ies that reported handling of missing data: three studies reported the software used (10.7%, CI 3.7 to 27.2), four Fig. 1 Flowchart of included studies Fig. 1 Flowchart of included studies rarely addressed in included studies. Within 28 stud- ies that reported handling of missing data: three studies reported the software used (10.7%, CI 3.7 to 27.2), four rarely addressed in included studies. Within 28 stud- ies that reported handling of missing data: three studies reported the software used (10.7%, CI 3.7 to 27.2), four Forty-four studies reported how missing data were handled (28.9%, 95% CI 22.3 to 36.6). The missing data item consists of four sub-items of which three were Forty-four studies reported how missing data were handled (28.9%, 95% CI 22.3 to 36.6). The missing data item consists of four sub-items of which three were Andaur Navarro et al. BMC Medical Research Methodology (2022) 22:12 Page 7 of 13 Funding (22) Supplemental Information (21) Implications (20) Interpretation (19b) Interpretation val (19a) Limitations (18) Model performance (16) Presentation (15b) Model specification* (15a) Unadjusted association* (14b) Model development (14a) Distribution* (13c) Demographics (13b) Flow of participants (13a) Differences* (12) Risk groups* (11) Performance measures (10d) Model building (10b) Handling of predictors (10a) Missing data (9) Study size (8) Blinding of predictors (7b) Predictors (7a) Blinding of outcome (6b) Outcome (6a) Details of treatment* (5c) Eligibitliy criteria (5b) Study setting (5a) Key dates (4b) Source of data (4a) Objectives (3b) Background (3a) Abstract (2) Title (1) 0 25 50 75 100 Adherence (%) Section Title Abstract Background Methods Results Discussion Other Fig. 2 Overall adherence per TRIPOD item. Discussion Characteristics of study participants were reported in 38/ 152 studies (25.0, 95% CI 18.8 to 32.4). Basic demo- graphics, at least age and gender (sub-item 13b.i), were provided in 117/152 studies (77.0%, CI 69.7 to 83.0), while summary information of the predictors (sub-item 13b.ii) was reported in 67/152 studies (44.1%, CI 36.4 to 52.0). Number of study participants with missing data for predictors (sub-item 13b.iii) was reported in 15 stud- ies (24.2%, CI 15.2 to 36.2). Unadjusted associations were reported in 41 out of the 74 studies that reported regres- sion-based models alongside with ML-models (41.9%, CI 31.3 to 53.3). The number of participants and events were described in 37 studies (24.3%, CI 18.2 to 31.7). In 31/ 152 studies, an explanation on how to use the devel- oped model to make predictions for new individuals was provided, often in the form of a scoring rule or online cal- culator (20.4%, CI 14.8 to 27.5). Flow of participants was reported in 6/152 studies (3.9%, CI 1.8 to 8.3) and model specification was reported in 6 out of 116 studies were this item was applicable (5.2%, CI 2.4 to 10.8). Model pre- dictive performance was completely reported in 9/ 152 studies (5.9%, CI 3.1 to 10.9). We conducted a systematic review of ML-based diagnos- tic and prognostic prediction model studies and assessed their adherence to the TRIPOD Statement. We found that ML-based prediction model studies adhere poorly to the reporting items of the TRIPOD Statement. Complete reporting in titles and abstracts is crucial to identify and screen articles. However, titles and abstracts were fully reported in less than 5% of articles. In addition, information about methods was infrequently reported. Complete and accurate reporting of the methods used to develop or validate a prediction model facilitates exter- nal validation, as well as replication of study results by independent researchers. For example, to enhance trans- parency and risk of bias assessment, it is recommended to report the number of participants with missing data and report how missing data were handled in the analy- sis. Handling of missing data was seldom reported, but this may be partially explained by the fact that some ML techniques can handle missing data by design (e.g. spar- sity aware splitting in XGBoost and surrogate splits in decision trees) [24, 25]. Other information (items 21 and 22) Availability of supplementary resources was mentioned in 93/152 studies (61.2, 95% CI 53.3 to 68.6). Funding information was reported in 42 studies (27.6%, CI 21.1 to 35.2). Title and abstract (item 1 and 2) Overall sample n=152 Reporting of measures used to assess and quantify the predictive performance was complete in 19 stud- ies (12.5, 95% CI 8.2 to 18.7). Though 106/152 studies (69.7%, CI 62.0 to 76.5) reported discrimination (sub- item 10d.i), only 19/152 studies (12.5%, CI 8.2 to 18.7) reported calibration (sub-item 10d.ii). Definitions of discrimination and calibration are stated in supplemen- tal material. Other performance measures (sub-item 10d.iii) such as sensitivity, specificity, or predictive val- ues, were reported in 124/152 studies (81.6%, CI 74.7 to 86.9). studies reported the variables included in the proce- dure (14.3%, CI 5.7 to 31.5) and no study reported the number of imputations (0%, CI 0.0 to 39.0). Predictor definitions were given in 32/ 152 studies (21.1%, CI 15.3 to 28.2), and justification of study size was reported in 27/152 studies (17.8%, CI 12.5 to 24.6). Model build- ing procedures, such as predictor selection and internal validation, were reported in 22/ 152 studies (14.5%, CI 9.8 to 20.9). Internal validation, a sub-item of item 10b, was one of the most reported sub-items across studies (91.4%, CI 85.9 to 94.9). studies reported the variables included in the proce- dure (14.3%, CI 5.7 to 31.5) and no study reported the number of imputations (0%, CI 0.0 to 39.0). Predictor definitions were given in 32/ 152 studies (21.1%, CI 15.3 to 28.2), and justification of study size was reported in 27/152 studies (17.8%, CI 12.5 to 24.6). Model build- ing procedures, such as predictor selection and internal validation, were reported in 22/ 152 studies (14.5%, CI 9.8 to 20.9). Internal validation, a sub-item of item 10b, was one of the most reported sub-items across studies (91.4%, CI 85.9 to 94.9). Andaur Navarro et al. BMC Medical Research Methodology (2022) 22:12 Page 8 of 13 Table 2 Adherence to TRIPOD items Table 2 Adherence to TRIPOD items Page 9 of 13 Page 9 of 13 Andaur Navarro et al. BMC Medical Research Methodology (2022) 22:12 Discussion (items 18–20) Overall interpretation of results was reported in 124/152 studies (81.6, 95% CI 74.7 to 86.9). Limitations of the study were reported in 144 /152 studies (94.7, 95% CI 90.0 to 97.3). An interpretation of model performance in the validation set in comparison with the develop- ment set was given in 14/19 studies (73.7%, CI 51.2 to 88.2). Potential clinical use and implications for future research was reported in 61/152 studies (40.1%, CI 32.7 to 48.1). Overall, most articles adhered to less than half of the applicable items considered essential for complete reporting. Authors may have avoided reporting specific details about methods and results because their objec- tive may be to explore the data and modeling technique accuracy, rather than build models for individualized predictions in “real world” clinical settings. However, high-quality reporting is also essential for reproduc- ibility and replication. Furthermore, most developed models were unavailable for replication, assessment, or clinical application. Only five studies referred to the TRIPOD Statement for reporting their research. Although TRIPOD was published and disseminated in 2015, it is infrequently used for reporting of ML-based prediction model studies. Discussion Also most studies divided a sin- gle dataset into three: training, validation and test set; the last is used for internal validation. The split sample approach for internal validation was among the most reported sub-items in our sample, but several methodo- logical studies and guidelines have long discouraged this approach [26]. Overall adherence per article Overall adherence of studies to items of the TRIPOD Statement ranged between 13.0 and 65.0% (median adherence =38.7% (IQR 31.0 to 46.5%)). The complete- ness reporting in prognostic model studies was higher (median adherence = 40.0% (IQR 33.3 to 46.8%)) than diagnostic model studies (median adherence = 35.7% (IQR 30.2 to 45.0%)) (Fig. 3). Moreover, median adher- ence was 40.6% (IQR 28.6 to 46.1%) in development (with internal validation) studies, compared to 37.9% (IQR 31.0 to 46.4%) in development with external validation studies. We stratified studies by type (diagnosis vs prognosis), aim (development vs development with external valida- tion), and year (2018 vs 2019). We included diagnostic model studies developed with deep learning if they used images in combination with demographic and clini- cal variables. Often, these studies use several numerical variables based on pixels or voxels and build prediction models based on multiple layers of statistical interaction. Both topics are challenging to report due to number of variables used and poor interpretability of interactions. Andaur Navarro et al. BMC Medical Research Methodology (2022) 22:12 Page 10 of 13 Fig. 3 Overall adherence per article This may explain why diagnostic ML-based model stud- ies were slightly worse reported compared to prognostic studies in our sample. However, we did not observe clear differences across stratified groups as most confidence intervals overlapped. This may explain why diagnostic ML-based model stud- ies were slightly worse reported compared to prognostic studies in our sample. However, we did not observe clear differences across stratified groups as most confidence intervals overlapped. the completeness of reporting of deep learning-based diagnostic model studies. Although they developed their own data extraction for reporting quality, authors found poor reporting of demographics, distribution of disease severity, patient flow, and distribution of alternative diagnosis [27]. These items were also inap- propriately reported in our study with a median adher- ence between 0 and 47.3%. Another systematic review that assessed studies comparing the performance of diagnostic deep learning algorithms for medical imag- ing versus expert clinicians reported the overall adher- ence to TRIPOD was poor with a median of 62% (IQR 45 to 69%) [28]. In line with our results, a study about the performance of ML models showed that 68% of included articles had unclear reporting [12]. Previous systematic reviews have shown poor report- ing of regression-based prediction model studies [7, 8, 10, 11]. Overall adherence per article One study assessed the completeness of report- ing in articles published in high impact journals during 2014 within 37 different clinical fields. In 146 predic- tion model studies, over half of TRIPOD items were not fully reported, obtaining an overall adherence of 44% (IQR 35 to 52%). Although authors excluded mod- els using machine learning, the review found poor reporting of the title, abstract, model building, model specification and model performance, similar to our study [7]. In a sample of prediction model studies published in general medicine journals with the top 7 highest impact factor, the overall reporting adherence was 74% before, and 76% after the implementation of the TRIPOD Statement. Authors included only predic- tion models developed with regression techniques but also found poor reporting of model building, specifi- cation, and performance [11]. A recent study assessed To our knowledge, this is the first systematic review evaluating the completeness of reporting of supervised ML-based prediction model studies in a broad sample of articles. We ran a validated search strategy and per- formed paired screening. We also used a contemporary sample of studies in our review (2018–2019). Though some eligible articles may have been missed, it is unlikely they would change the conclusions of this review. We used a systematic scoring-system enhancing the Page 11 of 13 Andaur Navarro et al. BMC Medical Research Methodology (2022) 22:12 Page 11 of 13 the completeness of reporting of ML-based studies might be even more challenging given the number of techniques and associated details that need to be reported. There are also practical issues, like terminology used, word limits, or journal requirements, that are acting as barriers to com- plete reporting. To overcome these barriers, the use of online repositories for data, script, and complete pipeline could help researchers share their models with enough details to make predictions in new patients and to allow external validation of the model. Further journal endorse- ment, training, and tailored guidelines might be required to improve the completeness of reporting. Our results will provide input and support for the development of TRI- POD-AI, an initiative launched in 2019 [17, 18]. We call for a collaborative effort between algorithm developers, researchers, and journal editors to improve the adoption of good scientific practices related to reporting quality. objectivity and consistency for the evaluation of adher- ence to a reporting guideline [22]. Acknowledgements Th h ld lik The authors would like to thank and acknowledge the support of René Spijker, information specialist. Conclusion ML-based prediction model studies currently do not adhere well to the TRIPOD reporting guideline. More than half of the TRIPOD items considered essential for transparent reporting were inadequately reported, espe- cially regarding details of title, abstract, blinding, model building procedures, model specifications and model per- formance. Whilst ML brings new challenges to the devel- opment of tailored reporting guidelines, our study serves as a baseline measure to define future updates or exten- sions of TRIPOD tailored to ML modelling strategies. Overall adherence per article We used the formal TRIPOD adherence form and checklist for data extrac- tion and assessment; however, these were developed for studies developing prediction models with regression techniques. Although we applied the option ‘not applica- ble’ for items that were unrelated to ML and items were excluded when calculating overall adherence, our results should be interpreted within this context. While some items and sub-items may be less relevant for prediction models developed with ML techniques, other items are more relevant for transparent reporting in these studies. For example, source of data (4a), study size (8), missing data (9), transformation of predictors (10a.i), internal validation (10b.iv), and availability of the model (15b) acquire new relevance within the context of ML-based prediction model studies. As ML techniques are prone to overfitting, we recommend extending item 10b of the TRIPOD adherence form to include a new sub-item specifically related to penalization or shrink- age techniques. New reporting items such as the hard- ware (i.e. technical aspects) that was used to develop or validate an algorithm in images studies are needed, as well as data clustering. New practices such as explain- ing models through feature importance plot or tuning of hyper-parameters could be also added to the exten- sion of TRIPOD for ML-based prediction models. Items such as testing of interaction terms (Item 10b-iv), unad- justed associations (14b), and regression coefficients (15a) require updating. Despite these recommendations, most TRIPOD items and sub-items are still applicable for both, regression and ML techniques and should be used to improve reporting quality. Authors’ contributions The study concept and design were conceived by CLAN, JAAD, PD, LH, RDR, GSC, and KGMM. CLAN, JAAD, TT, SN, PD, JM and RB conducted article screening and data extraction. CLAN performed data analysis and JAAD verified the under- lying data. CLAN wrote the first draft of this manuscript, which was critically revised for important intellectual content by all authors who have provided the final approval of this version. CLAN, the corresponding author, is the guaran- tor of the review. The corresponding author attests that all listed authors meet authorship criteria and that no others meeting the criteria have been omitted. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12874-021-01469-6. We identified nearly 25,000 articles with prediction and ML-related terms within 2 years, similar to previous systematic reviews about deep learning models [29, 30]. The literature has become saturated with ML-based stud- ies; thus, their identification, reporting and assessment becomes even more relevant. If studies are presented with- out essential details to make predictions in new patients, subsequent researchers will develop a new model, rather than validating or updating an existing model. Reporting guidelines aim to increase the transparent evaluation, rep- lication, and translation of research into clinical practice [31]. Some reporting guidelines for ML clinical prediction models have already been developed [32, 33]. However, these guidelines are limited and do not follow the EQUA- TOR recommendations for developing consensus-based reporting guidelines [34]. The improvement in reporting after the introduction of a guideline has shown to be slow [31]. We acknowledge that the machine learning com- munity developing predictive algorithm for healthcare might be unaware of the TRIPOD Statement. Improving Availability of data and materials learning over logistic regression for clinical prediction models. J Clin Epidemiol. 2019;110:12–22. https://doi.org/10.1016/j.jclinepi.2019.02.004 y The study protocol is available at doi: https://doi.org/10.1136/bmjopen-2020- 038832 . The search strategy is available in supplemental material; detailed extracted data are available upon reasonable request to the corresponding author. 13. Gravesteijn BY, Nieboer D, Ercole A, et al. Machine learning algorithms performed no better than regression models for prognostication in traumatic brain injury. J Clin Epidemiol. 2020;122:95–107. https://doi.org/ 10.1016/j.jclinepi.2020.03.005. 14. Glasziou P, Altman DG, Bossuyt P, et al. Reducing waste from incomplete or unusable reports of biomedical research. Lancet. 2014;383(9913):267– 76. https://doi.org/10.1016/S0140-6736(13)62228-X. Funding This study did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. GSC is supported by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre (BRC) and by Cancer Research UK program grant (C49297/A27294). PD is supported by the NIHR Oxford BRC. The views expressed are those of the authors and not necessarily those of the NHS, NIHR, or Department of Health. Page 12 of 13 Andaur Navarro et al. BMC Medical Research Methodology (2022) 22:12 Received: 22 July 2021 Accepted: 15 November 2021 20. Moher D, Liberati A, Tetzlaff J, et al. Preferred reporting items for sys- tematic reviews and meta-analyses: The PRISMA statement. Plos Med. 2009;6(7). https://doi.org/10.1371/journal.pmed.1000097. 21. Ouzzani M, Hammady H, Fedorowicz Z, Elmagarmid A. Rayyan-a web and mobile app for systematic reviews. Syst Rev. 2016;5(1):210. https://doi. org/10.1186/s13643-016-0384-4. Consent for publication Not applicable. 16. Collins GS, Reitsma JB, Altman DG, Moons KGM. Transparent reporting of a multivariable prediction model for individual prognosis or diagnosis (TRIPOD): the TRIPOD statement. Ann Intern Med. 2015;162(1):55. https:// doi.org/10.7326/M14-0697.i References 1. Moons KGM, Royston P, Vergouwe Y, Grobbee DE, Altman DG. Prognosis and prognostic research: what, why, and how? BMJ. 2009;338(7706):1317–20. https://doi.org/10.1136/bmj.b375. 1. Moons KGM, Royston P, Vergouwe Y, Grobbee DE, Altman DG. Prognosis and prognostic research: what, why, and how? BMJ. 2009;338(7706):1317–20. https://doi.org/10.1136/bmj.b375. 22. Heus P, Damen JAAG, Pajouheshnia R, et al. Uniformity in measuring adherence to reporting guidelines: The example of TRIPOD for assessing completeness of reporting of prediction model studies. BMJ Open. 2019;9(4). https://doi.org/10.1136/bmjopen-2018-025611. 2. Steyerberg EW, Moons KGM, van der Windt DA, et al. Prognosis Research Strategy (PROGRESS) 3: Prognostic Model Research. Plos Med. 2013;10(2). https://doi.org/10.1371/journal.pmed.1001381. 2019;9(4). https://doi.org/10.1136/bmjopen-2018-025611 23. Harris PA, Taylor R, Minor BL, et al. The REDCap consortium: Building an international community of software platform partners. J Biomed Inform. 2019;95:103208. https://doi.org/10.1016/j.jbi.2019.103208. 3. Riley RD, van der Windt D, Croft P, Moons KG, editors. Prognosis Research in Health Care: Concepts, Methods, and Impact: Oxford University Press; 2019. https://doi.org/10.1093/med/9780198796619. 001.0001. 24. Chen T, Guestrin C. XGBoost: a scalable tree boosting system. In: Pro- ceedings of the ACM SIGKDD International Conference on Knowledge Discovery and Data Mining. Vol 13–17-August-2016: Association for Computing Machinery; 2016. p. 785–94. https://doi.org/10.1145/29396 72.2939785. 4. Damen JAAG, Hooft L, Schuit E, et al. Prediction models for cardiovascular disease risk in the general population: systematic review. BMJ. 2016;353. https://doi.org/10.1136/bmj.i2416. 4. Damen JAAG, Hooft L, Schuit E, et al. Prediction models for cardiovascular disease risk in the general population: systematic review. BMJ. 2016;353. https://doi.org/10.1136/bmj.i2416. 5. Bi Q, Goodman KE, Kaminsky J, Lessler J. What is machine learning? A primer for the epidemiologist. Am J Epidemiol. 2019;188(12):2222–39. https://doi.org/10.1093/aje/kwz189. 5. Bi Q, Goodman KE, Kaminsky J, Lessler J. What is machine learning? A primer for the epidemiologist. Am J Epidemiol. 2019;188(12):2222–39. https://doi.org/10.1093/aje/kwz189. 25. Therneau TM, Atkinson EJ. An introduction to recursive partitioning using the RPART routines; 1997. 26. Austin PC, Steyerberg EW. Events per variable (EPV) and the relative per- formance of different strategies for estimating the out-of-sample validity of logistic regression models. Stat Methods Med Res. 2017;26(2):796–808. https://doi.org/10.1177/0962280214558972. 6. Mitchell T. Machine learning: McGraw Hill; 1997. 6. Mitchell T. Machine learning: McGraw Hill; 1997. 7. Heus P, Damen JAAG, Pajouheshnia R, et al. Poor reporting of multivari- able prediction model studies: towards a targeted implementation strategy of the TRIPOD statement. BMC Med. 2018;16(1):1–12. https://doi. org/10.1186/s12916-018-1099-2. 7. Heus P, Damen JAAG, Pajouheshnia R, et al. Competing interests GSC, RDR and KGMM are members of the TRIPOD Group. All authors have nothing to disclose. 17. Collins GS, Moons KGM. Reporting of artificial intelligence prediction models. Published online; 2019. https://doi.org/10.1016/S01406736(19) 302351. Ethics approval and consent to participate 15. Moons KGM, Altman DG, Reitsma JB, et al. Transparent reporting of a multivariable prediction model for individual prognosis or diagnosis (TRIPOD): explanation and elaboration. Ann Intern Med. 2015;162(1):W1– W73. https://doi.org/10.7326/M14-0698. Consent for publication Not applicable. Author details 1 Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands. 2 Cochrane Netherlands, University Medical Center Utrecht, Utrecht University, Utrecht, The Nether- lands. 3 Center for Statistics in Medicine, NDORMS, University of Oxford, Oxford, UK. 4 NIHR Oxford Biomedical Research Centre, Oxford University Hospitals NHS Foundation Trust, Oxford, UK. 5 Centre for Prognosis Research, School of Medicine, Keele University, Keele, UK. 18. Collins GS, Dhiman P, Andaur Navarro CL, et al. Protocol for development of a reporting guideline (TRIPOD-AI) and risk of bias tool (PROBAST-AI) for diagnostic and prognostic prediction model studies based on artificial intelligence. BMJ Open. 2021;11(7):e048008. https://doi.org/10.1136/ BMJOPEN-2020-048008. 19. Andaur Navarro CL, Damen JAAG, Takada T, et al. Protocol for a systematic review on the methodological and reporting quality of prediction model studies using machine learning techniques. BMJ Open. 2020;10(11):1–6. https://doi.org/10.1136/bmjopen-2020-038832. Received: 22 July 2021 Accepted: 15 November 2021 Received: 22 July 2021 Accepted: 15 November 2021 References Poor reporting of multivari- able prediction model studies: towards a targeted implementation strategy of the TRIPOD statement. BMC Med. 2018;16(1):1–12. https://doi. org/10.1186/s12916-018-1099-2. 27. Yusuf M, Atal I, Li J, et al. Reporting quality of studies using machine learning models for medical diagnosis: a systematic review. BMJ Open. 2020;10(3):e034568. https://doi.org/10.1136/bmjopen-2019-034568.i 8. Bouwmeester W, Zuithoff NPA, Mallett S, et al. Reporting and methods in clinical prediction research: a systematic review. Plos Med. 2012;9(5). https://doi.org/10.1371/journal.pmed.1001221. 28. Nagendran M, Chen Y, Lovejoy CA, et al. Artificial intelligence versus clinicians: systematic review of design, reporting standards, and claims of deep learning studies in medical imaging. BMJ. 2020;368. https://doi.org/ 10.1136/bmj.m689. 9. Collins GS, Mallett S, Omar O, Yu LM. Developing risk prediction models for type 2 diabetes: a systematic review of methodology and reporting. BMC Med. 2011;9. https://doi.org/10.1186/1741-7015-9-103. 29. Faes L, Liu X, Wagner SK, et al. A clinician’s guide to artificial intelligence: how to critically appraise machine learning studies. Transl Vis Sci Technol. 2020;9(2):7–7. https://doi.org/10.1167/tvst.9.2.7. 10. Collins GS, De Groot JA, Dutton S, et al. External validation of multivari- able prediction models: a systematic review of methodological conduct and reporting. BMC Med Res Methodol. 2014;14(1):40. https://doi.org/10. 1186/1471-2288-14-40. 30. Liu X, Faes L, Kale AU, et al. A comparison of deep learning performance against health-care professionals in detecting diseases from medi- cal imaging: a systematic review and meta-analysis. Lancet Digit Heal. 2019;1(6):e271–97. https://doi.org/10.1016/S2589-7500(19)30123-2. 11. Zamanipoor Najafabadi AH, Ramspek CL, Dekker FW, et al. TRIPOD statement: a preliminary pre-post analysis of reporting and methods of prediction models. BMJ Open. 2020;10(9):e041537. https://doi.org/10. 1136/bmjopen-2020-041537. 31. Simera I, Moher D, Hirst A, Hoey J, Schulz KF, Altman DG. Transparent and accurate reporting increases reliability, utility, and impact of your research: reporting guidelines and the EQUATOR network. BMC Med. 2010;8(1):24. https://doi.org/10.1186/1741-7015-8-24. j p 12. Christodoulou E, Ma J, Collins GS, Steyerberg EW, Verbakel JY, Van Calster B. A systematic review shows no performance benefit of machine Page 13 of 13 Andaur Navarro et al. BMC Medical Research Methodology (2022) 22:12 32. Luo W, Phung D, Tran T, et al. Guidelines for developing and reporting machine learning predictive models in biomedical research: a multidisci- plinary view. J Med Internet Res. 2016;18(12). https://doi.org/10.2196/jmir. 5870. 32. Luo W, Phung D, Tran T, et al. Guidelines for developing and reporting machine learning predictive models in biomedical research: a multidisci- plinary view. J Med Internet Res. 2016;18(12). https://doi.org/10.2196/jmir. 5870. 33. References Norgeot B, Quer G, Beaulieu-Jones BK, et al. Minimum information about clinical artificial intelligence modeling: the MI-CLAIM checklist. Nat Med. 2020;26(9):1320–4. https://doi.org/10.1038/s41591-020-1041-y. 34. Moher D, Schulz KF, Simera I, Altman DG. Guidance for developers of health research reporting guidelines. Plos Med. 2010;7(2). https://doi.org/ 10.1371/journal.pmed.1000217. 34. Moher D, Schulz KF, Simera I, Altman DG. Guidance for developers of health research reporting guidelines. Plos Med. 2010;7(2). https://doi.org/ 10.1371/journal.pmed.1000217. Publisher’s Note S i N i Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from:
https://openalex.org/W2560230696	https://europepmc.org/articles/pmc5241722?pdf=render	English	null	Activation of D2 Dopamine Receptors in CD133+ve Cancer Stem Cells in Non-small Cell Lung Carcinoma Inhibits Proliferation, Clonogenic Ability, and Invasiveness of These Cells	Journal of biological chemistry/The Journal of biological chemistry	2,017	cc-by	11,365	Activation of D2 Dopamine Receptors in CD133ve Cancer Stem Cells in Non-small Cell Lung Carcinoma Inhibits Proliferation, Clonogenic Ability, and Invasiveness of These Cells* Soumyabrata Roy‡, Kai Lu§, Mukti Kant Nayak¶, Avishek Bhuniya‡1, Tithi Ghosh‡1, Suman Kundu, Sarbari Ghosh‡, Rathindranath Baral‡, Partha Sarathi Dasgupta‡2, and Sujit Basu§3 From the ‡Department of Immunoregulation and Immunodiagnostics, Chittaranjan National Cancer Institute, Kolkata 700026, India, the §Department of Pathology, Ohio State University, Columbus, Ohio 43210, the ¶Division of Virology, National Institute of Cholera and Enteric Diseases, Kolkata 700010, India, the Cancer Biology and Inflammatory Disorder Division, Indian Institute of Chemical Biology, Kolkata 700032, India, and the Division of Medical Oncology, Department of Internal Medicine, Ohio State University, Columbus, Ohio 43210 From the ‡Department of Immunoregulation and Immunodiagnostics, Chittaranjan National Cancer Institute, Kolkata 700026, India, the §Department of Pathology, Ohio State University, Columbus, Ohio 43210, the ¶Division of Virology, National Institute of Cholera and Enteric Diseases, Kolkata 700010, India, the Cancer Biology and Inflammatory Disorder Division, Indian Institute of Chemical Biology, Kolkata 700032, India, and the *Division of Medical Oncology, Department of Internal Medicine, Ohio State University, Columbus, Ohio 43210 Edited by Xiao-Fan Wang Lung carcinoma is the leading cause of cancer-related death worldwide, and among this cancer, non-small cell lung carci- noma (NSCLC) comprises the majority of cases. Furthermore, recurrence and metastasis of NSCLC correlate well with CD133ve tumor cells, a small population of tumor cells that have been designated as cancer stem cells (CSC). We have dem- onstrated for the first time high expression of D2 dopamine (DA) receptors in CD133ve adenocarcinoma NSCLC cells. Also, activation of D2 DA receptors in these cells significantly inhib- ited their proliferation, clonogenic ability, and invasiveness by suppressing extracellular signal-regulated kinases 1/2 (ERK1/2) and AKT, as well as down-regulation of octamer-binding tran- scription factor 4 (Oct-4) expression and matrix metalloprotei- nase-9 (MMP-9) secretion by these cells. These results are of significance as D2 DA agonists that are already in clinical use for treatment of other diseases may be useful in combination with conventional chemotherapy and radiotherapy for better man- agement of NSCLC patients by targeting both tumor cells and stem cell compartments in the tumor mass. 85% of the cases (1, 2). The prognosis of NSCLC is poor, and the survival rate is only 15% after 5 years because many of these patients ultimately do not respond to chemotherapy and radio- therapy due to the presence of CSC (3–5). The CSC have the unique ability to promote tumor growth, recurrence, metasta- sis, and resistance to treatment (6, 7). This study was supported by Council of Scientific and Industrial Research Grant 21(0895)/12/EMR-II (to P. S. D.) and National Institutes of Health Grants R01DK098045, R01CA169158, and R01HL131405 (to S. B.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. crossmark THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 292, NO. 2, pp. 435–445, January 13, 2017 © 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. crossmark THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 292, NO. 2, pp. 435–445, January 13, 2017 © 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. ssmark THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 292, NO. 2, pp. 435–445, January 13, 2017 © 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 292, NO. 2, pp. 435–445, January 13, 2017 © 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide; PI, propidium iodide. 2 To whom correspondence may be addressed: Chittaranjan National Cancer Institute, West Bengal, Kolkata 700026, India. E-mail: partha42002@ yahoo.com. 1 Both authors contributed equally to this work. y 3 To whom correspondence may be addressed: Depts. of Pathology and Med- ical Oncology, Ohio State University, 166 Hamilton Hall, 1645 Neil Ave., Columbus, OH 43210. E-mail: sujit.basu@osumc.edu. j 4 Theabbreviationsusedare:NSCLC,non-smallcelllungcarcinoma;CSC,can- cer stem cell(s); D2 DA, D2 dopamine (DA) receptors; ERK1/2, extracellular signal-regulated kinases 1/2; MMP-9, matrix metalloproteinase-9; MTT, Activation of D2 Dopamine Receptors in CD133ve Cancer Stem Cells in Non-small Cell Lung Carcinoma Inhibits Proliferation, Clonogenic Ability, and Invasiveness of These Cells* Moreover, CD133, a surface glycoprotein with a five-trans- membrane domain, has been widely used as a surface marker to identify CSC in many human tumors including NSCLC (8–12). Although CD133 is expressed in various human tissues, its gly- cosylated epitopes specific for stem cells may be discordant or sometimes absent, making it difficult to identify this cell popu- lation (13). There may be transcriptional or post-translational modifications (14), leading to some degree of alteration in its expression, or variation in its expression in different tumors may be due to the use of antibodies from different clones (12). However, despite these limitations, recent results from differ- ent laboratories have shown significant association of CD133 (epitope-1) expression in a population of tumor cells with CSC characteristics in the brain, prostate, liver, and lung (15–18). There are now several reports that have further strengthened the concept of CD133ve tumor cells as CSC in NSCLC as these tumor cells possess the characteristics of CSC (18–20). In contrast to the CD133ve tumor cells, CD133ve tumor cells are more tumorigenic and resistant to radiation and anticancer drugs (18–20). These cells also have a greater ability to form anchorage-independent floating spheres, proliferation, and tumor mass than the CD133ve NSCLC cells. The CD133ve cells like CSC demonstrate significantly increased stemness, adhesion, motility, and expression of drug efflux gene (21–23) than CD133ve tumor cells. Importantly, the presence of CD133ve tumor cells correlates well with poor prognosis, decreased survival, and increased lymph node metastasis in NSCLC patients (24–26). Globally, lung cancers are considered to be the leading causes of cancer-related deaths, and non-small cell lung carcinoma (NSCLC)4 is the predominant type of lung cancer, occurring in Furthermore, in addition to CD133, aldehyde dehydrogenase 1 (ALDH1) is also used as a CSC marker in NSCLC (12, 27–30). j 4 Theabbreviationsusedare:NSCLC,non-smallcelllungcarcinoma;CSC,can- cer stem cell(s); D2 DA, D2 dopamine (DA) receptors; ERK1/2, extracellular signal-regulated kinases 1/2; MMP-9, matrix metalloproteinase-9; MTT, 435 JANUARY 13, 2017•VOLUME 292•NUMBER 2 JOURNAL OF BIOLOGICAL CHEMISTRY FIGURE 1. Percentage of cancer cells expressing stem cell marker CD133 and D2 DA receptors in human NSCLC adenocarcinoma cell lines (A549 and NCI-H23). A and B, forward scatter/side scatter (FSC/SSC) plots show the gated population. C, unstained A549 cells as a control. D, percentage of A549 cells expressing D2 DA receptors. E, unstained NCI-H23 cells as a control. F, percentage of NCI-H23 cells expressing D2 DA receptors. Activation of D2 Dopamine Receptors in CD133ve Cancer Stem Cells in Non-small Cell Lung Carcinoma Inhibits Proliferation, Clonogenic Ability, and Invasiveness of These Cells* These ALDH1A1-expressing NSCLC cells are also known for their clone formation ability, prolifera- tion, growth, and migration in vitro (28). Activation of D2 Dopamine Receptors in CD133ve Cancer Stem Cells in Non-small Cell Lung Carcinoma Inhibits Proliferation, Clonogenic Ability, and Invasiveness of These Cells* G, unstained A549 cells as a control. H, percentage of A549 cells expressing CD133. I, unstained NCI-H23 as a control. J, percentage of NCI-H23 cells expressing CD133. K, unstained A549 cellsasacontrol.L,A549cellsgroupedbydoublestainingofCD133andD2DAreceptors.M,unstainedNCI-H23asacontrol.N,NCI-H23cellsgroupedbydouble staining CD133 and D2 DA receptors. Results are representative of 3 independent experiments. D2 Dopamine Receptors Regulate Cancer Stem Cells D2 Dopamine Receptors Regulate Cancer Stem Cells eptors Regulate Cancer Stem Cells FIGURE 1. Percentage of cancer cells expressing stem cell marker CD133 and D2 DA receptors in human NSCLC adenocarcinoma cell lines (A549 and NCI-H23). A and B, forward scatter/side scatter (FSC/SSC) plots show the gated population. C, unstained A549 cells as a control. D, percentage of A549 cells expressing D2 DA receptors. E, unstained NCI-H23 cells as a control. F, percentage of NCI-H23 cells expressing D2 DA receptors. G, unstained A549 cells as a control. H, percentage of A549 cells expressing CD133. I, unstained NCI-H23 as a control. J, percentage of NCI-H23 cells expressing CD133. K, unstained A549 cellsasacontrol.L,A549cellsgroupedbydoublestainingofCD133andD2DAreceptors.M,unstainedNCI-H23asacontrol.N,NCI-H23cellsgroupedbydouble staining CD133 and D2 DA receptors. Results are representative of 3 independent experiments. cell population having CSC characteristics. Accordingly, we selected adenocarcinoma of the lung, the most common histo- logical type of NSCLC observed in patients, for our present study. The ALDH1 activity is mainly due to its ALDH1A1 (aldehyde dehydrogenase 1 family, member A1) isoform, an intracellular cytosolic isoenzyme that oxidizes intracellular aldehyde and controls several functions including drug resistance (30). Besides lung cancer, ALDH1A1 has also been used as a marker of CSC in breast and esophageal cancers (31, 32). NSCLC cells expressing high ALDH1 activity show the characteristics of CSC such as increased proliferation, self-renewal, and resis- tance to chemotherapy in vitro, are highly tumorigenic in immunocompromised mice, and maintain heterogeneity like the parent tumors (27). These ALDH1A1-expressing NSCLC cells are also known for their clone formation ability, prolifera- tion, growth, and migration in vitro (28). The ALDH1 activity is mainly due to its ALDH1A1 (aldehyde dehydrogenase 1 family, member A1) isoform, an intracellular cytosolic isoenzyme that oxidizes intracellular aldehyde and controls several functions including drug resistance (30). Besides lung cancer, ALDH1A1 has also been used as a marker of CSC in breast and esophageal cancers (31, 32). NSCLC cells expressing high ALDH1 activity show the characteristics of CSC such as increased proliferation, self-renewal, and resis- tance to chemotherapy in vitro, are highly tumorigenic in immunocompromised mice, and maintain heterogeneity like the parent tumors (27). Results H&E staining and Immunofluorescence staining of CD133, ALDH1A1, and D2 DA receptors in NSCLC patient tissues. A, representative H&E staining of NSCLC lung tissue. B–D and G–I, slides of each patient were co-stained either with DAPI (B) and ALDH1A1 antibody (C) or D2 DA receptor antibody (D) or with DAPI (G) and CD133 antibody (H) and D2 DA receptor antibody (I). After co-staining, images were acquired separately for each marker followed by merging. E, F, J, and K, co-localization of ALDH1A1 and D2 DA receptor (MERGE 1) (E), ALDH1A1, D2 DA receptor and DAP1 (MERGE 2) (F), CD133 and D2 DA receptor (MERGE 1) (J), and CD133 D2 DA receptor and DAP1 (MERGE 2) (K). DAPI is used as nucleus marker. Co-localization appears in yellow and is indicated by arrows in merge pictures. Scale bars, 10 mm. Results are representative of 35 independent experiments. D2 Dopamine Receptors Regulate Cancer Stem Cells D2 Dopamine Receptors Regulate Cancer Stem Cells D2 Dopamine Receptors Regulate Cancer Stem Cells FIGURE 2. H&E staining and Immunofluorescence staining of CD133, ALDH1A1, and D2 DA receptors in NSCLC patient tissues. A, representative H&E staining of NSCLC lung tissue. B–D and G–I, slides of each patient were co-stained either with DAPI (B) and ALDH1A1 antibody (C) or D2 DA receptor antibody (D) or with DAPI (G) and CD133 antibody (H) and D2 DA receptor antibody (I). After co-staining, images were acquired separately for each marker followed by merging. E, F, J, and K, co-localization of ALDH1A1 and D2 DA receptor (MERGE 1) (E), ALDH1A1, D2 DA receptor and DAP1 (MERGE 2) (F), CD133 and D2 DA receptor (MERGE 1) (J), and CD133 D2 DA receptor and DAP1 (MERGE 2) (K). DAPI is used as nucleus marker. Co-localization appears in yellow and is indicated by arrows in merge pictures. Scale bars, 10 mm. Results are representative of 35 independent experiments. The Clonogenic Ability of CD133ve NSCLC Tumor Cells Was Suppressed following Activation of D2 DA Receptors— Results from the colony-forming efficiency assay revealed sig- nificant inhibition of the clonogenic ability of CD133ve A549 NSCLC tumor cells (p 0.05) when these cells were treated with 1 or 10 M of specific DA D2 receptor agonist quinpirole (Fig. 4A). Results Similarly, when these cells were treated with 10 M AKT inhibitor (LY294002) or 10 M ERK1/2 inhibitor (U0126), there was also significant inhibition of this characteristic of CD133ve NSCLC cells when compared with untreated con- trols. These results thus indicate that these signaling pathways play critical roles in inducing clone formation by CD133ve NSCLC tumor cells (Fig. 4A). This observation was further val- idated from the sphere-forming ability of the tumor cells, a surrogate marker for stemness and the self-renewal ability of epithelial cancer cells (41). In addition, CD133ve tumor cells, when treated with quinpirole (1 or 10 M) in serum-free medium, tumor cell sphere formation showed a significant decrease in number and size (Fig. 4, B and C). sections co-expressed D2 DA receptors in the CSC of human primary lung adenocarcinoma patients (Fig. 2). Appropriate isotype-matched controls were used to determine the specific- ity of staining (data not shown). After that, we isolated and enriched the CD133ve popula- tion from the human NSCLC cell line (A549) to study the sig- nificance, if any, of this high expression of D2 DA receptors in these CD133ve CSC. Accordingly, experiments were under- taken with purified CD133ve NSCLC tumor cells (A549) to understand the biobehavior of this cell population following activation of D2 DA receptors present in these cells. D2 DA Receptor Stimulation Inhibits CD133ve NSCLC Tumor Cell Proliferation in Vitro—The 3-(4, 5-dimethyl-2- thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay showed significant inhibition of proliferation in purified CD133ve NSCLC tumor cells when these cells were treated with 1 or 10 M quinpirole, a specific D2 DA receptor agonist (Fig. 3A). However, this action of quinpirole was abolished when these cells were pre-treated with a specific D2 DA recep- tor antagonist, eticlopride (10 times more than the concentra- tion of quinpirole), thereby further confirming the role of D2 DA receptors (Fig. 3A). On the contrary, 1 or 10 M quinpirole treatment had no significant effects on the proliferation of CD133ve tumor cells (Fig. 3A). Furthermore, FACS analysis did not demonstrate a significant number of annexin V- and propidium iodide (PI)-positive tumor cells following treatment with 1 or 10 M quinpirole treatment, thereby indicating that this low concentration of D2 DA receptor agonist had no effects on the apoptosis of CD133ve NSCLC tumor cells (Fig. 3B). Results Association of CD133 and D2 DA Receptors in Human NSCLC—NSCLC is the most common type of lung cancer (2). Among NSCLC patients, adenocarcinoma is predominant in both men and women (40). Accordingly, we used human lung adenocarcinoma (NSCLC) patient tissue samples and cell lines A549 and NCI-H23 for our present study. At first, we determined the expression of D2 DA receptors in the CD133-expressing tumor cell population in NSCLC cell lines A549 and NCI-H23. After initial gating to exclude dead cells and debris (Fig. 1, A and B), our FACS analysis demon- strated that in A549 and NCI-H23 cells, 22 and 20%, respec- tively, of the total tumor cells expressed D2 DA receptors (Fig. 1, C–F), and 5 and 3.5%, respectively, of total A549 and NCI-H23 cells expressed CD133 (Fig. 1, G–J). Furthermore, in both A549 and NCI-H23 cells, more than 95% of CD133ve cells expressed D2 DA receptors (Fig. 1, K–N). On the contrary, less than 16 and 15%, respectively, of CD133ve cells in A549 and NCI-H23 expressed D2 DA receptors (Fig. 1, K–N). On the contrary, silencing of ALDH1A1 in these cells results in considerable loss of their stem cell-like characteristics (28). Importantly, reports from the clinics have indicated that ALDH1 expression is strongly associated with poor prognosis and lymph node metastasis in NSCLC (28, 29, 33). DA acts as a neurotransmitter regulating locomotor and behavioral functions (34), and reports from our laboratory have indicated that in addition, DA can inhibit vascular endothelial growth factor-A (VEGF-A), mediating angiogenesis by sup- pressing VEGFR-2 phosphorylation (35–38). Studies from our laboratory have further reported that D2 DA receptors can reg- ulate different functions of normal progenitor and stem cells such as endothelial progenitor and mesenchymal stem cells (38, 39). Therefore, it will be interesting to study the expression profile of D2 DA receptors in the CD133ve tumor cell popu- lation in NSCLC and investigate the regulatory role of this neurotransmitter, if any, on the biology of this specific tumor We next validated this association in patient tissue samples. Our confocal data demonstrated significant co-localization of D2 DA receptors with both stem cell markers, CD133 and alde- hyde dehydrogenase (ALDH1A1), in human primary lung ade- nocarcinoma (NSCLC) tissue sections. More than 90% of the CD133 and ALDH1A1ve tumor cells in human NSCLC tissue VOLUME 292•NUMBER 2•JANUARY 13, 2017 436 JOURNAL OF BIOLOGICAL CHEMISTRY FIGURE 2. Results , p 0.05 no treatment (NT) versus 1 M and no reatment versus 10 M, O. D., optical density. B, apoptosis was measured by the annexin V-PI staining assay following treatment of CD133ve tumor cells with or 10 M quinpirole. Panel a, forward scatter/side scatter (FSC/SSC) plots show the gated population. H indicates height. Panel b, unstained. Panel c, no reatment. Panel d, 1 M quinpirole. Panel e, 10 M quinpirole. Lower left (LL), annexin V-PI (viable cells); lower right (LR), annexin VPI (early apoptotic cells); upper left (UL) annexin V-PI; upper right (UR), annexin VPI (late apoptotic cells). Results are representative of 3 independent experiments. Error bars indicate mean S.D. FIGURE 3. Effects of D2 DA receptor stimulation by D2 dopamine agonist quinpirole on proliferation (A) and apoptosis (B) of CD133ve tumor cells purified (>95%) from A549 NSCLC cell line. A, proliferation measured by the MTT assay following treatment of CD133ve and CD133ve tumor cells with 1 or 10 M quinpirole (Quin) in the presence or absence of eticlopride (Eti), the D2 receptor-specific antagonist (10 times more the concentration of D2 receptor-specific agonist, quinpirole) for 72 h. Results are representative of 3 independent experiments. , p 0.05 no treatment (NT) versus 1 M and no treatment versus 10 M, O. D., optical density. B, apoptosis was measured by the annexin V-PI staining assay following treatment of CD133ve tumor cells with 1 or 10 M quinpirole. Panel a, forward scatter/side scatter (FSC/SSC) plots show the gated population. H indicates height. Panel b, unstained. Panel c, no treatment. Panel d, 1 M quinpirole. Panel e, 10 M quinpirole. Lower left (LL), annexin V-PI (viable cells); lower right (LR), annexin VPI (early apoptotic cells); upper left (UL) annexin V-PI; upper right (UR), annexin VPI (late apoptotic cells). Results are representative of 3 independent experiments. Error bars indicate mean S.D. CD133ve NSCLC tumor cells (43), we therefore determined the expression of Oct-4 in CD133ve NSCLC tumor cells. Untreated CD133ve NSCLC tumor cells expressed signifi- cant Oct-4 mRNA, as evident from real-time PCR (Fig. 6). Interestingly, when this tumor cell population was treated with a specific D2 DA receptor agonist, quinpirole, signifi- cant down-regulation of Oct-4 mRNA level was observed following treatment with 1 M (2.5-fold) or 10 M (10-fold) quinpirole. Results Inhibition of ERK1/2 and AKT in CD133ve NSCLC Tumor Cells following D2 DA Receptor Activation—The CD133ve tumor cell population has been reported to overexpress acti- vated ERK1/2 and AKT, and the inhibition of ERK1/2 and AKT significantly influences their clonogenic potential in colon can- cer cells (42). Our results also indicated the same in CD133ve NSCLC cells (Fig. 4A). To explore D2 DA receptor-mediated inhibition of the clonogenic ability of the CD133ve NSCLC tumor cells, we examined the effects of D2 DA receptor activa- tion on the status of ERK1/2 and AKT phosphorylation in this JANUARY 13, 2017•VOLUME 292•NUMBER 2 437 JOURNAL OF BIOLOGICAL CHEMISTRY D2 Dopamine Receptors Regulate Cancer Stem Cells FIGURE 3. Effects of D2 DA receptor stimulation by D2 dopamine agonist quinpirole on proliferation (A) and apoptosis (B) of CD133ve tu purified (>95%) from A549 NSCLC cell line. A, proliferation measured by the MTT assay following treatment of CD133ve and CD133ve tumor or 10 M quinpirole (Quin) in the presence or absence of eticlopride (Eti), the D2 receptor-specific antagonist (10 times more the concentrat eceptor-specific agonist, quinpirole) for 72 h. Results are representative of 3 independent experiments. , p 0.05 no treatment (NT) versus 1 reatment versus 10 M, O. D., optical density. B, apoptosis was measured by the annexin V-PI staining assay following treatment of CD133ve tumor or 10 M quinpirole. Panel a, forward scatter/side scatter (FSC/SSC) plots show the gated population. H indicates height. Panel b, unstained. Pa reatment. Panel d, 1 M quinpirole. Panel e, 10 M quinpirole. Lower left (LL), annexin V-PI (viable cells); lower right (LR), annexin VPI (early apopto upper left (UL) annexin V-PI; upper right (UR), annexin VPI (late apoptotic cells). Results are representative of 3 independent experiments. Error bar mean S.D. D2 Dopamine Receptors Regulate Cancer Stem Cells FIGURE 3. Effects of D2 DA receptor stimulation by D2 dopamine agonist quinpirole on proliferation (A) and apoptosis (B) of CD133ve tumor cells purified (>95%) from A549 NSCLC cell line. A, proliferation measured by the MTT assay following treatment of CD133ve and CD133ve tumor cells with or 10 M quinpirole (Quin) in the presence or absence of eticlopride (Eti), the D2 receptor-specific antagonist (10 times more the concentration of D2 eceptor-specific agonist, quinpirole) for 72 h. Results are representative of 3 independent experiments. Results Error bars indicate mean S.D. D2 Dopamine Receptors Regulate Cancer Stem Cells D2 Dopamine Receptors Regulate Cancer Stem Cells FIGURE4.EffectsofD2DAreceptorstimulationbyD2dopamineagonistquinpiroleonclonogenicity(A)andtumorsphereformation(B)byCD133ve tumor cells purified (>95%) from the A549 NSCLC cell line. A, following treatment with Akt inhibitor (LY294002) or ERK inhibitor (U0126) or 1 or 10 M quinpirole and culturing for 7 days, the colonies were fixed and stained with crystal violet and photographed. Magnification: 20. Results are representative of 3 independent experiments. B, following treatment with 1 or 10 M quinpirole and culturing for 7 days, the sphere formation was observed, and randomly chosen fields were then photographed. Magnification: 20. C, sphere-forming efficacy is expressed as numbers of spheres formed per 100 cells. Results are representative of 3 independent experiments. Editing of all original images was done without obscuring the actual differences. , p 0.05 no treatment versus 1 M and no treatment versus 10 M. Error bars indicate mean S.D. FIGURE4.EffectsofD2DAreceptorstimulationbyD2dopamineagonistquinpiroleonclonogenicity(A)andtumorsphereformation(B)byCD133ve tumor cells purified (>95%) from the A549 NSCLC cell line. A, following treatment with Akt inhibitor (LY294002) or ERK inhibitor (U0126) or 1 or 10 M quinpirole and culturing for 7 days, the colonies were fixed and stained with crystal violet and photographed. Magnification: 20. Results are representative of 3 independent experiments. B, following treatment with 1 or 10 M quinpirole and culturing for 7 days, the sphere formation was observed, and randomly chosen fields were then photographed. Magnification: 20. C, sphere-forming efficacy is expressed as numbers of spheres formed per 100 cells. Results are representative of 3 independent experiments. Editing of all original images was done without obscuring the actual differences. , p 0.05 no treatment versus 1 M and no treatment versus 10 M. Error bars indicate mean S.D. both the concentrations of quinpirole (1 M and 10 M) signifi- cantly inhibited the invasion of CD133ve NSCLC tumor cells through the Matrigel (Fig. 7, A and B). this population of tumor cells are well established (18–23). Only a few other reports had indicated the presence of D2 DA receptors in lung cancer cells (46) and inhibition of tumor growth following treatment with bromocriptine, a D2 DA receptor agonist (47). However, most of the other studies had been undertaken in large cell lung carcinoma and small cell lung carcinoma cells (48, 49). Results However, transfection of these tumor cells with D2 DA receptor siRNA abrogated the inhibitory effect of quinpirole, thereby confirming the inhibitory role of D2 DA receptor activation on OCT-4 expression in CD133ve NSCLC tumor cells (Fig. 6). population of tumor cells derived from NSCLC cell lines A549 and NCI-H23. Our results indicated significant inhibition of both ERK1/2 and AKT phosphorylation in the CD133ve pop- ulation of tumor cells following treatment with either 1 M or 10 M of the specific D2 DA receptor agonist, quinpirole (Fig. 5A), in CD133ve tumor cells from both NSCLC cell lines, A549 and NCI-H23. These results were further validated by using siRNA of D2 DA receptor. Our results indicated that quinpirole had no significant effects on ERK1/2 and AKT phos- phorylation when these cells were pretreated with D2 DA receptor siRNA, thereby confirming the role of D2 DA recep- tors (Fig. 5A). The concentrations of the scramble siRNA and D2 DA receptor siRNA were optimized to ensure that they did not affect cell viability (data not shown), but significantly inhib- ited D2 DA receptor expressions in the CD133ve NSCLC cells (Fig. 5B). Inhibition of CD133ve NSCLC Tumor Cells Invasion in Vitro following Activation of D2 DA Receptors—As expression of CD133 in NSCLC tumor cells correlates with invasion and metastasis in lymphnodes(25),wethereforeinvestigatedwhetherD2DArecep- tor activation in these CD133ve tumor cells in NSCLC has any effect on invasiveness of these cells. In our in vitro experiment, Down-regulation of Oct-4 Expression in CD133ve NSCLC Tumor Cells—Because octamer-binding transcription factor 4 (Oct-4) expression maintains the stem-like properties in 438 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 292•NUMBER 2•JANUARY 13, 2017 FIGURE4.EffectsofD2DAreceptorstimulationbyD2dopamineagonistquinpiroleonclonogenicity(A)andtumorsphereformation(B)byCD133ve tumor cells purified (>95%) from the A549 NSCLC cell line. A, following treatment with Akt inhibitor (LY294002) or ERK inhibitor (U0126) or 1 or 10 M quinpirole and culturing for 7 days, the colonies were fixed and stained with crystal violet and photographed. Magnification: 20. Results are representative of 3 independent experiments. B, following treatment with 1 or 10 M quinpirole and culturing for 7 days, the sphere formation was observed, and randomly chosen fields were then photographed. Magnification: 20. C, sphere-forming efficacy is expressed as numbers of spheres formed per 100 cells. Results are representative of 3 independent experiments. Editing of all original images was done without obscuring the actual differences. , p 0.05 no treatment versus 1 M and no treatment versus 10 M. Results The other study investigated the effects of D2 DA receptor agonist on myeloid-derived suppres- sor cells and angiogenesis in lung cancer (50). None of these reports mentioned the status of D2 DA receptors and their effects on the treatment-refractory CD133ve CSC in adeno- carcinoma of the lung, the most prevalent histological type of NSCLC (40). D2 DA Receptor Activation Inhibits MMP-9 Secretion by CD133ve NSCLC Tumor Cells—Matrix metalloproteinase-9 (MMP-9) plays a critical role in tumor cell invasion and metas- tasis (44). Our results indicated significant inhibition of MMP-9 concentration in the culture medium of D2 DA receptor agonist quinpirole-treated cells after 24 h in comparison with untreated controls (Fig. 7C). D2 DA Receptor Agonist, Quinpirole, Inhibited CD133ve A549 Non-small Cell Lung Cancer Tumor Cell Growth in Vivo—Similar to our in vitro experiments, on day 8, we also observed 47% inhibition of tumor growth in nude mice trans- planted with CD133ve NSCLC tumor cells after treatment with 10 mg/kg i.p. of D2 DA receptor agonist, quinpirole, for 7 days (Fig. 8). We did not observe any significant side effects after treatment with this low dose of quinpirole, which corrob- orated with previous reports (45). We demonstrate for the first time that stimulation of D2 DA receptors present in the CD133ve CSC of NSCLC by a D2 DA receptor agonist can inhibit their proliferation, clonogenic abil- ity, and invasiveness in vitro (Figs. 3A, 4, A–C, and 7, A and B), as well as suppressing tumor growth in vivo (Fig. 8). The under- lying mechanisms indicate that stimulation of D2 DA receptors suppresses ERK1/2 and AKT phosphorylation (Fig. 5A), result- ing in inhibition of the clonogenic ability of these cells. Recent results indicate that this property of CSC is regulated through the ERK1/2 and AKT pathways (42). Accordingly, it would be important to note that we and others had demonstrated D2 DA receptor-mediated inhibition of ERK1/2 and AKT activation or D2 Dopamine Receptors Regulate Cancer Stem Cells FIGURE 5. Effects of quinpirole on the expression of phospho-AKT and phospho-ERK1/2 in CD133ve tumor cells derived from A549 and NCI-H23 NSCLC cell line. A, the cell lysates prepared from cultured A549 and NCI-H23 cell lines at specific times after treatment were subjected to Western blotting using phospho-AKT (p-AKT) antibodies or phospho-ERK1/2 (p-ERK1/2) antibodies. For each of the experiments, the purified cells from both the cell lines were treated with 1 or 10 M quinpirole or D2 DA receptor siRNA plus 1 M quinpirole or D2 DA receptor siRNA plus 10 M quinpirole or scrambled siRNA plus 1 M quinpirole or scrambled siRNA plus 10 M quinpirole and cultured for 72 h. ERK, extracellular-signal-regulated kinases; Scram, scrambled; Quin, quinpirole. NT, no treatment. B, siRNA-mediated knockdown of D2 DA receptor expression. Results are representative of 3 independent experiments. D2 Dopamine Receptors Regulate Cancer Stem Cells FIGURE 5. Effects of quinpirole on the expression of phospho-AKT and phospho-ERK1/2 in CD133ve tumor cells derived from A549 and NCI-H23 NSCLC cell line. A, the cell lysates prepared from cultured A549 and NCI-H23 cell lines at specific times after treatment were subjected to Western blotting using phospho-AKT (p-AKT) antibodies or phospho-ERK1/2 (p-ERK1/2) antibodies. For each of the experiments, the purified cells from both the cell lines were treated with 1 or 10 M quinpirole or D2 DA receptor siRNA plus 1 M quinpirole or D2 DA receptor siRNA plus 10 M quinpirole or scrambled siRNA plus 1 M quinpirole or scrambled siRNA plus 10 M quinpirole and cultured for 72 h. ERK, extracellular-signal-regulated kinases; Scram, scrambled; Quin, quinpirole. NT, no treatment. B, siRNA-mediated knockdown of D2 DA receptor expression. Results are representative of 3 independent experiments. FIGURE 6. Effect of quinpirole on the expression of OCT-4 in CD133ve tumor cells derived from A549 NSCLC cell line. The transcripts encoding human OCT-4 were measured by real-time PCR and expressed as the -fold change after normalizing with respect to -actin. For each of the experiments, the purified cells were treated with 1 or 10 M quinpirole or D2 DA receptor siRNA plus 1 M quinpirole or D2 DA receptor siRNA plus 10 M quinpirole and cultured for 72 h. Results are representative of 3 independent experiments. , p 0.05 no treatment versus 1 M, no treatment versus 10 M. Error bars indicate mean S.D. FIGURE 6. D2 Dopamine Receptors Regulate Cancer Stem Cells Effect of quinpirole on the expression of OCT-4 in CD133ve tumor cells derived from A549 NSCLC cell line. The transcripts encoding human OCT-4 were measured by real-time PCR and expressed as the -fold change after normalizing with respect to -actin. For each of the experiments, the purified cells were treated with 1 or 10 M quinpirole or D2 DA receptor siRNA plus 1 M quinpirole or D2 DA receptor siRNA plus 10 M quinpirole and cultured for 72 h. Results are representative of 3 independent experiments. , p 0.05 no treatment versus 1 M, no treatment versus 10 M. Error bars indicate mean S.D. phosphorylation (38, 51–55). Our results indicated that inhibi- tion of ERK1/2 and AKT phosphorylation occurred on activa- tion of D2 DA receptors in progenitor and cancer cells (38, 51). Beaulieu et al. (52) had also demonstrated that activation of D2 DA receptors in neurons forms a signaling complex of adaptor molecule -arrestin, protein phosphatase 2 (PP2A), and AKT, resulting in dephosphorylation of AKT. Similarly, Chang et al. (54) showed that D2 DA receptor-mediated inhibition of ERK1/2 in adrenal cortical cells is through inhibition of intra- cellular cAMP. Clonogenic ability is an important criterion of the stemness of CD133ve NSCLC tumor cells, and OCT-4 regulates this Discussion Taken together, our results for the first time indicate signif- icant expression of D2 DA receptors in CD133ve tumor cells in human adenocarcinoma of the lung (NSCLC) tumor tissues and cell lines. Importantly, cancer stem cell characteristics of 439 JANUARY 13, 2017•VOLUME 292•NUMBER 2 JOURNAL OF BIOLOGICAL CHEMISTRY FIGURE 5. Effects of quinpirole on the expression of phospho-AKT and phospho-ERK1/2 in CD133ve tumor cells derived from A549 and NCI-H23 NSCLC cell line. A, the cell lysates prepared from cultured A549 and NCI-H23 cell lines at specific times after treatment were subjected to Western blotting using phospho-AKT (p-AKT) antibodies or phospho-ERK1/2 (p-ERK1/2) antibodies. For each of the experiments, the purified cells from both the cell lines were treated with 1 or 10 M quinpirole or D2 DA receptor siRNA plus 1 M quinpirole or D2 DA receptor siRNA plus 10 M quinpirole or scrambled siRNA plus 1 M quinpirole or scrambled siRNA plus 10 M quinpirole and cultured for 72 h. ERK, extracellular-signal-regulated kinases; Scram, scrambled; Quin, quinpirole. NT, no treatment. B, siRNA-mediated knockdown of D2 DA receptor expression. Results are representative of 3 independent experiments. D2 Dopamine Receptors Regulate Cancer Stem Cells D2 Dopamine Receptors Regulate Cancer Stem Cells D2 Dopamine Receptors Regulate Cancer Stem Cells FIGURE 7. Effects of quinpirole on the invasiveness of CD133ve tumor cells derived from A549 NSCLC cell line and secretion of MMP-9 from this cell population. A, in vitro invasion was determined by the Matrigel invasion assay. Purified CD133ve NSCLC tumor cells were seeded at a density of 10,000 cells/well in stem cell medium in the upper cell culture inserts, with 1 or 10 M quinpirole, and 20% serum-containing medium was placed in the lower chambers as chemoattractant. Relevant controls were kept. The invasion was measured after 24 h. Magnification: 20. Results are representative of 3 independent experiments. B, invasion index was determined according to the standard formula. , p 0.05, no treatment versus 1 M and no treatment versus 10 M. C, MMP-9 concentration in the culture supernatants from the upper chambers was measured by ELISA. Results are representative of 3 independent experiments. , p 0.05, no treatment versus 1 M and no treatment versus 10 M. Error bars indicate mean S.D. D2 Dopamine Receptors Regulate Cancer Stem Cells FIGURE 7. Effects of quinpirole on the invasiveness of CD133ve tumor cells derived from A549 NSCLC cell line and secretion of MMP-9 from this cell population. A, in vitro invasion was determined by the Matrigel invasion assay. Purified CD133ve NSCLC tumor cells were seeded at a density of 10,000 cells/well in stem cell medium in the upper cell culture inserts, with 1 or 10 M quinpirole, and 20% serum-containing medium was placed in the lower chambers as chemoattractant. Relevant controls were kept. The invasion was measured after 24 h. Magnification: 20. Results are representative of 3 independent experiments. B, invasion index was determined according to the standard formula. , p 0.05, no treatment versus 1 M and no treatment versus 10 M. C, MMP-9 concentration in the culture supernatants from the upper chambers was measured by ELISA. Results are representative of 3 independent experiments. , p 0.05, no treatment versus 1 M and no treatment versus 10 M. Error bars indicate mean S.D. characteristic of these cells (43). Our results indicate that acti- vation of D2 DA receptors significantly down-regulates OCT-4, and this correlates well with inhibition of tumor sphere forma- tion, a surrogate marker of stemness (41). VOLUME 292•NUMBER 2•JANUARY 13, 2017 VOLUME 292•NUMBER 2•JANUARY 13, 2017 JOURNAL OF BIOLOGICAL CHEMISTRY 440 Experimental Procedures Culture of NSCLC Cell Lines—Human NSCLC cell lines A459 and NCI-H23 procured from the National Center for Cell Sciences (Pune, India) were cultured in DMEM and RPMI 1640 (Life Technologies), respectively, with 10% FBS and penicillin/ streptomycin (50 units/ml). These cells were maintained at 37 °C with a supply of 5% CO2. Cell lines were authenticated by short tandem repeat analysis (61). Patient Tissue Samples and Confocal Imaging—Human NSCLC tissue slides (adenocarcinoma of the lung) were obtained from thirty-five de-identified patients following the Institutional Review Board (IRB) rules of the Ohio State Uni- versity and Chittaranjan National Cancer Institute. Character- istics of the patients are shown in Table 1. p Cell Proliferation by MTT Assay—Proliferation of CD133ve NSCLC cells in stem cell media was checked by the MTT assay as described previously (35). In brief, 50,000 cells/well were seeded in 96-well round bottom cell plates (Corning) contain- ing stem cell medium in the presence of 1 or 10 M quinpirole (Sigma) with or without eticlopride (10 times more than the concentration of quinpirole) and cultured for 72 h with the appropriate controls. The cells were pelleted, the medium was aspirated, and 20-l aliquots of MTT solution (5 mg/ml) (Sigma) were added and incubated for 4 h at 37 °C. The medium was finally removed by aspiration, the purple-colored formazan precipitate was dissolved in DMSO (100 l), and then absor- bance was measured at 550 nm using a microplate reader (BioTek). Slides were preblocked with 10% donkey serum at room tem- perature for 1 h. Thereafter, these slides were incubated with unconjugated primary antibodies for D2 DA receptor (Santa Cruz Biotechnology), CD133 (Miltenyi Biotec), or ALDH1A1 (Santa Cruz Biotechnology) overnight at 4 °C. On the next day, the slides were incubated with the respective secondary anti- bodies conjugated to fluorochrome for 1 h at room tempera- ture, washed in PBS, and mounted with fluorescence mounting gel and DAPI (Electron Microscopy Sciences), and then confo- cal microscopy was performed (Olympus FBV 1000) (62). All these antibodies have been previously validated by us and oth- ers (35, 38, 62–68). In Vitro Tumor Apoptosis Assay—In vitro apoptosis of tumor cells was determined using the apoptosis detection kit (BD Pharmingen) as per the manufacturer’s instructions. In brief, 1 105 CD133ve tumor cells were incubated with or without quinpirole (in different doses for 72 h) at 37 °C in a humidified atmosphere with 5% CO2. TABLE 1 Isolation and Purification of CD133ve Cells by Magnetic- activated Cell Sorting and Generation of Tumor Sphere—The expression of CD133 was determined by flow cytometry (FACSCalibur, BD Biosciences) using CD133/1-phycoerythrin. 1 107 cells were labeled with CD133/1 microbeads (Miltenyi Biotec), and the cell suspension was then loaded on a magnetic- activated cell sorting column. The effluent containing the cell population enriched with the CD133ve cell fraction was col- lected. The purity of cells (90%) was checked by flow cytom- etry after being labeled with CD133/1-phycoerythrin (Miltenyi Biotec). Mouse IgG1 phycoerythrin (Miltenyi Biotec) was used as isotype control. Thereafter, these purified CD133ve cells were suspended in stem cell medium containing serum-free DMEM/F12 supplemented with penicillin/streptomycin (50 units/ml), 20 ng/ml human recombinant epidermal growth fac- tor (hrEGF), 10 ng/ml human recombinant basic fibroblast growth factor (hrbFGF), and 1% B27 supplement (Invitrogen), and then cultured in 6-well plates (Corning) at different densi- ties as per the requirement varying from 1000 to 50,000 cells/ well. 1 or 10 M quinpirole (Sigma) was added to the medium. The sphere-forming efficiency was monitored following treat- ment by plating 1000 cells/well in 24-well cell culture plates and culturing them for 7 days with the appropriate controls. Sphere-forming efficiency was calculated by counting the number of tumor spheres formed in a given well and dividing this by the total number of living cells seeded in the well 100 (21). This and other experiments were carried out in pooled CD133ve NSCLC cells. therebysupportingtheinvitroresults(Fig.8).Theseresults,there- fore, suggest the possibility of selectively targeting the CD133ve CSC population in NSCLC with a D2 DA receptor agonist. Finally, D2 DA receptor agonists are already in use for the treat- ment of other disorders (59, 60). Therefore, these agonists, together with the current conventional anticancer agents and/or radiotherapy, may be beneficial in the management of NSCLC patients as this combination of therapeutic approaches will not only eradicate the bulk of tumor cells, but may also eliminate the population of CSC that imparts resistance to therapy. VOLUME 292•NUMBER 2•JANUARY 13, 2017 D2 Dopamine Receptors Regulate Cancer Stem Cells Interestingly, recent evidence indicates that ERK2 also phosphorylates OCT4 and thus suggests that regulation of OCT4 is through activation of ERK (56). AKT also phosphorylates OCT4 at threonine 235, and this action correlates with resistance to apoptosis and tumorigenesis (57). Therefore, down-regulation of OCT4 in CD133ve NSCLC stem cells following stimulation of D2 DA receptors in NSCLC cells may be due to inhibition of ERK1/2 and AKT phosphorylation following activation of D2 DA receptors. FIGURE 8. Effect of quinpirole on A549 tumor growth inhibition in nude mice. Freshly isolated CD133ve tumor cells (104) were mixed with Matrigel(1:1)andinoculatedsubcutaneouslyintherightflankofeachanimal. When the tumor reached 100 mm3, quinpirole (10 mg/kg) was administered intraperitoneally for consecutive 7 days. Control mice received only medium Matrigel. Thereafter tumor growth inhibition was noted. , p 0.05, no treat- ment versus quinpirole. Error bars indicate mean S.D. MMP-9 synthesis and release by tumor cells are associated with an invasive property of these cells leading to metastasis (44). Reports further indicate an association of CD133ve NSCLC cells with lymph node metastasis, and high levels of MMP-9 correlate significantly with invasiveness of A549 NSCLC cells (58). Therefore, our results demonstrating signif- icant inhibition of MMP-9 synthesis by CD133ve tumor cells following treatment with quinpirole may be the cause of inhi- bition of Matrigel invasion by these cells. FIGURE 8. Effect of quinpirole on A549 tumor growth inhibition in nude mice. Freshly isolated CD133ve tumor cells (104) were mixed with Matrigel(1:1)andinoculatedsubcutaneouslyintherightflankofeachanimal. When the tumor reached 100 mm3, quinpirole (10 mg/kg) was administered intraperitoneally for consecutive 7 days. Control mice received only medium Matrigel. Thereafter tumor growth inhibition was noted. , p 0.05, no treat- ment versus quinpirole. Error bars indicate mean S.D. Furthermore,invivoxeno-transplantedhumanNSCLCcellline A549alsoshowedsignificantinhibitionoftumorgrowthfollowing treatment with D2 DA receptor-specific agonist quinpirole, 441 JANUARY 13, 2017•VOLUME 292•NUMBER 2 JOURNAL OF BIOLOGICAL CHEMISTRY JANUARY 13, 2017•VOLUME 292•NUMBER 2 TABLE 1 Selection of patients Characteristics No. of patients Male (aged 45–68 years) 10 Female (aged 58–71 years) 25 Histological subtypes of adenocarcinoma 35 Histological staging Stage 1/II 29 Stage III 6 D2 Dopamine Receptors Regulate Cancer Stem Cells TABLE 1 Selection of patients D2 Dopamine Receptors Regulate Cancer Stem Cells D2 Dopamine Receptors Regulate Cancer Stem Cells D2 Dopamine Receptors Regulate Cancer Stem Cells The reaction was stopped with 2 N H2SO4 solution, and absorbance was mea- sured (450 nm) using a microplate reader (BioTek) (38). The plates were incubated for 24 h at 37 °C in 5% CO2. The cells that did not migrate or invade through the pores were removed with a cotton swab, and cells on the lower surface of the membrane were examined, stained with crystal violet, and counted under a microscope. Supernatant from the upper chamber was collected, stored at 80 °C, and used for the esti- mation of MMP-9. The invasion index was then calculated (74). mation of MMP-9. The invasion index was then calculated (74). ELISA of MMP-9—To measure the extracellular secretion of MMP-9, cell-free supernatants were quantified by ELISA. In brief, 96-well microtiter plates were coated with cell-free supernatants and incubated overnight at 4 °C. Plates were blocked with BSA (5%) for 2 h. After washing, 50 l of MMP-9 antibody (R&D Systems) diluted 1:1000 was added to each well and incubated for 45 min. Bound cytokine was detected with secondary IgG peroxidase (Sigma). The color was developed with 3,3, 5,5-tetramethylbenzidine (TMB) substrate solution (OptEIATM, BD Pharmingen). The reaction was stopped with 2 N H2SO4 solution, and absorbance was mea- sured (450 nm) using a microplate reader (BioTek) (38). Preparation of Whole Cell Protein Lysates and Immuno- blotting—To prepare whole cell lysates, CD133ve tumor cells were rinsed with PBS and disrupted with cell lysis buffer (20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 1% Triton X-100, 1 mM EDTA, 50 mM dithiothreitol, and 2 mM phenylmethylsulfonyl fluoride) and protease inhibitor cocktail (Sigma). The lysate was then spun down, cleared of debris by centrifugation, and assayed for total protein concentration using Bradford reagent (Bio-Rad). For immunoblotting analysis, protein samples were boiled in 1 SDS buffer, separated by SDS-PAGE, and trans- ferred to a PVDF membrane. The PVDF membrane was blocked with 0.2% I-BLOCK (Applied Biosystems) for 2 h at room temperature and incubated with the appropriate primary antibody overnight at 4 °C. Finally, after washing with Tris- buffered saline (containing 0.1% Tween 20), each blot was incu- bated with the corresponding HRP-conjugated secondary anti- body (Sigma), and the immunoblot signal was visualized by chemiluminescence reagents (Pierce) (72). Experimental Procedures Cells were then washed with cold PBS, and then suspended in binding buffer and incubated with annexin V-FITC and propidium iodide for 15 min at room tem- perature in the dark. Apoptotic cells were monitored by flow cytometric analysis (38). Flow Cytometry—Single cell suspension was stained with phycoerythrin-conjugated CD133/1 (Miltenyi Biotec, validated in several reports (69, 70)) and fluorescein isothiocyanate-con- jugated anti-DA D2 antibodies (Santa Cruz Biotechnology, vali- dated in our previous reports (38, 39)), either singly or in combi- nation, for 30 min as per the recommendation of the manufacturer. Following labeling, the cells were washed in FACS buffer (PBS, pH 7.4, with 1% FBS) and fixed in 1% paraformalde- hydeinPBS.ThenflowcytometrywasperformedusingCellQuest software (BD Biosciences). Appropriate negative isotype controls were used to rule out the background fluorescence. The percent- age of each positive population and mean fluorescence intensities were determined using quadrant statistics (38). siRNA Knockdown of D2 DA Receptor—Cells were plated at 50% confluence on six-well plates. D2 DA receptor siRNA (Santa Cruz Biotechnology) solution was diluted further with transfection medium (Santa Cruz Biotechnology), and then mixed with siRNA transfection reagent (Santa Cruz Biotech- nology) and added to the culture medium to achieve a final VOLUME 292•NUMBER 2•JANUARY 13, 2017 442 JOURNAL OF BIOLOGICAL CHEMISTRY D2 Dopamine Receptors Regulate Cancer Stem Cells conceived and designed the study, provided financial support, provided study material or patient tissue samples, and provided final approval of the study. Author Contributions—S. R., K. L., M. K. N., A. B., T. G., S. K., and S. G. performed collection and/or assembly of data; S. R., K. L., R. B., P. S. D., and S. B. performed data analysis and interpretation; S. R., P. S. D., and S. B. wrote the manuscript; P. S. D. and S. B. conceived and designed the study, provided financial support, provided study material or patient tissue samples, and provided final approval of the study. 1. Siegel, R., Naishadham, D., and Jemal, A. (2012) Cancer statistics. CA Cancer J. Clin. 62, 10–29 D2 Dopamine Receptors Regulate Cancer Stem Cells Primary antibodies used in this study were phospho-ERK1/2 from BD Transduc- tion Laboratories (catalogue number 612358; lot number 99125) and phospho-AKT (1:500) from Santa Cruz Biotechnol- ogy (catalogue number SC7985-R; lot number AL909). The molecular weight markers were procured from Bio-Rad. Xenotransplantation of Tumor in Nude Mice—Mice experi- ments were undertaken after approval by the Institutional Ani- mal Care and Use Committee. Nude mice (National Institute of Nutrition, Hyderabad, India) 4–6 weeks of age weighing 20–25 g were used for the study. CD133ve tumor cells from A549 NSCLC tumor cell culture were purified by magnetic bead sep- aration. Purified cells were further evaluated by flow cytometry to confirm 90% CD133ve tumor cells. Freshly isolated CD133ve tumor cells (104) were mixed with Matrigel (1:1) and inoculated subcutaneously in the right flank of each animal. When the tumor reached 100 mm3, animals were divided into two groups, each consisting of six animals. The control group received tumor cells Matrigel only, whereas the treated group received D2 DA receptor agonist quinpirole (10 mg/kg) intraperitoneally for consecutive 7 days. Tumor volume was measured (TV (mm3) d2 d/2, where d and D aretheshortest and longest diameter of the tumor) (62). Statistics—The results are expressed as mean S.D. Statisti- cal significance was determined using an unpaired Student’s t test, and the results were considered significant when p 0.05 (75). Quantitative Real-time PCR Analysis—Total RNA from treated and untreated CD133ve tumor cells was extracted using TRIzol reagent (Invitrogen). Total RNA (1 g) was reversely transcribed into cDNA using cDNA transcription kit (Applied Biosystems) and followed by quantitative real-time PCR analysis using the Universal SYBR Green Master Mix and Step One Plus Real-Time PCR System (Applied Biosystems). -actin was the endogenous control. The -fold expression of the target host gene was calculated by using the 2Ct formula (73). The primers used were as follows: OCT4forward, 5-TCGAGAACCGAGTGAGAGG-3; OCT4 reverse, 5- GAACCACACTCGGACCACA-3; -actin forward, 5-CTG- GAGAAGAGCTACGAGC-3; -actin reverse, 5-GGAT- GCCACAGGACTCCA-3. Author Contributions—S. R., K. L., M. K. N., A. B., T. G., S. K., and S. G. performed collection and/or assembly of data; S. R., K. L., R. B., P. S. D., and S. B. performed data analysis and interpretation; S. R., P. S. D., and S. B. wrote the manuscript; P. S. D. and S. B. D2 Dopamine Receptors Regulate Cancer Stem Cells siRNA concentration of 50 nmol/liter. Transfected cells were incubated at 37 °C for 48 h. siRNA concentrations were opti- mized to ensure that they did not affect cell viability and scram- ble (51). Scramble siRNA (Santa Cruz Biotechnology) was used as a control. Matrigel Invasion Assay—Invasion assays were performed according to a previously published protocol (74). Briefly, 1 104 CD133ve NSCLC cells in 200 l of serum-free medium containing 1 or 10 M quinpirole were added to the upper chamber of a Matrigel insert (BD Biosciences), and 20% serum containing medium or medium without serum was placed in the lower chamber. Matrigel Colony Formation Assay—The spheres collected by gentle centrifugation were dissociated by adding trypsin-EDTA followed by repeated pipetting. After determining cell viability by trypan blue staining, single cell suspension was seeded in DMEM with 10% FBS at a density of 4000 cells/well in 6-well plates that were precoated with Matrigel (BD Biosciences) and then treated with 1 or 10 M quinpirole. The colony formation ability was assessed after 7 days by counting the number of colonies (70 cells) under a microscope following crystal violet staining (Sigma-Aldrich) (71). To ascertain the effect of ERK and AKT inhibition on the colony-forming ability of CD133ve NSCLC cells in agarose, cells were treated with 10 M LY294002, AKT inhibitor (Cell Signaling Technology) or 10 M U0126, MEK1 inhibitor, and MEK2 inhibitor (42) was pro- cured from Merck Millipore. the lower chamber. The plates were incubated for 24 h at 37 °C in 5% CO2. The cells that did not migrate or invade through the pores were removed with a cotton swab, and cells on the lower surface of the membrane were examined, stained with crystal violet, and counted under a microscope. Supernatant from the upper chamber was collected, stored at 80 °C, and used for the esti- mation of MMP-9. The invasion index was then calculated (74). ELISA of MMP-9—To measure the extracellular secretion of MMP-9, cell-free supernatants were quantified by ELISA. In brief, 96-well microtiter plates were coated with cell-free supernatants and incubated overnight at 4 °C. Plates were blocked with BSA (5%) for 2 h. After washing, 50 l of MMP-9 antibody (R&D Systems) diluted 1:1000 was added to each well and incubated for 45 min. Bound cytokine was detected with secondary IgG peroxidase (Sigma). The color was developed with 3,3, 5,5-tetramethylbenzidine (TMB) substrate solution (OptEIATM, BD Pharmingen). D2 Dopamine Receptors Regulate Cancer Stem Cells Schiffer, D., Mellai, M., Annovazzi, L., Caldera, V., Piazzi, A., Denysenko, T., and Melcarne, A. (2014) Stem cell niches in glioblastoma: a neuro- pathological view. BioMed Res. Int. 2014, 725921 33. Alamgeer, M., Ganju, V., Szczepny, A., Russell, P. A., Prodanovic, Z., Ku- mar, B., Wainer, Z., Brown, T., Schneider-Kolsky, M., Conron, M., Wright, G., and Watkins, D. N. (2013) The prognostic significance of aldehyde dehydrogenase 1A1 (ALDH1A1) and CD133 expression in early stage non-small cell lung cancer. Thorax. 68, 1095–1104 16. Steinmetz, N. F., Maurer, J., Sheng, H., Bensussan, A., Maricic, I., Kumar, V., and Braciak, T. A. (2011) Two domains of vimentin are expressed on the surface of lymph node, bone and brain metastatic prostate cancer lines along with the putative stem cell marker proteins CD44 and CD133. Can- cers 3, 2870–2885 34. Alcaro, A., Huber, R., and Panksepp, J. (2007) Behavioral functions of the mesolimbic dopaminergic system: an affective neuroethological perspec- tive. Brain Res. Rev. 56, 283–321 17. Liu, H., Zhang, W., Jia, Y., Yu, Q., Grau, G. E., Peng, L., Ran, Y., Yang, Z., Deng, H., and Lou, J. (2013) Single-cell clones of liver cancer stem cells have the potential of differentiating into different types of tumor cells. Cell Death Dis. 4, e857 35. Basu, S., Nagy, J. A., Pal, S., Vasile, E., Eckelhoefer, I. A., Bliss, V. S., Man- seau, E. J., Dasgupta, P. S., Dvorak, H. F., and Mukhopadhyay, D. (2001) The neurotransmitter dopamine inhibits angiogenesis induced by vascu- lar permeability factor/vascular endothelial growth factor. Nat. Med. 7, 569–574 18. Bertolini, G., Roz, L., Perego, P., Tortoreto, M., Fontanella, E., Gatti, L., Pratesi, G., Fabbri, A., Andriani, F., Tinelli, S., Roz, E., Caserini, R., Lo Vullo, S., Camerini, T., Mariani, L., et al. (2009) Highly tumorigenic lung cancer CD133 cells display stem-like features and are spared by cisplatin treatment. Proc. Natl. Acad. Sci. U.S.A. 106, 16281–16286 36. Chakroborty, D., Sarkar, C., Mitra, R. B., Banerjee, S., Dasgupta, P. S., and Basu, S. (2004) Depleted dopamine in gastric cancer tissues: dopamine treatment retards growth of gastric cancer by inhibiting angiogenesis. Clin. Cancer Res. 10, 4349–4356 37. Sarkar, C., Chakroborty, D., Chowdhury, U. R., Dasgupta, P. S., and Basu, S. (2008) Dopamine increases the efficacy of anticancer drugs in breast and colon cancer preclinical models. Clin. Cancer Res. 14, 2502–2510 19. D2 Dopamine Receptors Regulate Cancer Stem Cells (2012) Aldehyde dehydro- genase 1A1 possesses stem-like properties and predicts lung cancer pa- tient outcome. J. Thorac. Oncol. 7, 1235–1245 8. Li, Z. (2013) CD133: a stem cell biomarker and beyond. Exp. Hematol. Oncol. 2, 17 9. Singh, S. K., Hawkins, C., Clarke, I. D., Squire, J. A., Bayani, J., Hide, T., Henkelman, R. M., Cusimano, M. D., and Dirks, P. B. (2004) Identification of human brain tumour initiating cells. Nature 432, 396–401 29. Barr, M., Macdonagh, L., Gray, S., O’Byrne, K., Cuffe, S., and Finn, S. (2016) 75P Inhibition and exploitation of aldehyde dehydrogenase 1 (ALDH1) as a cancer stem cell marker in cisplatin resistant NSCLC. J. Thorac. Oncol. 11, (suppl.) S87 10. Li, H., and Tang, D. G. (2011) Prostate cancer stem cells and their potential roles in metastasis. J. Surg. Oncol. 103, 558–562 30. Tomita, H., Tanaka, K., Tanaka, T., and Hara, A. (2016) Aldehyde dehy- drogenase 1A1 in stem cells and cancer. Oncotarget 7, 11018–11032 11. Zhu, Y., Karakhanova, S., Huang, X., Deng, S. P., Werner, J., and Bazhin, A. V. (2014) Influence of interferon- on the expression of the cancer stem cell markers in pancreatic carcinoma cells. Exp. Cell Res. 324, 146–156 31. Charafe-Jauffret, E., Ginestier, C., Iovino, F., Tarpin, C., Diebel, M., Es- terni, B., Houvenaeghel, G., Extra, J.-M., Bertucci, F., Jacquemier, J., Xerri, L., Dontu, G., Stassi, G., Xiao, Y., Barsky, S. H., et al. (2010) Aldehyde dehydrogenase 1-positive cancer stem cells mediate metastasis and poor clinical outcome in inflammatory breast cancer. Clin. Cancer Res. 16, 45–55 12. Roudi, R., Korourian, A., Shariftabrizi, A., and Madjd, Z. (2015) Differen- tial expression of cancer stem cell markers ALDH1 and CD133 in various lung cancer subtypes. Cancer Invest. 33, 294–302 13. Bidlingmaier, S., Zhu, X., and Liu, B. (2008) The utility and limitations of glycosylated human CD133 epitopes in defining cancer stem cells. J. Mol. Med. 86, 1025–1032 32. Yang, L., Ren, Y., Yu, X., Qian, F., Bian, B.-S.-J., Xiao, H.-L., Wang, W.-G., Xu, S.-L., Yang, J., Cui, W., Liu, Q., Wang, Z., Guo, W., Xiong, G., Yang, K., et al. (2014) ALDH1A1 defines invasive cancer stem-like cells and predicts poor prognosis in patients with esophageal squamous cell carcinoma. Mod. Pathol. 27, 775–783 14. Fargeas, C. A., Huttner, W. B., and Corbeil, D. (2007) Nomenclature of prominin-1 (CD133) splice variants? An update. Tissue Antigens 69, 602–606 15. D2 Dopamine Receptors Regulate Cancer Stem Cells 23. Tirino, V., Camerlingo, R., Franco, R., Malanga, D., La Rocca, A., Viglietto, G., Rocco, G., and Pirozzi, G. (2009) The role of CD133 in the identifica- tion and characterisation of tumour-initiating cells in non-small-cell lung cancer. Eur. J. Cardiothorac. Surg. 36, 446–453 2. Parsons, A., Daley, A., Begh, R., and Aveyard, P. (2010) Influence of smok- ing cessation after diagnosis of early stage lung cancer on prognosis: sys- tem review of observational studies with meta analysis. BMJ 340, b5569 3. Alberg, A. J., Ford, J. G., Samet, J. M., and American College of Chest Physicians (2007) Epidemiology of lung cancer: ACCP evidence based clinical practice guidelines (2nd edition). Chest 132, (suppl.) 29s–55s 24. Wang, W., Chen, Y., Deng, J., Zhou, J., Zhou, Y., Wang, S., and Zhou, J. (2014) The prognostic value of CD133 expression in non-small cell lung cancer: a meta-analysis. Tumour Biol. 35, 9769–9775 4. Danesi, R., Pasqualetti, G., Giovannetti, E., Crea, F., Altavilla, G., Del Tacca, M., and Rosell, R. (2009) Pharmacogenomics in non-small-cell lung cancer chemotherapy. Adv. Drug Deliv. Rev. 61, 408–417 25. Wu, H., Qi, X. W., Yan, G. N., Zhang, Q. B., Xu, C., and Bian, X. W. (2014) Is CD133 expression a prognostic biomarker of non-small-cell lung can- cer? A systematic review and meta-analysis. PLoS ONE 9, e100168 5. Gomez-Casal, R., Bhattacharya, C., Ganesh, N., Bailey, L., Basse, P., Gib- son, M., Epperly, M., and Levina, V. (2013) Non-small cell lung cancer cells survived ionizing radiation treatment display cancer stem cells and epi- thelial mesenchymal transition phenotypes. Mol. Cancer 12, 94 26. Qu H., Li, R., Liu, Z., Zhang, J., and Luo, R. (2013) Prognostic value of cancer stem cell marker CD133 expression in non-small cell lung cancer: a systematic review. Int. J. Clin. Exp. Pathol. 6, 2644–2650 6. Dawood, S., Austin, L., and Cristofanilli, M. (2014) Cancer stem cells: implications for cancer therapy. Oncology (Williston Park) 28, 1101–1107, 1110 27. Jiang, F., Qiu, Q., Khanna, A., Todd, N. W., Deepak, J., Xing, L., Wang, H., Liu, Z., Su, Y., Stass, S. A., and Katz, R. L. (2009) Aldehyde dehydrogenase 1 is a tumor stem cell-associated marker in lung cancer. Mol. Cancer Res. 7, 330–338 7. Nassar, D., and Blanpain, C. (2016) Cancer stem cells: basic concepts and therapeutic implications. Annu. Rev. Pathol. 11, 47–76 28. Li, X., Wan, L., Geng, J., Wu, C.-L., and Bai, X. References JANUARY 13, 2017•VOLUME 292•NUMBER 2 JOURNAL OF BIOLOGICAL CHEMISTRY 443 444 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 292•NUMBER 2•JANUARY 13, 2017 D2 Dopamine Receptors Regulate Cancer Stem Cells 5, e1502 45. Bozzi, Y., and Borrelli, E. (2006) Dopamine in neurotoxicity and neuro- protection: what do D2 receptors have to do with it? Trends Neurosci. 29, 167–174 46. Sokoloff, P., Riou, J.-F., Martres, M.-P., and Schwartz, J.-C. (1989) Pres- ence of dopamine D-2 receptors in human tumoral cell lines. Biochem. Biophys. Res. Commun. 162, 575–582 64. Al-Lamki, R. S., Wang, J., Yang, J., Burrows, N., Maxwell, P. H., Eisen, T., Warren, A. Y., Vanharanta, S., Pacey, S., Vandenabeele, P., Pober, J. S., and Bradley, J. R. (2016) Tumor necrosis factor receptor 2-signaling in CD133- expressing cells in renal clear cell carcinoma. Oncotarget 7, 24111–24124 47. Sheikhpour, M., Ahangari, G., Sadeghizadeh, M., and Deezagi, A. (2013) A novel report of apoptosis in human lung carcinoma cells using selective agonist of D2-like dopamine receptors: a new approach for the treatment of human non-small cell lung cancer. Int. J. Immunopathol. Pharmacol. 26, 393–402 65. Zhang, L., Liu, Y., Wang, X., Tang, Z., Li, S., Hu, Y., Zong, X., Wu, X., Bu, Z., Wu, A., Li, Z., Li, Z., Huang, X., Jia, L., Kang, Q., et al. (2015) The extent of inflammatory infiltration in primary cancer tissues is associated with lymphomagenesis in immunodeficient mice. Sci. Rep. 5, 9447 48. Senogles, S. E. (2007) D2 dopamine receptor-mediated antiproliferation in a small cell lung cancer cell line, NCI-H69. Anticancer Drugs 18, 801–807 66. Rappa, F., Cappello, F., Halatsch, M.-E., Scheuerle, A., and Kast, R. E. (2013) Aldehyde dehydrogenase and HSP90 co-localize in human glio- blastoma biopsy cells. Biochimie 95, 782–786 49. Ishibashi, M., Fujisawa, M., Furue, H., Maeda, Y., Fukayama, M., and Yamaji, T. (1994) Inhibition of growth of human small cell lung cancer by bromocriptine. Cancer Res. 54, 3442–3446 67. Li, T., Su, Y., Mei, Y., Leng, Q., Leng, B., Liu, Z., Stass, S. A., and Jiang, F. (2010) ALDH1A1 is a marker for malignant prostate stem cells and predictor of prostate cancer patients’ outcome. Lab. Invest. 90, 234–244 50. Hoeppner, L. H., Wang, Y., Sharma, A., Javeed, N., Van Keulen, V. P., Wang, E., Yang, P., Roden, A. C., Peikert, T., Molina, J. R., and Mukhopad- hyay, D. (2015) Dopamine D2 receptor agonists inhibit lung cancer pro- gression by reducing angiogenesis and tumor infiltrating myeloid derived suppressor cells. Mol. Oncol. 9, 270–281 68. Su, Y., Qiu, Q., Zhang, X., Jiang, Z., Leng, Q., Liu, Z., Stass, S. A., and Jiang, F. D2 Dopamine Receptors Regulate Cancer Stem Cells (2010) Aldehyde dehydrogenase 1A1-positive cell population is en- riched in tumor-initiating cells and associated with progression of bladder cancer. Cancer Epidemiol. Biomarkers Prev. 19, 327–337 51. Ganguly, S., Basu, B., Shome, S., Jadhav, T., Roy, S., Majumdar, J., Dasgupta, P.S.,andBasu,S.(2010)Dopamine,byactingthroughitsD2receptor,inhibits insulin-like growth factor-I (IGF-I)-induced gastric cancer cell proliferation via up-regulation of Krüppel-like factor 4 through down-regulation of IGF-IR and AKT phosphorylation. Am. J. Pathol. 177, 2701–2707 69. Mak, A. B., Blakely, K. M., Williams, R. A., Penttilä, P.-A., Shukalyuk, A. I., Osman, K. T., Kasimer, D., Ketela, T., and Moffat, J. (2011) CD133 Protein N-glycosylation processing contributes to cell surface recog- nition of the primitive cell marker AC133 epitope. J. Biol. Chem. 286, 41046–41056 52. Beaulieu, J.-M., Sotnikova, T. D., Marion, S., Lefkowitz, R. J., Gainetdinov, R. R., and Caron, M. G. (2005) An Akt/-arrestin 2/PP2A signaling com- plex mediates dopaminergic neurotransmission and behavior. Cell 122, 261–273 70. Liu, S., Li, N., Yu, X., Xiao, X., Cheng, K., Hu, J., Wang, J., Zhang, D., Cheng, S., and Liu, S. (2013) Expression of intercellular adhesion molecule 1 by hepatocellular carcinoma stem cells and circulating tumor cells. Gastro- enterology 144, 1031–1041.e10 53. Beaulieu, J.-M., Tirotta, E., Sotnikova, T. D., Masri, B., Salahpour, A., Gainetdinov, R. R., Borrelli, E., and Caron, M. G. (2007) Regulation of Akt signaling by D2 and D3 dopamine receptors in vivo. J. Neurosci. 27, 881–885 71. Cao, L., Zhou, Y., Zhai, B., Liao, J., Xu, W., Zhang, R., Li, J., Zhang, Y., Chen, L., Qian, H., and Wu, M., Yin, Z. (2011) Sphere-forming cell subpopula- tions with cancer stem cell properties in human hepatoma cell lines. BMC Gastroenterol. 11, 71 54. Chang, H.-W., Huang, C.-Y., Yang, S.-Y., Wu, V.-C., Chu, T.-S., Chen, Y.-M., Hsieh, B.-S., and Wu, K.-D. (2014) Role of D2 dopamine receptor in adrenal cortical cell proliferation and aldosterone-producing adenoma tumorigenesis. J. Mol. Endocrinol. 52, 87–96 72. Lu, K., Chakroborty, D., Sarkar, C., Lu, T., Xie, Z., Liu, Z., and Basu, S. (2012) Triphala and its active constituent chebulinic acid are natural in- hibitors of vascular endothelial growth factor-A mediated angiogenesis. PLoS ONE 7, e43934 55. Liu, J. C., Baker, R. E., Sun, C., Sundmark, V. C., and Elsholtz, H. P. (2002) Activation of Go-coupled dopamine D2 receptors inhibits ERK1/ERK2 in pituitary cells: a key step in the transcriptional suppression of the prolactin gene. J. Biol. Chem. 277, 35819–35825 73. D2 Dopamine Receptors Regulate Cancer Stem Cells European Respiratory Society International multidisciplinary classification of lung adenocarcinoma. J. Thorac. Oncol. 6, 244–285 stem-like cells from A549 cells through down-regulation of pAkt. Acta Biochim. Biophys. Sin. 46, 15–21 stem-like cells from A549 cells through down-regulation of pAkt. Acta Biochim. Biophys. Sin. 46, 15–21 p y 59. Goldberg, L. I. (1989) The role of dopamine receptors in the treatment of congestive heart failure. J. Cardiovasc. Pharmacol. 14, Suppl. 5, s19–s27 41. Dontu, G., Abdallah, W. M., Foley, J. M., Jackson, K. W., Clarke, M. F., Kawamura, M. J., and Wicha, M. S. (2003) In vitro propagation and tran- scriptional profiling of human mammary stem/progenitor cells. Genes. Dev. 17, 1253–1270 60. Denton, M. D., Chertow, G. M., and Brady, H. R. (1996) “Renal dose” dopamine for the treatment of acute renal failure: scientific rationale, experimental studies and clinical trials. Kidney Int. 50, 4–14 42. Wang, Y. K., Zhu, Y. L., Qiu, F. M., Zhang, T., Chen, Z. G., Zheng, S., and Huang, J. (2010) Activation of Akt and MAPK pathways enhances the tumorigenicity of CD133 primary colon cancer cells. Carcinogenesis 31, 1376–1380 61. Masters, J. R., Thomson, J. A., Daly-Burns, B., Reid, Y. A., Dirks, W. G., Packer, P., Toji, L. H., Ohno, T., Tanabe, H., Arlett, C. F., Kelland, L. R., Harrison, M., Virmani, A., Ward, T. H., Ayres, K. L., and Debenham, P. G. (2001) Short tandem repeat profiling provides an international reference standard for human cell lines. Proc. Natl. Acad. Sci. U.S.A. 98, 8012–8017 43. Chen, Y.-C., Hsu, H.-S., Chen, Y.-W., Tsai, T.-H., How, C.-K., Wang, C.-Y., Hung, S.-C., Chang, Y.-L., Tsai, M.-L., Lee, Y.-Y., Ku, H.-H., and Chiou, S.-H. (2008) Oct-4 expression maintained cancer stem-like prop- erties in lung cancer-derived CD133-positive cells. PLoS ONE 3, e2637 62. Chakroborty,D.,Sarkar,C.,Yu,H.,Wang,J.,Liu,Z.,Dasgupta,P.S.,andBasu, S.(2011)Dopaminestabilizestumorbloodvesselsbyup-regulatingangiopoi- etin 1 expression in pericytes and Krüppel-like factor-2 expression in tumor endothelial cells. Proc. Natl. Acad. Sci. U.S.A. 108, 20730–20735 44. Xu, D., McKee, C. M., Cao, Y., Ding, Y., Kessler, B. M., and Muschel, R. J. (2010) Matrix metalloproteinase-9 regulates tumor cell invasion through cleavage of protease Nexin-1. Cancer Res. 70, 6988–6998 63. Chavali, P. L., Saini, R. K. R., Zhai, Q., Vizlin-Hodzic, D., Venkatabalasu- bramanian, S., Hayashi, A., Johansson, E., Zeng, Z.-J., Mohlin, S., Påhlman, S., Hansford, L., Kaplan, D. R., and Funa, K. (2014) TLX activates MMP-2, promotes self-renewal of tumor spheres in neuroblastoma and correlates with poor patient survival. Cell Death Dis. D2 Dopamine Receptors Regulate Cancer Stem Cells Eramo, A., Lotti, F., Sette, G., Pilozzi, E., Biffoni, M., Di Virgilio, A., Con- ticello, C., Ruco, L., Peschle, C., and De Maria, R. (2008) Identification and expansion of the tumorigenic lung cancer stem cell population. Cell Death Differ. 15, 504–514 38. Chakroborty, D., Chowdhury, U. R., Sarkar, C., Baral, R., Dasgupta, P. S., and Basu, S. (2008) Dopamine regulates endothelial progenitor cell mobi- lization from mouse bone marrow in tumor vascularization. J. Clin. Invest. 118, 1380–1389 20. Hsu, H.-S., Huang, P.-I., Chang, Y.-L., Tzao, C., Chen, Y.-W., Shih, H.-C., Hung, S.-C., Chen, Y.-C., Tseng, L.-M., and Chiou, S.-H. (2011) Cucurbi- tacin I inhibits tumorigenic ability and enhances radiochemosensitivity in non-small cell lung cancer-derived CD133-positive cells. Cancer 117, 2970–2985 39. Shome, S., Dasgupta, P. S., and Basu, S. (2012) Dopamine regulates mobi- lization of mesenchymal stem cells during wound angiogenesis. PLoS ONE 7, e31682 21. Le, H., Zeng, F., Xu, L., Liu, X., and Huang, Y. (2013) The role of CD133 expression in the carcinogenesis and prognosis of patients with lung can- cer. Mol. Med. Rep. 8, 1511–1518 40. Travis, W. D., Brambilla, E., Noguchi, M., Nicholson, A. G., Geisinger, K. R., Yatabe, Y., Beer, D. G., Powell, C. A., Riely, G. J., Van Schil, P. E., Garg, K., Austin, J. H., Asamura, H., Rusch, V. W., Hirsch, F. R., et al. (2011) Interna- tional Association for the Study of Lung Cancer/American Thoracic Society/ 22. Eramo, A., Haas, T. L., and De Maria, R. (2010) Lung cancer stem cells: tools and targets to fight lung cancer. Oncogene 29, 4625–4635 444 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 292•NUMBER 2•JANUARY 13, 2017 444 JANUARY 13, 2017•VOLUME 292•NUMBER 2 D2 Dopamine Receptors Regulate Cancer Stem Cells Huang, C., Yang, L., Li, Z., Yang, J., Zhao, J., Dehui, X., Liu, L., Wang, Q., and Song,T.(2007)DetectionofCCND1amplificationusinglasercapturemicro- dissection coupled with real-time polymerase chain reaction in human esophageal squamous cell carcinoma. Cancer Genet. Cytogenet. 175, 19–25 56. Brumbaugh, J., Hou, Z., Russell, J. D., Howden, S. E., Yu, P., Ledvina, A. R., Coon, J. J., and Thomson, J. A. (2012) Phosphorylation regulates human OCT4. Proc. Natl. Acad. Sci. U.S.A. 109, 7162–7168 74. Han, J., Fujisawa, T., Husain, S. R., and Puri, R. K. (2014) Identification and characterization of cancer stem cells in human head and neck squamous cell carcinoma. BMC Cancer 14, 173 57. Lin, Y., Yang, Y., Li, W., Chen, Q., Li, J., Pan, X., Zhou, L., Liu, C., Chen, C., He, J., Cao, H., Yao, H., Zheng, L., Xu, X., Xia, Z., et al. (2012) Reciprocal regulation of Akt and Oct4 promotes the self-renewal and survival of embryonal carcinoma cells. Mol. Cell 48, 627–640 75. Lu, K., and Basu, S. (2015) The natural compound chebulagic acid inhibits vascular endothelial growth factor A mediated regulation of endothelial cell functions. Sci. Rep. 5, 9642 58. Liu, F., Cao, X., Liu, Z., Guo, H., Ren, K., Quan, M., Zhou, Y., Xiang, H., and Cao, J. (2014) Casticin suppresses self-renewal and invasion of lung cancer JANUARY 13, 2017•VOLUME 292•NUMBER 2 JANUARY 13, 2017•VOLUME 292•NUMBER 2 445 JOURNAL OF BIOLOGICAL CHEMISTRY
https://openalex.org/W4248643002	https://discovery.ucl.ac.uk/10091649/1/s12874-019-0889-3.pdf	English	null	Reporting guideline for priority setting of health research (REPRISE)	Research Square (Research Square)	2,019	cc-by	9,049	© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tong et al. BMC Medical Research Methodology (2019) 19:243 https://doi.org/10.1186/s12874-019-0889-3 Tong et al. BMC Medical Research Methodology (2019) 19:243 https://doi.org/10.1186/s12874-019-0889-3 Open Access Reporting guideline for priority setting of health research (REPRISE) Allison Tong1,2* , Anneliese Synnot3,4, Sally Crowe5, Sophie Hill3, Andrea Matus2, Nicole Scholes-Robertson1,2, Sandy Oliver6,7, Katherine Cowan8, Mona Nasser9, Soumyadeep Bhaumik10, Talia Gutman1,2, Amanda Baumgart1,2 and Jonathan C. Craig11 Abstract Background: Research priority setting with stakeholders can help direct the limited resources for health research toward priority areas of need. Ensuring transparency of the priority setting process can strengthen legitimacy and credibility for influencing the research agenda. This study aims to develop a reporting guideline for priority setting of health research. Methods: We searched electronic databases and relevant websites for sources (frameworks, guidelines, or models for conducting, appraising, reporting or evaluating health research priority setting, and reviews (including systematic reviews)), and primary studies of research priority setting to July 2019. We inductively developed a list of reporting items and piloted the preliminary guideline with a diverse range of 30 priority setting studies from the records retrieved. Results: From 21,556 records, we included 26 sources for the candidate REPRISE framework and 455 primary research studies. The REporting guideline for PRIority SEtting of health research (REPRISE) has 31 reporting items that cover 10 domains: context and scope, governance and team, framework for priority setting, stakeholders/ participants, identification and collection of priorities, prioritization of research topics, output, evaluation and feedback, translation and implementation, and funding and conflict of interest. Each reporting item includes a descriptor and examples. Conclusions: The REPRISE guideline can facilitate comprehensive reporting of studies of research priority setting. Improved transparency in research priority setting may strengthen the acceptability and implementation of the research priorities identified, so that efforts and funding are invested in generating evidence that is of importance to all stakeholders. Trial registration: Not applicable. Keywords: Priority-setting, Reporting, Transparency, Patient involvement Background or fail to address important needs of relevant stakeholders [1, 3–8]. For example, an analysis of 14 research priority setting partnerships involving patients with different medical conditions found that pharmacological interven- tions were prioritized only in 18% of the total priorities but 58% of the clinical trials in those fields evaluated pharmacological interventions [4]. Historically, the health research agenda has been largely investigator-driven with limited input from other stake- holders including patients, caregivers and the community [1, 2]. Given the evident mismatch between the research interests of patients and researchers, investment into health research may be misdirected to areas of low priority Globally, there have been calls for research priority setting with stakeholders to be done at all levels of health systems, jurisdictions, and health areas [9–12]. There is no consensus on the definition of research pri- ority setting but most definitions refer to a range of * Correspondence: allison.tong@sydney.edu.au 1Sydney School of Public Health, University of Sydney, Sydney, NSW, Australia 2Centre for Kidney Research, The Children’s Hospital at Westmead, Westmead NSW, Sydney 2145, Australia Full list of author information is available at the end of the article * Correspondence: allison.tong@sydney.edu.au 1Sydney School of Public Health, University of Sydney, Sydney, NSW, Australia 2Centre for Kidney Research, The Children’s Hospital at Westmead, Westmead NSW, Sydney 2145, Australia Full list of author information is available at the end of the article Purpose and context h f The purpose of REPRISE is to facilitate comprehensive and transparent reporting of health research priority setting exercises, in which there is direct involvement of stakeholders setting research priorities. The REPRISE guideline is flexible so that it may be used for a range of approaches. The scope of REPRISE does not cover approaches without direct involvement of stakeholders such as documentary analysis (e.g. evidence mapping), and econometrics methods (e.g. value of information). REPRISE is not intended for use to appraise the quality of priority setting studies, establish or evaluate criteria for research priorities (e.g. evidence gaps, prevalence of disease, economic considerations), and does not recom- mend a preferred approach. However, reviews of published research priority setting exercises have consistently demonstrated a lack of trans- parency because of suboptimal reporting [17, 21–26]. A systematic review of research priority setting in child- hood chronic disease, in which most studies were con- ducted in the UK, US, and Australia, found that methods for collecting and prioritizing research topics were reported in only 50 (60%) of the 84 studies included [23]. Another review of research priority setting exercises in Zambia reported that details about the process and the stakeholders involved were omitted in the majority of studies [22], and similarly, a review of studies in the Islamic Republic of Iran revealed that 22 (61%) of the 36 priority setting studies did not report methods and only listed the research priorities [21]. Inadequate description of the stakeholders and the methods makes it difficult to assess the validity of research priorities identified, and limits the ability to aggregate, analyze or compare research priorities that have been established [27]. Page 2 of 11 Page 2 of 11 Tong et al. BMC Medical Research Methodology (2019) 19:243 Page 2 of 11 activities that involve identifying, prioritizing, and achieving consensus on the research areas or questions of importance to stakeholders [13, 14]. The past two decades have seen increasing efforts to develop better ways to engage all relevant stakeholders, particularly pa- tients, in setting priorities for research across different health disciplines and populations [15–17]. Involving stakeholders in an explicit manner in research priority setting can help to: 1) ensure that funding decisions and research meet critical evidence gaps to inform decision making; 2) facilitate shared responsibility and account- ability in implementing the research agenda; 3) improve the relevance and legitimacy of research; and 4) ultim- ately achieve better health outcomes [12, 17]. strengthen legitimacy and credibility to support imple- mentation and maximise impact. A reporting checklist for research priority setting may facilitate more consistent and comprehensive reporting and enable researchers and end-users to better understand the processes taken in developing research priorities. The aim of this paper is to introduce the reporting guideline for priority setting of health research (REPRISE), describe its development and provide a rationale for the items included. REPRISE development We used the Enhancing the Quality and Transparency of Health Research (EQUATOR) toolkit [30], for developing the REPRISE Guideline and reported our approach based on the “Guidance for developers of health research report- ing guidelines” where possible [31]. We have also registered REPRISE with the EQUATOR Network. y A diverse range of methods are used to prioritize research given the different healthcare contexts, popula- tions, environments and resources available in which the priority setting is undertaken [14]. The process of research priority setting can be complex, political and value-laden. It can also be challenging to identify, address and integrate the different perspectives and values held by diverse stakeholders. While there is no consensus on what constitutes “successful” research priority setting, it has been advocated that processes must be fair, legitimate, informed by credible evidence, involve a broad spectrum of stakeholders, and be trans- parent [12, 13, 18–20]. REPRISE framework: content and rationale The REPRISE guideline includes ten domains and a total of 31 reporting items with a descriptor and examples pro- vided. (Table 1) The principles, rationale, and explanation for the domains are detailed below, which are based on the synthesis of sources listed in Additional file 3, priority setting studies, and discussion among the investigators. Context and scope (items 1–7) Establishing the context and scope is recommended as these “underpin the process of research priority setting,” [13] including the selection of relevant stake- holders and methods used. In terms of geographical scope, priority setting may be done at an institutional, local, national, or international level [13, 19, 21, 39– 41]; recognizing that each will have its own “sense of mandate, capacity, culture, and resources.” [39] Re- search priorities may address a specific condition, dis- ease or risk factor (e.g. cancer, mental health), population (e.g. elderly, adolescents), health system, re- search design, or interventions (e.g. vaccination) [5, 13, 19, 21, 24, 27, 37, 38, 40, 41, 44]. These can be decided upon based on the evidence [5, 38] and initial deliberations with stakeholders [38]. Identify the need for a guideline Systematic reviews have consistently shown the reporting of the process of research priority setting with stake- holders is highly variable and limited with many details omitted [21, 23, 25, 26, 32]. The need for a reporting guideline has also been identified through our workshops and forums at national and international meetings (e.g. Cochrane Colloquia and Symposia [12, 33–35]; James Lind Alliance [5]), and through our collective experiences of conducting, publishing, reviewing, and using research priority setting studies. We conducted a comprehensive search for frame- works, guidelines or models for conducting, appraising, reporting and evaluating health research priority setting, reviews (including systematic reviews) of research prior- ity setting studies, and primary research priority setting studies. We searched electronic databases including MEDLINE, Embase, CINAHL, PsycINFO from inception to 23rd July 2019 using sensitive search strategies Frameworks and guidelines are available for conducting and evaluating research priority setting, which mainly focus on criteria related to the process rather than the outcomes and impact of priority setting. There are no published guidelines for reporting priority setting for health research [5, 13, 18, 27–29]. Ensuring the transpar- ency of the process for research priority setting can Tong et al. BMC Medical Research Methodology (2019) 19:243 Page 3 of 11 preliminary guideline to report a research priority setting exercise in health communication and participation [45]. In the final stage, we piloted the guideline with research priority setting studies. We used a purposive sampling strategy to select 30 priority setting exercises from the total of 455 studies retrieved from the search to ensure a diverse range of health topics, regions, stakeholders involved, framework or methods used, and type of output. Using a standardized data extraction template with the reporting items from the REPRISE guideline, the investigators (two per study) independently assessed if the study reported on each item (yes/no), added comments, and suggested new reporting items that were not yet captured. The results are provided in Additional file 6. After completion, the investi- gators discussed the relevance and applicability of the items, clarity of the items, comprehensiveness of the descriptor and examples, and any new reporting items proposed. These were integrated into the final reporting guideline, which was reviewed and approved by all investigators. provided in Additional file 1. We used Medical Subject Heading (MeSH) terms and text words for research priorities and combined this with terms related to reporting, conduct and evaluation. Generating reporting items for the candidate checklist Generating reporting items for the candidate checklist We extracted items related to the process of priority- setting from the frameworks and systematic reviews included (the sources are listed in Additional file 2). We translated these into reporting items for the can- didate REPRISE reporting guidelines by grouping similar items and rephrasing the statements as a reporting item. We inductively developed the initial list of reporting items. This was reviewed by two other investigators (AB, AM) to ensure all relevant items were included in the list. The reporting items were compiled into 10 domains: context and scope, governance and team, framework for priority setting, stakeholders/participants, identification and collection of priorities, prioritization of research topics, output, evaluation and feedback, translation and implementa- tion, and funding and conflict of interest. (Add- itional file 4) We imported all sources (frameworks and systematic reviews) into HyperRESEARCH soft- ware for coding textual data, and AT conducted line- by-line coding of each source to the initial items. We generated a report of each reporting item and the corresponding coded text (content). We developed de- scriptors and examples based on the content of the sources and input from all the investigators. The sources that contributed to each reporting item, and examples of the original extracted items are shown in Additional file 5. Identify the need for a guideline We also searched Google Scholar, relevant organizational websites (e.g. WHO, EQUATOR, Cochrane, James Lind Alliance and PCORI), and reference lists of articles. The search results are shown in Additional file 2. From the 21,556 records retrieved, we identified 13 frameworks or guidelines for conducting or evaluating research priority guidelines [5, 13, 18–20, 28, 36–42] (none designed for reporting research priority setting), and 13 reviews of research priority setting [15–17, 21–27, 32, 43, 44], of which four were systematic reviews [23–26]. (Additional file 3) We also identified 455 primary research priority setting studies. provided in Additional file 1. We used Medical Subject Heading (MeSH) terms and text words for research priorities and combined this with terms related to reporting, conduct and evaluation. We also searched Google Scholar, relevant organizational websites (e.g. WHO, EQUATOR, Cochrane, James Lind Alliance and PCORI), and reference lists of articles. The search results are shown in Additional file 2. From the 21,556 records retrieved, we identified 13 frameworks or guidelines for conducting or evaluating research priority guidelines [5, 13, 18–20, 28, 36–42] (none designed for reporting research priority setting), and 13 reviews of research priority setting [15–17, 21–27, 32, 43, 44], of which four were systematic reviews [23–26]. (Additional file 3) We also identified 455 primary research priority setting studies. preliminary guideline to report a research priority setting exercise in health communication and participation [45]. p p [ ] In the final stage, we piloted the guideline with research priority setting studies. We used a purposive sampling strategy to select 30 priority setting exercises from the total of 455 studies retrieved from the search to ensure a diverse range of health topics, regions, stakeholders involved, framework or methods used, and type of output. Using a standardized data extraction template with the reporting items from the REPRISE guideline, the investigators (two per study) independently assessed if the study reported on each item (yes/no), added comments, and suggested new reporting items that were not yet captured. The results are provided in Additional file 6. After completion, the investi- gators discussed the relevance and applicability of the items, clarity of the items, comprehensiveness of the descriptor and examples, and any new reporting items proposed. These were integrated into the final reporting guideline, which was reviewed and approved by all investigators. Pilot testing the checklist The intended beneficiaries [13] may include patients, caregivers, or the general community who could benefit from the priority setting exercise, and the target audience are those who have the potential to implement or fund the research priorities identified [13, 17, 39–41]. The focus, content and type of research to be considered can deter- mine the scope of the priority setting exercise. In terms of the broad research areas, these generally span public health, health services, clinical research and basic science [24, 37, The preliminary REPRISE guideline was presented at the Australasian Cochrane Symposium, in which partici- pants used the guideline to assess the reporting of a research priority exercise, and provided feedback on the guideline [34]. We subsequently used the REPRISE guideline in two systematic reviews of research priority setting studies in childhood chronic conditions [23] and organ transplantation [26]. We also applied the Page 4 of 11 Tong et al. BMC Medical Research Methodology (2019) 19:243 Global, regional, national, city, local area, institutional/organizational level, health service Disease or condition specific, interventions, healthcare delivery, health system This may include the general population or a specific population based on demographic (age, gender), clinical (disease, condition), or other characteristics who may benefit from the research Policy makers, funders, researchers, industry or others who have the potential to implement the priorities identified Public health, health services research, clinical research, basic science Etiology, diagnosis, prevention, treatment (interventions), prognosis, health services, psychosocial, behavioral and social science, economic evaluation, implementation; this may not be pre-defined Interim, short-term, long-term priorities, plans to revise and update Those responsible for initiating, developing, and guiding the process for priority setting, and examples of structures include; Steering Committee, Advisory Group, Technical Experts Stakeholder group or role, institutional affiliations, country or region, demographics (e.g. age sex), discipline, experience, expertise 10 Describe any training or experience relevant to conducting priority setting Consultants or advisors, members with experience or skills relevant to the conducting priority-setting e.g. qualitative methods, surveys, facilitation C Framework for priority setting 11 State the framework used (if any) D Stakeholders or participants 12 Define the inclusion criteria for stakeholders involved in priority-setting James Lind Alliance, COHRED, CHNRI, Dialogue Model, no framework (general research priority setting) James Lind Alliance, COHRED, CHNRI, Dialogue Model, no framework (general research priority setting) need, feasibility, novelty, equity 24 State the method or threshold for excluding research topics/ questions Thresholds for ranking scores, proportions, votes; other criteria G Output 25 State the approach to formulating the research priorities Area, topic, questions, PICO (population, intervention, comparator, outcome) H Evaluation and feedback 26 Describe how the process of prioritization was evaluated Survey, workshop 27 Describe how priorities were fed back to stakeholders and/or to the public; and how feedback (if received) was addressed and integrated Public meetings or workshop, newsletters, website, email, online presentations I Implementation 28 Outline the strategy or action plans for implementing priorities Communication with target audience, via policies and funding 29 Describe plans, strategies, or suggestions to evaluate impact Integration in decision-making, funding allocation, review of relevant documents J Funding and conflict of interest 30 State sources of funding Name sources of funding for the priority-setting exercise; if relevant include the budget and/or cost 31 Declare any conflicts or competing interests State any conflicts of interest that may be at an individual level and/or at a contextual level (e.g. political issues, controversies) that may affect the process, output or implementation. Table 1 Reporting guideline for health research priority setting with stakeholders (REPRISE) (Continued) No Item Descriptor and/or examples Methods e.g. Delphi survey, surveys, nominal group technique, interviews, focus groups, meetings, workshops; Prioritization e.g. voting, ranking; Mode e.g. face-to-face, online; Criteria e.g. need, feasibility, novelty, equity Thresholds for ranking scores, proportions, votes; other criteria 31 Declare any conflicts or competing interests State any conflicts of interest that may be at an individual level and/or at a contextual level (e.g. political issues, controversies) that may affect the process, output or implementation. knowledge, decision-making skills, and ability and deliver the project. The leadership and management team is usually responsible for overseeing, developing and implementing the process for priority setting [5, 13, 19]. The leadership group may take the form of, for example, an Executive Committee, Advisory Group, Technical Ex- pert Group [13]. Members of the leadership team would generally be expected to contribute broad and relevant collective insights, harness their networks for engagement and partnership; and include a diversity of members to offer legitimacy to wider stakeholder networks (e.g. pa- tients, caregivers, researchers, policy makers, clinicians, representatives from other non-government or govern- ment organizations) [5, 21], and those with technical ex- pertise [5]. James Lind Alliance, COHRED, CHNRI, Dialogue Model, no framework (general research priority setting) Patients, caregivers, general community, health professionals, researchers, policy makers, non-governmental organizations, government, industry; specific groups including vulnerable and marginalized populations 12 Define the inclusion criteria for stakeholders involved in priority-setting 13 State the strategy or method for identifying and engaging stakeholders 14 Indicate the number of participants and/or organizations involved 15 Describe the characteristics of stakeholders 16 State if reimbursement for participation was provided E Identification and collection of research priorities 17 Describe methods for collecting initial priorities Partnership with organizations, social media, recruitment through hospitals Number of individuals and organizations, include number by stakeholder group Stakeholder group, demographic characteristics, areas of interest and expertise, discipline, affiliations Cash, vouchers, certificates, acknowledgement; what purpose e.g. travel, accommodation, honorarium Methods e.g. Delphi survey, surveys, nominal group technique, interviews, focus groups, meetings, workshops; prioritization e.g. voting, ranking; mode e.g. face-to-face, online; may be informed by evidence e.g. systematic reviews, reviews of guidelines/other documents, health technology assessment 18 Describe methods for collating and categorizing priorities 19 Describe methods and reasons for modifying (removing, adding, reframing) priorities 20 Describe methods for refining or translating priorities into research topics or questions 21 Describe methods for checking whether research questions or topics have been answered Taxonomy or other framework used to organize, summarise, and aggregate topics or questions Based on scope, clarity, definition, duplication, other criteria Reviewed by Steering Committee or project team Systematic reviews, evidence mapping, consultation with experts Page 5 of 11 Tong et al. BMC Medical Research Methodology (2019) 19:243 Table 1 Reporting guideline for health research priority setting with stakeholders (REPRISE) (Continued) No Item Descriptor and/or examples 22 Describe number of research questions or topics Number of priorities at each stage of the process F Prioritization of research topics/questions 23 Describe methods and criteria for prioritizing research topics or questions Methods e.g. Delphi survey, surveys, nominal group technique, interviews, focus groups, meetings, workshops; Prioritization e.g. voting, ranking; Mode e.g. face-to-face, online; Criteria e.g. James Lind Alliance, COHRED, CHNRI, Dialogue Model, no framework (general research priority setting) The membership and selection of stakeholders may need to take into consideration the need for equity [12, 36]. Also, it has been suggested that the involvement of individuals or organizations with experience in priority set- ting and relevant research skills can ensure a “high quality process.” [5, 13, 40] With regard to facilitators, neutrality and facilitations skills may be important to elicit input from diverse and mixed stakeholders [5, 36]. knowledge, decision-making skills, and ability and deliver the project. The leadership and management team is usually responsible for overseeing, developing and implementing the process for priority setting [5, 13, 19]. The leadership group may take the form of, for example, an Executive Committee, Advisory Group, Technical Ex- pert Group [13]. Members of the leadership team would generally be expected to contribute broad and relevant collective insights, harness their networks for engagement and partnership; and include a diversity of members to offer legitimacy to wider stakeholder networks (e.g. pa- tients, caregivers, researchers, policy makers, clinicians, representatives from other non-government or govern- ment organizations) [5, 21], and those with technical ex- pertise [5]. The membership and selection of stakeholders may need to take into consideration the need for equity [12, 36]. Also, it has been suggested that the involvement of individuals or organizations with experience in priority set- ting and relevant research skills can ensure a “high quality process.” [5, 13, 40] With regard to facilitators, neutrality and facilitations skills may be important to elicit input from diverse and mixed stakeholders [5, 36]. 41, 44]. The types of research questions that may be in- cluded can range from etiology, diagnosis, prognosis, treat- ment, to behavioural; and social science, economic evaluation and implementation [23, 24, 27, 39]. It is not ne- cessary for the type of research questions to be determined a priori. p Providing an estimated time frame that the priorities are expected to be valid or relevant may be relevant. This is because research priorities may evolve due to the develop- ment of new technology or interventions, emerging evi- dence, or changes to the health system or socio-political contexts [13, 17, 19, 37–39, 41, 42]. If there are plans to update the priority setting or to monitor the priorities for the need to update, these could be described. There has been suggestion of 3–5 year cycles of prioritization if the priority setting exercise is to be repeated [37, 38]. Framework for priority setting (item 11) and use a different approach, and not necessarily follow an existing or established framework. Some priority setting studies use or adapt frameworks to guide the process [17, 22, 23, 25, 44]. Common frame- works include the James Lind Alliance [5], Council on Health Research for Development Essential National Health Research (COHRED/ENHR) [19], Essential Na- tional Health Research (EHNR), Child Health and Nutri- tion Research Initiative (CHMRI) [41], and the Dialogue Model [36]. A summary of these frameworks is provided in Table 2. Some priority setting exercises may develop Governance and team (items 8–10) It has been argued that priority setting requires “cred- ible” [39] leadership to support acceptability and up- take. This may require leaders who are trusted by stakeholders and who have the necessary expertise, Tong et al. BMC Medical Research Methodology (2019) 19:243 Page 6 of 11 aRefers to developers Stakeholders or participants (items 12–16) BMC Medical Research Methodology (2019) 19:243 Tong et al. BMC Medical Research Methodology (2019) 19:243 Tong et al. BMC Medical Research Methodology (2019) 19:243 be respected in setting health research priorities” [41] can include patients, caregivers, clinicians, policy makers, representatives from non-governmental organizations [5, 18, 21, 39, 44]; and diverse groups, for example based on demographic or clinical characteristics, may need to be included in research priority setting [5, 18, 20, 36, 37, 39, 40, 42]. It has been emphasized that patients/caregivers (and if relevant the public) need to be directly involved in the priority setting process [5, 18, 38–40, 42], as they have direct experience of the health condition or context and often have different priorities to researchers and clinicians. There is also recognition of the need to involve individuals from vulnerable or marginalized groups, particularly in equity-focused research priority setting exercises [5, 12, 21, 36, 39]. some priority setting exercises, the initial list of research priorities is derived from literature or existing data ra- ther than consultation or engagement of stakeholders [47–49]. If a wide range of different initial research priorities are submitted or identified, it can be challenging to manage and synthesise to capture the diversity of views in a concise manner, whilst also retaining the context and nuances of the submissions. They may need to be organized, usually by collating and categorizing them into themes, topics or other relevant taxonomy [5, 22, 36, 38, 39, 41]; and by removing those that are “out-of- scope,” [5] or duplicative [39]. They may then be trans- lated into “indicative, researchable questions” [5] and edited for clarity [21, 27, 35, 36, 38]. Some priority setting exercises conduct cross checking of the priorities against the evidence (i.e. systematic reviews [5]) and evidence mapping [48, 50–52]. Multiple strategies may be used to engage stakeholders in the priority setting process, and this is namely through partnership with relevant stakeholder organiza- tions [5, 39]. The number and characteristics of the participants involved enables assessment of the degree of inclusivity, diversity and equity [5, 13, 39] in priority setting processes. The characteristics to specify may in- clude role and expertise, discipline, organizational affilia- tions, demographics (e.g. age, sex, socio-economics status, ethnicity), and clinical factors [13, 21–23, 36, 42, 44]. Sup- port for patients/caregivers involved in priority setting may include reimbursement for travel, arranging care for dependents, and time [5]. Prioritization of research topics/questions (items 23–24) Prioritization of research topics/questions (items 23 24) Prioritization techniques can include scoring, ranking, voting, and ordering, and these are usually embedded in similar methods and modes used for collecting priorities as outlined in Section E. Some frameworks and priority setting exercises use explicit criteria to prioritize ques- tions [13, 21, 22, 38, 39]. Examples include condition- related criteria (burden of disease, variation in care and outcome, evidence gaps), and research-related criteria (resources required, likelihood of success and impact) [37, 42]. The CHNRI method proposes criteria includ- ing: answerability, attractiveness (likely to be published in high-impact journals), novelty, potential for transla- tion, effectiveness (likely to identify better interventions), affordability, deliverability, sustainability, public opinion (acceptability to the general public), equity (leads to in- terventions that will be accessible to marginalized or vul- nerable populations), and cost and feasibility [27, 41]. Using specific criteria can facilitate a deliberative and ra- tional process, particularly when there is limited infor- mation [21]. It may be relevant to report the processes for selecting, defining and changing the criteria. Of note, the use of criteria can add complexity to the process, and strategies may be needed to avoid inadvertent exclusion of other stakeholder values that influence prioritization. Whilst assigning scores based on such cri- teria may be rational, there are concerns that it may give a false sense of objectivity. The method for excluding priorities at this stage i.e. based on a quantified thresh- old or other criteria should be provided. Any processes to appeal or challenge the results may be specified. Stakeholders or participants (items 12–16) Stakeholder involvement in priority setting can vary across the priority setting exercises. In some cases, they are involved in all key stages of the process and in others, they are consulted in specific steps and existing data or documents are used instead of consultation. Relevant stakeholders whose “values and interests should Table 2 Summary of frameworks for conducting health research priority setting Framework Year Organizationa Countrya Principles/values/ characteristics Stakeholders Scope Outline of process Output James Lind Alliance (JLA) [5] 2004 National Institute for Health Research (NIHR) UK Partnership Patients, caregivers, clinicians Diagnosis, intervention, care and support Gather priorities (survey) Process and verify Conduct Interim priority setting (survey) Conduct final priority setting (workshops using nominal group technique) Top 10 research questions for funders Council on Health Research for Development Essential National Health Research (COHRED/ENHR) [39] 2000 Council on Health Research for Development International Inclusivity, involvement of a broad range of stakeholders, multidisciplinary and cross- sectorial, partnership, par- ticipatory and transparent, systematic analysis of health needs, societal and profes- sional expectations Researchers, decision- makers, health ser- vice pro- viders, communities – Establish criteria Identify research areas (brainstorming, voting, nominal group technique, roundtable etc) Score against criteria (survey) – Child Health and Nutrition Research Initiative (CHNRI) [41, 46] 2007 Global Forum for Health Research International Systematic, fair, transparent Investors in health research, researchers, general public Fundamental, translation, implementation Discuss criteria Select useful and important criteria Score against criteria (survey) Elicit stakeholder input (reference group) Adjust scores with stakeholder input – Dialogue Model [36] 2007 VU University The Netherlands Participatory, respect for experiential knowledge, dialogue between different stakeholders, emergent and flexible design Patients, researchers, health professionals Explore (informal discussion) Consult (separate stakeholder consultations, focus groups, interviews, other methods) Prioritize (survey, focus group, Delphi technique) Integrate (meeting) – Table 2 Summary of frameworks for conducting health research priority setting Framework Year Organizationa Countrya Principles/values/ characteristics Stakeholders Scope Outline of process Output James Lind Alliance (JLA) [5] 2004 National Institute for Health Research (NIHR) UK Partnership Patients, caregivers, clinicians Diagnosis, intervention, care and support Gather priorities (survey) Process and verify Conduct Interim priority setting Top 10 research questions for funders ble 2 Summary of frameworks for conducting health research priority setting Child Health and Nutrition Research Initiative (CHNRI) [41, 46] 2007 Global Forum for Health Research Dialogue Model [36] 2007 VU University The Netherlands Page 7 of 11 Page 7 of 11 Tong et al. Stakeholders or participants (items 12–16) This may indicate to readers the degree to which the team was able to ensure inclusivity across the different groups. Of note, there is recognition that attention must be given to power dynamics, otherwise the engagement of disadvantaged and marginalized groups may lead to “presence without voice and voice without in- fluence.” [2] Therefore, it may be relevant to acknowledge and discuss how hierarchies and “asymmetries between stakeholders” [36] are addressed to maximize constructive and balanced interaction. For example, some groups, such as patients, may require additional time, training, resources, or other strategies to be able to engage; to have the opportunity to contribute meaningfully [36]. The number of research priorities identified at each stage vary widely [24]. Generally, 10 to 20 questions/ topics are included in the final set of priorities [5, 23–26, 38, 40]. Identification and collection of research priorities (items 17– 22) Different methods and approaches are available for col- lecting and selecting initial research priorities from stakeholders and developing the first list of priorities. This can be one or a combination of methods including interviews, focus groups, workshops, and surveys; and consensus methods (e.g. Delphi survey, nominal group technique); and these may be conducted through various modes such as face-to-face or online [5, 13, 23, 25–28, 37–39]. Documents such as systematic reviews, technical data, and other relevant reports may be used to identify the initial list of priorities [5, 13, 18, 21, 24, 39, 40]. In Page 8 of 11 Page 8 of 11 Tong et al. BMC Medical Research Methodology (2019) 19:243 Tong et al. BMC Medical Research Methodology (2019) 19:243 Implementation (items 28–29) Strategies to implement the research priorities could in- volve informing and garnering support from govern- ment, policy makers, and funding agencies to allocate funding and resources toward the priorities identified [13, 19, 22, 39], and working with researchers to develop proposals [5, 19, 39]. Assessing the impact of research priority setting is challenging but needs to be considered [5, 22]. This may include the impact on decision- making, allocation of funding and resources, and re- search output [18, 19, 38]. The REPRISE guideline may be used as a roadmap for reporting research priority setting studies, or to assess reporting of research priority setting studies as has been done in systematic reviews [25, 26]. REPRISE is focussed on the reporting of process or conduct and does not ad- dress in detail the values and criteria for establishing priorities, though these may be described in reporting the process of prioritizing research topics/questions. We did not conduct a Delphi survey, which has been used in other reporting guidelines to prioritize and achieve consensus on what reporting items should be included [30, 31]. Instead, we sought to be comprehensive, in- cluded all reporting items, and did not eliminate any Output (item 25) sources of funding and support is usually required. The resources required for research priority setting will de- pend on the size, scope, timeline, methods used, and personnel required [5, 19], and providing information about the budget may be useful for others who are plan- ning on conducting research priority setting. It is recom- mended that any relevant disclosures be stated for transparency, to allow assessment of potential political or commercial influences or undue bias [5, 13, 24]. For example, pharmaceutical companies may have close ties with patient organisations and clinicians, and the poten- tial influence this may have on the priority setting process would need to be addressed explicitly. This may be declared at an individual level, or at a process or con- textual level, for example, providing a narrative of any political issues, conflict or controversies that may affect the process, output or implementation of the priority setting exercise [20]. The output should be “clear and of value to the research community.” [5] The final priorities generated can range from having a specific structure i.e. the Population, Intervention, Comparator, Outcome (PICO) format [5, 37], to broader outputs such as topics or themes/areas [38]. It is possible that components of PICO are not spe- cified in original submission of priorities, or that it can- not be applied to some types of research questions [5]. In some circumstances, attempting to produce very technical research questions can potentially place non- researchers, who may include community members, pa- tients, caregivers, at a disadvantage, as they may feel un- able to articulate or consider the specific technical components. Also, consideration may need to be given to ensure that the contextual data and values around the questions are not missed. Some priority setting exercises seek to identify broader themes or areas and translate these into research questions after prioritization. Discussion The REPRISE Guideline is intended to facilitate trans- parent and comprehensive reporting of research priority setting studies that involve stakeholders. The guideline has 31 reporting items that cover 10 domains: context and scope, governance and team, framework for priority setting, stakeholders/participants, identification and col- lection of research priorities, prioritization of research topics, output, evaluation and feedback, translation and implementation, and funding and conflict of interest. The REPRISE guideline is flexible without being unduly prescriptive because different approaches for health re- search priority setting are necessary to ensure they are contextually appropriate, respect the underpinning values and criteria, and are feasible based upon resources available. By piloting the guideline with a broad selection of research priority setting studies, we have demon- strated the feasibility, acceptability and relevance of the REPRISE reporting guidelines. We emphasise that RE- PRISE is not designed for making judgements about the quality of the conduct in research priority setting studies. Evaluation and feedback (items 26–27) While there is no “gold standard” [37] approach for evaluating the process of research priority setting, process evaluation can provide information about the ac- ceptability, “reliability and usefulness” [37] of the process and results [5, 13, 37]. Stakeholder satisfaction with the process in terms of being able to engage and express opinions, and whether the priorities are considered meaningful and valid may be evaluated [13, 18]. Partici- pants and stakeholders could have an opportunity to re- view and provide feedback on the prioritized questions [22, 36, 39]; and having “revision or appeal” [18] mecha- nisms available to identify and address disagreements in a constructive manner [18] have been suggested. Competing interests Competing interests Allison Tong is a member of the Editorial Board of BMC Medical Research Methodology. The authors declare that they have no other competing interests. Funding AT is supported by a National Health and Medical Research Council Fellowship (APP1106716). The funding organization had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. Conclusions The REPRISE guideline has the potential to improve transparency in reporting research priority setting stud- ies. Improved explicitness in how research priority set- ting studies are conducted could strengthen legitimacy, confidence, and acceptability of the findings, and thereby support the implementation and impact of these efforts. Authors’ contributions AT contributed to the conception and design, acquisition of the data, analysis and interpretation of the data, and drafted the manuscript. AS contributed to the conception and design, acquisition of the data, analysis and interpretation of the data, and drafted the manuscript. SC contributed to the conception and design, acquisition of the data, analysis and interpretation of the data. SH contributed to the conception and design and interpretation of the data. AM acquisition of the data, analysis and interpretation of the data. NSR contributed to the acquisition of the data, analysis and interpretation of the data. SO contributed to design, acquisition of the data, analysis and interpretation of the data. KC contributed to the interpretation of the data. MN contributed to the design, analysis and interpretation of the data. SB contributed to the interpretation of the data. TG contributed to the interpretation of the data. AB contributed to the interpretation of the data. JCC contributed to the conception and design, interpretation of the data. All authors revised the article critically for important intellectual content. All authors read and approved the final manuscript. We acknowledge that there may be other potentially relevant items that could warrant further discussion, consideration, and evidence to support their inclusion in subsequent revisions of this framework. The items ad- dressing diversity and hierarchies amongst group mem- bers and the networks they represent, the criteria and degree of formality in decision making processes, and the medium of communication for sharing information and making decisions are all factors affecting good group decision making [53].. Additional factors, not addressed by the priority setting literature, are the size of a group making decisions, the time available for them to explore their knowledge to make choices or solve problems and the facilitation skills for managing constructive conflict. We seek further feedback from researchers, end-users and other stakeholders, to inform future efforts to refine and revise the guideline as needed. Funding and conflict of interest (items 30–31) There are different sources of funding that can affect a priority setting process. The funding and resources used to conduct the priority setting exercise and support the stakeholders (directly or indirectly) and whether the pri- ority setting exercise is connected to a funding source to support the identified research priorities. Reporting the Page 9 of 11 Page 9 of 11 Page 9 of 11 Page 9 of 11 Tong et al. BMC Medical Research Methodology (2019) 19:243 Tong et al. BMC Medical Research Methodology (2019) 19:243 Tong et al. BMC Medical Research Methodology (2019) 19:243 items based on judgement about relevance or import- ance. We believe this increases the practical utility of the REPRISE checklist considering the diverse range of methods and approaches that are used for research pri- ority setting exercises unlike other reporting guidelines which are based on study designs in one particular domain. items based on judgement about relevance or import- ance. We believe this increases the practical utility of the REPRISE checklist considering the diverse range of methods and approaches that are used for research pri- ority setting exercises unlike other reporting guidelines which are based on study designs in one particular domain. Consent for publication Not applicable. Consent for publication Not applicable. Author details 1Sydney School of Public Health, University of Sydney, Sydney, NSW, Australia. 2Centre for Kidney Research, The Children’s Hospital at Westmead, Westmead NSW, Sydney 2145, Australia. 3Centre for Health Communication and Participation, School of Psychology and Public Health, La Trobe University, Melbourne, Victoria, Australia. 4Cochrane Australia, School of Public Health and Preventive Medicine, Monash University, Melbourne, Victoria, Australia. 5Crowe Associates Ltd, Oxon, UK. 6Institute of Education, University College London, London, UK. 7Faculty of Humanities, University of Johannesburg, Johannesburg, South Africa. 8Katherine Cowan Consulting Ltd, East Sussex, UK. 9Peninsula Dental School, University of Plymouth, Plymouth UK 10The George Institute for Global Health New Delhi India Ethics approval and consent to participate Not required. Ethics approval and consent to participate Not required. Abbreviations 1. Macleod MR, Michie S, Roberts I, Dirnagl U, Chalmers I, Ioannidis JP, Al-Shahi Salman R, Chan AW, Glasziou P. Biomedical research: increasing value, reducing waste. Lancet. 2014;383(9912):101–4. CHNRI: Child Health and Nutrition Research Initiative; CINAHL: Cumulative Index for Nursing and Allied Health Literature; COHRED: Council on Health Research for Development; ENHR: Essential National Health Research; EQUATOR: Enhancing the Quality and Transparency of Health Research; JLA: James Lind Alliance; PCORI: Patient-centered outcomes research institute; REPRISE: Reporting guideline for priority setting of health research; WHO: World Health Organization 1. Macleod MR, Michie S, Roberts I, Dirnagl U, Chalm Salman R, Chan AW, Glasziou P. Biomedical resea reducing waste. Lancet. 2014;383(9912):101–4. 2. Pratt B. Towards inclusive priority-setting for global health research projects: recommendations for sharing power with communities. Health Policy Plan. 2019. https://doi.org/10.1093/heapol/czz1041 (ahead of print). 3. Tallon D, Chard J, Dieppe P. Relation between agendas of the research community and the research consumer. Lancet. 2000;355(9220):2037–40. 4. Crowe S, Fenton M, Hall M, Cowan K, Chalmers I. Patients', clinicians' and the research communities' priorities for treatment research: there is an important mismatch. Res Involv Engagem. 2015;1:2. Supplementary information Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s12874-019-0889-3. Additional file 1. Search strategies. Additional file 2. Search results. Additional file 3. Research priority setting: frameworks and reviews. Additional file 4. Preliminary REPRISE guideline. Additional file 5. Sources contributing to the reporting items. Additional file 6. Results of the pilot test. pp y Supplementary information accompanies this paper at https://doi.org/10. 1186/s12874-019-0889-3. Supplementary information accom Plymouth, UK. The George Institute for Global Health, New Delhi, India. 11College of Medicine and Public Health, Flinders University, Adelaide, South Australia, Australia. Received: 26 August 2019 Accepted: 17 December 2019 1. Macleod MR, Michie S, Roberts I, Dirnagl U, Chalmers I, Ioannidis JP, Al-Shahi Salman R, Chan AW, Glasziou P. Biomedical research: increasing value, reducing waste. Lancet. 2014;383(9912):101–4. 2. Pratt B. Towards inclusive priority-setting for global health research projects: recommendations for sharing power with communities. Health Policy Plan. 2019. https://doi.org/10.1093/heapol/czz1041 (ahead of print). 3. Tallon D, Chard J, Dieppe P. Relation between agendas of the research community and the research consumer. Lancet. 2000;355(9220):2037–40. 4. Crowe S, Fenton M, Hall M, Cowan K, Chalmers I. Patients', clinicians' and the research communities' priorities for treatment research: there is an important mismatch. Res Involv Engagem. 2015;1:2. Availability of data and materials Not applicable. All guidelines and publications used for this article are available in the public domain. Acknowledgements Not applicable. Page 10 of 11 Page 10 of 11 Page 10 of 11 Tong et al. BMC Medical Research Methodology (2019) 19:243 Tong et al. BMC Medical Research Methodology (2019) 19:243 5. JLA: The James Lind Alliance Guidebook Version 8 November 2018. In. Southampton, UK: National Institute for Health Research Evaluation, Trials and Studies Coordinating Centre University of Southampton; 2018. 5. JLA: The James Lind Alliance Guidebook Version 8 November 2018. In. Southampton, UK: National Institute for Health Research Evaluation, Trials and Studies Coordinating Centre University of Southampton; 2018. 28. PCORI: Workshop on methods for setting research priorities. Executive summary available at https://www.pcori.org/sites/default/files/Workshop-on- Methods-for-Setting-Research-Priorities1.pdf (accessed 24th July 2019). In. Baltimore: Patient-Centered Outcomes Research Institute Methodology Committee; 2012. 6. Corner J, Wright D, Hopkinson J, Gunaratnam Y, McDonald JW, Foster C. The research priorities of patients attending UK cancer treatment centres: findings from a modified nominal group study. Br J Cancer. 2007;96(6):875–81. 29. Greenhalgh T, Hinton L, Finlay T, Macfarlane A, Fahy N, Clyde B, Chant A. Frameworks for supporting patient and public involvement in research: systematic review and co-design pilot. Health Expect. 2019:1–17. 7. Jun M, Manns B, Laupacis A, Manns L, Rehal B, Crowe S, Hemmelgarn BR. Assessing the extent to which current clinical research is consistent with patient priorities: a scoping review using a case study in patients on or nearing dialysis. Can J Kidney Health Dis. 2015;2:35. 30. EQUATOR: Toolkit. How to develop and reporting guidelines. Available at http://www.equator-network.org/toolkits/developing-a-reporting-guideline/ (Accessed 1 February 2019). In. Oxford, UK: Enhancing the QUAlity and Transparency Of health Research (EQUATOR) Network; 2018. nearing dialysis. Can J Kidney Health Dis. 2015;2:35. 8. Nasser M, Clarke M, Chalmers I, Brurberg KG, Nykvist H, Lund H, Glasziou P. What are funders doing to minimise waste in research? Lancet. 2017; 389(10073):1006–7. 31. Moher D, Schulz KF, Simera I, Altman DG. Guidance for developers of health research reporting guidelines. PLoS Med. 2010;7(2):e10000217. 9. Ferri M, Davoli M. D'Amico R.\: involving patients in setting the research agenda in drug addiction. BMJ. 2013;347:f4513. 32. Reveiz L, Elias V, Terry RF, Alger J, Becerra-Posada F. Comparison of national health research priority-setting methods and characteristics in Latin America and the Caribbean, 2002-2012. Rev Panam Salud Publica. 2013;34(1):1–13. 10. Liberati A. Need to realign patient-oriented and commercial and academic research. Lancet. 2011;378(9805):1777–8. 33. Cochrane Colloquium Seoul, Korea. (2016) Improving transparency and accountability okf priority setting exercised: time for a reporting guideline? Acknowledgements Not applicable. On being a good listener: setting priorities for applied health services research. Milbank Q. 2003;81(3):363–88. 18. Sibbald SL, Singer PA, Upshur R, Martin DK. Priority setting: what constitutes success? A conceptual framework for successful priority setting. BMC Health Serv Res. 2009;9:43. 39. Okello D, Chongtrakul P. A manual for research priority setting using the ENHR strategy. Durban: The Council on Health Research fo Development (COHRED); 2000. 19. Montorzi G, De Haan S, Ijsselmuiden C. Priority setting for research for health. A management process for countries. In. Durban: Council on Health Research for Development; 2010. 40. Ranson MK, Benett SC. Priority setting and health policy and systems research. Health Res Policy Syst. 2009;7:27. 20. Nasser M, Welch V, Tugwell P, Ueffing E, Doyle J, Waters E. Ensuring relevance for Cochrane reviews: evaluating processes and methods for prioritizing topics for Cochrane reviews. J Clin Epidemiol. 2012;66(5): 474–82. 41. Rudan I, Gibson JL, Ameratunga S, El Arifeen S, Bhutta ZA, Black M, Black RE, Brown KH, Campbell H, Carneiro I, et al. Setting priorities in global child health research investments: guidelines for implementation of CHNRI method. Croat Med J. 2008;49(6):720–33. 21. Badakhshan A, Arab M, Rashidian A, Gholipour M, Mohebbi E, Zendehdel K. Systematic review of priority setting studies in health research in the Islamic Republic of Iran. East Mediterr Health J. 2018;24(8):753–69. 42. VanLare JM, Conway PH, Sox HC. Five next steps for a new national program for comparative-effectiveness research. N Engl J Med. 2010;362(11):970–3. 22. Chanda-Kapata P, Ngosa W, Hamainza B, Kapiriri L. Health research priority setting in Zambia: a stock taking of approaches conducted from 1998 to 2015. Health Res Policy Sys. 2016;14(1):72. 43. McGregor S, Henderson KJ, Kaldor JM. How are health research priorities set in low and middle income countries? A systematic review of published reports. PLoS One. 2014;9(10):e108787. 23. Odgers HL, Tong A, Lopez-Vargas P, Davidson A, Jaffe A, McKenzie A, Pinkerton R, Wake M, Richmond P, Crowe S, et al. Research priority setting in childhood chronic disease: a systematic review. Arch Dis Child. 2018; 103(10):942–51. 44. Swingler GH, Irlam JH, Macharia WM, Tietche F, Meremikwu MM. A systematic review of existing national priorities for child health research in sub-Saharan Africa. Health Res Policy Sys. 2005;3:7. 45. Synnot A, Bragge P, Lowe D, Nunn JS, O'Sullivan M, Horvat L, Tong A, Kay D, Ghersi D, McDonald S, et al. Acknowledgements Not applicable. Available at http://2016.colloquium.cochrane.org/workshops/improving- transparency-and-accountability-priority-setting-exercises-time-reporting (accessed 2nd July 2019). In. 11. Richards T, Montori VM, Godlee F, Lapsley P, Paul D. Let the patient revolution begin. BMJ. 2013;346:f2614. 12. Bhaumik S, Rana S, Karimkhani C, Welch V, Armstrong R, Pottie K, Dellavalle R, Dhakal P, Oliver S, Francis DK, et al. Ethics and equity in research priority- setting: stakeholder engagement and the needs of disadvantaged groups. Indian J Med Ethics. 2015;12(2):110–3. 34. Synnot A, Tong A, Craig JC, Hills S: Practical approaches to undertaking research priority setting in health, Workshop program and materials available at https://www.latrobe.edu.au/__data/assets/pdf_file/0009/687339/ ACC_Nov_15_prioritysettingworkshop_program_vFINAL_251115.pdf (Accessed 24th July 2019. In. Australian Cochrane Symposium. 25–26 November. Melbourne, Australia; 2015. 13. Viergever RF, Olifson S, Ghaffar A, Terry RF. A checklist for health research priority setting: nine common themes of good practice. Health Res Policy Sys. 2010;8:36. 14. Bryant J, Sanson-Fisher R, Walsh J, Stewart J. Health research priority setting in selected high income countries: a narrative review of methods used and recommendations for future practice. Cost Eff Resour Alloc. 2014;12:23. 35. Nasser M, Baumik S, Tong A, Welsh E, Synnot A, Crowe S, Morley R, Pardo Pardo J: Practical approaches to conducting an evidence-informed research priority setting exercise. Cochrane Colloquium 2015 Vienna, Australia available at https://abstracts.cochrane.org/2015-vienna/practical-approaches- conducting-evidence-informed-research-priority-setting-exercise (Accessed 1 February 2019). In. London, UK; 2015. 15. Oliver S, Gray J. A bibliography of research reports about patients', clinicians' and researchers' priorities for new research. In. James Lind Alliance: London; 2006. 16. Stewart RJ, Caird J, Oliver K, Oliver S. Patients' and clinicians' research priorities. Health Expect. 2011;14(4):439–48. 36. Abma TA, Broerse JEW. Patient participation as dialogue: setting researchagendas. Health Expect. 2010;13:160–73. 17. Tomlinson M, Chopra M, Hoosain N, Rudan I. A review of selected research priority setting processes at national level in low and middle income countries: towards fair and legitimate priority setting. Health Res Policy Syst. 2011;9:19. 37. Dubois RW, Graff JS. Setting priorities for comparative effectiveness research: from assessing public health benefits to being open with the public. Health Aff. 2011;30(12):2235–42. 37. Dubois RW, Graff JS. Setting priorities for comparative effectiveness research: from assessing public health benefits to being open with the public. Health Aff. 2011;30(12):2235–42. 38. Lomas J, Fulop N, Gagnon D, Allen P. On being a good listener: setting priorities for applied health services research. Milbank Q. 2003;81(3):363–88. 38. Lomas J, Fulop N, Gagnon D, Allen P. Acknowledgements Not applicable. Research priorities in health communication and participation: international survey of consumers and other stakeholders. BMJ Open. 2018;8(5):e019481. 24. Rylance J, Pai M, Lienhardt C, Garner P. Priorities for tuberculosis research: a systematic review. Lancet Infect Dis. 2010;10(12):886–92. 25. Tong A, Chando S, Crowe S, Manns B, Winkelmayer WC, Hemmelgarn B, Craig JC. Research priority setting in kidney disease: a systematic review. Am J Kidney Dis. 2015;65(5):674–83. 46. Rudan I. Global health research priorities: mobilizing the developing world. Public Health. 2012;126:237–40. 47. Dear RF, Barratt AL, Evans A, Simes J, Newsom J, Kent D, Crossing S, Holliday C, Segelov E, Hruby G, et al. Identifying and prioritising gaps in colorectal cancer trials research in Australia. Med J Aust. 2012;197(9):507–11. 26. Tong A, Sautenet B, Chapman JR, Harper C, MacDonald P, Shackel N, Crowe S, Hanson CS, Hill S, Synnot A, et al. Research priority setting in organ transplantation: a systematic review. Transpl Int. 2017;30(4):327–43. 48. Gierisch JM, Myers ER, Schmit KM, Crowley MJ, McCrory DC, Chatterjee R, Coeytaux RR, Kendrick A, Sanders GD. Prioritization of research addressing management strategies for ductal carcinoma in situ. Ann Intern Med. 2014; 160(7):484–91. 27. Terry RF, Charles E, Purdy B, Sanford A. An analysis of research priority- setting at the World Health Organization - how mapping to a standard template allows for comparison between research priority-setting approaches. Health Res Policy Syst. 2018;16(1):116. Page 11 of 11 Tong et al. BMC Medical Research Methodology (2019) 19:243 Tong et al. BMC Medical Research Methodology (2019) 19:243 49. Arora NK, Mohapatra A, Gopalan HS, Wazny K, Thavaraj V, Rasaily R, Das MK, Maheshwari M, Bahl R, Qazi SA, et al. Setting research priorities for maternal, newborn, child health and nutrition in India by engaging experts from 256 indigenous institutions contributing over 4000 research ideas: a CHNRI exercise by ICMR and INCLEN. J Glob Health. 2017;7(1):011003. 50. Lindson N, Richards-Doran D, Heath L, Hartmann-Boyce J. Setting research priorities in tobacco control: a stakeholder engagement project. Addiction. 2017;112(12):2257–71. 51. O'Neill B, Aversa V, Rouleau K, Lazare K, Sullivan F, Persaud N. Identifying top 10 primary care research priorities from international stakeholders using a modified Delphi method. PLoS One. 2018;13(10):e0206096. 52. Zimmerman EB, Cook SK, Haley AD, Woolf SH, Price SK, Berman D, DeLeire T, Etz R, Khalsa J, Knutson K, et al. A patient and provider research agenda on diabetes and hypertension management. Am J Prev Med. 2017;53(1):123–9. 53. Acknowledgements Not applicable. Oliver S, Hollingworth K, Briner B, Swann C, Hinds K, Roche C. Effective and efficient committee work: a systematic overview of multidisciplinary literatures. Evidence Base. 2018;2:1–28. Tong et al. BMC Medical Research Methodology (2019) 19:243 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W2945869885	https://www.int-arch-photogramm-remote-sens-spatial-inf-sci.net/XLII-2-W11/395/2019/isprs-archives-XLII-2-W11-395-2019.pdf	English	null	THE GEO-REFERENCED XIX CENTURY CARTOGRAPHY: AN ANALYSIS TOOL AND A PROJECT REFERENCE FOR THE PRESERVATION AND MANAGEMENT OF BUILT AND LANDSCAPE HERITAGE	The international archives of the photogrammetry, remote sensing and spatial information sciences/International archives of the photogrammetry, remote sensing and spatial information sciences	2,019	cc-by	9,396	The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W11, 2019 GEORES 2019 – 2nd International Conference of Geomatics and Restoration, 8–10 May 2019, Milan, Italy The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W11, 2019 GEORES 2019 – 2nd International Conference of Geomatics and Restoration, 8–10 May 2019, Milan, Italy THE GEO-REFERENCED XIX CENTURY CARTOGRAPHY: AN ANALYSIS TOOL AND A PROJECT REFERENCE FOR THE PRESERVATION AND MANAGEMENT OF BUILT AND LANDSCAPE HERITAGE. A. Cazzani 1 , R. Brumana 2, C.M. Zerbi 3 1 DASTU – Dipartimento di Architettura e Studi Urbani (Department of Architecture and Urban Studies), Politecnico di Milano, Italy – alberta.cazzani@polimi.it 2 DABC – Dipartimento di Architettura, Ingegneria delle costruzioni e Ambiente costruito (Department of Architecture, Built environment and Construction engineering), Politecnico di Milano, Italy – raffaella.brumana@polimi.it 3 PhD architect freelance, Studio Lunigiana15, Milan, and DASTU – Dipartimento di Architettura e Studi Urbani (Department of Architecture and Urban Studies), Politecnico di Milano, Italy – c.zerbi@lunigiana15.it Commission VI, WG VI/4 KEY WORDS: Preservation, Built heritage, Historic landscape, Innovative map tools, GEOPAN ATL@S APP, Permanencies, Transformations ABSTRACT: Considering built and landscape heritage, the paper demonstrates how it is necessary to conserve the historic stratification and to define new compatible plans and uses, identifying the ways to mitigate alteration impacts, requalify degraded areas, enhance natural, historic and cultural values, improve documentary and educational options, and manage new tasks and opportunities. Particularly, the historic complexity of built and landscape heritage, and the level of permanence can be recognized and evaluated by comparing multi-temporal historic and current maps, and surveying the present situation in order to define preservation strategies. Innovative tools (open source map registry, open source GIS data management) support the critical analysis of the maps, the representation of historic stratification, the evaluation of conservation levels, and the definition of heritage reuse and management. Moreover, innovative applications based on advanced Virtual Hub, when used to publish historical maps as Open Data (GEOPAN ATL@S APP), allow a larger public of non-expert users (tourists, citizens, bikers, students, etc.) to access the extraordinary richness of the historical map contents, and navigate across urban landscapes. Such APPs are thus becoming instruments of awareness with a strong pro-active capacity to stimulate new design plans encompassing local cultural identity and rediscovering traces of the past.1 Considering built and landscape heritage, the paper demonstrates how it is necessary to conserve the historic stratification and to define new compatible plans and uses, identifying the ways to mitigate alteration impacts, requalify degraded areas, enhance natural, historic and cultural values, improve documentary and educational options, and manage new tasks and opportunities. Particularly, the historic complexity of built and landscape heritage, and the level of permanence can be recognized and evaluated by comparing multi-temporal historic and current maps, and surveying the present situation in order to define preservation strategies. Innovative tools (open source map registry, open source GIS data management) support the critical analysis of the maps, the representation of historic stratification, the evaluation of conservation levels, and the definition of heritage reuse and management. Moreover, innovative applications based on advanced Virtual Hub, when used to publish historical maps as Open Data (GEOPAN ATL@S APP), allow a larger public of non-expert users (tourists, citizens, bikers, students, etc.) to access the extraordinary richness of the historical map contents, and navigate across urban landscapes. Such APPs are thus becoming instruments of awareness with a strong pro-active capacity to stimulate new design plans encompassing local cultural identity and rediscovering traces of the past.1 1 The present work was conceived and written jointly by the three authors. In particular, Alberta Cazzani developed section 3, Raffaella Brumana developed section 4 and Carlotta Maria Zerbi developed section 2. The authors collaborated in the writing of the Abstract, Introduction and Conclusions, and for the final revision of the manuscript. This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLII-2-W11-395-2019 \| © Authors 2019. CC BY 4.0 License. 395 Corresponding author 1. INTRODUCTION The most important monuments and historic centers are connected with architectures and cultural sites, parks and gardens, road systems, hydrographic features, agricultural settlements and natural/scenic areas. Natural and cultural features must be analyzed to manage, valorize and rehabilitate outstanding and ordinary landscapes in their dynamic changing. Considering the fact that our tangible and intangible heritage also involves social and economical aspects, in 2011 UNESCO adopted the Recommendation defining the “Historic Urban Landscape” as “the urban area understood as the result of a historic layering of cultural and natural values and attributes, extending beyond the notion of ‘historic centre’ or ‘ensemble’ to include the broader urban context and its geographical setting. This wider context notably includes the site’s topography, geomorphology, hydrology and natural features, its built environment, both historic and contemporary, its infrastructures above and below ground, its open spaces and gardens, its land use patterns and spatial organisation, perceptions and visual relationships, as well as all other elements of the urban structure. It also includes social and cultural practices and values, economic processes and the intangible dimensions of heritage as related to diversity and The concept of monument – as it is well known – has largely expanded during the 20th Century: from including few exceptional items of extraordinary value to built heritage, comprising “minor” works alongside sites of significant architectural and artistic importance, as historical and cultural documents of our past. Because of such new perspective of the value of the past, the preservation domain is being enlarged by a radical increase in the types of artefact considered worthy of protection and conservation. In addition to monumental high- style architecture - the traditional object of preservation - whole new categories of sites are now recognized as equally meritorious: cultural and natural landscapes, historic towns and villages, vernacular buildings and sites, industrial heritage, etc. It should also be remembered that seventy years ago the concept of landscape referred to the aesthetic perception of “Natural Beauty”. Today, this definition expanded to include human activities, so landscape now means “an area as perceived by people, whose character is the result of the action and interaction of natural and/or human factors” (European Landscape Convention, 2000). 1 The present work was conceived and written jointly by the three authors. In particular, Alberta Cazzani developed section 3, Raffaella Brumana developed section 4 and Carlotta Maria Zerbi developed section 2. The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W11, 2019 GEORES 2019 – 2nd International Conference of Geomatics and Restoration, 8–10 May 2019, Milan, Italy The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W11, 2019 GEORES 2019 – 2nd International Conference of Geomatics and Restoration, 8–10 May 2019, Milan, Italy The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W11, 2019 GEORES 2019 – 2nd International Conference of Geomatics and Restoration, 8–10 May 2019, Milan, Italy The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W11, 2019 GEORES 2019 – 2nd International Conference of Geomatics and Restoration, 8–10 May 2019, Milan, Italy Figure 1. Topography of the city of Monza and of I.R. Villa with corresponding Park and surroundings surveyed on site and draw in the scale of 1:10.000 by the I.R. First Lieutenant Engineer Geographer Giovanni Brenna, October 1845. y ( , , ) It is thus evident that, in order to set up a preservation and/or a design project, it is necessary to know the stratification of our heritage, not only the historical and constructive characters, but also the uses and users that followed one another in time. In this regard, the analysis of historical maps and the comparison with the current state are a significant method to recognize the degree of conservation or transformation of the land, and to define management and re-use criteria. This paper specifically considers the case study of the Royal Park of Monza and its context within the Milan metropolitan area, as addressed during a multidisciplinary Preservation Studio course providing geomatics, topography, preservation and conservation knowledge (Laurea Magistrale degree in Architecture, Politecnico di Milano)2. It demonstrates the methodological usefulness of one specific 19th Century historic map (also digitalized), the “Carta Topografica dei Contorni di Milano riconosciuta sul terreno e in parte rilevata e disegnata nella proporzionale scala di 1 a 25.000 dall’I.R. Primo Ingegnere Geografo Pensionato Giovanni Brenna”(i.e. Topographic map of Milan surroundings recognised on the ground and partially measured and drawn in the proportional scale of 1:25.000 by the I.R. First Engineer Geographer Pensioner Giovanni Brenna), usually known as Giovanni Brenna’s Map. Comparing in a comprehensive way – thanks also to innovative tools (open source map registry, open source GIS data management) – the digitalized version of Brenna’s map with other historic maps, the current ones, and a direct survey, makes it possible to recognize a lot of landscape features and historical components. 2 The Architectural Preservation Studio (Prof. Alberta Cazzani and Prof. Raffaella Brumana, with Arch. Carlotta Maria Zerbi PhD, Arch. Sara Caspani and Arch. Anna Turrina) consists of a Preservation course and a Survey Advanced Technique course. 3 The map was georeferenced by GIcarus Lab. of Politecnico di Milano- DABC. in the WGS 84 Web Mercator projection (EPSG:3857). Since the main aim for map georeferencing was its online publication, the spherical form of the Web Mercator projection, due to its simplicity in the calculation, allowed saving many computation cycles and enhance web visualization. The georeferencing was carried out using Ground Control Points measured using OpenStreetMap. The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W11, 2019 GEORES 2019 – 2nd International Conference of Geomatics and Restoration, 8–10 May 2019, Milan, Italy It is also possible to evaluate their level of conservation, transformation and maintenance, estimate at different scales the strengths, weaknesses, opportunities and threats (SWOT analysis), and develop a master plan focused on conserving the historic stratification of the architectural and landscape heritage, discovering and valorizing the traces of the past, by defining compatible reuses for today’s needs and users. Figure 1. Topography of the city of Monza and of I.R. Villa with corresponding Park and surroundings surveyed on site and draw in the scale of 1:10.000 by the I.R. First Lieutenant Engineer Geographer Giovanni Brenna, October 1845. This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLII-2-W11-395-2019 \| © Authors 2019. CC BY 4.0 License. 1. INTRODUCTION The authors collaborated in the writing of the Abstract, Introduction and Conclusions, and for the final revision of the manuscript. 395 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W11, 2019 GEORES 2019 – 2nd International Conference of Geomatics and Restoration, 8–10 May 2019, Milan, Italy 2. THE GEO-REFERENCED GIOVANNI BRENNA’S MAP: A TOOL TO READ AND ANALYZE BUILT AND LANDSCAPE HERITAGE The choice of scale is of particular interest: 1:25.000, large enough to make the size of villa gardens, bridges over rivers or vineyards recognizable. The Brianza territory, characterized by rolling hills and varying landscape, was a uniform system of architectural, landscaping and agricultural interventions; not only isolated and independent monuments, but a single and ordered set, in which villas, service buildings, gardens, tree- lined paths, woods, agricultural crops, enclosures and hunting lodges were strictly related, and in which aesthetic and productive purposes overlapped in an extraordinary landscape complex (Cazzani, 1994). In order to understand Brenna’s maps better, it is necessary to know his historical context. Giovanni Brenna lived in a time of great social and cultural innovations. In the field of cartography, those were the years when the first maps were created with scientific and professional methods. He received his professional training in the cartographic school of the French Army, directly related to the important cartographic school of the Cassini family who drew, between 1750 and 1789, the first geometric map (based on geodesic triangulation measurements) of the entire territory of the French Kingdom (the map was published only in 1815). In Italy, the first scientifically- surveyed map was the “Nuova Carta Geografica dello Stato Ecclesiastico” (i.e. New Geographic Map of the State of the Church), commissioned by Pope Benedict XIV, started by the Jesuit priest Roger Boscovich and finalized, after three years of work, by father Christopher Maire in 1755. The existing cartography so far did not circulate freely and was unfit for lay use; cadastral maps were issued by the Government mostly for fiscal purposes and were only accessible to civil servants. The Teresian cadastre, despite being a geometric particle cadastre and having a low level of geometric accuracy, provided somehow a representation of the territory: buildings, cadastral parcels and corresponding map numbers were indicated, and each individual parcel was hatched and coloured to show different crops, roads, rows, gardens, etc. Interestingly, subsequent 19th Century Lombard cadastral maps did not directly represent the territory but only showed the property borders and scant physical elements: in order to be interpreted, they required related “sommarioni” (registers) that reported more detailed information corresponding to each parcel. As for Lombardy, Giovanni Brenna’s native land, the territory underwent a detailed census of land ownerships during the years of Austrian rule, the so-called “Catasto Teresiano” (i.e. 2. THE GEO-REFERENCED GIOVANNI BRENNA’S MAP: A TOOL TO READ AND ANALYZE BUILT AND LANDSCAPE HERITAGE Teresian cadastre), which took place over a period of more than 40 years, between 1718 and 1755. Since the cadastral maps revealed a low level of precision and geometric reliability, the region was surveyed again between the end of 18th Century and the 1830s, using the latest scientific methods. In those years, the Brera Astronomical Observatory, an institution newly founded in Milan by father Boscovich and other Jesuit priests, got involved in the task. Between 1788 and 1791, upon request of the Austrian Government, the Brera astronomers mapped the area surrounding Milan and Mantua, which were then subject to the Austrian Empire. The geodesic triangulation network they developed was subsequently reused, its resolution was improved and its extent was broadened to cover the whole Lombard territory: so the “Carta Topografica del Regno Lombardo Veneto” (i.e. Topographic map of the Kingdom of Lombardy–Venetia) was published in 1833 by the Austrian I.R. Istituto Geografico Militare, the same military corps, to which Giovanni Brenna belonged 5. Hence the exceptional importance of Giovanni Brenna’s map. His survey returns an accurate territory representation, which soon proved to be essential for the perception of the variegated Brianza region and its configuration, of which, at the time, there were only general notions. This is particularly important considering the historical moment, characterized by Lombard society rapidly transforming and beginning to invest in industries and infrastructures: the local knowledge was for them a key-element for programming choices and investments. Through appropriate symbols specified in its legend, Brenna’s topographic map shows the land chorography, main and secondary watercourses (rivers, artificial canals, irrigation ditches …), the road network with its classification (roads accessible to vehicles, tree-lined streets, pedestrian paths …), bridges and railways. The map shows municipality boundaries, and meticulously reports, for each urban center, single buildings and the most relevant site designations; in the case of religious buildings, if they represent multi-parish headquarters, a parish see, or a simple prayer oratory. As for parks and gardens, the map returns the precise drawing of their compositional plans. Also the agricultural land is carefully described, showing parcel partitions, different cultivation types (fields, vineyards, rice fields, vegetable gardens, meadows, woods, …), the widespread presence of farmhouses, villas and aulic buildings (casini particolari), and the dense network of local and service roads connecting them. The location of cemeteries, that Napoleonic rules had moved out of urban centers, is also indicated. 2. THE GEO-REFERENCED GIOVANNI BRENNA’S MAP: A TOOL TO READ AND ANALYZE BUILT AND LANDSCAPE HERITAGE 396 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W11, 2019 GEORES 2019 – 2nd International Conference of Geomatics and Restoration, 8–10 May 2019, Milan, Italy The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W11, 2019 GEORES 2019 – 2nd International Conference of Geomatics and Restoration, 8–10 May 2019, Milan, Italy included, while technical data regarding the scale, production and engraver were printed on the map’s free outer margin, and the legend of symbols was not even on the same sheet, but was placed on the map container. The map was originally printed divided into 4 sheets, but an alternative edition was published with the map divided into 9 sheets (a format more suitable for commercial use), each of them named after the most significant city of the illustrated area. The “Carta dei Contorni di Milano” was surely a model Brenna turned to while planning his atlas maps (Donati, 2012). mountainous areas, longer and harder to be measured and drawn. The advent, in 1840, of the railway between Milan and Monza brought rapid and far-reaching changes in the territory, making it necessary to update some map sheets. Therefore, in 1842 new editions of the Monza and Milan surroundings sheets were published, while the release of the Cantù sheet was postponed because of the railway extension towards Como, and the survey works for the Como sheet remained unfinished. In fact, probably due to funding problems, Brenna’s plan remained unfinished compared to the original draft. The Brianza area was completed, though, and is now an extraordinary cartographic tool describing the historic landscape of that territory 4. mountainous areas, longer and harder to be measured and drawn. The advent, in 1840, of the railway between Milan and Monza brought rapid and far-reaching changes in the territory, making it necessary to update some map sheets. Therefore, in 1842 new editions of the Monza and Milan surroundings sheets were published, while the release of the Cantù sheet was postponed because of the railway extension towards Como, and the survey works for the Como sheet remained unfinished. In fact, probably due to funding problems, Brenna’s plan remained unfinished compared to the original draft. The Brianza area was completed, though, and is now an extraordinary cartographic tool describing the historic landscape of that territory 4. 4 For more information about Giovanni Brenna’s life and work, see Cazzani, 2009, Donati, 2012 and Viganò, 1983. 5 Further details about Lombard historical cartography and history of the Lombard landscape can be found in AA.VV., 1984, Bortolotti, 2002 and Negri, 1998. This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLII-2-W11-395-2019 \| © Authors 2019. CC BY 4.0 License. 2. THE GEO-REFERENCED GIOVANNI BRENNA’S MAP: A TOOL TO READ AND ANALYZE BUILT AND LANDSCAPE HERITAGE Born in Milan in 1779, Giovanni Brenna studied Mathematics and Drawing, both civil and military. At the young age of 21, he was employed as a draftsman by the Bureau of the French Corps of Engineers during Napoleon’s domination of Italy. In 1803, he was then recruited by the Corpo Topografico (Topographic Corps) of the Italian War Office. Promoted to First Lieutenant Engineer Geographer in 1810, Brenna continued working for about 30 years in the Topographic Corps, transformed, after the return of the Austrian in 1814, into the Imperial Regio Istituto Geografico Militare (Imperial Royal Military Geographic Institute) of Austrian High Command. Due to poor health conditions, he left the military career around 1830. Once retired, for the following twenty years (he died in 1854), he continued working as a cartographer, dedicating himself to the project of a 50 sheets-atlas of the area including the city of Milan, the Brianza, the city and lake of Como, and the city of Varese with its pleasant surroundings. Between 1833 and 1851 he published the already-mentioned “Carta Topografica dei Contorni di Milano […] “, divided into multiple sheets, each of which named after the most significant city in the corresponding area (Milan and its surroundings, Monza, Bollate, Verano, Cantù, Vaprio, Erba, Oggiono, …). To provide the necessary financial resources for his undertaking, right from the beginning, Brenna looked for subscribers to the maps: those would support the project when it became more and more expensive as the surveying works moved from flat to hilly or Detailed 19th Century maps represent an important resource to read and understand the built and landscape heritage. In particular, cadastral maps and military maps document many historic architectural and landscape components, and also functions and users, enabling a deeper understanding of the complexity of our heritage. Specifically, aforementioned Giovanni Brenna’s map, which was designed by the Lombard cartographer Giovanni Brenna in the mid-19th Century, is a fundamental document for studying the landscape history in the area of Brianza and North of Milan (Figure 1). The value and usefulness of this map are enhanced by the fact that it was recently georeferenced3. This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLII-2-W11-395-2019 \| © Authors 2019. CC BY 4.0 License. 5 Further details about Lombard historical cartography and history of the Lombard landscape can be found in AA.VV., 1984, Bortolotti, 2002 and Negri, 1998. 4 For more information about Giovanni Brenna’s life and work, see Cazzani, 2009, Donati, 2012 and Viganò, 1983. 2. THE GEO-REFERENCED GIOVANNI BRENNA’S MAP: A TOOL TO READ AND ANALYZE BUILT AND LANDSCAPE HERITAGE Lastly, the map indicates the presence of other urban and rural landscape components, such as enclosures (serragli), bird snares (roccoli); different types of industrial activities as brick kilns (fornaci), spinning mills (filande) and mills (mulini); and Another crucial map was published around 1830 by the I.R. Istituto Geografico Militare, that is to say, the “Carta dei Contorni di Milano, in quattro fogli fino a 25.000 metri al Nord e al Sud e a 40.000 metri all’Est e all’Ovest” (i.e. Map of Milan surroundigs, in 4 sheets, up to 25.000 meters North and South, and 40.000 meters East and West), to whose drafting Brenna himself probably took part before retiring. That was an innovative edition, because the cartouche and title that traditionally found place in the same area as the map were not This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLII-2-W11-395-2019 \| © Authors 2019. CC BY 4.0 License. 397 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W11, 2019 GEORES 2019 – 2nd International Conference of Geomatics and Restoration, 8–10 May 2019, Milan, Italy The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W11, 2019 GEORES 2019 – 2nd International Conference of Geomatics and Restoration, 8–10 May 2019, Milan, Italy Figure 2. Extract from the Brenna’s map of the Royal Park of Monza: it should be noted the abundance and precision of graphic details and denominations used to describe the territory’s landscape and architectural characteristics. and design projects. The European Landscape Convention recommends that landscapes to be identified and assessed, should not simply be subjected to judgment, but their features need be described with a view to undertaking projects. The purpose is to highlight multiple, widespread values and potentialities and to define the actions that each site requires in order to ensure widespread landscape quality. The actions might not only be about conservation, but also about regenerating degraded areas or involve substantial innovation (European Landscape Convention, 2000). The case study considered here is the Royal Park of Monza; the purpose of the study is to read and evaluate built and landscape heritage typologies and features involving historic, perceptive and constructive components, and to investigate – comparing old and current maps – the historic stratification. 2. THE GEO-REFERENCED GIOVANNI BRENNA’S MAP: A TOOL TO READ AND ANALYZE BUILT AND LANDSCAPE HERITAGE The case study considered here is the Royal Park of Monza; the purpose of the study is to read and evaluate built and landscape heritage typologies and features involving historic, perceptive and constructive components, and to investigate – comparing old and current maps – the historic stratification. Located north of the city of Monza, and northeast of the Royal Villa, the Royal Park of Monza (688 hectares) is the biggest fenced park in Europe. Requested by Eugène de Beauharnais, Viceroy of Italy, the park was designed, between 1806 and 1808, by the architect Luigi Canonica to extend the already existing Giardini Reali, the English garden of the Royal Villa, and to create a model country estate and a large game preserve for the hunting entertainment of the Viceroy and his court. Architect Canonica expanded the initial complex embedding the 17th Century villas of Counts Durini (Villa Mirabello and Villa Mirabellino) with annexed formal gardens, many farmhouses and mills, an extensive area of woodland and a large stretch of the river Lambro. 19th Century cartographic sources shows how the central part of the park was intended, since the beginning, for agricultural production, thanks to the presence of meadows, rye, corn and wheat fields irrigated with a rich network of artificial canals and ditches, fed by natural springs or by the river. Maps also reveal that there were woodland areas to the north and along the eastern rim, tree-lined avenues and vegetation architectures around buildings (formal gardens, a geometric designed orchard, …) or in support of the hunting entertainment (bird snare, pheasantry, …). An extended network of avenues crossed the park, connecting the most important buildings and providing perspective views on historic architectures or the surrounding landscape. Open to the public since 1919, in the first half of the 20th Century the park went through a period of changes and neglect. In the northern section, the most severely modified area of the park, a big part of the woodland was removed to build, in 1922, the renowned Autodrome, and in 1929 an exclusive golf course, still in business. In the central part, fields were replaced for some years by an Hyppodrome, and most of the buildings were abandoned and even now are no longer used. 2. THE GEO-REFERENCED GIOVANNI BRENNA’S MAP: A TOOL TO READ AND ANALYZE BUILT AND LANDSCAPE HERITAGE The southern part, with the Royal Villa and its garden, is still the best preserved area.6 Nowadays, the park is a very popular venue, used every day by both the population of neighbouring municipalities and by tourists as a leisurely destination in the weekends or during events organised by associations active in the park. Figure 2. Extract from the Brenna’s map of the Royal Park of Monza: it should be noted the abundance and precision of graphic details and denominations used to describe the territory’s landscape and architectural characteristics. all those infrastructural elements that prove how much the Lombard landscape was shaped by human hand, and became urbanised, for example land springs (fontanili), irrigation ditches (rogge), piers (darsene) and terracing (terrazzamenti) (Frigerio et al., 2010). The level of detail and clarity of his survey of landscape compositional elements makes of the Brenna’s map a precise and extremely valuable reference for the reading and studying of historic landscape and garden evolution (Figure 2). For a few specific sites, Brenna provided map enlargements in order to provide better representation of the different components and their relationships with surrounding areas. Besides the extraordinary 1:3000 enlargement of Villa Tittoni site in Desio, it is essential to mention the “Topografia della Città di Monza e I.R. Villa col rispettivo Parco e d’intorni rilevata d’in sul terreno e disegnata nella proporzione di 1 a 10.000 dall’I.R. Primo Tenente Ingegnere Geografo Giovanni Brenna, ottobre 1845” (i.e. Topography of the city of Monza and of Imperial Royal Villa with relative Park and surroundings surveyed on site and drawn in the proportion of 1:10.000 by the Imperial Royal First Lieutenant Engineer Geographer Giovanni Brenna, October 1845), unsurpassed photography of the Monza Royal Villa and its park (Figure 1). This map also had good success among traders and tourists, as it became frequently used by Milanese citizens who would plan, thanks to the recently built railway, little journeys to the nearby city of Monza, the doorway to the green Brianza. Although Brenna is neither a garden designer nor a landscape architect, but a topographer, his awareness and acuity in depicting the features of the area around Milan and Brianza, makes him a very interesting personality, who was able to describe, with great sensitivity and understanding, the landscape and architectural characters of his region. 6 For more information about the Monza Royal Park history, see De Giacomi, 1989, Mezzanotte, 1830 and Rosa, 2009. This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLII-2-W11-395-2019 \| © Authors 2019. CC BY 4.0 License. 3. FROM HISTORIC MAPS TO LANDSCAPE AND BUILT HERITAGE PRESERVATION, AND DESIGN PROPOSALS They could be emphasized with a new project aimed at preserving and enhancing the past, but they can at least be disclosed, through new communication tools. landscape, involving and linking cultural, natural, urban and peri-urban aspects (Figure 4). This research has shown how the use of archival and documentary sources and in particular of historical maps, as well as detailed surveys, is a fundamental condition for approaching a new project in a conscious way, but also for promoting and making known to the public the values and characters of the past that have now been erased or hidden. They could be emphasized with a new project aimed at preserving and enhancing the past, but they can at least be disclosed, through new communication tools. Figure 4. Master Plan proposal for the Royal Park of Monza: Brenna’s map (A) shows the clear presence of different types of crops, paths and water network, while the current aerial view (B) shows mostly meadows. The project wants to re-discover old, often hidden, traces and use them to re-create a new landscape: the old fields drawing is re-created with wood paths; each sector is filled with flowers and vegetable gardens (C-D). Where paths cross a tree or a land art installation, it becomes an exhibition platform or rest area (E). Figure 3. Royal Park of Monza case study: accurate reading and analysis of Brenna’s map (left) and comparison with current situation (right), pointing out the level of decay, transformation, maintenance and functions permanence. peculiarities of this important site clearly readable. The geo- referenced Giovanni Brenna’s map was considered the main reference to understand the complexity of Royal Park of Monza and its context: the accurate and detailed representation, abundance and precision of details and number of toponyms reported in the map, made it possible to underline and recognize the historic structural elements and functions of the site. 3. FROM HISTORIC MAPS TO LANDSCAPE AND BUILT HERITAGE PRESERVATION, AND DESIGN PROPOSALS Specifically, it was possible to identify buildings (villas, rural buildings, mills, doors and gates, other buildings, wall), road networks (main roads, secondary roads, paths), water networks divided into natural water (Lambro River) and artificial water (lake, main canals called roggia, secondary canals), agricultural areas and vegetation (cultivated fields, fields with vineyard, meadows, wood, hunting wood, formal garden, orchards, row of trees, isolated trees) and to compare all these elements with the current situation, recognizing the level of permanence or transformation of the different areas, components and features of the park. The direct survey was necessary to value the conservation grade, identifying the level of decay, transformation, maintenance and the functions (distinguished as original function, new compatible function, new incompatible function, abandoned) as illustrated by the Figure 3. Comparing the situation documented in Giovanni Brenna’s map with the current status it was possible to evaluate the Royal Park of Monza elaborating a SWOT analysis, involving cultural-architectural, landscape-ecological, social and economical aspects, identifying values, critical areas, potentialities in order to delineate a preservation and management strategy (Pereira Roders et al., 2019). It is important to remember that built and landscape heritage is a complex system consisting of elements in relation to each other and to the users: it is an expression of cultural identity that it is necessary to consider while defining a master plan focused on the definition of new compatible plans and uses, identifying the ways to mitigate alteration impacts, requalify degraded areas, enhance natural, historic and cultural values, improve documentary and educational options, manage new projects and opportunities, looking at social needs and functional options. Figure 4. Master Plan proposal for the Royal Park of Monza: Brenna’s map (A) shows the clear presence of different types of crops, paths and water network, while the current aerial view (B) shows mostly meadows. The project wants to re-discover old, often hidden, traces and use them to re-create a new landscape: the old fields drawing is re-created with wood paths; each sector is filled with flowers and vegetable gardens (C-D). Where paths cross a tree or a land art installation, it becomes an exhibition platform or rest area (E). The study of the Royal Park of Monza has demonstrated that many built and landscape heritage components documented by Giovanni Brenna are disappeared or hidden like the heterogeneous cultivation structures, the hunting wood settlement, the complex water and road network. 3. FROM HISTORIC MAPS TO LANDSCAPE AND BUILT HERITAGE PRESERVATION, AND DESIGN PROPOSALS At present, the park displays several conservation problems: the context is profoundly changed from a past agricultural and natural landscape, to the current urban and peri-urban landscape of the city of Milan; the agricultural and wood areas of the park are reduced and simplified; different transformations of the settlements have canceled important historic features, some current uses are incompatible with historic components, many buildings are abandoned or under-utilized, and the present maintenance level is too low, to make the complexity and the To define an active preservation and management plan about built and landscape heritage it is necessary to know the complexity of the site; this requires both direct knowledge from onsite surveys and visits, and indirect knowledge from documentation. Of course, the method used to read and analyze built and landscape heritage is directly linked to the purpose of the analysis. The targets underlie the methods selected, while the documents useful for the analysis and interpretation of the collected information form the basis for creating preservation This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLII-2-W11-395-2019 \| © Authors 2019. CC BY 4.0 License. 398 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W11, 2019 GEORES 2019 – 2nd International Conference of Geomatics and Restoration, 8–10 May 2019, Milan, Italy The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W11, 2019 GEORES 2019 – 2nd International Conference of Geomatics and Restoration, 8–10 May 2019, Milan, Italy The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W11, 2019 GEORES 2019 – 2nd International Conference of Geomatics and Restoration, 8–10 May 2019, Milan, Italy Figure 3. Royal Park of Monza case study: accurate reading and analysis of Brenna’s map (left) and comparison with current situation (right), pointing out the level of decay, transformation, maintenance and functions permanence. landscape, involving and linking cultural, natural, urban and peri-urban aspects (Figure 4). This research has shown how the use of archival and documentary sources and in particular of historical maps, as well as detailed surveys, is a fundamental condition for approaching a new project in a conscious way, but also for promoting and making known to the public the values and characters of the past that have now been erased or hidden. This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLII-2-W11-395-2019 \| © Authors 2019. CC BY 4.0 License. 4. APPS TO PROMOTE CULTURAL IDENTITY TOWARD A PROACTIVE DIMENSION A brokered Virtual Hub approach for the generation of web applications based on historical maps has been experimented (Previtali, 2018): an application (GEOPAN ATL@S APP) was developed to manage different historical maps (national, regional and urban maps) across Eu. It is based on a Virtual Hub adopted to publish historical maps as Open Data (Mazzetti, 2017): a hub works like the applications we use to access when we search and book flights or hotels, merging all the available data coming from the different proprietary sources of data. The development of applications and tools contributing to promote the cultural and local identity needs to be progressively tuned from passive to proactive services in order to better involve planners and citizens in heritage preservation: the vector based digitization of the Brenna’s Map, highlighting the layers upper described, is aimed to rise in this direction the reading of the historical maps by the citizens and professionals. The development of applications and tools contributing to promote the cultural and local identity needs to be progressively tuned from passive to proactive services in order to better involve planners and citizens in heritage preservation: the vector based digitization of the Brenna’s Map, highlighting the layers upper described, is aimed to rise in this direction the reading of the historical maps by the citizens and professionals. The aim is to show how to recognize – thanks also to innovative tools and APP – the historic and cultural significance of our built and landscape heritage and also to consider it like a current urban, architectural, landscape, and social resource. The historic complexity of built and landscape heritage and the level of permanence are recognizable and evaluable comparing multi- temporal historic and current maps and surveying the current situation in order to define preservation strategies. The aim is to show how to recognize – thanks also to innovative tools and APP – the historic and cultural significance of our built and landscape heritage and also to consider it like a current urban, architectural, landscape, and social resource. The historic complexity of built and landscape heritage and the level of permanence are recognizable and evaluable comparing multi- temporal historic and current maps and surveying the current situation in order to define preservation strategies. 4. APPS TO PROMOTE CULTURAL IDENTITY TOWARD A PROACTIVE DIMENSION Nowadays, the added value of the historic cartography in supporting research and analysis of the built environment in support of planning preservation actions is progressively recognized together with the growing of services and tools based on Open Data access to historical information for landscape analysis (Brumana, 2013). The availability of historical maps is increasing. The digitization of map collections by the Archives Bodies in Italy has been massively undertaken, as in the case of the National Archive of Milan that is progressively acquiring and publishing the maps, once digitalized, including the Brenna’s Map here considered: it is the case of the web site ‘Atlante dei Catasti Storici e delle Carte Topografiche della Lombardia’ (i.e. Atlas of the Historical Cadastres and Topographic maps of the Lombardy Region); through the DIVENIRE application it allows users to access the maps owned and conserved by the Archive, even if it is limited to the visualization, without georeferentiation and with no chart acquisition. Or across Europe as well: it is the case of theMaps data (WMTS-Web Map Tiled Services), created in a collaboration between different institutes, among which the Austrian State Archives, owner of the first and second military surveys drawing a picture of Europe in the 19th Century. Digitisation, georeferencing and development of the software to visualise the maps on different systems have been also increasing. The availability of different data sources, as in the case of historical maps, requires nowadays to exploit services to reduce the time of discovery and accessing such data, to maximizing the benefits of a massive use of them by the different users with a positive impact on heritage preservation. A brokered Virtual Hub approach for the generation of web applications based on historical maps has been experimented (Previtali, 2018): an application (GEOPAN ATL@S APP) was developed to manage different historical maps (national, regional and urban maps) across Eu. It is based on a Virtual Hub adopted to publish historical maps as Open Data (Mazzetti, 2017): a hub works like the applications we use to access when we search and book flights or hotels, merging all the available data coming from the different proprietary sources of data. 3. FROM HISTORIC MAPS TO LANDSCAPE AND BUILT HERITAGE PRESERVATION, AND DESIGN PROPOSALS The site lost textures and features that can be re-discovered and re-designed to valorize the old traces, and to create a new composite 399 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W11, 2019 GEORES 2019 – 2nd International Conference of Geomatics and Restoration, 8–10 May 2019, Milan, Italy The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W11, 2019 GEORES 2019 – 2nd International Conference of Geomatics and Restoration, 8–10 May 2019, Milan, Italy The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W11, 2019 GEORES 2019 – 2nd International Conference of Geomatics and Restoration, 8–10 May 2019, Milan, Italy Figure 5. The map search query service supported by the GEOPAN APP. A sample of the georeferenced maps nowadays available on the area selected: the Military Map (Carta Militare del Lombardo-Veneto) and the three historical cadasters (Catasto Teresiano, Catasto Lombardo-Veneto and Cessato Catasto). In order to promote the cultural and local identity and to involve people in heritage preservation it may also be useful to develop apps and innovative tools capable to make the complexity and the richness of our past clear. This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLII-2-W11-395-2019 \| © Authors 2019. CC BY 4.0 License. 4. APPS TO PROMOTE CULTURAL IDENTITY TOWARD A PROACTIVE DIMENSION Nowadays, the added value of the historic cartography in supporting research and analysis of the built environment in support of planning preservation actions is progressively recognized together with the growing of services and tools based on Open Data access to historical information for landscape analysis (Brumana, 2013). The availability of historical maps is increasing. The digitization of map collections by the Archives Bodies in Italy has been massively undertaken, as in the case of the National Archive of Milan that is progressively acquiring and publishing the maps, once digitalized, including the Brenna’s Map here considered: it is the case of the web site ‘Atlante dei Catasti Storici e delle Carte Topografiche della Lombardia’ (i.e. Atlas of the Historical Cadastres and Topographic maps of the Lombardy Region); through the DIVENIRE application it allows users to access the maps owned and conserved by the Archive, even if it is limited to the visualization, without georeferentiation and with no chart acquisition. Or across Europe as well: it is the case of theMaps data (WMTS-Web Map Tiled Services), created in a collaboration between different institutes, among which the Austrian State Archives, owner of the first and second military surveys drawing a picture of Europe in the 19th Century. Figure 5. The map search query service supported by the GEOPAN APP. A sample of the georeferenced maps nowadays available on the area selected: the Military Map (Carta Militare del Lombardo-Veneto) and the three historical cadasters (Catasto Teresiano, Catasto Lombardo-Veneto and Cessato Catasto). Digitisation, georeferencing and development of the software to visualise the maps on different systems have been also increasing. The availability of different data sources, as in the case of historical maps, requires nowadays to exploit services to reduce the time of discovery and accessing such data, to maximizing the benefits of a massive use of them by the different users with a positive impact on heritage preservation. acquisition to the publishing, are offered by the GEOPAN APP, as the map registry, open source webGIS data management, and Hub integration, in order to support the critical analysis of the maps, the representation of the historic stratification, the evaluation of the conservation level, the definition of heritage reuse and management. The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W11, 2019 GEORES 2019 – 2nd International Conference of Geomatics and Restoration, 8–10 May 2019, Milan, Italy The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W11, 2019 GEORES 2019 – 2nd International Conference of Geomatics and Restoration, 8–10 May 2019, Milan, Italy The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W11, 2019 GEORES 2019 – 2nd International Conference of Geomatics and Restoration, 8–10 May 2019, Milan, Italy Figure 7. Royal Park of Monza Itinerary map proposal: a sport ring pedestrian and bicycle itinerary, that connects several buildings currently existing in the park and makes readable traces from the past, documented by Giovanni Brenna’s map. The trails are built in calcestre (stabilized gravel), in three different shades of grey to differentiate existing (racetrack), recovery (old hippodrome) and new paths. large public of non-expert users (tourists, citizens, bikers, students) supporting the access to the extraordinary richness of the historical map heritage contents. Navigation itineraries performed across the rich landscapes and built environment of the Royal Park of Monza have been studied and proposed by the students of the Preservation Studio course. They can be uploaded on the GEOPAN APP and they can be integrated by the design ideas developed during the course. The objective is to foster the large public to choose itineraries of biking or walking across the time, across subjects dense of permanences, still readable, spanning from the ‘water’ theme, the anthropic canalizations (with the primary order represented by the ‘Roggias’ and secondary rich network order) derived from the Lambro river to harvest the cultivation areas, or addressed to feeding the mills presents in the Park, to the road and path network described, till to the ancient cultivated parcels traces, progressively disappeared, that can be re-discovered by the design ideas. Edit tools connected to the geolocation system allow the students, or APP users, to sketch itineraries on site or offsite, saving and exporting them within open source or proprietary GIS tools, or to share them among the visitors (Figure 6). Itinerary maps designed by the students is an outcome that allows citizen to appreciate the multi-temporal fruition of the Park choosing among different thematic itinerary subjects (Figure 7). Virtual museums developed on the built environment can be fed up via multi-temporal geodata hub, exploiting more informed narratives and immersive experiences transferring contents of the past (Brumana, 2018). Figure 7. The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W11, 2019 GEORES 2019 – 2nd International Conference of Geomatics and Restoration, 8–10 May 2019, Milan, Italy Royal Park of Monza Itinerary map proposal: a sport ring pedestrian and bicycle itinerary, that connects several buildings currently existing in the park and makes readable traces from the past, documented by Giovanni Brenna’s map. The trails are built in calcestre (stabilized gravel), in three different shades of grey to differentiate existing (racetrack), recovery (old hippodrome) and new paths. Figure 6. The navigation of the Brenna’s Map and the comparison with the current state of the art (Open Street Map), through the transparency percentage. A sample of the edit tool supporting itinerary map that can be sketched on-or off-site. case of the City of Strasburg that is carrying out an interesting Heritage&Development strategy, connecting the new planning of the ‘Deaux Rives’ to the historical maps): once tagged with the keyword both this cases can be brokered and discovered and accessed by the GEOPAN APP users in the world, together many others samples that could be added in the future: the objective is to make available best practices and case studies to enhance the heritage preservation within the planning activities and within the tourists and citizen making them aware of the richness a past still readable helping to undertake new cases. This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLII-2-W11-395-2019 \| © Authors 2019. CC BY 4.0 License. 4. APPS TO PROMOTE CULTURAL IDENTITY TOWARD A PROACTIVE DIMENSION The GEOPAN APP allows to perform semantic searches based on temporal and spatial subsetting, thanks to the linked endpoints of the Open Data without any duplication and with real time updating. The GEOPAN APP can be progressively implemented by new data sets: in this case different historical maps georeferenced in the area of the Park are available together with the Brenna Map, among which the Military Map (Carta Militare del Lombardo-Veneto) and the three historical cadasters (Catasto Teresiano, Catasto Lombardo-Veneto and Cessato Catasto), as illustrated by the Figure 5. The possibility to access the multi-temporal data published, allows the users to find the different published historical cartographies in the different periods, to read and highlight the transformations, permanencies and mutations respect the current state of the art through their comparison with the current map layers (i.e. Satellite Imagery, Urban Technical Map, Open Street Maps (OSM), UAV Orthophotos). The bet for the future is to transform the APP toward an instrument of awareness, rising with strong pro-active capacity to forge new generation of design plans able to take into account the cultural identity of the places aiming to rediscover the traces of the past; the systematic use of historical maps within the on coming planning and decision making can have an important role within the entire cycle of the Park heritage preservation, from the analysis till to the actions. The Lombardy Region is launching a Master Plan Tender on the Park, and it is including the access to such data sets, as in the case of the Brenna Map. ( ) p ) Innovative tools covering the entire process, from the p At the same time, the APP is also addressed to the fruition by a This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLII-2-W11-395-2019 \| © Authors 2019. CC BY 4.0 License. 400 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W11, 2019 GEORES 2019 – 2nd International Conference of Geomatics and Restoration, 8–10 May 2019, Milan, Italy 5. CONCLUSIONS Just to make an example, the word HUL (Urban Heritage Landscape) can be used to tag different on developing cases (the Case of the Monza Royal Park together with the on going 401 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W11, 2019 GEORES 2019 – 2nd International Conference of Geomatics and Restoration, 8–10 May 2019, Milan, Italy The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W11, 2019 GEORES 2019 – 2nd International Conference of Geomatics and Restoration, 8–10 May 2019, Milan, Italy protagonisti. Italia Settentrionale, Istituto Poligrafico e Zecca dello Stato, Roma, 225-226. more aware of the value of the past. According to the UNESCO Recommendation, now is the time to develop strategies for historic urban landscape management. De Giacomi, F., 1989. Il Parco Reale di Monza, Associazione Pro Monza, Monza. ACKNOWLEDGEMENTS The research leading to the results of this paper is partially funded under the ICT Policy Support Programme (ICT PSP) as part of the Competitiveness and Innovation Framework Programme by the European Community (CIP) GA n°620400. The authors would also thank the following Assistant Professors for researches carried out the within the ENERGIC-OD funded project (Polimi partner Sc. Res. R. Brumana): Dr. Arch. Daniela Oreni for the georeferentiation of the historical cadastres of the case study area and Dr. Ing. Mattia Previtali for the. georeferentiation of the Brenna’s map and the Military Map and for the Geopan APP implementation The research described here has been tested and developed within the Architectural Preservation Studio of the Laurea Magistrale (M.Sc.) degree in Architecture (Architecture - Built Environment - Interiors Programme) - Politecnico di Milano, Prof. Alberta Cazzani and Prof. Raffaella Brumana with Arch. Carlotta Maria Zerbi PhD, Arch. Sara Caspani and Arch. Anna Turrina during the academic years 2017/2018 and 2018/2019, involving over 100 students. Specifically the plans attached here were elaborated by: Ebrahim Abnar, Caterina Cameli, Melis Drogul, Monica Ferraboschi, Ilaria Furbetta, Du Jian, Zhao Jingxuan, Wang Mengwai, Luca Rizzo, Alberto Roncelli and Chiara Tassinari. De la Torre, M., 2002. Assessing the Values of Cultural Heritage: Research Report, The Getty Conservation Institute. Donati, M., 2012. Storie di Imprenditoria Lombarda. Giovanni Brenna, cartografo di Lombardia, Associazione Culturale Casimiro Teja, Eventi s.a.s, Cesano Maderno. Frigerio, F., Mappelli, L. & Viscardi, L., 2010. Il paesaggio agrario canturino: indicazioni per la salvaguardia e la valorizzazione”, Tesi di Laurea in Progettazione dell’Architettura Sostenibile, Facoltà di Architettura e Società, Politecnico di Milano, Relatore Lionella Scazzosi. GEOPAN atl@s APP http://geoserver.atlas.polimi.it/examples/ servlets/Geopan/D/Entry/selector2b.html (Accessed 12.03.2019). ‘Mapire - Historical Maps Online’ geoportal (https://mapire.eu/en/) (Accessed 12.03.2019). Mazzetti, P., Latre, M. Á., Ernst, J., Brumana, R., Brauman, S., & Nativi, S., 2015. Virtual hubs for facilitating access to open data, in EGU General Assembly Conference (Vol. 17). REFERENCES Mezzanotte, G., 1830. L’Imperial Regio Parco presso Monza, Monza. AA.VV., 1984. L’immagine interessata. Territorio e cartografia in Lombardia tra 500 e 800, Archivio di Stato di Milano, Nodo Edizioni, Como. Negri, G., 1998. Comprendere il paesaggio: studi sulla pianura lombarda, Electa, Milano. Archivio di Stato di Milano (http://asmilano.it/Divenire/home .html) (Accessed 12.03.2019). Pereira Roders, A. & Bandarin, F., 2019. Reshaping Urban Conservation. The Historic Urban Landscape Approach in Action, Springer Nature Singapore Pte Ltd. Boriani, M. & Cazzani, A., 2014. Cultural Heritage Preservation in Italy, in Casnati, G., The Politecnico di Milano in Armenia, Oemme Edizioni, Venezia, 33-42. Petzet, M., 2004. Principles of preservation: an introduction to the International Charters for Conservation and Restoration 40 years after the Venice Charter, in AA.VV., International Charters for Conservation and Restoration. Monuments & Sites, I. ICOMOS, München, 7-29. Bortolotti, L., 2002. Storia, città, territorio, Franco Angeli Edizioni, Milano. Brumana, R., Oreni, D., Cuca, B., Rampini, A., Pepe, M., 2013. Open access to historical information for landscape analysis in an SDI framework, International Journal of Agricultural and Environmental Information Systems, 4 (3), 18-40. IGI Global, https://doi.org/10.4018/ijaeis.2013070102, ISSN: 19473192. Previtali, M. & Latre, M.Á., 2018. A brokered Virtual Hub approach for the generation of web applications based on historical maps, Appl Geomat (2018) 10: 453. (https://doi.org/10.1007/s12518-018-0235-1, 453-472). Brumana, R., Oreni, D., Caspani, S., Previtali, M., 2018. Virtual museums and built environment: Narratives and immersive experience via multi-temporal geodata hub, Virtual Archaeology Review, 9 (19), 34-49. (https://doi.org/10.4995 /var.2018.9918). Recommendation on the Historic Urban Landscape, 2011. UNESCO, Paris (https://whc.unesco.org/uploads/activities/ documents/activity-638-98.pdf). Rosa, M., 2009. La Villa, i Giardini e il Parco di Monza nel fondo disegni delle Residenze Reali Lombarde, Skira. Cazzani, A., 1994. Il paesaggio delle grandi residenze briantee nei documenti cartografici e iconografici tra XVIII e XIX secolo: problemi di tutela e conservazione, in I giardini del Principe, Atti del IV Convegno Internazionale - Parchi e giardini storici, parchi letterari, Racconigi, 603-612. Sonkoly, G.., 2017. Historical Urban Landscape, Palgrave Macmillan. European Landscape Convention, 2000, Florence, Italy (https://rm.coe.int/1680080621). Cazzani, A., 2009. Brenna Giovanni, in Cazzato, V., Atlante del Giardino Italiano 1750-1940. Dizionario biografico di architetti, giardinieri, botanici, committenti, letterati e altri Viganò, A., 1983. Per una cartografia briantea: l’opera di Giovanni Brenna, in I quaderni della Brianza, anno VI, 30. This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLII-2-W11-395-2019 \| © Authors 2019. CC BY 4.0 License. This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLII-2-W11-395-2019 \| © Authors 2019. CC BY 4.0 License. 5. CONCLUSIONS The research presented in the paper tests how to recognize the historic and cultural significance of our built and landscape heritage – using historic maps and particularly the Giovanni Brenna’s map – and also to consider it like a current urban, architectural, natural, social resource. Brenna s map – and also to consider it like a current urban, architectural, natural, social resource. It is necessary now to create awareness among citizens, administrations and professionals of the importance of historic stratification before designing a new building/urban plan. In this regard, it is important to take into account that historical sources (documents, maps, surveys) are the main reference for restoration and conservation projects, but are often not considered enough in urban planning and new design projects. The same historical sources are often not known and understood by the public and specifically they are not identified. The development of innovative tools and app will connect the public to the past in an interactive way making the same public more interested in the design and planning process participation and Figure 6. The navigation of the Brenna’s Map and the comparison with the current state of the art (Open Street Map), through the transparency percentage. A sample of the edit tool supporting itinerary map that can be sketched on-or off-site. It is necessary now to create awareness among citizens, administrations and professionals of the importance of historic stratification before designing a new building/urban plan. In this regard, it is important to take into account that historical sources (documents, maps, surveys) are the main reference for restoration and conservation projects, but are often not considered enough in urban planning and new design projects. The same historical sources are often not known and understood by the public and specifically they are not identified. The development of innovative tools and app will connect the public to the past in an interactive way making the same public more interested in the design and planning process participation and The semantic based search tool allows to perform queries across Europe without borders, by integrating keywords within the metadata description supporting the Hub data management referenced to the historical or current open data linked to the hub. REFERENCES 402
W4380684916.txt	https://www.frontiersin.org/articles/10.3389/ftox.2023.1228175/pdf	en		Editorial: Chemical allergy and the relevance of new models	Frontiers in toxicology	2,023	cc-by	1,512	Editorial 14 June 2023 10.3389/ftox.2023.1228175 TYPE PUBLISHED DOI OPEN ACCESS Editorial: Chemical allergy and the relevance of new models EDITED AND REVIEWED BY François Huaux, Université Catholique de Louvain, Belgium CORRESPONDENCE Marc Pallardy 1, Valentina Galbiati 2 and Stefan F. Martin 3 1 Inﬂammation, Microbiome and Immunosurveillance, Inserm, Université Paris-Saclay, Orsay, France, Università degli Studi di Milano, Milano, Italy, 3University of Freiburg Medical Center, Freiburg, Germany 2 Valentina Galbiati, valentina.galbiati@unimi.it 24 May 2023 09 June 2023 PUBLISHED 14 June 2023 KEYWORDS RECEIVED ACCEPTED chemical allergy, toxicology, chemical allergens, adverse health effect, immuno response, sensitisation CITATION Pallardy M, Galbiati V and Martin SF (2023), Editorial: Chemical allergy and the relevance of new models. Front. Toxicol. 5:1228175. doi: 10.3389/ftox.2023.1228175 Editorial on the Research Topic Chemical allergy and the relevance of new models COPYRIGHT © 2023 Pallardy, Galbiati and Martin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Toxicology Chemical allergy represents an adverse health effect that results from an immune response activation following chemical exposure. Among the different forms of chemical allergies, the two most relevant for occupational toxicology are allergic contact dermatitis, and occupational rhinitis and asthma. Chemical allergens are low molecular weight molecules able to activate the immune system. To stimulate an immune response, chemical allergens function as haptens binding to carrier proteins. Several molecules can be involved in chemical allergy either as haptens or immune danger signals such as micro and nano-plastics, particles, pesticides, endocrine disruptors, drugs, fragrances, among others. The initiation and development of chemical allergy phenomena are of considerable interest and importance also for the toxicologist, who has the responsibility of identifying and characterizing the allergenic potential of chemicals and estimating the risk they pose to human health. This Research Topic aimed to combine interdisciplinary evidence on chemical allergy, covering the biological, pharmaceutical, toxicological, and clinical points of view. The purpose was to evaluate experimental evidence supporting the contribution of these interactions in chemical allergy. Allergic contact dermatitis is a widespread T cell-mediated inﬂammatory skin disease, but in vitro monitoring of chemical-speciﬁc T cells remains challenging. The work proposed by Curato et al., introduced short-term CD154/CD137 upregulation for the detection of human TNBS-speciﬁc CD4+ and CD8+ naive and memory T cells, combining a wellestablished technique for the generation of TNBS-induced T cell epitopes with recently developed AIM (activation-induced marker) assays for the detection of protein antigen and nickel-speciﬁc T cells. Peripheral blood mononuclear cells (PBMC) from healthy donor buffy coats were TNBS-modiﬁed and incubated with unmodiﬁed PBMC. Activated cells were sorted for re-stimulation and bulk T cell receptor (TCR) high-throughput sequencing (HTS). Among TNBS-speciﬁc CD4+ T cell clones and lines, 10/13 (77%) responded to TNBS restimulation with CD154 upregulation and the presence of different MHC II blocking antibody clones that prevented T cell activation, further conﬁrm antigen-speciﬁcity and conventional MHC II restriction for TNBS-speciﬁc CD4+ T cells. Results were similar for TNBS-speciﬁc CD137+CD8+ memory T cell clones and lines. In summary, the detection of TNBS-speciﬁc T cells by CD154/CD137 upregulation is a fast, comprehensive and quantitative method. Combined with TCR HTS, the mechanisms of chemical allergen recognition that underlie unusually frequent T cell activation can be assessed. 01 frontiersin.org Pallardy et al. 10.3389/ftox.2023.1228175 upon topical exposure to metals, representing a potential implication for the molecular mechanisms by which contact allergens cause skin sensitization. Since their discovery nearly 40 years ago, B-1 cells have continued to challenge the boundaries between innate and adaptive immunity, as well as myeloid and lymphoid functions. In the Hieronimus and Huaux review, the multifaced role of the B-1 cells is described, highlighting their different roles in both homeostatic and pathological conditions, comprising contactsensitivity-inducing chemicals. In addition to T cells, also B-1 cells are crucial in the development of contact sensitivity (CS) as supported by the evidence that the transfer of CS-B-1 cells is sufﬁcient to induce CS in untreated mice. After skin sensitization, the hapten-self protein conjugates forming the neo antigens can be drained to the peritoneum, where they encounter and activate B-1 cells. Stimulated B-1 cells migrate to the spleen and lymph nodes via IL-4-related signaling and CXCL13 chemoattraction to produce IgM antibodies speciﬁc to the conjugates. This novel IgM circulates in the serum and leads to the recruitment of T cells in lymphoid organs within few hours. Finally, T cells are activated in the spleen by dendritic cells, which are derived from Langerhans cells that have processed neoantigens in the skin. These T cells become CS-effector T cells and return to circulation 4 days after CS initiation. During the second encounter with the chemical, the induced IgM targeting anti-hapten/selfprotein conjugate is already circulating and is thus rapidly found in the skin. IgM produced by B-1 cells activates the classical complement pathway and promotes recruitment of T cells, including CS-effector T cells, to the skin for induction of inﬂammation. Taking into consideration this important evidence about the involvement of B-1 cells in CS, they should not be overlooked when developing new in vitro models for predicting the toxicity of chemical compounds. The immunological response in contact hypersensitivity (CHS) is incited by small electrophilic compounds, known as haptens, which react with endogenous proteins after skin absorption. However, the identity of hapten-modiﬁed proteins seen as immunogenic remains as yet largely unknown. Adduct measurements have mainly focused on the most abundant blood proteins, human serum albumin (Alb) and hemoglobin (Hb) and in the context of CHS, a large number of mainly in vitro studies have been conducted aiming at identifying the most reactive sites of relevant proteins. Despite the extensive mapping of the most reactive sites of relevant proteins, no protein-conjugates have been identiﬁed in vivo with the exception of the hapten-modiﬁed protein in the local lymph nodes of mice treated topically with the model hapten tetramethylrhodamine isothiocyanate (TRITC). The TRITC modiﬁcation was located on the N-terminal proline of the protein macrophage migration inhibitory factor (MIF). Ndreu et al. focused their attention on the investigation about the presence of this hapten-protein conjugate in blood samples from mice treated topically with TRITC, on TRITC modiﬁcations of Alb and Hb, as well as TRITC modiﬁcations of MIF other than the N-terminal proline. A proteomic approach was applied to characterize conjugate formation of the aforementioned proteins, using high resolution mass spectrometry (HRMS). No Hb and Alb conjugates were detected. Quantiﬁcation of both the TRITC-modiﬁed and unmodiﬁed N-terminal peptide of MIF in blood and lymph node samples gave interesting insights of MIF’s role in murine contact hypersensitivity. Incubation of MIF with four different haptens encompassing different reactivity mechanisms and potencies, showed adduct formation at different amino acid residues, suggesting that MIF can be the preferred target for a wide variety of haptens. The study provides essential progress toward understanding of hapten-protein conjugate formation in CHS and identiﬁes hapten-modiﬁed MIF as a potential biomarker for this condition. Lipids are an important constituent of skin and resulted modiﬁed in many skin diseases including psoriasis and atopic dermatitis. The direct effects of common metallic contact allergens on the lipid composition of skin remain an aspect to be better investigated. Knox et al. described the skin lipid proﬁles in the stratum corneum and viable epidermis of ex vivo human skin from a female donor upon exposure to three metal allergens (nickel, cobalt, and chromium). The time-of-ﬂight secondary ion mass spectrometry (ToF-SIMS), which allows for simultaneous visualisation of both the allergen and skin components such as lipids, has been used. Multivariate analysis using partial least squares discriminant analysis (PLS-DA) indicated that the lipid proﬁle of metal-treated skin was different to non-treated skin. Analysis of individual ions led to the discovery that cobalt and chromium induced increases in the content of diacylglycerols (DAG) in stratum corneum. Cobalt also induced increases in cholesterol in both the stratum corneum and viable epidermis, as well as monoacylglycerols (MAG) in the viable epidermis while chromium caused an increase in DAG in viable epidermis in addition to the stratum corneum. In contrast, nickel decreased MAG and DAG levels in viable epidermis. Results obtained in this study indicate that skin lipid content is likely to be altered Frontiers in Toxicology Author contributions This editorial introduction was led by VG. All authors contributed to the article and approved the submitted version. Conﬂict of interest The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. 02 frontiersin.org
https://openalex.org/W3080807994	https://www.frontiersin.org/articles/10.3389/fonc.2020.01016/pdf	English	null	Co-incidence of Human Papillomaviruses and Epstein–Barr Virus Is Associated With High to Intermediate Tumor Grade in Human Head and Neck Cancer in Syria	Frontiers in oncology	2,020	cc-by	7,153	ORIGINAL RESEARCH published: 20 August 2020 doi: 10.3389/fonc.2020.01016 Ishita Gupta 1,2†, Lina Ghabreau 3,4†, Hamda Al-Thawadi 1, Amber Yasmeen 5, Semir Vranic 1, Ala-Eddin Al Moustafa 1,2* and Mohammed I. Malki 1 Ishita Gupta 1,2†, Lina Ghabreau 3,4†, Hamda Al-Thawadi 1, Amber Yasmeen 5, Semir Vranic 1, Ala-Eddin Al Moustafa 1,2* and Mohammed I. Malki 1 1 College of Medicine, QU Health, Qatar University, Doha, Qatar, 2 Biomedical Research Centre, Qatar University, Doha, Qatar, 3 Pathology Department, Faculty of Medicine, University of Aleppo, Aleppo, Syria, 4 Syrian Research Cancer Centre of the Syrian Society Against Cancer, Aleppo, Syria, 5 Segal Cancer Centre, Lady Davis Institute for Medical Research of the Sir Mortimer B. Davis-Jewish General Hospital, Montreal, QC, Canada High-risk human papillomaviruses (high-risk HPVs) have been recently reported to be co-present with Epstein–Barr virus (EBV) in different types of human cancers including head and neck (HN), where they can cooperate in the initiation and/or progression of this cancer. Accordingly, we herein explored the prevalence of high-risk HPVs and EBV in 80 HN cancer tissues from the Syrian population using polymerase chain reaction, immunohistochemistry, and tissue microarray methodologies. We report that high-risk HPVs and EBV are present in 35/80 (43.7%) and 41/80 (51.2%) of our samples, respectively, and the most frequent HPV types are 33, 16, 18, 45, 52, 58, 35, 51, and 31, in this order. More signiﬁcantly, our data reveal that 25/80 (31.2%) of cancer cases are positive for high-risk HPVs as well as EBV, and their co-presence is associated with high/intermediate-grade squamous cell carcinomas. These data conﬁrm the co-presence of high-risk HPVs and EBV in HN cancers in the Syrian population of the Middle East and demonstrate that their co-incidence is linked to a more aggressive cancer phenotype. Thus, future studies are required to conﬁrm these data and elucidate the exact role of high-risk and EBV cooperation in human HN carcinogenesis. Edited by: Stéphane Hans, Hôpital Foch, France Reviewed by: Stefania Staibano, University of Naples Federico II, Italy Gaurisankar Sa, Bose Institute, India Correspondence: Ala-Eddin Al Moustafa aalmoustafa@qu.edu.qa †These authors have contributed Edited by: Stéphane Hans, Hôpital Foch, France Reviewed by: Stefania Staibano, University of Naples Federico II, Italy Gaurisankar Sa, Bose Institute, India Correspondence: Ala-Eddin Al Moustafa aalmoustafa@qu.edu.qa †These authors have contributed equally to this work Specialty section: This article was submitted to Head and Neck Cancer, a section of the journal Frontiers in Oncology Keywords: head and neck cancers, human papillomaviruses, Epstein–Barr virus, tumor grade, Syrian population Received: 06 January 2020 Accepted: 22 May 2020 Published: 20 August 2020 INTRODUCTION Head and neck (HN) cancer is a broad term that incorporates epithelial malignancies located in the paranasal sinuses, oral cavity, nasal cavity, pharynx, and larynx (1). HN cancer is one of the most common among both male and female worldwide, with around 650,000 new cases and 330,000 deaths each year assessed by the World Health Organization (2); notably, most of these deaths occur in developing countries (3). When it comes to cancer-related mortality, it is generally either directly attributed to metastasis, as in tumor involvement of critical organs, or caused indirectly due to therapeutic resistance and the adverse eﬀect of treatment on human organs (4, 5). Immunohistochemistry Immunohistochemical (IHC) procedures examining the expression patterns of E6 and LMP1, of HPV and EBV, were done using standard practices. Brieﬂy, slides were deparaﬃnized in graded alcohol, rehydrated, and boiled in 10 mM citrate buﬀer (pH 6.0) for antigen retrieval. Then, TMA slides were incubated for 35 min at 37◦C with primary monoclonal and polyclonal antibodies for E6 of HPV and LMP1 of EBV (clones 1–4 and clone C1P5 from Dako and Calbiochem, Canada, respectively) using an automated immunostainer (Ventana Medical System, Tuscon, AZ). Afterwards, staining procedures were achieved according to the manufacturer’s recommendations as slides were counterstained with hematoxylin prior to mounting. Negative controls were achieved by omitting primary antibody for E6 and LMP1. Following immunohistochemistry, two independent observers examined all TMA slides. The tumors were considered positive for E6 and LMP1 oncoproteins if cancer cells exhibited positivity ≥1% at any intensity (≥1+, scale 0–3+). On the other hand, several recent studies revealed that high- risk HPVs and EBV are co-present in human HN cancers especially oral (22–24). Moreover, it has been reported that the co-occurrence of high-risk HPVs and EBV in oral cancer is associated with a signiﬁcant increase in the invasiveness ability of cancer cells (25). We recently demonstrated that the co-presence of high-risk HPVs and EBV is linked to high/intermediate grade in diﬀerent types of human carcinomas including HN (5, 26, 27). Thus, it is evident that the co-presence of high-risk HPVs and EBV in high-grade human carcinomas could suggest a possible cooperation between their oncoproteins; however, there are only few studies regarding the co-presence of high-risk HPVs and EBV in the ME region focusing only on NPCs. Therefore, in this investigation, we assessed the presence of high-risk HPVs and EBV and their association with tumor phenotype in human HN cancer samples from Syria. Our study pointed out that high-risk HPVs and EBV are present in 43.7 and 51.2% of our samples, respectively, while co- incidence of these oncoviruses is 32.2%. More signiﬁcantly, we noted that the co-incidence of these oncoviruses is associated with high/intermediate-grade squamous cell carcinomas in the majority of positive cases. All TMAs also contained various cores representing positive and negative controls (e.g., cervical carcinoma and lymphatic tissues served as positive controls for HPV and EBV stains, respectively; normal HN tissues and epithelium were used as negative controls). Statistical Analysis Statistical assessments were achieved using IBM SPSS Statistics (version 22; SPSS Inc., Chicago, IL, USA) and R. Data were analyzed as non-parametric ﬁles. We used χ2-test with Yates correction to explore the signiﬁcance of the association between tumor grade and the co-incidence of high-risk HPVs and EBV. High-Risk HPV and EBV Detection g Eighty formalin-ﬁxed paraﬃn-embedded blocks of HN cancer (57 larynx, 19 lower lip, 3 upper lip, and 1 nasopharynx) from Syrian patients, 73 males and 7 females, with an average age of 54.51 years were used. The samples were obtained from the Pathology Department, Faculty of Medicine of Aleppo University, Syria. Tissue blocks and data used in this report were approved, in March 22, 2009, by the Ethics Committee Citation: Gupta I, Ghabreau L, Al-Thawadi H, Yasmeen A, Vranic S, Al Moustafa A-E and Malki MI (2020) Co-incidence of Human Papillomaviruses and Epstein–Barr Virus Is Associated With High to Intermediate Tumor Grade in Human Head and Neck Cancer in Syria. Front. Oncol. 10:1016. doi: 10.3389/fonc.2020.01016 Today, it is well-known that more than 20% of human cancers are estimated to be linked with microorganism infections including oncoviruses infection especially high-risk human August 2020 \| Volume 10 \| Article 1016 Frontiers in Oncology \| www.frontiersin.org HPVs and EBV in Human HN Cancer Gupta et al. papillomaviruses (high-risk HPV) and Epstein–Barr virus (EBV) (6–8). More speciﬁcally, it has been well-established that high- risk HPV infections are critical etiological factors in the development of human HN cancers, especially oral, as ∼40% of oral cancer cases are positive for high-risk HPVs, particularly types 16, 18, 31, 33, 35, 45, 52, and 58 worldwide including the Middle East (ME) region (7, 9). Additionally, it was pointed out that their presence is linked with vascular invasion and lymph node metastases in diﬀerent types of human carcinomas including cervical and HN (10–12). papillomaviruses (high-risk HPV) and Epstein–Barr virus (EBV) (6–8). More speciﬁcally, it has been well-established that high- risk HPV infections are critical etiological factors in the development of human HN cancers, especially oral, as ∼40% of oral cancer cases are positive for high-risk HPVs, particularly types 16, 18, 31, 33, 35, 45, 52, and 58 worldwide including the Middle East (ME) region (7, 9). Additionally, it was pointed out that their presence is linked with vascular invasion and lymph node metastases in diﬀerent types of human carcinomas including cervical and HN (10–12). of the Faculty of Medicine of Aleppo University, # 2009-007, Aleppo, Syria. One hundred nanograms of DNA was extracted from each sample using Qiagen GmbH kit (Hilden, Germany). These samples were analyzed for high-risk HPVs and EBV by PCR using primers for E6/E7 genes of high-risk HPV types (16, 18, 31, 33, 35, 45, 51, 52, and 58) in addition to primers for LMP1 and EBNA1 genes of EBV; meanwhile, primers for the GAPDH gene were utilized as an internal control (26, 28). This analysis was achieved as illustrated earlier by our group (5, 26, 28). Tissue Microarray Likewise, EBV is a human gamma herpesvirus that commonly infects more than 90% of the adult population (13). Persistent infection with EBV can cause infectious mononucleosis, and its latent state can lead to several types of human B-cell lymphomas and certain solid cancers, especially nasopharyngeal (14–17); additionally, EBV has been shown to be strongly associated with undiﬀerentiated nasopharyngeal carcinomas (NPCs). Several studies have detected the presence of EBV in HN squamous cell tumors implying its possible role in the development of malignancies throughout the upper aerodigestive tract (7, 18, 19). Moreover, it has been recently revealed that EBNA1 and LMP1 of EBV oncoproteins can enhance invasion of human cancer cells via the induction of epithelial-to-mesenchymal transition (EMT) (20, 21). Tissue microarray (TMA) building was realized as elucidated previously by our group (28, 29). Brieﬂy, HN cancer samples were inserted into a virgin paraﬃn TMA block using a non- automated tissue arrayer (Beecher Instruments, Silver Spring, MD) irrespective of pathological staging information. Three TMA cores of 1.0 mm in diameter were sampled from a cohort of 80 block tissue samples of Syrian HN cancer patients. Afterwards, to verify the histological diagnosis, 4-µm sections were cut and stained with hematoxylin and eosin (H&E). Then, slides of the completed blocks were used for immunohistochemistry assay. RESULTS TABLE 2 \| Presence of HPV types 16, 18, 31, 33, 35, 45, 51, 52, and 58 in human HN cancer. No. of cases High-risk HPVs 16 18 31 33 35 45 51 52 58 80 31/80 28/80 13/80 34/80 18/80 25/80 15/80 22/80 20/80 We note that HPV types 33, 16, 18, and 45 are the most common in HN cancer in the Syrian population. FIGURE 1 \| High-grade (Grade 3), non-keratinizing squamous cell carcinomas with basaloid appearance. Tumor cells were diffusely (>75%) and strongly (2+ and 3+ intensity on the scale 0–3) positive for both HPV (E6, upper images) and EBV (LMP1 protein) (lower images). The left-sided images are captured at 4× magniﬁcation, while the right-side ones are at 10× magniﬁcation (Olympus BX53). Expression of both proteins was assessed by immunohistochemistry using a semiquantitative approach. FIGURE 1 \| High-grade (Grade 3), non-keratinizing squamous cell carcinomas with basaloid appearance. Tumor cells were diffusely (>75%) and strongly (2+ and 3+ intensity on the scale 0–3) positive for both HPV (E6, upper images) and EBV (LMP1 protein) (lower images). The left-sided images are captured at 4× magniﬁcation, while the right-side ones are at 10× magniﬁcation (Olympus BX53). Expression of both proteins was assessed by immunohistochemistry using a semiquantitative approach. incidence of high-risk HPV types 16, 18, 31, 33, 35, 45, 51, 52, and 58 in a cohort of 80 HN cancer specimens from the Syrian population by PCR analysis, using speciﬁc primers for E6/E7 and LMP1 as well as EBNA1 genes of HPVs and EBV, respectively (5, 26, 28). Our data revealed that 35 (43.7%) and 41 (51.2%) of the 80 cancer samples are positive for high-risk HPVs and EBV, respectively (Table 1), and all of HPVs-positive specimens are co-infected with more than one HPV type. Regarding high-risk HPVs genotyping in these samples, our results pointed out that the most prevalent high-risk HPVs among the positive samples are types 33 (34/80), 16 (31/80), 18 (28/80), 45 (25/80), 52 (22/80), 58 (21/80), 35 (18/80), 51 (15/80), and 31 (13/80), as shown in Table 2. FIGURE 1 \| High-grade (Grade 3), non-keratinizing squamous cell carcinomas with basaloid appearance. Tumor cells were diffusely (>75%) and strongly (2+ and 3+ intensity on the scale 0–3) positive for both HPV (E6, upper images) and EBV (LMP1 protein) (lower images). RESULTS The left-sided images are captured at 4× magniﬁcation, while the right-side ones are at 10× magniﬁcation (Olympus BX53). Expression of both proteins was assessed by immunohistochemistry using a semiquantitative approach. Next, we investigated the co-presence of high-risk HPVs and EBV in our HN cancer samples by IHC and PCR analysis using monoclonal antibodies, for E6 and LMP1, as well as speciﬁc primers for these oncoproteins/genes, respectively; we found that 25 (31.2%) of the 80 cancer cases are positive for both high-risk HPVs and EBV with P < 0.001 (Table 1). Furthermore, we examined the relation between the co-presence of these oncoviruses and tumor grade in these samples. Our data revealed that the co-expression of E6 and LMP1 oncoproteins of high-risk HPVs and EBV, respectively, in the majority of cases (88%) is associated with high/intermediate (G3/G2)-grade invasive carcinoma form in comparison with high-risk HPVs+ or EBV+ alone cases or HPVs/EBV-negative cases, which are 4/10 (40%), 5/16 (31.2%), and 3/29 (10.2%) with P = 0.00328, 0.00018, and <0.001, respectively (Table 3 and Figures 1, 2), while it is important to highlight that cancer phenotype in the HPV+/EBV+ was not linked to a speciﬁc HPV type since all our positive cases were infected with more than one type of high- risk HPVs. Finally, normal HN tissues and epithelial cells, which served as controls, were shown to be negative for both high-risk HPVs as well as EBV. RESULTS In order to classify the presence of high-risk HPVs and EBV in human HN cancer tissues in the ME region, we explored the August 2020 \| Volume 10 \| Article 1016 Frontiers in Oncology \| www.frontiersin.org 2 HPVs and EBV in Human HN Cancer Gupta et al. TABLE 3 \| High-risk HPVs and EBV status in relation to tumor grade in HN cancer samples. EBV/HPVs status EBV+/HPVs+ (%) EBV+/HPVs− (%) EBV−/HPVs+ (%) EBV−/HPVs− (%) TUMOR GRADE High 5 (20.0) 1 (10.0) 2 (12.5) 1 (3.4) Intermediate 17 (68.0) 3 (30.0) 3 (18.7) 2 (6.8) Low 3 (12.0) 6 (60.0) 11 (68.7) 26 (89.6) Number of samples# 25 (31.2) 10 (12.5) 16 (20.0) 29 (36.2) We notice that 88% of HPVs- and EBV-positive cases are high/intermediate-grade carcinomas in comparison with EBV+/HPV−, EBV−/HPV+, and EBV−/HPV−, which are 40, 31.2, and 10.2% with a P-value of 0.00328, 0.00018, and <0.001, respectively. #The total number of samples examined in this study is 80. Values inside parentheses denote percentage. FIGURE 1 \| High-grade (Grade 3), non-keratinizing squamous cell carcinomas with basaloid appearance. Tumor cells were diffusely (>75%) and strongly (2+ and 3+ intensity on the scale 0–3) positive for both HPV (E6, upper images) and EBV (LMP1 protein) (lower images). The left-sided images are captured at 4× magniﬁcation, while the right-side ones are at 10× magniﬁcation (Olympus BX53). Expression of both proteins was assessed by immunohistochemistry using a semiquantitative approach. TABLE 1 \| High-risk HPVs and EBV detection in human head and neck cancers. Number of samples# HPVs+ EBV+ HPVs+/EBV+ Positive cases 35/80 41/80 25/80 (%)## (43.7) (51.2) (31.2) The presence of HPVs and EBV was found in 35 (43.7%) and 41 (51.2%) of the 80 cancer samples, respectively, while we observed that 25 (31.2%) of cancer cases are positive for both high-risk HPVs and EBV. The presence of these oncoviruses was conﬁrmed by PCR and IHC using speciﬁc primers for E6/E7 and LMP1 as well as EBNA1 genes of high-risk HPVs and EBV, in addition to monoclonal antibodies for E6 and LMP1, as illustrated in the Materials and Methods section. #The total number of samples examined in this study is 80. ##These two methodologies, PCR and IHC, were used to detect the presences of high-risk HPVs and EBV. TABLE 2 \| Presence of HPV types 16, 18, 31, 33, 35, 45, 51, 52, and 58 in human HN cancer. No. RESULTS of cases High-risk HPVs 16 18 31 33 35 45 51 52 58 80 31/80 28/80 13/80 34/80 18/80 25/80 15/80 22/80 20/80 We note that HPV types 33, 16, 18, and 45 are the most common in HN cancer in the Syrian population. incidence of high-risk HPV types 16, 18, 31, 33, 35, 45, 51, 52, and 58 in a cohort of 80 HN cancer specimens from the Syrian population by PCR analysis, using speciﬁc primers for E6/E7 and LMP1 as well as EBNA1 genes of HPVs and EBV, respectively TABLE 3 \| High-risk HPVs and EBV status in relation to tumor grade in HN cancer samples. EBV/HPVs status EBV+/HPVs+ (%) EBV+/HPVs− (%) EBV−/HPVs+ (%) EBV−/HPVs− (%) TUMOR GRADE High 5 (20.0) 1 (10.0) 2 (12.5) 1 (3.4) Intermediate 17 (68.0) 3 (30.0) 3 (18.7) 2 (6.8) Low 3 (12.0) 6 (60.0) 11 (68.7) 26 (89.6) Number of samples# 25 (31.2) 10 (12.5) 16 (20.0) 29 (36.2) We notice that 88% of HPVs- and EBV-positive cases are high/intermediate-grade carcinomas in comparison with EBV+/HPV−, EBV−/HPV+, and EBV−/HPV−, which are 40, 31.2, and 10.2% with a P-value of 0.00328, 0.00018, and <0.001, respectively. #The total number of samples examined in this study is 80. Values inside parentheses denote percentage. TABLE 3 \| High-risk HPVs and EBV status in relation to tumor grade in HN cancer samples. TABLE 1 \| High-risk HPVs and EBV detection in human head and neck cancers. -risk HPVs and EBV status in relation to tumor grade in HN cance #The total number of samples examined in this study is 80. We notice that 88% of HPVs- and EBV-positive cases are high/intermediate-grade carcinomas in comparison with EBV+/HPV−, EBV−/HPV+, and EBV−/HPV−, which are 40, 31.2, and 10.2% with a P-value of 0.00328, 0.00018, and <0.001, respectively. #The total number of samples examined in this study is 80. Values inside parentheses denote percentage. ##These two methodologies, PCR and IHC, were used to detect the presences of high-risk HPVs and EBV. TABLE 2 \| Presence of HPV types 16, 18, 31, 33, 35, 45, 51, 52, and 58 in human HN cancer. No. of cases High-risk HPVs 16 18 31 33 35 45 51 52 58 80 31/80 28/80 13/80 34/80 18/80 25/80 15/80 22/80 20/80 We note that HPV types 33, 16, 18, and 45 are the most common in HN cancer in the Syrian population. Frontiers in Oncology \| www.frontiersin.org DISCUSSION In this investigation, we explored, for the ﬁrst time, the incidence/co-incidence of high-risk HPVs and EBV in human HN cancer and the association of their co-presence with tumor grade in Syria, which can also be considered the ﬁrst study of this type in the ME region. We report that high-risk HPVs and EBV are present in 43.7 and 51.2%, respectively, in our Syrian samples, and the most frequent HPV types in HN cancer in Syria are 33, 16, 18, 45, 52, 58, 35, 51, and 31, correspondingly. Meanwhile, our data pointed out that 31.2% of the samples are positive for both high-risk HPVs and EBV. More signiﬁcantly, we report that the co-presence of these oncoviruses is associated with high/intermediate tumor grade in 88% of the samples in In this investigation, we explored, for the ﬁrst time, the incidence/co-incidence of high-risk HPVs and EBV in human HN cancer and the association of their co-presence with tumor grade in Syria, which can also be considered the ﬁrst study of this type in the ME region. We report that high-risk HPVs and EBV are present in 43.7 and 51.2%, respectively, in our Syrian samples, and the most frequent HPV types in HN cancer in Syria are 33, 16, 18, 45, 52, 58, 35, 51, and 31, correspondingly. Meanwhile, our data pointed out that 31.2% of the samples are positive for both high-risk HPVs and EBV. More signiﬁcantly, we report that the co-presence of these oncoviruses is associated with high/intermediate tumor grade in 88% of the samples in August 2020 \| Volume 10 \| Article 1016 Frontiers in Oncology \| www.frontiersin.org 3 Gupta et al. HPVs and EBV in Human HN Cancer cancer in the ME region since our samples include tissues from several HN locations. FIGURE 2 \| A case of moderately differentiated (Grade 2) squamous cell carcinoma exhibiting co-expression of E6 (upper images) and LMP1 (lower images) oncoproteins of HPV and EBV, respectively. Left-sided images are captured at 4× magniﬁcation, while the right-sided ones are at 10× magniﬁcation (Olympus BX53). Note that the expression of E6 protein was observed in >90% of cancer cells while LMP1 protein was expressed in ∼70% of cancer cells. Expression of both oncoproteins was assessed by immunohistochemistry using a semiquantitative approach. On the other hand, in 2012, Jalouli et al. DISCUSSION (40) examined the incidence and co-incidence of high-risk HPVs and EBV in 155 oral squamous cell carcinomas (OSCCs) from eight diﬀerent countries from Europe, Asia, Africa, and North America with a limited number of cases ∼20 cancer cases from each country including Sudan and Yemen from the ME region. They found that 35 and 55% of the samples are positive for HPVs and EBV, respectively. They reported that HPVs and EBV are co-present in 21% of all OSCCs. However, no clear conclusions can be drawn from this study due to the limited number of cancer cases from participant countries. Meanwhile, there are two recently published investigations from Europe regarding the presence of HPVs and EBV in human oral cancer; one from Poland showed that HPVs and EBV are co-present in 34.1% of cancer cases in comparison with HPV and EBV infections alone, which are 28.1 and 54.7%, respectively (23). In the second study from Finland, the authors reported that the co-incidence of HPV and EBV is 14% in the population of Finland (41). In comparison, our study focused on the presence/co-presence of HPVs and EBV in HN cancer in Syria with an acceptable number of samples, which allowed us to make an adequate conclusion about these oncoviruses in HN cancer in this country, revealing that 31.2% of the samples are HPVs+/EBV+, which is comparable with the study published from Poland. FIGURE 2 \| A case of moderately differentiated (Grade 2) squamous cell carcinoma exhibiting co-expression of E6 (upper images) and LMP1 (lower images) oncoproteins of HPV and EBV, respectively. Left-sided images are captured at 4× magniﬁcation, while the right-sided ones are at 10× magniﬁcation (Olympus BX53). Note that the expression of E6 protein was observed in >90% of cancer cells while LMP1 protein was expressed in ∼70% of cancer cells. Expression of both oncoproteins was assessed by immunohistochemistry using a semiquantitative approach. comparison to HPVs or EBV-positive alone and HPVs/EBV- negative samples. Regarding the most common HPV types in the Syrian population, the present data concur with our previous studies on HPVs in diﬀerent types of human carcinomas including cervical and breast in Syria where we found that HPV type 33 is the most frequent in these cancer tissues. In this context, HPV type 33 was reported to be the most common in breast cancer in Turkey (30). DISCUSSION Herein, it is important to highlight that the Syrian samples were collected from Aleppo province, which is located in the northern part of the country bordering Turkey. Accordingly, our data conﬁrm that speciﬁc types of high- risk HPV infection, in human cancers, are related to certain geographic locations, as it was demonstrated by a large number of investigations worldwide (7, 31–36). The co-presence of high-risk HPVs and EBV and their association with tumor phenotype in HN cancer is clearly demonstrated in our present study regardless of HPV type since all our HPV/EBV-positive cases are infected with more than one HPV virus strain. Our ﬁndings are in agreement with several investigations worldwide, including three from our group in addition to the Turkish and Moroccan studies; data of these reports pointed out that the co-presence of HPVs and EBV is associated with high-grade carcinomas in addition to positive axillary lymph nodes (5, 23, 25–27, 42, 43). Indeed, it has been reported that prevalence of poorly diﬀerentiated tumors is four times more frequent in HPV/EBV co-infection in comparison with EBV or HPV infection alone in oral cancer samples from Poland (23); in addition, the study pointed out that there is a signiﬁcant correlation between tumor dimensions in co-infected patients compared with single infection. However, a recent investigation in NPCs reported that 5-year overall survival is signiﬁcantly higher in HPV/EBV-positive patients in comparison with HPV/EBV- negative ones (41). Well, this could be due to radiation sensitivity as demonstrated by several investigations. Actually, earlier studies in HPV-positive cases of HNSCC found that the virus takes control of the cellular machinery for DNA repair, altering cell cycle distribution and causing hypoxia during radiation treatment (44, 45). On the other hand, numerous studies on the alteration of radiation response by EBV reported that LMP-1 blocks DNA repair by suppressing the phosphorylation and activity of DNA-dependent protein kinase, a key enzyme of non-homologous end-joining pathway in NPCs, and by repressing ATM, which ultimately modulates resistance of ionizing radiation-induced apoptotic cell death (46). Concerning the co-presence of HPVs and EBV in HN cancer in the ME region, Tatli Dogan et al. Frontiers in Oncology \| www.frontiersin.org DISCUSSION (37) published a study regarding the incidence of HPVs and EBV in NPCs in Turkey, they found that 72 of their 82 samples are positive for EBV and only one case revealed positive for HPVs; meanwhile, they reported that the highest rate of EBV positivity correspond with undiﬀerentiated NPCs. However, and in agreement with our investigation, one report from North Africa assessed the presence and co-presence of high-risk HPVs and EBV in 70 cases of NPCs from the Moroccan population (38). Their study revealed that 24 of the samples are positive for high-risk HPVs, and the most frequent HPV types are 31, 59, 16, 18, 33, and 35. They found that all their cancer cases are positive for EBV. Consequently, 24 (34%) Moroccan samples were positive for both high-risk HPVs and EBV, which are in their majority NPCs grade III and II. It is important to highlight that the Turkish and Moroccan studies in addition to another investigation from Iran with only 20 cancer cases (39) focused only on the co-presence of HPVs and EBV in NPCs; therefore, our investigation can be considered the ﬁrst study regarding the co-presence of these oncoviruses in HN August 2020 \| Volume 10 \| Article 1016 4 Gupta et al. HPVs and EBV in Human HN Cancer Apropos the mechanism of HPVs and EBV interaction, based on the fact that high-risk HPVs and EBV oncoproteins share diﬀerent downstream pathways, we assumed that oncoproteins (E5, E6/E7, LMP1, and EBNA1) of these oncoviruses can cooperate in the initiation and/or progression of several types of human carcinomas where the EMT event can play a crucial role in this procedure (47). Indeed, earlier investigations showed that E5 and E6/E7 oncoproteins of high-risk HPVs can enhance cell invasion and cancer progression via the induction of EMT in several types of human cancers including cervical and oral as well as NPCs (48–53). On the other hand, it has been reported by several investigations that LMP1, LMP2A, EBNA3C, and EBNA1 oncoproteins of EBV can enhance cancer progression via the modulation of EMT in human carcinomas including NPCs (20, 53–56). Meanwhile, our preliminary data showed that E6/E7 of HPV type 16 can cooperate with LMP1 of EBV to enhance EMT progression and consequently cell motility via the activation (phosphorylation) of Erk1/Erk2 and β- catenin (in preparation). ETHICS STATEMENT The studies involving human participants were reviewed and approved by Ethics Committee of the Faculty of Medicine of Aleppo University, # 2009-007. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. DISCUSSION Nevertheless, further studies are needed to elucidate the complete pathogenesis and role of the co- incidence of high-risk HPVs and EBV in human carcinomas including HN, especially since HPVs and EBV vaccines are currently available and under clinical trial, respectively (57– 59). This is a key step, which could possibly limit HPV and EBV infection and their associated cancers including HN malignancy initiation and development to a metastatic form, thus diminishing cancer-related mortalities especially in developing countries where oncoviruses-associated cancers are still considered major causes of death among both males and females in these countries. country and in combination with numerous investigations from the ME in general. country and in combination with numerous investigations from the ME in general. FUNDING This work was supported by Qatar University grants # QUHI-CMED-19/20-1 and GCC-2017-002 QU/KU. This work was supported by Qatar University grants # QUHI-CMED-19/20-1 and GCC-2017-002 QU/KU. ACKNOWLEDGMENTS Lastly, with regard to the number of specimens that we were able to amass from Aleppo, Syria, it is essential to conﬁrm our data using a larger number of samples from diﬀerent areas in this Lastly, with regard to the number of specimens that we were able to amass from Aleppo, Syria, it is essential to conﬁrm our data using a larger number of samples from diﬀerent areas in this The authors would like to thank Mrs. A. Kassab for her critical reading of the manuscript. The authors would like to thank Mrs. A. Kassab for her critical reading of the manuscript. DATA AVAILABILITY STATEMENT All datasets generated for this study are included in the article/supplementary material. AUTHOR CONTRIBUTIONS A-EA, SV, and HA-T conceived the study. LG provided the samples. IG, LG, SV, MM, AY, HA-T, and A-EA analyzed the data. All authors wrote and approved the ﬁnal version of the manuscript. All authors contributed to the article and approved the submitted version. A-EA, SV, and HA-T conceived the study. LG provided the samples. IG, LG, SV, MM, AY, HA-T, and A-EA analyzed the data. All authors wrote and approved the ﬁnal version of the manuscript. All authors contributed to the article and approved the submitted version. REFERENCES 7. Al Moustafa AE, Al-Awadhi R, Missaoui N, Adam I, Durusoy R, Ghabreau L, et al. Human papillomaviruses-related cancers. Presence and prevention strategies in the middle east and north African regions. Hum Vaccin Immunother. (2014) 10:1812–21. doi: 10.4161/hv. 28742 1. Daraei P, Moore CE. Racial disparity among the head and neck cancer population. J Cancer Educ. (2015) 30:546–51. doi: 10.1007/s13187-014-0 753-4 8. Al Moustafa AE, Cyprian FS, Al-Antary N, Yasmeen A. High-risk human papillomaviruses and Epstein-Barr virus presence and crosstalk in human oral carcinogenesis. In: Al Moustafa AE, editor. Development of Oral Cancer: Risk Factors and Prevention Strategies. Cham: Springer International Publishing (2017). p. 83–94. 2. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. (2015) 136:E359–86. doi: 10.1002/ijc.29210 3. Chaturvedi AK, Anderson WF, Lortet-Tieulent J, Curado MP, Ferlay J, Franceschi S, et al. Worldwide trends in incidence rates for oral cavity and oropharyngeal cancers. J Clin Oncol. (2013) 31:4550–9. doi: 10.1200/JCO.2013.50.3870 9. de Sanjosé S, Serrano B, Tous S, Alejo M, Lloveras B, Quirós B, et al. Burden of Human Papillomavirus (HPV)-related cancers attributable to HPVs 6/11/16/18/31/33/45/52 and 58. JNCI Cancer Spectrum. (2019) 2:pky045. doi: 10.1093/jncics/pky045 4. Al Moustafa A, Yasmeen A, Ghabreau L, Mohamed AH, Achkhar A. Brain metastases progression of breast cancer, breast cancer - carcinogenesis, cell growth and signalling pathways. In: Gunduz M, editor. London, UK: IntechOpen (2011). 10. Graﬂund, M. SB, Sigurdardóttir S, Karlsson M. HPV-DNA, vascular space invasion, and their impact on the clinical outcome in early- stage cervical carcinomas. Int J Gynecol Cancer. (2004) 14:896–902. doi: 10.1111/j.1048-891X.2004.014527.x 5. Al-Thawadi H, Ghabreau L, Aboulkassim T, Yasmeen A, Vranic S, Batist G, et al. Co-Incidence of Epstein-Barr virus and high-risk human papillomaviruses in cervical cancer of Syrian women. Front Oncol. (2018) 8:250. doi: 10.3389/fonc.2018.00250 11. Zuna R, Allen RA, Moore WE, Mattu R, Dunn ST. Comparison of human papillomavirus genotypes in high-grade squamous intraepithelial lesions and invasive cervical carcinoma: evidence for diﬀerences in biologic potential of precursor lesions. Mod Pathol. (2004) 17:1314–22. doi: 10.1038/modpathol.3800223 6. de Martel C, Ferlay J, Franceschi S, Vignat J, Bray F, Forman D, et al. Global burden of cancers attributable to infections in 2008: a review and synthetic analysis. Lancet Oncol. (2012) 13:607–15. doi: 10.1016/S1470-2045(12)70137-7 12. Umudum H, Rezanko T, Dag F, Dogruluk T. REFERENCES Glenn WK, Heng B, Delprado W, Iacopetta B, Whitaker NJ, Lawson JS. Epstein-Barr virus, human papillomavirus and mouse mammary tumour virus as multiple viruses in breast cancer. PLoS ONE. (2012) 7:e48788–e48788. doi: 10.1371/journal.pone.0048788 24. Huang WB, Chan JYW, Liu DL. Human papillomavirus and World Health Organization type III nasopharyngeal carcinoma: multicenter study from an endemic area in Southern China. Cancer. (2018) 124:530–6. doi: 10.1002/cncr.31031 25. Jiang R, Ekshyyan O, Moore-Medlin T, Rong X, Nathan S, Gu X, et al. Association between human papilloma virus/Epstein–Barr virus coinfection and oral carcinogenesis. J Oral Pathol Med. (2015) 44:28–36. doi: 10.1111/jop.12221 43. Broccolo F, Ciccarese G, Rossi A, Anselmi L, Drago F, Toniolo A. Human papillomavirus (HPV) and Epstein-Barr virus (EBV) in keratinizing versus non- keratinizing squamous cell carcinoma of the oropharynx. Infect Agents Cancer. (2018) 13:32. doi: 10.1186/s13027-018-0205-6 44. Weaver AN, Cooper TS, Rodriguez M, Trummell HQ, Bonner JA, Rosenthal EL, et al. DNA double strand break repair defect and sensitivity to poly ADP- ribose polymerase (PARP) inhibition in human papillomavirus 16-positive head and neck squamous cell carcinoma. Oncotarget. (2015) 6:26995–7007. doi: 10.18632/oncotarget.4863 26. Al Moustafa AE, Al-Antary N, Aboulkassim T, Akil N, Batist G, Yasmeen A. Co-prevalence of Epstein-Barr virus and high-risk human papillomaviruses in Syrian women with breast cancer. Hum Vaccin Immunother. (2016) 12:1936– 9. doi: 10.1080/21645515.2016.1139255 27. Malki MI, Gupta I, Fernandes Q, Aboulkassimc T, Yasmeen A, Vranic S, et al. Co-presence of Epstein–Barr virus and high-risk human papillomaviruses in Syrian colorectal cancer samples. J Hum Vaccin Immunother. (2020). doi: 10.1080/21645515.2020.1726680. [Epub ahead of print]. 45. Swick AD, Chatterjee A, De Costa A-MA, Kimple RJ. Modulation of therapeutic sensitivity by human papillomavirus. Radiother Oncol. (2015) 116:342–5. doi: 10.1016/j.radonc.2015.09.002 28. Darnel AD, Wang D, Ghabreau L, Yasmeen A, Sami S, Akil N, et al. Correlation between the presence of high-risk human papillomaviruses and Id gene expression in Syrian women with cervical cancer. Clin Microbiol Infect. (2010) 16:262–6. doi: 10.1111/j.1469-0691.2009.02774.x 46. Rödel F, Martin D, Balermpas P, Wieland U, Winkelmann R, Riekmann T, et al. Modulation of radiation sensitivity and antitumor immunity by viral pathogenic factors: Implications for radio-immunotherapy. Biochim Biophys Acta Rev Cancer. (2019) 1871:126–37. doi: 10.1016/j.bbcan.2018.12.006 29. Akil N, Yasmeen A, Kassab A, Ghabreau L, Darnel AD, Al Moustafa AE. High- risk human papillomavirus infections in breast cancer in Syrian women and their association with Id-1 expression: a tissue microarray study. Br J Cancer. (2008) 99:404–7. doi: 10.1038/sj.bjc.6604503 47. REFERENCES Tang Y, Zheng L, Yang S, Li B, Su H, Zhang LP. Epidemiology and genotype distribution of human papillomavirus (HPV) in Southwest China: a cross- sectional ﬁve years study in non-vaccinated women. Virol J. (2017) 14:84. doi: 10.1186/s12985-017-0751-3 19. Goldenberg D, Benoit NE, Begum S, Westra WH, Cohen Y, Koch WM, et al. Epstein-Barr Virus in head and neck cancer assessed by quantitative polymerase chain reaction. Laryngoscope. (2004) 114:1027–31. doi: 10.1097/00005537-200406000-00013 37. Tatli Dogan H, Kiliçarslan A, Dogan M, Süng,ü N, Güler Tezel G, Güler G. Retrospective analysis of oncogenic human papilloma virus and Epstein-Barr virus prevalence in Turkish nasopharyngeal cancer patients. Pathol Res Pract. (2016) 212:1021–6. doi: 10.1016/j.prp.2016.08.013 20. Gaur N, Gandhi J, Robertson ES, Verma SC, Kaul R. Epstein-Barr virus latent antigens EBNA3C and EBNA1 modulate epithelial to mesenchymal transition of cancer cells associated with tumor metastasis. Tumour Biol. (2015) 36:3051–60. doi: 10.1007/s13277-014-2941-6 38. Laantri N, Attaleb M, Kandil M, Naji F, Mouttaki T, Dardari R, et al. Human papillomavirus detection in moroccan patients with nasopharyngeal carcinoma. Infect Agents Cancer. (2011) 6:3–3. doi: 10.1186/1750-9378-6-3 21. Zuo LL, Zhang J, Liu LZ, Zhou Q, Du SJ, Xin SY, et al. Cadherin 6 is activated by Epstein-Barr virus LMP1 to mediate EMT and metastasis as an interplay node of multiple pathways in nasopharyngeal carcinoma. Oncogenesis. (2017) 6:402–402. doi: 10.1038/s41389-017-0005-7 39. Mirzamani N, Salehian P, Farhadi M, Tehran EA. Detection of EBV and HPV in nasopharyngeal carcinoma by in situ hybridization. Exp Mol Pathol. (2006) 81:231–4. doi: 10.1016/j.yexmp.2006.04.006 40. Jalouli J, Jalouli MM, Sapkota D, Ibrahim SO, Larsson PA., Sand L. Human papilloma virus, herpes simplex virus and Epstein Barr virus in oral squamous cell carcinoma from eight diﬀerent countries. Anticancer Res. (2012) 32:571– 80. 22. Deng Z, Uehara T, Maeda H, Hasegawa M, Matayoshi S, Kiyuna A, et al. Epstein-Barr virus and human papillomavirus infections and genotype distribution in head and neck cancers. PLoS ONE. (2014) 9:e113702. doi: 10.1371/journal.pone.0113702 41. Ruuskanen M, Irjala H, Minn H, Vahlberg T, Randen-Brady R, Hagström J, et al. Epstein-Barr virus and human papillomaviruses as favorable prognostic factors in nasopharyngeal carcinoma: a nationwide study in Finland. Head Neck. (2019) 41:349–57. doi: 10.1002/hed.25450 23. Drop B, Strycharz-Dudziak M, Kliszczewska E, Polz-Dacewicz M. Coinfection with Epstein-Barr Virus (EBV), Human Papilloma Virus (HPV) and Polyoma BK Virus (BKPyV) in laryngeal, oropharyngeal and oral cavity cancer. Int J Mol Sci. (2017) 18:2752. doi: 10.3390/ijms18122752 42. REFERENCES Human papillomavirus genome detection by in situ hybridization in ﬁne-needle aspirates of metastatic lesions August 2020 \| Volume 10 \| Article 1016 Frontiers in Oncology \| www.frontiersin.org 5 Gupta et al. HPVs and EBV in Human HN Cancer from head and neck squamous cell carcinomas. Cancer. (2005) 105:171–7. doi: 10.1002/cncr.21027 from head and neck squamous cell carcinomas. Cancer. (2005) 105:171–7. doi: 10.1002/cncr.21027 31. Abramowitz L, Jacquard A-C, Jaroud F, Haesebaert J, Siproudhis L, Pradat P, et al. Human papillomavirus genotype distribution in anal cancer in France: the EDiTH V study. Int J Cancer. (2011) 129:433–9. doi: 10.1002/ijc.25671 13. Niedobitek G, Meru N, Delecluse HJ. Epstein-Barr virus infection and human malignancies. Int J Exp Pathol. (2001) 82:149–70. doi: 10.1046/j.1365-2613.2001.iep0082-0149-x 32. García Muentes GD, García Rodríguez LK, Burgos Galarraga RI, Almeida Carpio F, Ruiz Cabezas JC. Genotypes distribution of human papillomavirus in cervical samples of Ecuadorian women. Revista Brasileira de Epidemiologia. (2016) 19:160–6. doi: 10.1590/1980-5497201600010014 14. Middeldorp JM, Brink AA, van den Brule AJ, Meijer CJ. Pathogenic roles for Epstein-Barr virus (EBV) gene products in EBV-associated proliferative disorders. Crit Rev Oncol Hematol. (2003) 45, 1–36. doi: 10.1016/S1040-8428(02)00078-1 33. Li Z, Liu F, Cheng S, Shi L, Yan Z, Yang J, et al. Prevalence of HPV infection among 28,457 Chinese women in Yunnan Province, southwest China. Sci Rep. (2016) 6:21039. doi: 10.1038/srep21039 15. Young LS, Rickinson AB. Epstein-Barr virus: 40 years on. Nat Rev Cancer. (2004) 4:757–68. doi: 10.1038/nrc1452 34. Wang L, Wang P, Ren Y, Du J, Jiang J, Jia X, et al. Prevalence of High- Risk Human Papillomavirus (HR-HPV) genotypes and multiple infections in cervical abnormalities from Northern Xinjiang, China. PLoS ONE. (2016) 11:e0160698. doi: 10.1371/journal.pone.0160698 16. Murata T, Tsurumi T. Switching of EBV cycles between latent and lytic states. Rev Med Virol. (2014) 24:142–53. doi: 10.1002/rmv.1780 17. Elgui de Oliveira D, Müller-Coan BG, Pagano JS. Viral carcinogenesis Beyond malignant transformation: EBV in the progression of human cancers. Trends Microbiol. (2016) 24:649–64. doi: 10.1016/j.tim.2016.03.008 35. Gheit T, Anantharaman D, Holzinger D, Alemany L, Tous S, Lucas E, et al. Role of mucosal high-risk human papillomavirus types in head and neck cancers in central India. Int J Cancer. (2017) 141:143–51. doi: 10.1002/ijc.30712 18. Gonzalez-Moles MA, Gutierrez J, Rodriguez MJ, Ruiz-Avila I, Rodriguez- Archilla A. Epstein-Barr Virus Latent Membrane Protein-1 (LMP-1) expression in oral squamous cell carcinoma. Laryngoscope. (2002) 112:482–7. doi: 10.1097/00005537-200203000-00014 j 36. REFERENCES Cyprian FS, Al-Farsi HF, Vranic S, Akhtar S, Al Moustafa A-E. Epstein-Barr virus and human papillomaviruses interactions and their roles in the initiation of epithelial-mesenchymal transition and cancer progression. Front Oncol. (2018) 8:111. doi: 10.3389/fonc.2018.00111 30. Gumus M, Yumuk PF, Salepci T, Aliustaoglu M, Dane F, Ekenel M, et al. HPV DNA frequency and subset analysis in human breast cancer patients’ normal and tumoral tissue samples. J Exp Clin Cancer Res. (2006) 25, 515–21. 48. Cheng Y-M, Chou C-Y, Hsu Y-C, Chen M-J, Wing L-YC. The role of human papillomavirus type 16 E6/E7 oncoproteins in cervical epithelial- mesenchymal transition and carcinogenesis. Oncol Lett. (2012) 3:667–71. doi: 10.3892/ol.2011.512 August 2020 \| Volume 10 \| Article 1016 Frontiers in Oncology \| www.frontiersin.org 6 Gupta et al. HPVs and EBV in Human HN Cancer epithelial-mesenchymal transition in metastatic nasopharyngeal carcinoma. Br J Cancer. (2011) 104:1160–7. doi: 10.1038/bjc.2011.38 epithelial-mesenchymal transition in metastatic nasopharyngeal carcinoma. Br J Cancer. (2011) 104:1160–7. doi: 10.1038/bjc.2011.38 49. Chamulitrat W, Sattayakhom A, Herold-Mende C, Stremmel W. Human papillomavirus 16 E6/E7-immortalized human gingival keratinocytes with epithelial mesenchymal transition acquire increased expression of cIAP-1, Bclx and p27Kip1. Exp Dermatol. (2009) 18:1067–9. doi: 10.1111/j.1600-0625.2009.00888.x 56. Kong Q-L, Hu L-J, Cao J-Y, Huang Y-J, Xu L-H, Liang Y, et al. Epstein-Barr virus-encoded LMP2A induces an epithelial-mesenchymal transition and increases the number of side population stem-like cancer cells in nasopharyngeal carcinoma. PLoS Pathog. (2010) 6:e1000940. doi: 10.1371/journal.ppat.1000940 50. Jung YS, Kato I, Kim HR. A novel function of HPV16-E6/E7 in epithelial- mesenchymal transition. Biochem Biophys Res Commun. (2013) 435:339–44. doi: 10.1016/j.bbrc.2013.04.060 57. Lee LY, Garland SM. Human papillomavirus vaccination: the population impact. F1000Research. (2017) 6:866. doi: 10.12688/f1000research.10691.1 51. Hatakeyama H, Mizumachi T, Sakashita T, Kano S, Homma A, Fukuda S. Epithelial-mesenchymal transition in human papillomavirus-positive and - negative oropharyngeal squamous cell carcinoma. Oncol Rep. (2014) 32:2673– 79. 58. Luxembourg A, Moeller E. 9-Valent human papillomavirus vaccine: a review of the clinical development program. Expert Rev Vaccin. (2017) 16:1119–39. doi: 10.1080/14760584.2017.1383158 59. Rajˇcáni J, Bánáti F, Szenthe K, Szathmary S. The potential of currently unavailable herpes virus vaccines. Expert Rev Vaccin. (2018) 17:239–48. doi: 10.1080/14760584.2018.1425620 52. Wakisaka N, Yoshida S, Kondo S, Kita M, Sawada-Kitamura S, Endo K, et al. Induction of epithelial-mesenchymal transition and loss of podoplanin expression are associated with progression of lymph node metastases in human papillomavirus-related oropharyngeal carcinoma. Histopathology. (2015) 66:771–80. Frontiers in Oncology \| www.frontiersin.org REFERENCES doi: 10.1111/his.12496 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 53. Chen X, Bode AM, Dong Z, Cao Y. The epithelial-mesenchymal transition (EMT) is regulated by oncoviruses in cancer. Faseb j. (2016) 30:3001–10. doi: 10.1096/fj.201600388R 54. Horikawa T, Yang J, Kondo S, Yoshizaki T, Joab I, Furukawa M, et al. Twist and Epithelial-mesenchymal transition are induced by the EBV oncoprotein latent membrane protein 1 and are associated with metastatic nasopharyngeal carcinoma. Cancer Research. (2007) 67:1970–8. doi: 10.1158/0008-5472.Can-06-3933 Copyright © 2020 Gupta, Ghabreau, Al-Thawadi, Yasmeen, Vranic, Al Moustafa and Malki. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 55. Horikawa T, Yoshizaki T, Kondo S, Furukawa M, Kaizaki Y, Pagano JS. Epstein-Barr Virus latent membrane protein 1 induces Snail and August 2020 \| Volume 10 \| Article 1016 Frontiers in Oncology \| www.frontiersin.org 7
https://openalex.org/W4389557503	https://www.e3s-conferences.org/articles/e3sconf/pdf/2023/90/e3sconf_icsdg2023_01004.pdf	English	null	Study on the Impact of Firecrackers on Atmospheric Pollutants during Diwali Festival in Tamil Nadu, India	E3S web of conferences	2,023	cc-by	6,894	Study on the Impact of Firecrackers on Atmospheric Pollutants during Diwali Festival in Tamil Nadu, India Abstract. The current study focuses on the impact of firecrackers emission on particulate matter (PM2.5 and PM10), carbon monoxide (CO), carbon dioxide (CO2), and total volatile organic compounds (TVOC) and the noise was analyzed on pre, during, and post-Diwali 2022 period (5 days) in a residential area of Tamil Nadu state of India. The findings demonstrated that, for 24-hour mean values of PM2.5 and PM10 concentrations over the study period were consistently higher than the NAAQS (National Ambient Air Quality Standard) permitted limit. On the day of Diwali, the mean CO concentration reaches 2.5 mg/m3, exceeding the NAAQS- recommended allowable limit of 2 mg/m3. The CO2 concentration during Diwali day is 1.8 times higher than the pre-Diwali day. In addition, the 24-hour mean concentration of TVOC during pre-Diwali, Diwali, and post-Diwali was found to be 915.21 µg/m³, 1513.52 µg/m³ and 1617.12 µg/m³ respectively which is quite higher than normal days. Furthermore, the average noise level in the study site during pre, during, and post-Diwali was found to be 57.71 dB (A), 75.5 dB (A), and 63.66 dB (A) respectively which were higher than the safer limit on the residential zone. As a result of the widespread usage of firecrackers during Diwali festivities, these pollutants are significantly increased, which may have a negative influence on the general public's health. Keywords: Air Quality; Diwali; Particulate matters; Gaseous pollutants; Noise E3S Web of Conferences 453, 01004 (2023) ICSDG 2023 E3S Web of Conferences 453, 01004 (2023) ICSDG 2023 E3S Web of Conferences 453, 01004 (2023) https://doi.org/10.1051/e3sconf/202345301004 Study on the Impact of Firecrackers on Atmospheric Pollutants during Diwali Festival in Tamil Nadu, India Shankar S1, Abbas G1, R.Nithyaprakash1, R.Naveenkumar2, Rakesh Mohanty S1, Sabarinathan A1, Karthick S1 1Department of Mechatronics Engineering, Kongu Engineering College, Erode, India 2Department of Mechanical Engineering, Kongu Engineering College, Erode, India Corresponding Author Email: shankariitm@gmail.com © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (https://creativecommons.org/licenses/by/4.0/). 1. Introduction The dense smoke restricts human visibility in the air to a greater extent, persisting for up to several hours, depending on the prevailing climatic conditions[28]. Several researchers have examined degradation in air quality due to the Diwali celebration in various regions of India[20, 27, 29-31], and have also reported an increase in particulate matters and pollutant gases, such as PM2.5, PM10,O3, NO2, SO2, and trace metals during the Diwali celebration.According to Attri et al., burning and huge fireworks displays during Diwali led to the creation of the potent secondary pollutant ozone at ground level [32, 33].Research on the ambient air quality in Lucknow during the celebrations of Diwali revealed a range of PM10, SO2, and NOx concentrations[30, 34]. When compared to their respective concentrations on pre-Diwali and regular days, the 24-hour average concentrations of NOx, SO2, and PM10 on Diwali day were found to be 6.59 times higher for NOx, 1.95 times higher for SO2, and 2.49 times higher for PM10[30, 34]. A study conducted in Thiruvananthapuram, India, on the impact of Diwali fireworks on the mass concentration of atmospheric black carbon, showed a threefold increase over average days[35]. According to research, the aerosol from fireworks contributes up to 23-33% of the ambient PM10. So, the population is impacted by the resulting acute cardiovascular ailments and chronic exposure to diseases [36, 37]. In India, the celebration of Diwali has been associated with an increase of 30–40% in cases of respiratory infections, breathlessness, and chronic obstructive pulmonary disease (COPD)[38]. The pollution that fireworks generate is diluted before it reaches people when they are set off at a higher altitude, which can improve people's health. On the other hand, ground-level fireworks immediately harm public health[39]. According to the Environmental Protection Agency (EPA), fine particulate matter (PM2.5) poses substantial health risks to people and can premature death, cause respiratory and cardiovascular disorders, and impact the neurological system[40]. During the Diwali season, noise pollution is produced in addition to air pollution. In Haridwar, the mean noise concentration in commercial and residential areas rises by 18.1 and 29.6% in comparison to non-festive days[41]. Mahecha et al. stated that during Diwali day, the mean noise level in Jaisalmer's residential and commercial zone is 72 dB and 73.74 dB respectively[42]. 1. Introduction The entire world is currently confronting significant difficulties in terms of the degradation of air quality due to an alarming rise in anthropogenic emissions brought on by rapid industrialization, motorization, urbanization, and a lack of sufficient understanding of air quality[1, 2]. High air pollution emission levels in India are a natural consequence of rising population density and rapid economic growth[3, 4]. Short-term air quality degradation events have recently attracted the attention of the scientific community and have become an important topic of discussion at all levels [5- 9].India is regarded as the land of festivals due to its ancient traditions and diverse culture in its various states. Diwali is a "Festival of Lights" that is observed throughout India and often falls in October or November of a year[10]. Since firecrackers are extensively used during this festival, it has a significant negative impact on the environment[11]. Firecrackers, which are made of oxidizing chemicals (such as nitrates of barium and strontium, potassium chlorate, potassium perchlorates, potassium nitrates, and iron oxide) and fuels, are burned as part of celebrations[12, 13]. Fireworks emit loud noises and hazardous substances into both indoor and outdoor surroundings. The explosion of firecrackers releases a variety of harmful gases and particles of various sizes[14]. Atmospheric pollutants such as particulate matter (PM2.5 and PM10), nitrogen dioxide (NO2),volatile organic compounds (VOC), carbon dioxide (CO2), carbon monoxide (CO), sulfur dioxide (SO2), and benzene, toluene, and Numerous different metals, such as cadmium, manganese, and aluminium, etc discharged in enormous quantities have been had been associated with substantial health risks as a result of fireworks activities[12, 15-19]. On the day of Diwali, the day before Diwali, and the day after Diwali, people use a lot of fire-sparklers and fire-crackers primarily at night to celebrate[20]. This firework also results in an increased number of particulate matter concentrations. Particles of diameter 10μm (PM10) and 2.5μm (PM2.5) are associated with cardiorespiratory problems and mortality [21-23]. Fireworks have a huge negative impact on the environment and people's health since they include roughly 75% potassium nitrate, 15% carbon, and 10% sulfur and trace elements [20, 24, 25]. They release trace gases such (as CO, NOx, SO2, and O3) as well as black carbon (BC) particulates during large-scale burning, which results in the E3S Web of Conferences 453, 01004 (2023) ICSDG 2023 https://doi.org/10.1051/e3sconf/202345301004 development of dense smoke clouds[26, 27]. 1. Introduction Even though there is more firework activity during the Diwali celebration in Tamil Nadu state, India, there has been less research reported in the state to understand the variations in air quality during the celebrations. The study was carried out at the residential site in Erode City which is situated in the western region of Tamil Nadu. Erode is frequently affected by air pollution caused by particle matter. For this study, continuous measurements of PM2.5, PM10, CO, CO2, TVOC, and noise levels during the Diwali period for 5 consecutive days on October, 2022. To understand the impact of firework activities on the air quality of Erode City throughout the Diwali celebration period, the particulate matter and trace gas concentrations on pre, during, and post-Diwali day were assessed and analyzed. The findings of this study would help understand the changes in urban air quality that have taken place in Erode, a city in western Tamil Nadu,India. 2.1. Description of study site 2.1. Description of study site The study was carried out at a residential site in Erode City which is located 11.3469° North and 77.7210° East in geographical coordinates is shown in Figure 1. It has a semi-arid climate with hot to sweltering temperatures throughout the year and relatively medium rainfall. Further, the sites are only 100 m from busy 2 2 E3S Web of Conferences 453, 01004 (2023) https://doi.org/10.1051/e3sconf/202345301004 E3S Web of Conferences 453, 01004 (2023) ICSDG 2023 ICSDG 2023 roadways where all vehicle types—light, medium, and heavy—travel at a fairly high density. Also, the study site is surrounded by commercial complexes and marketplaces. fFig.1.The map showing air sampling locations of study site (source:https,//maps.google.com) fFig.1.The map showing air sampling locations of study site (source:https,//maps.google.com) 2.2.Meterology of the site The micro-meteorological parameters like temperature (°C), relative humidity (%), and wind speed (m/s) over Erode during the study period for pre-Diwali, Diwali, and post-Diwali days were shown in Figure 2(a), 2(b) and 2(c).It was noticed that over Erode during the study period, the wind speed, relative humidity, and temperature varied from 0.7-3.7 m/s,55-98%, and 21-37°C respectively. These data were obtained from the Tamil Nadu Pollution Control Board (TNPCB), Government of Tamil Nadu, India. As noted in Fig. 2, the mean relative humidity and temperature on the days before Diwali were nearly identical to those on Diwali and the days after Diwali. On Diwali night and the days after Diwali, the average wind speed was reported to be somewhat minimum than the previous days. Fig.2 (a). Day and Night-time average of wind speed (m/s) over study area Fig.2 (a). Day and Night-time average of wind speed (m/s) over study area 3 3 E3S Web of Conferences 453, 01004 (2023) ICSDG 2023 E3S Web of Conferences 453, 01004 (2023) https://doi.org/10.1051/e3sconf/202345301004 Fig.2 (b). Day and Night-time average of relative humidity (%) over study area Fig.2 (c). Day and Night-time average of temperature(°C) over study area Fig.2 (b). Day and Night-time average of relative humidity (%) over study area Fig.2 (b). Day and Night-time average of relative humidity (%) over study area Fig.2 (c). Day and Night-time average of temperature(°C) over study area Fig.2 (c). Day and Night-time average of temperature(°C) over study area 2.3.Monitoring instrumentation Air Quality Sensor (Oizom Polludrone Air Quality Monitor (AQM), OIZOM Limited, Gujarat, India) was used to measure such PM2.5 (µg/m3), PM10 (µg/m3), CO (µg/m3), CO2 (ppm), TVOC (µg/m3) and Noise level (dB). The particulate matter (PM) sensor operates based on the working principle of the optical particle counter and light scattering method. Its CO2 sensor operates based on the non-dispersive infra-red principle and the CO sensor works based on the electro-chemical principle. The TVOC sensors operate based on the principle of the photoionization detector method which was used to quantify TVOCs. Air Quality Measurement (AQM) data were collected for a total of 5 days period in which 2 days before and 2 days after the Diwali Celebration.the abbrevations are elaborated for understanding purpose for the following CO - Carbon monoxide CO2 - Carbon dioxide TVOC - Total volatile Organic Compound PM - Particle Matter CO - Carbon monoxide CO2 - Carbon dioxide TVOC - Total volatile Organic Compound PM - Particle Matter 2.4.Sampling procedure The 24-hour continuous measurements of air pollutants and noise level from October 22 to October 26, 2022, were analyzed for three different specific time intervals, viz., Pre-Diwali, Diwali, and post-Diwali day to obtain a complete picture of the urban air quality during the Diwali season in Erode city, India. The major festival day (Diwali Day) was on October 24, 2022. Thus October 22 and October 23 were considered as pre-Diwali (before Diwali) and post-Diwali (after Diwali) days were October 25 and October 26 respectively. Sampling was done by installing OIZOM Polludrone AQM at the terrace of the buildings which is 15 m from the ground level. 4 E3S Web of Conferences 453, 01004 (2023) ICSDG 2023 E3S Web of Conferences 453, 01004 (2023) https://doi.org/10.1051/e3sconf/202345301004 3.1.Variation of particulate matter (PM2.5 and PM10) concentrations The average night-time and day-time concentrations of PM2.5 before, during, and after Diwali days have been shown in Figure 3.During the pre-Diwali period, the nightfall PM2.5 concentration (230.43697µg/m³) was reported to be greater than morning time (192.72µg/m³) and hourly variations of PM2.5 is shown in Figure 4. The higher day-time concentration of PM2.5 was reported on Diwali (348.63µg/m³) followed by post-Diwali (331.95µg/m³) day. The daytime PM2.5 levels before, during, and after-Diwali day were 3, 6, and 5 times higher than the permissible limit of the National Ambient Air Quality Standard (NAAQS) over residential areas (i.e., 60µg/m³). Similarly, higher night time PM2.5 levels were observed on Diwali (497.02µg/m³) followed by post- Diwali day (395.71µg/m³). The nightfall concentration of PM2.5 before, during, and after-Diwali day exceeds the NAAQS permissible limit by 3.8, 8, and 6 times which is extremely dangerous and can potentially cause serious lung impairments. Fig.3. Day-time and night-time PM2.5 concentration over study period Fig.3. Day-time and night-time PM2.5 concentration over study period Fig.4. Hourly Variations of PM2.5 Concentrations during study period Fig.3. Day-time and night-time PM2.5 concentration over study period Fig.3. Day-time and night-time PM2.5 concentration over study period Fig.4. Hourly Variations of PM2.5 Concentrations during study period Fig.4. Hourly Variations of PM2.5 Concentrations during study period The average night-time and day-time concentrations of PM10 before, during, and after Diwali days have been shown in Figure 5. Further, the night-time concentration of PM10 was reported to be 799.43µg/m³ which was higher than the daytime concentration (652.48µg/m³) during before-Diwali day.Further, a higher amount of day- time PM10 agglomerations were reported on Diwali (958.92µg/m³) followed by post-Diwali (937.57µg/m³) day and hourly variation of PM10 is shown in Figure 6. The daytime agglomeration of PM10 before, during, and after- 5 5 E3S Web of Conferences 453, 01004 (2023) ICSDG 2023 https://doi.org/10.1051/e3sconf/202345301004 Diwali day were found to be 6.52, 9.58, and 9.37 times higher than the NAAQS threshold limit over a residential area (i.e., 100µg/m³).In addition, the maximum night-time PM10 level was reported on Diwali (1005.76µg/m³) followed by post-Diwali day (964.71µg/m³). The nightfall concentration of PM10 before, during, and after-Diwali day was very higher than the threshold limit of NAAQS by 7.9, 10.05, and 9.64 times which is extremely threatening to public health and exacerbates cardiac and respiratory disorders[43]. E3S Web of Conferences 453, 01004 (2023) ICSDG 2023 https://doi.org/10.1051/e3sconf/202345301004 In this study, both PM2.5 and PM10 concentrations were higher at night time and minimum in daytime which is consistent with the findings of Chatterjee et al. over residential place in Brahmaputra plain[45]. As a result of the mixing height decreasing by 6°C along with a 6°C drop in temperature, the wind speed is reduced at night. During the night, the wind blows at a lesser speed, which prevents the particles from dispersing, increasing the concentration of particulate matter on the surface[53].On the contrary, stronger thermal convection during the daytime increases mixing height, and greater wind speed permits the aerosol particles to be dispersed, which in turn reduces surface particulate concentrations[54]. The observance of minimum particulate matter during daytime might be due to the meteorological condition of daytime being impeditive to the formation of the thermal inversion layer[54, 55]. 3.1.Variation of particulate matter (PM2.5 and PM10) concentrations The higher particulate matter agglomeration during pre-Diwali and post-Diwali might be due to the general populace beginning to celebrate two days before Diwali and continuing for two days. Fig.5. Day-time and night-time PM10 concentration over study period Fig.5. Day-time and night-time PM10 concentration over study period Fig.6. Hourly Variations of PM10 Concentrations during study period Fig.6. Hourly Variations of PM10 Concentrations during study period Fig.6. Hourly Variations of PM10 Concentrations during study period Fig.6. Hourly Variations of PM10 Concentrations during study period The concentration of PM2.5 and PM10 on Diwali day was 3 to 4 times higher than pre-Diwali period. On Diwali day, the aerosol mass increases since it is discharged into the air because of the spontaneous emissions from firecrackers and remains in the atmosphere for a maximum duration[44]. This is the reason PM2.5 and PM10 values were found to be greater in the post-Diwali days than pre-Diwali period. Thus, it is evident that the night-time use of fireworks had a significant impact on the particulate concentration over the next day[45-49].However, it is important to highlight that the cause of the increased particle concentration is not just firecrackers; other local activities including vehicle emissions and human activities are also to be responsible[50]. Similar studies were conducted in Varanasi, where Kumar et al. observed that the significant use of firecrackers on festival days increased aerosol surface mass loading by 56–121% for the year 2014 which is compatible with the current study[51]. Another important study done by Barman et al. in the year 2008 reported higher concentrations of PM10 particles over Lucknow during the Diwali celebration which showed a similar trend with this investigation[52]. 6 6 6 E3S Web of Conferences 453, 01004 (2023) ICSDG 2023 E3S Web of Conferences 453, 01004 (2023) ICSDG 2023 3.2. Variation of gas pollutants (CO, CO2& TVOC) concentrations On burning, firecrackers release toxic compounds in the form of smoke which contains carbon monoxide (CO) and carbon dioxide (CO2). The 24-hour concentration of CO in the study site during pre-Diwali, Diwali, and post- Diwali were found to be 1568.48µg/m³, 2507.82µg/m³, and 2017.45µg/m³ respectively and hourly variation of CO concentrations is shown in Figure 7. On Diwali day, the mean concentration of CO reaches 2.5 mg/m3 which is higher than the safer limit (2 mg/m3) recommended by NAAQS. The CO concentration gradually increases from pre-Diwali and it reaches peak concentration during Diwali day, again starting to decrease in trend which shows the influence of firework activities on ambient air quality during the Diwali celebration.The average night-time and day-time CO concentrations before, during, and after Diwali days have been shown in Figure 8. The highest daytime CO levels were recorded on post-Diwali (2955.45 µg/m3), Diwali (2132.12 µg/m3), and pre-Diwali (1706 µg/m3) days. During nightfall, the maximum level of CO concentration was determined on Diwali (7265.35µg/m³) followed by post-Diwali (2505.48µg/m³) and pre-Diwali (1829.13µg/m³) respectively.Thus, it is evident that the nightly use of fireworks had a significant impact on the next daytime CO concentration[38]. On Diwali day, an alarmingly high concentration of CO (7.2 + 1.3 mg/m3) was reported which is 3.6 times greater than the NAAQS threshold limit. This large amount of CO concentration emitted during Diwali day from the burning of crackers may result in several health issues like headaches, shortness of breath, loss of consciousness, and chest pain[56, 57]. Fig.7. Day-time and night-time CO concentration over study period Fig.7. Day-time and night-time CO concentration over study period 7 E3S Web of Conferences 453, 01004 (2023) E3S Web of Conferences 453, 01004 (2023) ICSDG 2023 https://doi.org/10.1051/e3sconf/202345301004 Fig.8. Hourly Variations of CO concentrations during study period Fig.8. Hourly Variations of CO concentrations during study period Generally, firecrackers are mainly made from black powder which is carbon and these components emit CO2 in the environment after the combustion of fireworks[18]. In this study, the 24-hour average concentration of CO2 in the study site during pre-Diwali, Diwali, and post-Diwali were found to be 910.12 ppm, 1715.54 ppm, and 1257.17 ppm respectively and hourly variation of CO2 concentrations is shown in Figure 9. The CO2 concentration during Diwali day is 1.8 times higher than the pre-Diwali concentration of CO2 which depicted the influence of firework activities on ambient air quality over the study region. 3.2. Variation of gas pollutants (CO, CO2& TVOC) concentrations The maximum day-time concentration of CO2 emission appeared on Diwali (1544.05 ppm) followed by post-Diwali (1481.84 ppm) and pre-Diwali (813.69 ppm). Similarly, the maximum night- time concentration of CO2 level was higher on Diwali (1867.85 ppm) and post-Diwali (1391.52 ppm) than pre-Diwali (978.81 ppm) days. On the other hand, the day-time concentration of CO2 emission was higher than the night-time during the post-Diwali day. This may be because the night-time use of fireworks had a significant impact on the CO2 concentration the next day.The average night-time and day-time CO2 concentrations before, during, and after Diwali days have been shown in Figure 10. Fig.9. Day-time and night-time CO2 concentration over study period Fig.9. Day-time and night-time CO2 concentration over study period 8 8 E3S Web of Conferences 453, 01004 (2023) E3S Web of Conferences 453, 01004 (2023) ICSDG 2023 https://doi.org/10.1051/e3sconf/202345301004 Fig.10. Hourly Variations of CO2 Concentrations during study period Fig.10. Hourly Variations of CO2 Concentrations during study period Variations of CO2 Concentrations during study period Furthermore, fireworks contain about 10% sulfur, 15% carbon, and 75% potassium nitrate and trace elements. On burning, these elements release the vapours of toxic organic chemicals in the form of volatile organic compounds (VOCs) into the atmosphere. During the study period, the 24-hour concentration of (Total Volatile Organic Compounds) TVOC during pre, during, and post-Diwali days were found to be 915.21µg/m³, 1513.52µg/m³, and 1617.12µg/m³ respectively and hourly variation of TVOC concentrations is shown in Figure 11. The results show that a gradual increase in TVOC concentration was reported from pre-Diwali to post-Diwali days which indicates the extensive of firecrackers.This could be due to the effect of Diwali night fireworks, which lasted till the next morning (post-Diwali day).Similar to PM2.5 and PM10, the concentration of TVOC was higher during night-time and minimum in the daytime except for post-Diwali day. The average night-time and day-time TVOC concentrations before, during, and after Diwali days have been shown in Figure 12. Because during the daytime of post-Diwali day, the wind speed was minimum than night time. Higher TVOC concentration during the night time may be due to climatic factors like high humidity, decreasing temperature, and calm winds during night-time which prevents the dispersion of pollutants, which eventually leads to higher concentration of pollutants combined with firecrackers emissions.The burning of firecrackers also emits harmful volatile organic chemicals and particulate particles, according to recent studies [36, 58-60]. 3.2. Variation of gas pollutants (CO, CO2& TVOC) concentrations According to a study by Fleischer et al., the use of sparklers like "Fountains" and "Blue lightning rockets" causes the production of highly harmful organic pollutants such as polychlorinated dioxins and furans[61]. Kulshrestha et al. found that numerous flying firecrackers generate highly toxic volatile organic compounds like dibenzofurans and polychlorinated dibenzodioxins into the lower troposphere during combustion[13]. In addition, the study conducted by Chatterjee et al. reported that organic carbon,VOCs, and Polycyclic aromatic hydrocarbons (PAHs) have also been found in fireworks emissions during Diwali celebrations[45]. Altogether, the volatile organic compounds were emitted due to the burning of firecrackers during Diwali celebrations which exacerbate the risks of cardio-pulmonary illness and central nervous systems (CNS) disorders[62]. 9 9 E3S Web of Conferences 453, 01004 (2023) E3S Web of Conferences 453, 01004 (2023) ICSDG 2023 https://doi.org/10.1051/e3sconf/202345301004 Fig.11. Day-time and night-time TVOC concentration over study period Fig.12. Hourly Variations of TVOC Concentrations during study period Fig.11. Day-time and night-time TVOC concentration over study period Fig.11. Day-time and night-time TVOC concentration over study period Fig.11. Day-time and night-time TVOC concentration over study period Fig.12. Hourly Variations of TVOC Concentrations during study period Fig.12. Hourly Variations of TVOC Concentrations during study period 3.3.Variation of noise level The average noise level in the study site during pre-Diwali, Diwali, and post-Diwali were found to be 57.71 dB, 75.5 dB, and 63.66 dB respectively and hourly variations of noise level study period is shown in Figure 13. The results indicate that noise level starts to increase from pre-Diwali days and reached a higher level during Diwali day and again starts to decline in trend. Thus it indicates that the bursting of firecrackers increased noise pollution during the Diwali celebration. The average night-time and day-time noise concentrations before, during, and after Diwali days have been shown in Figure 14. The maximum night-time noise level was reported on Diwali (91.29 dB) followed by post-Diwali (71.34 dB) day and pre-Diwali (62.09 dB) day. The noise level during night-time on pre, during, and post-Diwali days were respectively 2.02, 1.58, and 1.37 times higher than the permissible limit (45 dB) of ambient noise over residential areas during night-time. In addition, the maximum noise level in the daytime was reported on Diwali (85.02 dB) followed by post-Diwali day (65.98 dB) and pre-Diwali (53.34 dB) day. Furthermore, the noise level during the daytime on Diwali and post-Diwali days were 1.5 and 1.18 times higher than the permissible limit (55 dB) of ambient noise over residential areas during night time. The present findings are consistent with the study of Chanchpara et al. reported that noise level was reported to be maximum during night-time compared with the morning time[36]. This indicates that fireworks activities are extensively carried out during the night time.An increase in noise levels during Diwali celebrations can lead tosleep disturbance, high blood pressure, and temporary or permanent hearing loss [63]. 10 E3S Web of Conferences 453, 01004 (2023) E3S Web of Conferences 453, 01004 (2023) ICSDG 2023 https://doi.org/10.1051/e3sconf/202345301004 Fig.13. Day-time and night-time noise (dB) level over study period Fig.14. Hourly Variations of noise level during study period 4 C l i Fig.13. Day-time and night-time noise (dB) level over study period Fig.13. Day-time and night-time noise (dB) level over study period Fig.14. Hourly Variations of noise level during study period Fig.14. Hourly Variations of noise level during study period 4.Conclusion The current study investigates the impact of Diwali fireworks activities on air quality over a residential site at Erode City, Tamil Nadu state of India for five consecutive days from October 22 to October 26, 2022. This study shows that the fireworks activities had a strong effect in increasing particulate matter and gaseous pollutants concentration over the study site. Both PM2.5 and PM10 concentrations during pre, during, and post-Diwali days exceed the NAAQS permissible limit on the residential area. Furthermore, the night-time concentration of particulate matter was higher than daytime concentrations. This study shows that the nightfall concentration of PM10 before, during, and after-Diwali day was very higher than the threshold limit of NAAQS by 7.9, 10.05, and 9.64 times. Additionally, the nightfall concentration of PM2.5 before, during, and after-Diwali day exceeded the NAAQS permissible limit by 3.8, 8, and 6 times in the current study.The short-term high accumulation of PM2.5 and PM10 is a matter of serious issue for people because it can infiltrate deeply into the lungs and cause many respiratory and cardiovascular problems.On Diwali day, an alarmingly high concentration of CO (7.2 + 1.3 mg/m3) was found which is 3.6 times greater than NAAQS permissible limit. In this study, the 24-hour average concentration of CO2 in the study site during pre, during, and post- Diwali days were found to be 910.12 ppm, 1715.54 ppm, and 1257.17 ppm respectively.The maximum concentration of TVOC was observed to be 1849 µg/m³ during the night time of Diwali day.During the Diwali season, the accompanying climatic conditions (lowered night-time boundary layer height, lowering temperature, and low wind speed) cause an unwanted deposit of air pollutants close to the surface layer at the study location. During the study, the noise concentration was reported to be higher at in the night-time than daytime. REFERENCES REFERENCES 1. Fallahi, S., et al., Estimating solar radiation using NOAA/AVHRR and ground measurement data. Atmospheric Research, 2018. 199: p. 93-102.https://doi.org/10.1016/j.atmosres.2017.09.006 2. Tzanis, C.G., et al., Applying linear and nonlinear models for the estimation of particulate matter variability. Environmental Pollution, 2019. 246: p. 89-98.https://doi.org/10.1016/j.envpol.2018.11.080 3. Gurjar, B.R., K. Ravindra, and A.S. Nagpure, Air pollution trends over Indian megacities and their local-to-global implications. Atmospheric Environment, 2016. 142: p. 475- 495.https://doi.org/10.1016/j.atmosenv.2016.06.030 1. Fallahi, S., et al., Estimating solar radiation using NOAA/AVHRR and ground measuremen Atmospheric Research, 2018. 199: p. 93-102.https://doi.org/10.1016/j.atmosres.2017.09.006 1. Fallahi, S., et al., Estimating solar radiation using NOAA/AVHRR and ground measurement data. Atmospheric Research, 2018. 199: p. 93-102.https://doi.org/10.1016/j.atmosres.2017.09.006 2. Tzanis, C.G., et al., Applying linear and nonlinear models for the estimation of particulate matter variability. Environmental Pollution, 2019. 246: p. 89-98.https://doi.org/10.1016/j.envpol.2018.11.080 3 Gurjar, B R , K Ravindra, and A S Nagpure, Air pollution trends over Indian megacities and their Atmospheric Research, 2018. 199: p. 93-102.https://doi.org/10.1016/j.atmosres.2017.09.006 2. Tzanis, C.G., et al., Applying linear and nonlinear models for the estimation of particulate matter variability. Environmental Pollution, 2019. 246: p. 89-98.https://doi.org/10.1016/j.envpol.2018.11.080 p , p p g j 2. Tzanis, C.G., et al., Applying linear and nonlinear models for the estimation of particulate matter variability. Environmental Pollution, 2019. 246: p. 89-98.https://doi.org/10.1016/j.envpol.2018.11.080 3. Gurjar, B.R., K. Ravindra, and A.S. Nagpure, Air pollution trends over Indian megacities and their local-to-global implications. Atmospheric Environment, 2016. 142: p. 475- 495.https://doi.org/10.1016/j.atmosenv.2016.06.030 3. Gurjar, B.R., K. Ravindra, and A.S. Nagpure, Air pollution trends over Indian megacities and their local-to-global implications. Atmospheric Environment, 2016. 142: p. 475- 495.https://doi.org/10.1016/j.atmosenv.2016.06.030 4. Upadhyay, A., et al., Expected health benefits from mitigation of emissions from major anthropogenic PM2. 5 sources in India: Statistics at state level. Environmental Pollution, 2018. 242: p. 1817-1826.https://doi.org/10.1016/j.envpol.2018.07.085 5. Pope III, C.A. and D.W. Dockery, Health effects of fine particulate air pollution: lines that connect. Journal of the air & waste management association, 2006. 56(6): p. 709- 742.https://doi.org/10.1080/10473289.2006.10464485 6. Nastos, P.T., et al., Outdoor particulate matter and childhood asthma admissions in Athens, Greece: a time-series study. Environmental Health, 2010. 9(1): p. 1-9.https://doi.org/10.1186/1476-069X-9- 45 7. Singh, D., et al., Study of temporal variation in ambient air quality during Diwali festival in India. Environmental monitoring and assessment, 2010. 169: p. 1-13.https://doi.org/10.1007/s10661-009- 1145-9 8. Thakur, B., et al., Air pollution from fireworks during festival of lights (Deepawali) in Howrah, India-a case study. Atmosfera, 2010. 23(4): p. 347-365. 9. 4.Conclusion The noise level during the night-time on pre, during, and post-Diwali days were respectively 2.02, 1.58, and 1.37 times higher than the permissible limit and thus 11 11 E3S Web of Conferences 453, 01004 (2023) E3S Web of Conferences 453, 01004 (2023) ICSDG 2023 https://doi.org/10.1051/e3sconf/202345301004 depicted a higher amount of crack bursting during the night.A noteworthy finding from this study is that the average concentration of the air pollutants it observed remained high even on the day after Diwali, indicating that it may take longer than a day to clear the atmosphere after igniting firecrackers.Diwali celebrations result in the discharge of gaseous and particle pollutants that dwell in the atmosphere for a longer period, harming the ecosystem and the public's health. Thus, people should raise awareness to celebrate Diwali in an environmentally friendly manner. Acknowledgment We wish to thank the Indian Council for Medical Research (5/8-4/30/ENV/2020- NCD-II) for funding REFERENCES Bhuyan, P.K., et al., The role of precursor gases and meteorology on temporal evolution of O 3 at a tropical location in northeast India. Environmental Science and Pollution Research, 2014. 21: p. 6696-6713.https://doi.org/10.1007/s11356-014-2587-3 p g 10. Peshin, S.K., P. Sinha, and A. Bisht, Impact of Diwali firework emissions on air quality of New Delhi, India during 2013-2015. Mausam, 2017. 68(1): p. 111- 118.https://doi.org/10.54302/mausam.v68i1.438 11. Singh, A., P. Pant, and F.D. Pope, Air quality during and after festivals: Aerosol concentrations, composition and health effects. Atmospheric Research, 2019. 227: p. 220- 232.https://doi.org/10.1016/j.atmosres.2019.05.012 g , , , p , q y g , composition and health effects. Atmospheric Research, 2019. 227: p. 220- 232.https://doi.org/10.1016/j.atmosres.2019.05.012 p g j 12. Ravindra, K., S. Mor, and C. Kaushik, Short-term variation in air quality associated with firework events: a case study. Journal of Environmental Monitoring, 2003. 5(2): p. 260- 264.https://doi.org/10.1039/B211943A 13. Kulshrestha, U., et al., Emissions and accumulation of metals in the atmosphere due to crackers and sparkles during Diwali festival in India. Atmospheric Environment, 2004. 38(27): p. 4421- 4425.https://doi.org/10.1016/j.atmosenv.2004.05.044 p g j 14. Russell, M.S., The chemistry of fireworks. 2009: Royal Society of Chemistry. ssell, M.S., The chemistry of fireworks. 2009: R y y y y 15. Mandal, R., B. Sen, and S. Sen, Impact of fireworks on our environment. Indian Journal of Environmental Protection, 1997. 17(11): p. 850-853. 12 https://doi.org/10.1051/e3sconf/202345301004 E3S Web of Conferences 453, 01004 (2023) ICSDG 2023 E3S Web of Conferences 453, 01004 (2023) 16. Drewnick, F., et al., Measurement of fine particulate and gas-phase species during the New Year's fireworks 2005 in Mainz, Germany. Atmospheric Environment, 2006. 40(23): p. 4316- 4327.https://doi.org/10.1016/j.atmosenv.2006.03.040 17. Vecchi, R., et al., The impact of fireworks on airborne particles. Atmospheric Environment, 2008. 42(6): p. 1121-1132.https://doi.org/10.1016/j.atmosenv.2007.10.047 18. Wang, Y., et al., The air pollution caused by the burning of fireworks during the lantern festival in Beijing. Atmospheric Environment, 2007. 41(2): p. 417- 431.https://doi.org/10.1016/j.atmosenv.2006.07.043 19. Pachauri, T., et al., Characterization of major pollution events (dust, haze, and two festival events) at Agra, India. Environmental Science and Pollution Research, 2013. 20: p. 5737- 5752.https://doi.org/10.1007/s11356-013-1584-2 20. Pratap, V., et al., Analysis of air pollution in the atmosphere due to firecrackers in the Diwali period over an urban Indian region. Advances in Space Research, 2021. 68(8): p. 3327- 3341.https://doi.org/10.1016/j.asr.2021.06.031 p g j 21. Maciejewska, K., Short-term impact of PM2. 5, PM10, and PMc on mortality and morbidity in the agglomeration of Warsaw, Poland. 34. Kumar, S. and S.K. Dwivedi, Assessment of air quality in Lucknow, India during the festival of Diwali for four successive years amid the COVID-19 pandemic lockdown. Physics and Chemistry of the Earth, Parts A/B/C, 2023: p. 103439.https://doi.org/10.1016/j.pce.2023.103439 33. Tang, G., et al., Aggravated ozone pollution in the strong free convection boundary layer. Science of The Total Environment, 2021. 788: p. 147740.https://doi.org/10.1016/j.scitotenv.2021.147740 33. Tang, G., et al., Aggravated ozone pollution in the strong free convection boundary layer. Science of The Total Environment, 2021. 788: p. 147740.https://doi.org/10.1016/j.scitotenv.2021.147740 34. Kumar, S. and S.K. Dwivedi, Assessment of air quality in Lucknow, India during the festival of REFERENCES Air Quality, Atmosphere & Health, 2020. 13(6): p. 659- 672.https://doi.org/10.1007/s11869-020-00831-9 p g 22. Atkinson, R., et al., Epidemiological time series studies of PM2. 5 and daily mortality and hospital admissions: a systematic review and meta-analysis. Thorax, 2014. 69(7): p. 660- 665.http://dx.doi.org/10.1136/thoraxjnl-2013-204492 p g j 23. Beelen, R., et al., Long-term exposure to air pollution and cardiovascular mortality: an European cohorts. Epidemiology, 2014: p. 368-378.https://www.jstor.org/stable/24759129 24. Kotnala, G., et al., Variations in chemical composition of aerosol during Diwali over mega city Delhi, India. Urban Climate, 2021. 40: p. 100991.https://doi.org/10.1016/j.uclim.2021.100991 25. Kurwadkar, S., et al., Emissions of black carbon and polycyclic aromatic hydrocarbons: Potential implications of cultural practices during the Covid-19 pandemic. Gondwana Research, 2023. 114: p. 4-14.https://doi.org/10.1016/j.gr.2022.10.001 26. Kumar, K., et al., Episodic measurements of PM2 5 during crop residue burning and Diwali Periods at Delhi. Journal of Indian Geophysical Union, 2020. 24(4): p. 40-50. 27 S P l A l i f h i ll d i fi k f i l ‘Di li’ 27. Saxena, P., et al., Analysis of atmospheric pollutants during fireworks festival ‘Diwali’at a residential site Delhi in India. Measurement, analysis and remediation of environmental pollutants, 2020: p. 91-105. 28. Srivastava, S., et al., Long-term observation of black carbon aerosols at an urban location over the central Indo-Gangetic Plain, South Asia. Atmósfera, 2019. 32(2): p. 95- 113 h d 29. Barupal, T., et al., Evaluation of air quality change of udaipur city during banned crackers deepawali (2020) and unbanned crackers deepawali (2021). Biomaterials Journal, 2022. 1(3): p. 3-8. 30. Garg, A. and N.C. Gupta, Short-term variability on particulate and gaseous emissions induced by fireworks during Diwali celebrations for two successive years in outdoor air of an urban area in Delhi, India. SN Applied Sciences, 2020. 2: p. 1-14.https://doi.org/10.1007/s42452-020-03906-5 31. Gautam, S., et al. Short-term introduction of air pollutants from fireworks during Diwali in rural Palwal, Haryana, India: A case study. in IOP Conference Series: Earth and Environmental Science. 2018. IOP Publishing.DOI 10.1088/1755-1315/120/1/012009 32. Rathee, A. and S. Yadav, Health risk assessment using chemical signatures of fine and coarse particles collected at breathing level height during firework display in New Delhi, India. Human and Ecological Risk Assessment: An International Journal, 2022. 28(8): p. 893- 916.https://doi.org/10.1080/10807039.2022.2107480 , p p g j 34. Kumar, S. and S.K. Dwivedi, Assessment of air quality in Lucknow, India during the festival of Diwali for four successive years amid the COVID-19 pandemic lockdown. 33. Tang, G., et al., Aggravated ozone pollution in the strong free convection boundary layer. Science of The Total Environment, 2021. 788: p. 147740.https://doi.org/10.1016/j.scitotenv.2021.147740 34. Kumar, S. and S.K. Dwivedi, Assessment of air quality in Lucknow, India during the festival of Diwali for four successive years amid the COVID-19 pandemic lockdown. Physics and Chemistry of the Earth, Parts A/B/C, 2023: p. 103439.https://doi.org/10.1016/j.pce.2023.103439 REFERENCES Physics and Chemistry of the Earth, Parts A/B/C, 2023: p. 103439.https://doi.org/10.1016/j.pce.2023.103439 13 13 https://doi.org/10.1051/e3sconf/202345301004 E3S Web of Conferences 453, 01004 (2023) E3S Web of Conferences 453, 01004 (2023) ICSDG 2023 35. Babu, S.S. and K.K. Moorthy, Anthropogenic impact on aerosol black carbon mass concentration at a tropical coastal station: A case study. Current Science, 2001: p. 1208- 1214.https://www.jstor.org/stable/24106437 p j g 36. Chanchpara, A., et al., Pre-to-post Diwali air quality assessment and particulate matter characterization of a western coastal place in India. Environmental Monitoring and Assessment, 2023. 195(3): p. 413.https://doi.org/10.1007/s10661-023-11018-x 37. Brook, R.D., et al., Air pollution and cardiovascular disease: a statement for healthcare professionals from the Expert Panel on Population and Prevention Science of the American Heart Association. Circulation, 2004. 109(21): p. 2655-2671.https://doi.org/10.1161/01.CIR.0000128587.30041.C8 p 38. Saha, U., et al., Effects of air pollution on meteorological parameters during Deepawali festival over an Indian urban metropolis. Atmospheric Environment, 2014. 98: p. 530- 539.https://doi.org/10.1016/j.atmosenv.2014.09.032 p g j 39. Yerramsetti, V.S., et al., The impact assessment of Diwali fireworks emissions on the air quality of a tropical urban site, Hyderabad, India, during three consecutive years. Environmental monitoring and assessment, 2013. 185: p. 7309-7325.https://doi.org/10.1007/s10661-013-3102-x p p g 40. Basith, S., et al., The impact of fine particulate matter 2.5 on the cardiovascular system: A review of the invisible killer. Nanomaterials, 2022. 12(15): p. 2656.https://doi.org/10.3390/nano12152656 41. Sharma, V. and B. Joshi, Assessment of noise pollution during Deepawali festival in a small township of Haridwar City of Uttarakhand, India. The Environmentalist, 2010. 30: p. 216- 218.https://doi.org/10.1007/s10669-010-9265-x p g 42. Mahecha, G., et al., Noise pollution monitoring during Diwali festival in Golden city Jaisalmer of Rajasthan, India. The Environmentalist, 2012. 32: p. 415-419.https://doi.org/10.1007/s10669-012- 9404-7 43. Dominici, F., et al., Fine particulate air pollution and hospital admission for cardiovascular and respiratory diseases. Jama, 2006. 295(10): p. 1127-1134.doi:10.1001/jama.295.10.1127 44. VYAS, B. and A. Saxenna, Behavior of Atmospheric Air Pollutants during Diwali Fireworks Emission over Western Tropical Semi-Arid and Sub-Urban site, Udaipur, India. 2023.https://doi.org/10.21203/rs.3.rs-2527699/v1 p g 45. Chatterjee, A., et al., Ambient air quality during Diwali Festival over Kolkata-a mega-city in India. Aerosol and Air Quality Research, 2013. 13(3): p. 1133- 1144.https://doi.org/10.4209/aaqr.2012.03.0062 46. RAJpOOT, S., Impact Assessment of Diwali Fireworks Emissions on the Air at Special Locations in Delhi During Three Consecutive Years. Oriental Journal of Chemistry, 2023. 39(2). 47. Kumar, S. and S.K. REFERENCES Dwivedi, Assessment of air quality in Lucknow, India during the festival of Diwali for four successive years amid the COVID-19 pandemic lockdown. Physics and Chemistry of the Earth, Parts A/B/C, 2023. 131: p. 103439.https://doi.org/10.1016/j.pce.2023.103439 48. Kumar, S., P. Dwivedi, and S. Kumar, Air Quality During the Diwali Festival for Four Successive Years Amidst Covid-19 Pandemic Lockdown in India's Most Polluted City. Air Quality During the Diwali Festival for Four Successive Years Amidst Covid-19 Pandemic Lockdown in India's Most Polluted City, 2023.https://doi.org/10.1016/j.pce.2023.103439 49. Rani, N., S. Singh, and M.J. Kulshrestha, Measurement and Distribution Pattern of n-alkanes in Size-Segregated Aerosols During Diwali Festival in Delhi, India. Water, Air, & Soil Pollution, 2023. 234(4): p. 212.https://doi.org/10.1007/s11270-023-06220-z p p g 50. Huang, K., et al., Impact of anthropogenic emission on air quality over a megacity–revealed from an intensive atmospheric campaign during the Chinese Spring Festival. Atmospheric Chemistry and Physics, 2012. 12(23): p. 11631-11645.https://doi.org/10.1016/j.apr.2023.101888 51. Kumar, P., et al., Assessment of atmospheric aerosols over Varanasi: Physical, optical and chemical properties and meteorological implications. Journal of Atmospheric and Solar-Terrestrial Physics, 2020. 209: p. 105424. man, S., et al., Fine particles (PM. Journal of Environmental Biology, 2009. 30: p. 625 .https://doi.org/10.21203/rs.3.rs-2527699/v1 53. Jones, A.M., R.M. Harrison, and J. Baker, The wind speed dependence of the concentrations of airborne particulate matter and NOx. Atmospheric Environment, 2010. 44(13): p. 1682-1690. 54. Wang, J., et al., Impact of aerosol–meteorology interactions on fine particle pollution during China’s severe haze episode in January 2013. Environmental Research Letters, 2014. 9(9): p.094002. https://doi.org/10.1029/2022JD037986 14 https://doi.org/10.1051/e3sconf/202345301004 E3S Web of Conferences 453, 01004 (2023) ICSDG 2023 E3S Web of Conferences 453, 01004 (2023) 55. Sarkar, P., et al. Impact of bursting fireworks during Diwali in Durgapur suburb, India: A case study. in Proceedings of the 24th International Conference on Distributed Computing and Networking. 2023.https://doi.org/10.1145/3571306.3571440 p g 56. Goldsmith, J.R. and S.A. Landaw, Carbon monoxide and human health. Science, 1968. 162(3860): p. 1352-1359. 57. Stewart, R.D., The effect of carbon monoxide on humans. Annual review of pharmacology, 1975. 15(1): p. 409-423. 58. Klima, V., et al., Assessment of air pollution with polychlorinated dibenzodioxins (PCDDs) and polychlorinated dibenzofuranes (PCDFs) in Lithuania. Atmosphere, 2020. 11(7): p. 759 https://doi org/10 1080/10408398 2021 2012752 59. Liu, J., et al., Levels and health risks of PM2. 5-bound toxic metals from firework/firecracker burning during festival periods in response to management strategies. Ecotoxicology and Environmental Safety, 2019. 171: p. p g ( ) 62. Wolkoff, P., Volatile organic compounds. Indoor air, 1995. 3: p. 1-73. REFERENCES 406-413.https://doi.org/10.1007/s12647-023-00646-w y, p p g 60. Camilleri, R. and A.J. Vella, Effect of fireworks on ambient air quality in Malta. Atmospheric Environment, 2010. 44(35): p. 4521-4527.https://doi.org/10.1016/j.atmosenv.2010.07.057 p p g j 61. Fleischer, O., H. Wichmann, and W. Lorenz, Release of polychlorinated dibenzo-p-dioxins and dibenzofurans by setting off fireworks. Chemosphere, 1999. 39(6): p. 925- 932.https://doi.org/10.1016/S0045-6535(99)00019-3 61. Fleischer, O., H. Wichmann, and W. Lorenz, Release of polychlorinated dibenzo-p-dioxins and dibenzofurans by setting off fireworks. Chemosphere, 1999. 39(6): p. 925- 932.https://doi.org/10.1016/S0045-6535(99)00019-3 p g 62. Wolkoff, P., Volatile organic compounds. Indoor air, 1995. 3: p. 1-73. , , g p , p 63. Ralte, L., Assessment of noise pollution and its effects on human health in Aizawl City, Mizoram. 2014, Mizoram University. g p p 63. Ralte, L., Assessment of noise pollution and its effects on human health in Aizawl City, Mizoram. 2014, Mizoram University. 15
https://openalex.org/W3190165006	https://link.springer.com/content/pdf/10.1140/epjp/s13360-021-01747-8.pdf	English	null	Analysis of correlation and ionization from pair distributions in many-electron systems	The European physical journal plus	2,021	cc-by	9,367	Eur. Phys. J. Plus (2021) 136:763 https://doi.org/10.1140/epjp/s13360-021-01747-8 Eur. Phys. J. Plus (2021) 136:763 https://doi.org/10.1140/epjp/s13360-021-01747-8 Regular Article Analysis of correlation and ionization from pair distributions in many-electron systems S. López-Rosa1,2,a , J. C. Angulo2,3,b, A. L. Martín4, J. Antolín2 1 Departamento de Física Aplicada II, Universidad de Sevilla, 41012 Sevilla, Spain 2 Instituto Carlos I de Física Teórica y Computacional, Universidad de Granada, 18071 Granada, Spain 3 Departamento de Física Atómica, Molecular y Nuclear, Universidad de Granada, 18071 Granada, Spain 4 Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, 10115 Berlin, Germany S. López-Rosa1,2,a , J. C. Angulo2,3,b, A. L. Martín4, J. Antolín2 1 Departamento de Física Aplicada II, Universidad de Sevilla, 41012 Sevilla, Spain 2 Instituto Carlos I de Física Teórica y Computacional, Universidad de Granada, 18071 Granada, Spain 3 Departamento de Física Atómica, Molecular y Nuclear, Universidad de Granada, 18071 Granada, Spain 4 Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, 10115 Berlin, Germany Received: 21 January 2021 / Accepted: 10 July 2021 © The Author(s) 2021 Received: 21 January 2021 / Accepted: 10 July 2021 © The Author(s) 2021 Abstract Jensen–Shannon divergence is used to quantify the discrepancy between the Hartree–Fock pair density and the product of its marginals for different N-electron systems, enclosing neutral atoms (with nuclear charge Z = N) and singly-charged ions (N = Z ±1). This divergence measure is applied to determine the interelectronic correlation in atomic systems. A thorough study was carried out, by considering (i) both position and momentum conjugated spaces, and (ii) systems with a nuclear charge as far as Z = 103. The correlation among electrons was measured by comparing, for an arbitrary system, the double-variable electron-pair density with the product of the respective one-particle densities. A detailed analysis throughout the Periodic Table highlights the relevance not only of weightiness for the systems considered, but also of their shell structure. Besides, comparative computations between two-electron densities of different atomic systems (neutrals, cations, anions) quan- tify their dissimilarities, patently governed by shell-ﬁlling patterns throughout the Periodic Table. a e-mail: slopezrosa@us.es (corresponding author) b e-mail: angulo@ugr.es 0123456 1 Introduction The two-electron density Γ (r1, r2) is the probability density of any two electrons that are located at radii r1 and r2, respectively, and is a convenient starting point to study the spatial interaction between two electrons in an explicit manner [1]. This density provides a quantum- mechanical description of the distribution of electron pairs in the space and therefore rep- resents a central tool, in quantum chemistry, to study chemical binding [2] or electronic structure and correlation in molecular systems [3,4]. On the other hand, the exact interaction energy of a many-electron system is determined by the electron pair density, but is not well-approximated in standard Kohn–Sham density functional models [5,6]. Consequently, pair density functional theory is regarded as not only an extension of the density functional theory but a reduced density matrix theory as well [7,8]. The main feature of the pair density functional theory is that the pair density, as a basic a e-mail: slopezrosa@us.es (corresponding author) b e-mail: angulo@ugr.es 0123456 123 763 Page 2 of 16 763 Page 2 of 16 Eur. Phys. J. Plus (2021) 136:763 763 variable, essentially contains more information on the electron-electron interaction than the electron density [9,10]. The expectation value of an arbitrary two-particle operator can be obtained using only the electron pair density. In addition to this merit, the pair density functional theory has another, which is related to the expansion of the approximate functional. That is to say, in the pair density functional theory, the only required selection is the approximate form of the kinetic energy functional, because the exchange-correlation energy functional can rigorously be expressed by means of the pair density [11]. As a recent example multiconﬁguration pair density functional theory has been developed, as a way to combine the advantages of both wave function and density functional theories to provide a better treatment of strongly cor- related systems, and its results have been compared to those of some Kohn–Sham density functionals, including both traditional and modern functionals [12,13]. Additional applica- tions have been performed recently with a diversity of molecular systems, on the basis of their one- and two-electron RDMs (reduced density matrices), which comparison provides measures of electron-correlation and entanglement among systems. These measures display their sensitivity to the presence of electric ﬁelds [14]. 1 Introduction Therefore, given the above, it would be logical to assume that two-electron densities for atoms and molecules have been studied both intensively and extensively. However, this is not the case. The effort invested in interpretative studies of electron pair densities has been small as compared to that expended on such studies for one-electron densities. Indeed, there is a large amount of information coded inside monoelectronic densities; however, these densities do not give us any hint on how the position (or the momentum) of an electron conditions the positions (or the momenta) of the others. Besides it is worthy to note that within the Waller– Hartree theory, the two-electron density is related to the total X-ray scattering intensity which is accessible by experimental measurements [15,16]. One of the main difﬁculties rests in the interpretation of the electron pair density. Visual representation is a powerful analytical tool; however, being a function of 6 coordinates, graphical representation of the pair density is not feasible and manipulations to the two- electron density must be carried out to extract useful information. One such manipulation gives place to the intracule and extracule densities and many relevant results have been obtained in this framework [17–21]. It is important to note that these densities consider the pairs of electrons as a whole thing. Other simpliﬁcation consists in dealing with spherically averaged electron pair densities. Consequently, as a result of this reduction in dimensionality, some two-electron information is lost in the transformation. The concepts of uncertainty, randomness, disorder and delocalization are basic ingredients in the study, within an information-theoretic framework, of relevant structural properties for many different probability distributions appearing as descriptors of several chemical and physical systems and processes. The relevancy of these concepts has motivated new studies that pursue quantiﬁcation, giving rise to a variety of density functionals, such as Shannon entropy [22], Fisher information [23], disequilibrium [24], complexity [25], and many others. These information measures have been widely employed for describing the information content and behavior in a great variety of ﬁelds [26,27] and, in particular, for the study of many-electron systems [27–34] Following the usual procedures carried out within information theory for quantifying the uncertainty or disorder of individual distributions, some extensions have been made in order to introduce the concepts of ‘distance’ and ‘divergence’ between two distributions, as comparative measures of their dissimilarity. 1 Introduction Jensen–Shannon divergence (JSD) is a powerful divergence measure that represents the difference between the Shannon entropy of the mean density and the mean value of the indi- 123 Eur. Phys. J. Plus (2021) 136:763 Page 3 of 16 763 vidual entropies [35–37]. Its non-negativity arises from the convex character of the Shan- non entropy functional. This comparative measure has found deep applications in statistics and many other ﬁelds, including entanglement characterization [38], analysis of symbolic sequences or series, and in particular in the study of segmentation of DNA sequences [39]. JSD and some effective generalizations have been used for the study and comparison of one-electron densities [40–44]. For the ﬁrst time, to the best of our knowledge, this infor- mation measure of dissimilarity is applied to the study of two-electron density functions. Let us emphasize, however, recent applications of strongly related functionals (e.g. mutual information, cumulative residual entropies, interaction information) for uncoupled and inter- acting harmonic oscillators [45,46]. Additional applications include the cumulant part of two-electron RDMs, for the detection of van der Waals interactions between speciﬁc parts of molecular systems [47]. Other measures enclosing also the gradient of the density, as the relative Fisher information, were considered for the study of speciﬁc one-particle quantum systems (Morse potential, isotropic oscillator or hydrogen-like atoms) [48,49]. In this paper, small correlation effects in atoms are analyzed, on the basis of discrepancies between the Hartree–Fock one- and two-electron densities. In doing so, comparative den- sity functionals are employed as quantiﬁers of dissimilarity among the above densities. So differences arising from the exchange term are emphasized, as the main cause of them. A detailed analysis throughout the Periodic Table reveals the relevance not only of weightiness for the systems considered, but also of their shell structure. Besides, comparative computa- tions between two-electron densities of different atomic systems (neutrals, cations, anions) quantify the important differences patently governed by shell-ﬁlling patterns throughout the Periodic Table. Thereby, we use an information-theoretic functional that has shown its univer- sality and versatility in other computations: the Jensen–Shannon divergence (JSD) [35–37]. The organization of the paper is the following: in the next section, two-particle densities are deﬁned and presented for atomic systems and also the entropic divergence measure (JSD) we are going to use for comparing them. Numerical computations and results are presented in Sect. 3. 1 Introduction Finally, conclusions and open problems or future work are brieﬂy described in the last section. 2 Electron pair densities and related measures In terms of the N-electron wave function Ψ , and its Fourier transform Φ, the two-electron densities are deﬁned as Γ (r1, r2) = Ψ (x1, x2, . . . , xN)Ψ ∗(x1, x2, . . . , xN)dσ1dσ2dx3 . . . dxN (1) the position space, and Π( ) Φ( )Φ∗( )d d d d (2) Γ (r1, r2) = Ψ (x1, x2, . . . , xN)Ψ ∗(x1, x2, . . . , xN)dσ1dσ2dx3 . . . dxN (1) Γ (r1, r2) = Ψ (x1, x2, . . . , xN)Ψ ∗(x1, x2, . . . , xN)dσ1dσ2dx3 . . . dxN (1) the position space and (1) in the position space, and in the position space, and Π(p1, p2) = Φ(y1, y2, . . . , yN)Φ∗(y1, y2, . . . , yN)dσ1dσ2dy3 . . . dyN (2) (2) in the momentum space. The variables xi = riσi and yi = piσi are combined coordinates which include the spin. It is well known that the physical meaning of these densities regards the probability of ﬁnding an electron with a compatible state within the region r1dr1 if there is another electron with allowed/compatible quantum numbers within the region r2dr2, and similarly regarding the momentum regions p1dp1 and p2dp2. These densities are directly related to the electron correlations, as the compatibility of an electron state is in organically in the momentum space. The variables xi = riσi and yi = piσi are combined coordinates which include the spin. It is well known that the physical meaning of these densities regards the probability of ﬁnding an electron with a compatible state within the region r1dr1 if there is another electron with allowed/compatible quantum numbers within the region r2dr2, and similarly regarding the momentum regions p1dp1 and p2dp2. These densities are directly related to the electron correlations, as the compatibility of an electron state is in organically 123 763 Page 4 of 16 Eur. Phys. J. Plus (2021) 136:763 763 determined by the compatibility of its state with those of the others. They naturally give us quantiﬁers of correlation between electrons. 2 Electron pair densities and related measures The two-electron densities are going to be calculated by the Hartree–Fock approach and can be expressed as follows [50]: Γ (r1, r2) = 1 N −1 [Nρ(r1)ρ(r2) −Γx(r1, r2)] (3) (3) in the position space, and Π(p1, p2) = 1 N −1 Nγ (p1)γ (p2) −Πx(p1, p2) (4) (4) in the momentum space, respectively. The functions ρ(ri) and γ (pi) are the one-electron den- sities and Γx(r1, r2) and Πx(p1, p2) are the exchange densities in the position and momentum space respectively. Let us remark that, using Hartree–Fock functions, we are studying the Fermi correlation between same-spin electrons which arises from the antisymmetry of the wave function. In this sense, the term electron correlation, mentioned before, alludes to the statistical correlation. This point must be clariﬁed in order to not lead to confusion if one considers a correlated system as one beyond the Hartree–Fock approximation (the Löwdin deﬁnition of correlation energy). It should be noticed that the one-particle densities are marginal distributions of the two- particle ones, because the former are obtained from the latter by integration, as ρ(r1) = Γ (r1, r2)dr2 and γ (p1) = Π(p1, p2)dp2. Thus, the information content of the marginals is not enough to determine the double-variable distribution they are coming from. Different deﬁnitions of distance between two arbitrary distributions f (x) and g(x) appear, where both are deﬁned over the same domain. The interest in ﬁnding appropriate tools for ‘comparing’ two different systems or processes in terms of distribution functions is justiﬁed by the strong connection between some of the above mentioned information measures and many relevant physical and chemical properties of those systems. The term ‘distance’ should be understood as a measure of how dissimilar the two functions are, not necessarily verifying all mathematical properties required for a true distance, as the usual quadratic distance does. However, all of them keep as main distance properties the non-negativity, the symmetry (invariance under exchange of functions) and saturation (minimal zero value only for identical distributions). The relative entropy or Kullback–Leibler divergence (KL) [35] is one of the pioneering global measures of the difference between two arbitrary uni- or multivariate probability distributions. 2 Electron pair densities and related measures It expresses the amount of information supplied by the data for discriminating among the distributions, being a ‘directed divergence’ and therefore not symmetric: K L ( f, g) = f (x) ln f (x) g(x) dx (5) (5) Its applications for different procedures in obtaining minimum cross entropy estimations and the determination of atomic and molecular properties [29], among others, make it to constitute an essential tool within the information theory. Won and You introduced, some- what implicity, a closely related information measure between two or more distributions, the Jensen–Shannon divergence (JSD) [51,52]: J SD( f, g) = 1 2 K L f, f + g 2 + K L g, f + g 2 . (6) (6) Consequently, JSD represents the mean dissimilarity (understood in terms of the KL measure) of each density respect to the mean one. Consequently, JSD represents the mean dissimilarity (understood in terms of the KL measure) of each density respect to the mean one. 123 Eur. Phys. J. Plus (2021) 136:763 Page 5 of 16 763 This measure is strongly related to the Shannon entropy, which is deﬁned for an arbitrary uni- or multivariate probability distribution as S( f ) = − dx f (x) ln f (x). Shannon entropy is well known to play a relevant role within an information-theoretic framework because it constitutes a measure of spreading of the density over its domain [53]. Attending to the JSD deﬁnition given above, the Jensen–Shannon divergence can be also expressed in terms of the Shannon entropy as J SD( f, g) = S f + g 2 −1 2 [S( f ) + S(g)] . (7) (7) The JSD is characterized for quantifying the “Shannon entropy excess” of a couple of distributions with respect to the mixture of their respective entropies (mean density), deﬁned in Eq. (7) as a particular case of the so-called weighted generalized divergences [54] with identical weights 1/2. Apart from preserving the global character of the Shannon entropy, the JSD possesses the main properties required for a measure to be interpreted as an informational distance. In particular, its non-negativity is a consequence of the convexity of the S( f ) functional. 2 Electron pair densities and related measures Other important advantages of this divergence are that: (i) does not require the condition of absolute continuity for the probability distributions involved, (ii) weights of each density can be different from 1/2 as appearing in Eq. (7), and (iii) can be generalized for an arbitrary number of distributions. During the past years researchers have been interested in work towards parametric generalizations of these classical measures of information. The JSD also admits other kind of generalizations, as will be shown elsewhere. In this work we intend to perform an exhaustive analysis of atomic systems using JSD, on the basis of their two-electron densities, in the following two ways: (i) Differences between the pair distribution Γ (r1, r2) and the product of marginals ρ(r1)ρ(r2) reveal the existence of some interconnection between its variables and quan- tify the effects from the exchange density on the systems under consideration. With JSD those differences are obtained for atomic systems (neutrals and ions), in both conjugated spaces (Sect. 3.1). (ii) The ionization of a neutral system, by adding or removing an electron, modiﬁes its structural characteristics. This was studied by means of the JSD between the one-particle distributions of the neutral and the ionized systems [55]. Here, a step further is taken, dealing with divergences among electron-pair densities, also in position and momentum spaces (Sect. 3.2). 3 Numerical results Divergence measures allow us to quantify the differences between general systems, in par- ticular by means of electron atomic densities. In this section we intend to employ Jensen– Shannon divergence in order to analyse the particularities and characteristics of two-electron and one-electron densities. The Near-Hartree–Fock wavefunctions of Koga et al [56,57] are employed. Functions normalized to unity will be managed in what follows, for the sake of their interpretation as probability distributions. 3.1 Interelectronic correlation analysis 3.1 Interelectronic correlation analysis We will compute the Jensen–Shannon divergence between two-electron density and the product of one-electron densities, being one of the main reasons its property that its value is zero when the systems compared have no correlation. Thus, the Jensen–Shannon divergence 123 763 Page 6 of 16 Eur. Phys. J. Plus (2021) 136:763 Fig. 1 Jensen–Shannon divergence in position space (JSDr ) between the electron pair density and the product of monoelectronic densities, for neutral atoms with number of electrons N = 3−103. Labels are for systems with closed shells (red), closed subshells (blue) or anomalous shell-ﬁlling (green). Atomic units (a.u.) are used Fig. 1 Jensen–Shannon divergence in position space (JSDr ) between the electron pair density and the product of monoelectronic densities, for neutral atoms with number of electrons N = 3−103. Labels are for systems with closed shells (red), closed subshells (blue) or anomalous shell-ﬁlling (green). Atomic units (a.u.) are used acts as a measure of the disparity between two- and one-electron densities. These comparisons will be carried out on double variable distributions in both position (Γ (r1, r2) and ρ(r1)ρ(r2) respectively) and momentum (Π(p1, p2) and γ (p1)γ (p2) respectively) spaces. Let us remark that in this case, the last term in the JSD (see Eq. (7)) can be expressed as − S(Γ ) 2 + S (ρ) in position space, and similarly for the momentum space. The systems under study are neutral atoms with a number of electrons N ≤103 (nuclear charge Z = N), and singly-charged ions (Z = N ± 1) with N ≤54. Firstly,wecomputedtheJensen–Shannondivergencebetweentheelectronpairdensityand the product of the corresponding monoelectronic densities for neutral systems N = 2−103 in position space. The obtained results are shown in Fig. 1. It can be observed the decreasing trend of JSD when the nuclear charge of the system increases, which indicates that the correlation decreases as the number of electrons increases. This was an expected result, as the number of electrons is known to determine how strong or weak is the relevance of the correlation between the mentioned electrons. Thus, in the case that we have information about the position of an electron within a group of three, this knowledge becomes much more relevant than if we have a group of thirty electrons, when the information about just one does not tell us much more about the other twenty nine. – Closedshells: N = 10, 18, 36, 54, 86.Thusnoblegasesappearasminimasystematically. The same was observed in the K L divergence for systems up to N ≤36 [58]. 3.1 Interelectronic correlation analysis Nevertheless, additional remarks are in order. Comparing curves in position and momen- tum space, it is observed that (i) the aforementioned ‘changes of slope’ at N = 24, 29 in position space are displayed as maxima in momentum space; (ii) previous minimum N = 42 translates now to N = 43, also with outer d-subshell; (iii) instead of minimum N = 80 it is now observed the maximum N = 78. No extrema appear in the heavy systems region. Notice that just mentioned systems N = 24, 29, 42, 43, 46, 78 have a non-ﬁlled inner s- subshell and an outermost d-subshell (the inner one is empty for the system N = 46, clearly highlighted in both ﬁgures). As observed in Ref. [58] for KL, most JSD values are higher in position than in momentum space. The opposite applies for a few systems with anomalous shell-ﬁlling. Particularly relevant are the cases N = 24, 29 with half-ﬁlled 4s inner orbital and outermost 3d subshell (half-ﬁlled and completely ﬁlled, respectively). Their JSDp (Fig. 2) are greater than the respective JSDr (Fig. 1) by 17% and 25%. Such amount belongs to the range 8−13% for heavier systems with similar anomalies: N = 41, 42, 44, 45, 47 (subshells 5s4d), and N = 78, 79 (subshells 6s5d), all them with an inner half-ﬁlled s-orbital. It is concluded that, for most systems, correlation effects are weaker in momentum space, the opposite being true for the aforementioned anomalous systems, in a slight fashion with few exceptions. These results support and extend the conclusions obtained in Ref. [58] for systems up to N = 36, regarding the relative behaviors in position and momentum spaces. The above discussion was focused on neutral atoms and their properties, but in what follows we will consider also some of their corresponding ions. Let us now use the Jensen– Shannon divergence, JSD, in order to quantify the differences between the electron distribu- tions at the one- and two-electron levels, now for ionized systems. In doing so, Fig. 3 shows the JSD between the monoelectronic and the electron pair density of different ions, calculated in both conjugated spaces. First let us focus on Fig. 3a where singly-charged cations with number of electrons N = 2−54 have been taken into account. 3.1 Interelectronic correlation analysis Notwithstanding, some additional patterns are noticeable, most certainly related to the shell ﬁlling structure. This fact was emphasized in a previous work [58] for systems within the shorter range N ≤36, on the basis of the mutual information as quantiﬁed by the K L divergence among distributions, as here done by means of JSD up to N = 103. The trend is monotonous, without many exceptions for a long period, however some discontinuities appear when changing from a period to another indicating a transition to a new electronic shell. Each segment can be assigned to a particular shell, in the fashion the ﬁgure shows. Besides, some less relevant extrema appear, all of them related to anomalous shell ﬁlling systems. Some speciﬁc systems have been labelled, as follows: – Closedshells: N = 10, 18, 36, 54, 86.Thusnoblegasesappearasminimasystematically. The same was observed in the K L divergence for systems up to N ≤36 [58]. 123 123 Eur. Phys. J. Plus (2021) 136:763 Page 7 of 16 763 Eur. Phys. J. Plus (2021) 136:763 – (i) Anomalous shell-ﬁlling: minima N = 42, 46 (outer d-subshell), with valence subshell half-ﬁlled and ﬁlled, respectively. (ii) The same applies to N = 24, 29: in spite of not being extrema, the curve displays a ‘change of slope’ at those points, to be contrasted later with momentum space. (iii) Minor extrema appear for heavy systems, within a region of anomalies. – (i) Anomalous shell-ﬁlling: minima N = 42, 46 (outer d-subshell), with valence subshell half-ﬁlled and ﬁlled, respectively. (ii) The same applies to N = 24, 29: in spite of not being extrema, the curve displays a ‘change of slope’ at those points, to be contrasted later with momentum space. (iii) Minor extrema appear for heavy systems, within a region of anomalies. – Closed subshells: minima N = 48, 80 with ﬁlled d-subshell. – Closed subshells: minima N = 48, 80 with ﬁlled d-subshell. The Jensen–Shannon divergence between electron pair density and the product of mono- electronic densities in momentum space hardly provides additional information. The conclu- sions we arrive are similar than before, in fact, the curves in both spaces almost overlap each other, as it can be seen by comparing Fig. 2 (momentum space) with the previous one (note the identical scales of both ﬁgures). Thus the electron pair densities in momentum space are providing roughly the same information as the position ones. – As for neutral atoms, noble gases N = 10, 18, 36, 54 are displayed as lower peaks. On the other hand, alkalines N = 3, 11, 19, 37 appear as maxima. Similarly occurs, in position 3.1 Interelectronic correlation analysis – Pairs of extrema 23−24 and 28−29 (minimum–maximum) are due to the anomalous shell ﬁlling of both maxima N = 24, 29, with the inner half-ﬁlled 4s subshell, and with half-ﬁlled or ﬁlled (respectively) outermost 3d subshell. – Similarly the pair of consecutive extrema 41 −42 has inner half-ﬁlled 5s subshell, and outermost subshell 4d, half-ﬁlled for the maximum N = 42. – Similarly the pair of consecutive extrema 41 −42 has inner half-ﬁlled 5s subshell, and outermost subshell 4d, half-ﬁlled for the maximum N = 42. – Particularly interesting is the strong minimum N = 46, as discussed for neutrals, with empty 5s subshell and ﬁlled valence subshell (4d). – Particularly interesting is the strong minimum N = 46, as discussed for neutrals, with empty 5s subshell and ﬁlled valence subshell (4d). – A few of the above systems display a higher JSD in momentum than in position space: N = 24, 29, 42, 47 (with inner half-ﬁlled s-orbital and outermost half-ﬁlled or com- pletely ﬁlled d-orbital), as well as N = 25 (half-ﬁlled outermost d-orbital). In Fig. 3b, where the anions have been considered, exactly the same pattern is observed, but with a less elaborate structure, due to the fewer available systems as it was previously mentioned. Some alkaline earths have been labelled, but let us emphasize the ‘absence from the list’ of alkalines, which are displayed as maxima for cations. Position space JSD is almost systematically greater than the momentum one. The only extremely light exceptions are N = 41−42, with differences between spaces below 0.2%. 3.2 Atomic ionization 3.1 Interelectronic correlation analysis The curves show us a decreas- ing behaviour when N increases and, in this case, the momentum space roughly presents smaller values of JSD. However, an interesting pattern appears in the ﬁgure: cations corre- sponding to N = 10, 18, 23, 28, 36, 41, 46, 48, 54 have a minor difference, a lower value of JSD, in both conjugated spaces with the only exception N = 48 (extremely light mini- mum, only in position space). Among maxima (located at almost identical positions in both spaces), let us emphasize the presence of N = 24, 29, 42. Regarding these two collections, some comments are in order: – As for neutral atoms, noble gases N = 10, 18, 36, 54 are displayed as lower peaks. On the other hand, alkalines N = 3, 11, 19, 37 appear as maxima. Similarly occurs, in position 123 123 763 Page 8 of 16 Eur. Phys. J. Plus (2021) 136:763 Fig. 2 Jensen–Shannon divergence in momentum space (JSDp) between the electron pair density and the product of monoelectronic densities, for neutral atoms with number of electrons N = 3−103. Labels are for systems with closed shells (red) or anomalous shell-ﬁlling (green). Atomic units (a.u.) are used Fig. 2 Jensen–Shannon divergence in momentum space (JSDp) between the electron pair density and the product of monoelectronic densities, for neutral atoms with number of electrons N = 3−103. Labels are for systems with closed shells (red) or anomalous shell-ﬁlling (green). Atomic units (a.u.) are used space, with the closed-shell system N = 48 (minimum) and its neighbors N = 47, 49 (maxima). – Pairs of extrema 23−24 and 28−29 (minimum–maximum) are due to the anomalous shell ﬁlling of both maxima N = 24, 29, with the inner half-ﬁlled 4s subshell, and with half-ﬁlled or ﬁlled (respectively) outermost 3d subshell. – Pairs of extrema 23−24 and 28−29 (minimum–maximum) are due to the anomalous shell ﬁlling of both maxima N = 24, 29, with the inner half-ﬁlled 4s subshell, and with half-ﬁlled or ﬁlled (respectively) outermost 3d subshell. – Pairs of extrema 23−24 and 28−29 (minimum–maximum) are due to the anomalous shell ﬁlling of both maxima N = 24, 29, with the inner half-ﬁlled 4s subshell, and with half-ﬁlled or ﬁlled (respectively) outermost 3d subshell. 3.2 Atomic ionization Applying divergence measures to the analysis of ions will allow us to ﬁnd which electrons have a higher impact on the whole density. In order to do that, we inspect the Jensen–Shannon divergence between the pair densities of an ion (cation or anion) and its respective neutral, in both position and momentum spaces. For completeness, similar results on the basis of one-particle densities, previously obtained [55], are also displayed. This is shown in Figs. 4 and 5. Next results are to be interpreted as measures of the ‘amount of change’ on the electron cloud of a given system after its ionization takes place, either by ejecting or by capturing an Eur. Phys. J. Plus (2021) 136:763 Page 9 of 16 763 (b) (a) Fig. 3 Jensen–Shannon divergence, in position and momentum spaces, between the electron pair density and the product of one-particle densities, for a cations and b anions with number of electrons 3 ≤N ≤55. Labels are for systems with closed shells (red), closed subshells (blue) or anomalous shell-ﬁlling (green). Atomic units (a.u.) are used ( ) (a) (b) (b) Fig. 3 Jensen–Shannon divergence, in position and momentum spaces, between the electron pair density and the product of one-particle densities, for a cations and b anions with number of electrons 3 ≤N ≤55. Labels are for systems with closed shells (red), closed subshells (blue) or anomalous shell-ﬁlling (green). Atomic units (a.u.) are used electron. Thus, if the initial neutral system has N electrons, the resulting ion will have N −1 or N + 1 electrons, for the cation and the anion, respectively. This interpretation applies to both conjugated spaces, as well as to one-particle and electron pair densities. Figure 4 encloses curves of JSD between the electron distribution of a neutral system and that of its singly-charged cation, denoted in position and momentum spaces as JSDr(N, C) in Fig. 4a, and JSDp(N, C) in Fig. 4b, respectively. A simple function of the atomic ionization potential AIP needed for performing such process is also depicted. This function has been considered in order to better compare all curves, which display a variety of local extrema, most of them characterized accordingly with the shell-ﬁlling of neutrals and ions, as discussed below. 123 123 763 Page 10 of 16 Eur. Phys. J. Plus (2021) 136:763 (b) (a) Fig. – Rest of peaks (N = 23, 27, 41, 44) correspond to ‘d’ valence subshells. 3.2 Atomic ionization The same analysis has been carried out in momentum space, and the results are displayed in Fig. 4b. Conclusions are similar to those obtained from position-space divergences, and the differences are emphasized below: – Position-space peaks N = 3, 11, 19, 23, 37, 55 (enclosing all alkalines) remain in momentum space for both one- and two-particle comparisons. On the other hand, N = 31 is lost also in both cases. – N = 27, 45 are kept in the monoparticular case, while the slight shifts from maxima located at (27, 44) to (28, 45) are observed for electron pair divergences. – Maximum N = 49 disappears for momentum one-electron distributions, while N = 41, 47 for the two-electron ones. Notice that the half-ﬁlled 5s subshell of systems N = 41 and N = 47 becomes ﬁlled and empty, respectively, after ionization. – On the other hand, the disappearance of N = 31, 49 from the set of maxima in going from position to momentum space corresponds to systems which ‘p’ valence subshell becomes empty. Instead, the divergence at the one-electron level displays maxima N = 8, 52, characterized for getting a half-ﬁlled ‘p’ valence subshell after ionization. – Concerning asymptotic trends, it is observed that the monoelectronic JSD remains within a low interval for large N, while the electron pair divergence appreciably increases, roughly from N = 25 on. – Concerning asymptotic trends, it is observed that the monoelectronic JSD remains within a low interval for large N, while the electron pair divergence appreciably increases, roughly from N = 25 on. The differences between the JSD behaviors in position and momentum spaces arise from the asymptotic behaviors of the involved densities. This fact has been shown in previous studies on density functional analysis, particularly JSD among others [41]. In position space, the atomic one- and two-electron densities have an exponential decreasing behavior, so that the values of the involved integrals are mainly determined by the regions surrounding the nuclei, those almost unaffected by ionization. However, the lowspeed regions are associated with the outermost subshells, being consequently those mainly determining the JSD values. The differences between the valence subshells of the neutral and ionized systems are more clearly reﬂected in the pair distribution, as compared to the monoelectronic one. Let us analyse the structure of the JSD in each period comparing their extremal values to those of AI P. 3.2 Atomic ionization 4 Pair and one-electron Jensen–Shannon divergences between the neutral system with a number of electrons 3 ≤N ≤55 and its singly charged cation, and the inverse of square root of the atomic ionization potential (AIP), in a position and b momentum spaces. Atomic units (a.u.) are used (a) (a) (b) Fig. 4 Pair and one-electron Jensen–Shannon divergences between the neutral system with a number of electrons 3 ≤N ≤55 and its singly charged cation, and the inverse of square root of the atomic ionization potential (AIP), in a position and b momentum spaces. Atomic units (a.u.) are used Fig. 4 Pair and one-electron Jensen–Shannon divergences between the neutral system with a number of electrons 3 ≤N ≤55 and its singly charged cation, and the inverse of square root of the atomic ionization potential (AIP), in a position and b momentum spaces. Atomic units (a.u.) are used Let us start with the location of maxima of JSD in position space, when comparing a neutral of N electrons with the resulting cation of N −1 electrons, namely JSDr(N, C), as displayed in Fig. 4a. In the two-particle case, higher peaks are found at neutrals N = 3, 11, 19, 23, 27, 31, 37, 41, 44, 47, 49, 55. Within the range of N here considered, it is worthy to remark: – Alkalines N = 3, 11, 19, 37, 55 appear included, that is systems which outermost ‘s’ subshell becomes empty after ionization. – Alkalines N = 3, 11, 19, 37, 55 appear included, that is systems which outermost ‘s’ subshell becomes empty after ionization. – The same applies to the anomalous N = 47 regarding its valence subs – For systems N = 31, 49 the lost subshell is 4p and 5p, respectively. – Rest of peaks (N = 23, 27, 41, 44) correspond to ‘d’ valence subshells. 123 Eur. Phys. J. Plus (2021) 136:763 Page 11 of 16 763 – Comparing with the one-electron curve, almost identical structures are obtained regard- ing their extrema and monotonic trends. The most prominent differential feature is the systematic higher value of electron pair JSD as compared to the monoelectronic one. – Comparing with the one-electron curve, almost identical structures are obtained regard- ing their extrema and monotonic trends. The most prominent differential feature is the systematic higher value of electron pair JSD as compared to the monoelectronic one. 3.2 Atomic ionization – Alkalines N = 3, 11, 19, 37 remain as maxima, also for anions. Let us remark that the next alkaline (N = 55) is not included in the managed anion set. – The anion obtained from neutral N = 31 does not give rise to a maximum, contrary to the cation. – The anion obtained from neutral N = 31 does not give rise to a maximum, contrary to the cation. – Two sets of slight shifts are observed: (23, 27 and 41) to (24, 29 and 42), and (44 or 45, and 47) to (44 and 46). – Two sets of slight shifts are observed: (23, 27 and 41) to (24, 29 and 42), and (44 or 45, and 47) to (44 and 46). The only exception to the above common patterns is N = 49, shifted to N = 51 in the monoelectronic case, and lost for electron pair densities. In momentum space, Fig. 5b, (i) alkalines N = 3, 11, 19 are kept for anions, (ii) the one- electron shifts of maxima (8,23,27 or 28,41,45,47,52) to (7, 24, 29, 42, 44, 46, 51) include the two-electron ones, corresponding to N = 24, 29, 46, and (iii) in both cases, the new maximum N = 15 appears, as well as N = 33 at the one-electron level, that is systems with a half-ﬁlled ‘p’ valence subshell, as also N = 51 included in the above list of shifts. Differences between the global behaviors of the one- and two-electron curves appear justiﬁed in the same way as for neutral-cation transitions. That is, both densities decrease exponentially far from the nucleus in position space, while the asymptotic fall of momentum densities is much more slow. 3.2 Atomic ionization In Table 1, values of number of electrons N of the neutral system for which the Jensen–Shannon divergence display local extrema (maximum) are given. In addition, minimum values of the AI P are shown, which are associated to systems with a single electron in the valence subshell, making consequently such a subshell to disappear after ionization, and the resulting system to strongly differ from the initial one. This relevant difference is usually revealed in terms of a high divergence and dissimilarity between the initial and the ﬁnal system. There exist a strong correlation also with the structure displayed by the atomic ionization potential AI P in the NC ionization process in which an ‘s’ electron is removed, as shown in the corresponding column of the table. However, as it can be observed the same is not true when the removed electron is of ‘p’ or ‘d’ type. It appears natural to wonder ourselves about all the above features when the initial neutral system gets an additional electron, so becoming a singly-charged anion. The appropriate calculations have been performed, and the results obtained are displayed in Fig. 5. Conclusions for anions are much more limited, as compared to those obtained for cations. The reason is the absence of numerous systems from the list of anions, not included in the Ref. [56] considered for the present work. However, some interesting remarks are in order. 123 763 Page 12 of 16 Eur. Phys. J. Plus Eur. Phys. J. Plus (2021) 136:763 Table 1 Number of electrons N at local extrema for the atomic ionization potential AIP of neutral atoms and the Jensen–Shannon divergences in position (JSDr ) and momentum (JSDp) spaces, between the electron pair densities of a neutral system with a number of electrons 3 ≤N ≤54 and its singly charged anions (A) and cations (C). Atomic units are used Measure N −→C A −→N AIP (s) 3,11,19,23,28,37,47,55 (p,d) 5,8,13,16,31,34,49 JSDr (s) 3,11,19,23,27,37,41,44,47,55 3,11,19,24,29,37,42,44,46 (p,d) 31,49 JSDp (s) 3,11,19,23,28,37,45,55 3,11,19,24,29,46 (p,d) 49 15,49,53 In position space, Fig. 5a, maxima for the divergence neutral-anion are roughly the same as those of neutral-cation, for the one- and the two-electron distributions. In both cases, the following patterns are displayed: – Alkalines N = 3, 11, 19, 37 remain as maxima, also for anions. Let us remark that the next alkaline (N = 55) is not included in the managed anion set. 4 Conclusions and future work Jensen–Shannon divergence (JSD) has been employed as comparative functional for different many-electron atoms on the basis of their respective one- and two-electron distributions, enclosing neutral atoms (N = Z) and singly-charged ions (N = Z ± 1). Particularly JSD is applied to quantify the interelectronic correlation in atomic systems. A thorough study was carried out, by considering (i) both position and momentum conjugated spaces, and (ii) systems with a nuclear charge as far as Z = 103. The correlation among electrons was measured by comparing, for an arbitrary system, the double-variable electron pair density with the product of the respective one-particle densities. As expected, the absence of correlation translates into the extreme allowed value JSD = 0, 123 Eur. Phys. J. Plus (2021) 136:763 Page 13 of 16 763 Eur. Phys. J. Plus (a) (b) Fig. 5 Pair and one-electron Jensen–Shannon divergences between the neutral system with a number of electrons 3 ≤N ≤54 and its singly charged anion, in a position and b momentum spaces. Atomic units (a.u.) are used (a) (a) (a) (b) (b) (b) Fig. 5 Pair and one-electron Jensen–Shannon divergences between the neutral system with a number of electrons 3 ≤N ≤54 and its singly charged anion, in a position and b momentum spaces. Atomic units (a.u.) are used Fig. 5 Pair and one-electron Jensen–Shannon divergences between the neutral system with a number of electrons 3 ≤N ≤54 and its singly charged anion, in a position and b momentum spaces. Atomic units (a.u.) are used and any deviation from the above value is caused by the existence of correlation, more or less strong, accordingly with the relevance of the aforementioned deviations. The main conclusion is that the dependence on the number of electrons only governs general trends of JSD, being insufﬁcient as single variable to justify the structural patterns shown by those curves. Interelectronic correlation roughly decreases as the number of elec- trons increases, as revealed by the decreasing behavior of JSD in terms of N. However, there appear a number of local extrema, whose location and strength are clearly determined by shell-ﬁlling patterns. Specially relevant changes in JSD are observed just after adding an electron to a closed-subshell system. All this is valid in position and momentum spaces. The above comments on neutral atoms apply also for cations and anions, regarding struc- tural patterns. 4 Conclusions and future work Once again the interelectronic correlation, as measured by JSD, decreases along a given period (i.e. as adding electrons) but notably increases in passing to a new period. 123 763 Page 14 of 16 Eur. Phys. J. Plus (2021) 136:763 Apart from correlation, additional properties of atomic systems were analysed by means of JSD. Such is the case of ionization as described by the ‘resemblance’ among neutrals and their respective singly-charged ions, in terms of the corresponding electron pair densities. While structural patterns are similar in position and momentum spaces, the same is not true regarding monotonic behaviors. JSD in both spaces displays a decreasing trend for light systems, that remains in the position case for the whole set of systems. However, it turns out to an increasing one in momentum space for medium-heavy systems. So break of resemblance after ionization appears greater in momentum space than in the position one, for both cations and anions. Future work is planned by considering other functionals. For instance, relative Fisher information (containing also the gradient of the density) was exhaustively applied for the analysis of speciﬁc one-particle quantum-mechanical systems (Morse potential and harmonic oscillator [48], hydrogen-like atoms [49]). Additionally, numerous studies of atomic [59] and molecular [60] systems were performed by means of the quantum similarity index on the basis of their one-particle densities. On the other hand, a study of different divergences and similarity measures, particularly Jensen–Shannon divergence, between the exchange density and the product of the one particular densities could be interesting. Acknowledgements This work was supported in part by the Spanish MINECO project FIS2014-59311-P (coﬁnanced by FEDER). A.L.M., J.C.A. and J.A. belong to the Andalusian research group FQM-020, and S.L.R. to FQM-239. Funding Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. 4 Conclusions and future work If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. References Gadre, R.D. Bendale, S.P. Gejji, Analysis of atomic electron momentum densities: use of information entropies in coordinate and momentum space. Chem. Phys. Lett. 117, 138 (1985) 29. M. Ho, V.H. Smith, D.F. Weaver, C. Gatti, R.P. Sagar, R.O. Esquivel, Molecular similarity based on information entropies and distances. J. Chem. Phys. 108, 5469 (1998) K.D. Sen, C.P. Panos, K.C. Chatzisavvas, C.C. Moustakidis, Net Fisher information measure versus ionization potential and dipole polarizability in atoms. Phys. Lett. A 364, 286–290 (2007) 30. K.D. Sen, C.P. Panos, K.C. Chatzisavvas, C.C. Moustakidis, Net Fisher information measu ionization potential and dipole polarizability in atoms. Phys. Lett. A 364, 286–290 (2007) 31. S. Janssens, A. Borgoo, C. Van Alsenoy, P. Geerlings, Information theoretical study of chirality: enan- tiomers with one and two asymmetric centra. J. Phys. Chem. A 112, 10560–10569 (2008) 32. R.F. Nalewajski, Information principles in the theory of electronic structure. Chem. Phys. Lett. 372, 28 (2003) 33. J.C. Angulo, J. Antolín, Atomic complexity measures in position and momentum spaces. J. Chem. Phys. 128, 164109 (2008) 34. J.C. Angulo, J. Antolín, K.D. Sen, Fisher-Shannon plane and statistical complexity of atoms. Phys. Lett. A 372, 670–674 (2008) 35. S. Kullback, A. Leibler, On information and sufﬁciency. Ann. Math. Stat. 22, 79–86 (1951) 36. J.H. Lin, Divergence measures based on the Shannon entropy. IEEE Trans. Inf. Theory 37, 145–151 (1991) 37. B. Fuglede, F. Topsoe, Jensen-Shannon divergence and Hilbert space embedding. IEEE Int. Sym. Inf. Theo. 31, 71–73 (2004) 38. P.W. Lamberti, A.P. Majtey, A. Borras, M. Casas, A. Plastino, Metric character of the quantum Jensen– Shannon divergence. Phys. Rev. A 77, 052311 (2008) 39. I. Grosse, P. Bernaola-Galván, P. Carpena, R. Román-Roldán, J.L. Oliver, H.E. Stanley, Analysis symbolic sequences using the Jensen–Shannon divergence. Phys. Rev. E 65, 041905 (2002) 40. J.C. Angulo, J. Antolín, S. López-Rosa, R.O. Esquivel, Jensen-Shannon divergence in conjugate spaces: the entropy excess of atomic systems and sets with respect to their constituents. Phys. A 389, 899 (2010) 40. J.C. Angulo, J. Antolín, S. López-Rosa, R.O. Esquivel, Jensen-Shannon divergence in conjugate spaces: the entropy excess of atomic systems and sets with respect to their constituents. Phys. A 389, 899 (2010) 41. J. Antolín, J.C. Angulo, S. López-Rosa, Fisher and Jensen-Shannon divergences: quantitative comparisons among distributions. Application to position and momentum atomic densities. J. Chem. Phys. 130, 074110 (2009) 41. J. Antolín, J.C. Angulo, S. López-Rosa, Fisher and Jensen-Shannon divergences: quantitative comparisons among distributions. References 1. T. Koga, Electron-Pair Densities of atom. in Many-Electron Densities and Reduced Density Matri (Springer, New York, 2000) 1. T. Koga, Electron Pair Densities of atom. in Many Electron Densities and Reduced Density Matrices (Springer, New York, 2000) 2 M R d í M M Vi N d l M S là J M U ld X L E M i El i di i 2. M. Rodríguez-Mayorga, M. Via-Nadal, M. Solà, J.M. Ugalde, X. Lopez, E. Matito, Electron-pair dis bution in chemical bond formation. J. Phys. Chem. A 122, 1916–1923 (2018) 3. R.P. Sagar, N.L. Guevara, Mutual information and correlation measures in atomic systems. J. Chem. Phys. 123, 044108 (2005) 4. J. Cioslowski, Topology of Electron Correlation. in Many-Electron Densities and Reduced Density Matri- ces (Springer, New York, 2000) 5. P.W. Ayers, E.R. Davidson, Necessary conditions for the n-representability of pair distribution functions. Int. J. Quant. Chem. 1006, 1487–1498 (2006) 6. M. Levy, P. Ziesche, The pair density functional of the kinetic energy and its simple scaling property. J. Chem. Phys. 115, 9110–9112 (2001) y 7. Á. Nagy, Density-matrix functional theory. Phys. Rev. A 66, 022505 (2002 y 7. Á. Nagy, Density-matrix functional theory. Phys. Rev. A 66, 022505 (2002) 7. Á. Nagy, Density-matrix functional th 8. D.A. Mazziotti, Two-electron reduced density matrix as the basic variable in many-electron quantum chemistry and physics. Chem. Rev. 112, 244–262 (2012) 9. M. Higuchi, K. Higuchi, Pair density functional theory. Comput. Theor. Chem. 1003, 91–96 (2013) 9. M. Higuchi, K. Higuchi, Pair density functional theory. Comput. Theor. Chem. 1003, 91–96 (2013) 10. M. Higuchi, K. Higuchi, A pair density functional theory utilizing the correlated wave function. J. Phys. Conf. Ser. 150, 042056 (2009) 10. M. Higuchi, K. Higuchi, A pair density functional theory utilizing the correlated wave function. J. Ph Conf. Ser. 150, 042056 (2009) 10. M. Higuchi, K. Higuchi, A pair density functional theory utilizing the correlated wave function. J. Phys. Conf. Ser. 150, 042056 (2009) , ( ) 11. P. Ziesche, Attempts toward a pair density functional theory. Int. J. Quantum Chem. 60, 1361–1374 (1996) , ( ) 11. P. Ziesche, Attempts toward a pair density functional theory. Int. J. Quantum Chem. 60, 1361–1374 (1996) ( ) 11. P. Ziesche, Attempts toward a pair density functional theory. Int. J. Quantum Chem. 60, 1361–1374 (1996) 123 Page 15 of 16 763 Eur. Phys. J. Plus us (2021) 136:763 (2021) 136:763 12. References G. Li Manni, R.K. Carlson, S. Luo, D. Ma, J. Olsen, D.G. Truhlar, L. Gagliardi, Multiconﬁgurat pair-density functional theory. J. Chem. Theory Comput. 10, 3669–3680 (2014) 13. J.L. Bao, Y. Wang, X. He, L. Gagliardi, D.G. Truhlar, Multiconﬁguration pair-density functional the is free from delocalization error. J. Phys. Chem. Lett. 8, 5616–5620 (2017) y 14. M. Sajjan, K. Head-Marsden, D.A. Mazziotti, Entangling and disentangling many-electron quantum systems with an electric ﬁeld. Phys. Rev. A 97, 062502 (2018) 15. R. Benesch, V.H. Smith, Correlation and X-ray scattering. I. Density matrix formulation. Acta Cryst. 26, 579–586 (1970) 16. A.J. Thakkar, A.N. Tripathi, V.H. Smith, Molecular X-ray- and electron-scattering intensities. Phys. Rev. A 29, 1108–1113 (1984) 17. T. Koga, Y. Kasa, A.J. Thakkar, Accurate algebraic densities and intracules for heliumlike ions. Int. J. Quantum Chem. 46, 689 (1993) 18. J.M. Herbert, J.E. Harriman, Comparison of two-electron densities reconstructed from one-electron d sity matrices. Int. J. Quantum Chem. 90, 355 (2002) 19. C. Sarasola, J.M. Ugalde, R.J. Boyd, The evaluation of extracule and intracule densities in the ﬁrst-row hydrides, LiH, BeH, BH, CH, NH, OH and FH, from self-consistent ﬁeld molecular orbital wavefunctions. J. Phys. B: At. Mol. Opt. 23, 1095 (1990) y 20. J. Cioslowski, B.B. Stefanov, A. Tan, C.J. Umrigar, Electron intracule densities with correct elect coalescence cusps from Hiller–Sucher–Feinberg-type identities. J. Chem. Phys. 103, 6093 (1995) 21. A.J. Proud, D.E.C.K. Mackenzie, J.K. Pearson, Exploring electron pair behaviour in chemical bo using the extracule density. Phys. Chem. Chem. Phys. 17, 20194–20204 (2015) 22. C.E. Shannon, W. Weaver, The Mathematical Theory of Communication (University of Illinois Press, Urbana, 1949) 23. R.A. Fisher, Theory of statistical estimation. Proc. Cambridge Phil. Soc., 22, 700–725, Reprinted in Collected Papers of R.A. Fisher, edited by J.H. Bennet (University of Adelaide Press, South Australia) 1972, 15–40 (1925) ( ) 24. J. Pipek, I. Varga, Universal classiﬁcation scheme for the spatial-localization properties of one-particle states in ﬁnite, d-dimensional systems. Phys. Rev. A 46, 3148 (1992) 25. R. López-Ruiz, H.L. Mancini, X. Calbet, A statistical measure of complexity. Phys. Lett. A 209, 321–326 (1995) 26. B.R. Frieden, Science from Fisher Information (Cambridge University Press, Cambridge, 2004) 27. T.M. Cover, J.A. Thomas, Elements of Information Theory (Wiley, New York, 1991) 28. S.R. Gadre, R.D. Bendale, S.P. Gejji, Analysis of atomic electron momentum densities: use of information entropies in coordinate and momentum space. Chem. Phys. Lett. 117, 138 (1985) 28. S.R. References Application to position and momentum atomic densities. J. Chem. Phys. 130, 074110 (2009) 123 123 12 763 Page 16 of 16 763 Eur. Phys. J. Plus (2021) 136:763 Eur. Phys. J. Plus 42. J.C. Angulo, S. López-Rosa, J. Antolín, Generalized Jensen divergence analysis of atomic electron d sities in conjugated spaces. Int. J. Quantum Chem. 111, 297–306 (2011) 43. A.L. Martín, S. López-Rosa, J.C. Angulo, J. Antolín, Jensen-Shannon and Kullback–Leibler divergences as quantiﬁers of relativistic effects in neutral atoms. Chem. Phys. Lett. 75, 642 (2015) q y ( ) 44. J. Antolín, J.C. Angulo, S. Mulas, S. López-Rosa, Relativistic global and local divergences in hydrogenic d i i i d Ph R A 90 042511 (2014) q y 44. J. Antolín, J.C. Angulo, S. Mulas, S. López-Rosa, Relativistic global and local divergences in hydrogenic systems: a study in position and momentum spaces. Phys. Rev. A 90, 042511 (2014) y y p p y 45. S.J.C. Salazar, H.G. Laguna, R.P. Sagar, Higher-order statistical correlations in three-particle quantum systems with harmonic interactions. Phys. Rev. A 101, 042105 (2020) 46. S.J.C. Salazar, H.G. Laguna, R.P. Sagar, Higher-order information measures from cumulative densities in continuous variable quantum systems. Quantum Rep. 2, 560–578 (2020) 47. O. Werba, A. Raeber, K. Head-Marsden, D.A. Mazziotti, Signature of van der Waals interactions in the cumulant density matrix. Phys. Chem. Chem. Phys. 21, 23900 (2019) 48. T. Yamano, Relative ﬁsher information for Morse potential and isotropic quantum oscillators. J. Ph Commun. 2, 085018 (2018) 49. T. Yamano, Relative ﬁsher information of hydrogen-like atoms. Chem. Phys. Lett. 691, 196–198 (2018) 49. T. Yamano, Relative ﬁsher information of hydrogen like atoms. Chem. Phys. Lett. 691, 196 198 (2018) 50. P.O. Löwdin, Quantum theory of many-particle systems. I. Physical interpretations by means of density matrices, natural spin-orbitals, and convergence problems in the method of conﬁgurational interaction. Phys. Rev. 97, 1474 (1955) y , ( ) 51. A.K.C. Wong, M. You, Entropy and distance of random graphs with application to structural pattern- recognition. IEEE Trans. Pattern Anal. Mach. Intell. 7, 599 (1985) 52. C.R. Rao, T. Nayak, Cross entropy, dissimilarity measures, and characterizations of quadratic entropy. IEEE Trans. Inf. Theory 31, 589 (1985) y ( ) 53. C.E. Shannon, A mathematical theory of communication. Bell Syst. Tech. J. 27, 379 (1948) 3. C.E. Shannon, A mathematical theory of commu 54. L.E. Riveaud, D. Mateos, S. Zozor, P.W. Lamberti, Generalized divergences from generalized entropies. References Phys. A 68–76, 510 (2018) 55. S. López-Rosa, J. Antolín, J.C. Angulo, R.O. Esquivel, Divergence analysis of atomic ionization processes and isoelectronic series. Phys. Rev. A 80(1–9), 012505 (2009) 56. T. Koga, K. Kanayama, S. Watanabe, A.J. Thakkar, Analytical Hartree–Fock wave functions subject to cusp and asymptotic constraints: He to Xe, Li+ to Cs+, H- to I-. Int. J. Quantum Chem. 71, 491 (1999) 56. T. Koga, K. Kanayama, S. Watanabe, A.J. Thakkar, Analytical Hartree–Fock wave functions subject to cusp and asymptotic constraints: He to Xe, Li+ to Cs+, H- to I-. Int. J. Quantum Chem. 71, 491 (1999) 57. T. Koga, K. Kanayama, T. Watanabe, T. Imai, A.J. Thakkar, Analytical Hartree–Fock wave functions for the atoms Cs to Lr Theor Chem Acc 104 411 (2000) 57. T. Koga, K. Kanayama, T. Watanabe, T. Imai, A.J. Thakkar, Analytical Hartree–Fock wave functions the atoms Cs to Lr. Theor. Chem. Acc. 104, 411 (2000) 58. R.P. Sagar, H.G. Laguna, N.L. Guevara, Electron pair density information measures in atomic systems. Int. J. Quantum Chem. 111, 3497 (2011) 59. J.C. Angulo, J. Antolín, Atomic quantum similarity indices in position and momentum spaces. J. Phys. Chem. 126, 044106 (2007) 60. R. Carbó-Dorca, X. Girones, P.G. Mezey (eds.), Fundamentals of Molecular Similarity (Kluwer Aca- demic, Dordrecht, 2001) 123
https://openalex.org/W1994912684	https://ccsenet.org/journal/index.php/res/article/download/8459/6302	English	null	The Student's Attitude toward Use Platform as Learning Resources at University of Granada	Review of European studies	2,010	cc-by	6,509	Review of European Studies Review of European Studies Vol. 2, No. 2; December 2010 www.ccsenet.org/res The Student's Attitude toward Use Platform as Learning Resources at University of Granada Dr. Ahmad Alobiedat (Corresponding author) Faculty of Education, Tafila Technical University Tafila – Jordan P.O. Box 179 Zip code 66110- Jordan Tel: 962-77-727-1789 E-mail: alobiedat@yahoo.com Dr. Raji Saraierh Faculty of Education, Tafila Technical University Tafila – Jordan P.O. Box 179 Zip code 66110- Jordan Tel: 962-79-902-7886 E-mail: raji_awad_888@yahoo.com Dr. Raji Saraierh Faculty of Education, Tafila Technical University Tafila – Jordan P.O. Box 179 Zip code 66110- Jordan 1. Introduction The traditional education delivery system has been a classroom setting with a professor giving a lecture and students listening and writing notes. Interaction between the professor and student has been viewed as an essential learning element within this arrangement. However, innovations in educational technology use of platform as delivery mechanisms have challenged this paradigm. Advances in Information Technology (IT) are enabling little used educational delivery methods such as Distance Learning (DL) to gain new life. In addition, the advances in IT have ushered in a new paradigm, On-line Learning (OL). The result is that many universities have adopted distance and on-line education as the next logical step in educational delivery systems. These systems are being promoted as the educational pedagogy of the future. Some experts have gone as far as to predict that the "residential based model," that is, students attending classes at prearranged times and locations will disappear in the near future (Blustain, Goldstein, and Lozier 1999 and Drucker 1997). However, one overriding question that must be addressed is how will these new educational delivery approaches that move away from the basic face to face relationship between a professor and students impact student learning and student attitudes of learning. Most university students will participate in two distinct forms of networked communication. (Ofcom 2008) and (Crook 2008) in their study, universities will engage with their institutional learning platform, or virtual learning environment (VLE), while numerous reviews now suggest, they will also engage with those internet services collectively termed ‘Web 2.0 in particular social networking sites such as Facebook. VLEs now pervade all sectors of education (DfES 2005). Accordingly, their effective design is a matter of serious concern. Where a VLE is adopted, it has become clear for higher education that the resource is rarely used very creatively. Woods, Baker and Hopper (2004) surveyed over 800 members of faculty at 38 US institutions. The dominant use of VLEs was as a convenient repository for familiar course documents and resources. Morgan (2003) surveyed a similar number of US academics and closely interviewed 140 others. They report the same pattern, and comment: ‘faculty adopts course management systems principally to manage the more mundane tasks associated with teaching, especially teaching large classes’. Yet most platforms do provide tools and designs that allow tests, chat dialogue, discussions, blogs, FAQs, notice boards and so forth. Abstract The study aim to investigate students' attitudes towards use platform as learning resources at University of Granada (UGr), a survey of two hundred fifty-eight male and female undergraduate students from faculty of education, were randomly selected to participate in the study. The results of the study shows a positive attitude toward use of platform as learning resources and there was a significance difference, due to the gender, owning a personal computer (PC), and having access to the internet, and it was in favor of male students, and student who own a pc, and student whom do have access to the internet respectively. Keywords: Attitude, Platform, University of Granada, Learning resource, Information technology 1. Introduction Keywords: Attitude, Platform, University of Granada, Learning resource, Information technology 2. Literature Review Alternatives to traditional lecture style delivery of education have been offered for many years (Huang, 1996-1997). These alternatives, called online learning education, became available with the development of a reliable mail delivery system during the late nineteenth century (Ascough, 2002; Rumble, 2001). Distance learning has been defined as any approach to education delivery that replaces the same time, same place, face to face environment of a traditional classroom (Volery & Lord, 2000). According to this definition, distance education can take many forms such as mail correspondence, open and closed circuit audio and video presentations, telephone communications, and the increasingly popular Internet. With these alternatives, however, students are able to attain an education even if they live a great distance from an educational institution or when their respective campus does not offer desired courses (McLester, 2002). The advances in computer technology, particularly the development of the Internet, have improved the delivery of distance education. Many universities are developing and offering a multitude of courses designed to be taught using Internet services. With broadening horizons and the expanding utility of Internet resources available to universities and consumers, the potential for this form of education and lifelong learning has become more accessible. In fact, from 2003 to 2004, online learners in the United States increased from 1.98 million to 2.35 million (Allen & Seamen, 2005). Distance education is appealing for many reasons. Foremost, online courses do not require regular Attendance at scheduled lectures. Thus, those working fulltime or who have other responsibilities are drawn to online courses, as is the case with many non-traditional students. Another convenience of online education is the ability for students to learn materials at their own pace. Many online courses are presented in an asynchronous manner where materials are time and place independent allowing students to access course materials at any time (Newlin &Wang, 2002). In these instances, instructors can be contacted with a simple email, and students can receive feedback quickly. Online courses usually still have project due dates and examinations, but these courses generally provide students with flexible course options. Although the advantages of online education are appealing, there are also disadvantages. For those who have never taken an online course or who have little computer experience, an online course may be frightening. For example, Wang, Newlin, and Tucker (2001) found that many web based students expressed feelings of intense anxiety about the technology. 1. Introduction Malkowski, Thompson and Theis (2006) considered how far these were ISSN 1918-7173 E-ISSN 1918-7181 236 Vol. 2, No. 2; December 2010 Review of European Studies www.ccsenet.org/res actually taken up in a volunteer sample of 87 faculty members at a US university. Only 3 out of 13 interactive design features were used by more than half of this group: ‘faculty members primarily use a CMS to transmit information to students’ (171). To systematize what teachers create, Dutton, Cheong, and Park 2003). At many universities, the effectiveness use of the use platform, distance and on-line learning methodologies has not been well researched prior to adoption. To better understand how these learning technologies and its impact on student attitudes toward e-learning, this study was conducted at UGr. The goal of this research study is to understand faculty of education student's attitude toward use platform as learning resources at UGr. In addition, in this research paper investigative the effect use platform as learning resources on gender, owning PC, and access to the internet. Published by Canadian Center of Science and Education 2. Literature Review There are also disadvantages in online courses for individuals who need a great deal of structure. Online courses are frequently self paced and those lacking self discipline may struggle. However, these advantages and disadvantages are highly individual and should be considered by each individual learner. Assignments and homework is an important component of introductory instruction at the university level. An effective method of student assessment is necessary in university as well as all areas and levels of education. In the traditional formal classroom, assessment has been conducted with paper and pencil using questioning techniques such as multiple choice, constructed response, fill-in-the blank, and essay items. This type of testing has been used to assess the skills and knowledge of students since the early 1930s (Hatfield & Gorman, 2000). Advancements in technology have led to new methods of delivering information and student assessment. With the evolution of web-based technologies and the broad availability of computers, student assessment can now include computer-based forms. However, use platform homework system has some benefits. These benefits include obtaining students' results faster, having the ability to place grades into electronic format, measuring learning accurately, focusing on a student-centred environment, and costing less compared to paper-based exams (Bartlett, Reynolds, & Alexander, 2000; Dash, 2000; Oregon to Administer, 2001). Student using different kinds of the use platform s such as Module, WebCT, Blackboard as an e-learning tool has grown tremendously over the past several years as technology has been integrated into education and training. “E-learning” may be defined as instruction delivered electronically via the Internet, Intranets, or multimedia use platform s such as CD-ROM or DVD (Hall, 2003; O’Neill, Singh, & O’Donoghue, 2004). Since many users today have access to direct Internet connections, e-learning is often identified with web-based learning (Hall, 2003). Many writers refer to “e-learning,” “online learning,” “web-based learning” SWAD Use platform means in Spanish Web system to help in teaching (Sistema Web de Apoyo a la Docencia) (SWAD) interchangeably, an approach that ill be taken in this paper E learning can be implemented in a ariet of a s s ch as thro gh the 237 Review of European Studies Vol. 2, No. 2; December 2010 www.ccsenet.org/res use of self-paced independent study units, asynchronous interactive sessions (where participants interact at different times) or synchronous interactive settings (where learners meet in real time) (Ryan, 2001). 2. Literature Review Although some universities now days are offering an e-learning class that blended approaches that integrate online components into traditional classes, it still remains at an early stage of development. Consequently, developers and deliverers of online learning need more understanding of how students perceive and react to elements of e-learning (since student attitude and attitude is critical to motivation and learning) along with how to apply these approaches most effectively to enhance learning (Koohang & Durante, 2003). What we know about students learning is an important starting point for exploring the use of educational technology and the instructional design and success of use the use platform as an e-learning tool. The basis of effective use platform as an e-learning tool for learning is comparable to the foundation of effective learning in general. Among the many theories surrounding how people learn. Learning theory suggests that learning is promoted or enhanced (1) when students are actively involved in the learning, (2) when assignments reflect real-life contexts and experiences, and (3) when critical thinking or deep learning is promoted through applied and reflective activities (Bransford, Brown, & Cocking, 2000). The goal of this research is to achieve better understanding for student attitudes toward use platform as learning resources format at UGr, in addition, in this research paper investigative the effect use platform as learning resources on gender, owning a PC, and access to the internet. Numerous studies have demonstrated that a student’s active involvement in the learning process enhances learning, a process often referred to as active learning (Benek-Rivera & Matthews, 2004; Sarason & Banbury, 2004). Simply stated, active learning involves “instructional activities involving students in doing things and thinking about what they are doing” (Bonwell & Eisen, 1991, p. 5). Interactive instruction or “learning by doing” has been found to result in positive learning outcomes (Picciano, 2002; Watkins, 2005). Because many new technologies and web-based activities are interactive, using the platform as online coursework has the potential to create environments where students actively engage with material and learn by doing, refining their understanding as they build new knowledge (Johnston, Killion & Omomen, 2005; Pallof & Pratt, 2003). As Driscoll (2005) observes, that "When students become active participants in the knowledge construction process, the focus of learning shifts from covering the curriculum to working with ideas". And using technology tools ‘to think with’ facilitates working with ideas and learning from that process” (Scardamalia 2002). 2. Literature Review In addition to active involvement, students better understand and apply material when problems and situations are set in the context of real world issues and situations (Eble, 1988). Authentic situations and scenarios can provide a stimulus for learning, creating greater student motivation and excitement for learning, representing and simulating real-world problems and contexts, providing an important structure for student thinking (Quitadamo & Brown, 2001). Emphasizing authentic tasks in context rather than abstract out-of-context activities creates a greater likelihood of learning (Driscoll & Carliner, 2005). Technology and online instruction can facilitate learning by providing real-life contexts to engage learners in solving complex problems (Duffy & Cunningham, 1996; Honebein, 1996). The use of real-world situations has the potential to promote deep learning through the development of critical thinking skills. Critical thinking involves the active and skillful analysis, synthesis, and application of information to unique situations (Scriven & Paul, 2004). Learning retention and performance improves as students are required to apply what they have learned and then reflect upon the learning (Bransford, Brown, & Cocking, 2000). Online instruction has the potential to provide opportunities to promote reflective thought and deep learning through realistically integrating and applying principles learned. Online instruction, such as a simulation, thrusts learners into a learning experience, increasing engagement and providing activities that actively engage learners to analyze, synthesize, and evaluate information while constructing knowledge (Driscoll & Carliner, 2005). Built upon a foundation of learning theory, e-learning can potentially provide many important payoffs. To learners, online instruction offers the flexibility and convenience to complete learning units when and where a learner desires. Additionally, online education has been used to reduce costs and to provide an efficient, standardized way to deliver content (McDonald, 1999-2000; “E-learning,” 2003; “The Pay offs,” 2003). In addition to potential cost savings, e-learning has pedagogical potential beyond traditional methods related to the principles of learning discussed. For instance, multimedia capabilities can be used with learning exercises that allow learners to apply concepts realistically. Or, animation can help demonstrate concepts and events difficult to portray in traditional classes, which, in turn, can facilitate a more accurate communication of important ideas. E-learning can deliver “new” information not contained in traditional sources, effectively reinforcing other course information through offering examples, explanations, assessments, and exercises. 4. Significant of the study In the last two decades, Espania education system has been a gradual shift on the emphasis from teacher-centered approach to a student-centered approach. The implementation of the platform program provides further force to transform teacher's role from being the dispenser of knowledge to being the facilitator of learning. The use platform program as technology-supported education system and was introduced to help Espania fulfill the need for an IT-literate population. As the use platform program places major emphasis on the use of IT in classrooms, there is surely a need to shift from the conventional lecture-practice-recall teaching methods to methods where teachers engage students in activities that create opportunities to construct new knowledge and skills (Shelly et al, 2004). The teacher's role changes from that of the sole authority and a sage on the stage to that of a guide at the side and a facilitator (Rakes, 1996; Cifuentes, 1997). At the same time, the student's role shifts from being a passive and dependent learner to being an active and independent learner (Coombs & Wong, 2000; Hirumi, 2002). 3. Statement of the problem In search of better, more cost effective ways to deliver instruction and training, universities and corporations have expanded their use of e-learning. Although several studies suggest that online education and blended instruction (a “blend” of online and traditional approaches) can be as effective as traditional classroom models, few studies have focused on student use of the use platform as online instruction, particularly in the transition to online learning from traditional approaches. This study aimed to investigate the faculty of education student's at UGr their attitude toward use platform as learning resources, also investigate the effect of use platform as learning resources on the gender, owning PC, and access to the Internet.. 5. Research question of the study The first question: What is the attitude of UGr students' towards use platform as learning resources? The second question: Is there a statistical significance differences at the level of ( 0.05) in the attitude of UGr students' towards use platform as learning resources due to gender? The third question: Is there a statistical significance differences at the level of ( 0.05) in the students' attitude towards use platform as learning resources due to the owning PC? The fourth question: Is there a statistical significance differences at the level of ( 0.05) in the students' attitude towards use platform as learning resources due to access to the Internet? 2. Literature Review In this way, online instruction can potentially enhance learning compared to what can be accomplished i l l h (M E 2001) H th l t ti l di d t li it ti ISSN 1918-7173 E-ISSN 1918-7181 ISSN 1918-7173 E-ISSN 1918-7181 238 Review of European Studies Vol. 2, No. 2; December 2010 www.ccsenet.org/res takes place as users assimilate information, utilize software, apply knowledge to problem solving, and interact with the instructor and other learners (Laine, 2003). Other potential problems of e-learning that have been identified in previous research include a sense of learner isolation (Brown, 1996); learner frustration, anxiety, and confusion (Hara & Kling, 2000; Piccoli, Ahmad, & Ives, 2001); higher student attrition rates (Frankola, 2001; Laine, 2003; Ryan, 2001); the need for greater discipline, writing skills, and self-motivation; and the need for online users to make a time commitment to learning (Golladay, Prybutok, & Huff, 2000; Serwatka, 2003). 6.1 Sample of Study The study was conducted at faculty of education at UGr. A random sample of undergraduate male and female students (N= 258), the participants were asked to complete a questionnaire which included items to measure students' attitude toward use platform as learning resource. 6.2 Development of Instrument 6.2 Development of Instrument In study for Carnevale (2000) he found that distance education students look for many of the same things found in traditional courses including a knowledgeable professor, interaction with the professor, and additional features that create a feeling of community within the class. Schoech (2000) reported that the satisfaction, grades, and performance of students in a semester-long graduate social work course taught in a distance format were consistent with previous outcomes in traditional courses of similar content. A final factor in students' perceptions of online learning is personal contact, some students who learn online report feelings of isolation and loneliness, those students miss the social contact and face-to-face interaction that an institutional setting provides. Additionally, students who lack self-motivation dislike having to motivate themselves to do the coursework. The distance learner may have problems separating "work from home life, experiencing tensions in relations with their family and spouse" (Harrell, 1999, p. 270). 6.3.2 Question two: Is there statistical significance differences at the level of ( 0.05) in the attitude of UGr students' towards the use platform as learning resources due to gender? to answer the question, the means, standard deviations was computed for the students' attitude towards use platform as learning resources, according to the gender, therefore to detect the statistical significant differences between the mean for male and female attitude towards the use of platform as learning resources, independent-sample T test was used, and the results as shown in table 1 below. Insert Table ---1 --- Here It's clear from the table 1 above the result shows that there are significance differences, due to the gender attitude towards the use platform as learning resources; it was in favor of male students. The interpretation for this result, maybe due those male students prefers use platform method, because it makes learning convenient for them. In addition the nature of male student inpatient and they prefer speed in working with their assignment, the use of platform could create a learning environment that would help them patiently get the assignment done, several researchers studies found that attitudes toward technology differ significantly between males and females student, males indicating greater interest and knowledge with the use of technology, while female students perceived technology as less interesting and more difficult to understand and to use, than male students (Bame et al, 1993; Boser et al, 1998; Heywood, 2000). 6.2 Development of Instrument 6.2 Development of Instrument A 5-point Likert type scale with strongly agree; agree; undecided; disagree; and strongly disagree for the items of the questionnaire, was developed to measure students' attitude toward use platform as learning resource. The questionnaire consisted of two parts the first part consist of the demographic information, the second part consist twenty five items. To verify the reliability of the questionnaire the researchers distributed questionnaire on sample of forty five students out of the original sample and used the internal consistency Cronbach Alpha. The reliability of the scale was 0.84, and to verify the validity of the questionnaire in terms of objectivity, language, and the number of items, the questionnaire was submitted to panel of referees from faculties of education colleges to revise it, and report their remarks about the items a modification has been made according to the advised by the arbitrators. 6.3 Analysis and Discussions 6.3.1 Question one: to answer the question, what is the attitude of UGr students' towards use platform as learning resources? To answer the question, the means, and standard deviations, for the respondents of the study samples Published by Canadian Center of Science and Education 239 Review of European Studies Vol. 2, No. 2; December 2010 www.ccsenet.org/res was computed, the mean was 3.4 and std. deviation was 0.43, which this means a positive attitude from the students toward use of platform as learning resources. From above, the result indicate that use of platform as learning resources, will alleviate students learning in all of its form aspects, like help student to clarify the understanding, also help student enhanced their interaction between student each other and the faculty, the use platform also might help student organize their study time about the subject content, encouraged students to know more about the subject. Roblyer has found that the capacity to choose when to complete activities is the most important factor in positive student responses to online learning, because it grants students a measure of control over their learning, also community college students enrolled in distance education courses also responded positively to the online format (Roblyer, 1999). Undoubtedly, choice makes a significant difference in students' perceptions. Students who prefer online courses place greater value on their control of the pace of the course than on face-to-face interaction (Roblyer, 1999). 6.2 Development of Instrument For example, more males than females perceived their home as being more technological, also more males than females rated both parents' occupations as more technical, their study also mention that males indicated a greater interest and knowledge of computers than females 6.3.3 Question three: Is there statistical significance differences at the level of ( 0.05) in the attitude use platform as learning resources due to the owning PC? to answer the question, the means, standard deviations was computed for the students' attitude towards use platform as learning resources, according to the owning a pc, then to detect if there is a statistical significant differences. Independent-sample T test was used, and the results as shown in table 2 below. Insert Table --- 2 --- Here Insert Table --- 2 --- Here Table 2 above reflect that there is a statistical significance differences due to the owning a pc, it was in favor of student who own a pc, the interpretation this result it could be that owning a pc will give a great chance to the student to use available technology to them at all the times any time on their own pace, several studies found evidence of owing pc differences, virtually every study that did reported the males are more advantaged, males are more likely to own a computer (Schumacher & Morahan-Martin, 2001). Meanwhile Negative student responses to online learning are also due to owning a computer factor, particularly for students with part-time or full-time jobs, students who do not have an access to use pc or don’t have computers in their homes are often irritated by the additional time required to visit a computer lab (Crotty, 2000), a lack of convenience that contributes to working students' negative reactions. Students who own pc they do have the flexibility to use the platform, chats or other synchronous activities that occur at fixed times. Students whom don't own a pc they don't have the flexibility to use ISSN 1918-7173 E-ISSN 1918-7181 240 Review of European Studies Vol. 2, No. 2; December 2010 www.ccsenet.org/res platform, so those students will be forced to access the platform via computer lab schedule, computer lab availability, therefore their use and their attitude toward use the platform will decrease in general. (Carr, 2000). Other complaints due to don’t won a pc, include not having enough time to read and send e-mail and to perform related online activities, also the type of computer access students experience impacts their attitude toward online learning experiences. If students have a computer at home or in their dorm, they may find distance learning activities more convenient than students who have to locate an available computer. Also, an experienced computer user might be more comfortable with publicly available and unfamiliar hardware than a non-user. In a review of a longitudinal study involving more than 800 university students, McMahon, Gardner, Gray, and Mulhern (1999) reported that computer access accounts for 50% of the variance that exists among student attitudes toward online learning. 6.3.4 Question four: Is there a statistical significance differences at the level of ( 0.05) in the students' towards use platform as learning resources due to access to the internet? Insert Table --- 2 --- Here To answer the question the means, standard deviations was computed and, then to detect if there is a statistical significant differences. Independent-sample T test was used, and the results as shown in table 3 below. Insert Table --- 3 --- Here Table 3 above reflect that there is a statistical significance differences in function at the level of ( 0.05) in the attitude toward use platform as learning resources due to access to the internet, it was in favor of student whom do have access to the internet, this could be interpretation that access to the internet, give a chance to use the technology all the time any time on their own pace. With online learning, students control the factors, when, where, and what they learn, as well as how often and how quickly and this level of control is what creates satisfied students. In a recent study, nearly 68% of students were "satisfied" or "very satisfied" with using the Internet as the primary source of course materials (Beatty, 2000). Reasons for students' satisfaction ranged from accessibility and convenience to flexibility and student-teacher interaction. Even though technology-related experiences are as varied as the individuals who use computers to access internet, both men and women voice positive attitudes about online learning, student responses also reflect concerns they have due accessing the internet, about hardware issues such as modem speed and available memory. Complaints about the periodic slowness of Internet connections or server problems indicate that such difficulties frustrate students (Harrell, 1999). Computer hardware problems increase student concern about using computer to access internet and the quality of their online learning experience. When the connection is too slow, the server is down, or the memory is full, the computer experience becomes a hindrance to education. Also, students who may already lack confidence in computer equipment transfer their feelings of inadequacy to the learning experience. 7. Conclusion Things emerge in the study of students' attitudes toward use platform as learning resource: individual situations impact students' perceptions of virtual learning and students' individual characteristics make it difficult to define their perceptions conclusively. For example, some students have their own computers, while others rely on computer labs. Such variation in computer access can result in attitudinal attitudes differences. In addition, the purpose of computer use by students varies - distance education courses, for example, use computers in different ways than traditional classroom - which can also affect students' perceptions in general. A wide variety of achievement levels and attitudes exist among both e-learning and traditional learning. Although an illusory "typical learner" exists, a variety of factors, including students' gender, access to the internet, age, owning laptop, and motivation, could explain different reactions among the student population. Add to this the variety of modes that e-learning employs, and the data collected can be ambiguous at best. Whether students are involved in full-scale e-learning programs or experimenting online activities for a traditional class, their perception of the experience profoundly affects the process of education. Learning varies with each individual, as do preferences for the methods used to learn. Given the appropriate tools, students can become lifelong learners with a passion for knowledge. The challenge for educators is therefore the same as it has always have been: how to help students learn. The difference between the blackboard-bound classroom and the cyber-connected virtual classroom is just a matter of space, and integration therefore educators must learn how that space and integration helps to define student perceptions of future education. Findings from this research suggest future studies should focus on students' learning experiences as reflected in attitudes toward education Ascough, R. (2002). Designing for online distance education: Putting pedagogy before technology. Teaching Theology and Religion, 5, 1729. References 241 Published by Canadian Center of Science and Education Review of European Studies Vol. 2, No. 2; December 2010 www.ccsenet.org/res Bame, S, Petersen, N, & Wray, N.P. (1993). Variation in hemodialysis patient compliance according to demographic characteristics. Social Science & Medicine, 37(8), 1035-1043. Bartlett, J. E., II, Reynolds, K A., & Alexander, M. W. (2000). A tool for online learning. Journal of Online Learning, 11(3 & 4), 22-24. Beatty, P. T, & Mortera-Guiterrez, F. (2000). From research to practice in distance learning education: Strategies for fostering faculty development and improving instructional practice. ERIC Document Reproduction Service No. ED 437536. Benek-Rivera, & Matthews, V. (2004). Active learning with jeopardy: Students ask the questions. Journal of Management Education, 28(1), 104-118. Blustain, Harvey, Philip Goldstein, and Gregory Lozier. (1999). "Assessing the New Competitive Landscape," in Dancing with the Devil, Editors: Richard N. Katz and Associates, Jossey-Bass Publishers, San Francisco. Boser, R. Palmer, J, & Daugherty, M. K. (1998). Student's attitudes toward technology in selected technology education programs. Journal of Technology Education, 10(1), 4-19. Bransford, J. D, Brown, A. L., & Cocking, R. R. (Eds.). (2000). How people learn: Brain, mind experience, and school committee on developments in the science of learning. Commission on Behavioural and Social Sciences and Education of the National Research Council, National Academy Press. Brown, K. M. (1996). The role of internal and external factors in the discontinuation of off-campus students. Distance Education, 17(1), 44-71. Carnevale, D. (2000). Study assesses what participants look for in high-quality online courses. Chronicle of Higher Education, 47(9), A46. Retrieved April 6, 2002 from Academic Search Elite database. Carr, S. (2000). Two professors find that online chats are unpopular. The Chronicle of Higher Education, April 7, 31-32. Cifuentes, L. (1997). from sages to guides: a professional development study. Journal of Technology and Teacher Education, 5(1), pp. 67-77. Coombs, S.J. & Wong, P. (2000). Supporting student-centered learning with IT. In M.Williams (Ed), Integrating Technology into Teaching and Learning: concepts and application. Singapore, Prentice Hall, 230-249. Crook, C.K. (2008). Web 2.0 Technologies for Learning: The current landscape – Opportunities, challenges and tensions. Coventry: Becta. [Online] Available: http://partners.becta.org.uk/upload-dir/downloads/page_documents/research/web2_technologies_learning.pdf (March 3 2009) Crook, C.K. (2008). Web 2.0 Technologies for Learning: The current landscape – Opportunities, challenges and tensions. Coventry: Becta. [Online] Available: y [ ] http://partners.becta.org.uk/upload-dir/downloads/page_documents/research/web2_technologies_learning.pdf (March 3, 2009). Crotty, T. (2000 April 7). Constructivist theory unites distance learning and teacher education. [Online] Available: f http://edie.cprost.sfu.ca/it/constructivist-learning (May 12, 2001). References Dash, J. (2000). Computer ware introduces remotely hosted software for Internet testing service. Computer world, 34(88), 1, 4. DfES. (2005). DfES e-learning strategy: Harnessing technology -Transforming learning and childrens’ services. [Online] Available: http://www.dfes.gov.uk/publications/e-strategy/ (July 1, 2008). Driscoll, M. & Carliner, S. (2005). Advanced web-based training strategies. San Francisco: Pfeiffe Drucker, Peter. (1997). "An Interview with Peter Drucker," Forbes Magazine, March 10 pp.126-127. Duffy, T. M., & Cunningham, D. J. (1996). Constructivism: Implications for the design and delivery of instruction. In D. H. Jonassen (Ed.), Handbook of research for educational communications and technology. New York: Macmillan. Dutton, W.H., P.H. Cheong, and N. Park. (2003). The social shaping of a virtual learning environment: The case of a university-wide course management system. Electronic Journal of e-Learning 2, no. 1. Eble, K. (1994). Craft of teaching: A guide to mastering the professor's art (2ndedition), New York: Jossey-Bass. E-learning replaces classroom. (2003). Health Management Technology, 24(4), 50-51. Eble, K. (1994). Craft of teaching: A guide to mastering the professor s art (2ndedition), New York: Jossey-Bass. E-learning replaces classroom. (2003). Health Management Technology, 24(4), 50-51. E-learning replaces classroom. (2003). Health Management Technology, 24 Frankola, K. (2001). Why online learners drop out. Workforce, 10, 52-60. Frankola, K. (2001). Why online learners drop out. Workforce, 10, 52-60. Golladay R Prybutok V & Huff R (2000) Critical success factors for the online learner Journal of Computer Golladay, R., Prybutok, V. & Huff, R. (2000). Critical success factors for the online learner. Journal of Computer Information Systems, 40(4), 69-71. ISSN 1918-7173 E-ISSN 1918-7181 242 Vol. 2, No. 2; December 2010 Review of European Studies www.ccsenet.org/res Hall, B. (2003). FAQs about e-learning. Retrieved August 15, 2003, [Online] Available: www.brandonhall.com/public/faqs2/faqs2.htm. Hall, B. (2003). FAQs about e-learning. Retrieved August 15, 2003, [Online www.brandonhall.com/public/faqs2/faqs2.htm. Hara, N. & Kling, R. (2000). Students’ distress with a web-based distance education course: An ethnographic study of participants’ experiences. Information, Communication and Society, 3(4), 557-579. Harrell, W., Jr. (1999). Language learning at a distance via computer. International Journal of Instructional Media, 26 (3), 267-282. Hatfield, S. R., & Gorman, K. L. (2000). Assessment in education-the past, present, and future. Assessment in Business Education National Business Education Association Yearbook, 38, 1-10. Reston,VA: National Business Education Association. Haugen, S, LaBarre, J, & Melrose, J. (2001). Online course delivery: Issues and challenges. Issues in Information Systems, 2, 127-131. Heywood, J. (2000). Assessment in Higher Education. London: Jessica Kingsley Publishers Hirumi, A. (2002). References Student-centered, technology-rich learning environments (SCenTRLE): operationalizing constructivist approaches to teaching and learning. Journal of Technology and Teacher Education, 10(4), pp. 497-537. Honebein, P. C. (1996). Seven goals for the design of constructivist learning environments. In B. G. Wilson (Ed.), Constructivist learning environments: Case studies in instructional design. Englewood Cliffs, NJ: Educational Technology Publications. Huang, A.H. (1996-1997). Challenges and opportunities of online education. Journal of Educational Technology Systems, 25, 229-247. Johnston, J., Killion, J., & Oomen, J. (2005). Student satisfaction in the virtual classroom. The Internet Journal of Allied Health Sciences and Practice. 3. [Online] Available: http://ijahsp.nova.edu/articles/vol3num2/Johnston%20-%20Printer%20Version.pdf (January 26, 2007). Laine, L. (2003). Is e-learning effective for IT training? T +D, 57(6), 55-60. Laine, L. (2003). Is e-learning effective for IT training? T +D, 57(6), 55-60. McDonald, D. (1999-2000). Improved training methods through the use of multimedia technology. Journal of Computer Information Systems, 40(2), 17-20. McEwan, B. (2001). Web-assisted and online learning. Business Communications Quarterly, 64(2), 98-103. McLester, S. (2002). Virtual learning takes a front row seat. Technology and Learning, 22, 2436. McMahon, J, Gardner, J, Gray, C., & Mulhern, G. (1999). Barriers to computer usage: Staff and student perceptions. Journal of Computer Assisted Learning, 15, 302. Malkowski, S.R., M.E. Thompson, and J.G. Theis. (2006). External factors associated with adopting a CMS in resident college courses. Internet and Higher Education, 9: 163–74. Morgan. G. (2003). Course management system use in the University of Wisconsin system. Boulder, CO: EDUCAUSE Center for Applied Research (ECAR). Ofcom. (2008). Social networking: A quantitative and qualitative research report into attitudes, behaviors and use. [Online] Available: http://www.ofcom.org.uk/advice/media_literacy/medlitpub/medlitpubrss/socialnetworking/report.pdf (April 5, 2008). Oregon to Administer Standardized Tests on Computers. (2001). Electronic Education Report, 8(9), 2-7. Pallof, R., & Pratt, K. (2003). The Virtual Student: A Profile and Guide to Working with Online Learners. San Francisco, CA: Josey-Bass Publishers. Picciano, A.G. (2002). Beyond student attitudes: Issues interaction, presence, and performance in an online course. Journal of Asynchronous Learning Networks, 6, 20-41. Piccoli, G, Ahmad, R. & Ives, B. (2001). Web-based virtual learning environments: A research framework and a preliminary assessment of effectiveness in basic IT skills training. MIS Quarterly, 25(4), 401-425. Quitadamo, J. & Brown, A. (2001). Effective teaching styles and instructional design for online learning environments. National Educational Computing Conference, July 25-27, 2001 (Chicago,IL). [Online] Available: http://confreg.uoregon.edu/NECC2001/program/research_pdf/Quitadamo.pdf (January 10, 2006). Published by Canadian Center of Science and Education 243 Vol. 2, No. References 2; December 2010 Review of European Studies www.ccsenet.org/res Rakes, G. (1996). Using the Internet as a tool in a resource based learning environment. Educational Technology, 1: (5), pp. 52-56. Roblyer, M. D. (1999). Is choice important in distance learning? Journal of Research on Computing in Education, 32 (1), 157. Rumble, G. (2001). Reinventing distance education, International Journal of Lifelong Education, 2 Ryan, S. (2001). Is online learning right for you? American Agent & Broker, 73(6), 54-58. Sarason, Y, & Banbury, C (2004). Active learning facilitated by using a game-show format or who doesn't want to be a millionaire? Journal of Management Education, 28(4), 509-519. Scardamalia, M. (2002, April). Creative work with ideas: A luxury? Paper presented at the annual meeting of the American Psychological Association, New Orleans, LA. Schumacher, P., & Morahan-Martin, J. (2001). Gender, Internet and computer attitudes and experiences. Computers in Education, 17(1), 95-110. Schoech, D. (2000). Teaching over the Internet: Results of one doctoral course. Research on Social Work Practice, 10, 467-487. Serwatka, J. (2003). Assessment in on-line CIS courses. Journal of Computer Information Systems, 43(3), 16-20. Shelly, G. B., Cashman, T. J, Gunter, R. E. and Gunter, G. A. (2004). Teachers discovering computers - integrating technology into the classroom Thomson Course Technology, Boston, MA The pay-offs of e-learning go far beyond the financial. (2003). HR Focus, 80(10), 7. Volery, T. & Lord, D. (2000). Critical success factors in online education. The International Journal of Education Management, 14(5), 216-223. Volery, T. & Lord, D. (2000). Critical success factors in online education. The International Journal of Education Management, 14(5), 216-223. Wang, A.Y, Newlin, M.H, & Tucker, T.L. (2001). A discourse analysis of online classroom chats: Predictors of cyber student performance. Teaching of Psychology, (28), 222226. Watkins, R. (2005). Developing interactive e-learning activities. Performance Improvement, 44, 5-7. Watkins, R. (2005). Developing interactive e-learning activities. Performance Improvement, 44, 5-7. Table 1. The independent-sample T test analysis Variable Mean Std. Deviation t df Sig. Gender Male 4.2 0.46 12.98 256 0.000 Female 2.9 0.59 Table 2. The independent-sample T test analysis Variable Mean Std. Deviation t df Sig. Owning PC Yes 3.9 0.51 5 256 0.000 No 3.07 0.64 Table 3. The independent-sample T test analysis Variable Mean Std. Deviation t df Sig. Access Yes 3.8 0.53 3 256 0.01 No 3.1 0.64 Table 3. The independent-sample T test analysis ISSN 1918-7173 E-ISSN 1918-7181 ISSN 1918-7173 E-ISSN 1918-7181 244
https://openalex.org/W2419198271	https://europepmc.org/articles/pmc4898980?pdf=render	English	null	Effects of propolis and gamma‐cyclodextrin on intestinal neoplasia in normal weight and obese mice	Cancer medicine	2,016	cc-by	8,441	© 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Cancer Medicine 2016; 5(9):2448–2458 Cancer Medicine 2016; 5(9):2448–2458 doi: 10.1002/cam4.787 Correspondence Correspondence Darina L. Lazarova, The Commonwealth Medical College, Scranton, PA 18509. Tel: 570-504-9645; Fax: 570-504-9636; E-mail: dlazarova@tcmc.edu Effects of propolis and gamma-cyclodextrin on intestina neoplasia in normal weight and obese mice Youngjin Cho1,a, Linda Gutierrez2,a, Michael Bordonaro1, Daniel Russo1, Frank Anzelmi1, Jayde T. Hooven3, Carmine Cerra1 & Darina L. Lazarova1 1The Commonwealth Medical College, Scranton, Pennsylvania 18509 2Wilkes University, Wilkes-Barre, Pennsylvania 18766 3Penn State Hershey College of Medicine, Hershey, Pennsylvania 17033 1The Commonwealth Medical College, Scranton, Pennsylvania 18509 2Wilkes University, Wilkes-Barre, Pennsylvania 18766 3Penn State Hershey College of Medicine, Hershey, Pennsylvania 17033 Abstract Colon cancer, gamma-cyclodextrin, obesity, propolis, Western diet Obesity is associated with colorectal cancer (CRC). This effect might be at- tributed to adipokine-supported signaling. We have established that propolis suppresses survival signaling in CRC cells in vitro; therefore, we ascertained the ability of a propolis supplement to modulate intestinal neoplastic development in C57BL/6J-ApcMin/+/J mice in the lean and obese state. To induce obesity, mice were fed with a Western diet containing 40% fat. Since the propolis supplement includes gamma-cyclodextrin, the interventions included diets sup- plemented with or without gamma-cyclodextrin. The animals were administered the following diets: (1) control diet, (2) control diet/gamma-cyclodextrin, (3) control diet/propolis, (4) Western diet, (5) Western diet/gamma-cyclodextrin, and (6) Western diet/propolis. Western diet, resulting in obesity, accelerated neoplastic progression, as evidenced by the larger size and higher grade dysplasia of the neoplasms. In the context of normal weight, gamma-cyclodextrin and propolis affected neoplastic progression, as determined by the size of the lesions and their grade of dysplasia. A statistically significant decrease in the number of adenomas was detected in mice fed a control diet with the propolis supple- ment (61.8 ± 10.6 vs. 35.3 ± 7.6, P = 0.008). Although there was no significant difference in the polyp numbers between the six groups, the mice with the lowest number and size of adenomas were those fed a Western diet with gamma- cyclodextrin. This unexpected outcome might be explained by the increased levels of apoptosis detected in the intestinal tissues of these obese mice. We posit that butyrate derived from the metabolism of gamma-cyclodextrin may contribute to the decreased neoplastic burden in the context of obesity; however, future studies are required to address this possibility. ORIGINAL RESEARCH ORIGINAL RESEARCH Cancer Medicine Open Access Funding Information Funding Information This study was funded by a research grant from Manuka Health New Zealand Ltd and research funds of The Commonwealth Medical College. This study was funded by a research grant from Manuka Health New Zealand Ltd and research funds of The Commonwealth Medical College. Received: 15 March 2016; Revised: 28 April 2016; Accepted: 29 April 2016 Cancer Medicine 2016; 5(9):2448–2458 doi: 10.1002/cam4.787 aEqual contribution Received: 15 March 2016; Revised: 28 April 2016; Accepted: 29 April 2016 Received: 15 March 2016; Revised: 28 April 2016; Accepted: 29 April 2016 Animals All mice were maintained on a 12-hour light and dark cycle at the animal facility in The Commonwealth Medical College. The protocol for mouse work was approved by the Institutional Animal Care and Use Committee at The Commonwealth Medical College. Thirty-two male C57BL/6J-ApcMin/+/J mice (Jackson Laboratories, USA) at 5 weeks of age were randomly assigned to six intervention groups (n = 5–6). Three groups were fed a control diet (AIN-93G) without any supplements, with 6% gamma-cyclodextrin (Cavamax W8, Wacker Chemical Corporation), or with 8% propolis supplement containing gamma-cyclodextrin (Propolis with CycloPower Powder, Manuka Health New Zealand, Ltd, New Zealand). The amount of gamma-cyclodextrin (6%) in the second intervention diet was the same as the amount of gamma-cyclodextrin in the propolis powder-supplemented intervention diet. Three groups of mice were fed a Western diet without supplements, with 6% gamma-cyclodextrin, or with 8% propolis sup- plement containing gamma-cyclodextrin. The Western diet (cat # 100244, Dyets Inc, Bethlehem, PA) provides approximately 40% of total calories from fat. The diets were given at libitum for 8 weeks. Cages were equipped with mesh bottoms to prevent coprophagia. Body weight was recorded every other workday. Food consumption was assessed by weighing the food added to each cage, and by accounting for any spillage. At the end of the experiment, the mice were killed and tissues (intestines, liver, and epididymal fat) were collected for the analyses. The apoptotic effect of butyrate on CC cells can be further increased by propolis, a honeybee product [14]. Propolis is a complex mixture of flavonoids, terpenes, and caffeic acid phenethyl ester, and all of these com- pounds could exert anticancer effects [15]. We have ascertained that the combination of butyrate and propolis induces high levels of apoptosis in colon adenoma and carcinoma cells, but not in normal colonic cells [16]. Since propolis suppresses AKT and JAK/STAT pathways [14, 16] that might be activated in obesity, we reasoned that propolis may counteract intestinal neoplastic devel- opment and this effect might be most discernible in the context of obesity. To test this hypothesis, we uti- lized the mouse model of intestinal cancer C57BL/6J- ApcMin/+/J (JAX Strain 002020) that develops adenomas due to a truncating mutation in Apc [17, 18]. Introduction (adipokines) collaborate with cancer-initiating mutations to support neoplastic development in the large intestine. Adipose tissue and macrophages infiltrating this tissue secrete high levels of cytokines, including TNFalpha, inter- leukins, and WNT5A that activate AKT, ERK, JNK, and JAK/STAT signaling [3]. Recently, analyses of data from The Cancer Genome Atlas (TCGA) revealed that obese CC patients (with body mass index of 30 or greater) have a lower number of driver gene mutations than nor- mal weight CC patients [4]. One possible explanation for this observation is that neoplasm-promoting levels of Although the overall incidence of colorectal cancer has been declining in the past 35 years, the incidence of the disease has increased among 20- to 34-year olds and, by 2030, there could be up to a 90% increase in colon cancer (CC) incidence in this age group [1]. This shift in age of incidence is due in part to the increasing obesity among children and adolescents [2]. Although the mechanisms by which obesity predisposes to intestinal neoplasia are debated, one possibility is that obesity-associated cytokines 2448 Propolis and Cyclodextrin in Intestinal Neoplasia Y. Cho et al. survival pathways in obese individuals are maintained by adipokines, rather than driver gene mutations. in tumor multiplicity or increase their neoplastic bur- den. Day and colleagues have observed that the high-fat diet does not change the number of polyps; however, the ratio of large to small polyps is higher in mice fed a high-fat diet [22]. Utilizing a different dietary formulation, Baltgalvis and colleagues have reported that a Western diet increases the number of polyps in ApcMin/+ mice by 75% compared to AIN-76A (control diet) - fed mice [23]. Although obesity modulates the risk for at least 10 types of cancer [5], the link between obesity and CC risk might be modified by diet in a complex manner. Diet directly affects neoplastic development in the colon because the neoplasms are exposed to the luminal content, in which dietary agents and metabolites are present at high concentrations. For example, butyrate, a short-chain fatty acid, is a fermentation product of dietary fiber in the colon, and its physiological concentrations in the large intestine can reach up to 20 mmol/L [6–9]. Exposure of CC cells with mutations in the WNT pathway to physi- ological levels of butyrate hyperactivates WNT/beta- catenin-dependent gene transcription, and subsequently, leads to apoptosis [10]. Introduction These effects of butyrate are highly relevant since most CCs exhibit deregulated WNT/beta- catenin transcription due to mutations in the adenomatous polyposis coli (APC) gene, and this type of mutations is frequently the first to take place in intestinal neoplastic development [11]. This prevalent mutation profile may explain why human colon adenoma cells (i.e., LT-97) derived from the earliest stages of neoplastic development also exhibit apoptosis in the presence of butyrate [12]. Unlike neoplastic cells, normal colonocytes metabolize butyrate as a source of energy [13]; these cells do not hyperactivate WNT signaling (as they do not have muta- tions in the pathway) and do not commit to apoptosis in the presence of butyrate. The objective of our study was to ascertain whether the additional neoplastic burden in mice associated with diet-induced obesity is suppressed by a propolis supple- ment. We utilized Propolis with Cyclopower (Manuka Health New Zealand), which we have previously characterized in vitro [14, 16]. This propolis supplement is formulated with the oligosaccharide gamma-cyclodextrin; therefore, the oligosaccharide was included as a control supplement in the intervention diets. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Tissue analyses Intestines (small intestines and colons) were dissected, fixed in Histochoice MB (Electron Microscopy Sciences, Hatfield, PA) for 24 h, and transferred to 70% ethanol. Representative tissues with or without lesions were frozen in liquid nitrogen. Intestinal neoplasms were counted by three researchers, and the average number was calculated based upon these independent counts. To estimate the size of the lesions, pictures of the lesions were taken with AmScope MA-1000 camera on a Nikon SMZ645 dissecting microscope, images were obtained using Nikon ACT-1 software, and size (surface area) of the lesions was estab- lished with Image J. A minimum of 70% of all lesions per intestine were processed to obtain average size. For histological studies, we utilized the technique “Swiss roll” to analyze the entire length of the intestines [24]. Histopathological assessments were based upon hematoxy- lin and eosin-stained slides by two blinded for the inter- vention pathologists, and according to the nomenclature for intestinal neoplasia in mice [20]. Low- and high-grade dysplasia were determined as recommended by the nomen- clature for intestinal neoplasia in mouse models [20]. Cell culture and chemicals The human colorectal cancer cell line HCT-116, the normal human fetal colonic cell line CCD841CoN, and the murine fibroblast cell line 3T3-L1, were from the American Tissue Culture Collection. Cells were grown in alpha-MEM medium with 10% fetal bovine serum. Caffeic acid phene- thyl ester (CAPE)-enhanced propolis (Propolis with CycloPower) was a gift from Manuka Health New Zealand Ltd. For in vitro studies, propolis was dissolved in dimethyl sulfoxide at 100 mg/mL; the stock solution was kept at −80°C. Statistics Data were presented as mean ± standard deviation (SD). Unpaired Student’s t-test analysis was used to determine the significance of statistical differences in the number of mutations between mice on a control diet and mice on a Western type diet. Differences were considered sig- nificant at P < 0.05. Neoplastic burden (size/number of neoplasms) was analyzed by one-way analysis-of-variance (ANOVA), with Tukey’s post-test calculations to adjust for multiple comparisons with 95% confidence. Western blot analyses Western blot analyses were performed as reported [25]. The following antibodies were used: a) from Cell Signaling: anti-phospho-p44/42 Erk1/2 (Thr202/Tyr204), anti- phospho-Stat3 (Tyr705), anti-total Stat3, anti-Erk1/2, anti- cleaved Caspase 3 (#9661) that recognizes the large fragment (17/19kD) of activated Caspase 3 resulting from cleavage adjacent to Asp175; b) from Santa Cruz Biotechnology: anti-Ser473-phospho-Akt, anti-Akt, anti-phospho-cJUN, anti-c-Jun, anti-Bax; and c) from Sigma: anti-beta ACTIN. Antibodies were utilized at a dilution of 1:1,000, except the ant-Actin antibody utilized at 1:5,000 dilution. Lysates from control and neoplastic intestinal tissues were prepared with RIPA lysis and extraction buffer (# 89900), Halt protease inhibitor (# 87786), and Halt Phosphatase Animals Although APC mutations in humans are manifested by colorectal adenomas, ApcMin/+ mice develop predominantly small intestinal lesions [17, 19, 20], a phenomenon explained by the different number of stem cell divisions in the two tissue types in humans and mice [21]. Two research groups have reported that when the ApcMin/+ mice were fed different formulations of a Western diet, the mice exhibit increased adenoma size without changes 2449 © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Y. Cho et al. Propolis and Cyclodextrin in Intestinal Neoplasia inhibitor (# 78420) from ThermoScientific, USA. Tissues were homogenized in a Dounce tissue grinder with RIPA buffer containing protease inhibitors, phosphatase inhibi- tors, PMSF, and EDTA, lysed on ice for 40 min, and centrifuged at 4°C for 15 min. Western blotting analyses were performed with 30 μg of total protein as estimated by Bradford assays. Detection of signal was carried out with horseradish peroxidase conjugated secondary antibod- ies and chemiluminescence kits (Santa Cruz Biotechnology). Densitometry was performed using an Alpha Innotech MultiImage II, correcting for local background and nor- malizing for differences in ACTIN values. Exome sequencing and analysis Mouse exome sequencing was performed on genomic DNA from paraffin-embedded small intestines. DNA extraction, library preparation, Agilent SureSelect mouse (51 Mb) capture; HiSeq2500 PE100-125, and DNAnexus platform exome analysis (In/Del, SNV and CNV analysis) were performed by Otogenetics (Norcross, GA). All data were analyzed with BWA mapping software (to map low- divergent sequences against a large reference genome) and the Genome Analysis Toolkit (SNP/Indel pipeline, a software for analysis of high-throughput sequencing data, developed at the Broad Institute). Single-nucleotide poly- morphism (SNP) annotation was carried out against reference genome GRCm38/mm10. The database of somatic mutations was searched for mutations in genes associated with cancer (listed in COSMIC, a catalog of somatic mutations in cancer (http://cancer.sanger.ac.uk/ census/). © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Body weight and food consumption The average body weight gain in mice exposed to a control diet, control diet with gamma-cyclodextrin, and control diet with propolis supplement, was comparable (5.8 ± 1.9, 5.9 ± 1.7, and 6.1 ± 0.9 grams, respectively, one-way © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. 2450 Propolis and Cyclodextrin in Intestinal Neoplasia Y. Cho et al. Figure 1. (A) The mouse body weight was measured every other workday for the entire duration of the study. (B) The number of neoplasms per mouse was established by three independent researchers. (C) Average size of neoplasms per mouse in each group was measured as described in Materials and Methods. Only statistically significant differences are labeled (Tukey’s post hoc test, P < 0.05). C (control diet), CD (control diet with yclodextrin), CP (control diet with propolis), W (Western diet), WD (Western diet with cyclodextrin), and WP (Western diet with propolis). b d i ht d th kd f th ti d ti f th t d (B) Th b f l Figure 1. (A) The mouse body weight was measured every other workday for the entire duration of the study. (B) The number of neoplasms per mouse was established by three independent researchers. (C) Average size of neoplasms per mouse in each group was measured as described in Materials and Methods. Only statistically significant differences are labeled (Tukey’s post hoc test, P < 0.05). C (control diet), CD (control diet with cyclodextrin), CP (control diet with propolis), W (Western diet), WD (Western diet with cyclodextrin), and WP (Western diet with propolis). or Western diet with propolis (14.6, 13.0, 14.0 kcal/mouse/ day, respectively). between groups ANOVA: P = 0.960). The average body weight gain in mice exposed to a Western diet, Western diet with gamma-cyclodextrin, and Western diet with propolis supplement, was 9.0 ± 4.3, 9.1 ± 2.0, and 5.2 ± 1.0 grams, respectively, one-way between groups ANOVA: P = 0.052 (Fig. 1A). Between all six intervention groups, one-way ANOVA established significant differences in body weight gain [F(5, 26) = 3.2063, P = 0.022]. Food con- sumption was similar between the mice on a control, control-cyclodextrin, and control-propolis diet (10.3, 8.8, and 8.3 kcal/mouse/day, respectively). Similarly, food con- sumption did not differ significantly between the mice on a Western diet, Western diet with gamma-cyclodextrin, © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Neoplastic burden: number and size of intestinal neoplasms However, the percent of small and large adeno- mas exhibited inverse correlation with the size of the lesions (Figs. 2A, C). The groups on supplemented control p g Analyses of the mean size of the lesions were carried out between groups on control diets, groups on Western diets, as well as between all six dietary interventions (Fig. 1C). There were no significant differences in the neoplasm size between the three intervention groups on a control diet with or without supplements, as determined by one-way ANOVA [F(2, 13) = 1.5166, P = 0.256]. The mean size neoplasm in the group on a control diet without supplements was 5.64 ± 0.94 mm2, in the group on a control diet with gamma-cyclodextrin - 5.63 ± 0.90 mm2, and in the group on a control diet with a propolis sup- plement, 4.75 ± 1.07 mm2. There was a difference in neoplasm size between the three intervention groups on Western diets as determined by one-way ANOVA [F(2, 13) = 18.1197, P = 0.002]. A Tukey’s post hoc test revealed that the neoplasm size in the group on a Western diet with gamma-cyclodextrin (4.62 ± 0.82 mm2) was statisti- cally significantly lower than the neoplasm size in the group on a Western diet without supplements (8.00 ± 0.68 mm2, P = 0.001), and also than the neo- plasm size in the group on a Western diet with a propolis supplement (6.33 ± 1.07 mm2, P = 0.019). The neoplasm size in the group on a Western diet with a propolis sup- plement was also lower than that in the group on a Western diet without supplements (P = 0.021). One-way ANOVA that compared the effect of diet and supplements on the size of neoplasms in all six interventions revealed significant differences between the group means [F(5, 26) = 9.079, P = 0.001]. Figure 2. Number of neoplasms classified into three size categories: (A) small (<5 mm2), (B) medium (5–10 mm2), and (C) large (>10 mm2) in the six intervention groups: C (control diet), CD (control diet with cyclodextrin), CP (control diet with propolis), W (Western diet), WD (Western diet with cyclodextrin), and WP (Western diet with propolis). Only statistically significant differences are labeled (Tukey’s post hoc test, P < 0.05). Neoplastic burden: number and size of intestinal neoplasms Almost all neoplastic lesions were detected in the small intestine. There was a significant difference in the number of neoplasms between intervention groups on control diets as determined by one-way ANOVA [F(2, 13) = 6.7687, P = 0.0097], Figure 1B. The number of neoplasms in the group on a control diet without supplements was higher than in the group on a control diet with a propolis 2451 © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Propolis and Cyclodextrin in Intestinal Neoplasia Y. Cho et al. when compared to the neoplasm size in mice on a Western diet without supplements (Fig. 1C). supplement (61.8 ± 10.6 vs. 35.3 ± 7.6, Tukey’s post hoc test, P = 0.008). There were no significant differences in the number of neoplasms between the mice on a control diet and the mice on a control diet with gamma-cyclodextrin (43.6 ± 16.9), and between the two control diets with supplements (propolis or gamma-cyclodextrin). Among the groups on Western diets, there were no significant differences in the number of neoplasms [F(2, 13) = 1.4521, P = 0.270]; however, there were large fluctuations in the number of neoplasms among mice in the same interven- tion group; the mice on a Western diet without supple- ments had 47.6 ± 25.1 neoplasms, the mice on a Western diet with gamma-cyclodextrin exhibited 27.8 ± 15.9 neo- plasms, and the mice on a Western diet with a propolis supplement had 48.0 ± 23.0 neoplasms (Fig. 1B). Comparative analyses of all six interventions did not reveal statistically significant differences in the number of neo- plasms [F(5, 26) = 2.2663, P = 0.078], Fig. 1B. To ascertain differences in neoplastic progression among the six dietary intervention groups, we classified the neo- plasms into three size categories: small (<5 mm2), medium (5–10 mm2), and large (>10 mm2). This categorization is justified by the range of average size lesions in all six intervention groups (the range was determined by the smallest size of 4.6 ± 0.8 mm2, detected in mice fed a Western diet with gamma-cyclodextrin, and the largest size of 8.0 ± 0.7 mm2 in mice fed a Western diet). The percent neoplasms of medium size (5–10 mm2) did not differ in a statistically significant manner between the six dietary interventions [F(5, 26) = 0.4325, P = 0.822] (Fig. 2B). Neoplastic burden: number and size of intestinal neoplasms A B C 0 10 20 30 40 % lesions > 10 mm2 P<0.05 P<0.05 0 10 20 30 40 50 % lesions 5-10 mm2 0 20 40 60 80 C CD CP W WD WP C CD CP W WD WP C CD CP W WD WP % lesions < 5 mm2 P<0.05 A 0 20 40 60 80 C CD CP W WD WP % lesions < 5 mm2 P<0.05 B 0 10 20 30 40 50 % lesions 5-10 mm2 C CD CP W WD WP C 0 10 20 30 40 % lesions > 10 mm2 P<0.05 P<0.05 C CD CP W WD WP Figure 2. Number of neoplasms classified into three size categories: (A) small (<5 mm2), (B) medium (5–10 mm2), and (C) large (>10 mm2) in the six intervention groups: C (control diet), CD (control diet with cyclodextrin), CP (control diet with propolis), W (Western diet), WD (Western diet with cyclodextrin), and WP (Western diet with propolis). Only statistically significant differences are labeled (Tukey’s post hoc test, P < 0.05). When analyzing all six interventions, in addition to the above-mentioned differences between each three treatments (in the context of control or Western diet), a Tukey’s post hoc test to adjust for multiple comparisons with 95% con- fidence revealed differences (P < 0.01) between the mean values of neoplasm size in mice on all three control diets © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. 2452 Y. Cho et al. Propolis and Cyclodextrin in Intestinal Neoplasia the percent small neoplasms in mice fed a control with a propolis supplement (64.8 ± 8.2%, P = 0.030) (W vs. CP, Fig. 2A). and Western diets exhibited higher numbers of small (<5 mm2, Fig. 2A) neoplasms and lower numbers of large (>10 mm2, Fig. 2C) neoplasms compared to the respec- tive diets without supplements. the percent small neoplasms in mice fed a control with a propolis supplement (64.8 ± 8.2%, P = 0.030) (W vs. CP, Fig. 2A). Analyses of the percent neoplasms with surface area greater than 10 mm2 (Fig. 2C) detected a significant dif- ference among the six intervention groups [F(5, 26) = 3.0335, P = 0.028]. Neoplastic burden: number and size of intestinal neoplasms A Tukey’s post hoc test deter- mined that the percent large (>10 mm2) neoplasm was higher in mice fed a Western diet without supplements (23.5 ± 9.8%) compared to the percent large neoplasms in mice fed a control diet with a propolis supplement (8.2 ± 6.5%, P = 0.043) (W vs. CP, Fig. 2C). Also, the percent large (>10 mm2) neoplasm was higher in mice fed a Western diet without supplements (23.5 ± 9.8%) compared to that in mice fed a Western diet with gamma- cyclodextrin (11.8 ± 8.1%, P = 0.034) (W vs. WD, Fig. 2C). Analyses of the percent neoplasms smaller than 5 mm2 also detected a significant difference among the six inter- vention groups [F(5, 26) = 2.6228, P = 0.048] (Fig. 2A). A Tukey’s post hoc test revealed that the percent small (< 5 mm2) neoplasms was lower in mice fed a Western diet without supplements (42.3 ± 10.6%) compared to Grade of the intestinal lesions Our mouse model is limited to the precarcinoma stage. However, even at this stage, the size of adenomas strongly correlates with the grade dysplasia; large adenomas usually display high-grade dysplasia, and vice versa, smaller in size adenomas exhibit lower grade of dysplasia [26]. Since adding gamma-cyclodextrin and propolis to control or Western diets decreased the number of large (>10 mm2) adenomas (Fig. 2C), we ascertained whether the lower number of large neoplasms corresponds to a lower grade of dysplasia. Low-grade dysplasia was noted where branch- ing/elongation of the crypts with reduced interglandular stroma occurred with low nuclear/cytoplasmic ratio and elongated and crowded cell nuclei (Fig. 3A). High-grade dysplasia was characterized by high nuclear/cytoplasmic ratio, severe reduction of the interglandular stroma, cyto- logic and architectural changes, nuclei with loss of polarity with respect to the basal membrane, irregular nuclear Figure 3. Histopathologic evaluation and grading of intestinal lesions detected in ApcMin/+ mice exposed to dietary interventions. (A) Mice on a Western diet with gamma-cyclodextrin exhibited a higher number of adenomas of small size with less than six adenomatous glands. These glands look alike the surrounding normal ones with uniform nuclei with little dysplasia (A, arrow). (B) High-grade dysplastic adenomas as the ones developed in mice on a Western diet displayed severe disorganization of the glandular architecture. Abnormal nuclear changes such as hyperchromatism, increase of the nuclear/cytoplasm ratio, irregular and pseudostratificated nuclei, conspicuous nucleoli, and mitosis were evident. (C) Adenomas developed in mice on a Western diet frequently displayed intraluminal necrosis (arrows, C). Histological sections stained with hematoxylin and eosin are shown (400 × original magnification). (D) Percent neoplasms with low (l) or high (h) grade of dysplasia were calculated based upon the analyses of the HE-stained paraffin-embedded mouse intestines within each intervention group (n = 5–6). A B C D A C B A D Figure 3. Histopathologic evaluation and grading of intestinal lesions detected in ApcMin/+ mice exposed to dietary interventions. (A) Mice on a Western diet with gamma-cyclodextrin exhibited a higher number of adenomas of small size with less than six adenomatous glands. These glands look alike the surrounding normal ones with uniform nuclei with little dysplasia (A, arrow). (B) High-grade dysplastic adenomas as the ones developed in mice on a Western diet displayed severe disorganization of the glandular architecture. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Grade of the intestinal lesions Abnormal nuclear changes such as hyperchromatism, increase of the nuclear/cytoplasm ratio, irregular and pseudostratificated nuclei, conspicuous nucleoli, and mitosis were evident. (C) Adenomas developed in mice on a Western diet frequently displayed intraluminal necrosis (arrows, C). Histological sections stained with hematoxylin and eosin are shown (400 × original magnification). (D) Percent neoplasms with low (l) or high (h) grade of dysplasia were calculated based upon the analyses of the HE-stained paraffin-embedded mouse intestines within each intervention group (n = 5–6). 2453 © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Y. Cho et al. Propolis and Cyclodextrin in Intestinal Neoplasia Y. Cho et al. mice on a Western diet supplemented with gamma- cyclodextrin. We ascertained whether this effect is due to the ability of gamma-cyclodextrin to modify survival pathways in the intestinal neoplastic tissues. Western blot analyses with normal and neoplastic tissue lysates did not reveal consistent changes in Akt/pAkt, Stat3/pStat3, Erk/ pErk, and c-Jun/pc-Jun levels (data not shown). Next, we tested whether the smaller size and lower number of neoplasms in mice fed a Western diet with gamma- cyclodextrin are due to higher apoptosis in the intestinal tissues. The levels of the proapoptotic protein Bax and the cleaved Caspase 3 (a hallmark of apoptosis) were increased in the tissues of mice on a Western diet with gamma-cyclodextrin (Fig. 4A). membranes, and aberrant chromatin pattern (Fig. 3B). Among all intervention groups, the only signs of focal necrosis were observed in the neoplastic tissues of mice fed a Western diet without supplements (Fig. 3C). Among the control diets, in the group on a diet without sup- plements, all lesions were classified as high dysplasia, 5% were graded as low dysplasia in the control diet with gamma-cyclodextrin, and 10% were graded as low dysplasia in the control diet with a propolis supplement (Fig. 3D). Among the Western diets, the mice on a diet with gamma- cyclodextrin exhibited 20% of low grade lesions; whereas, the mice on a Western diet alone and a Western diet with propolis exhibited 11% and 4% low-grade neoplasms, respectively (Fig. 3D). Another observation of our in vivo analyses was that mice on a Western diet with propolis did not gain body weight to the same extent as mice fed a Western diet or a Western diet with gamma-cyclodextrin (Fig. 1A). To explain this observation at the molecular level, we analyzed © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Discussion We have reported that compared to normal weight patients, cancer genomes of obese microsatellite stable CC patients exhibit lower numbers of mutations in driver genes [4]. To test whether this trend is observed in a murine model of intestinal cancer, we undertook exome sequencing of obese (Western diet-fed) versus normal weight (control diet-fed) mice (Fig. 1A). The analysis confirmed the ger- mline Apc mutation in nine out of the 10 analyzed Apc Miin/+ mice. The inability to detect the Apc germline mutation in one of the samples was due to low data size based upon low quality of the sample derived from a paraffin-embedded intestine. We analyzed only mutations defined as moderate or high impact. High-impact variants have disruptive impact in the protein (e.g., truncations, loss of function, causing nonsense mediated decay); whereas, moderate impact variants are nondisruptive vari- ants that might change protein effectiveness (e.g., missense variants, in-frame deletions, etc.). We did not include low-impact variants in our analyses, since these mutations are mostly harmless or unlikely to change protein behavior (e.g., synonymous variants). Since the sequencing was performed on genomic DNA extracted from whole small intestines, we could not determine how many and what type of genes were affected in a single neoplastic lesion. All mice had multiple neoplasms; therefore, the number and type of affected genes refer to the sum of neoplastic lesions in each mouse. There was a high variation in the affected gene types between normal weight and obese mice; including Apc mutations, mice on a control diet exhibited moderate or high-impact mutations in 51 types of genes; whereas, obese mice exhibited such mutations in 36 types of genes. Seven affected genes were common for the two groups of mice, in addition to Apc, these mutations affected Akap9, Bcl3, Cdh11, Hsp90ab1, Hoxa13, Obesity is a growing health care problem, as it is a risk factor for 10 types of cancer [5, 27], including CC; however, the mechanisms for this association are still unclear [4, 28]. This study is the first translational step that follows upon our in vitro findings on how propolis modulates CC cells [14, 16], and it was designed to determine whether propolis influences intestinal neoplas- tic development in vivo, in the context of normal weight and obesity. Molecular studies In mice on a control diet, on average 12.2 ± 3.0 genes were affected by moderate- and high-impact muta- tions; in obese mice, 10.4 ± 3.6 genes were affected by such mutations. The number of mutations per mouse was more than the number of affected genes, since there were several mutations detected in some genes. Although the difference was not statistically significant, normal weight mice exhibited more mutations than obese mice (30.6 ± 6.6 vs. 23.2 ± 9.0, respectively, P = 0.177, Student’s t-test). the major obesity-related signaling pathways in the epididy- mal white fat tissues of mice fed Western diets with or without supplements. Western blot analyses revealed lower pErk1/2 levels in the epididymal white fat tissues of mice fed a Western diet with propolis (Fig. 4B); however, there were no consistent changes in the levels of Akt/pAkt, Stat3/pStat3, and pc-Jun/c-Jun (data not shown). We have previously reported that propolis hyperactivates ERK sign- aling in CC cells in vitro [16]. To address this discrepancy, we analyzed the response to propolis in two normal cell lines: murine fibroblast 3T3-L1 and human epithelial intestinal CCD841CoN. As we have reported, propolis augmented the levels of pERK1/2 in colorectal carcinoma HCT-116 cells; however, pERK1/2 levels were decreased in the normal cells (Fig. 4C). Molecular studies Among the three intervention groups on a Western diet, the size of neoplasms was statistically different in the Figure 4. A. A representative western blot analysis of Bax and cleaved Caspase 3 in “normal” (C) with no discernible lesions tissues, and neoplastic (N) tissues of mice on a Western diet without supplements (W) or supplemented with gamma-cyclodextrin (WD) or propolis (WP). B. A representative western blot analysis of ERK/pERK levels in the epididymal tissues of mice exposed to the three Western diets (W-Western, WD-Western and gamma- cyclodextrin, WP-Western and propolis). C. A representative western blot analysis of Erk/pErk levels in four cell lines exposed to mock treatment (M) or 100 μg/mL propolis (P) for 16 h. Actin is a loading control. Densitometry was performed for four sets of sample analyses for Caspase 3 and three sets for each Bax, pErk1/2, and total Erk1/2. Asterisks designate the statistically significant differences in Actin-normalized background-corrected average pixel values. Figure 4. A. A representative western blot analysis of Bax and cleaved Caspase 3 in “normal” (C) with no discernible lesions tissues, and neoplastic (N) tissues of mice on a Western diet without supplements (W) or supplemented with gamma-cyclodextrin (WD) or propolis (WP). B. A representative western blot analysis of ERK/pERK levels in the epididymal tissues of mice exposed to the three Western diets (W-Western, WD-Western and gamma- cyclodextrin, WP-Western and propolis). C. A representative western blot analysis of Erk/pErk levels in four cell lines exposed to mock treatment (M) or 100 μg/mL propolis (P) for 16 h. Actin is a loading control. Densitometry was performed for four sets of sample analyses for Caspase 3 and three sets for each Bax, pErk1/2, and total Erk1/2. Asterisks designate the statistically significant differences in Actin-normalized background-corrected average pixel values. 2454 Propolis and Cyclodextrin in Intestinal Neoplasia Y. Cho et al. and Nsd1. There was also a great variation in the types of genes affected within each intervention group. In the control diet group, in addition to the Apc mutations, only mutations in Akap9, Cdh11, Hoxa13, and Hsp90ab1 occurred in more than one mouse. Among the obese mice, in addition to the Apc mutations, only mutations in Akap9, Cdh11, and Hsp90ab1 occurred in more than one mouse. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Propolis and Cyclodextrin in Intestinal Neoplasia A similar effect of propolis on body weight in C57BL/6N mice fed a high- fat diet has been reported [31]; the authors demonstrated that propolis downregulates the expression of genes asso- ciated with lipid metabolism. Our analyses of major sign- aling pathways in the epididymal white fat tissues revealed suppressed pErk1/2 levels in mice fed a Western diet with propolis. It is not clear, however, whether the cell signal- ing changes are a cause for the suppressed body weight gain, or a result of this suppression. The lack of excessive adipose tissue could decrease the levels of the signaling pathway since obesity-associated cytokines support ERK activity [32]. However, propolis may also directly suppress ERK activity, and therefore, inhibit the formation of adi- pose tissues. For example, the levels of ERK signaling have a profound effect on adipocyte development [33], loss of ERK signaling inhibition leads to obesity in mice [34], and erk1-/- mice resist obesity induced by high-fat diet due to decreased adipogenesis and increased post- prandial energy expenditure [33]. We ascertained that the effect of propolis on ERK activity differs among normal and neoplastic cells; however, future studies are required to understand why propolis hyperactivates ERK signaling in neoplastic cells with APC and KRAS mutations (e.g., HCT-116 cells) and suppresses the same pathway in nor- mal cells (CCD841CoN and 3T3-L1 cells) and adipose tissues of mice fed a high-fat diet. The faster neoplastic progression in obese mice fed a Western diet could be due to a higher mutation rate, increased number of stem cells and stem cell divisions, increased levels of adipokines, epigenetic changes, and changes in the intestinal microbiota [35]. Some of these possibilities were addressed through exome sequencing analyses of normal weight (control diet-fed) and obese (Western diet-fed) mice. Although the average size of the neoplastic lesions in obese mice was larger than this in normal weight mice, the number and type of genes affected by moderate- and high-impact mutations were lower in obese mice. This result supports our findings that compared to normal weight patients, cancer genomes of obese CC patients exhibit fewer somatic mutations, and correspondingly lower numbers of mutations in driver genes [4]. The sequencing analyses did not reveal con- sistent differences in the type of affected genes. Propolis and Cyclodextrin in Intestinal Neoplasia Y. Cho et al. Propolis and Cyclodextrin in Intestinal Neoplasia Y. Cho et al. on a cyclodextrin-supplemented diet could be due to the apoptotic effects of butyrate. Analyses of the neoplasm size among the six interven- tion groups revealed that the mice fed a Western diet without supplements exhibited statistically larger size neo- plasms when compared to any other dietary intervention. This result confirms previous reports that a Western diet accelerates neoplastic progression [22, 29]. However, unlike previous reports [23], under our experimental conditions, the mice on a Western diet did not exhibit statistically higher number of neoplastic lesions compared to the mice fed a control-type diet. When the lesions were stratified in three categories by size, it became apparent that the mice on a Western diet had developed the highest number of large size neoplasms. The same analysis of neoplastic lesions stratified by size revealed a common trend among the six intervention groups. Thus, the intervention groups on supplements exhibited higher numbers of small neo- plasms (<5 mm2) and lower numbers of large neoplasms (>10 mm2) in the context of the control or Western diet. There were no statistically significant differences among the numbers of medium size adenomas. A possible inter- pretation is that both supplements slowed the microad- enoma to adenoma progression. However, whereas the propolis supplement was more potent in the context of a control diet, in the context of a Western diet, gamma- cyclodextrin was more potent in reducing the neoplastic burden. Specifically, in obese state, compared to the Western diet alone, the supplementation with gamma- cyclodextrin decreased both the size and number of lesions in a statistically significant manner, and the addition of propolis counteracted these effects. The lack of statistically significant difference between the numbers of neoplasms in mice fed a control diet and those fed a Western diet may arise from the fact in ApcMin/+/J mice, each somatic cell already carries a gatekeeping Apc mutation; therefore, we cannot determine the full impact of obesity on neo- plastic initiation rate. Our analyses revealed that unlike mice fed a Western diet without supplements or a Western diet with gamma- cyclodextrin, the mice on a propolis-supplemented Western diet did not gain excessive body weight. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Discussion Among the groups of mice on control diets, the groups exposed to supplements, gamma-cyclodextrin and propolis, decreased neoplastic progression, as determined by the mean size and number of all lesions, as well as by the grade of dysplasia. A statistically significant decrease in the number of adenomas was detected in mice fed a control diet supplemented with propolis, compared to mice fed a control diet without supplements. Compared to the mice on a control diet without supplements, the mice fed a control diet with gamma-cyclodextrin also exhibited a decrease in lesion number and in grade of dysplasia; however, these differences were not statistically significant. Among the three intervention groups on a Western diet, the differences in the number of neoplasms were not statistically significant; whereas, the differences in neoplasm size were statistically significant. The group with the lowest number/size of adenomas and the highest per- centage of lesions with low grade of dysplasia was the group on a Western diet supplemented with gamma- cyclodextrin. The group on a Western diet with propolis also exhibited a statistically significant decrease in the size of neoplasms when compared to the group of mice on a Western-type diet without supplements. 2455 © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Propolis and Cyclodextrin in Intestinal Neoplasia the presence of multiple mutations even at the early adenoma stage has already been confirmed in humans [36, 37]. 4. Bordonaro, M., and D. Lazarova. 2015. Hypothesis: obesity is associated with a lower mutation threshold in colon cancer. J. Cancer 6:825–831. 5. New Liver Cancer Report, New Links Emerge: Coffee Is Protective, Obesity Increases Risk, Available at www.aicr. org/press/press-releases/2015/liver-cancer-report-new-links- emerge-coffee-protective-obesity-risk.html (accessed 9 July 2015). Currently, gamma-cyclodextrin is generally recognized as safe (GRAS) by the U.S. Food and Drug Administration. The substance is added at 1% to 20% to food items such as dry mixes for beverages, soups, dressings, gelatins, instant coffee, cereals, snacks, dairy products, spreads, and is used as a carrier for vitamins and dietary supplements. The average daily intake of gamma-cyclodextrin from its uses in foods has been estimated at 4.1 g / person / day; whereas, the estimate at the 90th percentile consumption level is 9.0 g / person / day [38]. Propolis supplements are also available on the market; however, the constituents of propolis vary widely depending upon the geographic location where the honeybee product is collected. The utilized by us Propolis with CycloPower Powder, a product that will be commercially available in the second half of 2016, is tested and certified for the levels of eight bioac- tive compounds including caffeic acid phenethyl ester, chrysin, galangin, and pinocembrin. Therefore, the transi- tion to human clinical studies that explore the effects of these supplements on intestinal neoplastic burden is pos- sible; however, prior to such studies, expanded studies with a larger number of mice per intervention group and in an additional mouse model of intestinal cancer are warranted. 6. Cummings, J. H., E. W. Pomare, W. J. Branch, C. P. E. Naylor, and G. T. MacFarlane. 1987. Short chain fatty acids in human large intestine, portal, hepatic, and venous blood. Gut 28:1221–1227. 7. Macfarlane, G. T., G. R. Gibson, and J. H. Cummings. 1992. Comparison of fermentation reactions in different regions of the human colon. J. Appl. Bacteriol. 72:57–64. 8. Macfarlane, S., and G. T. Macfarlane. 2003. Regulation of short-chain fatty acid production. Proc. Nutr. Soc. 62:67–72. 9. Hijova, E., and A. Chmelarova. 2007. Short chain fatty acids and colonic health. Bratisl. Lek. Listy 108:354–358. 10. Lazarova, D. L., M. Bordonaro, R. Carbone, and A. C. Sartorelli. 2004. Linear relationship between Wnt activity levels and apoptosis in colorectal carcinoma cells exposed to butyrate. Int. J. Propolis and Cyclodextrin in Intestinal Neoplasia Cancer 110:523–531. 11. Fearon, E. R., and B. Vogelstein. 1990. A genetic model for colorectal tumorigenesis. Cell 61:759–767. 12. Lazarova, D., A. Lee, T. Wong, B. Marian, C. Chiaro, C. Rainey, et al. 2014. Modulation of Wnt activity and cell physiology by butyrate in lt97 microadenoma cells. J. Cancer 5:203–213. Acknowledgments This study was funded by a research grant from Manuka Health New Zealand Ltd and research funds of TCMC. We thank Dr. A. Suddes at Manuka Health New Zealand Ltd for providing Propolis with Cyclopower for our studies, and Victor Mallory for his care of the mice. 13. Roediger, W. E. W. 1982. Utilization of nutrients by isolated epithelial cells of the rat colon. Gastroenterology 83:424–429. 14. Drago, E., M. Bordonaro, S. Lee, W. Atamna, and D. L. Lazarova. 2013. Propolis augments apoptosis induced by butyrate via targeting cell survival pathways. PLoS ONE 8:e73151. Conflict of interest 15. Watanabe, M. A., M. K. Amarante, B. J. Conti, and J. M. Sforcin. 2011. Cytotoxic constituents of propolis inducing anticancer effects: a review. J. Pharm. Pharmacol. 63:1378–1386. None declared. Propolis and Cyclodextrin in Intestinal Neoplasia This could be due to the fact that neoplastic development in our experiment was limited to precarcinoma stage; whereas, the human exome analyses were performed on stage III colon carcinoma data from TCGA. However, The histopathology evaluation of the neoplasms revealed that the larger size lesions in mice fed a Western diet correlated with faster progression; only the neoplasms in these mice exhibited focal necrosis. On the other hand, the lowest rate of neoplastic progression in mice fed a Western diet supplemented with gamma-cyclodextrin was evidenced not only by the lowest number and the smallest size of neoplastic lesions but also by the histological evalu- ation of the tissues. This finding concurs with the obser- vation of increased levels of apoptotic markers in the intestinal tissues of mice fed a Western diet with gamma- cyclodextrin. In the intestines, gamma-cyclodextrin could be fermented to short-chain fatty acids, including butyrate [30], and when applied in vitro, butyrate induces apoptosis in cells with WNT/beta-catenin signaling-activating muta- tions, such as the mutations in Apc gene [10]. Therefore, the increased apoptosis in the intestinal tissues of mice © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. 2456 Y. Cho et al. References 1. Bailey, C. E., C. Y. Hu, Y. N. You, B. K. Bednarski, M. A. Rodriguez-Bigas, J. M. Skibber, et al. 2015. Increasing disparities in the age-related incidences of colon and rectal cancers in the United States, 1975– 2010. JAMA Surg. 150:17–22. 16. Bordonaro, M., E. Drago, W. Atamna, and D. L. Lazarova. 2014. Comprehensive suppression of all apoptosis-induced proliferation pathways as a proposed approach to colorectal cancer prevention and therapy. PLoS ONE 9:e115068. 2. Ogden, C. L., M. D. Carroll, B. K. Kit, and K. M. Flegal. 2014. Prevalence of childhood and adult obesity in the United States, 2011-2012. JAMA 311:806–814. 17. Moser, A. R., H. C. Pitot, and W. F. Dove. 1990. A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. Science 247:322–324. 3. Boland, C. R., and A. Goel. 2010. Microsatellite instability in colorectal cancer. Gastroenterology 138:2073–2087. 18. Su, L. K., K. W. Kinzler, B. Vogelstein, A. C. Preisinger, A. R. Moser, C. Luongo, et al. 1992. Multiple intestinal neoplasia caused by a mutation in 2457 © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Y. Cho et al. Propolis and Cyclodextrin in Intestinal Neoplasia the murine homolog of the APC gene. Science 256:668–670. 30. Guevara Zubillaga, M. A. Nutritional evaluation of α-cyclodextrin using canine, hamster, and in vitro models, doctoral thesis, Available at www.ideals.illinois. edu/handle/2142/29583, (accessed July 20, 2015). 19. Shoemaker, A. R., K. A. Gould, C. Luongo, A. R. Moser, and W. F. Dove. 1997. Studies of neoplasia in the Min mouse. Biochim. Biophys. Acta 1332:F25–F48. 31. Koya-Miyata, S., N. Arai, A. Mizote, Y. Taniguchi, S. Ushio, K. Iwaki, et al. 2009. Propolis prevents diet- induced hyperlipidemia and mitigates weight gain in diet-induced obesity in mice. Biol. Pharm. Bull. 32:2022–2028. 20. Boivin, G. P., K. Washington, K. Yang, J. M. Ward, T. P. Pretlow, R. Russell, et al. 2003. Pathology of mouse models of intestinal cancer: consensus report and recommendations. Gastroenterology 124:762–777. 21. Tomasetti, C., and B. Vogelstein. 2015. Cancer etiology. variation in cancer risk among tissues can be explained by the number of stem cell divisions. Science 347:78–81. 32. McArdle, M. A., O. M. Finucane, R. M. Connaughton, A. M. McMorrow, and H. M. Roche. 2013. Mechanisms of obesity-induced inflammation and insulin resistance: insights into the emerging role of nutritional strategies. Front. Endocrinol. (Lausanne) 4:52. 22. Day, S. D., R. T. Enos, J. L. McClellan, J. L. References Steiner, K. T. Velázquez, and E. A. Murphy. 2013. Linking inflammation to tumorigenesis in a mouse model of high-fat-diet-enhanced colon cancer. Cytokine 64:454–462. 33. Bost, F., M. Aouadi, L. Caron, and B. Binétruy. 2005. The role of MAPKs in adipocyte differentiation and obesity. Biochimie 87:51–56. 34. Lee, S. J., P. T. Pfluger, J. Y. Kim, R. Nogueiras, A. Duran, G. Pagès, et al. 2010. A functional role for the p62-ERK1 axis in the control of energy homeostasis and adipogenesis. EMBO Rep. 11:226–232. 23. Baltgalvis, K. A., F. G. Berger, M. M. Peña, J. M. Davis, and J. A. Carson. 2009. The interaction of a high-fat diet and regular moderate intensity exercise on intestinal polyp development in Apc Min/+ mice. Cancer Prev. Res. 2:641–649. 35. Irrazábal, T., A. Belcheva, S. E. Girardin, A. Martin, and D. J. Philpott. 2014. The multifaceted role of the intestinal microbiota in colon cancer. Mol. Cell 54:309–320. 24. Moolenbeek, C., and E. J. Ruitenberg. 1981. The “Swiss roll”: a simple technique for histological studies of the rodent intestine. Lab. Anim. 15:57–59. 36. Zhou, D., L. Yang, L. Zheng, W. Ge, D. Li, Y. Zhang, et al. 2013. Exome capture sequencing of adenoma reveals genetic alterations in multiple cellular pathways at the early stage of colorectal tumorigenesis. PLoS ONE 8:e53310. 25. Lazarova, D. L., M. Bordonaro, and A. C. Sartorelli. 2001. Transcriptional regulation of the vitamin D3 receptor gene by ZEB. Cell Growth Differ. 12:319–326. 26. Valarini, S. B. M., V. T. Bortoli, N. S. Wassano, M. F. Pukanski, D. C. Maggi, and L. M. Bertollo. 2011. Correlation between location, size and histologic type of colorectal polyps at the presence of dysplasia and adenocarcinoma. J. Colopractology 31:241–247. 37. Nikolaev, S. I., S. K. Sotiriou, I. S. Pateras, F. Santoni, S. Sougioultzis, H. Edgren, et al. 2012. A single- nucleotide substitution mutator phenotype revealed by exome sequencing of human colon adenomas. Cancer Res. 72:6279–6289. 38. Munro, I. C., P. M. Newberne, V. R. Young, and A. Bär. 2004. Safety assessment of gamma-cyclodextrin. Regul. Toxicol. Pharmacol. 39(Suppl 1):S3–S13. 27. Bhurosy, T., and R. Jeewon. 2014. Overweight and obesity epidemic in developing countries: a problem with diet, physical activity, or socioeconomic status? Sci. World J. 2014:964236. 28. Zeng, H., and D. L. Lazarova. 2012. Obesity-related colon cancer: dietary factors and their mechanisms of anticancer action. Clin. Exp. Pharmacol. Physiol. 39:161–167. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Supporting Information Additional supporting information may be found in the online version of this article: Table S1. The number of neoplasms per mouse is the average of three independent counts performed by three investigators blinded for the treatment. Table S1. The number of neoplasms per mouse is the average of three independent counts performed by three investigators blinded for the treatment. 29. Fan, K., N. Kurihara, S. Abe, C. T. Ho, G. Ghai, and K. Yang. 2007. Chemopreventive effects of orange peel extract (OPE). I: OPE inhibits intestinal tumor growth in ApcMin/+ mice. J. Med. Food 10:11–17. Table S2. Exome sequencing and analysis. Table S2. Exome sequencing and analysis. 2458 © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.
https://openalex.org/W2808572295	https://www.nature.com/articles/s41467-018-04625-7.pdf	English	null	Enhanced ocean-atmosphere carbon partitioning via the carbonate counter pump during the last deglacial	Nature communications	2,018	cc-by	12,663	1 GEOPS, Universities of Paris Sud and Paris-Saclay, CNRS, 91405 Orsay, France. 2 LSCE/IPSL Laboratoire des Sciences du Climat et de l’Environnement, CEA- CNRS-UVSQ, 91198 Gif-sur-Yvette, France. 3 Aix Marseille Univ, CNRS, IRD, INRA, Coll France, CEREGE, Aix-en-Provence, France. 4 Institute of Geological Sciences and Oeschger Center for Climate Change Research, University of Bern, 3012 Bern, Switzerland. Correspondence and requests for materials should be addressed to S.D.-A. (email: stephanie.duchamp@u-psud.fr) ARTICLE During the last ice age, the deep circuit was probably more isolated from the atmosphere due to increased sea-ice coverage6 and increased stratiﬁcation7,8. Meanwhile the STP, fueled by enhanced deposition of Fe-bearing dust that favored a more complete macronutrient uptake by phytoplankton, might have been more efﬁcient3,9,10. Therefore, reduced rates of vertical exchange combined with a more efﬁcient STP promoted the storage of CO2 in the ocean abyss, thereby contributing to lower atmospheric CO211,12. g Here, we explore the deglacial calciﬁcation pattern of cocco- lithophores, particularly the Noëlaerhabdaceae family, in a well- dated sediment core14, retrieved from the Chilean margin (MD07-3088; 46.1°S, 75.7°W, 1536 m water depth), at the tran- sition of AAIW and the Paciﬁc deep water (PDW)2 (Fig. 1 and Methods). This site is located within the Antarctic Circumpolar Current (ACC) under the direct inﬂuence of the northward transport of nutrient rich-SSW2 and relatively far from the main dust sources today, but likely also in the past22. As such, this core represents a suitable archive to document the leverage SO upwelling and associated changes in surface water chemistry might have exerted on SAZ coccolithophore productivity. We complement these observations with planktonic foraminifera records to provide a complete representation related to the integrated PIC accumulation and compare these data with reconstructed past changes in the buried POC:PIC ratio, sug- gested to reﬂect the C-rain ratio. As such, we qualitatively document the relative contribution of STP and CCP to the deglacial rise in atmospheric CO2 at a decadal timescale. Our study highlights that changes in biological export production in high southern latitudes operated synergistically with physical mechanisms thereby enhancing the transfer of carbon from the ocean to the atmosphere during the last deglacial. The reinvi- goration of the SO vertical mixing contributed to the release of respired carbon and regenerated nutrients to the SAZ that pro- moted planktonic calciﬁcation, thereby increasing the CCP, and concomitantly weakening the BCP. The collapse of vertical Δ14C and δ13C gradients in the SO suggests that more vigorous deep and mid-depth circulations would have reconnected the deep carbon reservoir to the surface during Heinrich Stadial 1 (HS1, 17.5–14.7 kyr BP) and the Younger Dryas (YD, 12.8–11.5 kyr BP), thus promoting the transfer of respired carbon to the surface ocean and the atmo- sphere13,14. ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04625-7 T he Southern Ocean (SO) is a key part of the global over- turning circulation as it witnesses the outcropping of carbon- and nutrient-rich Circumpolar Deep Water (CDW) in the Antarctic Zone (AZ), as a result of wind-driven (Ekman) upwelling1. A portion of these upwelled waters ﬂow southwards to feed the abyssal circuit to form Antarctic Bottom Water (AABW), while the remainder ﬂows to the North to feed Subantarctic Surface Waters (SSW) that mix with warm sub- tropical waters to form Antarctic Intermediate Waters (AAIW) and Subantarctic Mode Waters (SAMW), the mid-depth oceanic circuit that supplies nutrients to the low-latitude thermocline1,2. The STP, that is the net downward ﬂux of carbon associated with organic matter export, counteracts carbon evasion to the atmo- sphere, as a fraction of the photosynthetic biomass that ﬁxes dissolved inorganic carbon (DIC) in the sunlit ocean is exported and remineralized in the ocean interior. However, owing to iron (Fe) limitation on phytoplankton growth3,4, the pre-industrial SO STP was unable to fully compensate the CO2 outgassing and this area represented one of the main oceanic sources of natural CO2 to the atmosphere5. T contribution of the CCP, despite its fundamental role in the marine carbon cycle24. The production of particulate inorganic carbon (PIC) by calcifying plankton in the sunlit ocean and its eventual dissolution in the subsurface engenders a surface-to- depth alkalinity gradient, causing CO2 to be released back to the atmosphere24. As such, the CCP acts to partially offset the air-sea partitioning of carbon associated with the STP. p g Therefore, it is crucial to overcome these important short- comings by focusing on the production pattern of speciﬁc phy- toplankton groups from a broad range of locations within the SAZ and evaluating their impact on the carbon cycle. Cocco- lithophores are relevant for addressing this outstanding issue. This single-celled phytoplankton group has unique effects on the oceanic carbon cycle in that it uses DIC for both photosynthesis and calciﬁcation and accounts for a signiﬁcant proportion of the global marine export production25. Hence, coccolithophores contribute to both the STP and CCP and impact the strength of the BCP since modiﬁcations in calciﬁcation patterns related to changes in surface water chemistry modulate the POC:PIC ratio of sinking biogenic material with consequences for the air-sea partitioning of carbon26–28. ARTICLE These observations have been corroborated by sedi- mentary geochemical data suggesting that surface waters of the Subantarctic Atlantic and the Eastern Equatorial Paciﬁc (EEP), which derive from water upwelled in the SO, became a substantial source of CO2 during the last deglaciation15. Surprisingly, little is known about deglacial export production and STP patterns in the Subantarctic Zone (SAZ) outside the Atlantic sector. The weakening of the global STP efﬁciency has been documented during the early deglaciation (∼17.5– ∼14 kyr BP) using a global compilation of δ15N measurements and oxy- genation proxies16. Millennial-scale export production recon- structions based on organic (TOC, biomarkers) and inorganic (SiO2, CaCO3, bioBa) proxies9,10,17–21 provide insights into oceanic nutrient dynamics, ventilation changes, and export of particulate carbon across the deglaciation. However, these records remain sparse and mainly come from South Atlantic cores located downwind of Patagonia, the most prominent dust source region to the SO22. While they provide valuable case studies for testing the “Fe-hypothesis”3, these records are not necessarily repre- sentative of the entire SO. Particularly, the comparison with South Paciﬁc and Indian Oceans is not straightforward because hydrothermal and sedimentary Fe sources may play an important role in modulating productivity in these sectors23. \| DOI: 10.1038/s41467-018-04625-7\| www.nature.com/naturecommunications Enhanced ocean-atmosphere carbon partitioning via the carbonate counter pump during the last deglacial Stéphanie Duchamp-Alphonse1, Giuseppe Siani1, Elisabeth Michel2, Luc Beaufort3, Yves Gally3 & Samuel L. Jaccard 4 Stéphanie Duchamp-Alphonse1, Giuseppe Siani1, Elisabeth Michel2, Luc Beaufort3, Yves Gally3 & Samuel L. Jaccard 4 Stéphanie Duchamp-Alphonse1, Giuseppe Siani1, Elisabeth Michel2, Luc Beaufort3, Yves Gally3 & Samuel L. Jaccard 4 Several synergistic mechanisms were likely involved in the last deglacial atmospheric pCO2 rise. Leading hypotheses invoke a release of deep-ocean carbon through enhanced convec- tion in the Southern Ocean (SO) and concomitant decreased efﬁciency of the global soft- tissue pump (STP). However, the temporal evolution of both the STP and the carbonate counter pump (CCP) remains unclear, thus preventing the evaluation of their contributions to the pCO2 rise. Here we present sedimentary coccolith records combined with export pro- duction reconstructions from the Subantarctic Paciﬁc to document the leverage the SO biological carbon pump (BCP) has imposed on deglacial pCO2. Our data suggest a weakening of BCP during the phases of carbon outgassing, due in part to an increased CCP along with higher surface ocean fertility and elevated [CO2aq]. We propose that reduced BCP efﬁciency combined with enhanced SO ventilation played a major role in propelling the Earth out of the last ice age. 1 GEOPS, Universities of Paris Sud and Paris-Saclay, CNRS, 91405 Orsay, France. 2 LSCE/IPSL Laboratoire des Sciences du Climat et de l’Environnement, CEA- CNRS-UVSQ, 91198 Gif-sur-Yvette, France. 3 Aix Marseille Univ, CNRS, IRD, INRA, Coll France, CEREGE, Aix-en-Provence, France. 4 Institute of Geological Sciences and Oeschger Center for Climate Change Research, University of Bern, 3012 Bern, Switzerland. Correspondence and requests for materials should be addressed to S.D.-A. (email: stephanie.duchamp@u-psud.fr) \| DOI: 10.1038/s41467-018-04625-7\| www.nature.com/naturecommunications 1 NATURE COMMUNICATIONS\| (2018) 9:2396 Results and discussion leptoporus that have an afﬁnity for meso- to eutrophic conditions in the SE Paciﬁc38 (Fig. 2c, d). Besides, the structure of this record closely resembles those of ΔΔ14C and Δδ13C reconstructions14 that reﬂect increased rates of vertical mixing, thus supporting the notion that upper ocean productivity at site MD07-3088 was directly modulated by regional ocean circulation changes. Fur- thermore, the downcore coccolithophore export productivity record mimics coccolith abundances in the northern part of the and 12.8–11.1 ka BP), with highest abundances reported for HS1 and the YD (Fig. 2a). Within Subantarctic ecosystems located away from the main dust sources, phytoplankton growth is typically modulated by the supply of dissolved phosphate (PO43–) and nitrate (NO3–) via SO upwelling today32 but also in the past33,34. This is particularly the case for the Chilean margin, where river runoff35 and glacier erosion36 provide additional sources of micronutrients alleviating the limitation Fe is imposing on phytoplankton growth in the open SO34. Besides, macro- nutrient concentrations within the SSW were not perennially high37. Therefore, the most reasonable explanation is that coc- colith abundance primarily reﬂects the phytoplankton response to macronutrient supply. This assumption is corroborated by con- comitant higher abundances of H. carteri and to a lesser degree C. leptoporus that have an afﬁnity for meso- to eutrophic conditions in the SE Paciﬁc38 (Fig. 2c, d). Besides, the structure of this record closely resembles those of ΔΔ14C and Δδ13C reconstructions14 that reﬂect increased rates of vertical mixing, thus supporting the notion that upper ocean productivity at site MD07-3088 was directly modulated by regional ocean circulation changes. Fur- thermore, the downcore coccolithophore export productivity record mimics coccolith abundances in the northern part of the pp y The second outstanding feature of the micropalaeontological records relates to the ~50% distinct increases in the mean Noëlaerhabdaceae coccolith mass (Fig. 2i), coincident with decreasing local ΔΔ14C and Δ δ13C values (Fig. 2f, g)14, as well as with increasing surface water δ11B-based ΔpCO2 values in the EEP15 (Fig. 2j; Methods; Supplementary Fig. 1). Numerous studies related coccolith mass to the degree of coccolith calciﬁcation in Pleistocene and recent sediments39–41. Indeed, variations in coccolith mass can, under some circumstances, reﬂect variations in the thickness of an individual coccolith, and therefore relates to the calcite quota of a given cell27. Results and discussion Increase in SSW fertility and [CO2aq] during SO upwelling. In Fig. 2, we present coccolith abundance and mass (Fig. 2a, c, d, i) from sediment core MD07-3088 (Methods) that are compared to ΔΔ14C and Δδ13C reconstructions from the same core14 (Fig. 2f, g), coccolith abundances from the SE Paciﬁc29 (ODP 1233, Fig. 2b; Methods and Supplementary Fig. 1), biogenic opal ﬂux from the South Atlantic30 (TN057-13-4PC, Fig. 2h), δ11B-based ΔpCO2 from the EEP15 (ODP 1238, Fig. 2j; Methods; Supple- mentary Fig. 1), and local summer SST estimates14,31 (Fig. 2e), to better understand their relationships to productivity and ocean circulation patterns on a regional scale (Figs. 1 and 2). Cocco- lithophore calciﬁcation patterns are not affected by diagenetic alteration, with no evidence of coccolith dissolution (Methods and Supplementary Fig. 2). Noëlaerhabdaceae coccolith abun- dance features three distinct deglacial peaks (18.6–18.2, 17.7–16.2, Besides, these reconstructions neither document the con- tribution of speciﬁc phytoplankton groups, nor their respective leverage on the BCP strength. The sedimentary burial of biogenic opal and carbonate, often thought to reﬂect diatom and cocco- lithophore export production respectively, could be affected by changes in zooplankton abundance, grazing pressure, and/or changes in the degree of remineralization processes. More importantly, these reconstructions largely ignore the relative NATURE COMMUNICATIONS\| (2018) 9:2396 \| DOI: 10.1038/s41467-018-04625-7\| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04625-7 60°W 0° 28 26 26 26 24 24 22 22 20 20 18 18 18 16 16 14 14 12 12 10 10 10 8 8 6 4 2 0 Ocean Data View / DIVA 0 5 10 15 20 25 80°S 60°S 40°S 20°S 120°W 90°W 30°W Temperature [°C] @ Depth [m]=0 80°S 60°S 20°S 120°W 90°W 60°W 30°W 0° 1.75 1.75 1.75 1.75 1.5 1.5 1.5 1.5 1.5 1.25 1. Results and discussion The Polar Frontal and Subantarctic Zones are the regions between the PF and SAF, and between the SAF and the STF respectively. a Solid white circles symbolize the geographic location of sediment cores MD07-3088 (46.1°S, 75.7°W, 1536 m), TN057-13- 4PC30 (53.2°S, 5.1°E, 2850 m), and sites ODP 123329 (41.0°S, 74.4°W, 838 m) and 123815 (1.5°S, 82.5°W, 2203 m). The solid white square highlights the West Antarctic Ice Sheet Divide ice core56 (WDC, 79.5°S, 112.1°W, 1766 m above sea level) Fig. 1 Location of sites and modern ocean surface temperature and phosphate concentrations. Temperature (a) and phosphate (b) concentration ﬁelds are plotted with the Ocean Data View (ODV) software68 with WOA0969. Solid black lines represent the: Subtropical (STF), Subantarctic (SAF), Polar (PF) and Subantarctic Circumpolar Current (SACCF) Fronts70. The Polar Frontal and Subantarctic Zones are the regions between the PF and SAF, and between the SAF and the STF respectively. a Solid white circles symbolize the geographic location of sediment cores MD07-3088 (46.1°S, 75.7°W, 1536 m), TN057-13- 4PC30 (53.2°S, 5.1°E, 2850 m), and sites ODP 123329 (41.0°S, 74.4°W, 838 m) and 123815 (1.5°S, 82.5°W, 2203 m). The solid white square highlights the West Antarctic Ice Sheet Divide ice core56 (WDC, 79.5°S, 112.1°W, 1766 m above sea level) SAZ29 as well as opal ﬂuxes in the AZ at sites inﬂuenced by similar processes30. This suggests that the inferred changes in productivity were neither limited to a speciﬁc phytoplankton group nor a speciﬁc area, but rather highlight a regional sensi- tivity of phytoplankton growth to ocean circulation and nutrient supply from below. and 12.8–11.1 ka BP), with highest abundances reported for HS1 and the YD (Fig. 2a). Within Subantarctic ecosystems located away from the main dust sources, phytoplankton growth is typically modulated by the supply of dissolved phosphate (PO43–) and nitrate (NO3–) via SO upwelling today32 but also in the past33,34. This is particularly the case for the Chilean margin, where river runoff35 and glacier erosion36 provide additional sources of micronutrients alleviating the limitation Fe is imposing on phytoplankton growth in the open SO34. Besides, macro- nutrient concentrations within the SSW were not perennially high37. Therefore, the most reasonable explanation is that coc- colith abundance primarily reﬂects the phytoplankton response to macronutrient supply. This assumption is corroborated by con- comitant higher abundances of H. carteri and to a lesser degree C. Results and discussion 1.25 1 1 0.75 .75 0.75 0.5 0.5 .5 0.5 0.25 0.25 0.25 0.25 Ocean Data View / DIVA 0 0.5 1 1.5 2 40°S Phosphate [μmol/l] @ Depth [m]=0 STF SAF PF SACCF MD07-3088 TN057-13-4PC ODP 1238 WDC ODP 1233 a b 60°W 0° 28 26 26 26 24 24 22 22 20 20 18 18 18 16 16 14 14 12 12 10 10 10 8 8 6 4 2 0 Ocean Data View / DIVA 0 5 10 15 20 25 80°S 60°S 40°S 20°S 120°W 90°W 30°W Temperature [°C] @ Depth [m]=0 80°S 60°S 20°S 120°W 90°W 60°W 30°W 0° 1.75 1.75 1.75 1.75 1.5 1.5 1.5 1.5 1.5 1.25 1. 1.25 1 1 0.75 .75 0.75 0.5 0.5 .5 0.5 0.25 0.25 0.25 0.25 Ocean Data View / DIVA 0 0.5 1 1.5 2 40°S Phosphate [μmol/l] @ Depth [m]=0 STF SAF PF SACCF MD07-3088 TN057-13-4PC ODP 1238 WDC ODP 1233 a b cation of sites and modern ocean surface temperature and phosphate concentrations. Temperature (a) and phosphate (b) concentration ﬁelds are with the Ocean Data View (ODV) software68 with WOA0969. Solid black lines represent the: Subtropical (STF), Subantarctic (SAF), Polar (PF) and rctic Circumpolar Current (SACCF) Fronts70. The Polar Frontal and Subantarctic Zones are the regions between the PF and SAF, and between the the STF respectively. a Solid white circles symbolize the geographic location of sediment cores MD07-3088 (46.1°S, 75.7°W, 1536 m), TN057-13- 53.2°S, 5.1°E, 2850 m), and sites ODP 123329 (41.0°S, 74.4°W, 838 m) and 123815 (1.5°S, 82.5°W, 2203 m). The solid white square highlights the ntarctic Ice Sheet Divide ice core56 (WDC, 79.5°S, 112.1°W, 1766 m above sea level) 60°W 0° 28 26 26 26 24 24 22 22 20 20 18 18 18 16 16 14 14 12 12 10 10 10 8 8 6 4 2 0 Ocean Data View / DIVA 0 5 10 15 20 25 80°S 60°S 40°S 20°S 120°W 90°W 30°W Temperature [°C] @ Depth [m]=0 STF SAF PF SACCF MD07-3088 TN057-13-4PC ODP 1238 WDC ODP 1233 a a a Phosphate [μmol/l] @ Depth [m]=0 Fig. 1 Location of sites and modern ocean surface temperature and phosphate concentrations. Temperature (a) and phosphate (b) concentration ﬁelds are plotted with the Ocean Data View (ODV) software68 with WOA0969. Solid black lines represent the: Subtropical (STF), Subantarctic (SAF), Polar (PF) and Subantarctic Circumpolar Current (SACCF) Fronts70. \| DOI: 10.1038/s41467-018-04625-7\| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04625-7 between coccolith mass and the size normalized thickness index “SN”27,44 (Methods) (r2 = 0.73) as well as the coccolith aspect ratio “ARL”28 (Methods) (r2 = 0.63) (Fig. 3), which both document the degree of Noëlaerhabdaceae coccolith calciﬁcation. g Batch culture experiments provided conﬂicting responses related to coccolithophore calciﬁcation rates with studies reporting both depressed45,46 or elevated47 calciﬁcation under high [CO2aq]. However, in the geological record—when general selection for growth strategies28 and phenotypic plasticity naturally occurred and regulated the carbon acquisition within the cell48—more heavily calciﬁed coccoliths were systematically associated with increased atmospheric pCO227,28,49,50. Indeed, high pCO2 favors intracellular competitive reallocation of dissolved bicarbonate (HCO3−) from the site of photosynthesis (chloroplast) to the site of calciﬁcation (coccolith vesicle)48. Such processes may be relevant in coastal ecosystems such as the Chilean margin, where highly calciﬁed E. huxleyi morphotypes thrive under low-pH sea-surface conditions39. Therefore, it is most likely that more heavily calciﬁed coccoliths reported from site MD07-3088 at times when the upwelling of CO2-rich deep waters increased, reﬂect increasing surface [CO2aq] and highlight the equatorward advection of SSW together with AAIW/SAMW, thus supporting the mechanisms behind upper-ocean acidiﬁca- tion previously documented in the SAZ and the EEP during these time intervals15. The 50% increase in Noëlaerhabdaceae mass observed both during HS1 and YD occurred at times of ~2–3 µmol/L rises in SSW [CO2aq] in the SAZ15, which is in the exact same order of magnitude than the coccolith mass and [CO2aq] increases (50%, ~3 µmol/L respectively) previously documented for the penultimate deglaciation within the south- ernmost Paciﬁc28. We cannot exclude that increased tempera- tures during major upwelling phases (∼+4 °C during HS1, and ∼+3 °C during YD14,31), may have partially contributed in promoting coccolith production and calciﬁcation28,51, but warming was not always in phase with the coccolith patterns, and may thus be of secondary importance (Fig. 2). f b Reduced BCP due to increased CCP during SO upwelling. Sedimentary bromine (Br) and calcium (Ca) have been shown to be associated with biogenic organic carbon and carbonate in marine sediment records, respectively. Br is primarily associated with marine organic matter52 as it is directly involved in the marine biological cycle and in non-biological reactions in the water-column that implicate marine organic matter53. Calcium may be of detrital, biogenic or diagenetic origin. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04625-7 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04625-7 he Noëlaerhabdaceae family43 (Supplementary Fig. 2). W hat changes in coccolith mass typically reﬂect chang HOLOCENE LG LGIT Number of Noëlaerhabdaceae (×107 /g of sediment 0 5 10 15 20 25 30 Summer SST (°C) Martinez-Boti et al., 2015 Siani et al., 2013 Age cal kyrs BP 4 6 8 10 12 14 16 18 20 300 600 900 1200 1500 4 6 8 10 12 14 16 18 20 a f 0 1 2 3 4 5 6 7 8 Opal flux (g/cm2/kyr2) Anderson et al., 2009 h 1.5 2.0 2.5 3.0 3.5 Noëlaerhabdaceae mass (pg) i j –1.5 –0.9 –0.3 0.3 0.9 1.5 Siani et al., 2013 –40 –20 0 20 40 60 80 100 [CO2aq] conditions + – 0 3 6 9 12 15 0 5 10 15 20 Number of C. leptoporus (×106 /g of sediment) d e 0 10 20 30 40 50 Number of coccoliths number (×108 /g of sediment Saavedra-Pelittero et al., 2011 SO ventilation + – b c Fertility conditions – + 5 10 15 20 g ACR HS1 YD Oceanic Δ pCO2 Δδ 13C (‰) 14C B-P age (years) Number of H. carteri (×106 /g of sediment) Fig. 2 Southern Ocean productivity and circulation patterns during the last deglaciation. a, c, d Number of Noëlaerhabdaceae, H. carteri and C. leptoporus at site MD07-3088 (/g of sediment), with an error bar of ±1%. b Number of coccoliths at site ODP 1233 (/g of sediment)29. e Summer sea surface temperatures (SST, °C) using the Modern Analogue Technique14, 31. f 14C age difference between paired benthic and planktonic foraminifera (14C B-P)14. g Δ δ13C = δ13CG. bulloides – δ13CC. wuellerstorﬁ14. h Opal ﬂuxes at TN057-13-4PC30. i Noëlaerhabdaceae mass (pg) at site MD07-3088 with an error bar of ±3%. j Surface ocean ΔpCO2 reconstruction for ODP 123815. LG and LGIT are for Late Glacial and Last Glacial-Interglacial Transition, respectively. Smoothed curves (thick lines of a–e, g, and i) use a three-point moving average. Yellow shading marks periods of enhanced deep-water ventilation and resumption of SO upwelling during the last deglaciation, in conjunction with higher ocean surface fertility, [CO2aq], and sometimes increased SST conditions Fig. 2 Southern Ocean productivity and circulation patterns during the last deglaciation. a, c, d Number of Noëlaerhabdaceae, H. carteri and C. Results and discussion However, changes in coccolith mass may additionally be driven by changes in coccolith area associated to changes in cell sizes27,28,42 and as such coccolith mass must be size-normalized to represent the degree of coccolith calciﬁcation27,28. At site MD07-3088, we focus on changes that affected all narrowly restricted size classes within \| DOI: 10.1038/s41467-018-04625-7\| www.nature.com/naturecommunications NATURE COMMUNICATIONS\| (2018) 9:2396 3 ARTICLE \| DOI: 10.1038/s41467-018-04625-7\| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04625-7 At site MD07- 3088, the organic origin of Br is clear as testiﬁed by the overall excellent linear correlation with discrete TOC measurements (r2 = 0.87; Supplementary Fig. 3) as well as with the δ13C and C/ the Noëlaerhabdaceae family43 (Supplementary Fig. 2). We ﬁnd that changes in coccolith mass typically reﬂect changes in coccolith thickness (Fig. 3), suggesting that they document changes in the coccosphere calcite quota. Our assumptions are further corroborated by the clear positive relationship that exists NATURE COMMUNICATIONS\| (2018) 9:2396 \| DOI: 10.1038/s41467-018-04625-7\| www.nature.com/naturecommunications 4 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04625-7 ARTICLE Age cal kyrs BP 4 6 8 10 12 14 16 18 20 1.5 2.0 2.5 3.0 3.5 Noëlaerhabdaceae mass (pg) e g h Coccolith aspect ratio (ARL, dimensionless) 0.06 0.09 0.12 0.15 Coccolith aspect ratio (ARL, dimensionless) y = 0.0459x + 0.015 2.0 2.5 3.0 3.5 0.06 0.09 0.12 0.15 r 2= 0.001 r 2= 0.63 r 2=0.73 r 2= 0.35 y = 0.0539x + 8.4723 y=0.0794x + 0.0804 y =0.1226x – 0.0269 SN coccolith thickness (μm) 0.20 0.25 0.30 0.35 0.40 6 8 10 12 0.20 0.25 0.30 0.35 0.40 Thickness (μm) Thickness (μm) 2.0 2.5 3.0 3.5 0.20 0.25 0.30 0.35 0.40 Noëlaerhabdaceae mass (pg) 2.0 2.5 3.0 3.5 6 8 10 12 Noëlaerhabdaceae mass (pg) SN coccolith thickness (μm) 2.0 2.5 3.0 3.5 0.20 0.25 0.30 0.35 0.40 HOLOCENE LG LGIT 6 8 10 12 14 16 18 20 4 f i b a d c Area (μm2) Area (μm2) r 2= 0.001 y = 0.0539x + 8.4723 2.0 2.5 3.0 3.5 6 8 10 12 a Area (μm2) r 2= 0.35 y=0.0794x + 0.0804 Thickness (μm) 2.0 2.5 3.0 3.5 0.20 0.25 0.30 0.35 0.40 b Fig. 3 Noëlaerhabdaceae coccolith morphometrics, and their response to excepted [CO2aq] at site MD07-3088 over the last deglaciation. a–d Relationships between coccolith mass (pg) and coccolith area (a), thickness (b), as well as coccolith aspect ratio (ARL)28 (c) and SN thickness27 (d) (Methods section). e–i Coccolith morphometric changes during the deglaciation: e mass (pg), f area (μm2), g thickness (µm), h–i SN27 and ARL28 indices (Methods section). Coccolith mass show no relationship with coccolith area, but clear positive relationships with size normalized coccolith mass indices ARL and SN which indicates that the mass of coccoliths typically increases in proportion with their thickness. Therefore, changes in coccolith mass document changes in the degree of coccolith calciﬁcation (i.e. PIC/POC ratio). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04625-7 Obviously, the Noëlaerhabdaceae coccolith mass increases observed during enhanced SO upwelling associated with higher sea surface fertility conditions (yellow shading), document increased coccosphere calcite quota in response to SSW [CO2aq] rises during HS1 and YD b r 2=0.73 y =0.1226x – 0.0269 Noëlaerhabdaceae mass (pg) SN coccolith thickness (μm) 2.0 2.5 3.0 3.5 0.20 0.25 0.30 0.35 0.40 d Coccolith aspect ratio (ARL, dimensionless) y = 0.0459x + 0.015 2.0 2.5 3.0 3.5 0.06 0.09 0.12 0.15 r 2= 0.63 Noëlaerhabdaceae mass (pg) c d d c c free terrigenous material sourced from metamorphic and plutonic rocks of the Andes35 that contain negligible amounts of calcium- bearing minerals (plagioclases), while Ca and Ca/Ti report excellent linear correlations with CaCO3 (r2 = 0.75 and 0.73, respectively; Supplementary Fig. 3) thus excluding any Ca of terrigenous origin. As for other sites from the Chilean margin located well above the lysocline55 (Methods), it is thus reasonable to consider that Ca is primarily associated with biogenic carbo- nates, and more particularly planktonic calciﬁers. Indeed, bulk- sediment carbonate content ﬂuctuations mimic those observed for the CaCO3 produced by coccolithophores and planktonic foraminifera (Fig. 4, Supplementary Fig. 3; Methods). We suggest that, in our core, POC:PIC ratio changes in the sediments likely reﬂect changes in the C rain ratio (POC:PIC) (Methods). Therefore, the downcore Br/Ca ratio is used to provide an esti- mate of the strength of the STP relative to the CCP, which serves as a robust tool to reconstruct decadal changes in the BCP efﬁ- ciency (Fig. 4d). This record depicts highest values during the Late Glacial (19.6–17.7 ka), the Antarctic Cold Reversal (ACR; 14.1–12.0 ka) and the Mid Holocene (8.6–3.0 ka). On the other hand, signiﬁcant reductions in Br/Ca are observed at times intense SO upwelling prevailed, particularly during HS1 and the YD. These transient declines are mainly driven by changes in CaCO3 export during HS1 since the TOC record is characterized by increasing values during this speciﬁc time interval (Supple- mentary Fig. 3), while both increasing CaCO3 and relatively high yet decreasing TOC values might be associated during the YD. Furthermore, they match the pronounced peaks in surface ocean fertility (Fig. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04625-7 On the other hand, signiﬁcant reductions in Br/Ca are observed at times intense SO upwelling prevailed, particularly during HS1 and the YD. These transient declines are mainly driven by changes in CaCO3 export during HS1 since the TOC record is characterized by increasing values during this speciﬁc time interval (Supple- mentary Fig. 3), while both increasing CaCO3 and relatively high yet decreasing TOC values might be associated during the YD. Furthermore, they match the pronounced peaks in surface ocean fertility (Fig. 2a–d) as well as coccolith and planktonic for- aminifera abundances and masses that are well expressed by 3- fold to 10-fold and up to 20-fold increases in the overall amount of burial CaCO3 produced by coccolithophores and planktonic foraminifera respectively, and coincide with prominent rises in atmospheric pCO256 (Fig. 4). This increase in the CCP, associated with rising macronutrient availability and thus, enhanced fertility in the SAZ, is comparable to the 6-10 folds increase in deep-ocean PIC ﬂuxes previously documented for naturally iron-fertilized sites from the Polar Frontal Zone compared to non-fertilized ones24. In both cases, increased fertility is linked to a rise of about one order of magnitude of the PIC ﬂux, thus conﬁrming the important role of CCP in mediating the reduction of deep-ocean CO2 storage24. The impact of changing POC:PIC ratio (1/ρ) on pCO2 is shown in Fig. 5 for HS1 and the ACR, in cases for which 10 to 50% of the exported POC is preserved in the sediments (Methods). In all cases, primary production decreased sea surface [CO2aq], as 1/ρ is higher than the critical value of 0.54 for which the CCP would completely counteracts the STP. Nevertheless, N values of bulk organic matter (ranging from −24.9 to 20.05% and from 7.6 to 11.5, respectively) that cluster well within the typical ranges for well-preserved marine organic components54 (Supplementary Fig. 4). \| DOI: 10.1038/s41467-018-04625-7\| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04625-7 2a–d) as well as coccolith and planktonic for- aminifera abundances and masses that are well expressed by 3- fold to 10-fold and up to 20-fold increases in the overall amount of burial CaCO3 produced by coccolithophores and planktonic foraminifera respectively, and coincide with prominent rises in atmospheric pCO256 (Fig. 4). This increase in the CCP, associated with rising macronutrient availability and thus, enhanced fertility in the SAZ, is comparable to the 6-10 folds increase in deep-ocean PIC ﬂuxes previously documented for naturally iron-fertilized sites from the Polar Frontal Zone compared to non-fertilized ones24. In both cases, increased fertility is linked to a rise of about one order of magnitude of the PIC ﬂux, thus conﬁrming the important role of CCP in mediating the reduction of deep-ocean CO2 storage24. The impact of changing POC:PIC ratio (1/ρ) on pCO2 is shown in Fig. 5 for HS1 and the ACR, in cases for which 10 to 50% of the exported POC is preserved in the sediments (Methods). In all cases, primary production decreased sea surface [CO2aq], as 1/ρ is higher than the critical value of 0.54 for which the CCP would completely counteracts the STP. Nevertheless, free terrigenous material sourced from metamorphic and plutonic rocks of the Andes35 that contain negligible amounts of calcium- bearing minerals (plagioclases), while Ca and Ca/Ti report excellent linear correlations with CaCO3 (r2 = 0.75 and 0.73, respectively; Supplementary Fig. 3) thus excluding any Ca of terrigenous origin. As for other sites from the Chilean margin located well above the lysocline55 (Methods), it is thus reasonable to consider that Ca is primarily associated with biogenic carbo- nates, and more particularly planktonic calciﬁers. Indeed, bulk- sediment carbonate content ﬂuctuations mimic those observed for the CaCO3 produced by coccolithophores and planktonic foraminifera (Fig. 4, Supplementary Fig. 3; Methods). We suggest that, in our core, POC:PIC ratio changes in the sediments likely reﬂect changes in the C rain ratio (POC:PIC) (Methods). Therefore, the downcore Br/Ca ratio is used to provide an esti- mate of the strength of the STP relative to the CCP, which serves as a robust tool to reconstruct decadal changes in the BCP efﬁ- ciency (Fig. 4d). This record depicts highest values during the Late Glacial (19.6–17.7 ka), the Antarctic Cold Reversal (ACR; 14.1–12.0 ka) and the Mid Holocene (8.6–3.0 ka). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04625-7 The solid black arrows represent the effect of biogenic export production in the case of photosynthesis and calciﬁcation only. The dashed (or dot-dashed) black and grey arrows illustrate the inﬂuence of the biological pump (and particularly the CCP) during the HS1 relative to the ACR, i.e., when 1/ρ decreases by a factor of 1.8, assuming that 10 (or 50%) of the exported POC has been preserved within the sediment, i.e., under two probable export production conditions at site MD07-3088 6 NATURE COMMUNICATIONS\| (2018) 9:2396 \| DOI: 10.1038/s41467-018-04625-7\| www.nature.com/naturecommunications p y p p caused a decrease in surface-ocean alkalinity (ALK) (or in other deglaciat Increased effectivel ratio in t CO2 from Our st changes HOLOCENE LG ACR –1.5 –0.9 –0.3 0.3 0.9 1.5 Δδ 13C (‰) LGIT Marcott et al., 2014 Siani et al., 2013 0 0.5 1.0 1.5 2.0 2.5 3.0 Coccolith calcite mass (mg/g of sediment) 0.00 0.01 0.02 0.03 Br/Ca Atmospheric pCO2 (p.p.m.v.) 150 200 250 300 HS1 YD 4 6 8 10 12 14 16 18 20 4 6 8 10 12 14 16 18 20 b d CCP strength + – BCP efficiency + – 0.0 0.2 0.4 0.6 0.8 Calcite mass of Noëlaerhabdaceae (mg/g of sediment) a 0 2 4 6 8 10 12 Planktonic foraminifera calcite mass (mg/g of sediment) c e f SO ventilation + – Age cal kyrs BP Fig. 4 Sub efﬁciency the last de foraminife errors bar POC/PIC Suppleme f Atmosph a–c, e and and LGIT respective (associate [CO2aq]) d and subdu pCO2 2300 2280 2260 Alkalinity (μeq/kg) 2240 2220 19 Fig. 5 Inﬂ partial pre (1/ρ). Soli salinity (3 kg) and si effect of b calciﬁcatio illustrate t during the 1.8, assum within the at site MD 6 NATURE COMMUNICATIONS\| (2018)9:2396 \| D p Our study reveals the intrinsic link between BCP strength and changes in Southern Ocean circulation in coordinating the f 2300 2280 2260 Alkalinity (μeq/kg) 2240 2220 1950 1975 2000 2025 2050 ΣCO2 (μmol/kg) 1/ = POC/PIC 1/ = 13.5 1/ = 7.7 1/ = 2.7 1/ = 1.5 1/ = 0.54 200 220 240 260 280 300 320 340 380 Photosynthesis Calcification Fig. 5 Inﬂuence of soft tissue and carbonate counter pumps on the CO2 partial pressure of surface waters, as a function of the POC/PIC rain ratio (1/ρ). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04625-7 Solid black line isocontours represent pCO2 (μatm) for constant salinity (34‰), temperature (14 °C), and depleted phosphate (0.5 µmol/ kg) and silicate (3 µmol/kg) contents. The solid black arrows represent the effect of biogenic export production in the case of photosynthesis and calciﬁcation only. The dashed (or dot-dashed) black and grey arrows illustrate the inﬂuence of the biological pump (and particularly the CCP) during the HS1 relative to the ACR, i.e., when 1/ρ decreases by a factor of 1.8, assuming that 10 (or 50%) of the exported POC has been preserved within the sediment, i.e., under two probable export production conditions at site MD07-3088 2300 2280 2260 Alkalinity (μeq/kg) 2240 2220 1950 1975 2000 2025 2050 ΣCO2 (μmol/kg) 1/ = POC/PIC 1/ = 13.5 1/ = 7.7 1/ = 2.7 1/ = 1.5 1/ = 0.54 200 220 240 260 280 300 320 340 380 Photosynthesis Calcification d Fig. 5 Inﬂuence of soft tissue and carbonate counter pumps on the CO2 partial pressure of surface waters, as a function of the POC/PIC rain ratio (1/ρ). Solid black line isocontours represent pCO2 (μatm) for constant salinity (34‰), temperature (14 °C), and depleted phosphate (0.5 µmol/ kg) and silicate (3 µmol/kg) contents. The solid black arrows represent the effect of biogenic export production in the case of photosynthesis and calciﬁcation only. The dashed (or dot-dashed) black and grey arrows illustrate the inﬂuence of the biological pump (and particularly the CCP) during the HS1 relative to the ACR, i.e., when 1/ρ decreases by a factor of 1.8, assuming that 10 (or 50%) of the exported POC has been preserved within the sediment, i.e., under two probable export production conditions at site MD07-3088 Fig. 4 Subantarctic carbonate counter pump strength and biological pump efﬁciency coupled with circulation pattern and atmospheric pCO2 during the last deglaciation. a–c Noëlaerhabdacea, coccolith and planktonic foraminifera calcite masses (mg/g of sediment) at site MD07-3088, with errors bars of ±3% and ±20%, respectively. d Br/Ca ratio as an indicator of POC/PIC rain ratio and thus biological carbon pump efﬁciency (see Supplementary Fig. 3). e Δ δ13C = δ13CG. bulloides – δ13CC. wuellerstorﬁ14. f Atmospheric pCO2 from WDC56. Smoothed curves (thick lines of a–c, e and d) use three and eleven-point moving averages respectively. LG and LGIT are for Late Glacial and Last Glacial-Interglacial Transition, respectively. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04625-7 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04625-7 during HS1, the efﬁciency of the BCP was reduced compared to the ACR owing to lower POC:PIC ratios. The SAZ thus became a net source of CO2 during HS1 and the YD15, due to enhanced SO upwelling of aged, CO2-enriched deep waters14 and a con- comitant weakening of the BCP. Indeed, the increase in calcite production by coccolithophores and planktonic foraminifera caused a decrease in surface-ocean alkalinity (ALK) (or in other words, an increase in pCO2aq), thus promoting the net outgassing of carbon from the ocean interior to the atmosphere (Fig. 5). In such a scenario, the CCP would have contributed to weaken the marine BCP, with signiﬁcant impact on atmospheric pCO2 since the very beginning of the last deglaciation. The STP was not efﬁcient enough to offset the carbon release during the last deglaciation, in part due to the contribution of the CCP. Increased planktonic calciﬁcation in the SAZ has the potential to effectively amount to a reduction of the overall POC:PIC rain ratio in the SO (Fig. 5), with signiﬁcant impacts on the net ﬂux of CO2 from the ocean to the atmosphere24. production by coccolithophores and planktonic foraminifera caused a decrease in surface-ocean alkalinity (ALK) (or in other HOLOCENE LG ACR –1.5 –0.9 –0.3 0.3 0.9 1.5 Δδ 13C (‰) LGIT Marcott et al., 2014 Siani et al., 2013 0 0.5 1.0 1.5 2.0 2.5 3.0 Coccolith calcite mass (mg/g of sediment) 0.00 0.01 0.02 0.03 Br/Ca Atmospheric pCO2 (p.p.m.v.) 150 200 250 300 HS1 YD 4 6 8 10 12 14 16 18 20 4 6 8 10 12 14 16 18 20 b d CCP strength + – BCP efficiency + – 0.0 0.2 0.4 0.6 0.8 Calcite mass of Noëlaerhabdaceae (mg/g of sediment) a 0 2 4 6 8 10 12 Planktonic foraminifera calcite mass (mg/g of sediment) c e f SO ventilation + – Age cal kyrs BP 6 NATURE COMMUNICATIONS\| (2018)9 2396 production by coccolithophores and planktonic foraminifera caused a decrease in surface-ocean alkalinity (ALK) (or in other efﬁcient enough to offset the carbon release during the last deglaciation, in part due to the contribution of the CCP. Increased planktonic calciﬁcation in the SAZ has the potential to effectively amount to a reduction of the overall POC:PIC rain ratio in the SO (Fig. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04625-7 5), with signiﬁcant impacts on the net ﬂux of CO2 from the ocean to the atmosphere24. Our study reveals the intrinsic link between BCP strength and changes in Southern Ocean circulation in coordinating the HOLOCENE LG ACR –1.5 –0.9 –0.3 0.3 0.9 1.5 Δδ 13C (‰) LGIT Marcott et al., 2014 Siani et al., 2013 0 0.5 1.0 1.5 2.0 2.5 3.0 Coccolith calcite mass (mg/g of sediment) 0.00 0.01 0.02 0.03 Br/Ca Atmospheric pCO2 (p.p.m.v.) 150 200 250 300 HS1 YD 4 6 8 10 12 14 16 18 20 4 6 8 10 12 14 16 18 20 b d CCP strength + – BCP efficiency + – 0.0 0.2 0.4 0.6 0.8 Calcite mass of Noëlaerhabdaceae (mg/g of sediment) a 0 2 4 6 8 10 12 Planktonic foraminifera calcite mass (mg/g of sediment) c e f SO ventilation + – Age cal kyrs BP Fig. 4 Subantarctic carbonate counter pump strength and biological pump efﬁciency coupled with circulation pattern and atmospheric pCO2 during the last deglaciation. a–c Noëlaerhabdacea, coccolith and planktonic foraminifera calcite masses (mg/g of sediment) at site MD07-3088, with errors bars of ±3% and ±20%, respectively. d Br/Ca ratio as an indicator of POC/PIC rain ratio and thus biological carbon pump efﬁciency (see Supplementary Fig. 3). e Δ δ13C = δ13CG. bulloides – δ13CC. wuellerstorﬁ14. f Atmospheric pCO2 from WDC56. Smoothed curves (thick lines of a–c, e and d) use three and eleven-point moving averages respectively. LG and LGIT are for Late Glacial and Last Glacial-Interglacial Transition, respectively. Yellow shading marks periods of reinvigorated SO upwelling (associated with enhanced sea surface fertility conditions and higher [CO2aq]) during the last deglaciation, in conjunction to higher CCP strength and subdued biological pump efﬁciency, at times of increased atmospheric pCO2 2300 2280 2260 Alkalinity (μeq/kg) 2240 2220 1950 1975 2000 2025 2050 ΣCO2 (μmol/kg) 1/ = POC/PIC 1/ = 13.5 1/ = 7.7 1/ = 2.7 1/ = 1.5 1/ = 0.54 200 220 240 260 280 300 320 340 380 Photosynthesis Calcification Fig. 5 Inﬂuence of soft tissue and carbonate counter pumps on the CO2 partial pressure of surface waters, as a function of the POC/PIC rain ratio (1/ρ). Solid black line isocontours represent pCO2 (μatm) for constant salinity (34‰), temperature (14 °C), and depleted phosphate (0.5 µmol/ kg) and silicate (3 µmol/kg) contents. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04625-7 The sediment mostly receives carbonate- Age cal kyrs BP 4 6 8 10 12 14 16 18 20 1.5 2.0 2.5 3.0 3.5 Noëlaerhabdaceae mass (pg) e g h Coccolith aspect ratio (ARL, dimensionless) 0.06 0.09 0.12 0.15 2.0 2.5 3.0 3.5 SN coccolith thickness (μm) 0.20 0.25 0.30 0.35 0.40 6 8 10 12 0.20 0.25 0.30 0.35 0.40 Thickness (μm) 2.0 2.5 3.0 3.5 HOLOCENE LG LGIT 6 8 10 12 14 16 18 20 4 f i Area (μm2) Age cal kyrs BP 4 6 8 10 12 14 16 18 20 1.5 2.0 2.5 3.0 3.5 Noëlaerhabdaceae mass (pg) e g h Coccolith aspect ratio (ARL, dimensionless) 0.06 0.09 0.12 0.15 SN coccolith thickness (μm) 0.20 0.25 0.30 0.35 0.40 6 8 10 12 0.20 0.25 0.30 0.35 0.40 Thickness (μm) HOLOCENE LG LGIT 6 8 10 12 14 16 18 20 4 f i Area (μm2) g h Coccolith aspect ratio (ARL, dimensionless) 0.06 0.09 0.12 0.15 SN coccolith thickness (μm) 0.20 0.25 0.30 0.35 0.40 10 12 0.20 0.25 0.30 0.35 0.40 Thickness (μm) HOLOCENE LG LGIT 6 8 10 12 14 16 18 20 4 f i a (μm2) N values of bulk organic matter (ranging from −24.9 to 20.05% and from 7.6 to 11.5, respectively) that cluster well within the typical ranges for well-preserved marine organic components54 (Supplementary Fig. 4). The sediment mostly receives carbonate- N values of bulk organic matter (ranging from −24.9 to 20.05% and from 7.6 to 11.5, respectively) that cluster well within the typical ranges for well-preserved marine organic components54 (Supplementary Fig. 4). The sediment mostly receives carbonate- NATURE COMMUNICATIONS\| (2018) 9:2396 5 \| DOI: 10.1038/s41467-018-04625-7\| www.nature.com/naturecommunications ARTICLE \| DOI: 10.1038/s41467-018-04625-7\| www.nature.com/naturecommunications \| DOI: 10.1038/s41467-018-04625-7\| www.nature.com/naturecommunications Methods M t i l We obtained the Size Normalized Thickness index SN27,44 that considers coccolith thickness related to cell surface area as follow: SNthickness ðμmÞ ¼ ML CL ð Þ ´ S ½ þ CT ð2Þ ð2Þ Where ML is the mean coccolith length over the whole time serie, CL is the length of coccolith X in Sample A, S is the slope of the linear regression between coccolith length and coccolith thickness for all coccolith in Sample A, and CT is the original thickness of coccolith X in Sample A (i.e., coccolith mass/coccolith area ratio). We calculated the lateral cross-sectional aspect ratio ARL28 that considers coccolith thickness related to cell volume as follow: Where ML is the mean coccolith length over the whole time serie, CL is the length of coccolith X in Sample A, S is the slope of the linear regression between coccolith length and coccolith thickness for all coccolith in Sample A, and CT is the original thickness of coccolith X in Sample A (i.e., coccolith mass/coccolith area ratio). Age models of core MD07-3088 and ODP cores 1238 and 1233. The MD07- 3088 age model has been determined using SH1314 as the 14C Southern Hemi- sphere calibration curve57. In order to compare our micropaleontological and geochemical records with ODP sites 1233 and 1238 located in the South- Eastern Paciﬁc (SEP, 41.0°S, 74.4°W) and the Eastern Equatorial Paciﬁc (EEP, 1.5° S, 82.5°W) respectively, we established a common age model for these cores to test temporal phasing since the late glacial period. For ODP site 1233, we use the recently updated age model of ref. 58 and based on the reservoir 14C age estimates by ref. 14. The age model of ODP site 1238 is based on 10 AMS 14C dates obtained on planktonic foraminifera Neogloboquadrina dutertrei using a constant local sea- surface reservoir 14C age (RS) correction (ΔR = 72 ± 35 yr) based on previous regional estimates15. However, this approach did not consider the possible advection of old subsurface waters in particular during the deglaciation as sug- gested by previous studies at local and regional scales59,60. Since no independent RS estimates are available in literature for the EEP, other methods must be considered in order to obtain a robust common stratigraphic framework. Methods M t i l Hence, we ﬁrst compared the planktonic foraminifera δ13C records of the two cores versus con- ventional 14C age14,15, the δ13C record for ODP 1238 representing sub-surface record as it is measured on N. dutertrei (Supplementary Fig. 1). In general, the ﬁrst order (and most of the second order) changes in planktonic δ13C are recorded in both cores. These results also match co-existing benthic–planktonic foraminifera (B-P) 14C and δ13C differences in core MD07-3088, indicating variations in oceanic ventilation (see ref. 14 for extensive discussion) versus conventional 14C age. Similarly, the ventilation changes expressed in term of upwelling increases observed in core MD07-3088 were coeval with changes in surface ocean carbon content in the EEP15 (Supplementary Fig. 1). Through these comparisons, it is clear that enhanced mixing (between ~15 and ~13.1 14C ka, and between ~12 and ~10.5 14C ka) was characterized by a lower difference between planktonic and benthic carbon isotope signatures, and are globally synchronous with oceanic pCO2 changes. This ﬁnding supports the hypothesis that the planktonic foraminiferal records correspond to the water masses with the same history (SAW and SAMW) presenting similar radiocarbon contents. This allows us to deduce that EEP and SEP were characterized by similar reservoir 14C age changes at least since the last deglaciation. p ( ) We calculated the lateral cross-sectional aspect ratio ARL28 that considers coccolith thickness related to cell volume as follow: ARL dimensionless ð Þ ¼ TL=pAL ð3Þ ð3Þ where TL and AL are the thickness and the area of coccolith X in Sample A respectively. TL = ML/AL, i.e. coccolith mass (ML)/coccolith area (AL). Coccolith area and mass values as well as SNthickness and ARL values obtained herein are in the same order of magnitude than published data using similar birefringence-based methods27,28,39. where TL and AL are the thickness and the area of coccolith X in Sample A respectively. TL = ML/AL, i.e. coccolith mass (ML)/coccolith area (AL). Coccolith area and mass values as well as SNthickness and ARL values obtained herein are in the same order of magnitude than published data using similar birefringence-based methods27,28,39. Coccolith taxonomy and preservation. More than 96% of the assemblages were composed of ﬁve species: Emiliania huxleyi, Gephyrocapsa muellerae, Gephyrocapsa oceanica, Calcidiscus leptoporus, and Helicosphaera carteri. Methods M t i l As for modern settings, Emiliania and Gephyrocapsa, that constitute the Noëlaerhabdaceae family, repre- sent the most prominent genera (from 81 to 97% of the assemblages) and reﬂect the main patterns of the total coccoliths. For that reason, but also because smallest Emiliania and Gephyrocapsa from the SE Paciﬁc present a wide range of mor- photypes62 that are not easily classiﬁed under light microscope, we mainly con- sidered the Noëlaerhabdaceae family instead of Emiliania and Gephyrocapsa species. Besides, species assignations within the Noëlaerhabdaceae family are pri- marily based on size43, and all narrowly restricted size classes of Noëlaerhabdaceae present the same main patterns (Supplementary Fig. 2). Indeed, generally, <3 μm Noëlaerhabdaceae represent E. huxleyi type C and small Gephyrocapsa; 3–4 µm Noëlaerhabdaceae are associated to E. hyxleyi type B/C and G. muellerae; and >4 µm Noëlaerhabdaceae document E. huxleyi type A and B and G. oceanica patterns43,62. This study gathers speciﬁc morphological parameters of exactly 152,809 coccoliths that appear to reﬂect primary biomineralization features. The core MD07-3088 has been retrieved well above the lysocline. It is mainly made of homogenous ﬁne-grained material that, together with high sedimentation rates (∼300 cm/kyr during the Last Glacial and ∼60 cm/kyr during the deglaciation and the Holocene14), prevent post-depositional ﬂuid circulations. Besides, dissolution processes trigger a strong differential preservation of coccoliths keeping resistant specimens and losing delicate ones. The most delicate morphotypes belong to the Noëlaerhabdaceae family that represent the main coccolith of the assemblage. The smallest Noëlaerhabdaceae (<3 μm, i.e., mainly E. huxleyi type C and small This study gathers speciﬁc morphological parameters of exactly 152,809 coccoliths that appear to reﬂect primary biomineralization features. The core MD07-3088 has been retrieved well above the lysocline. It is mainly made of homogenous ﬁne-grained material that, together with high sedimentation rates (∼300 cm/kyr during the Last Glacial and ∼60 cm/kyr during the deglaciation and the Holocene14), prevent post-depositional ﬂuid circulations. Besides, dissolution processes trigger a strong differential preservation of coccoliths keeping resistant specimens and losing delicate ones. The most delicate morphotypes belong to the Noëlaerhabdaceae family that represent the main coccolith of the assemblage. The smallest Noëlaerhabdaceae (<3 μm, i.e., mainly E. huxleyi type C and small Gephyrocapsa) depict the same exact pattern as the larger ones, with higher masses when the oceanic carbon reservoir is reconnected to the surface waters and bring CO2-rich waters into the photic zone (Supplementary Fig. ARTICLE ARTICLE partitioning of carbon between the ocean interior and the atmosphere during the last glacial termination. As such, much more attention should be brought to the response of calcifying plankton at other sites within the Southern Ocean but also in low latitudes, to better quantify their relative contribution in the past global pCO2 budget. where A is the number of coccoliths per gram of sediment; Nc is the number of counted coccoliths (between 505 and 3900); Sf is the surface of the ﬂat beaker (3117 mm2) in which suspended sediments (and coccoliths) settle; No is the number of view ﬁelds (165); So is the surface of a view ﬁelds (0.01 mm2) and Ws is the weight of sediment that settled in the ﬂat beaker (between 0.018 and 0.043 mg). For each sample, abundance and morphometric analyses (length, width, area, mass) of individual coccoliths were automatically obtained with an average of 1591 coccoliths per sample, by the SYRACO software using automated microscope (Leica DM6000B). SYRACO performs pattern recognition under cross-polarized light using artiﬁcial neural networks61. It detects and classiﬁes most of the coccoliths present in the samples throughout the time series (mainly represented by Emiliania huxleyi, Gephyrocapsa muellerae, Gephyrocapsa oceanica, Calcidiscus leptoporus, and Helicosphaera carteri). Coccolith mass were directly deduced based on a quasi-linear relationship that exists between their brightness (birefringence in grey scale colors) and their thickness under cross-polarized light. Because this method applies on coccoliths thinner than 1.55 μm that exhibit grey scale colors61, we interpret only thickness and mass measurements for Noëlaerhabdaceae coccoliths. Indeed, their abundance and morphometric parameters show standard error of ±1% and ±3% in each sample respectively. Morphometric analyses for C. leptoporus and H. carteri that display third-order interference colors (and thus increasing standard errors), are only presented within the Supplementary Information (Supplementary Fig. 2). \| DOI: 10.1038/s41467-018-04625-7\| www.nature.com/naturecommunications Methods M t i l Material and site description. The CALYPSO core MD07-3088 was retrieved during the IMAGES PACHIDERME (MD 159) expedition by the French R/V Marion Dufresne off Southern Chile (46°04 S; 075°41 W), at a water depth of 1536 m, i.e., well above the modern lysocline (around 3700 m)55 (Fig. 1). The site is bathed by the upper layer of southward ﬂowing Paciﬁc Deep Water (PDW), at the boundary with northward ﬂowing AAIW2. The surface waters above the site are on the direct northward path of the SSW that is fed by the DIC- and nutrient-rich surface waters of the Antarctic Zone. These Antarctic and Subantarctic surface waters represent the source for AAIW/SAMW (i.e., intermediate depth waters), and have been hypothesized to be a major conduit through which high-latitude ocean changes are transmitted to the lower latitudes2. Core MD07-3088 has the advantage of being located within the Subantarctic Zone, well to the south and well to the north of the Tropical and Polar Frontal Zones, respectively, and has probably not been affected by potential shift of the Subtropical Front (STF) or the Sub- antarctic Front (SAF) in the past. The southern tip of Chile is the only continental mass intercepting the westerly winds within this latitude range, generating a zone of high precipitations that result in high ﬂuvial sediment supplies to the South Paciﬁc Ocean. Since any signiﬁcant sediment reworking is precluded at site MD07- 308814, the extremely high sedimentation rates recorded at site MD07-3088 (∼300 cm/kyr during the Last Glacial and ∼60 cm/kyr during the deglaciation and the Holocene14) provide a rare opportunity to study productivity patterns of the Subantarctic Zone with decennial to centennial resolution during the last glacial termination. Since coccolith mass are not independent of coccolith size, we calculated size- normalized thickness indices for all the Noëlaerhabdaceae coccoliths within each sample to verify that changes in coccolith mass represent changes in calciﬁcation, according to the two equations that exist so far27,28,44 (Fig. 3). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04625-7 Yellow shading marks periods of reinvigorated SO upwelling (associated with enhanced sea surface fertility conditions and higher [CO2aq]) during the last deglaciation, in conjunction to higher CCP strength and subdued biological pump efﬁciency, at times of increased atmospheric pCO2 6 NATURE COMMUNICATIONS\| (2018) 9:2396 NATURE COMMUNICATIONS\| (2018) 9:2396 \| DOI: 10.1038/s41467-018-04625-7\| www.nature.com/naturecommunications 6 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04625-7 Methods M t i l 2), while such conditions could have favored the dissolution of coccoliths in the water column. At last, the three main increases observed during the deglaciation in the mean Noëlaerhabdaceae coccolith mass, would not be biased by diagenetic overgrowth that would also affect C. leptoporus and to a lesser degree H. carteri, that generally depict however, reducing coccolith masses during these time intervals (Supplementary Fig. 2). Indeed, diagenetic processes (dissolution or overgrowth) Coccolith slides and morphometric measurements (SYRACO). Slides of 80 samples were prepared at GEOPS laboratory. Brieﬂy, ~0.03 g of sediment was diluted in 28 mL Luchon water (pH = 8, bicarbonate = 78.1 mg per liter, total dissolved solid = 83 g per liter) within a ﬂat beaker, and settled on a 12 × 12 mm coverslip for 4 h 30 min. The coverslip was then oven-dried at 70 °C, and mounted on slides with NOA74. This technique ensures a homogenous distribution of coccoliths and allows quantifying the amount of material per gram of sediment61 as follow: ( μ y y yp Gephyrocapsa) depict the same exact pattern as the larger ones, with higher masses when the oceanic carbon reservoir is reconnected to the surface waters and bring CO2-rich waters into the photic zone (Supplementary Fig. 2), while such conditions could have favored the dissolution of coccoliths in the water column. At last, the three main increases observed during the deglaciation in the mean Noëlaerhabdaceae coccolith mass, would not be biased by diagenetic overgrowth that would also affect C. leptoporus and to a lesser degree H. carteri, that generally depict however, reducing coccolith masses during these time intervals (Supplementary Fig 2) Indeed diagenetic processes (dissolution or overgrowth) A ¼ Nc ´ Sf ð Þ= No ´ So ´ Ws ð Þ ð1Þ p g g Supplementary Fig. 2). Indeed, diagenetic processes (dissolution or overgrowth) NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04625-7 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04625-7 would simultaneously impact all coccolith morphotypes, without any discrimination between morphotypes. would simultaneously impact all coccolith morphotypes, without any discrimination between morphotypes. high sedimentation rates, and homogeneous ﬁne-grained lithology, lead us to assume that post-depositional remineralization processes associated to (O2-rich) ﬂuid circulations within the sediments must be of secondary importance. More- over, it has been shown that it is in fact the oxygen exposure time that determines organic carbon degradation (i.e., ref. 63), and based on the considerations above, we infer that labile organic compounds must have been buried rapidly, minimizing the potential for selective alteration. There is no doubt that remineralization processes that occurred within the water column (and particularly the twilight zone), altered the downward ﬂux of POC, and thus the efﬁciency of carbon sequestration. However, the latitudinal distribution pattern of POC in surface sediments along the Chilean margin55 reﬂects satellite-derived surface-ocean chlorophyll concentra- tions64, which indicates that sedimentary TOC concentrations primarily reﬂect OC export rather than selective degradation processes within the water column. Besides, the high-latitude, iron-fertilized, near-shore ecosystem that characterize site MD07-3088, seems to be the perfect candidate to promote the sinking of organic matter to the deep seaﬂoor65–67. Therefore, in order to consider a wide range of POC transfer efﬁciencies65–67, we have tested the impact of BCP for HS1 and ACR, in cases where 10 to 50% of the exported POC is preserved within the sediments. Figure 5 indicates the inﬂuence of 1/ρ on seawater carbonate chemistry for cases ranging from photosynthetic processes to calciﬁcation processes only (solid black arrows) and for 10% to 50% of the TOC exported ﬂux preserved in core MD07-3088 sediments for the HS1 and ACR periods. Foraminifera abundance and mass data. Planktonic foraminifera assemblages were determined at the LSCE (Laboratoire des Sciences du Climat et de l’Envir- onnement) counting at least 300 specimens per sample. From three different depths (570, 950, and 990 cm), we weighted 30 individuals from the most abundant species (Neogloboquadrina pachyderma (sinistral and dextral coilings), Globigerina bulloides, Globorotalia inﬂata, Turborotalita quinqueloba and Globigerinita gluti- nata) for different sizes (>450 µm, 315–450 µm, 250–315 µm, 150–250 µm) to determine their mean weight. For this core, we obtained mean weights of: 7 ± 2 µg for N. pachyderma, T. quinqueloba, and G. glutinata, 18 ± 3 µg for G. bulloides, and 19 ± 5 µg for G. inﬂata. References 1. Marshall, J. & Speer, K. Closure of the meridional overturning circulation through Southern Ocean upwelling. Nat. Geosci. 5, 171–180 (2012). 2. Strub, P. T., Mesias, J. M., Montecino, V., Ruttlant, J., Salinas, S. Coastal ocean circulation off Western South America in The Global Coastal Ocean, Regional Studies and Syntheses, 273–315 (Wiley, Hoboken, NJ, 1998). 3. Martin, J. H., Gordon, R. M. & Fitzwater, S. E. Iron in Antarctic waters. Nature 345, 156–158 (1990). 4. Pollard, R. T. et al. Southern Ocean dee-water carbon export enhanced by natural iron fertilization. Nature 457, 577–580 (2009). 5. Morrison, A., Frölicher, T., Sarmiento, J., Upwelling in the Southern Ocean. Physics Today 68, 27–32 (2015). Total CaCO3 and organic carbon analyses. Total CaCO3 was determined at GEOPS laboratory using the vacuum-gasometric technique with a precision better than ±2%. 100 mg (±5) of crushed-dried sediments react with a few milliliters of HCl 6 N in a hermetic reaction chamber (22.4 cm3) that is connected to a man- ometer MANO MEX2-420 that measures the amount of outgassed CO2. The system is calibrated so that 100 mg of CaCO3 (100%) trigger a pressure rise to 1 bar. y y 6. Ferrari, R., et al. Antartic sea ice control on ocean circulation in present and glacial climates. Proc. Natl Acad. Sci. USA 111, 8753–8758 (2014). 7. Adkins, J. F., McIntyre, K. & Schrag, D. P. The salinity, temperature, and δ18O of the glacial deep ocean. Science 298, 1769–1773 (2002). 8. Watson, A. J., Vallis, G. K., Nikurashin, M. Southern Ocean buoyancy forcing of ventilation and glacial atmospheric CO2. Nat. Geosci. 8, 861–864 (2015). Total organic carbon and nitrogen contents together with organic matter δ13C analyses were obtained at the LSCE, using an Elementary Analyzer (Flash EA 1112) and the online continuous EA coupled with an Isotopic Ratio Mass Spectrometer (Finigan Delta + XP). The results are expressed in % C, % N, and in δ13C per mL (‰) against the international standard V-PDB (Vienna Pee Dee Belemnite). Error margin is deﬁned according to the source linearity checked for each run based on internal home-standard (ΔC < 0.03% and Δδ13C < 0.2‰). A aliquot of <250 μm of dry sediment is softly leached with ultra-pure HCl 6 N to remove carbonate and dry at 50 °C. The samples were then crushed in a pre-combusted glass mortar for homogenization prior to carbon, nitrogen content and δ13C analyses. 9. Kumar, N. References et al. Increased biological productivity and export production in the glacial Southern Ocean. Nature 378, 675–680 (1995). 10. Martinez-Garcia, A. et al. Iron fertilization of the subantarctic ocean during the last Ice Age. Science 343, 1347–1350 (2014). 11. Sigman, D. M., Hain, M. P. & Haug, G. H. The polar ocean and glacial cycles in atmospheric CO2 concentration. Nature 466, 47–55 (2010). 12. Jaccard, S. L., Galbraith, E. D., Martinez-Garcia, A. & Anderson, R. F. Covariation of deep Southern Ocean oxygenation and atmospheric CO2 through the last ice age. Nature 530, 207–210 (2016). g g 13. Skinner, L. C., Fallon, S., Waelbroeck, C., Michel, E. & Barker, S. Ventilation of the deep Southern Ocean and deglacial CO2 rise. Science 328, 1147–1151 (2010). XRF scanner measurements. The high-resolution elemental analysis of Br and Ca was performed using an Avaatech proﬁling X-ray ﬂuorescence (XRF) core scanner at Royal Netherland Institute for Sea Research (NIOZ) at a 1 cm downcore reso- lution. The external reproducibility of this core-scanner for Br and Ca in the range of the measurements is below 2% (1σ). 14. Siani, G. et al. Carbon isotope records reveal precise timing of enhanced Southern Ocean upwelling during the last deglaciation. Nat. Commun. 4, 2758 (2013). 15. Martinez –Boti, M. A. et al. Boron isotope evidence for oceanic carbon dioxide leakage during the last deglaciation. Nature 518, 219–222 (2015). 16. Galbraith, E. D. & Jaccard, S. L. Deglacial weakening of the oceanic soft tissue pump: global constraints from sedimentary nitrogen isotopes and oxygenation proxies. Quat. Sci. Rev. 109, 38–48 (2015). Sedimentary POC: PIC ratio vs POC: PIC rain ratio (1/ρ). It remains difﬁcult to evaluate the inﬂuence of changes in the TOC relative to the CaCO3 (POC/PIC ratio, 1/ρ) water column export and sedimentary burial on past pCO2 variability. Indeed, the amount of particulate organic and inorganic carbon in the sediments is not necessarily directly related to the fraction exported from the surface waters. While it is probably reasonable to assume that the CaCO3 accumulated in the sediment is representative of the PIC exported from the mixed layer to deep waters as core MD07-3088 was retrieved well above the lysocline (located around 3700 m depth nowadays55), it is probably not the case for TOC that might be more easily mineralized within the water column and upper sediments. However, at site MD07-3088, the combination of high sedimentary TOC contents (up to 1.9%), 17. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04625-7 For Globigerinella calida, Globigerina falconensis, G. ruber, Globigerina hexagonus (representing <2% in all samples), Neogloboquadrina dutertrei and Hastigerina digitata (representing <0.5% in all samples), we assumed a mean weight similar to G. bulloides. At last, for Globorotalia truncatulinoides, Globorotalia crassaformis, and Globorotalia hirsuta, we assumed a mean weight similar to G. inﬂata. From the assemblage and the mean weight of the different species, we estimated the planktonic foraminifera calcite mass for each sample, CaCO3pl.foram.mass in mg/g as follow: CaCO3pl:foram:mass ¼ N ´ 2split M ´ X i mi ´ Xi ð Þ ð4Þ ð4Þ Data availability. The data that support the ﬁndings of this study are available from the corresponding author (S.D.-A.) upon reasonable request. Data availability. The data that support the ﬁndings of this study are available from the corresponding author (S.D.-A.) upon reasonable request. where N is the total amount of determined foraminifera (≥300), split is the number of split done before establishing a planktonic assemblage, M is the total dry mass of the sample (g), mi the mean weight of the species i (mg), and Xi the percentage of the species in the sample. Received: 26 July 2017 Accepted: 8 May 2018 p p This approach is a ﬁrst order estimate of the foraminifera mass percentage as it does not fully take into account smaller species often <150 µm (such as G. uvula and partly T. quinqueloba) and juveniles. Besides, for 16 depths (covering LGM, HS1, ACR, YD and the Holocene), we weighted 6 to 60 specimens of G. bulloides (the most abundant foraminifera) from different size ranges (150–200, 200–250, 250–315, 315–355, 355–400, and 400–450 µm) in order to statistically characterize potential weight changes within a narrow size range. Mean weights for the different size classes decrease of about 20% from LGM to Holocene, and of about 7 and 18% during HS1 and YD respectively. If similar weight decreases are observed within the other planktonic species, the magnitude of the changes in the overall weight (∼20%) would be not sufﬁcient enough to signiﬁcantly change the estimated planktonic foraminifera mass ﬂux. Indeed, because of the drastic increases within the planktonic foraminifera abundance during these time intervals (more than one order of magnitude), ﬂuctuations in the planktonic foraminifera weights would imply changes in the ﬂux of planktonic foraminifera calcite mass that remain within the error bars ARTICLE ARTICLE NATURE COMMUNICATIONS\| (2018) 9:2396 7 \| DOI: 10.1038/s41467-018-04625-7\| www.nature.com/naturecommunications ARTICLE Glacial reduction and millennial-scale variations in Drake Passage throughﬂow. Proc. Natl Acad. Sci. USA 112, 13496–13501 (2015). 28. McClelland, H. L. O. et al. Calciﬁcation response of a key phytoplankton family to millennial- scale environmental change. Sci. Rep. 6, 34263 (2016). 59. Marchitto, T. M., Lehman, S. J., Ortiz, J. D., Fluckiger, J. & van Geen, A. Marine radiocarbon evidence for the mechanism of deglacial atmospheric CO2 rise. Science 316, 1456–1459 (2007). 29. Saavedra-Pellitero, M., Flores, J. A., Lamy, F., Sierro, F. J. & Cortina, A. Coccolithophore estimates of paleotemperature and paleoproductivity changes in the southeast Paciﬁc over the past ∼27 kyr. Paleoceanography 26, PA1201 (2011). 60. Umling, N. E., & Thunnel, R. C. Synchronous deglacial thermocline and deep- water ventilation in the eastern equatorial Paciﬁc. Nat. Commun. 8, 14203 (2017). 30. Anderson, R. et al. Wind-driven upwelling in the Southern Ocean and the deglacial rise in atmospheric CO2. Science 323, 1443–1448 (2009). 61. Beaufort, L., Barbarin, N. & Gally, Y. Optical measurements to determine the thickness of calcite crystals and the mass of thin carbonate particles such as coccoliths. Nat. Protoc. 9, 633–642 (2014). 31. Haddam, N. A., et al. Improving past sea surface temperature reconstructions from the Southern Hemisphere oceans using planktonic foraminiferal census data. Paleoceanography 31, 822–837 (2016). g p y 32. DiFiore, P. J. et al. Nitrogen isotope constraints on subantarctic bi h i t J G ph R 11 C08016 (2006) g p y 32. DiFiore, P. J. et al. Nitrogen isotope constraints on subantarctic biogeochemistry. J. Geophys. Res. 11, C08016 (2006). 62. Saavedra-Pellitero, M., Baumann, K. H., Flores, J. A. & Gersonde, R. Biogeographic distribution of living coccolithophores in the Paciﬁc sector of the Southern Ocean. Mar. Micropaleontol. 109, 1–20 (2014). biogeochemistry. J. Geophys. Res. 11, C08016 (2006). g y p y 33. Flores, J. A., Filippelli, G. M., Sierro, F. J., Latimer, J. The “White Ocean” hypothesis: a late Pleistocene Southern Ocean governed by coccolithophores and driven by phosphorus. Front. Microbiol. 3, 233 (2012). pp hypothesis: a late Pleistocene Southern Ocean governed by coccolithophores and driven by phosphorus. Front. Microbiol. 3, 233 (2012). 63. Hartnett, H. E., Keil, R. G., Hedges, J. I., and Devol, A. H. Inﬂuence of oxygen exposure time on organic carbon preservation in continental margin sediments. Nature 391, 572–574 (1998). 34. Chase, Z., McManus, J., Mix, A. C. & Muratli, J. ARTICLE ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04625-7 51. Saavedra-Pellitero, M., Baumann, K. H. & Ullermann, J. Marine isotope stage 11 in the Paciﬁc sector of the Southern Ocean, a coccolithophore perspective. Quat. Sci. Rev. 158, 1–14 (2017). 20. Lamy, F. et al. Increased dust deposition in the Paciﬁc Southern Ocean during glacial periods. Science 343, 403–407 (2014). g p 21. Gottschalk, J., et al. Biological and physical controls in the Southern Ocean on past millennial-scale atmospheric CO2 changes. Nat. Commun. 7, 11539 (2016). 52. Mayer, L. M., Schick, L. L., Allison, M. A., Ruttenberg, K. C. & Bentley, S. J. Marine vs. terrigenous organic matter in Louisiana coastal sediments: The uses of bromine:organic carbon ratios. Mar. Chem. 107, 244–254 (2007). 22. Mahowald, N. M., et al. Change in atmospheric mineral aerosols in response to climate: last glacial period, pereindustrial, modern, and doubled carbon dioxide climates. J. Geophys. Res. 11, D10202 (2006). dioxide climates. J. Geophys. Res. 11, D10202 (2006). 53. Ziegler, M., Jilbert, T., de Lange, G. J., Lourens, L. J., Reichart, G. J. Bromine counts from XRF scanning as an estimate of the marine organic carbon content of sediment cores. Geochem. Geophys. Geosyst. 9, Q05009 (2008). 23. Tagliabue, A., Aumont, O., Bopp, L. The impact of different external sources of iron on the global carbon cycle. Geophys. Res. Lett. 41, 920–926 (2014). p y y 54. Meyers, P. A. Preservation of elemental and isotopic source identiﬁcation of sedimentary organic matter. Chem. Geol. 114, 289–302 (1994). 24. Salter, I., et al. Carbonate counter pump stimulated by natural iron fertilization in the Polar frontal zone. Nat. Geosci. 7, 885–889 (2014). 55. Hebbeln, D., Marchant, M., Freudenthal, T. & Wefer, G. Surface sediment distribution along the Chilean continental slope related to upwelling and productivity. Mar. Geol. 164, 119–137 (2000). 25. Winter, A., Siesser, W. G. (eds) Coccolithophores. 242p (Cambridge University Press, Cambridge, 1994). g 26. Archer, D., Maier-Reimer, E. Effect of deep-sea sedimentary calcite preservation on atmospheric CO2 concentration. Nature 367, 260-263 (1994). p y 56. Marcott, S. A. et al. Centennial-scale changes in the global carbon cycle during the last deglaciation. Nature 514, 616–619 (2014). 27. Bolton, C. T. et al. Decrease in coccolithophore calciﬁcation and CO2 since the middle Miocene. Nat. Commun. 7, 10284 (2016). 57. Hogg, A. G. et al. SHCal13 Southern hemisphere calibration, 0–50,000 years cal BP. Radiocarbon 55, 1889–1903 (2013). 58. Lamy, F. et al. Acknowledgements p 41. Horigome, M. T. et al. Environmental controls on the Emiliania huxleyi calcite mass. Biogeosciences 11, 2295–2308 (2014). 41. Horigome, M. T. et al. Environmental controls on the Emiliania huxleyi calcit mass. Biogeosciences 11, 2295–2308 (2014). This research was ﬁnancially supported by the French INSU/LEFE - CHICO (2013-2016) project, the Swedish Research Council (VR-349-2012-6278) and the Swiss National Science Foundation (grants PP00P2-144811 and PP00P2_172915 to S.L.J). The research has been conducted within the framework of the international IMAGES program and the MD159- PACHIDERME/IMAGES cruise, with technical support from the Institute Paul Emile Vi (IPEV) W h k G I d h i d i f i if i ki 42. Henderiks, J. Coccolithophore size rules – reconstructing ancient cell geometry and cellular calcite quota from fossil coccoliths. Mar. Micropaleontol. 67, 143–154 (2008). 43. Young, J. R. et al. A guide to extant coccolithophore taxonomy. J. Nannoplankton Res. Spec. Issue 1, 1–125 (2003). Victor (IPEV). We express our thanks to G. Isgüder that assisted in foraminifera picking. 44. O’Dea, S. A., et al. Coccolithophore calciﬁcation response to past ocean acidiﬁcation and climate change. Nat. Commun. 5, 5363 (2014). Author contributions 45. Riebesell, U. et al. Reduced calciﬁcation of marine plankton in response to increased atmospheric CO2. Nature 407, 364–367 (2000). All authors contributed extensively to this work. S.D.-A. prepared the manuscript and collected the organic and inorganic carbon data together with the coccolith data. Coc- colith abundance and mass collection, using the SYRACO software, would not have been possible without L.B. and Y.G. E.M. and G.S. provided the XRF and foraminifera data. E.M., S.L.J., and G.S. particularly contributed to the redaction of the article. 46. Hoppe, C. J. M., Langer, G. & Rost, B. Emiliania huxleyi shows identical responses to elevated pCO2 in TA and DIC manipulations. J. Exp. Mar. Biol. Ecol. 406, 54–62 (2011). 47. Iglesias-Rodriguez, M. D. et al. Phytoplankton calciﬁcation in a high-CO2 world. Science 320, 336–340 (2008). ARTICLE Southern Ocean and glaciogenic nutrients control diatom export production on the Chile margin. Quat. Sci. Rev. 99, 135–145 (2014). 64. Thomas, A. C., Huang, F., Strub, P. T. & James, C. Comparison of the seasonal and interannual variability of phytoplankton pigment concentrations in the Peru and California current systems. J. Geophys. Res. 99(C4), 7355–7370 (1994). ( ) 35. Siani, G. et al. Late Glacial to Holocene terrigenous sediment record in the Northern Patagonian margin: paleoclimate implications. Paleogeogr. Paleoclimatol. Paleoecol. 297, 26–36 (2010). g g p Paleoclimatol. Paleoecol. 297, 26–36 (2010). 65. Buesseler, K. O. The decoupling of production and particulate export in the surface. Glob. Biogeochem. Cycles 12, 297–310 (1998). 36. Raiswell, R. et al. Contributions from glacially derived sediment to global iron (oxy-hydr)oxide cycle: implications for iron delivery to the oceans. Geochim. Et. Cosmochim. Acta 70, 2765–2780 (2006). 66. Dunne, J. P., Sarmiento, J. L. & Gnanadesikan, A. A synthesis of global particle export from the surface ocean and cycling through the ocean interior and on the seaﬂoor. Glob. Biogeochem. Cycles 21, GB4006 (2007). 37. Wang, X. T. et al. Deep-sea coral evidence for lower Southern Ocean surface nitrate concentrations during the last ice age. PNAS 114, 3352–3357 (2017). 67. Smetacek, V. et al. Deep carbon export from a Southern iron-fertilized diatom bloom. Nature 487, 313–319 (2012). 38. Saavedra-Pellitero, M., Flores, J. A., Baumann, K. H. & Sierro, F. J. Coccolith distribution patterns in surface sediments of Equatorial and Southeastern Paciﬁc Ocean. Geobios 43, 131–149 (2010). 68. Schlitzer, R. Ocean Data View http://odv.awi.de (2012). 69. Garcia, H. E., et al. in NOAA Atlas NESDIS 66 (ed. Levitus, S.) 216 pp (U.S. Government Printing Ofﬁce, Washington D.C., 2009). 39. Beaufort, L. et al. Sensitivity of coccolithophores to carbonate chemistry and ocean acidiﬁcation. Nature 476, 80–83 (2011). 70. Orsi, A. H., Whitworth, T. III & Nowlin, W. D. Jr. On the meridional extent and fronts of the Antarctic Circumpolar Current. Deep Sea Res. 42, 641–673 (1995). 40. Meier, K., Berger, C. & Kinkel, H. Increasing coccolith calciﬁcation during CO2 rise of the penultimate deglaciation (Terminaison II). Mar. 40. Meier, K., Berger, C. & Kinkel, H. Increasing coccolith calciﬁcation during CO2 rise of the penultimate deglaciation (Terminaison II). Mar. Micropaleontol. 112, 1–12 (2014). Micropaleontol. 112, 1–12 (2014). Additional information 48. Bolton, C. T. & Stoll, H. M. Late Miocene threshold response of marine algae to carbon dioxide limitation. Nature 500, 558–562 (2013). Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 018-04625-7. Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 018-04625-7. References Kohfeld, K. E., Le Quéré, C., Harrison, S. P. & Anderson, R. F. Role of marine biology in Glacial-Interglacial CO2 cycles. Science 308, 74–78 (2005). 18. Martinez-Garcia, A. et al. Links between iron supply, marine productivity, sea surface temperature, and CO2 over the last 1.1. Ma. Paleoceanography 24, PA1207 (2009). pp y p y surface temperature, and CO2 over the last 1.1. Ma. Paleoceanography 24, PA1207 (2009). 19. Muratli, J. M., Chase, Z., Mix, A. C. & McManus, J. Increased glacial-age ventilation of the Chilean margin by Antarctic Intermediate Water. Nat. Geosci. 3, 23–26 (2010). \| DOI: 10.1038/s41467-018-04625-7\| www.nature.com/naturecommunications NATURE COMMUNICATIONS\| (2018) 9:2396 8 Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 018-04625-7. 49. Henderiks, J. & Pagani, M. Reﬁning ancient carbon dioxide estimates: signiﬁcance of coccolithophore cell size for alkenone-based pCO2 records. Paleoceanography 22, PA3202 (2007). Competing interests: The authors declare no competing interests. Competing interests: The authors declare no competing interests. Competing interests: The authors declare no competing interests. 50. Hannisdal, B., Henderiks, J. & Liow, L. H. Long-term evolutionary and ecological responses of calcifying phytoplankton to changes in atmospheric CO2. Glob. Change Biol. 18, 3504–3516 (2012). Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ NATURE COMMUNICATIONS\| (2018) 9:2396 NATURE COMMUNICATIONS\| (2018) 9:2396 9 \| DOI: 10.1038/s41467-018-04625-7\| www.nature.com/naturecommunications © The Author(s) 2018 \| DOI: 10.1038/s41467-018-04625-7\| www.nature.com/naturecommunications ARTICLE material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party NATURE COMMUNICATIONS\| (2018) 9:2396 10 \| DOI: 10.1038/s41467-018-04625-7\| www.nature.com/naturecommunications
https://openalex.org/W3180595036	https://www.researchsquare.com/article/rs-690536/latest.pdf	English	null	Randomized Control Trial of Short Infusion of Low Dose Ketamine Vs Intravenous Morphine as Adjunct Analgesia for Acute Long Bone Fracture Pain in Emergency Department	Research Square (Research Square)	2,021	cc-by	5,626	Randomized Control Trial of Short Infusion of Low Dose Ketamine Vs Intravenous Morphine as Adjunct Analgesia for Acute Long Bone Fracture Pain in Emergency Department g y p Elisa Audrey Eddie Hospital Kuala Lumpur, Ministry of Health Malaysia: Kementerian Kesihatan Malaysia Ahmad Zulkarnian Ahmad Zahedi University of Malaya Medical Centre, University of Malaya: Universiti Malaya SABARIAH FAIZAH JAMALUDDIN (  drsabariahfj@yahoo.com ) Universiti Teknologi MARA Kampus Sungai Buloh https://orcid.org/0000-0002-3788-7915 Original research Keywords: Ketamine, Morphine, Pain Management, Bone fracture Posted Date: July 8th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-690536/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Background Ketamine is known as an alternative for pain control, but reports of emergency reactions limits its widespread use. We assessed the efficacy of short infusion of low dose ketamine (LDK) compared with intravenous morphine (MOR) as adjunct analgesia for acute long bone fracture pain. Trial Registration National Medical Research Register: https://www.nmrr.gov.my/ Registered 24 November 2017 ID:NMRR- 17-3184-38970 Original research Keywords: Ketamine, Morphine, Pain Management, Bone fracture Posted Date: July 8th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-690536/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/17 Page 1/17 Results Fifty-eight subjects were enrolled (MOR 27, LDK 31). Demographic variables and baseline NPRS scores MOR (8.33) vs LDK (8.84) were similar. The mean reduction of NPRS were significantly different between LDK (Mean= 3.1, SD=2.03) and MOR (Mean =1.8, SD 1.59), p= 0.009 at 30 minutes. Incidence of dizziness was reported higher in Ketamine group 19.4% (p=0.026). Conclusion When used as an adjunct, short infusion low-dose ketamine at 0.3mg/kg over 15minutes provides greater analgesic effect in comparison to intravenous morphine alone for acute long bone fracture pain but has higher incidence of dizziness. Methods Patients aged 18-60 years old, with acute long bone fracture and with numerical pain rating scale (NPRS) of 6 or more after 3mg intravenous morphine were eligible for enrolment. Subjects were consented and randomized to either short infusion LDK (0.3mg/kg) over 15 minutes or intravenous morphine (MOR) (0.1mg/kg) over 5 minutes. Evaluations of NPRS score and vital signs occurred at 15, 30, 60, 90 and 120 minutes. The primary outcome from this study was the mean reduction of numerical pain rating scale (NPRS) score from baseline and the mean time to achieve ³ 3 score reductions in NPRS. The secondary outcomes were the incidence of adverse events and mean consumption of rescue analgesia. Background Pain is a common cause of emergency department visits. 80% of patient with musculoskeletal injury has moderate to severe pain. Pain is the common cause of the emergency department visits and 80% of patients with musculoskeletal injury has moderate to severe pain [1].Each year millions of people from all over the world suffer from bone fractures due to incidence of traffic accidents. One of the most important measures in the management of such patients in emergency department is fixation and pain control. Page 2/17 Page 2/17 Malaysia is one of the countries that have highest traffic fatality rates in the world with 25 fatalities/100,000 population which is only second to Thailand in South East Asia[2]. Malaysia is one of the countries that have highest traffic fatality rates in the world with 25 fatalities/100,000 population which is only second to Thailand in South East Asia[2]. Tackling pain control in this group of patients has been a great challenge for many physicians. Currently the standard practice to treat fracture pain is with opioids[3]. Besides opioids, other medications to treat the acute pain are non-steroidal anti-inflammatory drugs, acetaminophen, and regional treatments. However, it is not recommended to administer opioids repeatedly to patient with acute pain, as it may lead to adverse effect such as hypotension and respiratory depression [4]. Additionally, many patients report inadequate pain control in the ED with opioids [5]. A study done in Malaysia in 2007 reported that only 26.5% of 85% patients in moderate and severe pain received analgesia in the emergency department despite the pain score being displayed on patients’ case notes. In emergency department in United States, a 0.1mg/kg dose of intravenous morphine was not adequate for controlling severe acute pain in most patients[6]. A post-operative pain study demonstrated that patients needed on average 12mg or a mean weight-based of 0.17mg/kg of morphine to achieve an acceptable level of pain reduction as determined by a 30mm change on a visual analogue scale.[7] Tackling pain control in this group of patients has been a great challenge for many physicians. Currently the standard practice to treat fracture pain is with opioids[3]. Besides opioids, other medications to treat the acute pain are non-steroidal anti-inflammatory drugs, acetaminophen, and regional treatments. Background Besides that, an epidemic of opioid misuse in the US has grown extensively to 300% increase in the past decade for opioid drug overdose death and a two-fold increase in emergency departments’ visits for opioid misuse and abuse[8]. Despite limited research for opioid overdose in Malaysia, a study showed that there is a higher prevalence of non-fatal overdose of opioids reported[9]. For this reason, alternative or adjunct to opioids therapy is needed. An alternative medication is ketamine. In Malaysia, intravenous Ketamine is used in Anaesthesiology Department as an analgesic adjuvant to general anaesthesia and patient controlled anaesthesia (PCA) such as major visceral surgery or hip surgery [10]. It is also used for analgesia and sedation for burns and repeated wound dressing especially in paediatric as it preserves the pharyngeal reflexes, cardiovascular stability and less respiratory depression [11]. Ketamine has also been used for pain control but in much lower dose that does not sedate patients. When it is given in sub dissociative doses, it prevents the development of increased pain sensitivity and opioid tolerance in a variety settings outside of ED [12– 14]. In the emergency department, Ketamine at a dose of (1 to 1.5mg/kg) has been commonly used for procedural sedation as well as an induction agent for rapid sequence intubation but its use as analgesia has been slower to gain momentum due to reports of emergence reactions (anxiety, nightmares, hallucinations, delirium) [15]. However, recent studies suggest the use of sub dissociative ketamine doses for acute pain control. Ketamine at sub dissociative dose of (0.1-0.6mg/kg) and particularly at a dose of (0.3mg/kg) gives analgesic effect while maintaining the airway reflexes is comparable with intravenous morphine for acute treatment of pain in ED [16, 17]. However, there is a higher incident of adverse effect such as emergence phenomenon occurred when ketamine is given as intravenous push. A recent study by Motov et al. compared intravenous push of low dose ketamine versus short infusion of low dose ketamine in 15 mins for undifferentiated pain such as abdominal, flank or low back pain in Emergency Department reported Page 3/17 Page 3/17 that by giving short infusion of low dose ketamine significantly reduce unreality adverse effect without compromising the analgesic effect [18]. To the best of our knowledge, there are limited studies regarding short infusion low dose ketamine in ED as adjunct analgesia for acute long bone fracture pain. Study Setting and Selection of Participants This study was conducted in a 620-bed hospital with an annual ED census of more than 160,000 visits. Enrolment occurred from May 2018 until February 2019. Patients were recruited at unplanned intervals during the study period, subject to availability of the investigators and patient consents. Patients were eligible for enrolment after they were assessed by attending emergency medicine residence or emergency physician on duty as having the following: were aged 18 years old to 60 years old, able to give consent, conscious (Glasgow Coma Scale [GCS] score = 15) with long bone fracture (femur, tibia/fibula, humerus and radius/ulna bone) and with numerical pain rating scale (NPRS) score greater or equal to 6 after 3mg of intravenous morphine given by the treating physicians in ED. Exclusion criteria consisted of altered mental status (GCS ≤ 14), pregnant, breast feeding patient, allergic to ketamine or morphine, hemodynamically unstable vital signs (systolic blood pressure < 90 or > 180mmHg, pulse rate < 50 or > 150 beats/min, and respiratory rate < 10 or > 30 breaths/min), and medical history of acute head injury or eye injury, seizure, intracranial bleed, renal or hepatic insufficiency, ischemic heart disease, cerebrovascular accident, asthma or chronic lung disease, drug or alcohol abuse, psychiatric illness. Background Therefore, we hypothesized that, when use as an adjunct, short infusion low dose ketamine has the analgesic effect comparable with IV morphine in acute long bone fracture in the emergency department. The primary outcome of effectiveness is measured by mean reduction of pain score. The amount and timing of administration rescue opioid analgesia was also be evaluated as outcome. The adverse events of intravenous morphine and short infusion of low dose morphine will be monitored. Study Design This was a single-center, prospective, randomized, single-blinded trial study comparing short infusion low dose ketamine (LDK) versus intravenous morphine as adjunct analgesia for acute long bone fracture pain in ED This was a single-center, prospective, randomized, single-blinded trial study comparing short infusion low dose ketamine (LDK) versus intravenous morphine as adjunct analgesia for acute long bone fracture pain in ED Study Protocol After written informed consent was obtained, each participant enrolled in the study was randomly assigned according to a predetermined randomization list that was generated using IBM SPSS Statistic 23.0 by the investigator. Participants were randomized to either short infusion LDK (0.3mg/kg) mixed in 100 mls normal saline solution given over 15 minutes or intravenous MOR (0.1mg/kg) in 10 mls normal saline solution over 5 minutes. ED pharmacist on duty who was independent of this study was alerted regarding the patients’ body weight for drug randomization and preparation, while the treating nurse who Page 4/17 was briefed and taught on administration of the intervention or control medication prior to intervention, administered the medication to eligible patients. This was a single-blinded protocol study; thus, the investigator, ED pharmacist, treating providers and statisticians were the only ones with the knowledge of the study arm to which patients was randomized. However, to minimize bias, the researcher had appointed one independent individual who was not a part of the study team (trained nurse) to assess vital signs and pain score after administration of medication. This individual was trained to assess vital signs and pain score prior to starting of this study. This independent individual was blinded from the treatment arm and recorded pain score, vital signs and adverse effects at baseline, 15 min, 30 min, 60 min, 90 min and 120 min. For participants reported of NPRS score ≥ 6 and still desiring pain medication 30 min after study drug administration, investigator offered intravenous fentanyl 1-2mcg/kg, maximum 100 mcg, as rescue analgesia. Data collection ended at 120 minute or upon patient admission or whichever came first. This study was approved by Malaysian Research and Ethics Committee (MREC) Malaysia and was registered with the National Medical Research Register (NMRR-17-3184-38970). Written and signed informed consent was obtained in accordance with institutional policy. Data Analysis Power analysis determined that a sample size of at least 20 subjects per group would achieve 80% power to detect a 2-point change in NPRS scores between treatment groups, with estimated groups of SDs of 2 for a 2-sided test with a significance level α of 0.05. We used repeated measures linear model with adjustments for treatment group, time and the group by time interactions. All analyses were performed with intention to treat. Data entry and analysis were done using Statistical Package for Social Science (SPSS) version 23.0 software. The statistical analysis included descriptive analysis such as mean, standard deviations, percentage, independent t-test and Chi-Square test. Statistical significance was set at P < 0.05 in the analyses. Results A total of 58 patients were enrolled in the study from May 2018 to February 2019; 27 in the morphine arm and 31 in the low-dose ketamine arm Demographic characteristics were similar between 2 groups including median age, sex, baseline vital signs, site of fractures and baseline NPRS scores MOR (8.3) vs LDK (8.9) were similar (Table 1). Page 5/17 Page 5/17 Table 1 Baseline Patients Characteristic Variable Morphine (n = 27) Low-dose ketamine (n = 31) P value Age (years) 25(12) * 27(17) * 0.487 Gender Male (n%) 24(46.2) 28(58.3) 0.861 Female (n%) 3 (50.0) 3 (50.0) Weight 64.44(12.3) # 66.23(12.7) # 0.593 Mechanism of injury Road Traffic injury (n%) 25(47.2) 28(52.8) ≥ 0.995 Fall (n%) 2(50.0) 2(50.0) Site of fracture Upper extremities (n%) 2 (28.6) 5 (71.4) 0.203 Lower extremities (n%) 26 (50.0) 26 (50.0) 0.90 Baseline NRS pain score 8.3 (1.3) # 8.9 (1.2) # 0.136 Note: Data not normally distributed presented as Median (IQR) # Data presented as mean (SD) Table 1 Baseline Patients Characteristic The primary outcome was the mean reduction in NPRS score from baseline between 2 groups (Table 2). Both treatment groups showed significant reduction from baseline. At 30 minutes, short infusion LDK group had significantly greater mean reduction in pain from baseline (Mean reduction = 3.1, SD = 2.05) in comparison with morphine (Mean reduction = 1.8, SD = 1.59), t (56) =-2.70, p = 0.009. At 60 minutes, the mean reduction of NPRS score were also significant in low-dose ketamine (Mean reduction = 3.5, SD = 2.17), in comparison with morphine (Mean reduction = 2.4, SD = 1.84), t (56) =-2.09, p = 0. 041.There was no significant difference in mean reduction of pain score found in other interval times. Results Page 6/17 Table 2 Comparison of mean NPRS between patients treated with IV morphine or Short infusion low-dose ketamine based on time Study Drug Time (minutes) Mean NPRS score (SD) Estimated marginal mean (95% CI) Morphine 0 8.3 (1.3) 7.84,8.83 15 7.1 (0.36) 6.34,7.79 30 6.6 (0.33) 5.86,7.18 60 5.9 (0.34) 5.29,6.64 90 5.4 (0.40) 4.57,6.17 120 5.1 (0.39) 4.29,5.86 Low-dose Ketamine 0 8.9 (1.2) 8.37,9.34 15 6.7 (0.35) 5.98,7.38 30 5.8 (0.32) 5.24,6.54 60 5.6 (0.33) 4.95,6.27 90 5.3 (0.40) 4.54,6.11 120 5.5 (0.39) 4.69,6.24 Repeated Measures ANOVA SD = Standard Deviation CI = Confidence Interval Table 2 Figure 2 showed the comparison of pain scores over all time points, which demonstrates the mean pain numeral rating scores in both groups. There was significant difference at 30 min between low-dose ketamine infusion and morphine (p = 0.009) Figure 2 showed the comparison of pain scores over all time points, which demonstrates the mean pain numeral rating scores in both groups. There was significant difference at 30 min between low-dose ketamine infusion and morphine (p = 0.009) The proportion of patients reporting a 3-point or more reduction in pain numeric rating scale score at 15 min and 30 min between low-dose Ketamine and Morphine were significantly different (p = 0.042) and (p = 0.014) (Table 3) Page 7/17 Table 3 Comparing mean reduction of NPRS score from baseline within patient treated between morphine and short infusion low-dose ketamine Time Morphine Mean (SD) Ketamine Mean (SD) Mean difference (95% CI) P value T15 1(2) 2(4) 0.113a T30 1.8(1.59) 3.1(2.05) -1.3 (-2.29, -0.34) 0.009b T60 2.4(1.84) 3.5(2.17) -1.1(-2.27, -0.08) 0.041b T90 3.0(2.03) 3.5 (2.63) -0.6 (-1.85, 0.70) 0.371b T120 3.3(2.23) 3.4 (2.47) -0.1 (-1.41, 1.14) 0.834b Table 3 Table 3 Comparing mean reduction of NPRS score from baseline within patient treated between morphine and short infusion low-dose ketamine Note: Data not normally distributed presented as median (IQR) Data not normally distributed presented as median (IQR) a Mann-Whitney test b Independent t test; SD=Standard Deviation; The median amount of fentanyl as rescue analgesia administered to both groups was not significantly different (morphine: 50mcg [IQR = 8] vs ketamine: 50 [IQR = 6]; p = 0.921). The proportion of rescue analgesia between both groups were not significantly different (p = 0.336) (Table 4). Table 4 Rates of rescue analgesia over time ime of rescue analgesia Morphine Ketamine Page 8/17 Table 4 Rates of rescue analgesia over time Time of rescue analgesia Morphine n = 27 n (%) Ketamine n = 31 n (%) P value a 30 min c 1 (3.7%) b 2 (6.4%) b ≥ 0.995 60 min 4 (14.8%) 3 (9.7%) 0.694 90 min 2 (7.4%) 5 (17.9%) 0.432 120 min 2 (7.4%) 2 (7.10%) ≥ 0.995 Total 9 (33.3%) 10 (32.3%) 0.366 a Fisher’s exact test b Frequency (percent) c No rescue analgesia given before 30 min No serious adverse events occurred in either drug group; these includes, respiratory distress, seizures, cardiac arrest or allergic reaction (Table 5). The prevalence of patient reported dizziness was statistically significantly observed in low-dose ketamine in comparison with morphine (p = 0.026). Fatigue and headache were also reported in low-dose ketamine. One patient in low-dose ketamine was reported to have hallucination but no intervention was required. Table 5 Adverse effects encountered in each group at any point throughout the study period Adverse effects Morphine n = 27 n (%) Ketamine n = 31 n (%) P value b Fatigue 0 3(9.7%) a 0.24 Dizziness 0 6(19.4%) 0.026 Headache 2(7.4%) a 2(7.1%) ≥ 0.995 Hallucinations 0 1(3.6%) ≥ 0.995 a Frequency (percent) b Fisher’s exact test Discussion reported that mean NPRS from baseline to 30 min between both treatment groups were not statistically significant different [17], interestingly our study demonstrated that low-dose ketamine provides safe and effective adjunct analgesia to acute long bone fracture patient, with mean reduction pain score (Mean = 3.1, SD = 2.05) in compare with morphine (Mean = 1.8, SD = 1.59), p = 0.009 was significant observed at 30 min and lasting up to 60 min. It is interesting to note that the proportion number of patients reporting a 3-point or more reduction in pain numeric rating scale score at 15 min and 30 min between LDK and MOR were statistically significant different (p = 0.042) and (p = 0.014) respectively. Even though these results differ from earlier studies, the authors reported that at 15 min, complete resolution of pain was observed in ketamine group compare to morphine group [17]. This effect maybe due to the synergic effect of morphine and ketamine combination which enhanced the analgesic effect [23]. The mean pain score plotted over time differed between both groups (Fig. 2); ketamine group has steeper slope reflecting the rapid reduction of pain score over time in comparable with the morphine group. However, the analgesic effect of low dose ketamine was unable to sustained after 60 min thus frequency of low-dose ketamine requiring rescue analgesia increased at 90 min. This finding contradicts with that of Beaudoin et al, who reported a greater pain reduction in patient who received ketamine over a 2-hour period [21]. Beaudoin et al. reported the median dose of rescue analgesia was 4.9mg to 6.1mg of intravenous morphine. In addition, the median time in which rescue analgesia was administered ranged between 54–143 minutes which may explained the observed reduction of pain score beyond 2 hours. In contrast with our study, the median dose rescue analgesia administered was 50mcg of intravenous fentanyl while the mean time rescue analgesia was administered ranged between 72–107 minutes. We strongly believed that if ketamine was infused over a longer period or ketamine was administered in repeated doses, perhaps the further decreased of pain score could have been observed over 60 min. There was no serious adverse event reported during the study but the incident of dizziness was reported more in the low-dose ketamine group (p = 0.026) in comparison with morphine. Discussion Our study has found that short infusion low-dose ketamine as an adjunct analgesia to intravenous morphine for patients with acute long bone fractures reduces the numerical pain rating scale (NPRS) score and the mean time to achieve ≥ 3 reductions in NPRS. Short infusion low-dose ketamine whether as adjunct to opioids or as single agent for analgesia is a promising alternative for acute long bone fracture pain management in ED. Its unique mechanism of actions that blocks non-competitive antagonist of the N-methyl-D-aspartic acid (NMDA) receptor and glutamate receptor antagonist that decreases sensitization at CNS and spinal cord level gives the effects of analgesia, hypnotic and amnestic[19] but adverse effects of emergence reactions limits its usage as analgesia in ED. Several studies have been published regarding ketamine as analgesia in ED setting. An out of hospital setting by Johansson et al [20] demonstrated a significant reduction of pain score in morphine-ketamine combination group by 5.4 points in comparison to 3.1 points of morphine alone. Jennings et al [14] demonstrated that morphine and ketamine combination group is superior than morphine group alone with mean pains score change of -5.6 (CI -6.2 to 5.0) and 3.2(CI -3.7 to -2.7) respectively. Beaudoin et al[21] demonstrated that IV morphine 0.1 mg/kg with IV ketamine at 0.3mg/kg had higher pain relief than ketamine at 0.15 mg/kg. Miller et al [22] compared low-dose ketamine with IV Page 9/17 Page 9/17 morphine in ED but concluded that ketamine at 0.3 mg/kg was not superior to morphine in maximum reduction of pain score. In addition, the author concluded that analgesic effect in low dose ketamine was significant within 5 minutes and provide moderate reduction in pain for 2 hours. Motov et al [18] demonstrated that short infusion low dose ketamine is associated with lower rates of unreality with no significant difference in analgesic efficacy (p < 0.001). In this prospective, randomized control trial study, we compared short infusion low dose with single dose morphine as adjunct analgesia for acute long bone fracture pain in ED. All patients were administered with 3 mg of morphine before randomization and enrollment into the study. This was an important component to the protocol because ketamine was not routinely administered as first-line analgesic. It was reserve to patient whose pain was refractory to morphine. Administering ketamine as the initial parental analgesic agent would not reflect the actual practice. Despite Motov et al. Discussion These findings are consistent with a previous study conducted by Ahern et al [24] of which 24 of 30 patients (80%) who received IV combination of hydromorphone 0.5mg and ketamine 15mg experienced dizziness being most Page 10/17 Page 10/17 common. Beaudoin et al [21] also reported that 9 of 20 (45%) combination of IV Morphine 0.1mg/kg and IV Ketamine 0.3mg/kg reported dizziness. By reviewing previous studies, there is a strong probability that the incidence of adverse effect reported in low-dose ketamine may be attributed to the rate of initial bolus administration and the dilution for ketamine. We believe that further trials of ketamine dose ranges and duration of infusion, may diminished the adverse effect of neuropsychological without compromising the analgesic effect. Some limitations need to be acknowledged. First, this was a single blinded study conducted in a single center. The investigators were not blinded from the study and the outcome may be skewed despite an independent individual who is not part of the study (trained staff nurse) who recorded the vital signs and pain score from the participants. Second, patient enrollment was subject to the availability of the investigators and the number of enrolled subjects may not represent the rest of the population of the country. Conclusion The evidence from this study points that, when used as an adjunct, short infusion low-dose ketamine at 0.3mg/kg over 15minutes provides greater analgesic effect in comparable with intravenous morphine alone for acute long bone fracture pain but has higher incidence of dizziness. Short infusion low-dose ketamine can be given in emergency department as analgesia on patients with long bone fractures for painful procedures such as applications of splints and wound irrigations. Future studies are highly recommended with a bigger sample-pooled size such as a superiority study design with multiple center sites due to its advantages in mean pain reduction from baseline in compare with intravenous morphine alone. Acknowledgements The authors thank all the staff of Ministry of Health Emergency and Trauma Department of Hospital Sungai Buloh for their relentless contribution and support for the study. Thanks also to Dr Mohd Zahir Amin bin Mohd Nazri, MBBS, MEmMed for the critical reviews and Ms Nadiah Sa’at for the statistical analysis reviews. We would like to thank the Director General Health of Malaysia for his permission to publish this article. Funding No funding has been received for the research, authorship, and/or publication of this article. Authors’ Contributions All the authors made substantial contributions to the concept or design of the work; acquisition, analysis, or interpretation of data; drafted the article or revised it critically for important intellectual content; and approved the version to be published. Each author participated sufficiently in the work to take public responsibility for appropriate portions of the content. Availability of data and materials Data are available on request to the corresponding author. Abbreviations BP Blood pressure CRC Clinical Research Centre ED Emergency department IBM International Business Machine MOR Morphine NPRS Numerical Pain Rating Scale GCS Glasgow Coma Scale Page 11/17 LDK Low-dose ketamine IV Intravenous MREC Medical Research and Ethics Committee NMDA N-methyl-D-aspartic acid NMRR National Medical Research Register PCA Patient control analgesia PSA Procedural sedation analgesia SPSS Statistical Package for the Social Sciences Ethics approval and consent to participate Page 12/17 Page 12/17 This study was approved by Medical Research and Ethics Committee (MREC) and registered with the National Medical Research Register (NMRR-17-3184-38970) on 24 November 2017. Written informed consent was obtained from patient included in the study. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the institution’s human research committee. Author details Faculty of Medicine, University Malaya, Jalan Universiti, Consent for publication Not applicable Competing interests The authors declare that there is no conflict of interest Author details The authors declare that there is no conflict of interest References Page 13/17 1. Thomas SH, Shewakramani S. Prehospital Trauma Analgesia J Emerg Med. 2008;35:47–57. 2. Abdelfatah A. 2016 Traffic Fatality Causes 1–19. 1. Thomas SH, Shewakramani S. Prehospital Trauma Analgesia J Emerg Med. 2008;35:47–57. 1. Thomas SH, Shewakramani S. Prehospital Trauma Analgesia J Emerg Med. 2008;35:47–57. 2. Abdelfatah A. 2016 Traffic Fatality Causes 1–19. 2. Abdelfatah A. 2016 Traffic Fatality Causes 1–19. 2. Abdelfatah A. 2016 Traffic Fatality Causes 1–19. 3. Lee EN, Lee JH, Seguin P, Branger B, Campion J, Mallédant Y 2016 The Effects of Low-Dose Ketamine on Acute Pain in an Emergency Setting: A Systematic Review and Meta-Analysis ed Z-L Ma PLoS One 11 e0165461. 4. Messenger DW, Murray HE, Dungey PE, Van Vlymen J, Sivilotti ML A 2008 Subdissociative-dose ketamine versus fentanyl for analgesia during propofol procedural sedation: A randomized clinical trial Acad. Emerg. Med. 15 877–86. 5. Cordell WH, Keene KK, Giles BK, Jones JB, Jones JH, Brizendine EJ. The high prevalence of pain in emergency medical care. Am J Emerg Med. 2002;20:165–9. 6. Bijur PE, Kenny MK, Gallagher EJ. 2005 Intravenous morphine at 0.1 mg/kg is not effective for controlling severe acute pain in the majority of patients Ann. Emerg. Med. 46 362–7. 7. Aubrun F, Langeron O, Quesnel C, Coriat P, Riou. B 2003 Relationships between measurement of pain using visual analog score and morphine requirements during postoperative intravenous morphine titration. Anesthesiology 98 1415–21. 8. Compton WM, Volkow ND. Major increases in opioid analgesic abuse in the United States: Concerns and strategies. Drug Alcohol Depend. 2006;81:103–7. 8. Compton WM, Volkow ND. Major increases in opioid analgesic abuse in the United States: Concerns and strategies. Drug Alcohol Depend. 2006;81:103–7. 9. Bazazi AR, Zelenev A, Fu JJ, Yee I, Kamarulzaman A, Altice FL. High prevalence of non-fatal overdose among people who inject drugs in Malaysia: Correlates of overdose and implications for overdose prevention from a cross-sectional study. Int J Drug Policy. 2015;26:675–81. 9. Bazazi AR, Zelenev A, Fu JJ, Yee I, Kamarulzaman A, Altice FL. High prevalence of non-fatal overdose among people who inject drugs in Malaysia: Correlates of overdose and implications for overdose prevention from a cross-sectional study. Int J Drug Policy. 2015;26:675–81. 10. Kurdi M, Theerth K, Deva R. Ketamine: Current applications in anesthesia, pain, and critical care. Anesth Essays Res. 2014;8:283. 10. Kurdi M, Theerth K, Deva R. Ketamine: Current applications in anesthesia, pain, and critical care. Anesth Essays Res. 2014;8:283. 11. References Clinical Trial Acad Emerg Med. 2014;21:1194–202. 17. Motov S, Rockoff B, Cohen V, Pushkar I, Likourezos A, McKay C, Soleyman-Zomalan E, Homel P, Terentiev V, Fromm. C 2015 Intravenous Subdissociative-Dose Ketamine Versus Morphine for Analgesia in the Emergency Department: A Randomized Controlled Trial Ann. Emerg. Med. 66 222– 229.E1. 17. Motov S, Rockoff B, Cohen V, Pushkar I, Likourezos A, McKay C, Soleyman-Zomalan E, Homel P, Terentiev V, Fromm. C 2015 Intravenous Subdissociative-Dose Ketamine Versus Morphine for Analgesia in the Emergency Department: A Randomized Controlled Trial Ann. Emerg. Med. 66 222– 229.E1. 18. Motov S, Mai M, Pushkar I, Likourezos A, Drapkin J, Yasavolian M, Brady J, Homel P, Fromm. C 2017 A prospective randomized, double-dummy trial comparing intravenous push dose of low dose ketamine to short infusion of low dose ketamine for treatment of moderate to severe pain in the emergency department Am. J. Emerg. Med.. 19. Gao M, Rejaei D, Liu H. Ketamine use in current clinical practice. Acta Pharmacol Sin. 2016;37:865– 72. 19. Gao M, Rejaei D, Liu H. Ketamine use in current clinical practice. Acta Pharmacol Sin. 2016;37:865– 72. 20. Johansson P, Kongstad P, Johansson A. The effect of combined treatment with morphine sulphate and low-dose ketamine in a prehospital setting. Scand J Trauma Resusc Emerg Med. 2009;17:61. 20. Johansson P, Kongstad P, Johansson A. The effect of combined treatment with morphine sulphate and low-dose ketamine in a prehospital setting. Scand J Trauma Resusc Emerg Med. 2009;17:61. 21. Beaudoin FL, Lin C, Guan W, Merchant RC. Low-dose Ketamine Improves Pain Relief in Patients Receiving Intravenous Opioids for Acute Pain in the Emergency Department: Results of a Randomized, Double-blind, Clinical Trial ed M B Mycyk. Acad Emerg Med. 2014;21:1193–202. 21. Beaudoin FL, Lin C, Guan W, Merchant RC. Low-dose Ketamine Improves Pain Relief in Patients Receiving Intravenous Opioids for Acute Pain in the Emergency Department: Results of a Randomized, Double-blind, Clinical Trial ed M B Mycyk. Acad Emerg Med. 2014;21:1193–202. 22. Miller JP, Schauer SG, Ganem VJ, Bebarta VS. Low-dose ketamine vs morphine for acute pain in the ED: A randomized controlled trial. Am J Emerg Med. 2014;33:402–8. 22. Miller JP, Schauer SG, Ganem VJ, Bebarta VS. Low-dose ketamine vs morphine for acute pain in the ED: A randomized controlled trial. Am J Emerg Med. 2014;33:402–8. 23. Adriaenssens G, Vermeyen KM, Hoffmann VLH, Mertens E, Adriaensen HF. Postoperative analgesia with i.v. patient-controlled morphine: Effect of adding ketamine. References Br J Anaesth. 1999;83:393–6. 23. Adriaenssens G, Vermeyen KM, Hoffmann VLH, Mertens E, Adriaensen HF. Postoperative analgesia with i.v. patient-controlled morphine: Effect of adding ketamine. Br J Anaesth. 1999;83:393–6. 24. Ahern TL, Herring AA, Stone MB, Frazee BW. Effective analgesia with low-dose ketamine and reduced dose hydromorphone in ED patients with severe pain. Am J Emerg Med. 2013;31:847–51. 24. Ahern TL, Herring AA, Stone MB, Frazee BW. Effective analgesia with low-dose ketamine and reduced dose hydromorphone in ED patients with severe pain. Am J Emerg Med. 2013;31:847–51. References Surgical and Emergency Medicine Services Unit. Medical Development Section of the Medical Development Division Health M of 2013 Pain Management Handbook. 11. Surgical and Emergency Medicine Services Unit. Medical Development Section of the Medical Development Division Health M of 2013 Pain Management Handbook. 12. Laulin J-P, Maurette P, Corcuff J-B, Rivat C, Chauvin M, Simonnet G. The role of ketamine in preventing fentanyl-induced hyperalgesia and subsequent acute morphine tolerance. Anesth Analg. 2002;94:1263–9. table of contents. 12. Laulin J-P, Maurette P, Corcuff J-B, Rivat C, Chauvin M, Simonnet G. The role of ketamine in preventing fentanyl-induced hyperalgesia and subsequent acute morphine tolerance. Anesth Analg. 2002;94:1263–9. table of contents. 13. Schmid RL, Sandler AN, Katz J. 1999 Use and efficacy of low-dose ketamine in the management of acute postoperative pain: A review of current techniques and outcomes Pain 82 111–25. 13. Schmid RL, Sandler AN, Katz J. 1999 Use and efficacy of low-dose ketamine in the management of acute postoperative pain: A review of current techniques and outcomes Pain 82 111–25. 14. Jennings PA, Cameron P, Bernard S, Walker T, Jolley D, Fitzgerald M, Masci. K 2012 Morphine and ketamine is superior to morphine alone for out-of-hospital trauma analgesia: A randomized controlled trial Ann. Emerg. Med. 59 497–503. 14. Jennings PA, Cameron P, Bernard S, Walker T, Jolley D, Fitzgerald M, Masci. K 2012 Morphine and ketamine is superior to morphine alone for out-of-hospital trauma analgesia: A randomized controlled trial Ann. Emerg. Med. 59 497–503. 15. Chudnofsky CR, Weber JE, Stoyanoff PJ, Colone PD, Wilkerson MD, Hallinen DL, Jaggi FM, Boczar ME, Perry. M a 2000 A combination of midazolam and ketamine for procedural sedation and analgesia in adult emergency department patients. Acad. Emerg. Med. 7 228–35. 15. Chudnofsky CR, Weber JE, Stoyanoff PJ, Colone PD, Wilkerson MD, Hallinen DL, Jaggi FM, Boczar ME, Perry. M a 2000 A combination of midazolam and ketamine for procedural sedation and analgesia in adult emergency department patients. Acad. Emerg. Med. 7 228–35. 16. Beaudoin FL, Lin C, Guan W, Merchant RC. Low-dose Ketamine Improves Pain Relief in Patients Receiving Intravenous Opioids for Acute Pain in the Emergency Department: Results of a 16. Beaudoin FL, Lin C, Guan W, Merchant RC. Low-dose Ketamine Improves Pain Relief in Patients Receiving Intravenous Opioids for Acute Pain in the Emergency Department: Results of a Page 14/17 Page 14/17 Randomized, Double-blind. Clinical Trial Acad Emerg Med. 2014;21:1194–202. Randomized, Double-blind. Figures Page 15/17 Page 15/17 Figure 1 Study flow for consented subjects. a Subjects were missing data because of transfer from the ED to ward. Study flow for consented subjects. a Subjects were missing data because of transfer from the ED to ward. Page 16/17 Page 17/17 Figure 2 Mean pain score over time. Figure 2 Mean pain score over time. Page 17/17 Page 17/17 Page 17/17
https://openalex.org/W4313125907	https://www.epj-conferences.org/10.1051/epjconf/202227408012/pdf	English	null	Holographic spacetime from lattice Yang-Mills theory	EPJ web of conferences	2,022	cc-by	2,929	Holographic spacetime from lattice Yang-Mills theory Niko Jokela1,2,∗, Arttu Pönni3,∗∗, Tobias Rindlisbacher3,∗∗∗, Kari Rummukainen1,2,∗∗∗∗, and Ahmed Salami1,2,† 1Helsinki Institute of Physics and Department of Physics, P.O. Box 64, FI-00014 University of Helsinki, Finland 2Micro and Quantum Systems Group, Department of Electronics and Nanoengineering, Aalto Univer- sity, Finland 3AEC, Institute for Theoretical Physics, University of Bern, Sidlerstrasse 5, CH-3012 Bern, Switzerland Abstract. Entanglement entropy is a notoriously diﬃcult quantity to compute in strongly interacting gauge theories. Existing lattice replica methods have suf- fered from a severe signal-to-noise ratio problem, making high-precision stud- ies prohibitively expensive. Our improved lattice method mitigates this situation and allows us to probe holographic predictions for the behavior of entanglement entropies in three- and four-dimensional Yang-Mills theories. We use this data for the numerical reconstruction of holographic bulk metrics. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). , 08012 (2022) th EPJ Web of Conferences 274 XV Quark Confinement and the Hadron Spectrum , 08012 (2022) th EPJ Web of Conferences 274 XV Quark Confinement and the Hadron Spectrum https://doi.org/10.1051/epjconf/202227408012 ∗e-mail: niko.jokela@helsinki.ﬁ ∗∗e-mail: arttuponni@gmail.com ∗∗∗e-mail: trindlis@itp.unibe.ch ∗∗∗∗e-mail: kari.rummukainen@helsinki.ﬁ †e-mail: ahmed.salami@helsinki.ﬁ 2 Entanglement as a probe of conﬁnement Let us ﬁrst recall the basic argument [1, 2] on why we expect some entanglement measure to indicate in which phase we are in at large-N. The idea is that in the deconﬁned phase of, say, SU(N) gauge ﬁeld theory, the degrees of freedom (dofs) that are eﬀective are colorful (e.g., gluons) and the entropy density scales as ∼O(N2) while in the conﬁned phase the eﬀective dofs are color singlets (e.g., glueballs) and the entropy density scales as ∼O(N0). Now, if one is at large-N, in particular, a quantity that captures dofs (such as an entanglement en- tropic c-function) acts as a sharp order parameter. Indeed, this expectation has been met in holographic works [1, 2], where the (derivative of the) holographic EE undergoes a transition from a ﬁnite to a vanishing value at around the scale of conﬁnement. Later this argument has been sharpened to a statement [3] that the holographic EE undergoes this transition when- ever there is an intrinsic energy scale in the system and so holographic EE probes the ﬁnite correlation length instead, beyond of which the EE is saturated. One goal in our program is to study entanglement c-function as extracted from lattice SU(N) Yang-Mills theory and explore if a precursor to a sharp order parameter emerges with increasing N. The determination of entanglement measures in gauge theories is a diﬃcult task, but with the so-called “replica trick”, a family of entanglement measures, Rényi en- tropies, becomes available with lattice Monte Carlo. In this work we will essentially follow the prescription [4]. However, its direct implementation suﬀers from a severe signal-to-noise ratio problem, which makes accurate determination of Rényi entropies very expensive. The improved method we have developed [5] is powerful enough to make meaningful comparison to holographic predictions. In addition, we will provide astounding evidence that the ﬁnite part of the entanglement entropy for strip subsystems in three dimensions scales linearly with the width, the slope matching with the Bekenstein-Hawking entropy of the dual black brane. , 08012 (2022) th EPJ Web of Conferences 274 XV Quark Confinement and the Hadron Spectrum , 08012 (2022) EPJ Web of Conferences 274 https://doi.org/10.1051/epjconf/202227408012 In this talk we reveal some results on our lattice exploration of entanglement entropies of bipartite pure systems in three- and four-dimensional bosonic pure glue Yang-Mills theories. As proxies for holographic suggestions for the behaviors of EEs (and Wilson loops) on what to expect, we consider supersymmetric versions of these ﬁeld theories at large-N. We will be stupeﬁed on how well the holographic expectations are met. 1 Introduction Color conﬁnement in QCD is a very hard problem. Since the phenomenon is non-perturbative in nature, its description has deﬁed theorists to date. There are heroic numerical works, but it is fair to say that we are still far from a complete picture. Instead of directly addressing conﬁnement in QCD, one often also seeks simple models and aims to gain some understanding that could be transferred back to QCD. One possible route is oﬀered via AdS/CFT or holography in short. In fact, one needs to break conformal symmetry in a controlled manner, a terminology better suited would be non-AdS/non-CFT correspondence. The holographic tools that are probes of conﬁnement include various loops (Wilson, ’t Hooft etc.), entanglement entropy (EE), and c-functions that count the number of degrees of freedom. In particular, entanglement c-functions seem quite intriguing, since they can also provide a clean score card on holography. We will mostly focus on this last probe. We are nowhere near solving conﬁnement even in holographic large-N ﬁeld theories, but the hope is that the methods we build using lattice formulation will feed insight also into conﬁnement in the long run. Our intermediate goals are rather challenging. They deal with also not-so-well-understood phenomena, entanglement of subsystems in gauge ﬁeld theories, especially at strong coupling. The study of entanglement measures in gauge theories is of course interesting in its own right. ∗e-mail: niko.jokela@helsinki.ﬁ ∗∗e-mail: arttuponni@gmail.com ∗∗∗e-mail: trindlis@itp.unibe.ch ∗∗∗∗e-mail: kari.rummukainen@helsinki.ﬁ †e-mail: ahmed.salami@helsinki.ﬁ © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). , 08012 (2022) th EPJ Web of Conferences 274 XV Quark Confinement and the Hadron Spectrum 3 Bulk reconstruction Once there is some data for a gauge ﬁeld theory in question, one can ask whether there is a dual gravity description from where this data could in principle be obtained. This is an inverse problem, applied AdS/CFT done in reverse. The entanglement measure data is in some sense the optimal starting point: the holographic candidate [6] only involves ﬁnding an extremal surface given a metric involving no assumptions on the matter content in the bulk. Of course, we are not able to construct all metric components. In particular, since our lattice method is naturally formulated in Euclidean signature, there are slim chances to get accurate predictions for time-dependent phenomena. Nevertheless, this limitation should not be taken as a discouragement, but perhaps be seen as an intermediate obstruction that can be circumvented via some analytic continuation to Lorentzian signature or with the help of bulk energy momentum tensor to ﬁx gtt, similarly to [7]. EE itself is not observable since it is UV-divergent. Some derived quantities are a better ﬁt for bulk reconstruction. In [8] it was proposed to use the derivative of the EE instead. Indeed, one can explicitly show that the derivative of EE with respect to the strip width, ∂ℓS EE, can be repackaged in a very convenient local expression with no explicit integrals, in terms of metric 2 2 , 08012 (2022) th EPJ Web of Conferences 274 XV Quark Confinement and the Hadron Spectrum , 08012 (2022) EPJ Web of Conferences 274 https://doi.org/10.1051/epjconf/202227408012 components evaluated at the bottom of the area functional whose extremal value captures the EE. The conﬁdence intervals for the metric components are thereby fed from the statistical errors in the ∂ℓS EE data in a very transparent manner. components evaluated at the bottom of the area functional whose extremal value captures the EE. The conﬁdence intervals for the metric components are thereby fed from the statistical errors in the ∂ℓS EE data in a very transparent manner. 4 Results The writings of the articles are underway, but here we will present some results that illustrate how well the method of extracting the entanglement entropy derivative from the lattice works. We will mostly focus on three-dimensional SU(2) Yang-Mills theory, so the relevant quantity in question is ∂S EE/∂ℓvs. ℓ, where ℓis the width of a strip subsystem. In three dimensions we mostly focus on ﬁnite temperature. In [5] and in the upcoming articles we also discuss four-dimensional YM theory with diﬀerent numbers of colors in more depth. In particular, one interesting focal point is the entanglement c-function. In Fig. 1 we depict preliminary results for the entanglement c- function C(ℓ) ∝ℓ3∂ℓS EE Figure 1. Comparison of our c-function results (black markers) for SU(3) in four dimensions with the results from [9]. This is at vanishing temperature. Figure 1. Comparison of our c-function results (black markers) for SU(3) in four dimensions with the results from [9]. This is at vanishing temperature. Let us now continue to three dimensions. In Fig. 2 we depict the derivative of the entan- glement entropy with respect to the strip width ∂S EE/∂ℓfor varying values of the temperature. We note two things. First of all, one ﬁnds that for small values of ℓ, one gets ∂ℓS EE ∼ℓ−7/3; this exponent is ﬁtted to good accuracy using a log-log scale. The exponent −7/3 is consis- tent with the expectation from D2-background, namely, that the ﬁnite part of EE scales as S EE ∼ℓ−4/3 [10]. The other observation is that ∂ℓS EE saturates to a constant in the large-ℓlimit. This is also expected since the RT surface is supposed to trace the black hole horizon and the area functional should scale linearly with ℓ. Indeed, we can associate this constant value with the Bekenstein-Hawking entropy of the black hole. We have, for example, checked that this scales as T 7/3, also expected for D2-brane background at ﬁnite temperature. Parts of the gravity metric can be readily reconstructed from the data, for example from that depicted in Fig. 2. In Fig. 3 we show radial-radial metric component of the metric, including the conﬁdence intervals. 4 Results This metric can then be used, in principle, to make some 3 3 , 08012 (2022) EPJ Web of Conferences 274 https://doi.org/10.1051/epjconf/202227408012 th XV Quark Confinement and the Hadron Spectrum 0.5 1 1.5 0 10 20 30 l [Λcr] \|∂A -1 ΔS/Δl [Λcr -2] fit: y = a (x-x0)-7/3+b →a=0.006(4) →b=23.58(7) →x0=0.039(10) →a=0.010(2) →b=11.85(8) →x0=0.033(3) →a=0.013(2) →b=6.85(8) →x0=0.029(3) →a=0.013(2) →b=4.39(9) →x0=0.029(3) data: V=6⨯962, β=48. V=8⨯962, β=48. V=10⨯962, β=48. V=12⨯962, β=48. Figure 2. The derivative of the entanglement entropy wrt. strip width ℓin three-dimensional SU(2) Yang-Mills theory. The diﬀerent curves represent increasing temperature (bottom-up). The ﬁt here is just to guide the eye, more reﬁned analyses reveal the IR (large-ℓ) ∂ℓS EE ∼T 7/3 and UV (small-ℓ) ∂ℓS EE ∼ℓ−7/3 behaviors. 0.5 1 1.5 0 10 20 30 l [Λcr] \|∂A -1 ΔS/Δl [Λcr -2] fit: y = a (x-x0)-7/3+b →a=0.006(4) →b=23.58(7) →x0=0.039(10) →a=0.010(2) →b=11.85(8) →x0=0.033(3) →a=0.013(2) →b=6.85(8) →x0=0.029(3) →a=0.013(2) →b=4.39(9) →x0=0.029(3) data: V=6⨯962, β=48. V=8⨯962, β=48. V=10⨯962, β=48. V=12⨯962, β=48. Figure 2. The derivative of the entanglement entropy wrt. strip width ℓin three-dimensional SU(2) Yang-Mills theory. The diﬀerent curves represent increasing temperature (bottom-up). The ﬁt here is just to guide the eye, more reﬁned analyses reveal the IR (large-ℓ) ∂ℓS EE ∼T 7/3 and UV (small-ℓ) ∂ℓS EE ∼ℓ−7/3 behaviors. predictions for other quantities. Alas, not all metric components can be constructed this way, especially the time-time component is seemingly out of reach due to lattice bound to Euclidean form. In addition, the metric as constructed is in the Einstein frame, while for example the holographic prescription for computing the Wilson loop requires the string frame metric. Nevertheless, by assuming the D2-brane ansatz for the dilaton eφ = (z/rp)5/4, with rp the curvature radius, one obtains a reasonable result for the static quark potential; see Fig. 3. The potential vanishes at some critical separation, corresponding to fundamental string breaking transition in the bulk. 0.1 0.2 0.3 0.4 T ℓ -1.5 -1.0 -0.5 α' V T2/3 rp5/3 Figure 3. Left: One of the metric components as reconstructed from the SU(2) lattice data for ∂ℓS EE at T/Tc = 2.646. Right: The prediction for the static quark potential given the metric on the left. In these plots the dashed curve is the maximum likelihood estimate of the data. 5 Discussion We presented ﬁrst results for the derivatives of the entanglement entropies for slab subsystems in bosonic SU(N) Yang-Mills theories in three and four dimensions using our improved lattice method. We showed that many non-trivial holographic predictions are consistent to numerical accuracy with our data. We also showed that one can reconstruct the corresponding gravity dual metric, including conﬁdence intervals. Lots of work is still needed in the construction of the dual geometry to large-N QCD, but our work has led us to the era of precision holography. In addition to improving our understanding on what the holography has to oﬀer to strongly coupled phases of gauge ﬁeld theories, there are several other entanglement measures wait- ing to be calculated on the lattice. Among these are entanglement entropies for multiparty systems, but also mixed state entanglement entropies. Other interesting extensions would be towards anisotropy and address the murky situation of whether there even exists an entangle- ment monotone [14]. , 08012 (2022) th EPJ Web of Conferences 274 XV Quark Confinement and the Hadron Spectrum https://doi.org/10.1051/epjconf/202227408012 Encouraged by the unreasonable success of the holographic expectations and by espe- cially the fact that the D2-brane background at ﬁnite temperature has its known limitations, we launched the computation of temporal Polyakov loops from the same lattice system. For small (but not too small) separation ℓof the quark pair, the D2-brane background implies that the q¯q-potential scales as ℓ−2/3 [10]. The lattice results that we obtain meet this expectation. In the large separation limit the standard expectation is that the fundamental string breaks around the hadronic scale and one ﬁnds a ﬂat potential as in Fig. 3. However, one can follow the prescription given in [11], i.e., ignore the string breaking and follow subdominant saddle for larger ℓand ﬁnally to the complex plane. In the large-ℓlimit, we ﬁnd ReV ∼ℓ−10/3 and ImV ∼1/ℓ. The data from the lattice we get is of course real but it is nevertheless consis- tent with the exponent −10/3. It is also consistent with the scaling that one expects from Debye screening e−mDℓ/ √ ℓ[12]. It would be interesting to dissect which one survives in the long run. For example, one could attempt to extract the imaginary part of the potential by following the real-time formalism in [13] and see if 1/ℓis obtained. Acknowledgments N. J. would like to thank the organizers of the XVth Quark Conﬁnement and the Hadron Spectrum 2022 conference in Stavanger, Norway, for the invitation to present this talk. The support of the Academy of Finland grants no. 1322307, 1320123, and 1345070 are acknowl- edged. 4 Results The black dashed curve is the median in the distribution of gzz while the dashed gray curves represent the 50% and 95% central conﬁdence intervals, respectively. -1.5 -1.0 -0.5 α' T2/3 r 0.1 0.2 0.3 0.4 T ℓ -1.5 -1.0 -0.5 α' V T2/3 rp5/3 Figure 3. Left: One of the metric components as reconstructed from the SU(2) lattice data for ∂ℓS EE at T/Tc = 2.646. Right: The prediction for the static quark potential given the metric on the left. In these plots the dashed curve is the maximum likelihood estimate of the data. The black dashed curve is the median in the distribution of gzz while the dashed gray curves represent the 50% and 95% central conﬁdence intervals, respectively. , 08012 (2022) th EPJ Web of Conferences 274 XV Quark Confinement and the Hadron Spectrum , 08012 (2022) EPJ Web of Conferences 274 [10] J.M. Maldacena, Phys. Rev. Lett. 80, 4859 (1998), hep-th/9803002 [11] J.L. Albacete, Y.V. Kovchegov, A. Taliotis, Phys. Rev. D 78, 115007 (2008), 0807.4747 [12] A. Dumitru, R.D. Pisarski, Phys. Rev. D 66, 096003 (2002), hep-ph/0204223 [13] Y. Burnier, O. Kaczmarek, A. Rothkopf, Phys. Rev. Lett. 114, 082001 (2015), 1410.2546 [14] C. Hoyos, N. Jokela, J.M. Penín, A.V. Ramallo, J. Tarrío, JHEP 10, 112 (2021), 2104.11749 [10] J.M. Maldacena, Phys. Rev. Lett. 80, 4859 (1998), hep-th/9803002 [ ] y ( ) p [11] J.L. Albacete, Y.V. Kovchegov, A. Taliotis, Phys. Rev. D 78, 115007 (2008), 0807.4747 [ ] , , y , ( ), p p / [13] Y. Burnier, O. Kaczmarek, A. Rothkopf, Phys. Rev. Lett. 114, 082001 (2015), 1410.2546 g y ( ) [12] A. Dumitru, R.D. Pisarski, Phys. Rev. D 66, 096003 (2002), hep-ph/0204223 [14] C. Hoyos, N. Jokela, J.M. Penín, A.V. Ramallo, J. Tarrío, JHEP 10, 112 (2021), 2104.11749 References [1] T. Nishioka, T. Takayanagi, JHEP 01, 090 (2007), hep-th/0611035 [2] I R Kl b D K t A M N l Ph B 796 274 (2008) 0709 2140 [1] T. Nishioka, T. Takayanagi, JHEP 01, 090 (2007), hep-th/0611035 [2] I.R. Klebanov, D. Kutasov, A. Murugan, Nucl. Phys. B 796, 274 (2008), 0709.2140 [1] T. Nishioka, T. Takayanagi, JHEP 01, 090 (2007), hep-th/0611035 [2] I.R. Klebanov, D. Kutasov, A. Murugan, Nucl. Phys. B 796, 274 (2008), 0709 [3] N. Jokela, J.G. Subils, JHEP 02, 147 (2021), 2010.09392 [4] P.V. Buividovich, M.I. Polikarpov, Nucl. Phys. B 802, 458 (2008), 0802.4247 [5] T. Rindlisbacher, N. Jokela, A. Pönni, K. Rummukainen, A. Salami, PoS LAT- TICE2022, 031 (2022), 2211.00425 [6] T. Nishioka, S. Ryu, T. Takayanagi, J. Phys. A 42, 504008 (2009), 0905.0932 [7] A. Saha, S. Karar, S. Gangopadhyay, Eur. Phys. J. Plus 135, 132 (2020), 1807.046 [8] N. Jokela, A. Pönni, Phys. Rev. D 103, 026010 (2021), 2007.00010 [9] A. Rabenstein, N. Bodendorfer, A. Schäfer, P. Buividovich, Phys. Rev. D100, 034504 (2019), 1812.04279 5 5 , 08012 (2022) EPJ Web of Conferences 274 https://doi.org/10.1051/epjconf/202227408012 th XV Quark Confinement and the Hadron Spectrum [13] Y. Burnier, O. Kaczmarek, A. Rothkopf, Phys. Rev. Lett. 114, 082001 (2015), 1410.2546 [14] C. Hoyos, N. Jokela, J.M. Penín, A.V. Ramallo, J. Tarrío, JHEP 10, 112 (2021), 2104.11749 6
https://openalex.org/W4290093479	https://zenodo.org/records/6967731/files/DTU3.pdf, https://zenodo.org/record/6511955/files/DTU3.pdf, https://zenodo.org/record/6570504/files/DTU3.pdf, https://zenodo.org/record/6967740/files/DTU3.pdf, https://zenodo.org/record/6554749/files/DTU3.pdf	es		Diccionario Temático de Ufología	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	112,911	Diccionario Temático de Ufología DTU Edición XXV aniversario - Versión digital En memoria de nuestro compañero y amigo Ricardo Caruncho (1961-1996) Matías Morey Ripoll (Coordinador de la obra) [AA]: Alejandro Agostinelli [AP]: Antonio Petit Gancedo [AR]: Ángel Rodríguez Álvarez [CL]: Carlos León Martínez [IC]: Ignacio Cabria García [ JA]: Julio Arcas Gilardi [ JAB]: Jordi Ardanuy Baró [ JM]: José Juan Montejo Aguilera [ JR]: José Ruesga Montiel [LG]: Luis R. González Manso [MB]: Manuel Borraz Aymerich [MF]: Martí Flò García [MG]: Matilde González García [MGC]: Miguel Guasp Carrascosa [MM]: Matías Morey Ripoll [PR]: Pere Redón Trabal [RC]: Ricardo Campo Pérez [VB]: Vicente-Juan Ballester Olmos Revisión de esta versión digital: Diego Zúñiga C. © De las dos primeras ediciones, Fundación Anomalía, 1997, 2000. © De esta tercera edición, los autores, 2022. Obra sujeta a una licencia Creative Commons Atribución-No comercialCompartir igual, CC BY-NC-SA. Presentación PRESENTACIÓN (2022) En 1997, cuando se cumplían cincuenta años desde el avistamiento de Kenneth Arnold del 24 de junio de 1947, acaecido cerca del monte Rainier, en el estado de Washington (hecho que se tiene convencionalmente como fecha de inicio de la era ufológica), la Fundación Anomalía, creada apenas un año antes, quiso conmemorar la efeméride con la publicación de un diccionario que, de alguna forma, estableciera cuál era el punto de la cuestión después de medio siglo de debates sobre una casuística tan controvertida. Han pasado ya veinticinco años desde la aparición del Diccionario Temático de Ufología (DTU), como así se le llamó, y en 2022 se celebra el septuagésimo quinto aniversario del caso Arnold, dos hitos que bien merecen ser recordados con una nueva edición de dicha obra. Por todo ello, el texto aquí recogido no difiere de la versión digital (de 2000) que estuvo tantos años colgada de la web de la Fundación, excepto en estos concretos aspectos: a) se han implementado enlaces en el PDF para facilitar la navegación entre voces; b) se han incorporado las fechas de fallecimiento o extinción tanto de las personas como de las organizaciones o publicaciones reseñadas; y c) se han incluido nuevas direcciones de Internet si las anteriores ya no están operativas. En todo lo demás, se ha mantenido el mismo contenido de la versión en papel de 1997, salvo los errores ya solventados en la segunda edición, y sin las imágenes que ilustraban el libro impreso. Finalmente, sólo nos resta agradecer la contribución de los coautores de la obra, sin la cual ésta no hubiese sido posible, y reiterarles mi más sentido reconocimiento por haberse atrevido a colaborar en un proyecto tan arriesgado y con tan pocos precedentes en la ufología española. Dejo al lector que juzgue por sí mismo si valió la pena su empeño, que para mí, veinticinco años después, considero que sólo me sigue mereciendo elogios. DTU Pese a que hemos sido tentados con elaborar una revisión completa del DTU, finalmente hemos desistido por la magnitud de la empresa y porque, a fin de cuentas, toda obra es fruto de su época, y reescribirla hoy en día supondría no sólo modificarla radicalmente (pues el tiempo transcurrido desde su publicación no ha pasado en vano, y ahora las perspectivas son muy diferentes a las existentes en 1997), sino también hacerle perder el carácter testimonial que posee a modo de ejemplo de cómo se planteaba la ufología a finales del siglo XX. No es que neguemos que no sean convenientes libros como éste en la actualidad, sino que en tal caso habría que redactar una obra ya completamente diferente, y no el DTU que todos conocemos. Matías Morey Ripoll Coordinador de la obra 2 Introducción INTRODUCCIÓN (1997) A lo largo de los cincuenta años que ahora nos sirven de excusa para editar la presente obra, lo que hemos venido en llamar Ufología ha llegado a crear un ingente y desordenado cuerpo doctrinal, fuertemente endogámico y poco accesible para los no iniciados, que nunca se ha caracterizado por su sistematización. Pocos han sido los esfuerzos que desde dentro de la comunidad ufológica se han dirigido a remediar la situación, y menos aún los que pueden considerarse dignos de alguna reseña. El fuerte dogmatismo proextraterrestre que imperó hasta los años ochenta lastró lastimosamente la mayoría de estas iniciativas, que hoy en día no tienen sino un valor puramente histórico o anecdótico. En los últimos tiempos hemos asistido a una renovación de los intentos codificadores que, con mayor o menor fortuna según los casos, han adoptado la forma de obras de consulta como The Encyclopedia of UFOs de Ronald D. Story, la UFO Encyclopedia de Jerome K. Clark o el Dizionario Ufologico del CISU. Naturalmente, se ha tratado de compilaciones muy alejadas tanto de las coordenadas culturales del mundo hispánico como de la idiosincracia propia de nuestra ufología, y, sobre todo, escritas en otras lenguas, de manera que han sido muy pocos los lectores españoles que han tenido acceso a ellas. Huérfanos por tanto de una obra de estas caracaterísticas en castellano, con la única excepción del Primer Diccionario Argentino de Ovnilogía de Ruth Gerstel (muy limitado en todos los aspectos), hemos creído que la mejora manera de presentar la recién creada Fundación Anomalía y de dar a conocer el estilo de hacer Ufología que pretende patrocinar era publicar por vez primera en España un Diccionario Temático de Ufología (DTU) que contribuyera por un lado a paliar las citadas carencias y, por otro, a divulgar esta disciplina de una manera diferente a como suele hacerse habitualmente. Sin más precedentes en nuestro país que el ya añejo Vocabulario Ufológico que hace quince años nuestro compañero Joan Plana dio a conocer por entregas en la revista Karma-7 (una especie de “abuelo espiritual” de esta obra que fue plagiado sin rubor en diversas ocasiones), el DTU se plantea cumplir sus objetivos a partir de la consideración de las diversas categorías de voces que lo integran. Así, en primer lugar, reúne lo que propiamente pueden definirse como términos ufológicos, es decir, todas aquellas palabras acuñadas en el seno de la Ufología o tomadas de otros ámbitos que constituyen el vocabulario usual para referirse a las realidades creadas o contempladas por los investigadores (abducción, humanoide, nave nodriza, ortotenia). Al igual que en otros aspectos de la cultura contemporánea, la fuerte influencia del idioma inglés -presente ya en el propio nombre de la disciplina- ha aconsejado en algunos casos reseñar entre paréntesis tras la adaptación española la palabra inglesa de la que procede. Igualmente, hemos aplicado la misma solución a ciertos sinónimos cuya cita independiente no parecía justificada. Como conceptos en sentido amplio, recoge el DTU tanto el relato de diversos hechos y episodios de la historia de la Ufología como el análisis de las 3 Introducción interacciones de ésta con los mundos de la ciencia, la cultura y la sociedad, y de las nuevas realidades que así se han generado. Cabe decir que se trata del grupo más numeroso de voces y el que posiblemente encuentre más interesante el lector, donde hallará abordadas por primera vez con detenimiento las últimas novedades surgidas en el mundillo ufológico. una panorámica general de las publicaciones de consulta imprescindible en este ámbito, tanto contemporáneas como desaparecidas pero de gran importancia histórica. Por último, el DTU incluye una decena de casos presentados de forma independiente y extensa, junto a otros expuestos o reseñados en sede de diversas entradas. Tratándose de un aspecto abordado hasta la saciedad en tantas y tantas obras sobre Ufología, no hemos creído adecuado inflar el diccionario con numerosas y prolijas descripciones de casuística, sino que hemos preferido incorporar únicamente una muestra de incidentes representativa de las diversas categorías posibles y que a su vez recogiera algunos de los sucesos de mayor relevancia histórica o social. Aquí encontrará el lector -quizás lamentablemente por primera vezlas referencias más actuales y creemos que objetivas de los hechos cuyo testimonio conforma el campo de estudio de nuestra disciplina, incluyendo las de algunos casos que aún no han podido ser satisfactoriamente explicados. En el apartado de biografías, las restricciones impuestas por el carácter divulgativo de la obra y por las limitaciones de espacio nos han aconsejado reducir a unas cincuenta el número de personalidades recogidas. Obviamente no están todos los que son, pero sí son todos los que están, entre los que el interesado encontrará tanto los clásicos imprescindibles como las figuras más reseñables de la actualidad, con especial mención de los autores hispanos, iberoamericanos y europeos, pocas veces citados en las habituales recopilaciones anglosajonas. Siempre que ha sido posible, la vida y obras del biografiado han sido complementadas con su opinión personal sobre el fenómeno OVNI, tomada de las fuentes más recientes. Como complemento a todo ello, un apéndice bibliográfico con más de doscientas cincuenta obras relaciona cada una de las referencias que aparecen numeradas entre corchetes al final de los artículos. Aquí hallará sin duda el buen aficionado material más que suficiente para ir profundizando por su cuenta en el, en demasiadas ocasiones, laberíntico terreno de los no identificados. Del mismo modo, el DTU dedica una especial atención a los grupos de aficionados y estudiosos, que han sido seleccionados con parecidos criterios, incluyendo a todos los actualmente existentes en España con cierta solera, y a los más relevantes de los países de mayor tradición ufológica. De acuerdo al espíritu de los tiempos que corren, y por vez primera en una obra de este tipo, se ha incluido en dichas entradas la dirección de las páginas web de aquellas organizaciones que ya se dan a conocer a través de Internet. Este dato está también presente en algunas de las voces dedicadas a las principales revistas ufológicas, donde el lector hallará Del mismo modo que cada artículo aparece acompañado de su bibliografía, junto a ésta se encuentran, también en medio de corchetes, las iniciales del autor o autores del mismo. Las únicas excepciones son las de aquellas voces muy breves (cuya 4 Introducción responsabilidad recae sobre quien esto suscribe) y las de las biografías, respecto a las cuales se ha creído oportuno no personalizar los comentarios vertidos. La lista completa de los redactores del DTU -a quienes desde aquí expreso mi más profundo agradecimiento, en especial a Luis R. González por su generosa contribución y por la revisión final del texto- es la siguiente, presentada por el orden alfabético de las abreviaturas: presente obra, para recordar la necesidad de incardinar la Ufología en el seno de los conocimientos establecidos como condición insoslayable para abonar sus avances. En una época en la que se nos predica que “la verdad está ahí afuera” sólo para intentar justificar lo que de ninguna manera puede tener cabida en mentes medianamente formadas, no resulta ocioso recordar cosas tan elementales. Deseamos, por tanto, que el DTU sea útil al menos para situar la cuestión en su justa perspectiva, independientemente de cuál sea luego la particular opción por la que cada uno se decante. DTU [AA]: Alejandro Agostinelli [AP]: Antonio Petit Gancedo [AR]: Ángel Rodríguez Álvarez [CL]: Carlos León Martínez [IC]: Ignacio Cabria García [ JA]: Julio Arcas Gilardi [ JAB]: Jordi Ardanuy Baró [ JM]: José Juan Montejo Aguilera [ JR]: José Ruesga Montiel [LG]: Luis R. González Manso [MB]: Manuel Borraz Aymerich [MF]: Martí Flò García [MG]: Matilde González García [MGC]: Miguel Guasp Carrascosa [MM]: Matías Morey Ripoll [PR]: Pere Redón Trabal [RC]: Ricardo Campo Pérez [VB]: Vicente-Juan Ballester Olmos Matías Morey Ripoll Coordinador de la obra Dicho esto, sólo nos resta desear al lector que disfrute con los contenidos del DTU de manera proporcional al gran esfuerzo que ha supuesto su confección, para lo cual le resultará útil dejarse guiar por las remisiones implícitas entre voces que hemos destacado en versalitas. En un mundo fatalmente interconectado, ningún elemento de la realidad puede subsistir aisladamente por sí mismo, ya se trate de un término de este diccionario o de la propia disciplina en la que se inserta. Sirva esta reflexión, así como la 5 Abducción A ABDUCCIÓN lugares apartados sino que los testigos son secuestrados en sus propias casas, pasando a través de paredes y ventanas, sin ni siquiera observar ningún OVNI. Desde mediados de los años setenta, primero en los EE.UU. y luego en todo el mundo, los ufólogos han ido encontrando cada vez con mayor frecuencia testigos que aseguran haber sido conducidos al interior de los OVNIs, pero a diferencia de los contactados de los años cincuenta, por la fuerza. Este tipo de casos recibió el nombre de abducciones a partir de un término legal anglosajón. Historia y evolución del concepto. Consideradas en sus inicios como el elemento definitivo que podría darnos la clave de todo el fenómeno OVNI, pronto se comprobó todo lo contrario. De hecho, hoy puede asegurarse que este fenómeno ha tomado vida propia, alejándose cada vez más del entorno ufológico tradicional. Resulta oportuno intentar ofrecer una visión cronológica de su desarrollo. Al principio, el casi único común denominador de los informes de abducción, era esa entrada forzada en el OVNI: ni las naves, ni los propios seres, ni siquiera el supuesto examen médico, eran similares ni se daban en todos los casos. Ya en la década de los 80, Thomas Bullard tras analizar casi 300 informes pudo señalar una tipología de seres (cabezones, con grandes ojos y de color gris) y un orden estructural estable del incidente: 1) Captura, 2) Examen, 3) Conferencia de los seres, 4) Paseo por la nave, 5) Viaje a otro mundo, 6) Teofanía, 7) Retorno, y 8) Secuelas. Quizá no casualmente ese es también el orden adecuado de contar una historia dramática: presentación de personajes, peligro y conflicto, explicación, suspense, clímax y nuevos capítulos. En los años 50 algunos investigadores como el mayor Keyhoe o Morris Jessup apuntaron la posibilidad de que los tripulantes de los platillos volantes tomasen también muestras humanas en su progresivo estudio del planeta (algo que ya habia predicho por su cuenta la ciencia ficción de los años 30). Y claro, pronto empezaron a surgir historias en esta línea de gentes que, o bien habían escapado por los pelos, o bien habían visto a otras personas ser secuestradas. Junto a ellas, y no siempre claramente separables, florecieron los relatos de contactados. Pero es en 1957 y desde las remotas selvas de Brasil cuando se añade el morboso componente sexual, con el conocido relato de Antonio Villas Boas y su apasionada noche de amor dentro de un OVNI junto a una extraterrestre muda. Una de las razones de su inmerecida credibilidad era la presunción racista de que un granjero del interior de Brasil era incapaz de Pero ese orden estructural pronto quedó desfasado, y en la década de los 90, han proliferado más bien los llamados visitantes de dormitorio, cuando las supuestas abducciones ya no ocurren en 6 Abducción XXX inventarse tal relato (a juzgar por los informes semejantes que han ido llegando desde aquel país en años posteriores no es precisamente imaginación lo que les falta a los brasileños -o será que "sus" extraterrestres son más fogosas que en otras partes del mundo-). estado ausente 5 días, los detalles revelados bajo hipnosis son muy escasos, y la aceptación inicial del caso vino motivada por ser el único donde parecía haber testigos independientes (los otros miembros de su equipo de leñadores). De todas formas, los escépticos mantienen que se trató de un montaje. Este tipo de casos ofrecen gran atractivo para los medios de comunicación y alcanzan rápida y gran difusión. Así, en pocos años, se recogen relatos parecidos en Italia (caso Zanfretta, con humanoides escamosos y altos), Inglaterra (caso Aveley, con distintos tipos de seres) e incluso España (caso Julio F., con humanos altos de cráneo hiperdesarrollado). Resulta importante destacar que ninguna de las abducciones citadas incluye seres semejantes a los posteriores grises. Incluso el debatido caso Hill que tuvo lugar en Septiembre de 1961 no alcanzaría gran aceptación hasta años más tarde. Su mayor notoriedad se debió no tanto a la propia abducción recordada mediante técnicas de hipnosis, sino al famoso "mapa estelar" visto dentro de la nave por Betty Hill y que parecía señalar que los OVNIs procedian de Zeta Retículi. Aunque hubo otros casos similares en EE.UU. durante estos años, apenas si ofrecían coincidencias más allá de la forzada visita al interior del OVNI. No es hasta 1973 cuando tiene lugar una abundante oleada de casos de humanoides en EE.UU y los ufólogos americanos se ven obligados a aceptar esta clase de informes (denominados asépticamente "Encuentros Cercanos del Tercer Tipo"). Pero al hacerlo, quedaron en el umbral casos como el de Pascagoula (Florida) donde dos pescadores fueron abducidos por una especie de momias unípedas, o el declarado fraude de Dionisio Llanca en Argentina que aseguró haber sido secuestrado por dos hombres y una mujer perfectamente humanos que lo llevaron al interior de un OVNI donde no fue examinado. Esta proliferación de casos quedó bruscamente interrumpida a finales de la década de los setenta durante varios años, período en que apenas se divulgaron avistamientos de OVNIs, y menos aún abducciones. Quizá por ello, la atención de los ufólogos, hasta entonces reticentes, acabó volviéndose hacia esos casos de alta extrañeza y peligrosa credibilidad. El Dr. Leo Sprinkle fue el pionero en la investigación de las abducciones mediante hipnosis regresiva manteniendo una actitud positiva hacia el fenómeno que lo llevó a organizar en 1980 la primera conferencia de abducidos, repetida después anualmente. Pero con él llegaron también las primeras complicaciones inesperadas, al informar de la gran cantidad de fenómenos paranormales que parecía rodear a los testigos, antes y después de sus abducciones. El 20 de Octubre de 1975 la cadena de televisión americana NBC emitió en horas de máxima audiencia una dramatización del caso Hill. Pocas semanas después las abducciones empezaron a aparecer en una abundancia sin precedentes y entre ellas una que años después sería incluso llevada al cine: el caso de Travis Walton. Pese a haber En 1979 aparecería The Andreasson Affair, el primero de una serie de libros donde el ufólogo Raymond E. Fowler 7 Abducción documentaría (¡a lo largo de casi 20 años!) la que sin duda es la más prolongada investigación ufológica jamás realizada. Y sin embargo, no ha podido ofrecer la menor prueba tangible y la única consecuencia real ha sido que el propio investigador ha descubierto ser ¡otro abducido!. Merece destacarse el libro inicial porque contiene la primera referencia a los llamados "implantes" que tanto darán que hablar en los años siguientes. repetirse. Ya no hay que complicarse la vida buscando nuevos testigos, los ya existentes pueden aportar sucesivas experiencias (y naturalmente, lo hacen). 3º) Supuestamente los exámenes médicos realizados por los extraterrestres dejarían a veces marcas o cicatrices que los testigos son luego incapaces de explicarse. De esta forma se facilita a los interesados una forma rápida de comprobar si han sido abducidos a pesar de no recordar nada, dando una excusa a cualquiera que crea haberlo sido para ponerse en contacto con el investigador más cercano, sin necesidad de complicarse avistando un OVNI. Pero fue con la publicación en 1981 del libro Missing Time que el fenómeno de la abducción se pondría de moda. Su autor, Budd Hopkins, un artista plástico metido a investigador, y sin la menor formación en psicoterapia, define 3 características que ampliaban enormemente el ámbito del fenómeno, facilitando su explosivo desarrollo (en número y tipo de casos): La proselitista e incansable labor de Hopkins y sus seguidores va conformando y delimitando el fenómeno, y así en 1987 Thomas Bullard puede definir un escenariotipo, en sucesivas etapas, que ya para entonces habían quedado obsoletas. Los cambios se suceden. 1º) Poniendo el énfasis en el llamado "tiempo perdido" (lapsos temporales entre varios minutos y algunas horas que los testigos son incapaces de justificar) defiende que cualquier persona que haya llegado a algún sitio más tarde de lo previsto podría haber sido abducida, ¡aunque ni siquiera recuerde haber visto un OVNI!. A partir de ese instante, el fenómeno abducción puede independizarse de los molestos límites materiales impuestos por los OVNIs como naves extraterrestres y comienzan a aparecer los llamados visitantes de dormitorio. Por un lado, aparece el libro de Whitley Strieber Communion (1987) donde este autor de novelas de terror relata sus propias y terroríficas experiencias como abducido, supuestamente reales, pero resistiéndose a darles una interpretación extraterrestre. El libro fue un éxito inmediato, y millones de ejemplares por todo el mundo mostraron a ese misterioso ser de ojos negros almendrados mirándonos desde los escaparates. Y poco después, la película del mismo título permitió divulgar aún más su historia. La avalancha subsiguiente llevó incluso a la organización de grupos de autoayuda para abducidos, donde estos intercambiaban libremente sus experiencias, realimentando sus creencias. 2º) Mediante hipnosis descubre que los testigos han sido sometidos a repetidas abducciones desde su más tierna infancia (incluso afectando a sucesivas generaciones de familias enteras). Ello implicaría una actuación metódica por parte de los alienígenas... y la necesidad de que el investigador realice un seguimiento continuo del testigo por si volvieran a Algo más tarde, y para no ser menos, Hopkins con su libro Intruders, añade una 8 Abducción nueva vuelta de rosca: el niño híbrido (mezcla de humano y extraterrestre) y los fetos perdidos (aparentes embarazos que desaparecen en pocas semanas, supuestamente implantados y extraídos por los extraterrestres). El impersonal examen médico de antaño se ha convertido en un acto cargado de sexualidad e impotencia. Y la descripción de los tripulantes (llamados "grises" por su color) se va pareciendo más y más a la caricatura de un feto. Aumenta la penetrante sensación de amenaza: ningún lugar es seguro, ni puertas ni ventanas detienen a los extraterrestres, que utilizan nuestro cuerpo y nuestra mente a su antojo. a cuatro de cinco síntomas sugerentes de abducción según estos investigadores. Por la misma época Hopkins saca a la luz el caso de Linda Napolitano, una mujer supuestamente secuestrada de un rascacielos en pleno Manhattan y con un testigo independiente del más alto nivel: Pérez de Cuéllar, por aquel entonces Secretario General de las Naciones Unidas. Como no podía ser de otra manera, éste lo niega. Y empiezan entonces a proliferar los casos donde se asegura haber visto en el interior de los OVNIs a seres humanos normales, aparentes aliados de los extraterrestres. De hecho, en los últimos años los propios abducidos han salido a la palestra (incluso la ya anciana Betty Hill) publicando sus relatos autobiográficos y divulgando nuevos escenarios de colaboración entre los militares y los OVNIs. Otro desarrollo inquietante son las abducciones de niños. Esta postura es llevada a su máxima expresión por otro ufólogo, David M. Jacobs, que en su libro Vida Secreta (1992), nos cuenta como las abducidas (la mayoría son mujeres) visitan salas llenas de grandes depósitos transparentes donde pueden verse cientos de niños híbridos, e incluso ¡ayudan a parir a otras abducidas!. Siguen añadiéndose nuevos elementos: la etapa unificadora de los "grises" empieza a disgregarse y aparecen unos seres más altos que se dedican a establecer vínculos emocionales con los testigos a través de la mirada. A estas alturas se ha ido abandonando ya cualquier intento de obtener pruebas materiales de lo que se afirma. Si acaso, y más como medida de tranquilidad para los abducidos, se instalan cámaras de video que nunca consiguen captar nada anormal o se "estropean" en el momento más inoportuno. Pero también en los últimos años han aparecido investigadores como el psiquiatra de Harvard John E. Mack o el Dr. Kenneth Ring que defienden una postura opuesta: el carácter beneficioso de las abducciones, ayudando a la humanidad en la siguiente etapa de la evolución, en concordancia con los mensajes de la llamada Nueva Era y cerrando el círculo de los antiguos y denostados contactados. Con estos investigadores, los niveles de credulidad y ausencia del menor interés en corroborar la realidad de los relatos alcanza un máximo dificilmente superable. Y el abandono del entorno ufológico se hace evidente cuando aceptan impasibles relatos de previas reencarnaciones o de espíritus alienígenas habitando en seres humanos. Por si no fuera suficiente, en 1991 aparece una encuesta que permite a Hopkins y compañía afirmar que, sólo en Estados Unidos, habría casi ¡cuatro millones de abducidos!... a partir de las respuestas de unos pocos individuos "aleatoriamente escogidos" que contestaron afirmativamente Hipótesis explicativas. En los primeros años, cuando el fenómeno abducción era debatido con un mínimo rigor, los 9 Abducción argumentos a favor de su realidad objetiva se centraban en el análisis de cada caso concreto. Pero cuando los escépticos (Klass, Roncoroni, Kottmeyer, etc.) empezaron a desmontar rigurosamente algunos de ellos (Travis Walton, Llanca, Hill, etc.) se abandonó esta táctica y se pasó a señalar las "importantes" similitudes que mostraban los distintos casos entre sí (aunque lo más evidente para cualquier lector eran las diferencias). Lo cierto es que, a pesar de los años transcurridos, no se conoce ningún caso que cuente con testigos independientes que confirmen al menos parte de lo que se cuenta. De hecho, más bien al contrario, se conocen varios casos en la literatura ufológica en que testigos fiables aseguran haber estado junto a los abducidos (dormidos o en trance) mientras estos aseguraban encontrarse a bordo de un OVNI (Magee 1978). Y en los supuestos casos de abducciones múltiples, la concordancia entre los distintos relatos deja mucho que desear. Se apela entonces a argumentos tan inmateriales como que los abducidos no buscan beneficios materiales (como si no bastasen los emocionales), que sus relatos presentan genuinas reacciones emotivas (lo que no dice nada sobre la objetividad de lo ocurrido), que no evidencian rasgos patológicos de personalidad (aspecto muy discutido, véase más adelante), o que tras lo sucedido experimentan importantes transformaciones en su forma de vida y creencias (quizá confundiendo causa y efecto). Con el paso de los años han ido argumentándose otras supuestas "evidencias". Comenzaron con las diversas marcas y cicatrices que presentan los abducidos, pero dado que podrían tener orígenes somáticos (a partir del propio inconsciente del protagonista) su valor ha sido siempre dudoso. Se pensó luego en los llamados implantes (supuestos mecanismos artificiales implantados por los extraterrestres en distintas partes del cuerpo de sus víctimas). Es cierto que en los ambientes ufológicos circulan fotos tomadas con rayos X, tomografias computarizadas o resonancias magnéticas, que parecen mostrar extraños nódulos u objetos en el cuerpo de los testigos, pero siempre desaparecen justo antes de ser extraídos (los extraterrestres se han adelantado....). A falta de algo material que analizar, es preferible pensar que se trata de errores del propio proceso informático que elaboró las imágenes o nódulos naturales de material biológico generados por el propio organismo. Finalmente se han llegado a mencionar hasta esos embarazos desaparecidos (v. ectogénesis), que sin embargo jamás han sido confirmados por ningún doctor. Muchos autores siguen insistiendo en el valor corroborativo de una serie de supuestos detalles, desconocidos para el público en general, y que aparecen repetidamente en los nuevos relatos. Pero dado que el rango de contenido de las abducciones ha crecido exponencialmente en los últimos años y gozan de gran popularidad en todos los medios de comunciación social, resulta dificil no encontrar paralelismos si se busca con empeño. Y pese a lo que nos cuentan en la serie televisiva Expediente X, ninguna abducción ha sido jamás investigada oficialmente por la policia, aunque el escéptico Philip J. Klass ofreció ya en 1987 una recompensa de 10.000 $ para la primera abducción confirmada por el FBI (como delito federal que es). 10 Abducción Entre los partidarios de la realidad de las abducciones existen dos tendencias: propio Lawson elaboró una hipótesis explicativa (v. Hipótesis del trauma natal) mucho más débil. En esta línea, algunos investigadores llegaron a relacionar la amplitud del fenómeno con los sentimientos de culpa derivados de la utilización masiva del aborto legal como sistema de control de natalidad en las sociedades occidentales a partir de 1970. a) La primera, agrupada en torno a Hopkins y Jacobs, defiende el carácter negativo de las abducciones, que consideran un programa de hibridación interplanetaria donde los extraterrestres utilizan esperma y ovulos humanos para sus propios fines, indiferentes a las repercusiones sobre sus víctimas humanas. c) Autores como Hilary Evans (1984) han destacado que algunas características de las abducciones apuntan a situaciones psicológicas ya conocidas, como la parálisis nocturna o las visiones hipnagógicas e hipnopómpicas por lo que las abducciones podrían darse en estados alterados de conciencia causados por simples situaciones de tensión emocional (fallecimientos, divorcios, operaciones médicas, depresiones, etc.). b) La segunda, liderada por Sprinkle y Mack, insiste en el carácter benéfico de las abducciones incluso para los propios testigos, ayudándolos en la metamorfosis de su conciencia humana hacia una conciencia cósmica (sea lo que sea eso). Esta visión espiritual y mística está ganando adeptos. En un punto intermedio estaría el proyecto DIANA iniciado en España. Entre los que defienden que las abducciones responden a estímulos inconscientes, tampoco faltan diversidad de posturas. Comentaremos algunas, por orden más o menos cronológico de aparición, sin entrar a valorarlas: d) El investigador australiano Keith Basterfield propuso en 1988 la hipótesis falsable de que los abducidos presentaban rasgos de lo que médicamente se define como "personalidad tendente a la fantasia", siendo personas inteligentes y con dotes artísticas, por lo general. Estudios posteriores parecen haber invalidado parcialmente dicha hipótesis, aunque habría que seguir profundizando en esta idea. a) Los más escépticos apuntaron desde un principio a una mezcla de fraudes y fabulaciones inconscientes obtenidas bajo hipnosis por investigadores predispuestos. Sin embargo, existen bastantes abducciones donde no se ha empleado la hipnosis y no siempre parece tratarse de fraudes. Algunos autores admiten la posible influencia de estímulos externos naturales: e) El Dr. Michael Persinger (1990) ha realizado experimentos con voluntarios que aportaron indicios de que las abducciones puedan deberse a disfunciones del lóbulo temporal del cerebro, estimuladas por campos electromagnéticos del entorno. Sin embargo, dichas experiencias están todavía en un estadio muy inicial. b) Lawson (1981) demostró con su estudio sobre abducidos imaginarios que cualquier persona dispone en su inconsciente de un cúmulo de elementos fácilmente combinables para obtener un relato de abducción creíble. Por desgracia, dicho experimento no ha sido repetido y falta un análisis más profundo. Luego el 11 Abenza Moreno, Joaquín f ) La última hipótesis verificable aparecida es la defendida por Albert Budden (1994-1995) atribuyendo las abducciones (y demasiadas cosas más, como los poltergeist, etc.) a la polución electromagnética del entorno que se concentraría en lugares determinados. [50, 116, 117, 131, 146, 153, 154, 161, 176, 195, 199, 200, 203, 219, 242][LG] poderes muy “terrestres” se están aprovechando de la inverosimilitud del fenómeno para ocultar siniestras intenciones. Es por ello que ahora, más que nunca, se torna difícil la solución de este enigma, y habrá que investigar mucho, y pasar muchos años antes de que todo el rompecabezas comience a encajar. No obstante, siempre tendremos los vestigios de “dioses” venidos de las estrellas en la antigüedad para estar seguros de que tras el actual “teatro” de la ufología subyace una causa tangible cuyo origen no es de este planeta. ABENZA MORENO, Joaquín (n. Murcia, 1961). Ingeniero industrial y actual presidente del Cife, entidad de la que fue fundador en 1976. Conocido principalmente por su faceta de investigador de campo tanto de la casuística ufológica como de la parapsicológica, ha sido el principal divulgador y estudioso del fenómeno OVNI en la Región de Murcia. Conferenciante y realizador de espacios radiofónicos sobre ufología y parapsicología, es autor de numerosos trabajos sobre estas materias, de entre los que destacan los dedicados a técnicas de investigación y aspectos metodológicos. En la actualidad (1996) presenta el programa de radio El último peldaño en Onda Regional de Murcia. ADAMSKI, George (1891-1965). Norteamericano de origen polaco, fue el primer contactado de la era moderna de los ovnis. Afirmaba que el 20 de noviembre de 1952 había mantenido un contacto en un desierto del estado de California con un ser procedente del planeta Venus que le habría comunicado un mensaje de tipo admonitorio ante la carrera armamentística iniciada tras la Segunda Guerra Mundial. A este encuentro seguirían una serie de viajes por los diversos planetas de nuestro Sistema Solar, todo ello relatado en una obra titulada Flying Saucers have landed (Los platillos volantes han aterrizado, 1953), que incluía numerosas fotografías de naves y seres alienígenas. Como es lógico, las pruebas aportadas por Adamski distaban mucho de ser contundentes y no pasaban de burdos montajes, como así dictaminó la Fuerza Aérea norteamericana. Este personaje -que no era, entre otras cosas, astrónomo, como algunos han pretendido- se habría aprovechado para obtener fáciles beneficios económicos del ambiente social que comenzaba a gestarse por aquellos años y que puso de manifiesto el filme Ultimátum a la Opinión sobre el fenómeno: El fenómeno OVNI, de cuya existencia no cabe la menor duda por la gran cantidad de pruebas que así lo demuestran, se ha convertido con el paso del tiempo en una caja de Pandora, de la cual cada uno saca los monstruos o los ángeles que le convienen. En muchos casos ha sustituido a la religión o a los ideales de vida clásicos que el devenir de la historia ha caducado. Cada vez aparecen fenómenos más extraños que son asociados irremediablemente al mundo de los “no identificados”. Y todo hace pensar que 12 Aetherius Society Tierra (1951), en el que un ser extraterrestre advertía a los terrestres sobre el peligro de las armas atómicas. Adamski no habría hecho otra cosa que inspirarse en ella para imaginar su falsa aventura mesiánica. Tras otro libro (Inside the Space Ships, 1955), pasó los últimos diez años de su vida como una gran celebridad, ofreciendo incontables charlas, entrevistas y apariciones en radio y televisión, dando la vuelta al mundo. Todavía hoy se publican libros defendiendo sus relatos (Zinsstag, 1983).[266][LG, RC] setenta de diversos grupos con orientación científica (GAIFE, CEFANC), en 1982 pasó a codirigir la CIU (Comisión de Investigaciones Ufológicas) junto a Guillermo Roncoroni y Alejandro Chionetti, y a editar, hasta su desaparición en 1986, la revista Ufo Press. Tras este último intento institucional por difundir el espíritu científico entre los ufólogos, colaboró desde 1990 con publicaciones como Cuadernos de Ufología y Conozca Más, al tiempo que se integraba en el CAIRP (Centro Argentino para la Investigación y Refutación de la Pseudociencia) como redactor y asesor editorial de El ojo escéptico. En 1994 abandonó este “escepticismo orgánico” para intensificar sus trabajos de investigación periodística, y desde entonces se ha decantado por el estudio de los nuevos movimientos religiosos (Nueva Era, sectas ufológicas, Ummo) desde el punto de vista de las ciencias sociales. AETHERIUS SOCIETY Grupo contactista inglés fundado en 1955 por George King. Aunque surgió en Londres, tiene su sede central en Los Ángeles y está ampliamente difundido en las naciones de habla inglesa. Se caracteriza por sus extravagantes prácticas religiosas, que incluyen la "carga de energía" de las montañas y cánticos diversos. Véase sectas ufológicas. Opinión sobre el fenómeno (tomada de una entrevista de Andrés Torres en Ufología Racional, año 1, nº2, 1996): ”El término «fenómeno OVNI» se utiliza para poder presentar a la masa de casos no explicados (que bien puden responder a distintas causas o estímulos conocidos) bajo un rótulo unificador que contribuya a crear la ilusión de que estamos ante un mismo fenómeno compacto. Es una expresión usada para cohesionar -en un plano imaginario- el mito popular de los extraterrestres que nos visitan (...) La fe en el OVNI es una nueva forma de religiosidad, que cada vez tiene menos que ver con experiencias personales extraordinarias (como las historias de encuentros cercanos tan comunes antes de los 80), y más con vivencias subjetivas (como las de los abducidos y contactados de los 90).” AGNÓPTENO Término propuesto por Félix Ares de Blas y Juan Carlos Imar (“¿O.V.N.I., OVNI u ovni?”, en Stendek nº 43, marzo de 1981) para substituir a OVNI y permitir referirse a la Ufología como agnoptenología, evitando así el anglicismo que supone. Está formado por las raíces griegas agnos (desconocido) y ptenos (volador). AGOSTINELLI, Alejandro César (n. 1963). Periodista argentino, principal divulgador crítico de los fenómenos extraños en su país. Fundador a finales de los 13 Alerta OVNI ALERTA OVNI estos se reúnan en una zona supuestamente propicia con la pretensión de que aparezcan no ya los ovnis, sino artefactos voladores tripulados por inteligencias extraterrestres; de esta manera la alerta ovni se convertiría en una hierofanía, en una manifestación de lo sagrado, en este caso de dioses o seres hipertecnificados y portadores de un mensaje transcendente (v. Sectas ufológicas). En ellas suele tener lugar un llamamiento a los “hermanos cósmicos”, como acto propiciatorio para la manifestación ovni. Habitualmente suelen ser convocadas por algún programa radiofónico especializado en “temas ocultos”, por individuos señalados dentro de la comunicación ufológica o por grupos de contactismo. En España las primeras concentraciones de este tipo fueron convocadas en 1977 por la desaparecida revista Mundo Desconocido. Entre las que gozaron de mayor repercusión social destaca la promovida por el programa El Gran Sabath, de Radio 4 de Radio Nacional de España en Cataluña. Miles de personas se congregaron en El Garraf, Barcelona, el 5 de junio de 1989. La enorme cantidad de coches que acudía al lugar formó un círculo y, tras la preceptiva “invocación a los hermanos cósmicos”, nada ocurrió, excepto una fortísima lluvia que deslució completamente el acto. El atasco de tráfico fue tan monumental como el que se iba a producir 19 días más tarde en Tenerife, Islas Canarias. En la noche de San Juan, Radio Nacional de España, a través de su programa Espacio en Blanco, había convocado la que se iba a convertir en la más multitudinaria alerta ovni celebrada en España. Unas 10.000 personas se congregaron en el Parque Nacional de Las Cañadas del Teide para contemplar el paso de los ovnis sobre la isla. Después de relajaciones, concentraciones y llamadas cósmicas, nada ocurrió. Mientras Una de las etapas del desarrollo de la mitología ufológica, desde las lejanas luces que se observaban a finales de los años 40 y durante los 50 hasta la actualidad era la salida en busca de cielos despejados (generalmente nocturnos) con el objeto de observar pasivamente a la búsqueda de OVNIs. En todos los países se constituyeron grupos de aficionados que salían por las noches “a la caza de OVNIs”...y generalmente los encontraban (aunque fuera en forma de confusiones astronómicas y con aviones). A veces incluso se organizaban noches de vigilia conjunta en amplias zonas del país, coordinadas por radio. Así, en España fueron famosas las “alertas OVNI” organizadas a final de la década de los 70 por Antonio José Alés. Otras veces, dichas salidas se planeaban de manera más seria y científica, como el Project Identification que desarrolló el Dr. Rutledge en Missouri (EE.UU.) a finales de esa misma década. El inevitable paso posterior era la búsqueda del contacto con las naves y seres procedentes de lejanos planetas a través de cita previa en un lugar propicio para ello. En esto se especializaron algunas sectas platillistas como el IPRI peruano, que luego dio paso a la misión rama. Haciendo uso del sentido común, las alertas ovni no tienen sentido alguno: si el fenómeno ovni por definición es desconocido y no sabemos cuándo ni dónde se va a manifestar, será una pérdida de tiempo concentrarse en un lugar y hora prefijados a la espera de contemplar algún portento celeste. Pero no es el sentido común el que predomina en estas reuniones; más bien el sentido de lo maravilloso. Mediante el llamamiento previo a través de los medios de comunicación se pretende que 14 Alucinación algunos se extasiaban ante el paso de varios aviones y varios bromistas enfocaban sus linternas desde las volcánicas laderas hacia la concurrencia, los organizadores aseguraban que se habían visto ovnis, pero en otros lugares del archipiélago. El mito ovni retomaba así el vuelo después de varios años en los que parecía haber decaído.[15, 210][LG, RC] por ejemplo: esquizofrenia, depresión mayor, estados maníacos, etc.). Otro apartado importante que hay que considerar es el de la sugestión, producida -por ejemplopor hipnosis, o la dificultad en distinguir la realidad del contenido imaginario propio. También hay que tener en cuenta la cuestión de las expectativas o creencias y las necesidades emocionales de las personas, que llevan a percibir un estímulo ambiguo como real, o bien adaptado a lo que se desea encontrar. Esto, unido a la influencia social y cultural, podría explicar el por qué personas sanas tienen también alucinaciones (v. también visiones hipnagógicas e hipnopómpicas). [MG] ALUCINACIÓN Podríamos definir las alucinaciones como percepciones sin objeto material. Constituyen los trastornos más característicos de la percepción y la imaginación. Aunque muy corrientes en las enfermedades mentales, no siempre son síntoma de problemas psiquiátricos, pues algunas personas fundamentalmente sanas pueden experimentarlas en ciertas situaciones o pueden ser provocadas bajo condiciones especiales. En este campo se puede hacer una clasificación fundamental entre: ANÁLISIS FOTOGRÁFICO Proceso seguido por los especialistas para determinar la autenticidad de los negativos y de los positivados de fotografías de supuestos OVNIs. Hasta la aparición del grupo norteamericano GSW (Ground Saucer Watch) a principios de la década de los setenta, este tipo de análisis se hacía -y se siguió haciendo en España por mucho tiempo- por profesionales de la fotografía con base en su experiencia, buena voluntad y pocos medios. Entre los estudiosos del fenómeno OVNI estos análisis se vieron influenciados, la mayor parte de las veces, por una notable capacidad de observación y conocimientos trigonométricos, como para posicionar los objetos de la toma fotográfica, calcular distancias y establecer tamaños en función de datos conocidos sobre objetos de referencia. Un ejemplo claro de esto es el trabajo de Óscar Rey Brea sobre las famosas fotos del caso de San José de Valderas. • Las producidas por un estímulo externo, aunque no percibido o interpretado tal como es, sino de forma distorsionada, de acuerdo a una construcción psicológica propia del individuo. • Las no fundamentadas en estímulos externos, ajenos al individuo. Las modalidades sensoriales más frecuentes son la visual y la auditiva (aunque puede haber alucinaciones olfativas, táctiles, cinestésicas, etc.) y se asocian a estados hipnagógicos e hipnopómpicos, fatiga, estrés, aislamiento sensorial, uso de ciertas drogas y estados, en general, de disminución de la conciencia (aparte de las que se registran en estados mentales patológicos, El GSW introdujo un refinado método de manipulación electrónica de imágenes 15 Ángeles de radar fotográficas mediante la utilización de un digitalizador de alta velocidad. El procedimiento inicial para efectuar el análisis con la ayuda del ordenador consiste en digitalizar la imagen, obtenida por exposición (scanning) de la foto con un dispositivo que mide la densidad fotográfica relativa, definida como el logaritmo de los coeficientes de luminosidad. Esto lo hacía el GSW con una cámara de televisión de gran sensibilidad, pero hoy existen en el mercado una gama de scanners de alta precisión que hacen más asequible el proceso para cualquier estudioso cualificado. Después de introducir la foto, el elaborador confecciona en su memoria una versión numérica del área examinada. Ésta es semejante a una red cuyas casillas son pixels, las unidades mínimas de imagen en una pantalla. A cada pixel se le asigna un valor basado en el nivel medio de gris contenido en la porción de la fotografía que comprenda. La escala de grises varía de 0 a 255, y por tanto existen 256 niveles de grises. Obtenida esta matriz numérica, luego puede aplicársele toda una amplia serie de herramientas informáticas de retoque fotográfico que permiten variados efectos: expansión/supresión de contraste, filtración espacial, intensificación y factorización de contornos, etcétera. copias en papel de los negativos, y no sobre copias de copias o copias en papel prensa, que invalidan cualquier análisis. [29, 43, 190, 232][ JR] ÁNGELES DE RADAR Véase Radar y OVNIs. ANILLO DE HADAS Los biólogos conocen con este nombre aquellas bandas de crecimiento diferencial de la vegetación que se pueden encontrar en prados, campos de cereales y en terrenos calcáreos con forma circular. En la Edad Media se atribuían a corros de hadas bailando de noche, de ahí su nombre. Hoy se sabe que la formación de estos anillos se debe a la maraña de hilillos microscópicos de algunos hongos en crecimiento. Generalmente el círculo no es perfecto debido a las condiciones del terreno, y al desecarse la vegetación debido a los cambios físico-químicos del suelo puede parecerse incluso a una quemadura. Como era de esperar, este tipo de marcas han sido muchas veces atribuídas al aterrizaje de un OVNI. Quizá el ejemplo más famoso sea el llamado caso Delphos (Kansas, EE.UU.) que en 1971 recibió el premio del periódico amarillista National Enquirer como el mejor caso del año. [207] [LG] De todas formas, la utilización de sofisticada tecnología digital no es sinónimo de garantía si no va acompañada de conocimientos fotográficos básicos sobre revelados, emulsiones, enfoques, etc. Así, por ejemplo, la distorsión de contornos puede ser atribuible a causas puramente ópticas, como un ligero desenfoque, distorsión atmosférica, imágenes situadas dentro del margen del infinito, movimientos de la imagen, velo de reverberación, o una combinación de todas ellas. Hay que resaltar que el proceso debe aplicarse sobre primeras ANOMALÍA 1. Véase Fundación Anomalía. 2. Concepto que hace referencia a una irregularidad, a una discrepancia con una regla. Se producen en todos los campos de la cultura humana, desde la gramática 16 Anomalia (declinaciones anómalas) a la física atmosférica (p. ej., refracciones especiales debidas a estados excepcionales de la atmósfera, como el fata morgana, ciertos tipos de espejismos, etc.). término, una anomalía es un hecho que no tiene solución alguna dentro del marco paradigmático aceptado. Acarreando este nivel de exigencia para ser considerado como anomalía, dista mucho el fenómeno OVNI de poder ser tenido por tal, ni siquiera valorando ese residuo inexplicado, ya que, como han puesto de manifiesto diversos autores, ello no implica necesariamente que sea inexplicable. Numerosas causas como encuestas defectuosas, ausencia de información clave, deformación testimonial de los hechos, han contribuído tradicionalmente a la persistencia del residuo. Después de todo, la existencia de ese resto depende de la potencia analítica de las armas con que le hagamos frente. [137][RC] La existencia de anomalías reconocidas no significa que el paradigma vigente en una disciplina científica deba ser abandonado; será su persistencia en el tiempo y el aumento de ámbitos donde se produce lo que dará paso a un tiempo de crisis que quizá desemboque en una revolución científica que cuestione el actual paradigma hasta que se establezca otro, ya sea por el mayor número de científicos que adopten el nuevo o por consenso. Pero, en principio, el reconocimiento de anomalías significa un acontecimiento muy importante para las disciplinas científicas: podría decirse que ante su presencia nos encontramos ante la violación de lo que era dado esperar de la naturaleza según el paradigma vigente. Pero no basta tal constatación para que la anomalía sea integrada en el conocimiento reconocido como válido; serán necesarios reajustes en el paradigma para que la anomalía sea reconocida previamente como problema científico. Tal reconocimiento prepara el cambio científico y la posibilidad de revoluciones científicas cuando la anomalía aumenta su campo de influencia. Según el físico y filósofo de la ciencia Thomas S. Kuhn (1994), la ciencia “parece ser un intento de obligar a la naturaleza a que encaje dentro de los límites establecidos y relativamente inflexibles que proporciona el paradigma”. Y “ninguna parte del objetivo de la ciencia normal está encaminada a provocar nuevos tipos de fenómenos: en realidad, a los fenómenos que no encajarían dentro de los límites mencionados frecuentemente ni siquiera se los ve”. ANOMALIA Revista anual editada por el grupo portugués Cnifo. ANOMALÍES Publicación trimestral francesa del grupo SOS Ovni. Dejó de publicarse en 1999, pero puede consultarse en http://files.afu.se/ Downloads/?dir=Magazines/France/ Anomalies ANTROPOLOGÍA DE LOS OVNIS La Antropología y la Sociología coinciden en considerar los ovnis como objeto de estudio en cuanto se trata de un fenómeno con una incidencia social, que forma parte importante de una tradición cultural y que ha adquirido características religiosas entre ciertos colectivos. Diferenciar una antropología de una Ateniéndonos al significado preciso del 17 Antropología de los ovnis sociología de los ovnis es más ficticio que real. Tradicionalmente la Antropología fue la ciencia de la cultura de los pueblos primitivos, mientras la Sociología estudiaba las sociedades complejas. Pero a medida que han desaparecido los países coloniales y que el mundo se ha industrializado, la Antropología ha pasado a estudiar los problemas de la civilización moderna y la Sociología ha empezado también a entrar en el campo de los países subdesarrollados. Si la Antropología se ha definido por la técnica de la observación participante -observar la sociedad desde dentro- frente a las técnicas cuantitativas de la Sociología, estas diferencias cada vez se están borrando más. Si acaso podemos mantener, aunque sea en una generalización excesiva, que los antropólogos se centran más sobre la cultura y los sociólogos sobre los procesos sociales. En este sentido, y por hacer alguna división, la Antropología se enfocaría más en la importancia de los ovnis como objeto cultural, mientras que la Sociología atendería preferentemente a la evolución de la opinión pública sobre el tema (v. Sociología de los ovnis). popular (del folklore, en terminología anglosajona), al tratarse de “un conjunto de creencias sobre materias controvertidas”. Para los folkloristas, el aspecto de la comunicación es el que inserta este fenómeno en el ámbito social y donde adquiere su significado como cultura popular. Así, cuando un testigo informa sobre una visión OVNI recibe una respuesta de la comunidad en forma de apoyo o rechazo, los ufólogos dan su versión especializada, la prensa especula sobre el caso y los científicos aportan su visión escéptica. Todo ello constituye una estructura de conflicto típica de la cultura popular, lo que la distingue del conocimiento oficial. La narrativa popular puede adquirir básicamente las formas de cuento, rumor, leyenda o mito. Lejos de la concepción arcaica del folklore, los estudiosos actuales de la cultura popular han redefinido los conceptos de rumor, leyenda y mito eliminando los prejuicios negativos que de ellos se tenía en el sentido de “historias no reales”. El antropólogo no entra en consideraciones sobre la realidad física del fenómeno, sino que estudia la dinámica de creación de la narración, su función social, su simbolismo, etc. El enfoque antropológico de los ovnis considera este fenómeno como un producto cultural, analizando sus varias facetas como rumor, como leyendas contemporáneas y como mito, estudiando el simbolismo contenido en él y la estructura de creencias de la sociedad. El antropólogo intenta comprender, tanto como explicar, un fenómeno que se desarrolla en una sociedad en un momento histórico, y por ello con una especial atención sobre su origen y significado. En su estadio más inmediato, el ovni constituye un rumor en cuanto proposición específica para creer que pasa de una persona a otra sin medios probatorios que la justifiquen, y se convierte en leyenda en cuanto presuntas ocurrencias de unos sucesos que contradicen el conocimiento oficial. En los últimos años, las leyendas urbanas -recientemente denominadas leyendas contemporáneasse han constituido en un campo específico de la cultura popular con un interés creciente entre los académicos. Sólo hace 25 años que los antropólogos y folkloristas empezaron a considerar los ovnis como un elemento de la tradición o cultura 18 Antropología de los ovnis Pero los ovnis son algo más que una leyenda contemporánea sobre visiones de objetos en los cielos y sobre secuestros de personas por extraterrestres. Los ovnis se han convertido en una subcultura alimentada por una comunidad de creyentes, seguidores y estudiosos, rodeada de una compleja simbología y una estructura de creencias de contenido mítico, según la cual los ovnis son astronaves de una civilización extraterrestre que visitan la Tierra. Este marco de creencias es tributario de fuentes tan diversas como la literatura y el cine de ciencia ficción, el pensamiento mágico occidental y la especulación ilustrada sobre la pluralidad de mundos habitados (v. ciencia ficción, extraterrestres en la literatura de; ciencia ficción, ufología y; cine y extraterrestres; extraterrestre). Este enfoque nos lleva al terreno de la mitología. Ufología a una religión científica: la Ufología como recreación de la vieja relación del hombre con la divinidad. Los contactados han encontrado en los extraterrestres una nueva forma de expresión de una religiosidad intuitiva y no institucionalizada, y buscan una comunión espiritual con los maestros superiores del espacio en experiencias que adquieren la categoría de lo místico. Sus contactos dan lugar a cuerpos doctrrinales que beben de numerosas fuentes culturales y religiosas en su inspiración, formándose auténticos cultos sincréticos. De acuerdo con sus mensajes, los Guías Extraterrestres dirigen el destino humano para evitar nuestra autodestrucción y con el fin de orientarnos en una evolución cultural hacia un hombre superior, revistiendo la forma de una intervención mesiánica. Algunas de estas doctrinas, de contenido escatológico, anuncian el fin del mundo y la salvación de los elegidos, que serán llevados en naves extraterrestres a otro planeta para fundar una nueva humanidad más perfeccionada. Las doctrinas de esta clase, en que se anuncia un futuro de felicidad que será traído por una fuente salvadora se denominan milenaristas por referencia al milenarismo cristiano primitivo. Los grupos milenaristas suelen anunciar un fin apocalíptico a plazo fijo, y las consecuencias de tales predicciones suelen ser la traslación de la transformación anunciada a un nivel mental o espiritual. El mito es un modo integrado de ver el mundo que aporta respuestas intelectuales, espirituales y psicológicas a las preguntas trascendentes sobre nuestros orígenes, nuestro destino y nuestra naturaleza. Los ovnis son un mito contemporáneo, pues responden a cuestiones sobre el origen de la humanidad, el lugar del hombre en el cosmos y la vida más allá de la muerte. En la subcultura de los ovnis se considera que los extraterrestres han jugado un papel fundamental en el origen de nuestra especie y a lo largo de su evolución biológica e histórica. Así, el suizo Erich von Däniken sostiene que la humanidad es el producto de un experimento genético realizado por seres de las estrellas, y el ufólogo Jacques Vallée cree que los ovnis son un “sistema de control” del devenir histórico de nuestra civilización. La idea de los extraterrestres como creadores de vida y vigilantes de la evolución supone una aproximación de la La simbología contenida en el mito de los extraterrestres aporta numerosas claves para comprender la cultura que lo sustenta. A partir de mediados del siglo XX, en un medio cultural de desarrollo tecnológico, fe en la ciencia, esperanza en el progreso y excitación con el dominio de la naturaleza y la conquista del espacio, los ovnis pasan a convertirse en un referente de las esperanzas 19 Aparición mariana e inquietudes de la civilización occidental. En las últimas décadas hemos contemplado la penetración de este mito en otras regiones del mundo antes culturalmente lejanas, que ha venido asociada a la progresiva permeabilidad de los valores occidentales y de la ideología desarrollista a escala planetaria. [184][IC] contenido de los mensajes, profecías y advertencias (donde se manifiesta normalmente un integrismo exarcebado, entremezclado con una preocupación por los detalles más nimios: vestimenta sacerdotal, etc.) y ni siquiera en la apariencia de la propia entidad (compárese a la Virgen de Guadalupe con la de Garabandal). Este fenómeno en su conjunto (y sus interesantes enseñanzas) ha sido largamente ignorado por los ufólogos en general, incapaces de asimilar que incluso en la actualidad sigan produciéndose aparentemente decenas de casos similares todos los años. Algunos han señalado como el número de apariciones marianas se disparó en los años 1947 y 1954 (oleada francesa) lo que es claramente significativo. Pero, excepto aquellos partidarios de la hipótesis demoníaca sobre los OVNIs, rara vez se ha pasado más allá del comentario de casos concretos como el famoso "milagro solar" de Fátima. APARICIÓN MARIANA Dícese de la experiencia sufrida por uno o varios testigos (por lo general mujeres jóvenes) donde estos afirman ver a un ser femenino que se identifica como la madre de Jesús, solicitando sus rezos y dando mensajes casi siempre apocalípticos. Dichos encuentros acostumbran a repetirse varias veces, llegando a acudir miles de peregrinos. Sin embargo, los únicos que ven a la Virgen son los testigos iniciales, debiendo los demás conformarse en el mejor de los casos con manifestaciones luminosas o de luces brillantes, que en algunas ocasiones (Fátima -Portugal-, Zeitoun -Egipto-) han llegado a fotografiarse. Recordemos que según la tradición católica, la Virgen María ascendió a los cielos en su cuerpo mortal. Muchos ufólogos han escrito sobre OVNIs en la Biblia, pero solo Erich von Däniken (1974) ha dedicado todo un libro a su equivalente moderno. Fue Jacques Vallée (1965) uno de los primeros en mencionar las similitudes de lo ocurrido en Fátima con los OVNIs modernos, tendencia que ha culminado en el libro de Joaquim Fernandes (1981) donde se presenta a una Virgen María claramente extraterrestre. Esta postura ofrece el atractivo de conjugar el secular mundo de la ciencia y la racionalidad con el mundo mágico y místico de la religión. Pese a conocerse decenas de relatos similares desde principios de este segundo milenio, la Iglesia sólo ha reconocido oficialmente ocho apariciones marianas en el último siglo y medio. La principal evidencia a favor de la veracidad de dichas apariciones se deriva de las conversiones y de las curaciones aparentemente milagrosas que acontecen entre los peregrinos. Pero incluso estas últimas han sido cuestionadas seriamente por los escépticos. Lo indiscutible es que no existe ninguna consistencia entre los distintos sucesos: ni en la forma de llegada o partida, ni en el Hilary Evans (1984, 1987) ha sido quien más ha explorado desde una perspectiva unitaria (no extraterrestre) los encuentros con seres no humanos. En España, Miguel Peyró (1979) publicó varios 20 APRO trabajos sobre las apariciones de El Palmar de Troya. [72, 85, 156, 173, 178] [LG] decidió disolver la organización y donar sus archivos al Cufos, aunque éstos finalmente fueron a parar a un grupo contactista, pese a la animadversión siempre mostrada por los Lorenzen hacia los movimientos de este tipo. [MM] APRO (Aerial Phenomena Research Organization). La primera asociación ufológica del mundo, fundada en enero de 1952 en Sturgeon Bay, Wisconsin (EE.UU.), por Leslie James ( Jim) Lorenzen y su esposa Coral. En sus comienzos era más un club de creyentes en el origen extraterrestre de los OVNIs que una entidad dedicada a la investigación, y de hecho ideológicamente nunca se apartó demasiado de esas posturas iniciales. Ya en junio de 1952 salió a la venta el primer número de The A.P.R.O. Bulletin, todo un clásico entre las publicaciones de casuística. Después de diversos cambios de sede motivados por la movilidad laboral de los Lorenzen, en 1960 la organización fijó definitivamente su domicilio en Tucson, Arizona. A diferencia del Nicap, con el que rompería relaciones en 1962, el APRO defendía la legitimidad de los casos con humanoides y nunca creyó en la existencia de una conspiración gubernamental para ocultar la verdad sobre el tema. El centro vivió sus mejores momentos durante los años sesenta, cuando alcanzó los 1.500 afiliados, llegó a contar con J. Richard Greenwell como empleado con dedicación exclusiva, y los libros del matrimonio Lorenzen cosechaban gran popularidad. Sin embargo, la escisión del Mufon en 1969 y su posterior éxito determinaron el principio del declive del APRO, que se agudizó con la mala imagen ofrecida en la investigación del caso de Travis Walton, por las acusaciones de ocultar los resultados negativos del examen poligráfico al que se sometió el abducido. Tras la muerte de Jim (1986) y Coral (1988)Lorenzen, la dirección del APRO ARCAS GILARDI, Julio (n. Santander, 1951). Durante 1966 inicia su actividad en el campo de la Ufología y un año después funda con un grupo de estudiantes la primera asociación privada del norte de España dedicada al estudio del tema, Ciove, cuya filosofía se situaba en la línea del origen inteligente de los OVNIs como manifestación de tecnologías extraplanetarias. Con una destacada vocación por sumar colaboraciones, participa en representación de este grupo en los primeros intentos de coordinar el estudio de la Ufología en el país: Primera reunión de investigadores y grupos privados (1971), Federación de Centros del Norte de España (1972) y Coordinadora de Estudios sobre el fenómeno OVNI (1978, 1979 y 1980). En el año 1971 publica un primer boletín informativo, Tiempo y Espacio, que se transformaría poco después en la revista Vimana. En 1987, en colaboración con José Ruesga Montiel, pasa a ser co-editor de la segunda época de Cuadernos de Ufología, plataforma desde la cual ambos impulsan la celebración de dos encuentros de carácter internacional (OVNIS: experiencias y análisis, 1990, y Jornadas Internacionales, 1991) y la creación del Colectivo Cuadernos. En 1996 se convierte en fundador y presidente del patronato de la Fundación Anomalía. Opinión sobre el fenómeno (adaptado de una entrevista en Paraciencias, 21 Área 51 junio de 1995): “Con la perspectiva del tiempo, sin duda el mejor maestro, tiendes a contemplar esos esfuerzos casi filantrópicos de los inicios como los intereses denodados de la juventud, con poca información pero mucha y buena voluntad (...) Sin duda de aquellas primeras manifestaciones se dio paso a un trabajo e interés por el fenómeno más ponderado, en el que la pasión inicial dejaba lugar a la reflexión de ideas y planteamientos, con el fin de encontrar respuestas razonables y que pudieran sustentarse en pruebas. Casi nos convertimos en escépticos, pero en la acepción menos evangélica del término. El estudio de las presuntas evidencias de los fenómenos y observaciones demuestra que existen explicaciones razonables, dentro del mundo de los fenómenos físicos o de la intervención humana, que pueden aclarar muchos de los presuntos misterios convertidos hoy en auténtico mito de las postrimerías del siglo XX”. pruebas de vuelo de uno de los aparatos. Con posterioridad a su intervención televisiva, “Dennis” y tres amigos suyos, entre los que se encontraba John Lear (v. Matrix, informe), acudieron repetidamente hasta los límites de la “S-4” para observar y filmar algunos de esos vuelos de prueba, hasta que el personal de seguridad del Área 51 les detuvo y les obligó a abandonar el lugar. Al día siguiente, “Dennis” fue despedido, y en noviembre de 1989 decidió volver al programa televisivo para dar a conocer su verdadera identidad y el resto de la historia. Robert Scott Lazar (ése es su verdadero nombre) pensó que así protegería su vida de posibles atentados que buscasen acallar sus denuncias. Por su parte, otro testigo, conocido sólo como “Jarod-2”, sostiene haber ayudado a diseñar los simuladores de vuelo de las naves construidas por el gobierno norteamericano con la tecnología extraterrestre procedente de los artefactos investigados en el Área 51. Afirma además haber hablado con un alienígena llamado Jarod que se comunicaría con un extraño dialecto del húngaro. ÁREA 51 Ninguna de estas manifestaciones cuenta con pruebas que las respalden al margen de los testimonios precitados, si bien parece ser cierto que alrededor de la base existe un intenso dispositivo de seguridad que disuade a los curiosos de permanecer en sus inmediaciones. Las razones de dicho celo, no obstante, podrían ser más prosaicas. Cierta casuística ufológica registrada en la zona admitiría ser explicada en términos de proyectos aeronáuticos secretos que tendrían Groom Lake como base de pruebas u operaciones. De hecho, esta instalación acogió ya en su momento los U-2 y los SR-71 Blackbird, y se ha insinuado que el substituto de este último, el avión de tecnología stealth (invisible al radar) Aurora, o el Northrop Denominación popular de una importante base secreta de la Fuerza Aérea de los EE.UU. situada junto al lago seco Groom, dentro del campo de pruebas de Nellis, en el desierto de Nevada. Dicho complejo, que no figura en los mapas oficiales, se puso de actualidad en marzo de 1989 cuando un tal “Dennis” apareció en la televisión americana con el rostro oculto y la voz deformada. Según relató, era un científico que desde diciembre de 1988 estaba trabajando en la “S-4”, una instalación al sur del Área 51, examinando los sistemas de propulsión de nueve platillos volantes que ahí se custodiaban. Por lo visto, había contemplado en cierta ocasión las 22 Ares de Blas, Félix .1995): “Hoy pienso que el ovni está formado por una multitud de fenómenos complejos: TR-3A Black Manta (las características y existencia de los cuales no han sido aún oficialmente comunicadas) podrían estar operando desde la llamada Área 51. [20][MM] 1) Indudablemente hay una gran componente antropológica: estamos ante un mito urbano muy interesante de estudiar. De hecho es una situación única: por primera vez en la historia de la humanidad tenemos ante nosotros los datos para saber con exactitud cómo han nacido y evolucionado un conjunto de sectas que en su conjunto constituyen una nueva religión (...). Pienso que el conocimiento detallado de su evolución nos permite entender mejor el nacimiento de otras religiones que nos son muy próximas, como, por ejemplo, el cristianismo. ARES DE BLAS, Félix (n. Madrid, 1947). Ingeniero superior de telecomunicaciones, investigador por oposición en el UAM-IBM Madrid Scientific Center y del Proyecto ERIPS. Doctor en Informática y profesor de la Facultad de Informática de San Sebastián, amén de periodista científico. Fue creador del Grupo Aleph (1967) y de Eridani AEC (1968). Con David G. López fundó la delegación en Madrid del Cei de Barcelona (1971), y, más tarde, Arifo (1985) y Arp (1988). Ha sido uno de los más firmes defensores de una colaboración efectiva entre todos los investigadores españoles y un activo investigador de campo y de gabinete. Sus contribuciones, en especial el Estudio de la Oleada 1968-1969 (con David G. López, Eridani AEC, Madrid, 1970) y las Bases para una modelación teórica del fenómeno Ovni (con D.G. López y Ángel Salaverría Garnacho, en Actas del I Congreso Nacional de Ufología, Stendek-CEI, Barcelona, 1978), aún no han sido superadas pese al tiempo transcurrido. Fue un adelantado a nivel mundial al plantear hipótesis de corte sociológico como explicación de los ovnis. Escéptico militante desde principios de los ochenta (es asesor del Csicop), tras su paso por la presidencia de Arp-Lar (1988-93) editó por su cuenta hasta 1996 la publicación aperiódica El investigador escéptico. Cuadernos de debate racionalista. 2) Es posible que detrás del ovni haya un fenómeno todavía no bien comprendido. Aunque es más probable lo contrario, es decir: que detrás del fenómeno ovni no se esconda ningún fenómeno físico extraño. 3) No descarto totalmente la idea de que algunos casos -una ínfima minoría- tengan un origen en civilizaciones extraterrestres, pero la probabilidad de que ello sea así es muy próxima a cero.” ARIFO Siglas de Alternativa Racional para la Investigación del Fenómeno OVNI. Véase ARP. ARMAS SECRETAS Véase Hipótesis sobre los OVNIs. Opinión sobre el fenómeno (tomada de Papers d’Ovnis nº 21-22, sep./oct 23 Arnold, caso ARNOLD, caso Segunda Guerra Mundial, los Horten Ho.IX (Gotha Go229), que el ejército americano había descubierto una vez acabada la misma (Petit, 1995). Aunque antes del 24 de junio de 1947 se habían producido algunos casos de observaciones extrañas asociadas posteriormente a los platillos volantes, es este día cuando un piloto privado de avionetas llamado Kenneth Arnold observó 9 extraños aparatos volando en formación cerca del Monte Rainier, en el estado de Washington, al noroeste de los EE.UU. Había despegado a las 14:00 h. de Chehalis para llegar a Yakima. Poco después de las 15:00 h. vio un resplandor a su izquierda, al norte del citado monte, que delató la presencia de una hilera de nueve objetos parecidos a aviones volando de norte a sur, a unos 9.500 pies de altura, que parecían saltar entrando y saliendo de la formación, al tiempo que reflejaban la luz solar como un espejo. Estaban en la misma parte del cielo en la que había visto el resplandor y calculó que volaban a más de 1.300 km/h. Carecían de cola y la parte trasera formaba un ángulo convexo; tendrían unos 50 pies de envergadura. Curiosamente Arnold no mencionó en ningún momento que lo observado fueran “platillos volantes”, sólo que los objetos se movían como platos lanzados contra el agua, dando saltos. Fue suficiente para que un periodista de un diario local llamado William Bequette informara erróneamente de tal suceso afirmando que los objetos avistados tenían forma de platillo, cuando Arnold sólo había hecho mención al modo de desplazamiento. La leyenda de los platillos volantes nacía de un error de interpretación de las palabras de quien se iba a convertir en el primer testigo de la aparición de “platillos volantes” en los cielos del planeta. Por otro lado, la descripción aportada por Arnold de los objetos coincide plenamente con ciertos prototipos aeronáuticos alemanes de la Aunque el caso no es nada impresionante (testigo único y repeater, observación lejana, etc.), la historia le ha investido de un significado especial, lo que inevitablemente ha obligado a todos los escépticos a buscar una explicación para el mismo. Y no se ponen de acuerdo: confusión con aviones (Hynek, Van Kampen), prototipos secretos (Vesco, Petit), luces tectónicas (Devereux), globos (Martin Gardner), alucinaciones (Billig), nubes de montaña (Menzel), bólido (Klass), espejismos (Steuart Campbell), bandada de cisnes (Kottmeyer), etc. [177] [LG, RC] ARP Siglas de Alternativa Racional a las Pseudociencias. Asociación de los autodenominados escépticos españoles, inscrita oficialmente como tal en marzo de 1988. Según su declaración de principios, "trata de fomentar la investigación crítica de las supuestas manifestaciones de lo paranormal y los hechos presuntamente situados más allá de los límites de la ciencia siempre desde un punto de vista racionalista, científico y responsable, y dar a conocer los resultados de tales investigaciones a la comunidad científica y al público en general" (v. Escepticismo). Su origen estuvo en el grupo ARIFO (Alternativa Racional para la Investigación del Fenómeno OVNI) constituido por un grupo de ufólogos vascos, nucleados en torno a Félix Ares de Blas y Luis Alfonso Gámez Domínguez. En aquella primera época tuvieron gran difusión sus 24 Astronautas de la Antigüedad, teoría de los ASTRONAUTAS DE LA ANTIGÜEDAD, teoría de los enfrentamientos públicos con los periodistas Juan José Benítez y Manuel Toharia, al segundo de los cuales llegarían a incorporar a sus filas con el paso del tiempo. Su órgano informativo oficial fue desde el principio el boletín La Alternativa Racional (LAR) y ya a fines de 1987 acordaron ampliar su campo de actuación a todas las pseudociencias, cambiando el nombre del grupo por el que actualmente tienen. Con el paso de los meses el movimiento fue creciendo y apareciendo grupos similares en Madrid, Cataluña, etc., por lo general con una actitud muy combativa que algunos consideraron excesiva, pero que es comprensible en cualquier organización naciente. Dentro del campo ufológico sus aportaciones más importantes corresponden a la primera época; últimamente sólo merece destacarse que fue en las páginas de LAR donde apareció la confesión de José Luis Jordán Peña como autor del fraude Ummo. También han denunciado las falsedades del caso Roswell. También llamada de los antiguos astronautas, de los dioses del espacio o simplemente "astroarqueología", esta teoría sostiene que seres de otros planetas visitaron la Tierra en el pasado, realizaron experimentaciones genéticas para provocar un salto evolutivo en la especie humana, transmitieron la sabiduría a las antiguas civilizaciones y controlaron su devenir histórico. Las pruebas en que se apoyan los proponentes de esta teoría son variadas: referencias a seres celestiales en los textos sagrados y en los mitos, crónicas antiguas de hechos maravillosos, dibujos e inscripciones de difícil interpretación de épocas prehistóricas e históricas, monumentos megalíticos, tecnología de antiguas civilizaciones, leyendas, etc. Las referencias a visitantes del cosmos en el pasado se remontan a las obras de Blavatsky y Charles Fort, pero en su versión moderna aparecen con la era de los platillos volantes, a principios de la década de los cincuenta. Los autores pioneros de esta teoría fueron Adamski y Leslie, Jessup, Wilkins y Agrest. En 1961 Louis Pauwels y Jacques Bergier mencionan estas ideas en su best-seller El retorno de los brujos (1962), y posteriormente Robert Charroux, Paul Misraki, Peter Kolosimo, Andrew Thomas y otros dedicaron numerosas obras a exponer supuestas pruebas sobre la presencia extraterrestre en el pasado. Pero es al suizo Erik von Däniken a quien se debe el boom de la arqueología fantástica en los años setenta, sobre todo por sus primeros libros, titulados en España Recuerdos del futuro y Regreso a las estrellas. En 1975 se estrenó una película documental que unía ambos títulos. El En 1993 ARP organizó en Pamplona el I Congreso Nacional sobre Pseudociencia, y en septiembre de 1994 fue una de las siete organizaciones fundadoras del Consejo Europeo de Organizaciones Escépticas. Para 1997 está previsto que ARP acoja en España el Congreso Internacional Europeo de este tipo de organizaciones. Dirección: ARPLAR, Apartado de correos 1516, 50080Zaragoza (España). En Internet: https:// www.escepticos.es [LG] ASTROARQUEOLOGÍA Véase Astronautas de la antigüedad, teoría de los. 25 Astroarqueología primer libro español sobre este tema se debe al escritor Eugenio Danyans, y posteriormente el autor español más reconocido en esta materia ha sido Andreas Faber Kaiser, fallecido en 1994. de encuentro cercano propuesto por Hynek, pues es indiferente la distancia a la que se halle el testigo con tal de que exista constancia, cuando menos testimonial, del descenso del OVNI. Precisamente el interés vertido por algunos ufólogos sobre esta categoría reside en la posibilidad de examinar los diversos tipo de efectos que tal aproximación al suelo puede haber dejado en la tierra, plantas, animales, vehículos y hasta en los testigos; consecuencias que podrían ser más fácilmente objeto de una investigación técnica que los avistamientos de cuerpos o luces distantes. Desde un punto de vista científico, la teoría que pretende que los restos arqueológicos, mitos, etc. son debidos a visitantes extraterrestres de la antigüedad no resiste un análisis crítico. Las pruebas sobre las que aquellos autores se basan admiten explicaciones más simples y racionales, y la interpretación que hacen de los mitos y leyendas ignora completamente el contexto cultural en que se produjeron. Una tendencia común entre los autores de esta tendencia es subestimar la capacidad tecnológica y la complejidad cultural de las civilizaciones antiguas. Aún peor, en autores como Däniken se encuentran numerosas inexactitudes, ocultación de las pruebas que contradicen sus tesis, citas incorrectas y un torrente de afirmaciones sin fundamento. Sin embargo, este enfoque no se impuso sino tras vencer muchas dificultades, pues hasta finales de los años sesenta el concepto de aterrizaje (como el de encuentro cercano) se encontraba muy ligado a los relatos de los contactados y a la incredulidad que suscitaban entre los ufólogos. Fue Jacques Vallée quien con su catálogo de observaciones tipo-I (conocido como Magonia, 1961-69) llamó la atención de los investigadores sobre los aterrizajes, al tiempo que recuperaba para la Ufología los primeros casos acaecidos durante la oleada de la nave aérea de 1896-97. Paralelamente, Vallée realizó el estudio estadístico de unos 300 incidentes tipo-I registrados durante la oleada francesa de 1954, de los cuales dedujo las llamadas tres leyes de los aterrizajes (Vallée, 1967): Es sorprendente el volumen de literatura "astroarqueológica" que se ha producido entre los años setenta y ochenta, toda ella de la misma tendencia. Sólo se puede destacar, en cambio, un libro crítico publicado en España sobre este tema: Los dioses vinieron de las estrellas, de Ernst von Khuon (editor) [70, 71, 128, 188].[IC] ATERRIZAJE 1ª) Los fenómenos descritos tenían una simetría de revolución (discoidales) y un diámetro real de unos cinco metros. Aproximación de un OVNI al suelo, posándose sobre él o suspendiéndose a una altura máxima de 15 m., y permaneciendo inmóvil al menos por un instante. 2ª) La famosa ley horaria: el carácter predominantemente nocturno de esta casuística. El concepto de aterrizaje se corresponde con el tipo-I de la clasificación de Vallée y no coincide necesariamente con el 3ª) La distribución geográfica de los sucesos era inversamente proporcional a la 26 Aurora boreal densidad de población (la “ley negativa”). Mas, a pesar de ello, lo cierto es que el análisis de los casos de aterrizaje no ha permitido avanzar en el conocimiento de los OVNIs. Además, la sequía de casuística que trajeron los años ochenta, la polarización de la ufología americana en las abducciones y el auge en Europa de las hipótesis psicosociológicas han provocado el casi total abandono de este tipo de estudios, que no suscitan ya el interés de antaño. [28, 30, 38, 251, 252][MM] Pese a la rotundidad empleada aquí por el investigador francés -que habla nada menos que de leyes-, no puede dejar de señalarse que, tras la revisión de esta oleada realizada por Barthel y Brucker (1979), existen serias dudas sobre la realidad de muchos de los sucesos que comprendió, por lo que los resultados obtenidos por Vallée deben mesurarse con mucha prudencia. Espoleado por estos estudios, VicenteJuan Ballester Olmos inició en España una similar labor de recopilación de casuística, que en 1971 dio sus frutos en forma de un catálogo de 100 incidentes tipoI localizados en la Península Ibérica, publicado primero en Stendek (núm. extra, julio 1971) y Flying Saucer Review (cuarto núm. especial, agosto 1971) y luego, junto a algunos análisis estadísticos básicos, como apéndice de la edición española (1972) de la obra de Vallée Pasaporte a Magonia. El siguiente paso del autor fue la publicación de OVNIS: el fenómeno aterrizaje (1978), voluminosa monografía que marcó un hito en la literatura ufológica hispana por su exhaustividad y rigor metodológico. En 1987 aparece la Enciclopedia de los encuentros cercanos con OVNIS -coescrita con J.A. Fernández Peris-, que, pese al cambio terminológico, supone la culminación del estudio de Ballester sobre los aterrizajes. En ella se recogen y comentan los catálogos LANIB (LANdings in the IBerian peninsula), de casos positivos, y NELIB (NEgative Landings in the Iberian peninsula), con un total de 230 y 355 entradas, respectivamente. Las conclusiones de los autores se resumen en dos puntos: existe un fenómeno desconocido por estudiar, y su investigación puede arrojar beneficios científico-técnicos a la sociedad. AURORA POLAR Las auroras son fenómenos luminosos que se producen en ocasiones en el cielo de las regiones polares bajo la forma de cortinas, arcos, bandas y chorros de luz. Se deben a la luminiscencia de la alta atmósfera al ser alcanzada por partículas electrizadas procedentes del Sol, aceleradas y desviadas hacia los polos por el campo magnético terrestre. Reciben el nombre de auroras boreales las que se producen en las regiones nórdicas y el de auroras australes las que tienen lugar en el hemisferio sur. Ocasionalmente pueden ser visibles desde latitudes más bajas. En las noches del 13 de marzo de 1989 y del 8 de noviembre de 1991, por ejemplo, llegaron a observarse auroras boreales desde algunos puntos de la geografía española. Aunque no es habitual, algunas observaciones del fenómeno acaban bajo la etiqueta OVNI, ya sea por tratarse de auroras de aspecto atípico o, simplemente, por haber sido observadas en lugares donde son un fenómeno infrecuente con el que los observadores no están familiarizados. No obstante, es en sus búsquedas retrospectivas de apariciones de OVNIs donde los ufólogos se han tropezado más a menudo con este 27 Aviones fantasmas tipo de fenómenos. Así, descripciones de “globos y columnas de fuego” como las observadas el 9 de octubre y el 2 de noviembre de 1730 desde diversos lugares de la Península Ibérica, han sido interpretadas a veces en clave ufológica. Lo cierto es que, a menudo, se trata de una auténtica reinterpretación, pues algunos de los relatos de épocas pasadas no hacen sino reflejar la interpretación fantástica que se hizo del fenómeno en su día. Muchas descripciones de combates aéreos de ejércitos fantasmales, a veces adorrnadas con un sorprendente lujo de detalles, se refieren con toda probabilidad a manifestaciones de auroras boreales. [60, 113][MB] sobre "aviones fantasma". Pronto relatos similares se extendieron por todo el norte de Suecia, Noruega y Finlandia. Generalmente se mencionaban extraños fenómenos luminosos (comportándose como aeroplanos), aunque también observaciones cercanas de aviones con comportamientos anómalos: vuelos a baja altura sin dirección concreta, en plena noche o en medio de fuertes tormentas. Por aquel entonces se sospechó de los rusos (cuya aviación era prácticamente inexistente) pero algunos historiadores posteriores han hablado de misiones secretas de entrenamiento procedentes de la Alemania nazi, todavía bajo la prohibición establecida en el Tratado de Versalles de tener aviación militar. De todas formas, estas teorías no parecen explicar toda la complejidad de lo ocurrido, considerando la tecnología de la época en los paises circundantes y las grandes distancias a cubrir. La investigación sigue todavía en marcha: Archives for UFO Research (AFU), PO Box 11027, S-600 11 Norrköping. Suecia. AVIONES FANTASMAS Normalmente se denomina así a un conjunto de fenómenos luminosos y avistamientos de "aeroplanos" que tuvieron lugar durante los años 30 sobre Escandinavia. En la actualidad, los ufólogos suecos han localizado más de 1000 informes de estas características, desde que John A. Keel publicase una serie de artículos en la revista inglesa FSR en 1970 exponiendo sus primeras investigaciones al respecto. Suponen además el primer ejemplo de implicación oficial, pues los avistamientos fueron detalladamente documentados por la policía e investigadores militares y están todavia archivados en los War Archives de Estocolmo, a disposición de cualquier analista serio. Desde entonces, los casos de "aviones fantasma" han pasado a un segundo plano ante la proliferación de platillos volantes, aunque continúan apareciendo. Recordemos el conocido como Barón Negro que aparentemente sobrevoló los cielos de París en distintas ocasiones hace pocos años, o los crecientes rumores sobre aviones "espantalluvia" notificados por agricultores de toda España durante la última sequía. [125][LG] En Diciembre de 1933, sólo 8 años después de que el comandante Byrd realizase el primer vuelo sobre el Polo Norte (8/5/1926), empezaron a llegar desde la frontera entre Noruega y Suecia cercana al paralelo 66 un creciente número de informes AVISTAMIENTO Observación repentina y sorprendente de un objeto o fenómeno que por sus características, condiciones de observación o 28 AVRO intepretación personal, no es adecuadamente identificado por el testigo. Hace referencia en sentido estricto a observaciones lejanas, tanto diurnas como nocturnas, y, por tanto, son casos en general de baja extrañeza, aunque evolucionen hasta convertirse en otro tipo de observación más cercana al testigo. Véase clasificación de Hynek.[RC] AVRO Véase Prototipos experimentales. DTU 29 Ballester Olmos, Vicente-Juan B BALLESTER OLMOS, Vicente-Juan OVNI en poder del Ejército del Aire español (1992). (n. Valencia, 1948). Diplomado en programación de ordenadores, profesionalmente ejerce como gerente en el Departamento de Seguros, Beneficios y Pensiones de Ford España. Fundador del Ceoni (1968), del ENI (Equipo Nacional de Investigación, 1969) y del Consejo de Consultores de Stendek (1978). Editor jefe de Upiar (1983-85) y primer investigador europeo becado por la Fund for UFO Research (1982). Es asesor científico de Cuadernos de Ufología, director de investigaciones del Cei y Vicepresidente 2º de la Fundación Anomalía. Es autor de más de 200 ensayos y artículos publicados en las más prestigiosas revistas internacionales, y de cinco libros sobre la materia: Ovnis: el fenómeno aterrizaje (1978, 79 y 84), Los OVNIS y la Ciencia, con Miguel Guasp (1981 y 89), Investigación OVNI (1984), Enciclopedia de los encuentros cercanos con OVNIS, con Juan Antonio Fernández Peris (1987), y Expedientes Insólitos (1995). Asimismo, ha publicado informes técnicos y ha participado en diez obras colectivas editadas en Gran Bretaña, Alemania y EE.UU. Ballester Olmos constituye el ejemplo por antonomasia de la búsqueda de la racionalidad y el rigor en la ufología española, en la que tanto ha influido. Tradicionalmente especializado en casos de aterrizajes, desde 1989 ha centrado su atención en los incidentes con intervención de las FF.AA., y a él se debe en gran medida la desclasificación de los informes Opinión sobre el fenómeno: Llevo treinta años de investigación pura y dura, cubriendo sus principales facetas: encuesta, análisis e historia, lo cual creo que me permite hablar con autoridad. Puedo afirmar que la observación de fenómenos -luminosos o no- transitorios y por sorpresa, que el espectador no puede aclarar, evoca importantes transformaciones por parte del testigo, que cree ver o lo que no ocurre, o lo que ocurre de forma más simple. Este testimonio alterado es tan común que lo encontramos en gente de a pie al tiempo que en narraciones de pilotos y otros profesionales. El observador modifica su visión en función de muchos parámetros, y lo hace inadvertidamente. Por ello, una investigación detallada permite explicar la inmensa mayoría de los avistamientos. Y para el resto... realmente ya no sé qué pensar, porque por lo antedicho no necesariamente lo que se informa como extraño ha sido verdaderamente extraño. Mi esperanza actual de que exista un genuino fenómeno OVNI es muy baja. Subsisten, sin embargo, experiencias curiosas que siguen incitándome a estudiarlas BANCHS, Roberto Enrique (n. City Bell, Argentina, 1952). Cursó estudios superiores en la Facultad de Arquitectura y Urbanismo de la Universidad de Belgrano, graduándose en Urbanismo y 30 Bases de OVNIs XXX Planeamiento. Doctor en Psicología Social, Máster en Metodología de la Investigación, Postgraduado en Clínica Sistémica y profesor de medios audiovisuales aplicados a la enseñanza, ha realizado también estudios de sociología, biótica y etnología. Interesado por la investigación ufológica desde 1965, fue fundador y director de CEFAI (Centro de Estudios de Fenómenos Aéreos Inusuales) de Buenos Aires, desde donde organizó el II Simposio Nacional de Investigaciones sobre Ovnis (1979). Ha sido igualmente director de la revista Atom, consultor de Ovnis: Un desafío a la ciencia y asiduo colaborador de revistas como Phénomènes Spatiaux, Stendek, Data-Net, The APRO Bulletin o Cuadernos de Ufología. Aunque últimamente se ha centrado en el estudio de la cauística de humanoides en la Argentina, ha abordado las diversas facetas del fenómeno OVNI en sus cinco libros aparecidos hasta el momento: Fenómenos Aéreos Inusuales (CEFAI, Buenos Aires, 1973), Las evidencias del fenómeno Ovni (Ed. Rodolfo Alonso, Buenos Aires, 1980), Los Ovnis y sus ocupantes (Ediciones Tres Tiempos, Buenos Aires, 1991), Ovnis Peregrinos del silencio (CEFAI, Buenos Aires, 1991) y Fenómenos Aéreos Inusuales: Un enfoque biopsicosocial (Leuka, Buenos Aires, 1994). Últimamente se ha vinculado también este concepto con las actividades de grises y asimilados.Véase Triángulos mortales y Zona caliente. BAVIC, línea Denominación dada por Aimé Michel a la ortotenia que, según él, unía las ciudades francesas de Bayona y Vichy -de cuyas letras iniciales toma el nombrepasando por otros cuatro puntos donde durante la oleada gala de 1954 se habían observado OVNIs en el mismo día. Análisis posteriores han permitido atribuir esta alineación al puro azar. BENÍTEZ LÓPEZ, Juan José (n. Pamplona, 1946). Licenciado en periodismo por la Universidad de Navarra, desde 1965 trabajó como reportero en La Verdad de Murcia, Heraldo de Aragón de Zaragoza y La Gaceta del Norte de Bilbao y como redactor-jefe en la Hoja del Lunes de esa misma ciudad. En 1973 empieza a escribir reportajes sobre OVNIs para ese penúltimo rotativo, labor que se intensificará a raíz de su destino como enviado especial en Sudamérica, donde se relaciona, entre otros, con el grupo contactista peruano IPRI, experiencias que recoge en sus primeros libros Existió otra humanidad (1975) y OVNIs: SOS a la Humanidad (1975). En 1979 abandonó el periodismo activo para dedicarse profesionalmente a su vocación de escritor, lo que le ha llevado a publicar una veintena de títulos sobre Ufología y materias afines, en los que ha defendido acérrimamente la HET en su versión más clásica y simple y donde ha criticado hasta el insulto personal a quienes discrepan de sus Opinión sobre el fenómeno: Los ovnis han devenido en un objeto cultural producto de la confusión que tiende hacia una construcción colectiva estimulada por hechos materialmente diferentes. BASES DE OVNIS Supuestas instalaciones extraterrestres en nuestro planeta (submarinas o subterráneas) desde donde surgirían los OVNIs. 31 Bioastronomía ideas. Partidario del dogma de la fiabilidad absoluta de los testigos, ha destacado más como comunicador que como estudioso del fenómeno. Entre su producción estrictamente ufológica cabe reseñar la trilogía sobre fotografías de OVNIs formada por Los visitantes, Terror en la Luna y La gran oleada (1982), y OVNIS: Materia reservada (1994), obra consagrada a cuestionar por sistema el proceso de desclasificación de los expedientes OVNI del Ejército del Aire. astrónomos se manifiesta un cierto optimismo sobre las posibilidades de vida inteligente en el universo, explicitado en la famosa ecuación de Frank Drake: N=R* fp ne fl fi fc L En donde N es el número de civilizaciones galácticas capaces de comunicarse con nosotros, R* es la proporción de formación de planetas, fp la fracción de estrellas que tienen sistemas planetarios, ne el número medio de planetas habitables, fl la fracción de dichos planetas con vida, fi la proporción de tales planetas con vida inteligente, fc la fracción de los mismos en los que se llega a la fase de comunicación interestelar, y L la duración de una civilización técnicamente avanzada. Mucho se ha discutido sobre los parámetros astronómicos y biológicos de la ecuación del Dr. Drake, pero habría que llamar la atención también sobre los parámetros culturales y tecnológicos, que tampoco son tan claramente favorables al desarrollo de una ciencia y una tecnología orientada al contacto interestelar en plazos de tiempo coincidentes a través de las enormes distancias interestelares. Recordemos, por ejemplo, que la civilización china dispuso de grandes adelantos tecnológicos que sin embargo no dieron lugar a un método científico como el desarrollado por Occidente siglos después. Opinión sobre el fenómeno (extraída y adaptada de una entrevista en Expedientes Secretos, nº 3, 19 octubre 1996): “Es un tema tan importante y trascendental a todos los niveles que el ser humano siente un rechazo natural. Durante años, se creyó que la Tierra era el centro del universo y todo giraba a su alrededor. Hoy sabemos que eso no es cierto. Con el fenómeno OVNI pasa lo mismo. Sabemos que no estamos solos, que somos seguramente la población más pequeña y ridícula del universo, y eso al ser humano no le gusta (...). Hay muchas pruebas de la existencia de OVNIs que nos son escondidas por los gobiernos. Hay incluso películas. Pero, sobre todo, hay una información extraordinaria de muchos testigos, tanto militares como civiles, que para mí son tan creíbles como cualquier prueba física. Esa información se nos oculta porque aceptar que hay civilizaciones más avanzadas que la nuestra obligaría a revisar todo nuestro sistema y nuestra propia situación en el universo y... ¡eso no interesa!” Las distintas estimaciones se sitúan en torno al millón de civilizaciones avanzadas sólo en nuestra galaxia (Sagan y Shkolvskii, 1966). De ser cierto ese orden de magnitud, la gran paradoja fue señalada hace años por el físico Enrico Fermi: “¿Dónde están los otros?”. En cambio, entre los biólogos existen muchas más dudas, hasta el extremo de que algunos (Erben, 1984) consideran posible que la vida inteligente no haya BIOASTRONOMÍA Parte de la radioastronomía que se ocupa de la posible recepción (v. Seti) y emisión (v. Ceti) de mensajes interestelares. Entre los 32 Blue Book, proyecto aparecido sino una sola vez... la nuestra. principalmente con la dirección del mayor Héctor Quintanilla (1963-1969), la creciente popularidad de los OVNIs y las presiones y críticas recibidas por parte de la opinión pública, la prensa, los políticos y las organizaciones ufológicas privadas motivaron que los militares solicitasen en 1966 un estudio independiente sobre la cuestión a la Universidad de Colorado, que puso el encargo bajo la dirección del profesor Edward Condon. Apoyándose en las conclusiones y recomendaciones del informe Condon, las Fuerzas Aéreas dieron por cerrado el proyecto el 17 de diciembre de 1969, al no estar justificado “ni en la seguridad nacional ni en el interés de la ciencia”. Las conclusiones del informe final presentado poco después fueron que los OVNIs no representaban una amenaza para la seguridad nacional y que no existía evidencia de que “los casos considerados como «no identificados» representen desarrollos tecnológicos más allá del nivel del conocimiento científico actual”. Los documentos del proyecto fueron desclasificados y hoy pueden ser consultados en los Archivos Nacionales de Washington. En el informe estadístico final se recogieron 12.618 casos, de los cuales 701 habían permanecido sin identificar. Con la desaparición del proyecto Blue Book se esfumó también la implicación oficial de la Fuerzas Aéreas norteamericanas en la investigación ufológica, pese a la opinión de los sectores “conspiranoicos” de que todo ello no fue sino una tapadera para el “auténtico” y secreto programa OVNI. [208, 235] [IC] En donde sí existe más coincidencia entre todos ellos es en considerar que el fenómeno OVNI no representa bajo ningún concepto la visita de seres extraterrestres a nuestro planeta. Las simples razones económicas sumadas a las limitaciones impuestas por la barrera insuperable de la velocidad de la luz impiden radicalmente los vuelos interestelares en plazos compatibles con la duración de la vida humana, y menos todavía en la cantidad defendida por los ufólogos más crédulos. [79, 189, 212][LG] BLUE BOOK, proyecto (Proyecto Libro Azul). Último y más famoso de los proyectos de investigación que los militares norteamericanos emprendieron para conocer si los OVNIs eran o no una amenaza para la seguridad nacional de los EE.UU. Con el nombramiento de Edward J. Ruppelt como responsable del proyecto Grudge, éste fue reactivado y cambió su nombre en marzo de 1952 por el de proyecto Blue Book. Bajo su dirección (1952-54) gozó de medios materiales y humanos como nunca volvería a tener, pero apenas fue capaz de gestionar el gran volumen de informes que recibía para su análisis. Tras abandonar Ruppelt el proyecto, la investigacion decayó en una rutina y sus sucesores emprendieron una línea más escéptica que la que había caracterizado a aquél. Según la historiografía tradicional de la Ufología, el proyecto Blue Book actuó desde entonces como una excusa de las Fuerzas Aéreas para explicar el fenómeno OVNI de cualquier manera posible y en función siempre de las necesidades políticas del Pentágono. En los años sesenta, período que se corresponde BOLAS DE FUEGO VERDE Véase Twinkle, proyecto. 33 Bólido BÓLIDO trayectoria desde su posición de observación. Tampoco faltan casos en que algún testigo ha indicado una duración de hasta varios minutos, en contraste con las estimaciones más moderadas del resto de observadores del mismo meteoro. Los meteoros son una fuente habitual de avistamientos de presuntos OVNIs, especialmente los más brillantes, o bólidos (v. también Reentrada de Satélite). El observador en busca del contacto con los hermanos del Cosmos que avista un punto luminoso, con o sin estela apreciable, desplazándose a gran velocidad por el firmamento, creerá descubrir una nave extraterrestre donde otros reconocerán una simple estrella fugaz. Algunos ufólogos han querido ver en estos informes anómalos minoritarios la evidencia de que un determinado bólido debió estar acompañado simultáneamente de verdaderos OVNIs. También hay quien va más allá y opta por descartar la posibilidad de un bólido cuando halla el más mínimo detalle discordante en alguno de los testimonios. Asumir que el fenómeno sobrevoló la vertical de los lugares de observación, o que las horas indicadas por cada uno de los testigos son totalmente fiables, son errores frecuentes en los análisis de este tipo de casos. No obstante, otras veces es la espectacularidad del fenómeno, más que la predisposición del observador, la que resulta determinante. Es el caso, por ejemplo, de los avistamientos de grupos de luces brillantes surcando el cielo majestuosamente, visibles desde una extensa área geográfica. Los fragmentos luminosos de un bólido de estas características pueden ser interpretados por algunos de los observadores como una auténtica escuadrilla de aeronaves o incluso como las ventanillas iluminadas de un único y gigantesco "cohete". Aunque hay determinadas fechas a lo largo del año en que suelen ser más frecuentes ("lluvias de estrellas"), los meteoros son fenómenos impredecibles. Por consiguiente, salvo en casos en que haya un registro gráfico muy completo o se produzca la caída y posterior recuperación de un meteorito, el único criterio de identificación disponible es la comprobación de que las características informadas por todos o, al menos, la gran mayoría de los testigos, sean compatibles con las de un meteoro. Lo cierto es que, en la práctica, ilusiones visuales y de perspectiva, errores y exageraciones hacen que entre los testimonios del paso de un bólido siempre haya algunos que contengan descripciones atípicas y estimaciones erróneas de tiempos y distancias. Mientras son visibles, los meteoros suelen encontrarse a alturas de más de 20.000 metros, siguiendo trayectorias continuas más o menos descendentes y extinguiéndose en cuestión de segundos. Así y todo, algunos testigos (incluidos pilotos profesionales) han llegado a temer una colisión al subestimar radicalmente la distancia. Otros los han visto ascender, confundidos por la perspectiva de la Como ocurre con otras clases de confusiones, las debidas a observaciones de meteoros también han sido asociadas a muy diversos "efectos" sobre el observador y el entorno. Al respecto hay que resaltar que en los casos de avistamientos multitudinarios abarcando un amplio territorio aumenta la probabilidad de que el bólido sea observado por algún testigo particularmente sugestionable o coincida con algún suceso 34 Borraz Aymerich, Manuel llamativo o insólito, como un corte de suministro eléctrico en algún lugar o el descubrimiento de una huella "extraña" en días posteriores. [47, 112] [MB] Pionero de la ufología española. Funcionario de Correos, fue autor en 1955 de uno de los primeros libros editados en España sobre el fenómeno: Astronaves sobre la Tierra (Ed. Oromí, Barcelona), donde aparecía expuesta su célebre teoría sobre el ciclo bienal y se apostaba decididamente por el origen marciano de los platillos volantes. El prestigio ganado con esta obra entre los incipientes aficionados le permitió nuclear el grupo de barceloneses con el que cofundó en 1958 el Cei, del cual fue su primer presidente hasta 1962. Persona desconfiada y dogmática, impulsó durante su mandato la publicación del Boletín del Centro de Estudios Interplanetarios (1959-61), primero en su género en España, al tiempo que mantenía las primeras disputas ideológicas con otros socios de la entidad. Tras descollar como uno de los primeros exponentes de la ufología científica con trabajos sobre sistemas de propulsión (Bol. CEI nº 3, marzo de 1959) o estadísticas de casuística (Bol. CEI nº 9, octubre de 1961), en su última etapa en activo pareció inclinarse hacia posturas mesiánicas y esotéricas. Abandonó de hecho la Ufología en marzo de 1962, después de dejar la presidencia del CEI por las crecientes divergencias con otros miembros, hecho que supuso el práctico colapso de la entidad hasta su relanzamiento en 1967. BORRAZ AYMERICH, Manuel (n. Barcelona, 1961). Ingeniero Superior de Telecomunicación, viene colaborando en la revista Cuadernos de Ufología desde principios de los años ochenta, aportando revisiones y análisis de muy diversos casos. Entre los asuntos tratados destacan las observaciones con explicaciones astronómicas (OVNIs: historias increíbles con explicaciones creíbles, CEI, Barcelona, 1997) y los avistamientos multitudinarios de los años setenta en las islas Canarias (Los gigantes de Gáldar, CdU., Santander, 1992). Opinión sobre el fenómeno (extraída de su recopilación OVNIs: historias increíbles con explicaciones creíbles): "A pesar de que siempre habrá algún que otro caso enigmático, lo que la gente tiene en mente al pensar en «el fenómeno OVNI» no constituye ningún desafío científico. En líneas generales, se «ven» OVNIs porque se habla de los OVNIs, y se habla de los OVNIs porque se «ven» OVNIs. El único punto de contacto entre el conductor que viaja al anochecer y se siente «perseguido» por Venus y el bromista que truca una foto de un disco volador es el hecho de que se «inspiran» en unos mismos lugares comunes. Unas referencias comunes que tienen auténtica categoría de mito moderno y que ellos, a su vez, están contribuyendo a alimentar". BUFORA (British UFO Research Association). Asociación ufológica británica, la única de ámbito nacional existente en el Reino Unido. Fue creada en 1959 bajo el nombre de London UFO Research Organization (LUFORO), denominación que conservó hasta que en 1962 constituyó junto a otros siete grupos la BUFORA propiamente BUELTA SAURA, Eduardo (Paterna, 1916-Barcelona, 1973). 35 BUFORA dicha. Por sí misma o a través de los grupos regionales afiliados, cuenta con una densa red de investigadores que cubre toda Gran Bretaña y que a finales de los años ochenta había recopilado ya unos 15.000 informes, codificados informáticamente en un archivo central. La asociación editaba cada dos meses (hasta 1997) la revista UFO Times, antes conocida como BUFORA Journal. Bianualmente, en los años impares, la BUFORA viene celebrando desde 1981 un congreso internacional que reúne a los más prestigiosos especialistas británicos y extranjeros. Dirección: 16 Southway, Burgess Hill, Sussex, RH15 9ST, Gran Bretaña. En Internet: http:// www.bufora.org.uk [MM] DTU 36 Cabello de ángel C CABELLO DE ÁNGEL CABRIA GARCÍA, Ignacio Denominación otorgada a las excrecencias relacionadas con el paso de OVNIs consistentes en finos hilos de apariencia transparente, de corta vida, que se volatilizan en contacto con el suelo. Con precedentes de siglos pasados recogidos por Charles Fort, el primer caso moderno se registró en Depues Ferry (Pennsylvania, EE.UU.) el 10/11/1949. En Europa, Aimé Michel (1963) recogió las primeras denuncias de tal fenómeno. Estudios relacionados con apariciones marianas, especialmente los realizados por investigadores lusos como Joaquim Fernandes, ponen de manifiesto que en las apariciones de Fátima este fenómeno ya estuvo presente, de ahí la denominación hilos de la Virgen que también reciben. En Jacques Vallée (1967) se documenta este fenómeno en relación al caso francés de Oloron (17/10/1952), donde la permanencia de estos hilos sobre árboles y casas se prolongó durante varias horas. Su composición era similar a los silicatos, haciéndose untuosa al tacto y fácilmente volátil. Su originalidad y el hecho de coincidir en ocasiones con la presencia de OVNIs en el cielo hizo que el cabello de ángel fuera considerado una de las evidencias de la realidad material de aquéllos. No obstante, parece que su origen debe buscarse en las finas hebras que algunos arácnidos producen para desplazarse volando a grandes distancias, si bien no en todos los casos ha sido posible hallar arañas entre las muestras recogidas. [110, 162, 250][ JR] (n. Santander, 1955). Antropólogo y funcionario del Ministerio de Asuntos Exteriores español, se inició en la Ufología en 1976 en el seno del Ciove cántabro, desde donde participó en las diversas actividades divulgativas organizadas por el grupo. Durante su residencia en Barcelona se vinculó al Cei y fue miembro del comité de redacción de la revista Antropologies. Desde 1987 es asesor de Cuadernos de Ufología, tribuna desde la que ha dado a conocer sus trabajos sobre la antropología del fenómeno OVNI (“OVNIS y extraterrestres como sistemas de significación”, nº 9-10, 1990; “El mito de la venida de los extraterrestres: génesis y estructura”, nº 11, 1991) y su vertiente histórica (“Sesma, Saliano, UMMO y la Ballena Alegre: una historia del contactismo español”, nº 3, 1988; “Historia del interés social por los OVNIS en España. Medios de comunicación y movimiento ufológico”, nº 6, 1989). En esta última faceta es autor de una obra sin parangón en su género: Entre Ufólogos, Creyentes y Contactados. Una historia social de los OVNIs en España (Cuadernos de Ufología, Santander, 1993), la más completa visión histórico-crítica de la ufología española y auténtico hito en la literatura ufológica nacional. Opinión sobre el fenómeno (tomada de Cabria, 1993, op. cit.): “No es cierto que la ufología estudie lo que no tiene cabida en otras clasificaciones de lo natural -lo no identificado-, sino que estudia 37 Calidad, índice de aquellas cosas a las que se atribuye un determinado significado: el origen extraterrestre. Esto no es una boutade. Aunque no está dicho de manera explícita en las definiciones de lo que son los OVNIS, sí se encuentra de forma implícita. (...) . Sólo desde un punto de vista sociologista puedo intentar una definición del fenómeno OVNI: es todo aquello que en nuestra cultura popular se considera relacionado con un origen extraterrestre; o para ser más provocativo: OVNI es lo que la gente llama OVNI. (...) De acuerdo con esta significación permanente de «extraterrestre» que el fenómeno OVNI ha llevado asociada, la delimitación del objeto de estudio de la ufología ha ido evolucionando de acuerdo con lo que los especialistas han considerado verosímil como atribuible a una civilización exterior.” así como “La verdad sobre el caso 22/6/76” (en Espacio y Tiempo, nº 47, enero 1995) y “El ovni de la discordia” (en Enigmas nº 5, marzo 1996), ambos sobre las espectaculares experiencias con misiles observadas desde el Archipiélago Canario (v. Canarias, casos de). Opinión sobre el fenómeno: “Aunque no me decanto completamente por ninguna de las hipótesis explicativas tradicionales, pienso que es fundamental la influencia de los medios de comunicación en la generación, desarrollo y supervivencia de la creencia ovni. El fenómeno ovni sería un mito contemporáneo, al margen de que en alguna ocasión y en casos muy concretos se haya manifestado algún fenómeno enigmático”. CANARIAS, casos de CALIDAD, índice de Las Islas Canarias han poseído tradicionalmente cierta popularidad ufológica como zona especialmente propicia para la observación de supuestos fenómenos catalogables como OVNIs. No obstante, esto no pasa de ser una leyenda o un simple rumor. Entre los casos más relevantes del archipiélago canario destacan los que a continuación se exponen. Puede encontrarse abundante información general sobre la casuística canaria en González (1995) y Campo (1996). Véase Índices de Ballester-Guasp. CAMPO PÉREZ, Ricardo (n. Santa Cruz de Tenerife, 1965). Integrante de la llamada “cuarta generación” de ufólogos, licenciado en Filosofía e investigador canario del fenómeno OVNI desde 1988. Es representante de Cuadernos de Ufología en el archipiélago y colaborador de la Fundación Anomalía y el Cei de Barcelona. Compilador de la casuística canaria en el catálogo denominado CATCAN (Catálogo Canario), ha investigado y reencuestado los casos insulares más significativos. Fruto de ello es su ensayo “El fenómeno ovni en Canarias” (en Cuadernos de Ufología nº 19-20, 1996), El 22 de junio de 1976, a las 22:30 h., se observa desde todas las islas, excepto Fuerteventura y Lanzarote -las más orientales-, un espectacular fenómeno luminoso semiesférico que daba la impresión de apoyar su base en el horizonte. Previamente se vio ascender un punto luminoso que "quedó estático" o girando 38 Canarias, casos de según algunos testimonios. Otras versiones hablan de dos formas rojizas que giraban y se movían en zig-zag, tal y como cita el expediente desclasificado por el Ejército del Aire español. La diferencia de un testimonio a otro depende, con toda seguridad, del momento en que fue divisado, en función de la orografía, condiciones meteorológicas, etc. Como es sabido, el suceso desde el principio adquirió injustificadamente tintes de encuentro cercano, al ser centrado en uno solo de los testigos, aquél que ofreció una versión más subjetiva de lo observado (seres de tres metros en el interior de una esfera, "misiones elevadas", etc.). Sus características y las circunstancias que rodean el caso permiten afirmar que se trató de algún tipo de prueba balística o experiencia con cohetes en aguas internacionales del Atlántico. Málaga-Tenerife, y un tercero de Air France en la aerovía Casablanca-Agadir. Un cuarto avión también pudo contemplarlo: un T-12 del 461 Escuadrón en el que viajaba, desde Lanzarote a la base aérea de Gando, el General de División Carlos Dolz Espejo, a la sazón General Jefe de la Zona Aérea de Canarias, junto a otros mandos inferiores. Simultáneamente fue observado desde suelo canario, en concreto desde diferentes localidades de Gran Canaria y Tenerife. Igualmente fue contemplado por el buque escuela de la Armada española Juan Sebastián Elcano cuando se encontraba al oeste de Lanzarote en viaje de maniobras desde Cádiz a Las Palmas. Los testigos militares que viajaban en el T-12 afirmaron que el gran semicírculo tenía unos recuadros rectangulares de color verde botella en su centro geométrico. La dirección de observación, como en el resto de casos similares, fue oeste. La explicación más plausible parece ser algún tipo de experiencia con cohetes en el Atlántico. El 19 de noviembre de 1976, a las 19:05 h., empezó a divisarse desde diversos aviones que sobrevolaban terreno peninsular y varias localidades canarias, al igual que desde algunos barcos cercanos a las islas, un fenómeno de carácter luminoso tan espectacular como el del día 22 de junio. Comenzó viéndose un punto brillante mayor que una estrella de 1ª magnitud que, después de efectuar dos o tres giros en espiral, según uno de los testigos de alta mar, fue agrandándose hasta adquirir un diámetro aparente de unas dos o tres veces el disco lunar (gigantesco, según algunos testimonios). En ese momento su forma era semicircular, aparentando tener su base sobre el horizonte. Su duración puede cifrarse en unos 25 minutos. Fueron recogidos testimonios desde el espacio aéreo peninsular, concretamente desde diversos aviones comerciales en vuelo; entre ellos cabe destacar tres Boeings 727: uno de Spantax que hacía la ruta Santiago de Compostela-Madrid, otro de Iberia en vuelo La más conocida de las observaciones canarias es la de 5 de marzo de 1979. Se inició aproximadamente a las 18:30 h. con unas líneas serpenteantes de diversos colores; "un extraño atardecer", señaló el juez informador del Ejército del Aire en el informe final desclasificado por el Mando Operativo Aéreo. Posteriormente es vista ascender una estela de color blancoamarillento que comienza a expanderse enormemente hasta que, de forma paulatina, ya totalmente de noche a ras de suelo, se diluye y desaparece; en ese momento vuelven a verse líneas semejantes a las del principio. Todas las islas canarias pudieron contemplar el espectáculo luminoso a la misma hora -centrándose su fase más espectacular alrededor de las 20:05 h.- y en la misma dirección, aproximadamente oeste. 39 Carballal, Benito Manuel La hipótesis más realista para explicar el fenómeno luminoso es que se trató de una prueba con misiles o cohetes en el Atlántico, a centenares de kilómetros al oeste de las islas (v. Ingenios astronáuticos).[46, 56, 99][RC] ortodoxos, se han centrado en la denuncia de timos y fraudes esotéricos, y de supuestas tramas que emplearían a los OVNIs para encubrir pruebas aéreas secretas. Otros libros de Manuel Carballal: Secuestrados por los OVNIs (1992), Los peligros del esoterismo (1995) y La ciencia frente al misterio (1995) CARBALLAL, Benito Manuel Opinión sobre el fenómeno (tomada de esta última obra): ”Tras años de intensa investigación, me consta plenamente que el fenómeno OVNI es un hecho incuestionable (...) pero la reflexión lógica me impide aceptar la HET clásica como viable para explicar los OVNIs (...) En mi opinión, efectivamente los OVNIs no son astronaves extraterrestres, pero resulta incuestionable que SÍ SON (...): manifestaciones desconocidas de fenómenos naturales, inteligencias de mundos paralelos, fenómenos místicos, manipulaciones terrestres...” (n. A Coruña, 1967). Investigador y escritor paracientífico, con estudios de Electrónica, Teología y Criminología. En 1984 creó en su ciudad natal el grupo Fénix, cuyas primeras actividades se centraron en la Parapsicología. En los años siguientes recorrió media España en busca de todo tipo de fenómenos paranormales, desde personas con poderes psíquicos hasta sectas y ritos satánicos (tema de su primer libro: El síndrome del Maligno, 1990, escrito con Gabriel Carrión). Su interés por el fenómeno OVNI se inició a partir de un episodio de abducción sufrido por unos de sus amigos en 1986 y, sobre todo, por un avistamiento del que fue testigo en Montserrat junto a Javier Sierra y el contactado Luis José Grífol (23/07/1987). Tales experiencias le condujeron a especializarse en el seguimiento de varios grupos contactistas (Sixto Paz, hermanos Bongiovanni, caso Daro: v. Cuadernos de Ufología nº 16-17 y 18, 1994 y 1995). Asiduo conferenciante y colaborador habitual de las revistas comerciales paracientíficas, destacan también sus entrevistas a pilotos (OVNIs y pilotos, 1995) y la recopilación de la casuística gallega que realizó junto a otros investigadores (catálogo CATAG: Cuadernos de Ufología nº 13, 1992). Ya profesionalizado como reportero de lo paranormal, sus investigaciones más recientes, que no siempre han seguido cauces CASOS POSITIVOS Y NEGATIVOS En general, se conocen por el nombre de positivos aquellos incidentes para los que no ha podido hallarse una explicación convencional satisfactoria (OVNIs estrictos); mientras que reciben la denominación de negativos los que sí han podido explicarse cabalmente (OVIs). Las relaciones entre ambos grupos parecen venir marcadas por la llamada indiscernibilidad, definida inversamente- por Ballester Olmos como la supuesta cualidad de unos avistamientos (los casos positivos) sobre otros (los negativos) por la que las propiedades de los primeros son intrínsecamente diferentes de las de los segundos y, en consecuencia, tienen origen y naturaleza distintos (Ballester, 1995). En definitiva, de lo que se trata es de averiguar si el primer grupo posee 40 Catálogos de casuística CATÁLOGO DE CASUÍSTICA características específicas que permitan determinar con claridad si hay o no un fenómeno OVNI con sustantividad propia y diferenciar éste de la casuística OVI. Fichero de casos creado conforme a principios específicos y uniformes de realización contenidos en las directrices de una lista de incidentes que describe el material de la colección. En un sentido más específico, lista de casos OVNI presentados cronológicamente y preparada según una o varias características concretas, sean éstas de orden intrínseco (forma, número, etc.) o externo (geográficas, temporales, etc.). Para dilucidar la cuestión se ha propuesto compilar muestras de ambos tipos de sucesos y cotejarlas estadísticamente, cosa que lleva a considerar el problema de la pureza de los catálogos que se manejan y a la necesidad de proceder a la depuración y filtrado de los mismos antes de efectuar las comparaciones. Ésta es la filosofía que subyace, por ejemplo, en el catálogo Unicat, según la cual el ruido de fondo existente en los catálogos ufológicos es la causa de las distribuciones estadísticas similares que aparecen hoy al comparar repertorios de casos positivos y negativos. No obstante, otros autores se oponen a este tipo de estrategias por entender que lo único que así se consigue, por operar con filtros cargados de subjetividades, es un resultado acorde al estereotipo de OVNI que ha creado la comunidad ufológica. Junto a esta idea normalmente se sostiene la nimiedad de las diferencias existentes entre ambas categorías o la consideración de que éstas no existen y que tanto los OVNIs como los OVIs pertenecen a un mismo conjunto. Pese a que dentro de la Ufología es considerada como una de las actividades más importantes que puede desarrollarse, la confección de repertorios de casuística no presenta caracteres homogéneos respecto a sus finalidades ni en relación a los criterios o campos descriptivos empleados. Aunque un catálogo debería servir como herramienta de trabajo para la investigación, a menudo, y tal vez por los magros resultados que se obtienen, se ve más como un fin en sí mismo que no como un medio para avanzar en el conocimiento. En consecuencia, sus propósitos -al margen del puro coleccionismo- dependen a menudo de las concretas tesis o presunciones que su autor pretenda resaltar. La variedad de criterios para la selección de los sucesos es muy grande, si bien generalmente se reducen a consideraciones territoriales (catálogos nacionales y regionales) o relativas a la tipología de los incidentes agrupados (encuentros cercanos, casos fotográficos, con intervención militar, etc.). En este sentido, la determinación de los campos plasmados en los listados dependerá en gran medida de las finalidades y de los criterios adoptados, por más que suelen resultar ineludibles las menciones a la fecha, hora, lugar y fuente del suceso. Pese a la capital importancia de hallar atributos específicos del fenómeno, no es un tipo de debate que se haya prodigado entre los ufólogos. Ello probablemente se deba en gran medida a que no existe ningún estudio que haya podido establecer con claridad tales diferencias ni parece sencillo encontrarlas. El último intento en este sentido (Morey, 1996) involucra técnicas propias de la física de sistemas dinámicos, lo que da una medida de lo rebuscado que resulta resaltar las supuestas características particulares de los OVNIs. [35, 112, 167][MM] 41 Catálogos de casuística PRINCIPALES CATÁLOGOS DE CASUÍSTICA UFOLÓGICA Nombre Tipo Autor Fecha AEROCAT Casos aeronáuticos (ESP) Plana 1985/I ANDALCAT Andalucía Ruesga 1993/P Becassine EC-3r tipo Breysse 1986/I CASUIB Islas Baleares Morey 1996/P CATAG Galicia Carballal 1992/P CATCAN Canarias Campo 1991/I CATIB España y Portugal CEI 1989/P Principado de Asturias Objetos caídos (ITA) Comunidad de Madrid León y Souto 1994/P Stilo 1996/I Montejo y Padial 1990/I ITACAT EC (ITA) Verga / CISU 1985/I LANIB EC ibéricos Ballester y Fernández 1987/C Magonia Tipo I Vallée 1996/C Intervención militar EC negativos ibéricos Ballester y Plana 1989/I Ballester y Fernández 1987/C TRACAT Huellas (ITA) Verga / CISU 1985/I UFOCAT General Saunders / CUFOS 1980/C UFODATA Dinamarca Andersen / SUFOI 1980/P UNICAT Alta calidad Smith 1984/I USOCAT Objetos subacuáticos (ITA) Bianchini 1996/I CIOPA CRASHCAT FAECOM MILICAT NELIB Notas: C: concluido y publicado; P: publicado y aún en desarrollo; I: iniciado 42 CAUS La elaboración de catálogos fue una labor muy ligada a las pretensiones cientifistas de los ufólogos de los años sesenta y setenta. Tras una época de escasa actividad en esta materia, en los últimos tiempos las posibilidades brindadas por los ordenadores le han proporcionado un nuevo impulso. [233][MF, MM] obra de Eduardo Buelta, primer presidente de la asociación. Después de unos años de escasa actividad, a finales de 1967 un grupo de personas entró en contacto con aquel CEI para plantear el proyecto de una refundación. Comenzó así una nueva etapa marcada por la aprobación de nuevos estatutos, la entrada en servicio del actual local social y la publicación, entre 1970 y 1981, de la revista Stendek. Dado que materialmente no se heredó nada del antiguo CEI, se procedió a crear un archivo, organizar una biblioteca y editar un sencillo boletín para los socios. En aquellos años se organizaron multitud de actos culturales y sociales, sin olvidar la investigación de todos los casos ocurridos en España. El amplio y polifacético archivo se ha convertido con el tiempo en un verdadero banco de datos, base del CATIB (Catálogo Ibérico), a disposición de quienes se han vinculado al CEI. La biblioteca contiene más de 600 volúmenes relacionados con el tema OVNI y posee una sección de mecanoscritos de trabajos de investigación originales no publicados y una hemeroteca con varios miles de revistas y boletines procedentes de todo el mundo. Entre 1970 y 1974, además, se mantuvo activa en la capital de España la delegación CEI-Madrid, integrada por jóvenes universitarios que desarrollaron diversos trabajos de investigación de campo y de gabinete. CAUS Véase Just Cause. CEFO (Coordinadora de Estudios del Fenómeno OVNI). Federación de grupos (RNC, CIOVE, IIEE, ADEM) e investigadores creada en diciembre de 1978 en Huesca como respuesta al supuesto elitismo del Consejo de Consultores de Stendek. Tras celebrar reuniones en Zaragoza (1979) y Sevilla (1980), desapareció a finales de 1981 a consecuencia del descenso del interés popular por los OVNIs y por una serie de disensiones internas. [55][MM] CEI (Centro de Estudios Interplanetarios). Entidad decana de la ufología española fundada en octubre de 1958 en Barcelona por -entre otros- Eduardo Buelta, Antonio Ribera y Màrius Lleget. Durante su primera época, el CEI editó, entre enero de 1959 y diciembre de 1961, nueve números de un Boletín -el primero en su género en España- en el que se dio a conocer el estudio de la relación entre las oleadas OVNI y las oposiciones de Marte, Tras una etapa intermedia de baja actividad entre los años 1985 y 1993, a partir de enero de 1994 el CEI reinició sus actividades con renovado vigor, volviéndose a programar actos públicos y a editar un nuevo boletín: Papers d’Ovnis. El CEI sigue en la actualidad preparando nuevos proyectos, como la publicación del contenido de su biblioteca, al que seguirá el 43 CEONI de la hemeroteca. También ha editado, a partir de los fondos de su archivo, una colección de monografías de las que hasta ahora han aparecido nueve títulos y que a finales de 1996 se completará con otros ocho más. En este momento está apoyando a la Fundación Anomalía y se encuentra integrado en el staff de Cuadernos de Ufología. El CEI cerró su local en 2013, y sus fondos documentales fueron donados al AFU sueco, aunque mantiene presencia en Internet: https://www.el-cei.org [PR] colaboradores, nucleado por Ballester Olmos, que algún tiempo después Aimé Michel bautizaría con el nombre de “Escuela Valenciana de Ufología”. [MM] CEONI CERTIDUMBRE, índice de CEREALOGISTAS Denominación que reciben los estudiosos de los círculos aparecidos en campos de cultivo. Véase Círculos en campos de cereales. Véase Índices de Ballester-Guasp. (Círculo de Estudios sobre Objetos No Identificados). Entidad de investigación ufológica creada por Vicente-Juan Ballester Olmos en Valencia en febrero de 1968, si bien venía ya funcionando informalmente desde un año antes con el nombre de Comisión de Estudios sobre ONI. Tal asociación fue fundada en el seno del Colegio Mayor Alejandro Salazar, que proporcionaba la infraestructura material y el soporte económico, razón por la cual se exigía poseer estudios universitarios para ser miembro de la misma. Estructurada en cinco secciones (casuística contemporánea, casos en la antigüedad, teorías explicativas, hipótesis extraterrestres y fotografías), su principal actividad consistió en organizar en noviembre de 1968, por primera vez en España, un “Seminario de Información sobre Objetos No Identificados” en un campus universitario y con una intervención a cargo de un militar del Ejército del Aire. En noviembre de 1972 el CEONI decidió disolverse a causa de la ruptura del nexo legal que le unía con el Colegio Mayor y por la excesiva carga burocrática-administrativa que empezaba a generar. En su lugar se optó por crear un grupo informal de asesores y CETI (Contact with Extra-Terrestrial Intelligence). Contacto con inteligencias extraterrestres. Supone adoptar una postura activa, emitiendo nuestros propios mensajes para que sean captados por posibles seres inteligentes extraterrestres. Ya en el siglo XIX, científicos como Karl Gauss, Joseph von Littrow o Charles Cros discutieron posibles formas de enviar señales a la Luna o Marte (principalmente la demostración gráfica del teorema de Pitágoras) mediante enormes plantaciones de árboles, fuegos o espejos. Cuando se abandonaron las esperanzas de vida inteligente en los planetas próximos, la enormidad de las distancias interestelares sólo dejó abierta la posibilidad del empleo de señales electromagnéticas, y así en noviembre de 1974 se envió desde el radiotelescopio de Arecibo el primer mensaje deliberado al espacio en dirección a la constelación de Hércules, a 25.000 añosluz de distancia. A efectos puramente testimoniales, en los únicos cuatro satélites 44 CETI artificiales que han abandonado de momento nuestro sistema solar (los Pioneer 10 y 11, y los Voyager 1 y 2, lanzados en la década de los setenta) se incluyeron, respectivamente, una famosa placa (considerada pornográfica por algunos al incluir las figuras de un hombre y una mujer desnudos) y un videodisco con imágenes y música de todos los rincones del planeta. consideran que este argumento es una simple “falacia homocéntrica” o llegan a considerarlo “irrelevante” (Budd Hopkins). Algunos, como Stanton Friedman, opinan que se trata de misiones de recogida de datos no preocupadas por el contacto con seres inferiores. La excusa más común son los terribles efectos que causaría en la humanidad dicha revelación, extrapolando exageradamente los disturbios causados en distintas partes del mundo cuando se ha dramatizado como real la famosa novela de H.G. Wells La guerra de los mundos, o los efectos negativos que sobre los aborígenes del Nuevo Mundo tuvo su descubrimiento por parte de los europeos. Nadie, sin embargo, menciona el caso de Japón, país muy atrasado y aislacionista a ultranza hasta que en 1853 el comandante americano Perry forzó su apertura a Occidente, y que en la actualidad se ha convertido en líder mundial en muchos campos. Además esta excusa choca de frente con el gran número de casos acumulados a través de los años y la enorme variedad de seres descritos. Resulta irracional esperar que todos ellos apliquen tan estrictamente esa especie de Primera Directiva (a lo Star Trek) de “no interferencia”, que ninguno en todos estos años haya optado por un contacto abierto. Porque, a mayor abundamiento, eso sería éticamente reprobable: si los extraterrestres superiores existen, ¿cómo permiten que tantos humanos sufran y mueran de hambre y enfermedades?. La principal crítica a este tipo de esfuerzos es el enorme tiempo que puede tardar una respuesta (como mínimo decenas de años). Pero si todos escuchamos y nadie emite, el fracaso está asegurado. Aunque, de todas formas, llevamos emitiendo involuntariamente desde hace décadas otro tipo de mensajes electromagnéticos: programas de radio y de televisión (hay quien cree que ello tal vez explique por qué nadie ha respondido). ¿O sí? Para los creyentes en la HET, los OVNIs son precisamente naves extraterrestres que nos visitan. No se trata ya de simples mensajes, sino de un contacto físico y en persona. Aparece entonces en toda su crudeza el llamado Problema del NoContacto, que podríamos parafrasear así: ¿por qué tras tan largo viaje y casi cincuenta años -por lo menos- de observaciones, sigue sin existir un contacto abierto y claro? Los ufólogos partidarios de la HET han adoptado distintas posturas ante este tema. Los más crédulos simplemente niegan el problema: sí existe un contacto, pero es a nivel de las más altas esferas y se mantiene oculto al público en general, beneficiando a los de siempre. O bien, en las famosas palabras de Charles Fort, “somos propiedad”. Parece claro que, de ser así, nadie explota esa propiedad. Otros, como James Otras alternativas manejadas en los ambientes ufológicos serían el efecto “Catflap” (trampilla de gato) defendido por Aimé Michel: en el momento en que una civilización alcanza la tecnología necesaria para el primer viaje interestelar puede literalmente ocupar toda la galaxia en pocos millones de años. Por tanto, ya estarían aquí McCampbell, 45 Chupacabras antes que nosotros, sólo que seríamos incapaces de percibirlos. También se ha hablado de reserva animal o manicomio galáctico. Pero la más original sin duda corresponde a Delillo y Marx, que en 1979 presentaron su Teoría Turística. Si los OVNIs estuvieran tripulados por turistas se explicarían muchos de sus peculiares comportamientos, incluso hasta las más aberrantes abducciones (¿turismo sexual?). Pueblo de 9/04/1954), si bien fue también hallada de forma independiente por Eduardo Buelta, quien la presentó en su libro Astronaves sobre la Tierra (Ed. Oromí, Barcelona, 1955) y la fue desarrollando en artículos posteriores. Otros autores europeos como Aimé Michel en 1954 y Jacques Vallée (1967) apoyaron en su momento estas mismas ideas. Según Buelta, la coincidencia de las oleadas con las oposiciones de Marte (cuando el Planeta Rojo se encuentra más próximo a la Tierra) era causada por los viajes de exploración de los marcianos a nuestro mundo, opinión que se veía reforzada por el llamado “desplazamiento hacia el Este” de las mismas, tal como se había comprobado en las oleadas de 1950 (costa del Pacífico), 1952 (costa occidental de EE.UU.), 1954 (Francia e Italia) y 1956 (Asia). Siguiendo estas pautas, Buelta predijo (Boletín del CEI nº 1, enero 1959) una nueva tanda de avistamientos para febrero de 1959 en Australia y Filipinas, cosa que de acuerdo al propio Cei (Boletín nº 6, junio 1959) se habría visto más tarde confirmado por una masiva presencia de OVNIs en esos países. Buelta aún pronosticó una última oleada para 1961 en el Pacífico, la que debería cerrar el ciclo exploratorio extraterrestre tras dar la vuelta al mundo; pero en el nº 9 del Boletín (octubre de 1961) el propio autor reconoció el fracaso de su predicción. En cambio, para los escépticos la falta de contacto abierto y manifiesto es la piedra de toque que demuestra la inexistencia de seres extraterrestres. Resulta inconcebible recorrer las inmensas distancias interestelares para luego no darse a conocer. [LG] CHUPACABRAS Véase Mutilaciones de ganado. CICLO BIENAL Según algunos autores, período de dos años y dos meses (equivalente al intervalo entre dos oposiciones de Marte) que mediaría entre las sucesivas oleadas de avistamientos OVNI. El descubrimiento del ciclo bienal fue fruto, por una parte, de los primeros estudios estadísticos sobre la casuística ufológica y, por otro, de la creencia en el origen marciano de los platillos volantes, que había sido propuesto por Donald E. Keyhoe en 1949 al mismo tiempo que la hipótesis extraterrestre. Históricamente le corresponde a Óscar Rey Brea el hallazgo de esta correlación (expuesta por primera vez en un artículo aparecido en el periódico Pese a que incluso en tiempos recientes han existido intentos de resucitar la validez del ciclo bienal (o de su mitad, 13 meses: v. Ritrovato, 1995), lo cierto es que no existen datos empíricos que actualmente lo avalen ni pueden considerarse fiables los análisis estadísticos que en su momento permitieron deducir esta suposición. No obstante, como 46 Ciencia ficción, extraterrestres en la literatura de ha afirmado Ares de Blas, las tesis marcianas de Buelta, Rey Brea y otros tuvieron el mérito de ser al menos falsables (verificables), característica que no poseen ni la HET en general ni muchas otras hipótesis explicativas que posteriormente han formulado los ufólogos. [55, 201, 250][MM] CIENCIA FICCIÓN, extraterrestres en la literatura de como ganado. Incluso en la actualidad muchos ufólogos defienden que éste es realmente el caso. De todas formas, dentro del tema de la invasión extraterrestre la ciencia ficción ha desarrollado infinidad de subgéneros (desde su ausencia absoluta en la ciencia ficción rusa, hasta las invasiones benignas de Arthur C. Clarke -El fin de la infancia (1953)-, las cómicas -Marciano vete a casa (1955) de Fredric Brown-, etc.) que no parecen tener ninguna relevancia en el fenómeno OVNI moderno. La mayoría de los autores considera exagerado retrotraer los inicios de la ciencia ficción más allá de Verne y Wells. De hecho, antes del siglo XIX los seres extraterrestres que aparecen en la literatura no eran genuinos seres extraños, sino simples copias terrestres con matices utópicos o exageraciones satíricas. Pero sirvieron para instaurar la premisa de que los extraterrestres son superiores a los humanos (Kepler, Voltaire, etc.). La idea de un extraterrestre real sólo pudo originarse tras la asimilación del concepto de evolución. El primer escritor de este tipo con gran repercusión en el público podría ser Camile Flammarion (La pluralidad de mundos habitados, 1862; Astronomía popular, 1880), quien contribuyó indudablemente a la imagen colectiva sobre los extraterrestres con su defensa de la vida en el moribundo planeta Marte y sus canales. A partir de los años 30 la ciencia ficción en Estados Unidos alcanzó gran difusión popular en novelas baratas pulp, dominadas fundamentalmente por las space operas, aventuras siderales donde los seres extraterrestres que aparecen tienen apariencia casi humana. Han tenido una ascendencia directa y evidente sobre el contactismo, y quizá de forma más sutil en todo el fenómeno OVNI porque generalmente se caracterizaba a las razas extraterrestres como amorales, siendo los seres humanos la única especie inteligente con escrúpulos respecto a la manipulación o destrucción de seres vivos. A este estilo pertenecen las contribuciones españolas al género que escribieron George H. White, pseudónimo de Pascual Enguidanos (Saga de los Aznar, 1955-58) y nada menos que Antonio Ribera (El gran poder del espacio, 1957; Ellos, 1958; Los comandos de la humanidad, 1961). Otro tipo de influencia (que resultaría duradera) está representada por H. G. Wells en sus dos conocidas novelas La guerra de los mundos (1898) y Los primeros hombres en la Luna (1901). En ambas, la apariencia de los extraterrestres es claramente repulsiva y sus intenciones, sobre todo en la primera, no pueden ser más perversas, ya que pretenden colonizar la Tierra y tratar a la raza humana Volviendo a la historia de la ciencia ficción, se considera Una Odisea Marciana de Stanley Weinbaum (1934) como el primer retrato complejo, humanizado y plausible de un ser extraterrestre con personalidad propia que pasa a ser el centro del relato. En torno a la Segunda Guerra Mundial la atención de los autores se trasladó a los problemas de comunicación 47 Ciencia ficción, Ufología y fundamental sigue siendo la incapacidad de la ciencia ficción para presentar la extrañeza esencial de todo ser extraterrestre no solo a nivel formal sino también cerebral. Los intentos más logrados pueden ser el océano inteligente de Solaris (1961) de Stanislaw Lem, la nube estelar de The Black Cloud (1957) de Fred Hoyle, o la parte final de 2001: Una Odisea del Espacio (1968). Aunque, desde luego, siempre con mucha más imaginación y extrañeza que las ideas defendidas por los ufólogos extraterrestristas. [37, 64, 67, 169][LG, MM] (lingüística y política) -Primer Contacto de Murray Leinster (1945)-. Al finalizar el conflicto, el boom editorial de la ciencia ficción abre los limites temáticos y permite una mayor diversidad tipológica, y volviendo hasta cierto punto a los orígenes, los extraterrestres se utilizan para evaluar o criticar las actitudes y valores humanos (militarismo, sexo -Los amantes (Philip José Farmer, 1952)-, racismo, religión -Un caso de conciencia ( James Blish, 1958)-, etc. A partir de 1964 se inicia una nueva fase histórica, la llamada New Wave, caracterizada por una mayor atención por los aspectos formales y al "espacio interior" del hombre antes que al espacio exterior. La nueva corriente hizo desaparecer a los extraterrestres de las obras de ciencia ficción, salvo celebradas excepciones como La mano izquierda de la oscuridad (Ursula K. Le Guin, 1969). Cerrado hacia 1970 el experimentalismo de esta etapa, asistimos hasta la actualidad a una nueva diversificación del tratamiento de la figura del alienígena. Por un lado, algunos autores han recuperado el cultivo de la space opera (C.J. Cherryh, Orson Scott Card) incluso en España (Ángel Torres y el binomio J. RedalJ.M. Aguilera) y por otro, los extraterrestres son el eje central de muchas otras novelas. Así, encontramos obras sobre "primeros contactos" como En el océano de la noche (Gregory Benford, 1977), El texto de Hércules ( Jack McDevitt, 1986), o Contacto (Carl Sagan, 1985); sobre relaciones entre diversas especies (Marea estelar, David Brin, 1983); sobre extrañas tecnologías (Mundo anillo, 1970, Larry Niven), o Cita con Rama, 1973 (y sus secuelas) de A.C. Clarke; etc. etc. CIENCIA FICCIÓN, Ufología y La difusión del fenómeno OVNI ha estado íntimamente ligada a la ciencia ficción. El primer gran divulgador del fenómeno de los platillos volantes en general, y de la observación fundacional de Kenneth Arnold en particular, fue Ray Palmer, director de una de las más importantes revistas de ciencia-ficción de la época, Amazing Stories. Durante los años anteriores a 1947 este personaje aumentó considerablemente la circulación de su revista incluyendo en la misma una serie de historias, supuestamente basadas en hechos reales, y que se han llegado a conocer como el misterio Shaver, mostrando como todos somos manipulados a distancia por los rayos de unos malignos robots habitantes de enormes cavernas subterráneas. Con este precedente de teorías conspirativas y paranoicas (que tan perniciosa influencia siguen teniendo aún hoy), Palmer reconoce en el avistamiento de Arnold un nuevo filón, y a principios de 1948 deja su anterior trabajo y funda la primera revista dedicada a los fenómenos extraños, Fate, en cuyo número inicial aparece el primero de varios Pese a la amplia diversidad que hemos tratado de poner de manifiesto el punto 48 Ciencia ficción, Ufología y artículos firmados por el propio Arnold que acabaría siendo contratado como uno de esos nacientes ufólogos, llegando a investigar y defender la verosimilitud del infausto fraude de la isla Maury. (creado en 1934). Naturalmente los platillos volantes han figurado asiduamente en este tipo de publicaciones desde sus inicios. Y han traspasado géneros interviniendo, por ejemplo, en las aventuras de Tintín dibujadas por Hergé (Vuelo 714 para Sidney) hasta acabar naturalmente convirtiéndose en los verdaderos protagonistas como en las tiras francesas de Jacques Lob y Robert Gigi, Le Dossier des Soucoupes Volantes (1972), Ceux Venus d' Ailleurs (1973) y O.V.N.I., Dimension Autre (1975). Incluso el propio Von Däniken ha divulgado sus teorías astroarqueológicas mediante comics. Un tipo distinto de influencia mutua es la que señala el autor francés Bertrand Méheust en su obra Science-fiction et soucoupes volantes (1978). Inspirado por la forma en la que la famosa nave aérea estadounidense de 1896/97 reflejaba hasta en sus más pequeños detalles el relato de Julio Verne Robur el Conquistador (1886), Méheust analiza la ciencia ficción europea (principalmente de las décadas de 1920 y 1930) encontrando los más sorprendentes precedentes, tanto en los temas generales (formas, evoluciones, luces, efectos en los testigos y en la maquinaria, secuestros, etc.) como en elementos muy concretos y específicos de conocidos incidentes OVNI. La coincidencia es tan absoluta que el autor sólo puede concluir que ambos fenómenos (los relatos de ciencia ficción y los OVNIs) se nutren de un fondo arquetípico común manipulado por un desconocido "Agente X". Podemos disentir en la conclusión, pero los hechos son innegables. Pero la ciencia ficción siempre ha negado los platillos volantes. Isaac Asimov y Arthur C. Clarke han escrito muchos artículos denunciando la manía platillista. Y cuando los escritores de ciencia-ficción han utilizado elementos ufológicos en sus relatos, por lo general lo han hecho de forma irónica, simbólica o meramente oportunista. Por ejemplo, Robert Heinlein explota los miedos a la invasión extraterrestre en su Amos de títeres (1951) y el Mesías de Gore Vidal (1954) se inicia con un análisis de los OVNIs como portentos, anticipando en cierta manera las teorías de Jung. Más recientemente, el fenómeno OVNI ha vuelto a inspirar a los autores de cienciaficción hacia dos líneas diferentes. Por un lado, hacia hipótesis alternativas a la extraterrestre como pueden ser los OVNIs como seres vivos en Dream Maker de W.A. Harbinson (1991); la de armas secretas nazis en la tetralogía de W.A. Harbinson, Projekt Saucer: Génesis (1980), Inception, Phoenix y Millenium (1995); o la paranormal en Visitantes milagrosos de Ian Watson (1978). Y por otro, sacando partido a las últimas modas ufológicas como los OVNIs estrellados o las abducciones: The Ogden enigma de Gene Snyder (1980), Majestic de Siguiendo en esta línea, es evidente que la literatura de ciencia ficción con su difusión minoritaria parece incapaz de tener gran influencia sobre el inconsciente colectivo. Por ello, debemos prestar más atención a los aspectos audiovisuales y gráficos como principales responsables de la propagación mundial de motivos e imágenes ufológicas. Por lo que respecta a los cómics, baste recordar que el más famoso superhéroe, Superman (creado en 1939), es un extraterrestre, indistinguible de los humanos excepto cuando pone en práctica sus poderes superiores. Otros héroes galácticos son Buck Rogers (creado en 1929) y Flash Gordon 49 CIFE componentes que operan como especialistas a un nivel más informal. Dirección: Apdo. postal 94, 30600-Archena (Murcia), España. Internet: https://www.orm.es/programas/ elultimopeldano/ [MM] Whitley Strieber (1989), The UFO Conspiracy de David Bischoff (1990), La Conspiración del Juicio Final de Sidney Sheldon (1991). [6, 140, 141][LG] CIFE CINE Y EXTRATERRESTRES (Centro Investigador de Fenómenos Extraños). Entidad de estudio y divulgación del fenómeno OVNI creada en Archena (Murcia) en 1976 por Joaquín Abenza y otros tres jóvenes investigadores, ninguno mayor de 16 años. En sus inicios funcionó como asociación juvenil en el seno de la Delegación de Juventud de Archena, organismo que, gracias a la colaboración de su representante Andrés Rodríguez, aportó la infraestructura necesaria para el mantenimiento del grupo. Pese a este modesto origen, la intensa labor divulgativa de los primeros años propició una extraordinaria expansión pronto traducida en siete delegaciones en Murcia y otras cinco en las principales capitales de España. En esa época, segunda mitad de los setenta, el CIFE llegó a contar con más de 200 socios, repartidos en cinco secciones autónomas sobre Ufología, Parapsicología, Astroarqueología, Astronomía y Tecnología Aplicada. Como asociación defensora de la HET y volcada en la investigación de campo, el CIFE se ha distinguido siempre más por su faceta divulgativa que por los desarrollos teóricos que ha propiciado, aspecto resaltado desde 1981 con la publicación del boletín Estigia, revista de periodicidad irregular sobre Ufología y Parapsicología, y por la constante participación de sus asociados en emisiones radiofónicas (El último peldaño, Onda Regional de Murcia, que desde 1991 ha organizado diversas alertas ovni). En los últimos tiempos el grupo cuenta con una veintena de La aparición de seres de otros mundos sobre una pantalla cinematográfica es casi tan antigua como el mismo cine. En 1902, George Meliès incluía una fantasiosa visión de belicosos y saltarines selenitas en su versión del clásico de Julio Verne Viaje a la Luna, anticipando al mismo tiempo el tratamiento que más adelante recibiría la figura del alienígena. En efecto, la hostilidad manifestada en su debut sería el principal referente para toda una larguísima lista de películas posteriores. Pese a la precocidad del filme de Meliès, no es hasta mitad de siglo cuando vuelven a aparecer en las carteleras títulos protagonizados por alienígenas. El reencuentro se produce con El enigma de otro mundo (1951) -que cuenta con un digno remake: La cosa (1982)-, que presenta ya el tema de los platillos estrellados y las perversas intenciones de sus monstruosos tripulantes, todo lo cual inspirará dos corrientes paralelas dentro de la perspectiva del extraterrestre maligno. Por un lado, y abonado por los temores de la Guerra Fría, la década de los cincuenta conocerá un aluvión de producciones sobre la invasión de la Tierra con consecuencias dramáticas para la reputación de los alienígenas. La guerra de los mundos (1953 y 1980), La invasión de los ladrones de cuerpos (1956, 1978 y 1994), La Tierra contra los platillos volantes (1956) y otras muchas películas contribuyeron a crear un estereotipo de extraterrestre poco o nada 50 Cine y extraterrestres distinto del comunista de los peores tiempos del macartismo. Alguno de estos filmes aportaron además elementos que en décadas venideras sazonarían los relatos de abducidos y contactados, como Invasores de Marte (1953 y 1986), donde aparece ya el tema del secuestro y posterior control mental de los abducidos mediante sofisticadas tecnologías. Los excesos cometidos y la decadencia de los principios ideológicos que lo sustentaban acabaron con este estilo de producciones, que pese a todo fue cultivado todavía en los años sesenta en una serie de delirantes películas japonesas (Invasión extraterrestre, 1968) y resucitó en los noventa (Independence Day y Mars Attacks!, 1996). Otra tendencia, que aún pervive, obviaba el problema logístico de la invasión y se centraba en el aspecto repulsivo y amenazador de los visitantes de otros EXTRATERRESTRES EN LA TELEVISIÓN Dejando a un lado aquellas teleseries de carácter costumbrista donde la atención se centra en las más o menos cómicas vicisitudes de la convivencia entre humanos y extraterrestres (Alf, De otro mundo, Alien Nación), la tipología televisiva se reduce a dos grandes categorías: las series de invasiones y las de viajeros galácticos. Las primeras han experimentado una gradual escalada belicista desde que en 1968 la iniciativa privada del incomprendido David Vincent se bastaba para detener a Los invasores. En 1973, OVNI nos mostraba ya la implicación de las autoridades en la defensa del planeta, pero apostando por las tesis secretistas: el cuartel general se camuflaba en unos estudios de cine. La serie V (1985) prescindía de tantos subterfugios y planteaba una ocupación militar en toda regla por parte de unos visitantes que enarbolaban una bandera roja, reflejo de la mentalidad reaganista entonces imperante. Los periplos espaciales, en cambio, apuestan por un escenario cambiante en el que en cada episodio aparecen nuevos ejemplares exobiológicos. Lo de menos es el medio empleado para desplazarse: haciendo autostop en California (Starman), en una nave de guerra (Galáctica), en una estación espacial (Perdidos en el espacio), en la Luna salida de órbita (Espacio 1999), o en una cabina de teléfonos (Doctor Who). Dentro de este estilo de producciones hay que resaltar Star Trek (1966-69) y sus continuaciones La próxima generación (1989-94), Espacio profundo nueve (1993-99), Voyager (1995-2001) y Enterprise (2001). Aunque la primera entrega ya contó con guionistas de la talla de Harlan Ellison y Theodore Sturgeon, La próxima generación fue de excepcional calidad visual y argumental, superando en popularidad a la serie madre, todo lo contrario que la siguiente secuela, muy criticada por los trekkies. Fuera de clasificación queda Investigación OVNI, emitida en 1979, que narraba las idas y venidas de dos oficiales del ejército del aire americano en pos de los casos recopilados en el proyecto BLUE BOOK. Realización de escasa calidad, pretendía compensar sus deficiencias exagerando espectacularmente la descripción de los incidentes y dejando al final de cada entrega una puerta abierta para la interpretación platillista de los mismos. A partir de 1993, algunos episodios de EXPEDIENTE X retomarían, ya con más medios, el tema de la investigación ufológica, pero esta vez narrando hechos imaginarios tenidos por "verídicos". 51 Cine y extraterrestres planetas. Su cultivo arranca también de los años cincuenta (The Blob, 1958 y 1988), pero eclosionó definitivamente a partir de Alien (1979) de Ridley Scott y sus secuelas (Aliens, 1986, Alien 3, 1992 y Alien Resurrección, 1997). Filmes como Inseminoid (1980), Xtro (1982), Hidden (1987), Depredador (1987) y Species (1995) nos fueron describiendo los aspectos más íntimos de la biología alienígena, aproximando, a veces más de lo deseable, el cine de ciencia ficción al de terror "gore". 1980; Esfera, 1998) o de las consecuencias que tienen para los humanos el descubrimiento de otras formas de vida más avanzadas y absolutamente incomprensibles (2001, una odisea del espacio, 1968; Solaris, 1971 y 2002). Pese a la notable calidad de algunas de estas producciones, el distanciamiento numinoso y casi divino con el que en ellas se muestra a los alienígenas casa mal con su explotación comercial, por lo que ningún otro filme ha seguido el ejemplo de los citados. Pero al margen de lo que tradicionalmente han dado por supuesto todas estas películas, es posible hallar ejemplos de extraterrestres bondadosos. El primer título de estas características es Ultimátum a la Tierra (1951) de Robert Wise, que narra las dificultades que un alienígena de corte adamskiano encuentra para transmitir a la Humanidad una advertencia sobre el peligro de las armas nucleares (y todo ello antes de Adamski). La pasión, muerte y resurrección del visitante parecen calcadas de la más pura ortodoxia cristiana, y se verán fielmente reproducidas en E.T. (S. Spielberg, 1982), la obra más conocida de este subgénero. A partir de ella, otras cintas intentarán con menor fortuna artística y comercial seguir esta nueva senda: Starman (1984), Cocoon (1985), Enemigo mío (1985) y Abyss (1989), pero esta corriente pronto degenerará en un infantilismo excesivo -ya presente en la producción de Spielberg- que liquidará sus posibilidades de evolución (Exploradores, 1985; Nuestros maravillosos aliados, 1988). Por último, cabe señalar la existencia de películas de inspiración ufológica, esto es, cuyo argumento se apoya en casos supuestamente reales. La primera y más significativa de todas es Encuentros en la tercera fase (S. Spielberg, 1977, y "nueva edición" en 1980), título que responde a la desafortunada traducción de Close Encounters of the Third Kind, lo que ha provocado no pocas confusiones terminológicas (v. Clasificación de Hynek). Protagonizada por Richard Dreyfuss y un Françoise Truffaut que viene a representar a Jacques Vallée, en ella se va haciendo un repaso casi didáctico a la tipología de avistamientos de Hynek (que colaboró en el guión y aparece fugazmente en la conclusión del filme) con abducción incluida, hasta llegar a la apoteosis final del contacto directo con los visitantes de otro mundo. Producción más lograda visual que argumentalmente, parecía destinada a propiciar una nueva oleada de casuística que finalmente sólo se produjo en lugares muy concretos, posible síntoma del agotamiento del tema OVNI para el público y los medios de comunicación. Sea como fuere, se trata de la más costosa y celebrada incursión cinematográfica en la materia. Las restantes películas de este subgénero, mucho más modestas, se adhieren de pleno a los mitos Un tercer grupo de películas tienen en común unos orígenes literarios y, curiosamente, la ausencia del extraterrestre, que se manifiesta sólo a través de su tecnología (Planeta prohibido, 1956; Stalker, 52 CIOVE contemporáneos de la ufología americana: los platillos estrellados (Hangar 18, 1981; Roswell, 1994) y las abducciones. Así, el caso Hill fue narrado en The Ufo incident (1975), en tanto que Comunión (1989) y Fuego en el cielo (1993) plasman respectivamente en imágenes las desacreditadas experiencias de Whitley Strieber y Travis Walton.[39, 248, 259][MM] Cantabria, junto a un creciente interés por la divulgación pública, mediante ciclos de conferencias primero y simposiums anuales después, el primero de los cuales se organizó, con asistencia de representantes de diversos centros de todo el país, en 1975. La divulgación se amplió con la edición de un primer boletín, Tiempo y Espacio (1970), y, posteriormente, con la publicación de la revista Vimana (1978-81). CIOVE participó activamente en todos los intentos de coordinar grupos y esfuerzos individuales. Así, en 1971 algunos de sus miembros se reunieron en Madrid con otros integrantes de la “segunda generación” de investigadores; en 1973 creó, junto a CEADI de San Sebastián y el Grupo Charles Fort de Valladolid, la Federación de Centros del Norte de España; y en 1978, con RNC, IIEE, ADEM de Zaragoza y algunos investigadores privados, la Cefo, presentada como respuesta al sensacionalismo entonces imperante. CIOVE (Centro Investigador de Objetos Volantes Extraterrestres). Grupo ufológico privado fundado en 1967 en Santander por cinco jóvenes estudiantes: Julio Arcas, Juan José de la Riva, José Felipe Arronte, Pablo Caloca y J. Irigaray. Buen ejemplo de agrupación surgida al calor de la carrera espacial de los años sesenta y de los distintos movimientos juveniles, sus ideas iniciales entroncaban plenamente (ya en su denominación) con la HET, la interpretación más generalizada en su época, de la que se irán separando paulatinamente hasta alcanzar posiciones más críticas. La actividad de CIOVE se vio interrumpida como grupo a mediados de los años ochenta, cuando, superando la dinámica asociacionista de los primeros tiempos, el esfuerzo de sus componentes pasó a centrarse en la edición de Cuadernos de Ufología. [ JA] Los primeros contactos internacionales se establecieron con un astrónomo aficionado argentino, el jesuita Benito Reyna, director del observatorio de Adhara, con el que mantuvieron una intensa correspondencia. En España, pronto pasaron a relacionarse con la naciente nómina de grupos que fueron apareciendo en las postrimerías de los años sesenta: CEI y CEOSI de Barcelona, Eridani de Madrid, CEONI de Valencia, RNC de Sevilla y CIFA de Algorta. La actividad del CIOVE se orientó en dos frentes principales: la recopilación de casuística y el estudio in situ de las observaciones producidas en CÍRCULOS EN CAMPOS DE CEREALES Figuras geométricas de progresiva complejidad y origen desconocido que empezaron a aparecer en los campos de cereales de Inglaterra en el año 1980, formadas por aplastamiento de las plantas en un esquema circular con bordes definidos. La investigación de este fenómeno extraño (que pronto recibió el apelativo de 53 CISU Cereología) ofrece apasionantes y reveladores paralelismos (y contrastes) con la Ufología y su mundillo. En este caso disponemos de evidencias físicas innegables, lo que faltan son testigos de cómo aparecieron. En todos los años transcurridos apenas si se conocen un par de incidentes fiables donde alguien observase el proceso. distinguir los círculos genuinos de los falsos... pese a sus repetidos fracasos en conseguirlo. Sin embargo, a pesar de las evidencias repetidas de que se trata de obras humanas (aunque también es cierto que nadie ha sido detenido jamás, pese a las recompensas ofrecidas), el fenómeno ha seguido repitiéndose todas las primaveras, cada vez con figuras más complejas hasta convertirse en una verdadera forma de arte anónimo y atracción turística, llegando a aparecer incluso en la carátula de un disco de Led Zepellin. Mientras las huellas fueron pocas y limitadas a formas circulares podía admitirse un origen natural como el defendido por el Dr. Meaden y su "vórtice de plasma". Uno de las mayores dificultades era explicar por qué no se habían conocido casos semejantes hasta entonces. Algunos ufólogos quisieron establecer paralelismos con los nidos OVNI descritos en Australia durante los años 60, mas no pasaban de tener un remoto parecido. Pero tras algunos años de figuras elementales, conforme su existencia fue divulgándose a la opinión pública, las figuras que aparecían empezaron a hacerse más y más complejas (hileras de círculos, anillos, grandes combinaciones, incluso rectángulos y ¡fractales!) y la hipótesis del origen natural tuvo que ser abandonada. Obviamente el entorno bucólico donde tenían lugar los hechos invitaba a interpretaciones ecológicas y místicas en la línea de la Nueva Era y la hipótesis Gaia (los círculos serían abstrusas señales de la "Madre Tierra"). Y algunos ufólogos defendieron también un origen extraterrestre. Como es natural, la idea se ha extendido a otros países gracias a los medios de comunicación social y han proliferado las imitaciones, pero sin alcanzar la maestría original. Y pronto empezó a hablarse de círculos en otros tipos de cultivos, en el hielo, y hasta ¡en la arena de las playas!. [17, 73, 218][LG] CISU (Centro Italiano Studi Ufologici). Principal organización ufológica italiana, creada en diciembre de 1985 por una serie de estudiosos italianos que procedían del CUN (Centro Unico Nazionale). Su primera directiva la formaron Antonio Chiumiento, en calidad de presidente, Gian Paolo Grassino, como secretario, Edoardo Russo, Paolo Fiorino, Maurizio Verga y Gianni Settimo, uno de los pioneros de la ufología italiana y editor de la publicación Clypeus. Pero en el otoño de 1991 se hizo público que dos jubilados confesaban ser autores de muchas de las huellas. Ciertamente había una inteligencia detrás de las formaciones circulares... pero era terrestre. Entre los creyentes se consideró una maniobra de desinformación, señalando que con métodos experimentales como la radiestesia y el análisis de muestras eran capaces de El CISU tiene como finalidad la promoción del estudio científico del fenómeno OVNI, así como la difusión de información sobre el tema, que desarrolla muy activamente en sus distintas publicaciones y trabajos. Su revista, UFO54 Clark, Jerome Keith Rivista di informazione ufologica, edita dos números anuales de 40 páginas con un enfoque muy serio y preciso tanto de la casuística como de las aportaciones teóricas. Es una publicación de referencia para los interesados que busquen objetividad en el tratamiento del fenómeno. Editan además diversos boletines como Notizie UFO, sobre la actualidad en Italia y otros países e información del Centro y sus actividades, Rassegna Casistica sobre las investigaciones de incidentes que realiza el CISU, así como el Notiziario Archivio Stampa, selección mensual de artículos de prensa y revistas no especializadas italianas. histórica compuesta por UFOs in the 1980s (1990), The emergence of a Phenomenon: UFOs from the beginning through 1959 (1992), y High Strangeness:UFOs from 1960 through 1979 (1996). Entre sus trabajos más conocidos se encuentra el ITACAT (catálogo italiano de encuentros cercanos) y el TRACAT (catálogo de casos italianos con huellas en el suelo), ambos compilados por Maurizio Verga. Dirección: Casella Postale 82, 10100 Torino, Italia. En Internet: http:// www.cisu.org [ JA] Opinión sobre el fenómeno (tomada de Ronald D. Story (ed.), 1980: The Encyclopedia of UFOs): ”Simpatizo con el afán de teorizar sobre los OVNIs; después de todo las teorías son una manera de intentar dar sentido a las cosas. Pero no debemos bajo ninguna circunstancia tomar nuestras teorías demasiado en serio, ni nunca darles más relevancia que la que concedemos a los hechos observados (por muy extraños e inconvenientes que sean) del fenómeno OVNI. Durante mis peores momentos he llegado a sospechar que los OVNIs podrían representar algo tan alejado de nosotros que nuestros intentos por entenderlo serían comparables a los esfuerzos de una hormiga para comprender los principios de la física nuclear”. CLARK, Jerome Keith CLASIFICACIÓN DE HYNEK (n. Canby, Minnesota, 1946). Periodista ufológico estadounidense, autor de numerosos trabajos de tipo histórico sobre el fenómeno OVNI. Antiguo editor asociado de la revista Fate, ostenta desde 1985 el mismo cargo en el Iur, desde donde ejerce una decisiva influencia en la moderna ufología americana. Durante los años setenta fue uno de los principales postulantes de la hipótesis paraufológica, con libros como The Unidentified (Warner Paperbacks, New York, 1975), de la que -en uno de sus acostumbrados cambios de opiniónrenegaría años más tarde para decantarse por la HET. Es igualmente autor de la trilogía The UFO Encyclopedia, obra enciclopédica e La más popular de las clasificaciones de observaciones OVNI fue propuesta por Josef Allen Hynek en su obra The UFO Experience (1972). En primera instancia, dividía los sucesos en encuentros cercanos (a menos de 200 yardas / 180 metros) y avistamientos lejanos. Estos últimos podían ser: • Luces nocturnas (LN): visiones de luces durante la noche no explicables en términos convencionales. • Discos diurnos (DD): objetos ovalados o en forma de disco y apariencia metálica. • Radar/visual (RV) o radar/óptico: 55 Clasificación de Vallée CLASIFICACIÓN DE VALLÉE detección por radar de un cuerpo simultáneamente observado por testigos. Con posterioridad otros autores han hablado de Discos nocturnos y de casos Sólo radar (Radar-only), cuando no existe comprobación visual de lo apreciado por este instrumento. Tipología de avistamientos presentada por Jacques Vallée (y su esposa Janine) en la obra Fenómenos insólitos del espacio (1967). Estaba basada en la dinámica y aspecto exterior de los objetos, y comprendía cinco tipos principales: Los encuentros cercanos, por su parte, se clasificaban de la siguiente manera: T. I: A) Objeto en el suelo o a la altura de los árboles. • Encuentros cercanos de primer tipo (ECI): observación cercana en la que el OVNI no interacciona con el medio ni con los testigos. B) Objeto en la superficie o en la proximidad de una masa líquida. C) Objeto en la proximidad del suelo, con gestos de los ocupantes o efectos de luz. • Encuentros cercanos de segundo tipo (EC-II): existe interacción con el medio en forma de huellas o efectos físicos sobre los testigos, animales, plantas, aparatos eléctricos, etc. T. II: A) Forma cilíndrica vertical rodeada de nubes en movimiento. B) Cilindro vertical inmóvil que origina objetos secundarios. • Encuentros cercanos de tercer tipo (ECIII): informes en los que se menciona la presencia de entidades en las cercanías del OVNI. C) Puro con objetos secundarios que no dejan o no vuelven al cuerpo principal. Igualmente, en lo que representa una total desvirtuación de los conceptos originales, estas categorías han sido más tarde completadas (sobre todo en los EE.UU) con los EC-IV, las abducciones, los EC-V, los casos de comunicación entre los testigos y las entidades, e incluso los EC-0, los incidentes de los que no se tendría noticia. T. III: A) Objeto oscilante en caída libre. B) Objeto que se desplaza con una parada, pero sin variar la altura. C) Objeto que se para, cambia de forma, de luminosidad o que libera un pequeño objeto. D) Evoluciones de objetos o movimientos particulares de un objeto. Aunque todavía son habitualmente usadas por los ufólogos, las categorías ideadas por Hynek distan mucho de ser plenamente satisfactorias. La heterogeneidad de los criterios empleados y la concepción pro-extraterrestrista que subyace en ella impiden que esta clasificación sea tan objetiva como se pretende. [120][MM] T. IV: A) Objeto de trayectoria normal, continua. B) Objeto cuyo vuelo está afectado por una aeronave. C) Objetos en formación. 56 Clipeología D) Objeto en progresión ondulante, zigzagueante. 254][MM] T. V: A) Gran objeto luminoso. CLIPEOLOGÍA B) Punto-origen con inmovilidad prolongada. C) Punto-origen movimiento. de Término de origen italiano que designa el estudio de las observaciones de OVNIs supuestamente ocurridas en la Antigüedad o, en general, antes del siglo XX. Se ha formado a partir de la expresión latina clipei ardentes (escudos de fuego), que era empleada por autores romanos para referirse a fenómenos celestes desacostumbrados que hoy algunos interpretan como OVNIs. [MM] rápido Confusa y con criterios diferenciadores entre clases más bien caprichosos, su único mérito consistió en ser la primera clasificación creada para ordenar la creciente casuística, pues su aplicación práctica fue mínima y pronto fue abandonada en favor de la clasificación de Hynek. CNIFO Posteriormente (1990), el propio Vallée ideó una nueva clasificación, claramente inspirada en la de Hynek, recogida en su libro Confrontations. Dicho sistema pretende catalogar cualquier tipo de anomalía, no sólo los avistamientos OVNI, de acuerdo a las siguientes categorías: (Comissão Nacional de Investigação do Fenómeno OVNI). Asociación ufológica portuguesa, fundada en 1984 como resultado de la unión de diversos investigadores y grupos preexistentes (CEAFI, CECOP). Dividida territorialmente en cuatro zonas (norte, norte-centro, centro-sur y sur), tiene por objetivos la recogida y evaluación científica de informaciones sobre fenómenos aeroespaciales de naturaleza aún no determinada, su difusión entre la comunidad universitaria como medio de estimular eventuales estudios e hipótesis, y la divulgación y discusión de los resultados de sus investigaciones a través de coloquios y simposios o de su anuario Anomalia. Edita desde 1993 la citada publicación anual, y, desde 1995, la revista trimestral CNIFO Newsletter. En 1997 el CNIFO tiene previsto celebrar un simposio en el ámbito universitario (“Ciencias de Frontera o realidades sutiles”) en el que se presentará públicamente la nueva Sociedad Portuguesa de Exploración Científica (Dirección: Rua AN1: Anomalías sin efectos físicos duraderos. AN2: Anomalías con efectos físicos perdurables. AN3: asociadas. Anomalías con entidades AN4: Anomalías con interacción entre los testigos y las entidades. AN5: Anomalías que dejan secuelas en los testigos en forma de lesiones o muerte. La tipología se completaba con clasificaciones de maniobras, trayectorias y encuentros cercanos formuladas en paralelo a la expuesta, amén de un triple método para concretar la credibilidad de los sucesos.[250, 57 Cohetes fantasmas de Sá da Bandeira, 331, 3º, s/ 31 e 32, 4000Porto. Dicha sociedad se mantuvo activa hasta 2001, y posteriormente sus fondos pasaron al Centro Transdisciplinar de Estudos da Consciência de la Universidade Fernando Pessoa de Oporto: http:// cela.ufp.pt/[MM] más cercana, siendo el máximo alcance de las V-2 alemanas unos 370 Kms). Tras dragar el lago no se encontró nada. Las observaciones continuaron (incluyendo algunos posibles avistamientos de radar) hasta alcanzar más de 1.600 informes, debidamente conservados para la posteridad en los War Archives de Estocolmo. Conforme avanzaba el verano, estos fenómenos parecieron ir desplazándose hacia el sur, y así en Septiembre se divulgaron avistamientos de fenómenos similares en Suiza, Bélgica e incluso Portugal, Grecia y hasta la India. COHETES FANTASMAS Término con el que los estudiosos del fenómeno OVNI se refieren a una serie de fenómenos extraños observados en los cielos de Europa, justo después de la Segunda Guerra Mundial, durante 1946. Esta oleada supone el verdadero inicio de la era moderna de actividad OVNI, que seguiría en 1947, ya sobre Estados Unidos, con el famoso caso Arnold. Los ufólogos han recibido estos informes anteriores a 1947 como prueba evidente de que los platillos volantes no eran una simple moda americana impulsada por el miedo al comunismo y la bomba atómica. Según esta argumentación, los visitantes alienígenas se adaptarían en cada momento a las formas y características de la época. Desde los escudos ardientes de los romanos (clipeología), pasando por la nave aérea del cambio de siglo, los aviones fantasma de la década de 1930, los cohetes fantasma de 1946 y hasta los platillos volantes de 1947 en adelante. No obstante, ello parece imponer a los extraterrestres un excesiva carga de sofisticación y esfuerzo sin motivos plausibles. [82][LG] Se inició en Febrero de 1946 sobre Finlandia con una "actividad meteórica poco habitual" pero acabó extendiéndose por toda Escandinavia. Pronto los periódicos acuñaron el término "cohetes fantasma" para explicar esos meteoros en forma de misíl. Conforme aumentaba el número de casos, se acrecentaban las sospechas de que pudiera tratarse de pruebas de cohetes realizadas por la Unión Soviética sobre el Báltico. El 9 de Julio llegaron a contabilizarse hasta 250 avistamientos en toda Suecia (entre ellos la única foto conocida) de lo que parece haber sido (con la perspectiva de los años pasados) un raro ejemplo de bólido diurno. Tal avalancha motivó la creación de la primera comisión oficial establecida en el mundo para investigar este tipo de fenómenos. Pese a los medios empleados resultó imposible llegar a ninguna conclusión; ni siquiera el 19 de Julio cuando uno de esos "cohetes" se estrelló en el lago Kölmjärv (a más de 700 kms de la posible base de lanzamientos rusa COLECTIVO CUADERNOS Véase Cuadernos de Ufología. COLEGIO INVISIBLE Denominación dada, en forma un tanto extravagante, por Jacques Vallée en su 58 Componente psíquico obra de mismo título (1975) a un cierto número de hombres de ciencia que deseaban conocer la naturaleza del fenómeno OVNI y, especialmente, determinar si sus orígenes eran imputables o no a alguna inteligencia, pero permaneciendo en el anonimato. Este nombre, sugerido ya por Hynek en su libro The UFO Experience (1972), está tomado del que se daban a sí mismos los científicos que debían trabajar en secreto antes de la autorización de la Royal Society por Carlos II de Inglaterra a principios de 1660.[120, 253][ JR] aprendidos a lo largo de su vida. Ante algo desconocido, que no es posible referir a nada preexistente, el sujeto tenderá a buscar una explicación acorde con su experiencia cultural y social, es decir, basándose en la información de que disponga, incluyendo mitos y creencias. Para lograr esto es frecuente que realice una “recreación”, añadiendo detalles, recuerdos, explicaciones, etc., sobre todo a medida que pasa el tiempo desde la observación original y según el impacto que haya podido tener su testimonio en su círculo familiar y personal y/o en los medios de comunicación. Paolo Toselli distingue en sus estudios sobre OVIs entre el factor “expectativas del testigo” -pues, básicamente, existe una exposición selectiva a la información (tendemos a ver y oír lo que queremos)- y el factor “influencia social”, ya que, hoy en día, hay ya un gran folklore social establecido sobre este tema y todo el mundo ha oído hablar de los OVNIs (asimilados a naves interplanetarias) y sus características. Incluso se podría aludir a algún tipo de inconsciente colectivo, propiciado por la literatura y el folklore populares, tal como brillantemente expone el investigador francés Bertrand Méheust (1978 y 1985). COMPONENTE PSÍQUICO 1. En la década de los setenta, este término se reservaba para los aspectos paranormales del fenómeno OVNI, que autores como Vallée (1975) llegaban a considerar fundamentales: “creo que por cada caso que me presenten donde los datos sean puramente físicos puedo encontrar otro con la misma credibilidad de los testigos, huellas, fotos, etc., y que incluya elementos paranormales” (Fuller, 1980: p. 404). Ello dio lugar a las hipótesis paraufológicas. [91, 253][LG] 2. Factor personal que aporta el testigo de un acontecimiento inusual que puede ser calificado como dentro del contexto ufológico. Otra cuestión anexa es el concepto de “realidad psíquica” del sujeto, en el sentido de que cree haber visto algo que es irreal y producido por su mente, en respuesta a necesidades de tipo personal (angustia, conflictos vitales, deseo de protagonismo y de atraer la atención de los demás) o, incluso, de tipo colectivo (acudiendo a explicaciones de tipo místico, religioso, mesiánico, etc.). También se puede hablar de mecanismos relacionados con trastornos del sueño (visiones hipnagógicas e hipnopómpicas), sobre todo en los casos de abducciones y de visitantes de Ante una experiencia de naturaleza desconocida y que no se ajusta a modelos establecidos, puede suceder que el sujeto haga un intento de interpretación de un estímulo, en principio externo, esto es, no producto de su mente. La psicología cognitiva establece que el sujeto integra los acontecimientos o estímulos externos de forma que se adapten a sus esquemas mentales o patrones de conocimientos 59 Condon, informe dormitorio. [159, 160][MG] Saunders fueron despedidos, y poco después éste último escribiría (con Roger Harkins) una especie de versión alternativa del informe Condon titulada UFOs? Yes! Where the Condon Comittee Went Wrong (1968). CONDON, informe En marzo de 1966, el llamado comité O’Brien, encargado de revisar el proyecto Blue Book, aconsejó a las Fuerzas Aéreas estadounidenses encargar un estudio científico sobre los OVNIs a alguna universidad de prestigio. Finalmente se encomendó éste a la Universidad de Colorado, que colocó como director del mismo al Dr. Edward U. Condon, un prestigioso físico de la misma universidad, aunque fue Robert J. Low quien actuó de hecho como coordinador del proyecto. Junto a un numeroso grupo de especialistas de diversas áreas contratados al efecto, ofrecieron su colaboración los más importantes investigadores norteamericanos y las organizaciones Apro y Nicap, en un intento de influir en lo posible en las conclusiones finales del mismo. En noviembre de 1968 el equipo de Condon concluyó el trabajo, oficialmente titulado The Final Report of The Scientific Study of Unidentified Flying Objects, que fue presentado para su revisión a un comité de la Academia Nacional de Ciencias. El 8 de enero de 1969 esta entidad aprobó la metodología y las conclusiones alcanzadas y remitió el estudio a las Fuerzas Aéreas. El informe en sí, de 1.465 páginas, constaba de siete capítulos, el primero de los cuales -escrito personalmente por Condoncontenía las conclusiones y recomendaciones del estudio, resumidas en que ningún elemento procedente de la investigación de los OVNIs había aportado nada al conocimiento científico y que posteriores esfuerzos de este tipo no podían justificarse con la esperanza de que la ciencia avanzaría gracias a ellos. El capítulo segundo esbozaba el desarrollo del proyecto y las clases de pruebas analizadas, que eran tratadas con más detalle en el siguiente. El cuarto capítulo estaba consagrado a la revisión de 59 casos, 23 de los cuales quedaban sin explicación, mientras que el quinto pasaba revista a la historia de los OVNIs y a los proyectos oficiales de investigación en otros países. El sexto apartado era una colección de ensayos sobre cuestiones relativas a la percepción y a aspectos físicos de los OVNIs, y cerraba el informe un capítulo formado por apéndices con documentos de carácter oficial e histórico. Enseguida el proyecto topó con diversas dificultades, derivadas del tiempo perdido en elaborar estudios preliminares, la imposibilidad de revisar casos antiguos y la ruptura con el NICAP causada por algunos comentarios negativistas de Condon. En julio de 1967, los investigadores del proyecto Saunders y Levine encontraron un memorándum de Low, escrito antes de la adjudicación del contrato a la Universidad de Colorado, en el que sugería presentar el tema al gran público como un estudio totalmente objetivo pero como un trabajo sin expectativas de éxito ante la comunidad científica. En desacuerdo con este enfoque, entregaron copia del mismo al NICAP y a James McDonald, que no dudaron en presentarlo como prueba del supuesto fraude que con él se quería cometer. Levine y 60 Consejo de consultores de Stendek CONTACTADO De acuerdo con las recomendaciones del estudio, las Fuerzas Aéreas cancelaron el proyecto Blue Book en diciembre de 1969, acabando así con la investigación oficial que se había desarrollado durante más de 20 años. A pesar de las críticas que los simpatizantes de la HET han vertido durante décadas sobre el informe Condon, éste constituye una de las referencias científicas más importantes para valorar la verdadera dimensión del fenómeno OVNI. [97][IC, MM] En definición de Javier Sierra y José Juan Montejo (1988), persona o colectivo que mantiene voluntariamente algún tipo de relación, más o menos continuada, con OVNIs o con entidades asociadas al fenómeno, o ambas a la vez, recordando los hechos y mensajes -si los hubiera- de modo consciente. Véanse Contactismo y Sociología de los ovnis. CONTACTISMO CONSEJO DE CONSULTORES DE STENDEK Conjunto de creencias y doctrinas de las personas y grupos que dicen estar en contacto con seres extraterrestres. Grupo informal creado en mayo de 1978 por iniciativa de Vicente-Juan Ballester Olmos para conjuntar al amparo del Cei y de Stendek al sector de los titulados universitarios con interés por el fenómeno OVNI. Desapareció en 1982 al hacerlo también Stendek, dejando tras de sí 121 comunicaciones internas mayoritariamente elaboradas por Ballester. [31, 55][MM] El contactismo ha sido una materia tradicionalmente excluida por los investigadores del ámbito de la Ufología, pues su carácter marcadamente místico y religioso encajaba mal con las concepciones cientifistas y fisicalistas de los ufólogos clásicos. Del mismo modo, su escasa incidencia social le ha privado de la atención de los antropólogos y sociólogos, con las excepciones de la obra de Festinger et al. When profecy fails (1956) y del estudio de Balch y Taylor sobre “Bo y Peep”. Sólo en los últimos tiempos algunos jóvenes investigadores (como Javier Sierra y Manuel Carballal) han centrado su interés, a falta de otros aspectos más asequibles del fenómeno OVNI, en la problemática del contacto con extraterrestres. CONSPIRACIONISMO Véase Encubrimiento gubernamental. CONSPIRANOICO Adjetivo formado por la contracción de “conspiración” y “paranoico” que en Ufología se aplica a los defensores a ultranza de la existencia de conjuras gubernamentales encaminadas a ocultar la verdad sobre el fenómeno OVNI. Origen y desarrollo. El contactismo, sin embargo, es un fenómeno con precedentes bastante antiguos, pues -obviando al místico sueco Enmanuel Swedenborg (1688-1772) y la cuestión de la comunicación en general con otras inteligencias no humanas (ángeles, 61 Contactismo demonios, espectros)- parece registrar sus primeros ejemplos en algunas sesiones de espiritismo celebradas entre 1860 y 1873 en Massachusetts (EE.UU.), durante las cuales se habría entrado en contacto con habitantes de Marte. De hecho, como se verá más adelante, el espiritismo es una de las principales fuentes de las que ha bebido el contactismo, que surgirá como tal en 1952, coincidiendo con la publicación de algunas obras “sobrenaturalistas” sobre los platillos volantes que pueden considerarse precedentes de la hipótesis paraufológica. No obstante, y pese a que será común que los contactados daten sus primeras experiencias con anterioridad, no es desdeñable la influencia que tuvo el film Ultimátum a la Tierra (Robert Wise, 1951) en la aparición de los pioneros del contactismo, cuyo mensaje apenas difiere del “arrepentíos y dejad las pruebas nucleares” que transmite la película (v. Cine y extraterrestres). Tal fue el caso de George Adamski, que en 1952 dio a conocer sus encuentros con venusinos que inauguraban el contactismo y daban paso a una legión de imitadores como Bethurum, van Tassel, King (v. Aetherius Society), Angelucci o Fry. Todos ellos, y muchos otros, convirtieron los años cincuenta en la edad dorada del contactismo americano y mundial, que fue objeto de atención constante por parte de los medios de comunicación. El fenómeno decayó en los años sesenta para reactivarse en los setenta con las crecientes expectativas de un contacto inminente con los extraterrestres. Los años ochenta y el desinterés social por los OVNIs que trajeron volvieron a provocar la desaparición de los grupos menos arraigados, que han vuelto a proliferar en las postrimerías del siglo cuando el milenarismo de muchos de ellos se ha acentuado. Estas últimas muestras de contactismo son a menudo difícilmente separables de las abducciones en unos casos y de los visitantes de dormitorio en otros, experiencias que pueden actuar como detonantes de las vocaciones contactistas. El actual declive de la hipótesis extraterrestre ha condicionado también la existencia de ejemplos de contactismo con seres no propiamente alienígenas (los “ángeles de Paiporta”, entre otros) y la evolución de algunos contactados hacia el curanderismo u otras tradiciones irracionalistas. Modalidades del contacto. La mecánica del contacto con los supuestos seres extraterrestres puede presentar múltiples variantes que, siguiendo a Cabria (1993), se clasifican de la siguiente manera: a) Contacto visual, basado en el avistamiento de OVNIs, cuyo grado más bajo de implicación serían las sesiones nocturnas de observación (v. Alerta ovni), y su grado más alto el del contacto visual con cita previa acordada a través de la tabla ouija, la psicografía (escritura automática) o las psicofonías. Estos mismos medios (tomados del espiritismo) pueden aparecer en la más común de estas variantes, la observación activa, consistente en la comunicación mediante señales luminosas, esperando o no algún otro tipo de mensaje. b) El contacto físico, por su parte, puede presentarse en muy diversas formas que culminarían con la abducción del contactado. Antes, es posible que se dé el contacto físico con intercambio simbólico, en el que se entrega un objeto que posee algún significado especial (la “piedra de Marte” de Alberto Sanmartín), o el contacto físico verbal con transmisión de un mensaje estructurado, al estilo de Adamski y tantos otros. 62 Contraencuesta evidentemente no se produce hace que, ante la perspectiva nihilista que supondría abandonar la creencia, se opte antes por negar la evidencia y trasladar la catástrofe profetizada bien a una nueva fecha o a un futuro indeterminado, bien a un plano meramente espiritual, afirmándose que la transformación predicha sucederá en el alma de los creyentes. Precisamente, la adhesión a la perspectiva espiritualista del cambio anunciado ha llevado a algunos contactados a reorientar sus preocupaciones hacia funciones de utilidad social, desmarcándose de sus doctrinas originales. [55, 86][MM] c) Contacto mental, la modalidad propia de los contactados que se consideran más evolucionados, caracterizado por la ausencia de otros medios que no sean los puramente psíquicos, que incluyen desde oír voces en la cabeza hasta la posesión por entidades extraterrestres. El mensaje contactista. Las doctrinas contactistas, ya sean de individuos o de sectas platillistas, se caracterizan por seguir una línea ecléctica que aúna elementos de muy diversa procedencia, tomados de las corrientes apocalípticas, la teosofía, el espiritismo, el esoterismo, el conspiracionismo, e incluso de la cienciaficción. Según Cabria, esta aparente desestructuración de las creencias contactistas se debe también a que el contacto es vivido por sus practicantes como una experiencia religiosa caracterizada por tratarse de una vía personal de comunicación mística, y por tanto difícilmente sometible a un control doctrinal que la formalice. Por este camino se llega a entender el contactismo como una respuesta intuitiva y espiritualista a la artificiosidad de las formas religiosas establecidas, como un verdadero fenómeno de revitalización religiosa. CONTRAENCUESTA Véase Reencuesta. CREDIBILIDAD Objetividad del testigo de la observación de un OVNI, entendida en términos de su aptitud para reflejar en el testimonio la realidad de lo acontecido. Bajo el nombre de probabilidad es, al igual que la extrañeza, uno de los dos conceptos propuestos por Hynek (1972) en su matriz para la depuración de la casuística (v. test de extrañeza-credibilidad). No obstante, a diferencia de aquélla la credibilidad ha resultado mucho más difícil de cuantificar e incluso de denominar, pues junto al término empleado por Hynek hay que reseñar los de confiabilidad (Olsen, Banchs), objetividad (Saunders), credibilidad (Poher, Adell, Ballester y Guasp) y peso (Smith). No se registra tampoco consenso respecto al método que debe seguirse para medirla, ya que existen tantos como investigadores proponentes y ni siquiera está Igualmente, el contactismo conlleva siempre una promesa de salvación de tipo milenarista, basada en la catástrofe final (y subsiguiente evacuación de los creyentes a otro planeta) que sobrevendrá y/o en la subversión del orden social que propugna. Subversión que se manifiesta en un rechazo del materialismo y la lógica imperantes en el mundo moderno que inclina a los contactados hacia la pureza espiritual y corporal, lo cual se traduce muchas veces en un tradicionalismo incluso reaccionario. El elemento apocalíptico conduce en algunos casos a fijar fechas concretas para el cataclismo anunciado, que cuando 63 CSICOP claro si la escala de medida debe ser aditiva o progresiva. Pese a que en 1966 Olsen ya realizó los primeros estudios de cuantificación, la extrema subjetividad presente tanto en el objeto como en los autores de tal medición ha impedido avanzar en el análisis del problema. Por otro lado, la idoneidad del testigo que pretende determinarse con los distintos índices de credibilidad no parece ser en sí misma suficiente -ni aun en combinación con otros factores- para facilitar la explicación de un incidente ni para asegurar, en caso contrario, que nos hallemos ante un fenómeno desconocido. La confirmación de estos extremos depende más de la apreciación conjunta de cada suceso que del valor que pueda arrojar un parámetro abstracto. [31, 120][MM] encuentros; 5) publicar artículos que examinen las pruebas de lo paranormal; 6) no rechazar las pruebas de una forma apriorística, antes de investigarlas, sino examinarlas objetiva y cuidadosamente. Para ello cuenta con un amplio panel de miembros y consultores técnicos o científicos de la talla de Isaac Asimov (†), Martin Gardner, Murray Gell-Mann, Stephen J. Gould, Marvin Minsky, Carl Sagan (†), Steven Weinberg, etc., siendo su presidente-fundador el filósofo Paul Kurtz. En su seno funcionan cinco subcomités: Astrología, Comunicación Electrónica, Salud, Parapsicología y OVNIs. Durante sus casi 20 años de existencia, el CSICOP se ha visto envuelto en muchas controversias: desde el "efecto Marte" en Astrología hasta el "Experimento Alfa" y los enfrentamientos legales de Uri Geller con James Randi en Parapsicología. Dentro de la Ufología destacan los trabajos de Philip Klass para desenmascarar el fraude de Majestic-12 y los debates sobre el síndrome de falso recuerdo y su relación con abducciones, o el caso Roswell. CSICOP Siglas de Committe for the Scientific Investigation of Claims of the Paranormal, que en castellano podría traducirse como "Comité para la Investigación Científica de los Supuestos Hechos Paranormales". Constituido el 1 de mayo de 1976 durante el encuentro anual de la American Humanist Association (Asociación Humanística Americana) dedicado en aquella ocasión a "El nuevo irracionalismo: anticiencia y pseudociencia", sus objetivos declarados son: 1) coordinar grupos de personas interesadas en el examen crítico de supuestas pruebas de lo paranormal y otros fenómenos en los márgenes de la ciencia, así como contribuir a la educación de los consumidores; 2) preparar bibliografías de material publicado donde se examinen detalladamente estas cuestiones; 3) incentivar la investigación objetiva e imparcial en las áreas que se necesite; 4) organizar conferencias y En la actualidad sus actividades se extienden desde EE.UU. a 28 países de todo el mundo, donde existen organizaciones con fines similares pero con carácter autónomo e independiente (v. Escepticismo y arp). Desde 1983 el CSICOP organiza una conferencia anual itinerante en distintas ciudades de los EE.UU. y recientemente ha inaugurado su cuartel general y biblioteca con más de 50.000 volúmenes en la ciudad de Amherst (Nueva York). Su órgano oficial de difusión, el Skeptical Inquirer, se empezó a publicar semestralmente a finales de 1976 bajo el nombre de The Zetetic, para adoptar su 64 Cuadernos de Ufología denominación actual en el tercer número. Desde enero de 1995 es una publicación bimestral de unas 65 páginas en inglés. Dirección: CSICOP/SKEPTICAL INQUIRER, Box 703, Amherst, NY 14226-0703 USA. En Internet: https:// skepticalinquirer.org/[LG] Ignacio Cabria García (1993). En 1997 se inició la tercera época de la revista, ya como órgano de difusión de la Fundación Anomalía. CdU. ha organizado eventos públicos divulgativos como “OVNIs: Experiencias y análisis” (diciembre 1990) y “Jornadas Internacionales” (octubre 1991), ambos en Santander. Desde 1991 los miembros de su staff se han reunido anualmente, dando origen en 1994 al denominado Colectivo Cuadernos, impulsor de la Fundación Anomalía. Dirección: Apartado de correos 5.041, 39080-Santander (España). Dejó de publicarse en 2012, pero puede consultarse en http://files.afu.se/Downloads/ ?dir=Magazines/Spain and Catalonia/ Cuadernos de Ufologia (Fundacion Anomalia)[ JR] CUADERNOS DE UFOLOGÍA (CdU.). Publicación especializada fundada en marzo de 1983 por José Ruesga Montiel en Sevilla. En su primera época se publicaron 17 números de aparición trimestral. Desde 1987 Julio Arcas Gilardi y Matilde González García, antiguos miembros del Ciove de Santander, refuerzan la línea editorial, pasando a ser una revista con formato de libro, de 106 páginas y una muy cuidada presentación. Actualmente ha alcanzado una importante proyección internacional que le ha valido estar considerada como una de las tres mejores revistas especializadas en Ufología del mundo. La postura editorial, si bien crítica, ha mantenido un equilibrio entre tendencias irreconciliables que le ha valido la denominación de "la línea de enmedio". Sus contenidos se han destacado por los amplios dossieres que a lo largo de sus 20 números editados en su segunda época han abordado temas como las abducciones, Ummo, las tesis psicosociales, la ufología andaluza, los OVNIs en la CEI, las observaciones de pilotos, etcétera. La labor editorial de CdU. se ha complementado con la edición de una monografía con trabajos del Dr. Willy Smith y Manuel Borraz Aymerich (1992), así como con el libro Entre ufólogos, creyentes y contactados. Una historia social de los OVNIs en España del antropólogo CUASI-ATERRIZAJE Aproximación de un OVNI al suelo sin llegar a posarse en él. Véase Aterrizaje. CUESTIONARIO Formulario para la recogida sistemática de datos sobre casuística. Los cuestionarios ufológicos tienen su origen en los primeros proyectos militares estadounidenses de investigación, de donde pasaron a los organismos civiles, que los han ido adaptando a sus diversas necesidades. Así, junto a cuestionarios para la encuesta general de todo tipo de casos (en España unificados en los del CEI-RNC) es posible hallar otros especializados para los incidentes con humanoides, luz sólida, con pilotos como testigos, etc. Los cuestionarios suponen una gran ayuda para la investigación de 65 CUFOS campo puesto que permiten una recogida exhaustiva de la información por medio de unos modelos normalizados que facilitan luego un mejor análisis de los datos y de los propios hechos. Naturalmente, lo deseable es que sean confeccionados de la manera más objetiva posible, pero es casi inevitable que en realidad reflejen muchas de las presunciones establecidas respecto a la naturaleza del fenómeno que se pretende estudiar, y que puedan incluso a conducir el relato del testigo si se utilizan rígidamente. El CEI ha editado una extensa recopilación a nivel mundial de todo tipo de cuestionarios: Selección de cuestionarios de observación (CEI, Barcelona, 1995). [MM] electromagnéticos de los OVNIs, encuentros cercanos de tercer tipo y otras materias. Al concluir la década de los setenta, la disminución del número de avistamientos y del interés popular por el tema acarrearon problemas económicos al CUFOS, que se vio imposibilitado para seguir pagando el sueldo de Hendry. Con la retirada de éste por motivos laborales e ideológicos (The UFO Handbook apareció en 1979), el grupo se mantuvo gracias a la fama de Hynek, quien a principios de 1985 nombró a Jerome Clark nuevo editor del IUR, con lo cual la publicación ganó en amplitud temática y en número de suscriptores, sobre todo a partir de la reactivación del tema OVNI a finales de los ochenta. CUFOS En 1985 el CUFOS se instaló en Glenview (Illinois), y poco después, tras la muerte de Hynek en 1986, Mark Rodeghier asumió la presidencia por expresa voluntad de aquél. La organización fue rebautizada como “J. Allen Hynek Center of UFO Studies”, aunque se conservaron las mismas siglas. Con el aumento de sus fondos documentales causado por la recuperación de los archivos del Nicap, el centro estableció su nueva sede en Chicago, desde donde -bajo la dirección del profesor Michael D. Swords- se reinició en 1989 (y hasta 2006) la publicación del Journal of UFO Studies, con carácter de anuario y un claro afán de rigor científico. Dirección: 2457 West Peterson Avenue; Chicago, Illinois 60659. En Internet: http: // www.cufos.org [MM] ( J. Allen Hynek Center for UFO Studies). Centro ufológico estadounidense creado en 1973 por Josef Allen Hynek y Sherman J. Larsen a partir de un pequeño grupo, el PEG (Public Education Group), que éste había fundado poco antes. Tuvo su sede inicialmente en el domicilio de Hynek en Evanston (Illinois), desde donde se editó el llamado CUFOS News Bulletin, de irregular periodicidad. A fin de relanzar la publicación y el propio centro, se contrató a Allan Hendry como investigador con dedicación exclusiva y editor del International UFO Reporter (IUR) -la nueva revista del CUFOS a partir de noviembre de 1976-, centrada en la casuística investigada por el propio grupo, que contaba para estos menesteres con una línea telefónica de 24 horas diarias y conexión directa con las autoridades. A finales de los setenta aparecieron igualmente tres números del Journal of UFO Studies, más orientada hacia el análisis científico del fenómeno, y una serie de monografías sobre efectos CULTOS EXTRATERRESTRES Véase Sectas ufológicas. DTU 66 Däniken, Erich Anton von D DÄNIKEN, Erich Anton von Pero no me pregunten qué son los OVNIs. Simplemente no lo sé. Considerando que el problema de los OVNIs ha cautivado a tan gran número de personas, pienso que debería ser investigado científicamente. Sin detenernos demasiado en si efectivamente hay o no OVNIs. Quizás la respuesta se encuentre en la psicología o en algún lugar del cerebro humano.” (n. Zofingen, 1935). Escritor suizo de gran popularidad en los años setenta. Dedicado profesionalmente a la hostelería, abandonó esta ocupación para exponer en numerosos libros sus tesis sobre los astronautas de la Antigüedad, llegando a crear escuela y a convertirse en la máxima figura mundial de la llamada astroarqueología. Prolífico escritor y conferenciante, destacan entre sus libros Recuerdos del futuro (1968, trad. española de 1970), Regreso a las estrellas -escrito en la cárcel por problemas fiscales- (1970, trad. de 1971), El oro de los dioses (1972, trad. de 1974), y una curiosa incursión en el tema de las apariciones marianas: Las apariciones (1974, trad. de 1975). En 1973 fundó la Ancient Astronaut Society, entidad dedicada al estudio y divulgación de la supuesta presencia de extraterrestres en épocas pretéritas. Pese a lo descabellado de sus ideas y a que éstas fueron rápidamente olvidadas al pasar la moda editorial que las aupó a la fama, todavía se mantenía activo a finales de los años ochenta con obras como Todos somos hijos de Dios (1988) y Los ojos de la esfinge (1990). DARNAUDE ROJAS-MARCOS, Ignacio (n. Sevilla, 1931). Licenciado en Ciencias Económicas, ha sido pionero y el teórico por excelencia de la ufología andaluza. Su actividad ufológica ha conocido altibajos y, pese a sus numerosos contactos con organizaciones españolas y extranjeras, no se ha caracterizado por su presencia en actos públicos ni por su prodigalidad en los medios escritos. En 1956 comenzó a reunir todo tipo de informaciones sobre OVNIs, que se encuentran hoy ordenadas y clasificadas en uno de los archivos más voluminosos de la ufología española. Se ha interesado igualmente por las apariciones marianas, de las que ha reunido una magnífica bibliografía, y ha sido la primera persona en afrontar un estudio sistemático de la documentación “ummita”, fruto del cual es el monumental Catálogo descriptivo de los documentos del criptogrupo Ummo (ummocat). Existen pocas contribuciones salidas de su pluma, entre ellas el artículo Opinión sobre el fenómeno (adaptada de Ronald D. Story (ed.), 1980: The Encyclopedia of UFOs): ”Hoy soy de la opinión de que de hecho existe algo que llamamos OVNIs. Estoy convencido de que en ocasiones suceden a nuestro alrededor cosas extrañas para las que de momento no tenemos ninguna explicación razonable. 67 67 Debunker Palabra inglesa que podríamos traducir por "desmitificador". Dentro del mundillo ufológico siempre ha tenido unas connotaciones peyorativas, aplicándose por parte de los creyentes a todos aquellos que cuestionan la realidad del fenómeno OVNI en general y de los platillos volantes en particular. Entre los más conocidos acusados por esta práctica están Donald Menzel, Philip J. Klass; James Oberg y Robert Sheaffer. Sólo este último ha sido traducido al castellano: Veredicto OVNI (1994). [RC] fabricación humana; si estos constasen en los catálogos como genuinos OVNIs, tergiversarían e invalidarían cualquier resultado de tipo estadístico, habitual en el estudio analítico de este enigma. De esta forma, la tarea previa de análisis de cualquier conjunto de observaciones misteriosas consistirá en obtener la mayor cantidad posible de información de cada incidente a la búsqueda de una explicación, de un escenario alternativo habitualmente trivial en el que encuadrar la mayor cantidad posible de episodios. Se trata, en suma, de separar y distinguir claramente entre el ruido (casos explicados) y la señal (casos OVNI genuinos). Entre los estudios más divulgados siguiendo esta metodología se encuentra el realizado por Allan Hendry, quien fuera director del Cufos de Estados Unidos. La pregunta central era: “¿qué toma la gente por ovnis?” Después de un largo y duro trabajo de seguimiento individual de 500 casos catalogados como presuntos ovnis que llegaron al Center durante 1978, Hendry verificó que el 93% quedaba aclarado convenientemente. Sólo el 7% quedó por identificar; estos podrían catalogarse, en principio, como OVNIs. El resto del porcentaje se divide de la siguiente forma: DEPURACIÓN DE CASUÍSTICA • El 28% de estos casos se debió a estrellas y planetas. “Ovnis: todas las respuestas” (en Año Cero nº5, diciembre de 1990), donde repasa las múltiples hipótesis explicativas, y el trabajo inédito Los motivos del no-contacto extraterrestre. Opinión sobre el fenómeno (extraída de Boletín Informativo Andaluz nº2, 1972): “Nos encontramos ante hechos que superan el marco de las ciencias y que se encuentran muy relacionados con lo paranormal. Soy defensor de la hipótesis extraterrestre, pero en una acepción más amplia.” DEBUNKER • El 27% fueron aviones reactores y aviones publicitarios, muy frecuentes en EE.UU. Los estudiosos del fenómeno OVNI desde premisas analíticas y racionalistas se percataron desde el principio de la necesidad de separar “el trigo de la paja”, usando una expresión vulgar. Los catálogos ufológicos suelen contener gran número de observaciones que llegan a conocimiento del investigador con la etiqueta OVNI. Sin embargo, la gran mayoría son identificables como muy diversos tipos de observaciones astronómicas, atmosféricas y de ingenios de • El 12% fueron causados por meteoros luminosos. • El 26% restante fue debido a diversas causas, como globos sonda, satélites artificiales, etc. 68 Desapariciones misteriosas Esta estrategia de análisis podría poner de manifiesto la existencia de un fenómeno desconocido en el estado actual de la ciencia, aunque en último término abocaría al dilema de la falacia del residuo y al de la indistinguibilidad estricta entre ambos subconjuntos, ya que no existe un método para aclarar si ese conjunto final de casuística inexplicada es realmente de naturaleza desconocida o si obedece a causas externas, como encuestas defectuosas, falta de información, etc., es decir, el hecho de que los casos inexplicados se diferenciasen de los explicados no probaría que se trata de casos absolutamente inexplicables. acuñar la famosa frase "Somos propiedad". Todos hemos oído hablar del regimiento inglés desaparecido en Gallípoli en 1915; de los cinco aviones torpederos desaparecidos el 5 de Diciembre de 1945 cerca de las costas de Florida; del barco Mary Celeste encontrado sin tripulantes cerca de Gibraltar; de ciertos astronautas rusos desaparecidos en el espacio; y hasta de la famosa teleportación de los esposos Vidal de Argentina a México. Pues bien, investigaciones posteriores no tan divulgadas han encontrado explicaciones convencionales a todos estos casos (generalmente fraudes). Frente a la depuración de casuística también se ha propuesto el uso de filtros (o más bien simples comparaciones sistemáticas de las características de los avistamientos con las que son típicas de los fenómenos conocidos por la ciencia actual) a través de los cuales contrastar la información enigmática procedente de los casos ovni, a la búsqueda de episodios que superen esas condiciones en mayor o menor medida rigurosas. Quedaría a la lógica y precisión del investigador el diseño de esos filtros que permitan obtener la pepita de oro de un caso genuinamente inexplicable. De cualquier manera, la depuración de casuística es la herramienta habitual que permite obtener el producto ufológico en apariencia enigmático. [112][RC] Si inicialmente todos estos sucesos eran considerados por sus descubridores como ejemplos de la intervención de seres superiores (dioses o demonios), la divulgación del fenómeno OVNI permitió una realimentación mutua, por la que los ubicuos platillos volantes se convirtieron en los inmediatos responsables de cualquier suceso similar en tierra, mar o aire (Gaston 1973). Una muestra clara de dicha interrelación sería la también desaparecida revista ufológica Stendek, cuyo nombre se deriva de las últimas palabras pronunciadas por el vuelo del Lancastrian Star Dust que supuestamente desapareció para siempre minutos antes de aterrizar en Santiago de Chile el 2 de Agosto de 1947. La más publicitada fuente de desapariciones misteriosas es naturalmente el conocido "Triángulo de las Bermudas". Han sido muchos los ufólogos que han defendido la existencia de una base extraterrestre bajo esas aguas del Atlántico. Y no contentos con ello, algunos como Antonio Ribera en su libro Los doce triángulos de la muerte (refritando las teorías de Ivan T. Sanderson), han llegado a encontrar zonas similares repartidas DESAPARICIONES MISTERIOSAS Término con el que se engloban todo tipo de desapariciones de vehículos (barcos, aviones, submarinos, etc.) y/o personas que, a primera vista, resultan inexplicables. Ya el pionero Charles Fort recopiló informes sobre extrañas desapariciones, llegando a 69 Desclasificación periódicamente por todo el globo terrestre. Lo absurdo de dichas teorías ha sido puesto de manifiesto en distintas ocasiones (Kusche 1977, 1980) sin alcanzar por desgracia la difusión merecida (v. triángulos mortales). [96, 138, 139, 193][LG] ser ocultado a los investigadores, lo que provocó que el Jefe del Estado Mayor del Aire ( JEMA) elevara a la JUJEM una propuesta de desclasificación que, sin embargo, no prosperó. A partir de mediados de los años ochenta, y con más intensidad desde 1990, Vicente-Juan Ballester Olmos y Joan Plana Crivillén mantuvieron estrechos contactos con autoridades civiles y militares tratando de convencerles de que la cuestión OVNI tenía que dejar de ser reservada. Finalmente, y fruto de estas gestiones, en mayo de 1991 el Coronel Jefe de la Sección de Seguridad de Vuelo propuso la desclasificación al JEMA. Adoptada esta decisión, todo el material archivado se entregó al Mando Operativo Aéreo (MOA) para que propusiera la desclasificación en términos profesionales. El 14 de abril de 1992, la JUJEM procedió a rebajar al nivel de “reserva interna” toda esa documentación, dejando al JEMA la facultad de hacerla pública si no comprometía la Seguridad Nacional. DESCLASIFICACIÓN Al igual que en todos los países, el Ejército del Aire español posee documentación sobre avistamientos de fenómenos OVNI que ha venido gestionándose desde la Sección de Seguridad de Vuelo de la División de Operaciones del Estado Mayor. La excepcionalidad española reside en que, desde 1992, estos archivos están siendo desclasificados, esto es, liberados de su anterior carácter de secreto y facilitados al público. Históricamente, tal clasificación tuvo su origen en una nota de prensa publicada el 5 de diciembre de 1968 por el Ministerio del Aire solicitando a los ciudadanos que informaran a las autoridades de cualquier visión de objetos volantes extraños. A dicho material se le otorgaba internamente el tratamiento de “confidencial”. En 1976, la difusión sensacionalista por parte del periodista J.J. Benítez de unos pocos e incompletos informes que le habían sido entregados para que comprobara que no se disponía de nada extraordinario provocó una verdadera repulsión en medios del Cuartel General del Aire que abortó cualquier nueva relación con ufólogos y propició que el 3 de marzo de 1979 la Junta de Jefes de Estado Mayor ( JUJEM) declarase el tema “materia reservada”. Poco antes, en enero de ese mismo año, Pere Redón solicitó a S.M. el Rey don Juan Carlos que intercediera para que el asunto dejase de De septiembre de 1992 a enero de 1997 la desclasificación ha abarcado 74 archivos o expedientes con un total de 97 informes distintos ocurridos de 1962 a 1995, comprendiendo 1.819 páginas de interesantes legajos, trazas de radar, dictámenes de jueces investigadores, declaraciones de testigos, etc. Se ha encontrado una explicación convincente para el 90% del total de informes, cuyas causas más comunes son los estímulos astronómicos (Venus, principalmente) y los globos estratosféricos. El 10% restante permanece todavía bajo la etiqueta de presunto no identificado, a la espera de los análisis que se siguen praticando sobre estos casos. Entre ellos se encuentran los de Canarias (24/11/1974), Quintanaortuño (1/1/1975), Las Bárdenas Reales 70 Desmaterialización (2/1/1975), Argoncillo (20/7/1978) o el registro de un eco radar y posterior observación visual desde un caza interceptador el 17 de noviembre de 1979. Pero cuando proliferan los llamados visitantes de dormitorio (entidades que se materializan en el interior de las casas) y son los propios testigos los que pasan a través de puertas y paredes, resulta inaceptable cualquier explicación externa y debemos apelar a la Psicología. [223, 260] [LG] En la actualidad, la cifra de 62 expedientes que recopiló el MOA en 1992 ha aumentado sustancialmente gracias al empeño puesto en la búsqueda de nuevas referencias por el nuevo responsable del proceso, el Tte. Col. Enrique Rocamora, cuyas gestiones han permitido sacar a la luz diversos casos de los que inicialmente no se tenía constancia. [34, 35, 40][VB] DETECCIÓN DE OVNIS La detección de OVNIs por excelencia es atribuible a la intervención del radar, presente ya en las primeras denuncias de estas observaciones en EE.UU. y la vieja Europa. Históricamente, la primera tentativa de esta clase fue la protagonizada por el proyecto Magnet canadiense, que entre noviembre de 1953 y agosto de 1954 dispuso de una caseta de madera equipada con un contador de rayos gamma, un magnetómetro, un gravímetro y un receptor de radio. De cualquier forma, la detección clásica proviene de la observación de supuestas alteraciones electromagnéticas (v. efectos electromagnéticos) al paso de los OVNIs, lo que llevó a investigadores sudamericanos a desarrollar detectores en la década de los sesenta que incluso llegaron a comercializarse entre aficionados. La idea base era muy simple y consistía en un circuito cerrado en el que se intercalaba una brújula, que al detectar un cambio en el campo magnético rompía el circuito y hacía saltar una alarma, a veces acústica, otras luminosa. Como es de suponer, la fiabilidad de tal medio de detección era muy discutible. DESMATERIALIZACIÓN Se han descrito casos donde el OVNI surge o desaparece sin movimiento de aproximación o distanciamiento. Ello ha llevado a algunos ufólogos a defender la existencia de universos paralelos y/o la trasmisión de materia como forma de desplazamiento de los mismos. En esta línea coincidiría la pseudocientífica propuesta ummita de los IBOZOO UU (v. Ummo). Existe una famosa foto tomada en Oregón el 22/11/1966 donde supuestamente se vería un OVNI materializándose repetidamente en un mismo fotograma.... se ha demostrado que era una señal de tráfico vibrando (Wieder 1993). Cuando esa materialización o desmaterialización (súbita o paulatina) se extiende también a los casos de ocupantes (a veces como si se tratase del teletransportador de Star Trek) se hace más díficil aceptar su materialidad y se han propuesto explicaciones parapsicológicas o incluso de proyección de imágenes por mecanismos como la inducción telepática, hipnótica o por microondas. Existe incluso una tentativa de clasificación por tipos (Scornaux 1979). La iniciativa con más relevancia y medios fue sin duda el Project Starlight International (dependiente de la Association for the Understanding of Man, Inc.), que 71 DIANA, proyecto intentó encontrar a principios de los años setenta una forma directa de resolver científicamente el misterio de los OVNIs. La filosofía científica del proyecto no estaba basada en una profunda recopilación de observaciones, sino en algo que sus promotores (con Ray Stanford a la cabeza) denominaban un camino más corto. Las metas de estos investigadores eran la detección (fotográfica o electrónica), el rastreo y el contacto mediante avisos luminosos con objetos volantes no identificados. La instrumentación utilizada para ello consistía en el llamado “UFOVECTOR” (UFO Video Experiment Console for Transitional-Over Response), compuesto por: 1) sistema de transmisión de láser modulado por cámara de vídeo, 2) sistema de precisión de grabación de los registros del magnetómetro, 3) telescopio (catadrióptico) Schmidt-Cassegrain de 8 pulgadas, 4) unidad de TV para grabaciónreproducción, 5) telescopio astronómico. Otros instrumentos con los que contaba el proyecto era una segunda unidad de TV, un micrófono parabólico de 2 m. para el estudio de los sonidos emitidos por el OVNI, y otras cámaras situadas en diferentes puntos para poder realizar estudios de triangulación. Pese a que en un principio se cosecharon resultados supuestamente positivos, el poco éxito de la iniciativa provocó que Stanford sufriera repentinos trances en los que era aparentemente poseído por entidades extraterrestres (sic). [105] [ JR] Agresión Nocturna Alienígena". Se pretendía un estudio en profundidad de las experiencias de visitantes de dormitorio, aquellas donde no existe una alusión directa y comprobable al fenómeno OVNI. Tras algunas conferencias y notas preliminares no se ha presentado públicamente ningún informe de resultados o conclusiones definitivas. [58, 108] [LG] DICCIONARIO TEMÁTICO DE UFOLOGÍA (DTU). La presente obra. En noviembre de 1994, Matías Morey Ripoll propuso al Colectivo Cuadernos, a través de su boletín interno, la realización de un diccionario especializado en términos y conceptos ufológicos que sirviese para difundir la Ufología en vísperas de la conmemoración de su cincuenta aniversario. En abril de 1995 el proyecto recibió un nuevo impulso por parte de Ignacio Cabria García, quien se encargaría de elaborar las primeras listas de voces. En abril de 1996, Morey y Cabria presentaron una primera selección conjunta de términos, completada en septiembre del mismo año con la incorporación de las personalidades, grupos, revistas y casos que hoy recoge el DTU. Con Morey como coordinador del grupo, se inició entonces la fase de redacción de las distintas voces por parte de los miembros del Colectivo Cuadernos, concluida en mayo de 1997. En septiembre de 1996 el proyecto fue subvencionado por la Consejería de Cultura y Deportes del Gobierno de Cantabria y en julio de 1997 por Caja Cantabria. El diccionario, de una amplitud temática inusitada para una obra ufológica, cuenta con 351 entradas que se corresponden con 247 artículos independientes. En octubre de 2000 se DIANA, proyecto Proyecto iniciado en Agosto de 1990 por los ufólogos Javier Sierra y Josep Guijarro en colaboración con miembros de la Sociedad Española de Parapsicología. Son las iniciales de "Delirio Individual de 72 Disco diurno confeccionó una versión digital en formato PDF, y en 2022, coincidiendo con el vigésimo quinto aniversario de su publicación, la presente edición. [MM] DISCO DIURNO Véase Clasificación de Hynek. DISTRIBUCIÓN GEOGRÁFICA Y TEMPORAL DE LA CASUÍSTICA Véase Estadísticas de casuística. DRAKE, ecuación de Véase Bioastronomía. DREAMLAND En inglés, "tierra de los sueños". Supuesto nombre en clave por el que también se conoce el Área 51. DTU 73 EBD E EBD cilindros transparentes que algunos abducidos aseguran haber visto a bordo de las naves extraterrestres. [121][LG] Siglas de Entidad Biológica Desconocida, usadas por aquellos ufólogos que defienden la existencia de seres de origen desconocido que nos visitan, pero que ante el desprestigio de la HET no se atreven a atribuirles un origen extraterrestre. EFECTOS DE LOS OVNIS Se denominan así todos los cambios, efectos y reacciones aparentemente ocasionados por OVNIs sobre el entorno físico y biológico terrestre. Todos los informes de este tipo se agrupan como encuentros cercanos de segundo tipo (v. Clasificación de Hynek). Al tratarse de los únicos casos donde se dispone de evidencias (supuestamente independientes de los testigos) analizables y contrastables por terceros resultan un elemento vital para la solución del fenómeno OVNI. Si dichas pruebas fueran suficientes demostrarían la realidad objetiva del fenómeno y permitirían, cuando menos, descartar las hipótesis psicosociológicas. Sin embargo, no siempre bastan las evidencias físicas, recordemos el ejemplo opuesto de los círculos en campos de cereales: aquí se dispone de pruebas objetivas indiscutibles, pero su origen sigue siendo tan discutido como el de los OVNIs. Ya en el caso Arnold se consideró como validación adicional el testimonio de un trampero cuya brújula osciló en todas direcciones mientras un OVNI lo sobrevolaba horas antes en la misma zona. De hecho, según los últimos datos disponibles (1987), el catálogo de huellas elaborado por Ted Phillips recoge ya más de 3.000 casos. Pero si consideramos EBE Siglas de Extraterrestrial Biological Entities, entidades biológicas extraterrestres, denominación empleada en los documentos del Majestic-12 y en el informe Matrix para designar a los alienígenas. ECTOGÉNESIS Término referido a la idea de fetos desarrollándose fuera del vientre materno. La primera mención de esta posibilidad aparece en la fábula moral para niños The Water Babies escrita en 1863, aunque no alcanzó gran debate hasta los años veinte del siglo XX. Quedó permanentemente establecida como una imagen cultural cuando Aldous Huxley inició su famosísimo relato Un Mundo Feliz con una visita al Centro de Incubación y Condicionamiento de la Central de Londres. En Ufología se refiere a los supuestos fetos híbridos que tras desarrollarse las primeras semanas en el vientre de algunas abducidas, son extraídos y terminan de desarrollarse en decenas de 74 Efectos de los OVNIs todo tipo de evidencias materiales el número de incidentes aumentaría considerablemente; así mencionaremos, aunque se tratan bajo otros epígrafes, el cabello de ángel, la detección por radar, las fotografías, los implantes en los abducidos, etc. , etc. observaciones de OVNIs. El tema del apagón de dimensiones colosales causado por visitantes alienígenas fue anticipado en una conocida película de ciencia ficción, Ultimátum a la Tierra (The Day The Earth Stood Still, 1951). Más populares aún, si cabe, son las alteraciones del funcionamiento del motor y el alumbrado de los vehículos atribuidas a los OVNIs. Puede considerarse que hicieron su aparición en la oleada francesa de 1954, pero hubo que esperar a 1957 para que se impusieran en la escena americana. En la noche del 2 al 3 de noviembre de dicho año, en un plazo de menos de tres horas, se habrían sucedido hasta diez avistamientos OVNI dentro de un radio de veinte millas de la localidad tejana de Levelland: en la mayoría de estos casos, los encuentros habrían estado acompañados de fallos en el motor y las luces de los vehículos. Pasemos pues revista a los distintos tipos de efectos mencionados en la literatura ufológica: Efectos electromagnéticos. Bajo esta denominación (abreviadamente, "efectos EM") se han venido englobando efectos muy variopintos, tales como interferencias radioeléctricas, detención de relojes, funcionamiento anómalo y paro de motores de explosión, debilitamiento y extinción del alumbrado de vehículos, interrupciones del suministro eléctrico, desviaciones de la aguja en brújulas, etc. Nos detendremos brevemente en dos de los efectos más clásicos. Se ha especulado con diversas hipótesis físicas para explicar la acción de los OVNIs sobre los motores y los sistemas eléctricos, apelando a fenómenos de ionización del aire, intensos campos magnéticos, etc. Estas tentativas se han visto frustradas por dificultades como las planteadas por la heterogeneidad de los casos y la ausencia de pautas claras en los efectos informados y sus circunstancias. Por otro lado, suponiendo que las hipótesis fueran correctas, tanto los vehículos como las personas deberían experimentar efectos concomitantes de los que -generalmente- no se tiene noticia. Los testimonios sobre paros de motores diesel complican el panorama, pues las hipótesis explicativas que se centran en el sistema eléctrico del vehículo sólo son aplicables a motores de explosión. Lo que sí puede constatarse es que detrás de muchos casos no hay más que averías comunes, errores de La época de oro de los apagones atribuidos a la actividad OVNI se sitúa a finales de los cincuenta y en los años sesenta. El incidente más famoso, el "gran apagón de la costa nordeste", tuvo lugar en Norteamérica el 9 de noviembre de 1965 y afectó durante horas a millones de personas, incluidos los habitantes de la ciudad de Nueva York. Coincidió con algunas observaciones de OVNIs. En opinión de muchos ufólogos, los OVNIs serían capaces de producir sobrecargas o bien de absorber energía de centrales y líneas eléctricas, hasta el punto de que hay quien ha hecho extensiva esta explicación a apagones que no han venido acompañados de avistamientos. No obstante, todo parece indicar que la gran mayoría de casos pueden explicarse como averías y percances fortuitos, pese al misterio añadido que suponen algunas de las 75 Efectos de los OVNIs conducción propiciados por el nerviosismo de los observadores, testimonios malinterpretados e incluso fraudes de todo tipo. [97][MB] plantas cercanos aparecen con ramas rotas o derribados, con quemaduras o deshidratados. • Cráteres: depresiones en el terreno. Efectos mecánicos y sobre el entorno. Se clasifican en tres apartados distintos: • Huellas de pisadas: pequeñas marcas relacionadas con las huellas principales y que se atribuyen a los supuestos tripulantes del OVNI. a) Primarios. Huellas. Según la clasificación de Ted Phillips se distinguen cuatro tipos: • Extracciones de terreno o vegetación. • Anillos, huellas circulares realizadas por presión, quemadura o deshidratación. Los mayores cambios se localizan en el perímetro exterior mientras el área central parece intocada. Pueden citarse entre los casos más conocidos: Quarouble, Francia (10/09/1954); Valensole, Francia (1/07/1965); Turís, España (25/07/1979); Rosedale, Australia (30/04/1980); Transen-Provence, Francia (8/02/1981). • Nidos: huellas ovaladas donde la vegetación aparece aplastada y arremolinada (v. Nidos OVNI). c) Residuos y artefactos. A veces se informa de residuos sólidos (incluyendo filamentos: v. cabello de ángel), líquidos o en polvo. Y en contadas ocasiones se mencionan fragmentos o artefactos, hasta llegar a la prueba definitiva pero inalcanzable: los platillos estrellados. Otro tipo de residuo que merece especial mención es la radioactividad. Aunque resulta absurdo que alguien inteligente tripule una nave con pérdidas radiactivas, ha sido práctica habitual investigar casos llevando un contador Geiger, quizá tratando de descubrir datos sobre la propulsión de los OVNIs. Ello dio lugar a gran cantidad de alarmas infundadas, aunque también es cierto que determinados efectos fisiológicos podrían haber sido provocados por algún tipo de radiación, aunque no existan rastros sobre el terreno. Como ejemplos de los casos más famosos se encuentran: Roswell, EE.UU. (2/07/1947); Ubatuba, Brasil (09/1957); Trancas, Argentina (21/10/1963); San José de Valderas, España (1/06/1967). • Círculos: huellas circulares cuya totalidad aparece quemada, deshidratada o aplastada. • Irregulares: determinada. huellas sin forma Como ejemplos más conocidos destacaremos: Socorro, EE.UU. (24/04/1964); Delphos, EE.UU. (2/11/1971); Quintanaortuño, España (1/01/1975). b) Secundarios. Marcas y daños a la vegetación. Se distinguen cinco tipos: • Marcas de aterrizaje: supuestas marcas dejadas por elementos estructurales del OVNI mientras permanecía posado. Se han encontrado relacionadas con todos los tipos de huellas del apartado anterior, dispuestas generalmente en forma triangular o rectangular. • Daños a la vegetación: los árboles y 76 Efectos de los OVNIs Parecería que ante tal cúmulo de evidencias físicas resultaría innegable la consideración de los OVNIs como naves espaciales. Quizá, pero no desde una perspectiva científica. Incluso los cada vez más complejos análisis con instrumentos sofisticados han sido incapaces de ofrecer una prueba definitiva (p. ej., para una crítica del caso Trans-en-Provence, investigado por el Gepan con los más avanzados medios, véase Evans, 1997). Como bien señala Maurizio Verga, la vinculación entre la huella y el supuesto OVNI la establece el propio ser humano (en muchos casos, las huellas han sido vistas sólo por el testigo, o encontradas por un investigador a la búsqueda de cualquier indicio misterioso). Ni siquiera hace falta recurrir a fraudes aunque, consciente o inconscientemente, sean abundantes-, pues existen otras alternativas: 1) fenómenos naturales, como el rayo en bola, pueden dejar huellas verdaderas; 2) huellas naturales preexistentes (p. ej.: anillos de hadas) que el testigo o el investigador atribuyen al OVNI. Porque además, pese a los intentos de sistematización, lo cierto es que las huellas conocidas son casi tan variadas como los incidentes en que aparecen; lo que es especialmente sospechoso en las que se refieren a las supuestas patas de aterrizaje. inconveniente es que tal clase de reacciones no dejan rastros duraderos más allá del relato del testigo, aunque a veces los animales manifiesten el mismo tipo de secuelas fisiológicas que los seres humanos. El interés de estos casos reside en dos razones fundamentales. Por un lado, se supone que los animales no comparten nuestras fantasías, luego si reaccionan es porque existía realmente un estímulo. Además, como poseen sentidos más sensibles que los humanos podrían ofrecer datos sobre características no percibidas por nosotros. Pero la gran dificultad reside en que es el propio testigo quien apunta la relación causa-efecto entre la visión del OVNI y la reacción del animal, lo cual no deja de ser una interpretación subjetiva, especialmente cuando se trata de animales de compañía muy sensibilizados con sus dueños. Algunos ufólogos como James McCampbell han especulado con que las microondas emitidas por los OVNIs podrían explicar algunas de estas reacciones. Pero tampoco podemos olvidar que parece probada la sensibilidad de los animales a los terremotos (v. hipótesis tectónicas). Como bibliografía sobre este apartado, véase el catálogo de la Flying Saucer Review, comenzado en el vol. XVI-1 (enero/febrero 1970) y concluido en el vol. XVIII-3 (mayo/junio 1972). Efectos sobre animales. Para ser precisos, más que hablar de efectos sobre los animales habría que hablar de las supuestas reacciones de éstos ante la aparición de un OVNI. Muchas veces la primera indicación de la presencia de un OVNI es un comportamiento anómalo en los animales presentes (perros, caballos, ovejas, aves, etc.). También se conoce el denominado efecto Oz cuando lo que el testigo menciona es el extraño silencio del entorno campestre, sin ruido de pájaros o insectos. El principal Efectos fisiológicos. En cualquier avistamiento de un OVNI podemos encontrar descripciones de luces brillantes y multicolores en distintas intensidades; ausencia de sonidos, zumbidos, silbidos o hasta ruidos violentos; olores extraños a ozono, azufre u otras muchas substancias; escalofríos, golpes de calor o dolores de cabeza; parálisis temporal total o parcial; etc., etc., incluso se conocen casos en que los testigos manifiestan haber sentido un sabor 77 Efectos de los OVNIs metálico en la boca. El inconveniente de todos estos efectos fisiológicos es que pocas veces dejan secuelas lo suficientemente duraderas como para ser certificadas médicamente, ni tampoco existe la seguridad de que no existiesen con anterioridad al avistamiento o sean una “simple” respuesta psicosomática del organismo, no causada por algo externo. Los más frecuentes son ojos irritados, pérdida temporal de visión o audición, quemaduras de variada intensidad, daños por radiación (incluyendo muertes) y también curaciones. Destacados sucesos de este tipo son los de Michalak, Falcon Lake, Canadá (20/05/1967); Dr. X, Francia (2/11/1968); Anolaima, Colombia (4/07/1969); Vidal, Alborache, España (30/07/1975); Jaraba, España (15/10/1978); Cash-Landrum, Huffman, EE.UU. (29/12/1980). Componente psíquico). Dado que esos llamados “poderes paranormales” no han sido verificados por la Ciencia y resultan contradictorios con nuestros conocimientos actuales, es evidente que menos aún han podido contrastarse vinculados al fenómeno OVNI, pese a su aceptación a nivel popular. Como ejemplos de casos más conocidos destacaremos: Hill, EE.UU. (19/09/1961); Valensole, Francia (1/07/1965); Dr. X, Francia (2/11/1968); Imjärvi, Finlandia (7/01/1970). [33, 84, 112, 155, 180][LG] ENCUBRIMIENTO GUBERNAMENTAL Según ciertos autores partidarios de la HET, ocultación por parte de las autoridades tanto de las agencias y programas secretos para la investigación de los OVNIs como de las diversas pruebas que obrarían en su poder y que demostrarían la realidad material del fenómeno. Por extensión abarca también las actividades gubernamentales de desinformación que tendrían como objeto desprestigiar a la Ufología y a los ufólogos para preservar el secreto sobre estos temas. Efectos psicológicos. Muchas veces son los efectos psicológicos los más evidentes y los únicos que puede analizar el investigador. De hecho, muchos defienden que una forma de diferenciar entre OVI y OVNIs es la reacción emocional de los testigos, argumento que también se ha empleado en relación con las abducciones. Pero las reacciones de temor o incluso pánico se dan igualmente en los casos con una explicación convencional (Hendry, 1979) y siempre queda la duda de si las secuelas psicológicas que puedan encontrarse estaban ya presentes antes del avistamiento o incluso lo originaron (v. Psicología del testigo OVNI). Históricamente, la primera expresión de ideas conspiracionistas fue formulada por el editor Ray Palmer cuando aludió oscuramente a un sabotaje como causa del accidente aéreo que acabó con la vida de los oficiales encargados de la encuesta del fraudulento incidente de la isla de Maury (31/07/1947). Desde entonces, toda muestra de implicación de las autoridades en estos temas ha ido acompañada de la sospecha de ocultación de informaciones, a lo que sin duda ha contribuido el carácter de materia reservada que sobre ellas recae, por comprensibles razones de seguridad, en la mayoría de países del mundo. En los Efectos parafísicos. Con creciente frecuencia a partir de los años sesenta los testigos manifiestan haber sufrido una serie de efectos paranormales (telepatía, levitación, etc.) tanto durante el incidente como anterior o posteriormente (v. 78 Encuentro cercano EE.UU., las iniciales conclusiones proextraterrestristas del Proyecto Sign y su desautorización por el alto mando y por el Proyecto Grudge (v. Sign y Grudge, proyectos) fueron inevitablemente interpretadas como una maniobra oscurantista que buscaba preservar el secreto sobre el origen alienígena de los OVNIs. Ello se traduciría después en un enorme recelo hacia la labor del Proyecto Blue Book (del que se dijo que era sólo una tapadera para encubrir el auténtico programa oficial de investigación al que irían a parar los casos auténticamente inexplicables) y hacia las conclusiones escépticas que arrojó (v. Condon, informe). investigación (MJ-12) que desarrolla proyectos clandestinos (Aquarius, Sigma, Snowbird), que custodia pruebas irrefutables sobre los OVNIs (platillos estrellados, cadáveres de ocupantes) y que emplea técnicas de desinformación contra los investigadores (contra William Moore, según su versión de los hechos). En la actualidad se viene observando un incremento de las ideas conspiracionistas en los EE.UU. (v. Matrix, informe) que tiene también su reflejo en otros países. En el caso americano, es un fenómeno que corre paralelo al auge de las ideas ultraderechistas de grupos opuestos al Gobierno Federal. En España, los más acérrimos partidarios de la HET han denunciado sin mucho éxito la supuesta existencia de una conspiración militar para alterar el contenido de los informes que el proceso de Desclasificación de la materia OVNI ha sacado a la luz pública. [103, 215, 224, 225][MM] No obstante, nunca nadie ha aportado pruebas creíbles del encubrimiento de las actividades ufológicas gubernamentales, que, en cierto modo, sí parece existir en relación a ensayos aeronáuticos de nuevos aviones militares (v. Área 51); pero desde una perspectiva completamente inversa: desacreditar avistamientos indeseados resulta más sencillo si lo que se denuncia haber observado se considera un OVNI y no un prototipo experimental. ENCUENTRO CERCANO Véase Clasificación de Hynek. Curiosamente, uno de los aspectos siempre presentes al hablar de ocultación oficial es el de que, según se dice, correrían rumores sobre su inminente desaparición. Desaparición que podría ser gradual y venir precedida por una “concienciación” de la opinión pública en forma de pequeñas filtraciones o incluso de películas de Hollywood que presentasen la cara amable de los extraterrestres. Esta tesis fue la esgrimida por los defensores del fraude de Majestic-12 para hacer valer la autenticidad de los documentos aportados. Es en este engaño donde más claramente aparecen reunidos todos los elementos de las hipótesis conspiracionistas: un grupo ultrasecreto de ENCUENTROS EN LA TERCERA FASE 1. Película dirigida en 1977 por Steven Spielberg, cuyo título original es Close Encounters of the Third Kind (véase Cine y extraterrestres). 2. Con esta misma expresión se alude a veces, incorrectamente, a los encuentros cercanos de tercer tipo; véase Clasificación de Hynek. ENCUESTAS SOCIOLÓGICAS 79 Encuestas sociológicas sobre ovnis SOBRE OVNIS hacerse mención al fenómeno OVNI como enigma de la ciencia, de tal forma que ha llegado a convertirse en una convención. Lo cierto es que, en un primer acercamiento al problema, este tiene la apariencia de un enigma relevante para las ciencias físicas. Si partimos de la confiabilidad de los testigos que han informado experimentar estas visiones, nos encontramos con un fenómeno de consistencia física, y que, a pesar del tiempo dedicado a su estudio por cierto número de investigadores de prestigio, no ha podido explicarse en su totalidad. No obstante, se podría aducir que este tipo de actuaciones han sido fragmentarias y esporádicas, insuficientes por tanto para otorgar el rango de enigma al fenómeno OVNI. Véase Sociología de los ovnis. ENIGMAS Habitualmente se entiende por tal un dicho o una cosa de difícil comprensión y entendimiento. En los tiempos antiguos era común su uso, como por ejemplo el conocido enigma de la esfinge de Tebas, que descifrara Edipo. Pero es en el terreno científico donde tienen particular importancia. Una de las facetas más importantes de la ciencia normal, constituida o establecida, es la resolución de pequeños problemas o puzzles que permitan reforzar el paradigma en el cual se encuentran inscritas, aunque tengan escasa relevancia práctica. En una primera impresión parece ser evidente que al menos una pequeña parte de la casuística ufológica no tiene explicación. Dando esto por cierto, el fenómeno OVNI no sería un enigma, ya que como ha señalado Kuhn (1994; 71) los enigmas son aquellos problemas que una comunidad científica supone que tienen solución. Bajo este punto de vista, el o los fenómenos habitualmente encuadrados en el fenómeno OVNI pasarían a ser simples hechos inconexos, sin significación y relevancia para la ciencia actual, o quizá demasiado problemáticos para dedicar a ellos un tiempo que no redundará en un mejor conocimiento del campo científico particular. Es más, esos problemas insolubles pueden tener gran importancia social (en el caso de los OVNIs, simple popularidad) pero científicamente no se consideran merecedores de atención porque no pueden ser abordados desde el paradigma establecido. Dentro de los periodos de ciencia normal los enigmas no solucionables pueden ser valorados de tres formas distintas, dependiendo del proceso histórico en el que se encuentre en un momento dado el paradigma: 1) Que no sea relevante para el desarrollo y permanencia del paradigma establecido y se deseche. 2) Que se dedique a su estudio más tiempo y recursos económicos en un intento de resolverlo, convirtiéndose en un problema fundamental para esa disciplina científica. 3) A pesar de esta inversión en trabajo humano y recursos, el problema puede permanecer irresoluto, convirtiéndose en una anomalía. Son diversos los fenómenos de la naturaleza que podrían dar lugar a ser En la terminología ufológica suele 80 Entidades interpretados como OVNIs, según el patrón de la mitología popular, como por ejemplo los rayos en bola y las luces de terremotos (v. hipótesis tectónicas) desde el punto de vista físico; en relación con la personalidad de los testigos destaca el síndrome de transgresión de la realidad. Es probable que cierto número de casos OVNI no explicados puedan deberse a alguna de estas causas. [137][RC] origen común: en la mente humana. El único criterio diferenciador aplicable a los supuestos tripulantes de los OVNIs sería el componente tecnológico. Históricamente, aunque el comportamiento aparentemente inteligente de los OVNIs hizo que la idea de que estuviesen tripulados por seres vivos fuera asumida casi desde el principio (aunque algunos como Ivan T. Sanderson los consideraron meros animales y otros hablaron de naves teledirigidas a distancia), los informes sobre sus posibles ocupantes eran recibidos en cambio con gran reticencia. Por ejemplo, el americano Proyecto Blue Book clasificó automáticamente como fraudes todos los casos de entidades, en una exagerada reacción frente a las sospechosas historias de contactados que proliferaron en la década de los cincuenta. En Europa la oleada francesa de 1954 marcó el punto de inflexión, pero en Estados Unidos grupos como el Nicap no aceptaron este tipo de casos hasta después del famoso caso Socorro en 1964. Este tipo de informes parecían muy prometedores por dos motivos. Primero, las posibilidades de una interpretación errónea por parte del testigo se reducían al mínimo, quedando sólo tres alternativas a eliminar (fraude, alucinación y locura). Y segundo, el volumen de información disponible era mucho mayor y detallado, llegando en ocasiones a incluir huellas o efectos físicos. ENTIDADES Numerosos han sido los vocablos utilizados para referirse a los seres descritos en el interior o en las cercanías de los OVNIs. Desde los términos descriptivos (humanoides, ocupantes, tripulantes, pilotos....) hasta los claramente identificadores (extraterrestres, uránidas, marcianos...) pasando por todos aquellos vagos y generales (seres, entidades...) o las burdas traducciones del inglés (alienígenas). En general, el uso de cada término supone una toma de postura a priori conforme a la hipótesis preferida por el ufólogo de turno. Incluso voces como "ser" o "entidad" reflejan una cierta idea de materialidad que está muy lejos de estar plenamente confirmada por los hechos. Lo cierto es que los supuestos tripulantes de los OVNIs son sólo parte de los muchos seres (antropomorfos o no) que parecen haberse relacionado con los humanos a través de los siglos. Desde monstruos como los yeti o los bigfoot a los dioses, ángeles y demás figuras religiosas (v. apariciones marianas) pasando por los fantasmas, espíritus, hadas, duendes, etc., etc. Aunque estos distintos subgrupos han sido estudiados siempre separadamente, quizá convendría plantearse la posibilidad de un No fue hasta la década de los setenta (tras la aparición de un número especial de la Flying Saucer Review inglesa en 1966) que se elaboraron las principales tipologías de humanoides tratando de encontrar pautas homogéneas. Así proliferaron los estudios estadísticos sobre los casos de este tipo en cada país (Brasil, Argentina, Australia, etc.) 81 Eridani AEC o a nivel mundial. Incluso el MUFON americano llegó a formar el Humanoid Study Group dirigido por David Webb y Ted Bloecher. Madrid, etc.), y la edición de su órgano difusor, Boletín Eridani, con sólo dos números aparecidos. Las relaciones entre los dos grupos mayoritarios se fueron deteriorando por diferencias acerca de si el asunto Ummo debía ser estudiado por Eridani o no, y en junio de 1971 los contrarios a dicha opción y partidarios de un estudio serio del fenómeno OVNI en general, encabezados por Félix Ares y David Gustavo López, se separaron de la agrupación. Hasta ese momento había contado con no más de 40 socios, que tras la escisión se vieron reducidos a la tercera parte. Su actividad se vio casi reducida a la tertulia semanal, aunque cobró nuevo protagonismo en los años siguientes con investigaciones sobre las caras de Bélmez de la Moraleda ( Jaén) en 1972 y sobre nuevos incidentes ufológicos en 1974-75 (Talamanca del Jarama y Autocine de Barajas, en Madrid, etc.). En su última época Eridani logró recuperar casi su número inicial de socios, colaboró con la CONIE (Comisión Nacional de Investigación del Espacio), y finalmente se disolvió en octubre de 1975. [ JM] Sin embargo, pronto se hizo evidente la imposibilidad de llegar a ninguna conclusión fiable dada la bajísima calidad de las investigaciones de campo y la enorme diversidad de seres descritos. Y con la aparición del fenómeno de las abducciones el campo de los simples encuentros con entidades perdió atractivo y se ha limitado a la pura catalogación de nuevos incidentes. Y lo que es peor, la realización de estudios estadísticos sobre casos de humanoides negativos (es decir, explicados) ha demostrado que resulta imposible separar unos de otros con seguridad (Banchs 1980, González 1984). [1, 36, 81, 100][LG] ERIDANI Agrupación de Estudios Cosmológicos Entidad fundada en Madrid el 17 de enro de 1970 por José Luis Jordán Peña, Félix Ares de Blas y ocho personas más. Fue una de las consecuencias de la reunión convocada por Vicente Juan Ballester Olmos en Galapagar (Madrid) el 12 de octubre de 1969. Surgió de la fusión de varios grupos existentes en la capital, principalmente Aleph y el antiguo ICSADE. Casi toda su actividad ufológica se desarrolló durante su primer año y medio de existencia, destacando la continuación del análisis sobre el flap de 1968-69, el apoyo a Herbert S. Williamson para un Congreso Mundial de Ufología en Madrid que nunca se celebró, varias encuestas sobre supuestos avistamientos (Monasterio de Yuste en Cáceres, Ucero en Soria, Galapagar en ESCEPTICISMO Dentro del ambiente de las mal llamadas "paraciencias" se denomina así al movimiento organizado que, apelando a la Razón, insiste en la necesidad de que sean aquellos que realizan las afirmaciones más extraordinarias quienes deben facilitar pruebas suficientes de lo que afirman, más allá de toda duda razonable. Para estos escépticos los verdaderos enemigos de la Razón no son necesariamente las personas crédulas, sino aquellos que se benefician personalmente de que los demás sean 82 Escritura automática crédulos. Ufología procesal, Zona caliente. Siempre han existido personas que han denunciado, con mayor o menor éxito, las creencias irracionales de los demás, pero ante la supervivencia de tantas tonterías de la Edad de Piedra en plena Era Atómica, precisamente cuando más patentes son los logros (y riesgos) del pensamiento científico, y más necesaria es la formación racional de las personas, desde hace algunos años muchos de ellos han decidido adoptar una postura activa, organizándose en grupos y asociaciones por todo el mundo, contando entre sus filas con científicos y filósofos de prestigio (v. csicop y arp). ESTIGIA Boletín de periodicidad irregular editado desde 1981 por el grupo español Cife. EVANS, Hillary (Shrewsbury, 1929 - Londres, 2011). Ufólogo inglés, principal representante de las corrientes psicosociológicas en el Reino Unido. Estudió literatura en Cambridge y trabajó como publicista antes de pasar a regentar la Mary Evans Picture Library. Miembro del Consejo del Bufora (también lo fue de la Society for Psychical Research) y cofundador de la Association for the Scientific Study of Anomalous Phenomena. Es coordinador del Project Bolide, una base de datos internacional de intercambio de información acerca de los rayos en bola. Interesado especialmente por el estudio psicológico del testigo OVNI y por los aspectos socioculturales del fenómeno, ha escrito diversos libros sobre la materia, aunque sólo cuenta con uno traducido al castellano: Visiones, apariciones, visitantes del espacio (Kier, Buenos Aires, 1990). Junto con John Spencer fue el recopilador de la fundamental obra colectiva UFOS 1947-1987: The 40 years search for an explanation (Fortean Tomes, London, 1987). Naturalmente, cualquier movimiento naciente de denuncia que desea hacer oír su voz trae consigo actitudes intransigentes, posturas muy agresivas, descalificaciones generalistas, etc.. Son los defectos inevitables de una lucha quijotesca contra los modernos charlatanes pseudocientíficos y contra los medios de comunicación social que difunden ese consumismo alienante sin la menor reserva. Como recordaba Carl Sagan, si los hábitos del escepticismo estuvieran ampliamente difundidos, se aplicarían también a quienes verdaderamente disfrutan del poder en nuestra sociedad, y eso sí que no les interesa. [LG] ESCRITURA AUTOMÁTICA Véase Contactismo. Opinión sobre el fenómeno (extraída de su artículo “Aspectos psicológicos de la Ufología”, en Cuadernos de Ufología nº 7, 2ª época, enero 1990): “En mi caso, siempre he propugnado que una parte sustancial de los avistamientos OVNI se deben a fenómenos naturales, aunque ESTADÍSTICAS DE CASUÍSTICA Véase Casos positivos y negativos, Catálogo de casuística, Ciclo bienal, Depuración de casuística, Falacia del residuo, Ley horaria, Oleada, 83 Evidencia física desconocidos para la ciencia (...). Además, existen fenómenos no naturales, conocidos normalmente por la ciencia, capaces de explicar estos avistamientos.(...) Pienso que aquellas personas que afirman tener encuentros con la Virgen María o con entidades extraterrestres se encuentran en un estado alterado de conciencia, en el cual salen de su estado de realidad habitual y se abren a la influencia de su propio inconsciente.” vida en la Tierra, intentando averiguar si en las mismas o semejantes condiciones ésta pudiera haber aparecido fuera de ella. De hecho, los primeros balbuceos de esta disciplina se dieron con Arrhenius, quien en 1908 pretendió solventar la cuestión apuntando la hoy inaceptada posibilidad de que la vida se esparciera entre las estrellas en forma de esporas (panespermia). Descartadas por ineficientes otras sustancias (fluoruro de hidrógeno, silicio, amoníaco a bajas temperaturas), la opinión general basa la vida en la química del carbono y, por tanto, en la molécula del ADN. El origen de esta última se acepta también que se encuentra en el ARN, cuya procedencia, en cambio, es muy discutida. Dejando a un lado las hipótesis de sistemas genéticos más primitivos de los que derivaría, los aminoácidos requeridos podrían haberse formado espontáneamente en la atmósfera reductora (de hidrógeno, metano y amoníaco) de los primeros tiempos de la Tierra, tal como Miller y Urey demostraron en 1953. La presencia de las mismas substancias obtenidas entonces, y en muy parecida proporción, en algunos meteoritos como el de Murchison (condritas carbonáceas) y en las nubes de polvo interestelar, hicieron pensar en la posibilidad de una tendencia generalizada en el cosmos hacia la creación de compuestos orgánicos. EVIDENCIA FÍSICA Véase Efectos de los OVNIs. EXOBIOLOGÍA Rama de la Biología que tiene como objeto de estudio las formas de vida que pudieran darse fuera de la Tierra. Aparece vinculada a la Ufología únicamente en la medida en que se admita la validez de la HET como explicación general o parcial de la casuística. Ante la imposibilidad material de examinar seres vivos de otros mundos, la exobiología ha incluido dentro de su ámbito el problema -aún irresuelto- del origen de la ¿VIDA EN MARTE? El 7 de agosto de 1996, la NASA anunció la posible detección de formas de vida fósiles en el meteorito AH 84001, que se supone procedente de Marte. Al parecer, 16 millones de años atrás el impacto de un asteroide en el Planeta Rojo envió al espacio una serie de materiales que hace unos 13.000 años cayeron sobre la Antártida, donde se recuperaron en 1984. En ellos, de una antigüedad aproximada de 3.500 millones de años, se encontraron restos de hidrocarburos aromáticos policíclicos, considerados de origen biológico, junto a estructuras filiformes parecidas a los estromatolitos terrestres. La subsiguiente polémica sobre cómo interpretar estos hallazgos sigue viva hoy en día [158]. 84 Expediente X No obstante, investigaciones recientes descartan una primitiva atmósfera tan reductora como se pensaba, de manera que ahora se señalan como posible fuente de esos aminoácidos primigenios los cometas, meteoritos, el polvo interestelar o los humeros submarinos. propuesto el análisis de las longitudes de onda del infrarrojo medio procedentes de planetas extrasolares (sobre cuya existencia existe hoy ya probada evidencia) para detectar así las marcas espectrales del agua, el ozono y el dióxido de carbono, compuestos siempre presentes en un mundo que sustente vida. Ello se conseguiría anulando el brillo de la estrella mediante técnicas de interferometría, lo que permitiría recibir la débil luz reflejada por los planetas. [8, 16, 18, 157, 217][MM] Sea como fuere, lo cierto es que la vida apareció en la Tierra muy tempranamente, hace unos 3.500-3.800 millones de años, lo que algunos han interpretado como una prueba de su naturaleza altamente probable. Tal precocidad ha motivado especulaciones sobre la posibilidad del florecimiento de la vida en Marte durante el período cálido en el que el agua corría por su superficie, durante los primeros 500-1.000 millones de años. Si esto fue así (como sugieren recientes estudios sobre meteoritos de origen marciano y los análisis in situ de la sonda Pathfinder), no puede descartarse que algunas especies sobrevivieran el tiempo suficiente para formar comunidades en el interior de rocas o en los fondos de lagos helados, cuyos fósiles podrían haber perdurado hasta hoy. La presencia actual de materia orgánica en el suelo marciano, sin embargo, fue totalmente descartada por las sondas Viking en 1976-77. Algo más lejos, la capa de hielo de agua que recubre Europa, luna de Júpiter, ha hecho especular sobre la existencia de un océano bajo la misma que pudiese albergar organismos termófilos. Por su parte, Titán, el mayor satélite de Saturno, posee una densa atmósfera de nitrógeno y metano muy parecida a la de la Tierra primitiva, la cual en combinación con agua, aportada por cometas o liberada por impactos meteoríticos, se ha demostrado que podría sintetizar aminoácidos, si bien las bajas temperaturas allí reinantes no suponen condiciones aptas para la vida. EXPEDIENTE X Serie televisiva norteamericana (X-Files, 1993-2000) que narra las peripecias de dos agentes del FBI ocupados en la investigación de casos con implicaciones ufológicas y parapsicológicas. Debido al gran éxito alcanzado por la serie, este término se ha convertido en un atractivo reclamo publicitario y por extensión ha pasado a designar cualquier clase de suceso fuera de lo normal. Parece que la particularidad que diferenciaría este tipo de informes de los fenómenos paranormales de toda la vida es que están enfocados desde la perspectiva oficial. Y cuando se habla de oficialidad suele existir la connotación de secreto y de ocultación de información (v. encubrimiento gubernamental).[IC, MM] EXTRAÑEZA Característica de un informe OVNI que señala el número de elementos de información contenidos en el mismo que desafían una explicación en términos físicos ordinarios. Fuera del sistema solar, hace poco se ha 85 Extraterrestre La consideración de la extrañeza de un incidente ha sido empleada como criterio para distinguir los OVNIs de los OVIs – finalidad ya presente en los primeros trabajos sobre la cuestión, obra de Jacques Vallée–, pero no fue hasta Hynek (1972) cuando este concepto cobró carta de naturaleza. A él se debe la definición de la misma, que otros autores han tratado de dotar de contenido cuantitativo siguiendo dos posibles caminos. El primero de ellos consiste en considerar una escala progresiva que mesure cada suceso en función de cada una de las circunstancias inexplicables que comprenda, ordenadas de menor a mayor extrañeza. Ésta fue la línea seguida en 1968 por Saunders para calibrar los casos del Ufocat, que se completaba con una escala de objetividad que, representada perpendicularmente a la de extrañeza, servía para mostrar gráficamente el interés potencial de cada suceso (v. Test de extrañeza-credibilidad). La sencillez de este método inspiró posteriormente los trabajos de Poher, Banchs y Adell (Ballester y Guasp, 1989), básicamente variaciones sobre el mismo tema basadas en la consideración de a lo sumo diez parámetros posibles. En los años ochenta, Jean-Luc Overal y el proyecto Unicat propusieron incrementar el número de “índices de extrañeza” hasta 105 y 70, respectivamente (Smith, 1992), tomando en consideración todas las posibles anomalías que, a su juicio, podía contener un informe OVNI. En cualquier caso, todos los métodos expuestos pecan de cierta subjetividad a la hora de la selección de los elementos definidores de la extrañeza, reflejo de las diferentes convicciones personales sobre la naturaleza del fenómeno OVNI. [31, 120, 230][MM] EXTRATERRESTRE Dícese de todo aquello que se origina y proviene de fuera de la Tierra, especialmente de los supuestos seres inteligentes de otros planetas. La creencia en la existencia de vida consciente en otros astros se remonta a la Antigüedad (Anaxágoras, Plutarco, Lucrecio), si bien no es sino a partir del Renacimiento cuando surge, con los orígenes del pensamiento científico, la reflexión sobre la pluralidad de mundos habitados, fruto del devastador efecto que causaron las ideas copernicanas. Si la Tierra ya no era el centro del universo, nada impedía creer que la humanidad tampoco constituía el centro de la creación. Así, pensadores como Nicolás de Cusa, Fontenelle, Swedenborg, Hervás y Panduro o Giordano Bruno se dedicaron a poblar el cosmos con toda clase de seres, por lo general física y moralmente más perfectos que el hombre, al tiempo que la literatura hacía lo propio en las obras de Kepler, Bergerac, Godwin o Wilkins (v. Ciencia ficción, extraterrestres en la literatura de). Las reflexiones de todos estos autores transmiten un cierto optimismo sobre la existencia y el talante de los extraterrestres, cosa que explica la enorme curiosidad popular que desató en 1835 el anuncio del hallazgo de formas de vida en la Luna (v. recuadro). Pero sería Marte el astro que, El segundo enfoque es el propuesto por Ballester y Guasp (v. Índices de Ballester-Guasp), que se diferencia del anterior en que la escala empleada es aditiva y no depende tanto de la presencia o no de un determinado factor como de la consideración conjunta de los diversos parámetros posibles. 86 Extraterrestre EL “ENGAÑO LUNAR” La fascinación de la opinión pública por los seres extraterrestres no es exclusiva del siglo XX. A fin de estudiar las constelaciones australes, John Herschel (hijo de William Herschel, descubridor de Urano) se trasladó en 1835 a Ciudad del Cabo con un importante equipo de observación. Esta circunstancia fue aprovechada por Richard Adams Locke, periodista del New York Sun y escritor de ciencia ficción, para redactar toda una serie de imaginativas crónicas sobre los supuestos hallazgos de Herschel en la Luna, posteriormente conocidas como el “engaño lunar”. Empezó atribuyendo una capacidad de resolución inusitada al telescopio sudafricano, para pasar a glosar toda la exuberante y pintoresca flora y fauna que aparentemente habitaba el satélite. Yendo un poco más lejos, relató el descubrimiento de unos seres de aspecto humano, pero con alas de murciélago, cuyos gestos, y en especial sus movimientos de manos y brazos, daban a entender que se trataba de seres racionales. Llegados a este punto, el New York Sun era ya el diario de mayor venta del mundo. Para poner fin a la fraudulenta serie de artículos, Locke escribió que un descuido de los astrónomos había dejado expuesto al sol el gran espejo del telescopio, lo que había provocado el incendio y la destrucción del instrumento. después del descubrimiento de “canales” sobre su superficie, dispararía toda suerte de especulaciones entre los científicos (Flammarion, Lowell), los literatos (Wells, Burroughs) e incluso los ufólogos (v. Ciclo bienal). concepciones han fundamentado no pocos episodios de histeria colectiva, el primero de los cuales se registró el 30 de octubre de 1938 cuando la versión radiofónica de la novela de Wells –dirigida por Orson Welles– llegó a provocar muertes al ser interpretada literalmente por la audiencia. Pese a los beatíficos mensajes de los contactados y de las películas de S. Spielberg, la leyenda negra extraterrestre ha cobrado nuevos bríos con la desaparición de la amenaza comunista y el resurgimiento de las corrientes conspiracionistas (v. Encubrimiento gubernamental), como lo prueba, por ejemplo, la reproducción del pánico y el caos en el cincuentenario de la emisión de Orson Welles, celebrado por Radio Braga de Portugal con una nueva invasión ficticia. Es sobre todo a partir de la novela de H.G: Wells La guerra de los mundos (1897) cuando estas perspectivas optimistas y amables se trocarán por visiones oscuras y terribles de nuestros posibles vecinos estelares. El colonialismo practicado por las potencias europeas en el resto del mundo invierte sus términos en esta obra, que presenta a los marcianos cometiendo las mismas tropelías que las naciones imperialistas. La ciencia ficción posterior, la belicosa primera mitad del siglo XX y la Guerra Fría se encargarán de mantener vivo este estereotipo, que conocerá su apoteosis en las paranoicas y reaccionarias películas de los años cincuenta (v. Cine y extraterrestres) y en las implicaciones estratégicas que el NICAP descubría detrás de cada avistamiento OVNI. Estas Tras la Segunda Guerra Mundial, la cuestión de la existencia de otros mundos habitados ha reverdecido gracias a las expectativas abiertas por la ciencia en general y por la astronáutica en particular. De un lado, no han cesado los esfuerzos de la Bioastronomía para detectar posibles 87 Extraterrestre mensajes interestelares, y por otro se ha consolidado el cultivo de la Exobiología. No obstante, lo que realmente ha supuesto un hito en su desarrollo –al menos a nivel popular– ha sido la aparición de la Ufología y su dogmática adhesión, hasta los años ochenta, a la hipótesis extraterrestre. De hecho, uno de los pocos resultados prácticos cosechados por los ufólogos ha consistido en difundir en amplias capas de la población la creencia en constantes visitas de alienígenas a la Tierra. [23, 54, 186][ JR, MM] DTU 88 Falacia del residuo, argumento de la F FALACIA DEL RESIDUO, argumento de la. seguridad no convierte a una hipótesis especulativa que introduce naves espaciales de civilizaciones extraterrestres y vuelos semejantes de la imaginación".[265][RC] Las falacias son conclusiones que han sido ilegítimamente obtenidas a partir de ciertas premisas. En la comunidad ufológica internacional no existe consenso en torno al tanto por ciento de casuística factible de explicación, situándose entre el 90 y el 95% el volumen total que puede ser reducido a causas conocidas por la ciencia actual. El resto -entre un 5 y un 10%- constituye un resto o residuo que ha sido valorado de diversas formas. Los defensores de la realidad de un fenómeno no reductible a los conocimientos actuales han argumentado que ese residuo prueba precisamente esa realidad, ya que se trata de casos que han sido sometidos a profundos análisis sin hallar explicación definitiva. En cambio, para los investigadores más escépticos, como Félix Ares de Blas, esta valoración es una simple falacia, dado que esa escasa relevancia cuantitativa no puede ser aducida como prueba de una realidad ajena a la ya conocida; lo normal, lo significativo, es el alto tanto por ciento de hechos no enigmáticos. Todas las ciencias cuentan con un pequeño porcentaje de hechos anómalos, sin que por ello provoquen el abandono de la teoría dominante que constituye la ciencia establecida. John Ziman (1981) argumenta que el hecho de que "una clase particular de conceptos científicos no sea capaz de explicar un determinado conjunto de observaciones no es base suficiente para falsar casi toda nuestra comprensión científica de otros muchos fenómenos y con FALSOS OVNIS Véase OVI. FALSO RECUERDO, síndrome del Se denomina así el conjunto de recuerdos sobre acontecimientos que nunca ocurrieron pero que sin embargo, terapeutas ineptos y con poca o ninguna formación profesional, empleando técnicas tales como hipnotismo, regresiones, drogas, interpretación de sueños e imaginación dirigida, consiguen inculcar con facilidad en las mentes de sus pacientes más sugestionables. Estas memorias falsas se hacen tan vívidas y reales para aquellos pacientes que las asumen que los convierten en unos testigos muy convincentes, incluso durante juicios penales. Esta moda psiquiátrica se inició a principios de la década de los noventa en Estados Unidos centrada especialmente en los aparentes abusos sufridos por los niños dentro del entorno familiar, los cuales supuestamente habrian sido reprimidos y olvidados por completo aflorando en su edad adulta con distintos síntomas, algunos tan elementales como dolores de cabeza. Poco después se extendió su campo de 89 Fata morgana acción a otros supuestos abusos sexuales de origen satánico ya en el entorno escolar. Y claro, también aparecieron personas que habrían sufrido abusos sexuales de naturaleza extraterrrestre: las abducciones. localizar un presunto tráfico no identificado detectado en un radar, pasando por el conductor que viaja de noche por una carretera solitaria y se ve sorprendido por una Luna suspendida sobre las copas de los árboles. Los efectos atmosféricos sobre la propagación de la luz y las ilusiones de la percepción contribuyen a veces a volver irreconocibles los astros observados, tanto en lo que respecta a su aspecto (forma, color, luminosidad) como a sus movimientos aparentes. En los últimos años se han ido anulando muchas de las sentencias dictadas en base a estas únicas pruebas referidas a casos de supuestos abusos familiares y/o satánicos. Queda pendiente algo similar dentro del ámbito ufológico respecto a las abducciones. [94, 95, 152][LG] En este sentido, la dispersión atmosférica es responsable del color anaranjado o rojizo de los astros cerca del horizonte. La refracción atmosférica, por su parte, puede dar lugar a muy diversos efectos, como el centelleo de las estrellas (eventualmente acompañado de destellos de diversos colores), que algún observador puede interpretar como un movimiento de rotación. También puede originar cambios de luminosidad y pequeños desplazamientos aparentes e incluso distorsionar, fraccionar o desdoblar la imagen de un astro muy cercano al horizonte. Por otro lado, la interposición de nubes puede atenuar y extinguir el brillo de los astros, así como crear juegos de luces y sombras con el disco lunar. En ocasiones, el observador tiene la impresión de que tales variaciones de intensidad luminosa se deben a sendos movimientos de acercamiento o alejamiento. En otras ocasiones, nubes en movimiento cerca de algún astro pueden crear la ilusión de que es este último, en lugar de las nubes, el que se desplaza. FATA MORGANA Véase Fenómenos ópticos. FENÓMENOS ASTRONÓMICOS En ocasiones, las estrellas y planetas más brillantes, la Luna, e incluso el Sol poniente, han sido tomados por OVNIs (en cuanto a los meteoros, v. Bólido). De hecho, las confusiones de tipo astronómico resultan ser las más habituales, en particular las debidas al planeta Venus. También son las más fáciles de desentrañar si se dispone de la información completa sobre el avistamiento, ya que las posiciones de los astros pueden calcularse con exactitud recurriendo a tablas de efemérides o a programas astronómicos para ordenador. Las expectativas del observador y las circunstancias que rodean la observación juegan un importante papel en estos casos. El amplio abanico de situaciones abarca desde el aficionado a los OVNIs que se asoma a la ventana para escrutar el cielo y se fija en una estrella particularmente brillante, hasta el piloto militar que observa el planeta Venus a su "misma altura" tras recibir el encargo de Otras ilusiones de movimiento no tienen relación con factores atmosféricos. El llamado efecto autocinético, que se pone de manifiesto cuando se observa un punto luminoso en ausencia de un marco de referencia, puede dar lugar a que el 90 Fenómenos atmosféricos observador perciba una estrella o un planeta describiendo continuos movimientos desordenados. Cabe destacar también la ilusión de desplazamiento de un astro debido al cambio de posición del observador, hasta el extremo de que algunos conductores pueden llegar a sentirse "acompañados", y hasta "perseguidos", por la Luna. con el tema OVNI hay que destacar en primer lugar las nubes. Algunas nubes, por su forma (como en el caso de los característicos cúmulos lenticulares) o por el aspecto que les confiere la iluminación solar (con efectos espectaculares durante el crepúsculo), han dado lugar a confusiones. A veces, el hecho de que una nube se presente en solitario o bajo una apariencia muy diferente de las de su entorno contribuye a llamar la atención del observador. Algunos ufólogos, basándose en observaciones de este tipo, han sustentado la creencia de que las nubes podrían servir de camuflaje a naves no terrestres. Adicionalmente pueden mencionarse, aunque su papel aquí es muy marginal, las llamadas nubes nacaradas o irisadas, situadas a alturas mucho mayores que las nubes comunes, y visibles sólo con el sol ya oculto. Lo mismo puede decirse de las nubes noctilucientes, constituidas por partículas en suspensión procedentes del espacio exterior o de erupciones volcánicas. La proximidad de un astro al horizonte potencia muchos de los factores citados: no solamente los efectos debidos a la refracción y la dispersión de la luz, sino también alguna que otra ilusión perceptiva, como la que lleva a atribuir a un cuerpo celeste un mayor tamaño aparente cuando está cerca del horizonte. Nótese además que un astro a muy baja elevación suele ser percibido como algo inhabitual. En ocasiones se atribuye al astro avistado supuestos efectos, tales como alteraciones de diversa índole en los testigos, interferencias en la radio, huellas en el terreno, ecos en el radar, etc., sin auténtica relación con la observación. Un capítulo aparte merecerían, por el contrario, las nubes de origen artificial, que han originado todo tipo de avistamientos extraños. Nos referimos a las estelas de condensación producidas por aviones, misiles y cohetes, así como las nubes de sustancias liberadas en la atmósfera con fines experimentales o durante la puesta en órbita de ingenios astronáuticos. Aparte de permitir una identificación del OVNI en ciertos incidentes, las comprobaciones astronómicas también resultan muy útiles en otros casos para obtener datos complementarios (como conocer las condiciones de iluminación solar durante la observación) y para contrastar la fiabilidad de la información (por ejemplo, si se menciona la fase de la Luna, verificar si se corresponde con la de la fecha asignada).[47, 112][MB] Cerraremos este primer apartado con una referencia a las termales, una especie de burbujas de aire caliente formadas por convección que tras alcanzar cierta altura pueden pasar a ser arrastradas por el viento. Su tamaño supera en ocasiones los centenares de metros. Aunque no son visibles a simple vista, pueden aparecer en fotografías infrarrojas (Balester y Guasp, 1989), dando pie a algunos equívocos. A destacar que también pueden FENÓMENOS ATMOSFÉRICOS De la extensa gama de fenómenos atmosféricos susceptibles de ser relacionados 91 Fenómenos forteanos aparecer en las pantallas de radar. FENÓMENOS FORTEANOS Un segundo gran apartado de fenómenos atmosféricos de interés al hablar de OVNIs comprende los fenómenos ópticos. Aquí quedan englobados tanto los espejismos (por extensión, también los que afectan a las ondas radáricas) como los halos y otros efectos ópticos (espectro de Brocken, rayo verde, etc.). En la literatura especializada han existido precursores importantes, que más de treinta años antes del llamado “período oficial de los platillos volantes”, centraron su personalísimo interés en la marejada de esos mundos extraordinarios que Pauwels y Bergier definieron como “realismo fantástico”. Un tercer apartado se refiere a los fenómenos eléctricos atmosféricos. Remitimos al lector a las explicaciones correspondientes al controvertido rayo en bola y a la aurora boreal (fenómeno que nos adentraría ya en el campo de la astronomía). También puede citarse el fuego de San Telmo, una débil descarga eléctrica producida por inducción eléctrica de una nube sobre objetos terminados en punta, tales como chimeneas, veletas, árboles, mástiles de embarcaciones, alas de aviones, etc. Generalmente se produce con tiempo tormentoso y se manifiesta bajo la forma de una llama pálida de color azulado, acompañada de un zumbido o chisporroteo. Por lo que respecta al rayo convencional, hay que mencionar que, en ocasiones, deja en el terreno huellas que se prestan a interpretaciones fantásticas, como cuando produce surcos, agujeros y canales ramificados que, en suelos arenosos, pueden albergar tubos de sílice fundida (fulguritas). En una sociedad alterada por los descubrimientos tecnológicos, como la aplicación de la telefonía sin hilos, las primeras grabaciones musicales, la luz eléctrica y otros, el burgués Charles Hoy Fort (1874-1932), vivía en un pequeño apartamento de la ciudad de Nueva York, saciando su más que notable sed de archivero y recopilador, en las amplias colecciones de periódicos, revistas y semanarios de la Biblioteca municipal. Allí encontró materia para sus reflexiones, más filosóficas que científicas, reuniendo toda una amplísima colección, en miles de fichas, de hechos totalmente imposibles para un mundo cada vez más tecnificado. Lluvias de peces, de ranas, de carne. Apariciones de naves, extraños cometas, ruedas luminosas en el mar Rojo. Inscripciones en meteoritos. Balas en cráneos de mamut. Huellas de animales fabulosos que se evaporan sin motivo aparente. Desapariciones inexplicables de barcos y multitud de personas en insólitas circunstancias. Soles verdes y lunas azules y toda clase de pequeñas criaturas del bosque. Un universo poblado de fenómenos naturales opuestos y de intervenciones de seres desconocidos, que a su inquieta mirada califica como los “hechos condenados”, y sobre los cuales comenta en su primera obra (El libro de los condenados, 1919, recopilación selectiva de sus más de Los anteriores apartados no agotan todas las posibilidades que ofrecen los fenómenos atmosféricos. Minitornados y diversas variedades de torbellinos, por ejemplo, pueden dar lugar a ciertas observaciones equívocas y dejar huellas insólitas. Algunas de estas perturbaciones, no todas bien estudiadas, se acompañan de fenómenos eléctricos y efectos luminosos y caloríficos. [31, 57][MB] 92 Fenómenos forteanos veinticinco mil fichas que guardaba en cajas de cartón): estimo, pues, que todo lo que comúnmente se denomina «existencia» y que yo llamo intermediaridad, es una casiexistencia ni real ni irreal, sino expresión de una tentativa apuntando a lo real, o a la penetración de una existencia real. “Por los condenados, entiendo los excluidos. Pero por los excluidos entiendo también todos aquellos que, un día, excluirán a su vez. Pues el estado común y absurdamente llamado existencia es un ritmo de infiernos y de paraísos. Y los condenados no seguirán siéndolo, ya que la salvación precede a la perdición, y nuestros malditos andrajosos serán un día melosos ángeles que, mucho más tarde todavía, volverán a marcharse al mismo lugar de donde habían venido... “...Ved, pues, como la ciencia tiende, al principio, a negar tanto como puede las relaciones exteriores a esta Tierra. Mi libro, precisamente, es una suma de datos sobre esas relaciones. Sostengo que mis datos han sido malditos, no en consideración a un mérito o demérito, sino conforme a una tentativa general de aislamiento de esta Tierra. “...Vivimos una seudoexistencia cuyas apariencias todas participan de su irrealidad esencial. Pero ciertas apariencias se aproximan más que otras al estado positivo. Concibo todas las «cosas» como ocupando gradaciones, etapas seriales entre lo positivo y lo negativo, entre la realidad y la irrealidad. Ciertas apariencias son más constantes, más individuales o más estables que otras. “Si alguien era, hace cien años, lo bastante crédulo como para creer que caían piedras del cielo, se le espetaba este razonamiento: no hay piedras en el cielo; por lo tanto ninguna piedra puede caer. Nada más razonable, más científico o más lógico podía sostenerse sobre un tema cualquiera. El único inconveniente era que la premisa mayor era falsa o intermedia entre lo real y lo no real. “No soy un realista. No soy un idealista. Soy un intermediarista. Nada es real, pero nada es irreal, y todos los fenómenos son aproximaciones de una o de otra parte entre la realidad y la irrealidad. De manera que toda nuestra casiexistencia es un estado intermedio entre lo real y lo irreal. Pero en esta apresurada suma, la Realidad es un aspecto del estado positivo. “Por realidad, designo lo que no se confunde con algo de otra cosa, lo que es parcialmente otra cosa, lo que no es una reacción a algo o una imitación de algo. Un héroe real sería alguien que no fuese parcialmente cobarde, o cuyas acciones o motivo no se confundiesen con la cobardía... “En 1722, una comisión de la que formaba parte Lavoisier, fue designada por la Academia de Ciencias francesa para examinar un informe sobre la caída de una piedra del cielo en Luce. Entre todas las tentativas de positividad en lo que al aislamiento se refiere, no conozco noción más ardientemente defendida que la del noparentesco terrestre. Lavoisier analizó la piedra de Luce. La explicación exclusivista quería, en aquella época, que ninguna piedra cayese del cielo: objetos luminosos parecían aterrizar, y se recogían piedras ardientes en su punto de caída. Sólo el rayo alcanzando a una piedra podía calentarla o fundirla. “...En términos de metafísica general, “La piedra de Luce mostraba señales de 93 Fenómenos ópticos fusión. El análisis de Lavoisier «probó irrefutablemente» que aquella piedra no había caído, sino que había sido alcanzada por el rayo. Oficialmente, las caídas de piedras fueron condenadas, y la explicación del rayo fue la norma de la exclusión. recopilando los trabajos del archivero y muchos de sus “hechos condenados”: Lluvia de ranas, de peces, de sangre, de carne, de barro. Soles verdes, Lunas azules. Nieve negra. Animales fabulosos. Huellas de gigantes..., hechos admitidos y hechos condenados que Charles Fort consideraba claramente excluidos por la Ciencia: “La ciencia moderna ha excluido falsamente, a falta de estándares positivos. Ha excluido fenómenos que, según sus seudoestándares, tenían tanto derecho a la existencia como los elegidos”.[88] [ JA] ”...Desde el comienzo, las vestales de la ciencia han combatido, llorado, aullado, maldecido las relaciones externas, con los mismos pretextos... ya ahí, o de arriba abajo. “Progreso significa violación. “Mantequilla y sangre. Carne de buey y una piedra cubierta de inscripciones”. FENÓMENOS ÓPTICOS Hasta aquí unos apuntes someros de los pensamientos-razonamientos de esa “intermediaridad” en la cual, aparentemente, se movía como un escualo nuestro Charles Hoy Fort y que él publicaba en la segunda década del siglo XX en su obra El Libro de los Condenados. Los rayos de luz se desvían (refracción) cuando atraviesan oblicuamente capas de aire a distinta temperatura. Como consecuencia, la imagen de objetos y luces distantes puede llegar a verse desplazada y distorsionada de forma apreciable, efecto óptico que se conoce como espejismo. El pensamiento y trabajo de Fort tuvo su repercusión entre los críticos de la época demostrando que tales cuestiones coincidían con el descubrimiento de teorías tan impensables como la de la relatividad de Einstein o los “quanta” de Planck. Cuatro años después, publicó su segundo trabajo, Tierras Nuevas, donde elaboraba insólitas afirmaciones astronómicas, oponiéndose a la velocidad de la luz o al paralaje de las estrellas, avanzando de algún modo la teoría de las “supercuerdas”. A ese libro le sucedería Lo!, en el año 1931 y Talentos salvajes en 1932, tras la desaparición del escritor. Tanto su personalidad, como las ideas que trabajó a lo largo de su vida, atrajeron el interés de sus coetáneos, hasta el punto que un grupo de intelectuales norteamericanos fundó a su mayor gloria la Sociedad Charles Fort, que publicaría la revista Doubt (Duda), Es bien conocido el típico espejismo del desierto o espejismo inferior. Aunque más infrecuente, también existe el espejismo superior, es decir, aquel en que la imagen de un objeto queda por encima de su posición real. Se produce cuando se dan condiciones de inversión de temperatura, o sea cuando existen capas de aire cuya temperatura es mayor que la de capas inferiores. Los rayos de luz se curvan como si fueran "reflejados" por las capas más calientes. Las imágenes de astros, luces lejanas, elementos del paisaje, barcos, aviones, etc., situados cerca del horizonte, pueden presentarse deformadas en sentido vertical, ya sea comprimidas, expandidas (la "fata morgana" sería un caso muy especial), mutiladas, invertidas o repetidas (espejismos 94 Fenómenos ópticos múltiples). En el caso de un espejismo superior suele darse un alcance óptico extendido, permitiendo observar objetos que en condiciones normales quedarían más allá del horizonte, esto es, bajo el mismo. No es sorprendente pues que los espejismos aparezcan involucrados en determinadas observaciones de presuntos OVNIs. habitual es que sólo sea visible una parte del conjunto. Sin duda, los elementos más susceptibles de ser tomados por OVNIs son los parhelios o falsos soles (paraselenes, si se trata de un halo lunar), situados a la misma altura que el Sol. A destacar que algunas porciones del halo presentan forma de cruz, lo que permite explicar ciertas visiones de cruces luminosas sucedidas en tiempos pasados. Cabe señalar que las ondas electromagnéticas utilizadas en los radares también pueden experimentar efectos similares, no sólo debido a inversiones de temperatura sino también a inversiones en la variación de la humedad con la altura (v. Radar y OVNIs). Algunos fenómenos suelen ser observables desde la cima de una montaña o desde un avión en vuelo. El subsol no es más que el reflejo del Sol en una nube de cristales de hielo situada por debajo del nivel del observador. Este fenómeno luminoso es visible en la misma dirección del Sol, a la misma elevación bajo el horizonte que la elevación del Sol sobre el horizonte. Por el contrario, el espectro de Brocken aparece en dirección opuesta al Sol, al tratarse de la sombra del observador proyectada sobre la niebla o sobre una nube. A veces se presenta rodeado de uno o varios anillos luminosos (glorias), debido a la difracción de la luz en las gotas de agua. Nótese que un observador que viaje en avión y vea alguno de los anteriores efectos tendrá la impresión de que el fenómeno lo "acompaña", desplazándose a su misma velocidad. El centelleo de las estrellas y la coloración anaranjada del Sol poniente son buena muestra de otros efectos ópticos atmosféricos que alteran la apariencia de los objetos (v. Fenómenos astronómicos). Dependiendo de las condiciones meteorológicas, los rayos del Sol, de la Luna, e incluso de otras fuentes de luz, pueden producir peculiares efectos luminosos. La posición y las características de tales efectos los hacen fácilmente reconocibles, al menos para el observador bien informado. En este sentido, puede descartarse que el familiar arco iris se preste a confusiones. Tampoco parece que la corona que se observa en ocasiones en torno al Sol o la Luna pueda llegar a sorprender a muchos observadores. Ahora bien, otros fenómenos, como el halo, el subsol, el espectro de Brocken o el rayo verde, pueden constituir auténticas rarezas para el lego. En ocasiones, a la puesta (salida) del Sol puede observarse un último (primer) destello, en forma de rayo verde. Se trata de un efecto combinado de la refracción y la dispersión atmosféricas, que sólo es observable en un horizonte despejado y con una atmósfera limpia. También puede ser visto en otros astros brillantes, como la Luna o Venus. Aunque ufológicamente hablando no tiene mucho interés, el fenómeno puede llegar a aparecer como un ingrediente más en alguna confusión de tipo astronómico. El halo consiste en un complejo despliegue de anillos, arcos y otros elementos luminosos, originados por reflexión y refracción de la luz solar en los cristales de hielo que constituyen algunas nubes. Lo 95 Fernandes, Joaquim Por último, no puede dejar de señalarse que hay infinidad de situaciones en que los efectos luminosos más triviales pueden desconcertar a un observador desprevenido y dar lugar a informes y evidencias de supuestos OVNIs (Ballester, 1984). También son efectos ópticos algunas imágenes "fantasmas" e imágenes distorsionadas que aparecen en determinadas fotografías y filmaciones. [29, 216] [MB] (n. Valencia, 1959). Investigador ufológico, es Ingeniero Técnico en explotaciones agropecuarias y en hortofruticultura y jardinería. Se interesó por el fenómeno OVNI en 1974, pasando a especializarse en la investigación de campo y la recogida de casuística de su ámbito geográfico, que ha ido recopilando en el Catálogo OVNI de la Comunidad Valenciana. Ha contribuido con notables revisiones de casos ya clásicos, como el de Morón de la Frontera (11/05/1970), y publicó el primer estudio bibliográficocrítico en España sobre el tema OVNI, que bajo el título Treinta años de literatura Ovni en España (1950-1980) apareció como apéndice de la obra de V.J. Ballester Olmos Investigación OVNI (Plaza & Janés, Barcelona, 1984). Con este último ha sido también coautor de la Enciclopedia de los encuentros cercanos con OVNIS (Plaza & Janés, Barcelona, 1987) y de los catálogos LANIB y NELIB (v. aterrizaje). En 2000 publicó El expediente Manises, magna obra consagrada a la explicación y al análisis monográfico de este significativo incidente, que había sido premiada en 1998 con el Premio Ricardo Caruncho de la Fundación Anomalía. Entre sus aportaciones a revistas especializadas podrían destacarse “Los informes de abducciones: análisis de las presuntas experiencias en el interior de los Ovnis” (en Cuadernos de Ufología nº 4, 2ª época, 1988) y “¿Son los OVNIs fenómenos naturales?” (ibídem, nº 5, 2ª época, 1989), ambos coescritos con Ballester Olmos. FERNANDES, Joaquim (n. Oporto, 1946). Investigador y divulgador del fenómeno OVNI en Portugal, licenciado en Historia y profesor de Ciencias de la Comunicación en la Universidade Fernando Pessoa de Oporto. Se sintió atraído por la Ufología en 1975, año en el que inicia la publicación (hasta 1985) de una columna sobre el tema en el diario Jornal de Notícias de su ciudad natal y en que se encarga de la edición de la revista del CEAFI Insólito (1975-1981). En 1978 coorganizó el I Congreso Ibérico de Ovnilogía y publicó su primer libro, Ovnis em Portugal, que fue seguido en 1982 por Intervenção Extraterrestre em Fátima (Livraria Bertrand, Lisboa), escrito con Fina d’Armada y reeditado en 1995 con el título de As Aparições de Fátima e o fenómeno OVNI, todo un clásico sobre las apariciones marianas. En 1984 cofundó la Cnifo, dentro de la cual ejerce de editor del anuario Anomalia, y actualmente es uno de los promotores de la Sociedade Portuguesa de Exploração Científica (SPEC, creada en 1997). FLAP En su acepción moderna, período en el que se produce un incremento repentino de observaciones de OVNIs distribuidas en un corto espacio de tiempo (pocas semanas) o FERNÁNDEZ PERIS, Juan Antonio 96 Flying Saucer Review en áreas o zonas muy localizadas (provincias, regiones). Se contrapone así a la oleada, que tendría un carácter más amplio. la FSR se convirtió en uno de los primeros foros de las hipótesis paraufológicas y psicosociales. Tras el abandono de Bowen por problemas de salud, en 1982 Gordon Creighton se hizo cargo de la publicación, lo que conllevó un nuevo cambio de filosofía, esta vez hacia las ideas demonológicas y conspiracionistas del nuevo editor. Pese a que la FSR ha seguido difundiendo interesantes materiales procedentes de todos los rincones del globo, en la actualidad su influencia se ha visto mermada por los excesos paranoicos de Creighton, que han provocado también un descenso sustancial del número de lectores. En diciembre de 1996 apareció la versión española de la FSR, difundida junto a la revista Enigmas. Dirección: FSR Publications Ltd, PO Box 585, Rickmansworth, WD3 1YJ, UK. En Internet: http://www.fsr.org.uk/ [MM] Sin embargo, históricamente fue este término el que primeramente designó lo que hoy conocemos como oleadas. La palabra procede de la jerga militar estadounidense, donde "flap" (literalmente: aleteo) designaba un alto grado de excitación y confusión. De ahí se tomó por la USAF para aludir a las acumulaciones de avistamientos, especialmente si iban acompañadas de amplia publicidad; lo que se oponía a las llamadas "concentraciones", donde el fenómeno se presentaba sin ninguna especial difusión por los medios de comunicación. [MM] FLYING SAUCER REVIEW (FSR). Revista ufológica británica publicada desde el año 1955 en Londres. Apareció inicialmente de forma trimestral y editada por el ex-piloto de la RAF Derek Dempster, quien en 1956 sería substituido por Brinsley le Poer Trench. Tres años más tarde, cuando fue nombrado editor Waveney Girvan, la revista cambió a bimensual y experimentó un incremento en su circulación e influencia. Al morir Girvan en 1964, Charles Bowen se hizo cargo de la misma y dio paso al período más floreciente de la FSR, durante el cual contó con los trabajos de las más prestigiosas firmas de la ufología mundial e incluyó casuística de los cinco continentes. A diferencia de Trench y Girvan, próximos a posiciones contactistas, Bowen orientó la línea editorial hacia los aspectos más insólitos del fenómeno (criaturas extrañas, hombres de negro, etc.), que se fueron alternando en sus páginas con informes tradicionales. En este sentido, FOLKLORE Y OVNIS Véase Antropología de los ovnis. FOO-FIGHTER (cazas fantasmas o kraut fireballs) Denominación dada por los aviadores militares aliados durante la Segunda Guerra Mundial a las esferas luminosas que seguían a sus aviones en el curso de las misiones de guerra. El nombre parece derivar de fighter (caza) y de foo (corrupción del francés feu), que en el argot militar americano también significaría "fuego" (Ribera, 1966, citando a Wilkins, 1954), aunque existen otras interpretaciones. De aspecto inmaterial, de color cambiante, del rojo al anaranjado y del blanco al rojo, por lo general no sobrepasaban los 50 cm. de diámetro, con rangos de velocidades entre 300 y 800 km/h 97 Fort, Charles H. y no lograban ser detectados por el radar. Podían presentarse solos o en formación de hasta veinte objetos. Fueron observados en los últimos meses de la contienda al oeste del Rin, en Japón y en Truk Lagoon (Pacífico central). El primer avistamiento documentado fue el realizado el 23 de noviembre de 1944 por la tripulación de un B-29 de la 415ª Escuadrilla de Cazas Nocturnos de los EE.UU., estacionada en Dijon, Francia. científicos elaboró una serie de libros sobre fenómenos inexplicables, a los que desde entonces se les conoce como fenómenos forteanos. Los únicos traducidos al castellano son El Libro de los Condenados (1969) y Nuevos mundos (1985), llenos a rebosar de datos anómalos y curiosas teorías e hipótesis, pero sin ningún intento de evaluación o depuración. Tras la muerte de Fort, la recogida de datos fue continuada por la Fortean Society, fundada en 1931 y que editaba la revista Doubt, ambas desaparecidas en los años setenta. En la actualidad el testigo ha sido recogido por la International Fortean Organization, que publica The INFO Journal en EE.UU. y la revista Fortean Times en Gran Bretaña. Dirección: Fortean Times, FREEPOST (SW6096), Bristol BS21 OBR, Gran Bretaña. En Internet: https:// subscribe.forteantimes.com/ [88] En cuanto a la naturaleza de estos objetos, inicialmente se les consideró armas secretas alemanas producidas en Wiener Neustadt, Alemania, destinadas a anular o desorientar el radar e interrumpir las corrientes electromagnéticas (Vesco, 1969), hasta el punto de que los ingleses habrían contado entre 1943 y 1944 con una organización encargada de su estudio, según se confirmaría oficialmente en 1966 (Edwards, 1966). Pero parece que en el bando contrario también el Oberkommando der Luftwaffe creó el "Sonderbüro nº 13", cuyas actuaciones estuvieron bajo el nombre cifrado de operación Uranos, con propósitos semejantes (Durrant, 1972). Se ha apuntado igualmente la posibilidad de que se trataran de fuegos de San Telmo o de rayos en bola propiciados por la continua actividad de numerosas aeronaves militares que cargaría el aire con contaminantes y electricidad estática (Klass, 1968).[76, 77, 129, 191, 257, 262][ JR] FOTOGRAFÍA INFRARROJA En la década de los setenta, y con el auge alcanzado por las “sesiones de observación del cielo”, muchos aficionados y grupos, en busca de datos experimentales de tipo físico e influenciados por proyectos como el Starlight norteamericano, pasaron a utilizar durante sus vigilias nocturnas películas fotográficas infrarrojas que, virtualmente, presentaban la posibilidad de captar imágenes de los fenómenos OVNI no visibles al ojo humano. Se partía de la presunción de que tales fenómenos podían emitir ciertas radiaciones no visibles en el espectro convencional y que ese tipo de información quedaba registrada en películas sensibles a la radiación infrarroja. Por ello, los aficionados que dedicaban muchas horas nocturnas a sesiones de observación FORT, Charles H(oy) (1874-1932). Periodista y escritor norteamericano que a partir de abundantísimas notas tomadas de periódicos, revistas y hasta de artículos 98 Fotografías de OVNIs pusieron de moda el uso de emulsiones especiales, una para realizar negativos en blanco y negro (Kodak high speed infrared) y otra para registrar el color en esa franja del espectro (Kodak Ektachrome infrared), que una vez reveladas se convertían en diapositivas enmarcadas. la película infrarroja a los focos de calor ha sido fuente inagotable de formación de imágenes espurias tanto durante las tomas como en el almacenamiento anterior o posterior a las mismas, por lo que el análisis de esta clase de fotografías exige el más estricto cumplimiento de todo tipo de garantías que permitan asegurar el rigor metodológico en su obtención. [31, 256][CL, JA, MM] El empleo de esta técnica permitía, según sus practicantes, captar las imágenes de numerosos “OVNIs invisibles” simplemente disparando la cámara al azar. Ciertamente las tomas así obtenidas mostraban vaporosas formas circulares de gran tamaño pero, en contra de una opinión muy extendida entre los grupos de investigadores de campo, en absoluto correspondían a OVNIs, sino a “termales”. Se conocen con este nombre las masas globulares que se forman en zonas en las que ha habido un rápido enfriamiento de los alrededores y que, al encontrarse a temperatura superior a la del medio que las rodea, se elevan hasta una altura en la que se alcanza el punto de saturación del vapor de agua en ellas contenido y allí se estabilizan. Dicha altura depende de la temperatura del interior de la nube, pero suele rondar los 600 m. El tamaño de estas masas oscila entre los 100 y los 400 m. y su velocidad de ascensión entre unos pocos cm/seg hasta más de 30 m/seg. Las burbujas térmicas, al estar más calientes que el medio circundante, despiden mayor cantidad de radiación calorífica que el aire de los alrededores, y por tanto destacan con claridad en cualquier imagen tomada con película infrarroja, que al ser revelada muestra una irregular forma circular “luminosa” que no ha sido apreciada por el ojo del fotógrafo. De esta forma se generaban muchas de las tomas fervientemente defendidas como pruebas de la existencia de fenómenos físicos invisibles identificados con OVNIs.. FOTOGRAFÍAS DE OVNIS Véase Análisis fotográfico. FRATELLANZA COSMICA (Centro de Estudios Fraternidad Cósmica). Grupo contactista italiano fundado en 1962 por Eugenio Siragusa. En 1988, ya bajo la dirección de los hermanos Bongiovanni, cambió su nombre por Nonsiamosoli, título de la publicación del centro. Véase sectas ufológicas. FRAUDES El fraude se define como una acción contraria a la verdad y la rectitud. La Ufología, por su propia naturaleza, ha estado sometida desde sus orígenes en los años 40 a todo tipo de manipulaciones y engaños perpetrados con diversos fines. Dos son sus principales clases: testimoniales y fotográficos. Los primeros son aquellos relatos que en el transcurso de la investigación de los supuestos hechos relatados se comprueban falsos en su totalidad o en parte. Los de tipo fotográfico tradicionalmente consisten en hacer pasar un objeto común hábilmente camuflado o Por otro lado, la extrema sensibilidad de 99 Fuego de san Telmo FUNDACIÓN ANOMALÍA disimulado por algo extraño; ha sido habitual la utilización de técnicas de trucaje fotográfico en los negativos. Las razones para perpetrar estos engaños pueden ser por beneficio económico, deseo de probar la creencia personal en la realidad de la existencia de los extraterrestres, prestigio social entre los ambientes de aficionados, bromas infantiles, montajes periodísticos, etc. Son innumerables los fraudes que se han puesto al descubierto en el transcurso de la investigación del fenómeno OVNI. La Ufología es también terreno donde prosperan los pícaros. [103, 166, 229][RC] Primera fundación creada en Europa para la investigación científica de carácter ufológico. Fue constituida en Santander por iniciativa del Colectivo Cuadernos el 12 de julio de 1996, y sus fines son la promoción del estudio, desde una perspectiva racional y basada en la metodología científica, del fenómeno de los denominados objetos volantes no identificados, así como de las materias conexas, y su influencia sociocultural; la preservación y gestión de archivos, bibliografía y legados culturales relativos a estas materias; la divulgación de su labor mediante sus órganos de difusión y por cualquier otro medio; y la concesión de becas de ayuda a la investigación. FUEGO DE SAN TELMO Véase Fenómenos atmosféricos. Su primer patronato lo integraron Julio Arcas Gilardi -como presidente-, Vicente-Juan Ballester Olmos, Matilde González García, Luis R. González Manso, Matías Morey Ripoll, Antonio Petit Gancedo y José Ruesga Montiel, a quienes se unió en 1999 Ricardo Campo Pérez, en 2004 Martí Flò García, y en 2010 Pedro Ojalvo. Tenía como órgano de difusión la revista Cuadernos de Ufología. FUEGOS FATUOS Llamas erráticas que se forman a poca distancia del suelo por la inflamación de los gases emanados de las materias orgánicas en descomposición. De color generalmente azulado, su tamaño no suele ser mayor de unos pocos centímetros. Tienen su origen en la acción de bacterias anaerobias que descomponen la materia vegetal en compuestos como el metano y el fosfuro de hidrógeno, que entran en combustión en presencia de oxígeno. Por este motivo, los fuegos fatuos son comunes en cementerios y zonas cenagosas, de ahí que el llamado gas de los pantanos (básicamente metano) haya sido propuesto en ocasiones como explicación para algunos avistamientos de OVNIs.[MM] En 2010 cambió su nombre a Fundación Íkaros (http://www.ikaros.org.es), aunque en diciembre de 2011 se acordó su disolución y, tras cumplir con los trámites administrativos, se extinguió finalmente en 2013. Sus fondos bibliográficos se destinaron a la Biblioteca Pública de Cantabria, donde en la actualidad pueden consultarse.[ JR] DTU 100 Gas de los pantanos G GAS DE LOS PANTANOS principal del grupo es la investigación de campo, tratando de buscar la explicación más racional posible a cada caso. Destacan en su haber, entre otras, las investigaciones realizadas en torno al caso de un supuesto aterrizaje en el poblado de Las Medianas (Almonte, Huelva) y la presencia de supuestos humanoides en la playa de Los Bateles (Conil, Cádiz), incidente en el que el GEIFO demostró la existencia de un fraude. La entidad se organiza en tres departamentos: Información, Investigación de Campo, y Estudio y Clasificación. Dirección: C/ Tolosa Latour, 5, Bloque 1, 3º C; 11007-Cádiz (España). Hoy en día mantiene su presencia en Internet: https:// geifo.es.tl/ [AR] 1. Véase Fuegos fatuos. 2. Famoso episodio de la historia de la ufología estadounidense ocurrido en el marco de una oleada de observaciones sobre el estado de Michigan durante el mes de marzo de 1966. Requerido el Dr. Hynek como asesor del proyecto Blue Book para dar una explicación de los múltiples avistamientos, indicó ante la insistencia de la prensa la posibilidad de que algunos de ellos pudieran tener su origen en la combustión del gas de los pantanos. Estas palabras se interpretaron con carácter general y absoluto, lo que provocó que Hynek fuese objeto de todo tipo de burlas y críticas en los medios de comunicación al no ajustarse de ninguna forma esta explicación a las descripciones de los testigos. Al margen del descrédito cosechado ante la opinión pública por las Fuerzas Aéreas y su asesor, estos hechos contribuyeron a que Hynek reorientara su inicial postura escéptica hacia una aceptación de la realidad del fenómeno OVNI. [MM] GENERACIONES DE UFÓLOGOS Divisiones cronológicas que en España suelen hacerse respecto a los ufólogos. Así, se habla de “primera generación” en relación a los investigadores que se iniciaron en la Ufología en los años cincuenta (Buelta, Lleget, Ribera, Rey Brea), y de “segunda generación” refiriéndose a quienes lo hicieron durante los sesenta (Arcas, Ares, Ballester, Redón). La “tercera generación” comprendería a los ufólogos activos desde la década de los setenta (Benítez, Cabria, González, Plana); aunque en ocasiones se obvían y dicha etiqueta se reserva para los aparecidos a finales de los ochenta y en los noventa (Campo, Carballal, Montejo, Sierra), GEIFO (Grupo Español de Investigación del Fenómeno OVNI). Asociación ufológica, creada en Cádiz en 1981 y legalizada en 1985, que tiene como ámbito de acción territorial la Comunidad Autónoma de Andalucía. Su fundador y presidente es Ángel Rodríguez Álvarez. La actividad 101 Geónida que otros autores con mayor rigor denominan “cuarta generación”. [35, 55] [MM] información completa de fuentes y sus autores. Su director, Fouéré, prefería los artículos en profundidad, dudando de la idoneidad de las herramientas estadísticas como útil imprescindible para comprender la casuística OVNI. Se incluían también trabajos sobre metodología, avisando ya sobre la influencia que de hecho podía tener el investigador y su cultura ufológica en la recopilación de los testimonios. Solicitaba igualmente una investigación seria, sin apriorismos ni posiciones de partida, considerando la difundida HET como exclusivamente una hipótesis más. Phénomènes Spatiaux editó dos números extraordinarios durante su existencia: Objets Volants Non Identifiés. Le plus grand problème scientifique de notre temps? (1969), del físico e investigador norteamericano James E. McDonald, y una completa tipología de humanoides preparada por el brasileño Jader U. Pereira: Les Extraterrestres (1974). GEÓNIDA Sinónimo, poco intraterrestre. usado, de GEPA (Groupement d’Etude de Phénomènes Aériens). Entidad francesa de investigación ufológica fundada en 1962 por René Hardy. Se trataba de una escisión de la Comission Internationale d’Enquêtes Ouranos, el primer centro de estudios francés, creado en 1951 por Marc Thirouin. Los entonces miembros de Ouranos Hardy, Fouéré y otros estimaban que faltaba competencia científica y había escasa seriedad en las reuniones de su equipo directivo, lo que causó continuas crisis entre 1961 y 1962. La enfermedad de Thirouin y su obligada ausencia de París dieron como resultado la creación del GEPA y la integración en su seno del colectivo más serio de estudiosos de Ouranos. El GEPA supo rodearse de científicos y técnicos que asesoraron muy positivamente al propio René Fouéré, hombre culto y de gran probidad intelectual, fallecido el 14 de enero de 1990. [ JA] René Fouéré se convertiría, hasta poco antes de su fallecimiento, en el animador de sus labores, centradas en la publicación Phénomènes Spatiaux, de la que llegaron a aparecer 51 números entre 1964 y 1977. La estructura de la revista, que comenzó a editarse en versión ciclostilada (seis números) como “Boletín del GEPA” hasta que bajo la presidencia del general Lionel Max Chassin (1964-1970) cambió de nombre y de formato, presentaba sus contenidos con una orientación seria, mediante informes bien detallados e GEPAN (Groupe d’Etudes des Phénomènes Aérospatiaux Non Identifiés). Organismo oficial francés dedicado a la investigación del fenómeno OVNI. Fue creado en el seno del CNES (Centre National d’Etudes Spatiales) el primero de mayo de 1977, como un pequeño negociado formado por su primer director, el ingeniero aeronáutico Claude Poher, y una secretaria; si bien contaba igualmente con un grupo de consejeros científicos que asesoraban y supervisaban su 102 Globos sonda labor. El GEPAN recibía informes de la Gendarmería, la Fuerza Aérea y Aviación Civil, a partir de los cuales analizaba los casos y emitía una Note Technique, de las que se publicaron un total de 18, la última de ellas en 1983. Bajo la dirección de Poher se puso especial énfasis en la elaboración de una metodología rigurosa y objetiva para el estudio de los OVNIs, también expuesta en diversas notes techniques.Tras la dimisión de éste en octubre de 1978, la dirección fue confiada al experto en estadística Alain Esterlé, quien hasta su substitución por JeanJacques Velasco en septiembre de 1983 promovió una intensa actividad social y editorial gracias al incremento de la dotación presupuestaria, que incluso permitió contar con diez empleados trabajando a jornada completa. En noviembre de 1988 el GEPAN cambió su nombre por SEPRA (Service d’Expertise des Phénomènes de Rentrée Atmosphérique) y fue ubicado dentro del departamento del CNES llamado ESO (Environnement Spatial Terrestre). La plantilla se vio reducida a su director y una secretaria y sus objetivos fueron orientados hacia el seguimiento de la reentrada de satélites en la atmósfera y la elaboración de informes sobre fenómenos aéreos no identificados, aunque ya no con el carácter de exclusividad de antaño. En los últimos tiempos se ha acusado a Velasco de seguir una línea oscurantista que ha convertido la organización en una mera excusa para el resto de las administraciones francesas, que remiten por sistema al SEPRA cualquier consulta ufológica que se les formula. proponiendo la creación de un “centro europeo para la observación de OVNIs” (2 de diciembre de 1993). La moción sugerida aconsejaba a tales efectos aumentar el ámbito de actuaciones del SEPRA a todo el territorio de la Unión Europea, aunque finalmente dicha propuesta fue apartada del orden del día de la eurocámara y no volvió ya a ser planteada. En septiembre de 2005 el SEPRA fue substituído por el GEIPAN (Groupe d'Etude et d'Information sur les PAN): https://geipan.fr/[ JA, MM] GLOBOS SONDA Un globo sonda consta de una bolsa fabricada con material diverso (papel, tejido o plástico) llena de un gas más ligero que el aire para que pueda elevarse, y cuya finalidad suele ser transportar una carga, generalmente instrumentos científicos de medida. El globo estalla cuando alcanza una determinada altitud, por lo que suele situarse entre el globo y la carga un paracaídas que frene el descenso de ésta preservándola; para facilitar su localización, ambos suelen tener colores llamativos o ir cubiertos de sustancias reflectantes. Debe tenerse también en cuenta que, incluso cuando ha tenido lugar la puesta de sol, a altitudes entre 15.000 y 20.000 metros la superficie del globo continúa recibiendo y reflejando la luz solar. Todos los archivos de grupos dedicados a la investigación OVNI contienen casos sobre la observación de globos sonda confundidos con OVNIs; suelen ser casos gratificantes, dado que permiten concluir la encuesta como caso resuelto. A consecuencia del impacto que tuvo sobre la opinión pública la oleada belga de 1989-91, la Comisión de Energía, Investigación y Tecnología del Parlamento Europeo elaboró a instancia de los representantes belgas un informe Las observaciones de globos sonda, debido a que en ocasiones duran varias horas, propician que sean contempladas por 103 Green Bank, ecuación de GREEN BANK, ecuación de numerosas personas (incluso de localidades diferentes), y permiten asimismo alertar a los investigadores, quienes posteriormente pueden confrontar sus impresiones personales con otros testigos y constatar la propensión de éstos a percibir en ocasiones detalles del objeto contemplado (ventanas, luces e incluso figuras antropomórficas) allí donde evidentemente no existen, lo cual apoyaría el criterio de quienes mantienen cómo la proclividad del testigo por “ver” lo que “cree que tiene que ver” desvirtúa numerosos episodios. Nombre por el que también se conoce la ecuación de Drake (v. Bioastronomía), por encontrarse reunidos sus autores en ese observatorio. GREENWOOD, Barry Véase Just Cause y Majestic-12. GRISES La innumerable casuística sobre este tipo de confusiones se inicia ya en la Segunda Guerra Mundial, cuando los alemanes lanzaron durante el bombardeo de Europa por los Aliados numerosos globos como contramedidas electrónicas mediante las cuales confundir a los bombardeos guía (denominados “pathfinder”), encargados de señalar el objetivo a las grandes formaciones de bombarderos que les seguían (de forma similar a la “Operación Window” de los Aliados lanzando tiras de papel de estaño para interferir los métodos de detección a distancia de los alemanes); lo que pudo ser motivo de observaciones de posibles FooFighters. Según los defensores de las abducciones, se trataría de los humanoides más comúnmente implicados en estos incidentes. Se les supone un color de piel grisáceo (de ahí el nombre), una altura de 1 a 1,25 m., gran cabeza con enormes ojos negros y unas intenciones más bien perversas (v. Matrix, informe). A menudo se apunta el sistema de Zeta Reticuli como su lugar de origen (v. Hill, caso). Constituyen uno de los más infantiles delirios de la ufología americana contemporánea. [MM] GROOM LAKE Base militar estadounidense situada a 120 millas al noroeste de Las Vegas (Nevada) al borde del lago seco del mismo nombre, aunque no aparezca en los actuales mapas oficiales. Es más conocida como Área 51. Otro caso OVNI, el de Roswell, es uno de los que ha mantenido siempre una tenaz controversia respecto a la naturaleza de los restos que allí se recogieron. Los defensores del globo sonda aportan dos posibilidades: la de los globos lanzados por los japoneses durante la Guerra Mundial, y la que lo relaciona con el Proyecto Mogul. [164, 261][AP] GRUDGE, proyecto Nombre que recibió el proyecto Sign a partir de febrero de 1949. Véase Sign y Grudge, proyectos. 104 Guasp Carrascosa, Miguel GSW GUIJARRO TRIADÓ, Josep (Ground Saucer Watch). Grupo norteamericano, creado en 1957, que destacó internacionalmente en los años setenta por su especialización en técnicas de análisis fotográfico. (n. Terrassa, 1967). Periodista e investigador de la llamada cuarta generación de ufólogos. Siendo muy joven funda la Unión Autónoma de Investigadores Parapsicológicos (1983), y a partir del Primer Simposio Nacional de Ufología (Barcelona, 1988) comienza a especializarse en esta materia. Con Javier Sierra inició en 1990 el proyecto Diana, centrado en la investigación de las experiencias de visitantes de dormitorio. Ha presentado diversos programas en la televisión local de Terrassa, conduce programas de radio y ha colaborado en la elaboración de la enciclopedia Más Allá de los OVNIs (1992). En 1994 publicó su primer libro: Infiltrados: seres de otras dimensiones entre nosotros (Sangrilá, Barcelona). En la actualidad es el responsable de la seción de OVNIs de la revista Más Allá de la ciencia. GUASP CARRASCOSA, Miguel (n. Valencia, 1953). Licenciado en Ciencias Físicas por la Universidad de Valencia. Ha sido colaborador honorífico del Departamento de Mecánica Celeste y Astronomía de la misma universidad y actualmente es físico adjunto del Servicio de Protección Radiológica de la Generalitat Valenciana. Integrante del Ceoni de Valencia (1968-1972) y miembro activo de la revista DATA-NET (EE.UU.) hasta 1972. Colaboró con el Institute of Parascience y la Spiritualist Association of Great Britain para el simposium sobre paraciencias celebrado en Cambridge en junio de 1974. Asesor-editor de Upiar, junto con Pedro Redón del CEI dio cuerpo al CATIB (Catálogo Ibérico de casos OVNI). Es coautor, con Ballester Olmos, de Los OVNIS y la Ciencia (1981 y 89). Su principal contribución a la Ufología fue su Teoría de procesos de los Ovnis (ed. del autor, Valencia, 1973) y la serie de artículos en Stendek que la presentaron. Prácticamente retirado del tema desde principios de los ochenta, su último artículo ha sido “Efectos radiofisiológicos presuntamente vinculados a observaciones de Ovnis” (Cuadernos de Ufología, nº 13, 2ª época, 1992). Opinión sobre el fenómeno (extraída de un artículo de Papers d’OVNIS nº 35-36, 1996): “El denominado fenómeno OVNI es, en realidad, un montón de «fenómenos» juntos y, consecuentemente, no existe una sola explicación que se amolde a la totalidad de esta compleja materia. Resulta evidente que fenómenos atmosféricos, astronómicos, meteorológicos e incluso aeronáuticos pueden explicar una gran parte de las manifestaciones que se producen alrededor de nuestro planeta, pero ¿hay alguna que sugiera la intervención de un fenómeno inescrutable? Desde mi punto de vista sí, y es a estos NO IDENTIFICADOS (que no necesariamente objetos) a los que creo debemos de centrar nuestra atención.” DTU 105 Haines, Richard Foster H HAINES, Richard Foster tecnología, las ciencias sociales y la religión. El grueso de las pruebas parece apuntar hacia una fuente de avanzada inteligencia. Sin embargo, no está todavía claro dónde podría tener su origen tal inteligencia. Es probable que la ciencia tradicional no sea capaz de explicar adecuadamente el fenómeno en todas sus dimensiones. De todas formas, se requiere un enfoque conjunto multidimensional y multidisciplinar que implique a todas las naciones de la Tierra.” (n. Seattle, 1937). Ufólogo estadounidense, doctor en psicología experimental y fisiología por la Michigan State University. Entre 1964 y 1988 trabajó en el Ames Research Center de la NASA, del que llegó a ser jefe de la Oficina de Factores Humanos, y en la actualidad ejerce como consultor en el área de iluminación. Su labor en el campo ufológico se ha centrado en el análisis fotográfico, los aspectos psicológicos de los avistamientos y las observaciones de pilotos. Es autor, entre otros libros, del celebrado Observing UFOs: An Investigative Handbook (Nelson Hall, Chicago, 1980), uno de los mejores manuales para la investigación del fenómeno, y editor de UFO Phenomena and the Behavioral Scientist (The Scarecrow Press, Metuchen, 1979), una compilación de textos sobre la sociología y la psicología de los OVNIs. Su interés por los avistamientos desde aeronaves se ha plasmado en Melbourne Episode-Case Study of a Missing Pilot y Advanced Aerial Devices Reported During the Korean War (LDA Press, Los Altos, 1987 y 1990, respectivamente), así como en la confección del Aircat, catálogo mundial de observaciones de pilotos, con 3.600 entradas registradas entre 1923 y 1991. HENDRY, Allan (n. 1950). Astrónomo estadounidense y una de las figuras más destacadas de la ufología de los años setenta. A mediados de esa década ingresó en el Cufos como editor del Iur (cuyo primer número lanzó en noviembre de 1976) e investigador con dedicación exclusiva, empeños en los que despuntó por su orientación racionalista y su preocupación por la metodología. Sus pesquisas ufológicas pronto le hicieron ver el carácter banal de la inmensa mayoría de los avistamientos y la necesidad de desarrollar nuevos métodos para el estudio del fenómeno que tuviesen en cuenta este hecho. Estas ideas, expuestas en su libro The UFO Handbook (Doubleday and Co., New York, 1979) resultaban insólitas en el contexto norteamericano de la época, en el que Hendry se convirtió en una figura incomprendida a medio camino de la ufología tradicional y del escepticismo que siempre rechazó. En febrero de 1981, los Opinión sobre el fenómeno (extraída de Jerome Clark, 1990, UFOs in the 1980s):”El fenómeno OVNI representa uno de los desafíos verdaderamente importantes de nuestro tiempo y promete brindar numerosos avances para la ciencia, la 106 Hessdalen, proyecto XXX problemas económicos del CUFOS obligaron a prescindir de sus servicios, y para 1982 Hendry, desengañado, había abandonado ya completamente la Ufología. colores: en ocasiones parecían una bala con la punta hacia abajo o un balón de color blanco o blanco-amarillento, con una luz roja en la parte superior o inferior. Más raramente eran multicolores: rojo, verde, azul, amarillo, etc. En otras ocasiones eran varias las luces observadas juntas, guardando las distancias, a menudo dos amarillas o blancas con una roja al frente. Eran observadas preferentemente al atardecer o en horas nocturnas. A pesar de los numerosos avistamientos, las autoridades locales no parecían mostrar demasiado interés por tan curioso fenómeno, y fueron dos grupos de investigación ufológica los que dieron los primeros pasos para aclarar su origen: el UFO-Norway y el UFO-Sweden. En ellos destacan los ufólogos Erling Strand, Leif Havik y Hâkan Ekstrand, que pusieron en marcha el Proyecto Hessdalen a partir del 3 de junio de 1983. El 27 de agosto fue presentado al III congreso internacional del BUFORA, en Gran Bretaña. Durante el otoño de ese mismo año se llevan a cabo los preparativos, dándolo a conocer a los habitantes del valle, que reportaron múltiples observaciones. Por fin, se lleva a cabo una investigación de campo de estas misteriosas luces del 21 de enero al 26 de febrero de 1984, apoyados por el Ejército noruego y las universidades de Oslo y Bergen. El propio profesor y ufólogo Josef Allen Hynek visitó las instalaciones del proyecto en su segunda fase, proponiéndoles organizar un grupo internacional formado por científicos de prestigio que no pudo materializarse debido a la muerte de Hynek. Opinión sobre el fenómeno (tomada de Ronald D. Story (ed.), 1980: The Encyclopedia of UFOs): ”Puesto que los últimos treinta años de investigación ufológica han constituido una frustración científica, mi perenne esperanza ha sido explotar cualquier técnica o sistema efectivo que pudiera apoyar el testimonio humano a estos efectos. En el mejor de los casos, los medios habituales (...) sólo han conducido a la ambigüedad. Si los investigadores quieren ahondar en el potencial científico que representan los casos radar-visuales, los incidentes con huellas físicas, o la base física (si la hay) de los sucesos con humanoides, será necesario idear nuevos procedimientos para sostener los relatos de los partidarios de los OVNIs. De lo contrario, nos enfrentaremos a otros treinta años de promesas y frustraciones intentando trazar la distinción entre los estímulos OVNI denunciados y la creciente mitología ufológica.” HESSDALEN, proyecto Hessdalen en un valle noruego situado en el centro del país en el que a partir de diciembre de 1981 sus escasos habitantes (no llegaban a 200) comenzaron a observar numerosas luces misteriosas. Algunas eran avistadas incluso hasta tres o cuatro veces al día. Podían permanecer estáticas durante más de una hora o moverse lentamente hasta pararse bruscamente. Se observaban en cualquier lugar, en el tejado de las casas, en el campo, en las alturas o en las faldas de las montañas. Presentaban diferentes formas y Según consta en los informes que los citados grupos han dado a conocer, el fenómeno reaccionaba emitiendo destellos cuando se le enfocaba con un rayo láser y podía ser detectado en radar cuando no era visible a simple vista. Las trazas de radar 107 HET HILL, caso indicaban velocidades comprendidas entre los 0 y los 30.000 km./h. Aunque no se pudo establecer una correlación precisa entre ambos hechos, parece ser que el fluido eléctrico se debilitaba ante el paso de alguna de las misteriosas luces. Se recogieron más de 180 informes de luces extrañas, aunque solo 4 fueron consideradas de alta extrañeza y elevada calidad de información. Algunos de los experimentos llevados a cabo con el radar mostraban reflexión similar a la de los objetos sólidos. No se obtuvieron registros con el contador Geiger ni con el sismógrafo, pero aun así se han propuesto diferentes hipótesis explicativas, sin confirmación científica definitiva: bolas de plasma, luces de terremoto, fluctuaciones en los campos magnéticos, etc. La noche del 19 de Septiembre de 1961, el matrimonio racial mixto formado por Barney y Betty Hill regresaban en automóvil de unas cortas vacaciones en Canadá. Notaron una extraña luz que los seguía y que acabaría convirtiéndose en un objeto con dos hileras de ventanillas iluminadas donde, observando a través de unos prismáticos, Barney Hill pudo vislumbrar unas figuras con uniformes negros que los observaban. Salieron huyendo en el coche por la desierta carretera pero escucharon un extraño sonido rítmico y empezaron a sentirse amodorrados y somnolientos. Los despertó de nuevo el extraño sonido y se encontraron conduciendo unos 50 kms más adelante. Después de aquel encuentro cercano Betty empezó a tener gran curiosidad por el tema OVNI, leyó algunos libros y acabó poniéndose en contacto con la base aérea más cercana y con el grupo ufológico NICAP. Entonces tuvo varias pesadillas en noches sucesivas referidas a su encuentro con el OVNI donde ambos eran capturados y examinados dentro del mismo por unos extraños hombrecillos. Tras la visita de algunos investigadores del NICAP intentando reconstruir lo sucedido, se dieron cuenta de que habían tardado unas dos horas más de lo previsto y fue entonces cuando se sugirió la hipnosis, pero sólo después de más de dos años el hipnoterapeuta Dr. Benjamin Simon pudo realizar varias regresiones con ambos pacientes. Una segunda campaña de investigación fue llevada a cabo en 1985 con mayor apoyo oficial. Sin embargo, los resultados obtenidos no fueron concluyentes ya que se observó una cantidad mucho menor de luces que en el año anterior; además el tiempo fue malo y no fue posible realizar investigaciones de campo en el mismo número que en 1984. Los responsables del proyecto han afirmado que se encuentran ante algo desconocido y es necesario desarrollar investigaciones que permitan entrever la naturaleza de las luces del Valle de Hessdalen. Mientras, el misterio continúa. En Internet: http://www.hessdalen.org [241][RC] Esas entrevistas constituyeron el primer caso conocido de lo que posteriormente se llamó abducción. Según lo revelado bajo hipnosis (en línea con las posteriores pesadillas de Betty), ambos se habían visto obligados a detener el vehículo frente a un HET Siglas de Hipótesis extraterrestre. 108 Hill, caso grupo de seres con uniformes situados en medio de la carretera, quienes a continuación los condujeron al interior del OVNI, donde los examinaron en cuartos distintos. Betty afirmó incluso haber pasado por una extraña prueba de embarazo a la que seguiría una breve y absurda charla (en inglés) con quien parecía ser el jefe, durante la cual éste le enseñó un libro y una especie de mapa estelar que se convertiría en el elemento que mayor credibilidad aportó a todo el caso (v. cuadro). Al terminar los devolvieron a su automóvil y les recomendaron que olvidasen todo. La versión de Barney Hill era mucho más simple y el propio Dr. Simon pensaba que EL MAPA ESTELAR Betty Hill dibujó bajo hipnosis un croquis del mapa estelar que le habría mostrado el jefe de los seres que los secuestraron. Consistía en unos 26 puntos, 15 de los cuales uniría luego mediante líneas gruesas que representaban rutas comerciales regulares y líneas punteadas para las expediciones, pero sin poder identificar ninguno de ellos con una estrella conocida, ni siquiera nuestro Sol. De hecho, la propia Betty Hill llegó después a encajarlos con un croquis estelar de la constelación de Pegaso donde por aquella época se detectó uno de los primeros quásares conocidos (CTA-102). Pero el gran espaldarazo que parecía suponer un impresionante respaldo científico al mapa (y por consiguiente, a la totalidad del relato) llegó en 1974 cuando tras varios años de trabajo y empleando datos astronómicos conocidos algunos sólo con posterioridad a la publicación del mapa, la profesora Marjorie Fish logró pacientemente elaborar un modelo tridimensional de las estrellas con características habitables situadas en un radio de 33 años-luz en torno al Sol (unas 256). Y aseguró haber identificado entre ellas todas las 15 estrellas unidas por rutas espaciales. El trabajo se publicó en el número de Diciembre 1974 de la revista Astronomy. Según su interpretación los seres procederían de las estrellas Zeta 1 y Zeta 2 Retículi. Ninguna otra interpretación incluye a todas las estrellas de características similares a la nuestra sin excluir ninguna; algunos cálculos estadísticos hablan de una posibilidad entre mil y otros la elevan mucho más. Los escépticos apuntan distintas críticas. Según Carl Sagan, la única razón por la que parece existir cierto parecido entre el mapa y los cálculos de Fish se debe a la forma en que se han trazado las líneas de unión. Si se dibujan sólo los dos grupos de puntos, sin líneas que ayuden a la vista, el parecido desaparece. De hecho, han existido otras interpretaciones posteriores (la más destacada fue realizada por el astrónomo aficionado Charles W. Atterberg) lo que demuestra que existen muchos grupos de estrellas que pueden encajar con el croquis de Betty Hill. Incluso algunos han encontrado paralelismos entre el mismo y ¡el mapa local de carreteras de la zona de New Hampshire donde vivian los Hill!. Además existen muchas dudas sobre la precisión y exactitud (a nivel de coordenadas estelares) que pueden tener los datos extraídos bajo hipnosis, respecto de un mapa que sólo pudo ser visto por la testigo durante pocos segundos. La puntilla definitiva a la propuesta de la Sra. Fish fue el descubrimiento de que la estrella Zeta Retículi era binaria. Es decir, que debería haber sido excluida de la selección inicial al no ofrecer características habitables. 109 Hipnosis regresiva HIPNOSIS REGRESIVA podría haber absorbido muchas de sus ideas oyendo a Betty contar sus sueños en los meses posteriores al encuentro. Existían además flagrantes contradicciones como cuando, en una gráfica comparación, Barney Hill equiparó la nariz de los seres con la del famoso -y narizotas- actor americano de la época, Jimmy Durante, mientras Betty apenas si mencionaba dos aberturas verticales. Técnica consistente en aplicar el procedimiento hipnótico en determinados sujetos con el fin de hacer aflorar recuerdos de sucesos olvidados por la mente consciente, a la vez que se va retrocediendo en el tiempo, pudiendo llegar hasta la infancia. También se ha utilizado este sistema en investigaciones sobre supuestas “vidas anteriores”. Existen bastantes elementos dudosos en este caso. Algunos escépticos han rastreado muchos de los detalles del relato hasta los libros leidos por Betty Hill tras el incidente, así como hasta algunos episodios televisivos de ciencia ficción de la época. Parece demostrado que el punto luminoso observado al inicio del suceso, considerado por Betty Hill como un OVNI, era en realidad el planeta Júpiter. También existen serias dudas sobre los horarios y el tiempo transcurrido, que podría explicarse suficientemente por todas las paradas y revueltas por carreteras secundarias a baja velocidad. Son muchos los problemas de la hipnosis regresiva y la opinión profesional del propio Dr. Simon fue que todo había sido un sueño. En Ufología, la técnica de la regresión hipnótica se ha utilizado, fundamentalmente, en investigaciones sobre incidentes de abducción, ya desde el caso Hill, tal como relata John Fuller en su libro El viaje interrumpido (1970), pero no se popularizó hasta los años ochenta, en que se multiplicaron los informes de secuestros por alienígenas y principalmente a raíz de las publicaciones de Budd Hopkins (con sus investigaciones sobre tiempo perdido, fenómeno muy frecuente en los contactos OVNI) y W. Strieber. En España se han dado varios casos, los más importantes los de Julio F. y Próspera Muñoz. En ambos se utilizó este tipo de técnica por parte de algunos investigadores. Para complicar (o aclarar) aún más el asunto, en los años posteriores al fallecimiento de su marido (en 1969) Betty Hill confesó haber experimentado numerosos sucesos paranormales a lo largo de su vida, y llegó a localizar una zona de aterrizaje OVNI cerca de su domicilio, donde los observa frecuentemente. Quienes la han acompañado sólo han visto luces identificables. La historia original alcanzó gran difusión en un libro (Fuller 1966) que sería llevado a la televisión en 1975, generando a su vez una oleada de relatos similares. [92, 133, 221, 224][LG] Los resultados al emplear este sistema eran, aparentemente, muy espectaculares, pues los sujetos relataban vivencias de todo tipo: secuestros, exámenes físicos, contactos con diferentes tipos de seres, visitas en la infancia, etc., que parecían estar ocultos en la memoria. Sin embargo, es preciso hacer notar la poca fiabilidad de este sistema (y de la hipnosis en general) para dotar de credibilidad a las narraciones así obtenidas. Ya la propia definición de la hipnosis la considera un fenómeno psicofisiológico caracterizado por una fuerte sugestibilidad del sujeto. En la literatura sobre hipnosis está bien descrito cómo actúa sobre fenómenos 110 Hipótesis sobre los OVNIs vegetativos y sobre los psíquicos (ilusiones, alucinaciones, desdoblamiento, experiencias “fuera del cuerpo”, etc.), a la vez que es frecuente la asociación con fenómenos parapsicológicos como telepatía y clarividencia (Milan Ryzl: Hipnosis y PES, 1970). ufólogos. Hipótesis del arma secreta. Se conoce por arma secreta aquella cuya existencia se mantiene oculta, ya sea como elemento de disuasión frente a un potencial enemigo, o porque sus características la convierten en algo excepcional, capaz de superar a otras armas convencionales destinadas al mismo fin, constituyendo una baza estratégica o táctica en un posible conflicto bélico. La circunstancia de mantener su existencia en secreto determina que un eventual testigo no pueda asociarla o identificarla con algo habitual o conocido, sobre todo si posee una forma inusual y presenta en consecuencia una imagen rara. Dado que los OVNIs suelen desplazarse en el medio aéreo, suelen ser prototipos de aviones secretos los más asociados con aquellos. En este contexto, la hipótesis del arma secreta posee dos facetas: a) considerar que los OVNIs son en sí mismos armas secretas, y b) que existen armas secretas que pueden ser tomadas por OVNIs. Pero a consecuencia de la hipnosis, y por su mismo estado de extrema sensibilidad a la sugestión cuando se está en esta situación, el sujeto es muy permeable a la influencia del hipnotizador, implícita o explícita, y a las expectativas de la situación, así como a sus creencias (tanto del hipnotizador como del propio sujeto). Los trabajos de Elisabeth Loftus (1979) y de Ernest Hilgard (1977) sobre el funcionamiento de la memoria demuestran la elaboración de falsos recuerdos, bien por creación del sujeto (confabulación), bien por sugestión externa, de tal forma que el hipnotizador no puede diferenciar entre la memoria real y la creada. También cabría señalar que las personas fácilmente hipnotizables y con facilidad para experimentar regresiones son también las que podrían caber dentro de la denominación de personalidad propensa a la fantasía (v. Hipótesis sobre los OVNIs), que tienden a confundir realidad con ficción, por lo que alentar esta práctica no parece muy prudente. [25, 92, 114, 151][MG] Ya en la observación de Kenneth Arnold (24/6/1947) nos encontramos con posibles prototipos secretos, y así lo manifestaron entonces el Secretario de la Casa Blanca y un general norteamericano (Cabria, 1993), quienes asociaron las aeronaves de dicha observación a proyectos experimentales. Las aeronaves descritas por Arnold han sido comparadas repetidamente durante los últimos cincuenta años con diferentes modelos de aviones, rozando más o menos acertadamente la verosimilitud, tales como el Chance Vought XFU-5 (conocido por su forma como Flap jack o Flying Pancake), las alas volantes Northrop XB-35 e YXB-49, o el F-84 Thunderjet, y existen documentos militares de la época (estudio de la Air Intelligence Division nº 203, apéndice A, con fecha 10/12/1948) HIPÓTESIS SOBRE LOS OVNIS Las hipótesis explicativas sobre el fenómeno OVNI han sido legión a lo largo de la historia de la Ufología (según Ignacio Darnaude, más de trescientas), por lo que nos limitamos aquí a presentar la reseña de aquellas con mayor trascendencia histórica o con más amplio predicamento entre los 111 Hipótesis sobre los OVNIs CASUÍSTICA En los años que transcurren desde finales de los cuarenta hasta el presente, existen bastantes incidentes atribuibles a prototipos secretos, como los que a continuación se recuerdan: • El caso de William A. Rhodes, que fotografió un objeto volante en Phoenix, Arizona (10/7/1947), también puede corresponder a un prototipo secreto de avión del tipo “ala volante”; en las imágenes se aprecia lo que parecen una cabina y derivas marginales en el extremo de las alas. Las fotos aparecieron en la prensa local y los originales fueron prácticamente “confiscados” por militares. • Otro posible prototipo secreto es el fotografiado por el periodista Bruno Ghibaudi, en la costa italiana del Adriático cerca de Pescara (27/4/1961), volando a poca altura sobre el mar procedente del sudeste, aparentemente de unos ocho a diez metros de longitud, que guarda gran semejanza con los “aviones de superficie” Ekranoplan, desarrollados con fines militares por ingenieros soviéticos desde el segundo lustro de los años cincuenta. • El caso de Kecksburg, Pensilvania (9/12/1965), es parecido; descrito como una bola incandescente que atravesó el cielo de varios estados durante su trayectoria, es recordado por los testigos próximos al lugar del impacto en dicha localidad como una “estrella de cuatro puntas” (similar a un ala volante Horten), siendo inmediatamente acordonada la zona por fuerzas militares especiales que retiraron los restos. que los comparan con el ala volante Horten Ho.IX (Greenwood, 1996) (v. Arnold, caso). De igual forma, la revisión retrospectiva de noticias de prensa en busca de posibles observaciones OVNI condujo al hallazgo de la denominada oleada de cohetes fantasma de 1946 sobre Escandinavia, atribuida en aquellas fechas a pruebas soviéticas con equipos capturados a los alemanes tras la Segunda Guerra Mundial, dado que procedían del este en dirección oeste. primer prototipo de los cuales voló en el lago seco Groom (18/6/1981). A finales de 1983 fueron declarados operativos y actuaron desde Tonopah en la Base de Nellis, Nevada (conocida como Área 51). Los rumores sobre el proyecto hicieron que el Departamento de Defensa admitiera su existencia diez años después de iniciarse, y tras cinco años de actividad de los aviones como grupo de combate organizado (Grupo Táctico 4450) en los EE.UU. y en Gran Bretaña (Base aérea de Louisiana). El desarrollo del B-2 Spirit es similar, si bien la existencia del prototipo que sirvió de banco de pruebas (denominado Tacit Blue) no se hizo pública hasta abril de 1996; sólo fue volado por cinco pilotos y se retiró del servicio en 1986, habiéndose mantenido en secreto durante todos estos años (aunque su forma se parece más al YF-23, aún en desarrollo, que al B2 Spirit). Por su parte, la existencia de los actuales aviones stealth (furtivos) apoya la tesis de los prototipos secretos confundidos con OVNIs. El F-117 Nighthawk surge de un proyecto secreto convocado en 1975, al que concurrieron Lockheed y Northrop, y que dio origen a su predecesor el Have Blue, cuyo éxito en las pruebas de vuelo (en 1976) llevó al desarrollo del programa “Senior Trend” (1977) y a la fabricación de los F-117A, el Durante los diez últimos años, otros 112 Hipótesis sobre los OVNIs países han emprendido también programas de aviones furtivos: el grupo ruso Mig-Mapo con el proyecto 1.42 y Japón por medio de su Instituto Técnico de Investigación y Desarrollo diseñando el caza FI-X. OVNI se encuentra algún tipo de inteligencia, lo difícil es determinar si es o no exterior al propio testigo. El gran atractivo de la HET es su omnipotencia; con las elucubraciones adicionales necesarias es capaz de explicarlo todo, aunque sea apelando a la famosa tercera Ley de Arthur C. Clarke: “cualquier tecnología superior resultará indistinguible de la magia”. Pero, desde una perspectiva científica, ése es a la vez su punto más débil: una hipótesis que no es falsable ni predictiva es simplemente inútil. La necesidad de mantener un secreto de índole militar da lugar a situaciones como la que tuvo lugar cerca de Bakersfield (California), cuando un F-117 (aún proyecto secreto) se estrelló, provocando un incendio que tardó dieciséis horas en extinguirse y que obligó a acordonar la zona, que fue declarada área de seguridad nacional durante tres semanas, al cabo de las cuales los medios de comunicación pudieron contemplar los restos de un veterano F-101 Voodoo cuidadosamente esparcidos (Pinillos, 1995). En definitiva, no es desdeñable suponer que parte de las observaciones OVNI corresponden en realidad a la observación fortuita de prototipos secretos. Mención aparte requieren las aeronaves discoidales (v. prototipos experimentales) que se han diseñado con carácter experimental y que hemos de distinguir de los aviones dotados de ala circular o de aviones de exploración dirigidos por control remoto. [55, 104, 182][AP] Es oportuno recordar que la HET no fue la primera. En una encuesta realizada en agosto de 1947 ...ni se menciona. Por aquel entonces, las opciones más plausibles para los ciudadanos eran las alucinaciones o espejismos (29%), las armas secretas americanas (15%), los fraudes (10%) y especialmente la ignorancia (33%). Fue el mayor Donald E. Keyhoe quien en un famoso artículo publicado en 1950 llamó la atención del gran público sobre esta posibilidad, y gracias a su persistencia en años posteriores logró convertirla en casi la única explicación admisible a nivel popular. Junto a él, autores como Frank Edwards o los esposos Lorenzen (v. Apro) creyeron leer en los datos disponibles por las sucesivas oleadas de aquella década (1952, 1954, 1958, etc.) un evidente programa por etapas de reconocimiento y espionaje de nuestro planeta, previo quizá a una invasión (la idea de la hostilidad de los OVNIs reaparece periódicamente, aunque el total de muertes humanas atribuibles a los mismos, incluso aceptando sin pestañear todos los casos que se mencionan en la literatura ufológica, es inferior a las muertes censadas por ataques de ovejas; y, desde luego, siempre menor que el número de suicidios motivados por la mera creencia en esos salvadores Hipótesis extraterrestre. La idea de que los OVNIs son naves tripuladas por seres inteligentes de origen extraterrestre es, con mucho, la postura más popular. Sin embargo, merece la pena recordar a autores como Trevor Constable (1958) e Ivan T. Sanderson (1967) que, admitiendo su origen ajeno a la Tierra, consideraban a los OVNIs como simples animales atmosféricos. Aunque estas ideas podían ser defendibles en los primeros años, en cuanto empezaron a proliferar los aterrizajes y los casos de humanoides fueron pronto descartadas. Resulta evidente que detrás del fenómeno 113 Hipótesis sobre los OVNIs extraterrestres). Aviones fantasmas, Cohetes fantasmas, Nave aérea de 1896-97). Y se empezaron a señalar reveladores paralelismos con otras parcelas del conocimiento humano (v. Apariciones marianas). En aquellos años de pleno desarrollismo y de total optimismo tecnológico, las extrapolaciones más simplistas daban por descontado el establecimiento de bases terrestres en la Luna y Marte antes del año 2000, así que nadie podía extrañarse de que otros seres ya hubieran llegado hasta aquí. Los primeros sospechosos fueron precisamente los planetas más cercanos, Venus y especialmente Marte, pero pronto quedaron descartados ante los avances de la astronáutica terrestre, que proporcionaron descorazonadoras fotografías. Ganímedes, el mayor satélite de Júpiter, todavía se menciona de vez en cuando a nivel popular, aunque la mayoría ha dado ya el salto a distancias interestelares o incluso galácticas. Los años se convirtieron en décadas y el “inminente” contacto definitivo no llegaba. A mediados de la década de los setenta (y quizá vinculado a la crisis enérgetica de aquellos años) un creciente desencanto ante la falta de un contacto definitivo y la ausencia de pruebas innegables llevó a la aparición de explicaciones más complejas como la hipótesis del sistema de control (queriendo justificar las incongruencias en un hipotético plan del propio fenómeno para negarse a sí mismo: Vallée, 1975) o las hipótesis paraufológicas (ante la proliferación de aspectos paranormales en los relatos de los testigos: Clark y Coleman, 1975). También tuvo lugar una reacción contraria y dentro de las propias filas de la Ufología empezaron a aparecer investigadores que cuestionaban la existencia real del fenómeno, principalmente en Europa (Scornaux, 1976; Monnerie, 1977), aunque también en EE.UU. (Hendry, 1979). También contribuyó a su popularidad el fracaso de otras hipótesis para explicar la persistencia, la apariencia y los comportamientos del fenómeno OVNI. Pero está clara la inutilidad de pretender una respuesta única para algo tan complejo. Así, en el caso de la HET, pronto se hizo evidente lo sospechoso de que las supuestas visitas extraterrestres se hubieran iniciado justo en la misma época en que nosotros los terrícolas dabamos los primeros pasos interplanetarios. Ello llevó a distintos autores (Desmond Leslie, Brinsley Le Poer Trench, Eugenio Danyans, etc.) a bucear en el pasado más o menos reciente a la búsqueda de posibles visitas extraterrestres (v. Astronautas en la Antigüedad, teoría de los). Lo máximo que pudieron obtener fueron algunas sugerentes posibilidades. Otros creyeron ver lo que se llamó el caracter “mimético” del fenómeno, pues en cada época se describían artefactos distintos, un poco por delante de la tecnología terrestre del momento (v. El renacimiento de la HET vino en los años ochenta, asociado a la aparición del novedoso fenómeno de las abducciones, que inmediatamente se vinculó con los OVNIs y sus supuestos tripulantes extraterrestres. Y al mismo tiempo, la falta de pruebas definitivas llevó a muchos ufólogos, principalmente norteamericanos, a centrar su atención en los rumores sobre platillos estrellados. Todo este desarrollo que acabamos de mencionar ha quedado limitado sólo a nivel de los propios ufólogos, la gente en general apenas sí distingue dichos 114 Hipótesis sobre los OVNIs matices. estructural y procedentes de tantísimos puntos de la galaxia (v. Tipologías de humanoides y Tipologías de OVNIs). En palabras de John Keel (1969): “sólo quedan dos opciones: o bien cada objeto fue construido específicamente y utilizado en esa única ocasión, o bien ninguno de los objetos existen realmente como tales”. Las estimaciones más conservadoras (Evans, 1987) hablan de unos 200.000 casos en 40 años, lo que supone unos 5.000 sucesos al año. Pero según otros cálculos (Sagan, 1972), incluso admitiendo un único caso al año y un millón de civilizaciones tecnológicas en nuestra galaxia, ello exigiría 10.000 lanzamientos por civilización y año... sólo la cantidad de metal consumido representaría la masa de más de medio millón de estrellas. Ante estos números, está claro que la supuesta persistencia de los extraterrestres en visitarnos supone un desmesurado interés en nosotros, y ello, aparte de halagar nuestro ego, resulta una contradicción lógica (Sagan, 1972): “si hay muchas, el desarrollo de una civilización como la nuestra debe ser muy común. No llamaríamos la atención. Y si no somos tan normales, entonces no habrá suficientes civilizaciones avanzadas capaces de enviar visitantes”. Aunque siempre es peligroso extrapolar a partir del único ejemplo de vida inteligente que conocemos, la mera constatación de que después de 5.000 millones de años éste siga siendo un tema opinable indica que las probabilidades de que seamos visitados por seres procedentes de otras estrellas no son muy altas (v. más detalles en Bioastronomía). Los grandes problemas de la HET son el espacio y el tiempo. Espacio en el sentido de que la distancia media entre las estrellas es tan enorme que ninguna analogía terrestre permite dar una idea clara al respecto (¿quién puede asimilar en todas sus consecuencias magnitudes como que la segunda estrella más cercana está 60.000 veces más lejos de nosotros que Júpiter?). Y tiempo, en el doble sentido de que los viajes interestelares serían muy, muy largos (con las dificultades económicas, técnicas y emocionales que ello conlleva) y que resulta casi imposible la coincidencia temporal de civilizaciones extraterrestres con un grado similar de desarrollo. Sin embargo, y aunque algunos puedan sorprenderse, ningún científico niega la posibilidad de visitas extraterrestres; la gran objeción es el volumen de tráfico que proclaman los defensores de los OVNIs. Se han llegado a mencionar estimaciones de hasta ¡50 millones de casos! Incluso cuando se admite que en torno al 90% de los informes se refieren a OVIs, también se llega a calcular que sólo conocemos una de cada 38.400 visitas a nuestro planeta (Giraud, 1979) porque muchos incidentes ocurren en zonas deshabitadas donde no existen cauces para su denuncia y publicación. Como decía el Dr. Hynek, es una “embarazosa abundancia”. Resulta inadmisible que lleguen tantos viajeros con tipología tan grotesca en naves de pródiga variedad Porque además, aflora entonces la otra gran dificultad de la HET, el problema del no-contacto (v. Ceti y Seti). Resulta a todas luces absurdo recorrer las enormes distancias interestelares para luego no darse a conocer... pero tampoco pasar precisamente desapercibidos. Si admitimos la realidad objetiva de tantos relatos el fenómeno OVNI se manifiesta como un fenómeno “celoso” que parece seleccionar muy cuidadosamente dónde, cuándo y ante quién aparecer, lo que imposibilita su análisis científico y lo convierte en una cuestión de 115 Hipótesis sobre los OVNIs fe. [65, 80, 98, 112, 124, 127, 132, 165, 211, 222, 253][LG] visos de realidad. Poco antes, Joseph Alexandre Saint-Yves d’Alveydre había publicado La misión de la India en Europa y la cuestión del Mahatma y su solución (1886), donde se cita por primera vez la existencia en Oriente de un reino subterráneo llamado Agharta que está gobernado por “el Rey del Mundo”, salvaguarda espiritual y político de la Humanidad. Ferdinand Ossendowski, en Bestias, hombres, dioses (1923), reiterará, ya en el siglo XX, muchos de los conceptos vertidos por d’Alveydre. Simultáneamente, el pintor Nicolás Roerich recogerá en su periplo de 1924-28 por la India, Mongolia y el Tíbet diversas leyendas sobre el reino de Shamballá (sinónimo de Agharta), divulgadas en su crónica Altai-Himalaya: Diario de viaje (1930) y en obras posteriores. Hipótesis intraterrestre. Esta hipótesis alude a la existencia en el interior de la Tierra de una raza superior dotada de grandes poderes, descendientes de los atlantes o herederos de conocimientos secretos y desconocidos, que les permitirían utilizar como vehículos de transporte lo que nosotros llamamos OVNIs. La creencia en la existencia de seres no humanos habitando el subsuelo de la Tierra es muy antigua y aparece ya en los primeros testimonios escritos que se conocen. Más modernamente, el fundamento literario para el desarrollo de las teorías intraterrestres quedó asentado durante los siglos XVII, XVIII y XIX. En esos años se publicaron, correlativamente, Narración de un viaje desde el polo Ártico al Antártico, a través del centro del Mundo (anónimo, 1723), Lamékis, o los viajes extraordinarios de un egipcio en la Tierra interior (Mouhy, 1727), Nicolás Klim en el subterráneo (Ludwig Baron von Holberg, 1741), y Vida y aventuras de Peter Wilkins (Robert Paltock, 1751). Mediado el siglo XIX aparece la novela de Julio Verne Viaje al centro de la Tierra (1864), y, hacia finales, otras dos novelas que tratan de mundos subterráneos habitados: Lo que verdaderamente hay bajo nuestros pies (Beneath Your Very Boots, 1889) de Charles James Cutcliffe Hyne, y La raza futura (The Coming Race, 1871) de Lord Bulwer Lytton, obra de gran repercusión posterior, pues confiere a los intraterrestres un poder llamado “Vril” que seis años más tarde fue recogido por Helena Petrova Blavatsky (fundadora de la Sociedad Teosófica) en sus libros Isis sin velo (Isis Unveiled, 1877) y La doctrina secreta (The Secret Doctrine, 1888), pero otorgándole Hasta aquí hemos expuesto la génesis de la información conocida sobre el mundo subterráneo. Tras la Segunda Guerra Mundial el interés subsiste y continúan apareciendo libros, unos ilustrativos como el de Sprague de Camp y Ley (1967), y otros tomando partido, como Agharta del estadounidense Robert Ernst Dickhoff (1951), quien menciona una red de túneles que atraviesan todo el orbe comunicando la Antártida con Brasil, éste con el Himalaya, y así sucesivamente. La difusión durante los años setenta de la supuesta existencia de túneles “tallados” en Colombia renovó el interés por el tema, y aparecen títulos que convierten esta hipótesis en una cuestión ligada a una ideología muy concreta y con sectas o sociedades místico-religiosas contemporáneas (Stilo, 1994), tales como La crónica de Akakor (1976) de Karl Brugger, donde se refiere la existencia de colonias nazis en antiguas ciudades subterráneas del Brasil; Reinos en el interior 116 Hipótesis sobre los OVNIs otro apartado. de la Tierra (Kingdoms within Earth, 1985) de Norma Cox, defendiendo tesis sobre la pureza de raza en el Sur de los EE.UU.; o El viajero de Aghartha (1989) de Abel Posse, sobre la búsqueda por los alemanes durante los años treinta y cuarenta de la energía “Vril”. Con la divulgación del fenómeno OVNI a partir de 1947, muchos de sus más místicos creyentes los han considerado mensajeros de estos reinos subterráneos a los que se accedería a través de puertas dimensionales invisibles para los no iniciados, como el cerro Uritorco (Argentina), el monte Shasta (EE.UU.), o las pirámides de Güimar (Canarias) (v. también Tierra hueca, teoría de la). [234, 239][AP, LG] La aparición de estas propuestas (surgidas y fenecidas con los años setenta) responde en buena medida a la incapacidad de la HET clásica para dar adecuada respuesta a la progresiva complejidad del fenómeno OVNI en esa década, que vino marcada sobre todo por la aceptación por parte de la comunidad ufológica de los encuentros cercanos de tercer tipo (v. clasificación de Hynek) y por la proliferación de supuestos aspectos paranormales en los relatos de los avistamientos. La aparición de entidades junto a los OVNIs y la posibilidad de que interactuaran con los testigos no sólo contradecía las concepciones “militaristas” o “exploratorias” hasta entonces vigentes sino que evocaba sugerentes paralelismos con mil y una tradiciones culturales de encuentros con inteligencias no humanas (ángeles, demonios, hadas). En este contexto, la paraufología supuso en primera instancia la recuperación de muchos elementos folkloristas u ocultistas antes no considerados en relación a los OVNIs: la transmogrificación de John Keel, las leyendas celtas de Vallée, las apariciones marianas, etc. En segundo lugar, la apertura hacia la interpretación simbólica de la casuística, que dio pie a que Clark y Coleman (1975) formulasen las llamadas leyes de la paraufología, a saber: 1) el fenómeno OVNI es subjetivo y su contenido de tipo simbólico, y 2) las manifestaciones objetivas no son sino subproductos generados psicocinéticamente a partir de procesos inconscientes. Este último postulado avanza ya el tercer paso dado por las ideas paraufológicas: si la HET y su fisicalismo implícito no solucionan el problema, cabe entonces recurrir a otros planteamientos diferentes; cosa que, Hipótesis paraufológica (parafísica o psíquica). Propiamente no puede hablarse de una sola hipótesis paraufológica, más bien alude este término a un conjunto de explicaciones, muy diversas entre sí, que sólo tendrían en común oponerse a la hipótesis extraterrestre y hacerlo mediante la invocación de elementos no físicos (parapsicológicos, psíquicos o “parafísicos”). El punto de partida indiscutible de todas ellas es la obra de Vallée Pasaporte a Magonia, originalmente publicada en 1969, pero de hecho cuentan con precedentes muy remotos; entre ellos Jung (1958), las tesis sobrenaturales de Meade Layne (Flying Discs. The Ether Ship Mistery and Its Solution, 1950), George Unger (Flying Saucers: Physical and Spiritual Aspects, 1958) y Frank E. Stranges (Danger from the Stars, 1960), el espiritismo de Hermann Oberth (Katechismus der Uranidem, 1966) y otros, e incluso Charles Fort (The Book of the Damned, 1919). Entre estas ideas debe contarse igualmente la hipótesis del sistema de control, la más popular de ellas, que por su complejidad se analiza en 117 Hipótesis sobre los OVNIs tratándose de fenómenos desconocidos, pasa necesariamente por la importación de ideas y conceptos de la Parapsicología. De ahí el nombre de estas tesis y la habitual cohabitación durante los años setenta de ufólogos y parapsicólogos en las mismas asociaciones. sociables). La combinación de todos estos factores de forma asistemática y caótica y la hetereogeneidad -cuando no contradicciónde las diferentes hipótesis elaboradas determinaron la corta vida de esta escuela y su escasa herencia para la Ufología posterior. No obstante, las tesis paraufológicas tuvieron el mérito de romper con el monopolio de la HET y propiciar una sana pluralidad de pareceres que con el tiempo desembocaría en el resurgir de alguna de sus ideas en el seno de la HPS, de la cual constituyen un precedente inmediato. [55, 65, 123, 224, 252][MM] • Frecuentes experiencias “fuera del cuerpo”, apariciones, visiones hipnagógicas e hipnopómpicas, con sueños vívidos y muy realistas. • Dicen tener poderes sobrenaturales o parapsicológicos (telepatía, precognición, etc.). • De niños vivían en un mundo imaginario propio mantenido en secreto. En investigaciones realizadas para determinar si el grupo de población tendente a la fantasía experimenta un número mayor de experiencias de encuentros cercanos (Spanos, 1993), se demuestra que los grupos de alta intensidad de creencia en los OVNIs manifestaban que dichos incidentes estaban relacionados con el sueño o procesos oníricos con mucha mayor frecuencia que los grupos de baja intensidad de creencia. Es decir, entre los creyentes en los OVNIs aquellos con más tendencia a la fantasía eran los que tenían propensión a generar este tipo de experiencias y, sobre todo, éstas se interpretaban como sucesos reales cuando aparecían en situaciones de baja estimulación sensorial (confusión entre imágenes internas y sucesos externos). [MG] Hipótesis de la personalidad propensa a la fantasía. Se describe así a cierto tipo de personas (aproximadamente el 4% de la población) que son -generalmente- muy imaginativas, de forma que fantasean continuamente y experimentan plenamente tales fantasías como reales (Wilson y Barber, 1985). Son excelentes sujetos hipnóticos, sensibles a sugestiones de todo tipo. Todo esto no quiere decir que sean personalidades patológicas, sino todo lo contrario. Hipótesis psicosociológicas. Como alternativa a la hipótesis del origen extraterrestre de los fenómenos OVNI, surgió en Francia, a finales de los años 70, otra clase de explicación de los mismos que pone en duda dicha procedencia. Aunque con precedentes en la obra de C.G. Jung Sobre cosas que se ven en el cielo (1961) y en las hipótesis paraufológicas, el introductor y principal promotor de esta teoría fue Michel Monnerie, a través de sus En diversos estudios se han establecido las características de este tipo de personalidad, distintas a los otros grupos de población. De ellas, las más importantes son: • Personalidad predominante entre médiums, psíquicos y visionarios religiosos. • Plenas facultades mentales (no son psicóticos, sino individuos sanos y 118 Hipótesis sobre los OVNIs creación del mito, al estimular temores y fantasías, realimentar el interés por el problema y contribuir a la formación de creencias y opiniones a favor de la existencia de los ovnis. A veces son, incluso, el fenómeno desencadenante de las observaciones en gran número, como sucede en las llamadas oleadas. libros Et si les OVNI n’existaient pas? (1977) y Le naufrage des extraterrestres (1979); posteriormente secundado por otros investigadores como Jacques Scornaux, Thierry Pinvidic, Claude Maugé, Paolo Toselli, Bertrand MÉheust y otros. El enunciado original desarrollado por Monnerie consiste, en síntesis, en lo siguiente: el mito del origen extraterrestre de los OVNIs está ampliamente aceptado en nuestra cultura. Los testigos perciben un suceso u objeto aéreo extraño, no reconocido por ellos, que tratan de explicar de acuerdo con sus conocimientos. Influenciado por dicho mito OVNI, y a partir de un nivel establecido de emoción, angustia o extrañeza, el inconsciente del observador elabora un escenario ovni donde poder interpretar lo percibido. Por ello, la estructura normativomental o los condicionantes culturales del testigo están estrechamente ligados al marco de referencia de esta manifestación anómala. Consiguientemente, el centro de la atención del investigador pasa del supuesto suceso anómalo al observador, como fuente fiable de datos del acontecimiento. Puesto que la capacidad de percepción sin errores y la fidelidad de la memoria de los testigos es muy dudosa, en la mayoría de los casos de informes OVNI estos quedarían explicados recurriendo a esta causa (distorsiones de la percepción y de la memoria, e interpretación de un estímulo ambiguo bajo el prisma social dominante: v. Transgresión de la realidad, síndrome de). Así, Claude Maugé llega a decir que “la ufología funciona como un sistema de creencias”. En sus formulaciones más modernas, las hipótesis psicosociológicas (actualmente dominantes en la ufología europea) destacan que cuantitativamente la gran mayoría de casos pueden ser explicados de manera convencional, y que cualitativamente incluso los “mejores” casos OVNI terminan teniendo una explicación banal; sin que ese hetereogéneo grueso de la casuística tenga en común nada más que el mito ovni como referente social. El residuo restante, igualmente diverso en sus manifestaciones, presentaría pocos o nulos elementos de originalidad respecto al conjunto principal de incidentes. Siendo esto así, la Ufología no sería una disciplina científica en sí misma; sino, a lo sumo y dado su carácter pluridisciplinar, una forma de referirse al estudio científico de los casos explicables. [55, 12, 165][MG] Hipótesis del sistema de control. Según ha expuesto Jacques Vallée en sus obras Pasaporte a Magonia (1969, trad. esp. de 1972), El Colegio Invisible (1975, trad. de 1981) y Messengers of Deception (1979), el fenómeno OVNI sería en realidad un mecanismo de control social que mediante la manipulaión del inconsciente colectivo pretende dirigir la evolución humana hacia determinadas metas. Se trata de una de las llamadas hipótesis paraufológicas que, pese a su gran carga especulativa, ha cosechado más adeptos entre los ufólogos. A esto habría que añadir el papel y la importancia decisiva de los medios de comunicación, pues constituyen la fuente principal de datos e información para el público y son responsables, en parte, de la Parte del hecho de que la naturaleza 119 Hipótesis sobre los OVNIs contradictoria del fenómeno obedece a un doble y deliberado propósito: por un lado, ocultar su verdadera función y, por otro, impedir la comprensión lógica del problema en beneficio de una percepción puramente metalógica del mismo, cosa que tendría como efecto una serie de modificaciones del acervo imaginario y mítico de la humanidad. Para Vallée, ésta se rige fundamentalmente por esos componentes no racionales, de suerte que controlar la imaginación humana supone dirigir su conducta. Las observaciones de OVNIs en tiempos recientes serían sólo una modalidad de un variado tipo de manifestaciones anómalas (hadas, demonios, seres y hechos extraordinarios) que habrían operado en todas las épocas de la historia, guiándola hacia ignoradas metas. En la actualidad, el efecto observable de este mecanismo de control sería la difusión de la creencia en la vida extraterrestre. especula sobre la existencia de un antiguo supercomputador, superviviente de otra era, que produciría apariciones de monstruos, OVNIs y demás para evitar ser localizado. Igualmente, Bertrand Méheust sostuvo en una primera época que las analogías entre folklore, ciencia ficción y el fenómeno OVNI eran debidas a ideas y conceptos introducidos por los extraterrestres en las mentes de las personas. En España, desde 1994 el iiee auspicia con el Proyecto Delfos una “reacción” contra esos supuestos mecanismos de control social. [252, 253][MM] Hipótesis tectónicas. También conocida como TST (Tectonic Strain Theory, en inglés: teoría de la tensión tectónica), pretende explicar muchas observaciones de OVNIs como bolas eléctricas generadas por los terremotos. Entre sus principales defensores destacan Michael A. Persinger, neurofisiólogo de la Laurentian University en Ontario (Canadá), y Paul Devereux de Gran Bretaña. La hipótesis tectónica sostiene que durante los terremotos -o justo antes de que ocurran- las rocas afectadas acumulan y descargan energía por torsión y compresión. La energía así liberada (que también puede proceder de los flujos habidos en las aguas subterráneas) puede manifestarse en forma de luces redondas con movimientos irregulares, que serían confundidas con OVNIs, o también como una forma de energía electromagnética que afectaría a los lóbulos temporales del cerebro, provocando alucinaciones que, por ejemplo, se nutrirían de la imaginería ufológica. Persinger ha propuesto este modelo para explicar los síntomas presentes en quienes dicen haber sido víctimas de una abducción (sensaciones de flotar y volar, angustia, miedo), y experimentos con personas que voluntariamente se sometieron Vallée confiesa no poder determinar si dicho mecanismo es explicable en términos de genética, psicología social o fenómenos ordinarios; ni si es espontáneo o artificial y, en este último caso, si está dirigido por una inteligencia alienígena, por un grupo secreto o por agencias gubernamentales. Tales inconcreciones, la imposibilidad de falsar estas tesis y su escaso valor explicativo (como mucho resolverían el llamado problema del no-contacto) han minado la aceptación de la propuesta de Vallée, que ha sido muy criticada desde las posiciones de la HET clásica. No obstante, otros autores han secundado ideas semejantes, como John Keel, quien en ocasiones se ha referido a una radiación procedente del espacio que “programaría” el comportamiento de la raza humana; y que en The Eight Tower (1975) 120 Hombres de negro a la acción de campos magnéticos han revelado los mismos efectos. imaginarios. En ambos casos, parte de la culpa debe imputarse al propio Lawson, que quiso extender sus hipótesis más allá de lo razonable, señalando una serie de paralelismos ciertamente exagerados. En síntesis, la hipótesis del trauma natal pretende explicar las características que presentan las abducciones apelando a la teoría psicoanalítica (ya superada) del Dr. Grof, que defendía el trauma del nacimiento como responsable de futuras neurosis. En concreto, Lawson enumera una gran cantidad de similitudes y parecidos entre los rasgos descritos en las abducciones (e incluso durante meros encuentros con humanoides) y el proceso de desarrollo fetal hasta el nacimiento: semejanza entre los seres descritos y los fetos, la matriz como equivalente del interior del OVNI y el tubo de luz como reflejo del mismo acto de nacer, similitudes entre la placenta y el cordón umbilical con características de los OVNIs, etc., etc. Los partidarios de la TST se han apoyado en las posibles correlaciones entre los seísmos y los lugares donde se dan frecuentes avistamientos de OVNIs. Aparte del propio Persinger, ha destacado en esta labor el geofísico John Derr, del Instituto Geológico de EE.UU. en Alburquerque (Nuevo México). Como muestra, en ese mismo estado se informó entre 1951 y 1952 de numerosos no identificados sobre zonas que, durante el mismo período, sufrieron tres terremotos con una intensidad entre 4 y 5 en la escala de Richter. En España, se apuntó la correlación entre los casos que integran el flap gallego de 1995-96 y varios movimientos sísmicos ocurridos al mismo tiempo en Galicia. Sin embargo, los críticos de la TST argumentan que las hipótesis tectónicas no bastan para explicar determinados aspectos del fenómeno OVNI (como los encuentros cercanos, los aterrizajes, etc.) y que en ocasiones los terremotos se producen en zonas muy alejadas de donde se observan OVNIs, no explicándose el proceso de transmisión de la energía que da lugar a las luces sísmicas cuando el epicentro se halla a cientos o miles de kilómetros de la zona de los avistamientos. [5, 74, 174, 209][ JM] El principal reparo que puede señalarse a esta hipótesis es que el propio niño durante la gestación y al nacer no tiene consciencia de su propia imagen, ni de los detalles que lo rodean. En cualquier caso, una prometedora alternativa realmente testable (deberían aparecer diferencias entre los abducidos que hubieran tenido un nacimiento normal y aquellos otros nacidos mediante cesáreas) nunca sufrió realmente la prueba definitiva, pues fue rechazada sólo por posturas personales. [236][LG] Hipótesis del trauma natal. Recibe este nombre la hipótesis elaborada por el profesor de literatura inglesa Alvin H. Lawson en 1982 para tratar de ofrecer una explicación convencional a los cada vez más frecuentes relatos de abducción. Se presentó como la primera hipótesis falsable científicamente, pero en el mundillo ufológico no corrió mejor suerte que su anterior propuesta (1980) señalando las similitudes entre dichos relatos y los obtenidos hipnotizando a “abducidos” HOMBRES DE NEGRO Las historias sobre los Hombres de Negro dan vida y justificación a mucha de la paranoia y de las teorías conspirativas que rodean el mundillo de la ufología popular. 121 Hopkins, Budd Fue Albert Bender, según confesión propia, el primer ufólogo visitado por estos siniestros personajes. Bender era en 1953 editor en EE.UU. del boletín ufológico Space Review y creía haber encontrado la solución al misterio OVNI, pero tan pronto como empezó a comentarlo por ahí, dijo haber sido visitado en su casa por tres hombres vestidos de negro cuyas amenazas lo llevaron a cerrar su revista y retirarse de la Ufología. Gray Baker popularizó esta historia en su libro They Knew Too Much About Flying Saucers (1956), y en 1962 Bender volvió a la carga con una obra prologada por Baker, Flying Saucers and the Three Men, donde confesaba que los hombres de negro eran extraterrestres que le habían impedido divulgar sus verdaderas intenciones, al parecer nada amistosas. Desde entonces, un cierto número de investigadores y testigos aseguran haber sufrido encuentros similares con personas (vestidas siempre con ropas oscuras) de un comportamiento muy peculiar que profieren vagas amenazas y les ordenan silenciar lo que saben o han visto. HOPKINS, Budd (Wheeling, 1931 - Nueva York, 2011). Pintor y escultor estadounidense especializado en el estudio de las abducciones. Se inició en la Ufología a raíz de ser testigo de un avistamiento en 1964, lo que le llevó a ingresar en el NICAP y a desarrollar diversas investigaciones de campo. La publicación de una de ellas le supuso empezar a recibir casuística relacionada con episodios de tiempo perdido, y de ahí pasó a investigar los casos en que abducciones previamente no recordadas salían a la luz. Éste ha sido el tema de sus dos libros más populares: Missing Time: A Documented Study of UFO Abductions (Marek Publishers, New York, 1981) e Intrusos (Edaf, Madrid, 1988), en los que apuesta por la realidad de estas experiencias y las explica en términos de experimentos genéticos alienígenas. Principal propagandista de esta línea de pensamiento, en 1989 constituyó la Intruders Foundation (Fundación Intrusos) con el objetivo de llamar la atención de los científicos sobre la cuestión, establecer una red de hipnólogos y terapeutas en numerosas ciudades de los EE.UU., y publicar un boletín trimestral dedicado a la investigación de las abducciones. Su última obra, Witnessed: The true story of the Brooklyn Bridge UFO Abductions (Simon & Schuster, New York, 1996) aborda el caso de Linda Napolitano, una abducción con aparentes testigos independientes. John Keel puede considerarse el más influyente propagandista de la mitología de los Hombres de Negro en los últimos años. Dado que estos seres hacen visitas a domicilio, conducen llamativos coches negros y realizan amenazas telefónicas, deberían ser más fáciles de verificar o capturar que un elusivo OVNI, pero han resultado ser tan insustanciales como los fantasmas. Ello junto con su ridículo comportamiento, como de gangsters de película mala (amenazan de palabra pero raramente inflingen daño material o físico, y resultan tan exitosos eliminando evidencias como los alienígenas en suprimir las memorias de los abducidos), apuntan a que se trata de simples elementos folklóricos. [102, 126, 136, 204, 263][LG, RC] Opinión sobre el fenómeno (tomada de Jerome Clarke, 1990, UFOs in the 1980s): “Desde el principio he mantenido pocos prejuicios sobre el fenómeno OVNI, y como cualquier investigador con inclinaciones científicas 122 HPS haría, he ido simplemente a donde los datos me han conducido. Inicialmente tenía mis reservas sobre los relatos de abducciones, pero la clara preponderancia de gente creíble que informa de estos sucesos y las pruebas físicas que lo acompañan me persuadieron de que estos relatos eran generalmente ciertos. Los datos dejan manifiestamente claro que las abducciones -como toda clase de avistamientos- tienen tanto una dimensión física como una dimensión paranormal”. Blue Book. Con un brillante historial como astrónomo a sus espaldas, sus éxitos en programas de seguimiento óptico de satélites le valieron el encargo de revisar los numerosos incidentes que la Fuerza Aérea iba recopilando, frente a los cuales adoptó una postura escéptica. No obstante, a partir del desafortunado episodio del gas de los pantanos (1966) inició un cambio de pensamiento hacia posturas más abiertas a admitir la realidad del fenómeno OVNI. Tras las conclusiones negativas del informe Condon (1969), que marcan el fin de su contrato con la USAF, Hynek sigue investigando por su cuenta el tema y escribe The UFO Experience (Henry Regnery, Chicago, 1972), donde critica duramente el trabajo de la Universidad de Colorado y se inclina claramente por la tesis de la realidad objetiva de los OVNIs. En 1973 funda, con Sherman J. Larsen, el Cufos, desde donde editará el IUR y, tiempo después, el Journal of UFO Studies. Junto a Jacques Vallée publica en 1975 The Edge of Reality (Henry Regnery, Chicago, 1975), una discutida incursión en las ideas paraufológicas de su colega francés, a las que Hynek se mostró receptivo en sus últimos años. Su último libro, The Hynek UFO Report (Dell, New York, 1977; traducido al castellano: El informe Hynek, J. Vergara editor, Buenos Aires, 1979), es una revisión y comentario sobre los archivos del Proyecto Blue Book. Personalidad idolatrada hasta la mitificación, se le ha considerado el paradigma viviente de la conversión desde el escepticismo a la aceptación de la existencia material de los OVNIs. HPS Siglas lógica. de Hipótesis psicosocio- HUELLAS Véase Efectos de los OVNIs. HUMANOIDE Término acuñado en el seno de la Ufología para designar las entidades de apariencia antropomorfa asociadas a los OVNIs, bien por aparecer en sus proximidades, bien, en ausencia de estos, por presumírseles un origen extraterrestre (v. Tipologías de humanoides) HYNEK, Josef Allen (Chicago, 1910-Scottsdale, 1986). La más conocida figura de la ufología mundial, doctor en astronomía por la Universidad de Chicago y asesor astronómico de la USAF, para la que trabajó desde 1948 en el Proyecto Grudge primero y más tarde en el Proyecto Opinión sobre el fenómeno (adaptada de una entrevista en Lumières dans la Nuit, nº 168, octubre de 1977): ”La HET ha de luchar contra un gran inconveniente, a saber, que se ven 123 Hynek, Josef Allen demasiados OVNIs. La Tierra sólo es una mota de polvo en el universo, ¿por qué hemos de ser honrados con tantas visitas? Me inclino más a pensar en términos de algo metaterrestre, una especie de realidad paralela. Tengo la impresión de que los OVNIs anuncian un inminente cambio en nuestros paradigmas científicos. Mucho me temo que los OVNIs están relacionados con ciertos fenómenos psíquicos. Y digo «mucho me temo» porque en el CUFOS intentamos investigar este problema desde la perspectiva de las ciencias físicas. Pero sería absurdo seguir sólo un camino excluyendo todos los otros.” DTU 124 Identificación I IDENTIFICACIÓN IMPLANTE Véase Depuración de casuística. Se denominan así en la jerga abduccionista unos supuestos mecanismos artificiales introducidos por los extraterrestres en distintas partes del cuerpo de sus víctimas. Jacobs (1992) ha llegado a apuntar cinco posibles utilidades: para control mental, emisores para la localización de los testigos, telemedidores de parámetros fisiológicos, transmisores para la comunicación, e incluso generadores de los cambios moleculares necesarios para transportar a seres humanos a través de las paredes. IIEE (Instituto de Investigaciones y Estudios Exobiológicos). Asociación ufológica creada en 1975 en Barcelona por una serie de personas unidas por vínculos de tipo laboral procedentes de la empresa Olivetti y del sector de aeropuertos. Formó parte de la Cefo, si bien desde 1981 ha trabajado aislado del resto de la ufología española. Se ha especializado en labores de investigación de campo, con más medios que los habituales para un grupo de estas características, pero de manera aislacionista y sin aceptar las revisiones de las encuestas realizadas por el centro, que defiende la naturaleza extraterrestre de la mayoría de los incidentes estudiados. Siempre bajo la presidencia de Ramón Navia, entre mayo de 1980 y junio de 1985 el IIEE editó un boletín trimestral llamado Espacio Compartido, centrado en la casuística investigada por sus propios socios. En los últimos tiempos el grupo parece orientado hacia las tesis conspiracionistas y parafísicas del llamado Proyecto Delfos, una especie de llamamiento a la rebelión contra el “sistema de control” que representarían los OVNIs. A partir de 2003 estableció una delegación en Santiago de Chile, que sigue presente en Internet: https://www.iiee.cl [MM] Los primeros implantes (Larson 1975, Andreasson 1977) eran introducidos y extraídos a través de las fosas nasales, imagen llevada luego al cine en la película de Arnold Schwarzenegger Desafío Total (1990) con su gigantesco implante nasal. Pero pronto quedó claro lo peligroso (para el testigo) de estos procedimientos tan poco asépticos y las inquietantes interpretaciones freudianas a que podrían dar lugar, así que se han ido trasladando a otras zonas del cuerpo. Recientemente Strieber ha declarado: "los implantes se encuentran más frecuentemente en la parte izquierda del cuerpo, y las cicatrices en la parte derecha (...) Ningún implante mostraba una cicatriz en las cercanías". Aunque ya en 1991 Budd Hopkins prometió presentar un implante a la comunidad científica, sólo han circulado fotos mostrando extraños nódulos u objetos 125 Implicación oficial en el cuerpo de los testigos, pero que siempre desaparecen justo antes de ser extraídos. Alcanzó gran difusión el caso de Richard Price que, 30 años después de su abducción, recobró algo que le habían implantado en el pene. Examinado en los laboratorios del MIT resultó ser colágeno formado por el propio organismo en torno a unas fibras de algodón, seguramente procedentes de la ropa interior del testigo. Sólo muy recientemente se han documentado otros supuestos análisis, pero sin que hayan podido ser verificados por investigadores independientes, ni se haya evidenciado una estructura claramente artificial. Ante esta falta de evidencias tangibles, algunos creyentes han llegado a afirmar que "muchos de los implantes se colocan en los cuerpos astrales". Coartada perfecta. El fenómeno OVNI ha suscitado la atención oficial casi exclusivamente por su vinculación con la defensa de la soberanía del espacio aéreo, y de hecho, con la excepción de los proyectos estadounidenses de los años 50 y 60 (Twinkle, Sign y Grudge, Blue Book), no ha recibido un tratamiento específico sino que se ha limitado a ser un aspecto más de aquélla. Incluso en el caso americano, casi nunca ha propiciado la creación de entidades estables dedicadas exclusivamente a su estudio, pues los citados proyectos se desarrollaron en el seno de organismos (el ATIC primero y el FTD después) con competencias mucho más extensas y diversas. No obstante, sí que existen en algunos países iberoamericanos servicios dependientes de las Fuerzas Armadas más o menos consagrados al análisis de la casuística ufológica, como el Grupo de Trabajo Ovni del CITEFA argentino, la Comisión Receptora e Investigadora de las Denuncias OVNI (CRIDOVNI) en Uruguay, y el Comité Especial Ovni de la Agencia Estatal de la Defensa Civil portorriqueña. En el resto de países occidentales este cometido viene desempeñado por las unidades encargadas de la defensa del espacio aéreo o por sus secciones de inteligencia. En realidad, la idea no es nueva. Como siempre ya había sido predicha por la ciencia ficción. Los neurólogos hace tiempo que han abandonado esa simplista posibilidad del control mental a través de implantes, pese a aquella famosa aparición televisiva en la década de los sesenta del español Dr. José Delgado deteniendo la carga de un toro enfurecido en un coso taurino. Sin embargo, ofrece un atractivo tan insuperable para todos aquellos que creen en conspiraciones gubernamentales que es creciente el número de personas que aseguran ser sujetos de control a distancia por parte de siniestros personajes, en este caso nada extraterrestres. [109, 121, 134, 181][LG] En el terreno puramente civil, es de destacar la existencia del Gepan francés (el actual SEPRA), cuyo ámbito de actuaciones pretendió ampliarse en diciembre de 1993 por iniciativa del Parlamento Europeo (B3-1990/90 y A3-0389/93) a toda la Europa comunitaria, aunque finalmente dicha propuesta no prosperó. De forma parecida, la ONU estuvo tentada de crear un organismo similar en 1977, cuando Sir Eric Gairy, primer ministro de la isla de Granada, auspició un debate sobre la materia en noviembre de ese año. A resultas de ello, el IMPLICACIÓN OFICIAL Expresión con la que se alude al conjunto de actividades de los organismos gubernamentales relacionadas con OVNIs. 126 Índices de Ballester-Guasp 14 de julio de 1978 diversos ufólogos (Vallée, Hynek, Saunders, Stringfield, Poher) disertaron sobre su conveniencia ante un subcomité especial del Comité de la ONU para los Usos Pacíficos del Espacio Exterior. Finalmente, el 8 de diciembre se aprobó un documento de trabajo que acogía favorablemente la propuesta pero que nunca llegó a desarrollarse.[187][MM] de movimientos anómalos, aparición de incongruencias físico-espaciales, seguimiento tecnológico, encuentro cercano, presencia de seres y hallazgo de huellas o producción de efectos. La letra Σ expresa la extrañeza y se define como n / 7, siendo n el número de los anteriores factores positivos que se encuentran en una observación ufológica. El rango de sigma varía desde 0 a 1. ÍNDICES DE BALLESTER-GUASP El índice de credibilidad de un suceso, representado por π, se expresa de acuerdo con una fórmula que relaciona seis elementos (número de testigos, profesión, relación interpersonal, relación geográfica, actividad y edad) con unos pesos relativos, de forma que la aplicación aritmética de los valores da también un margen de credibilidad de 0 a 1. Perfeccionando los conceptos de extrañeza y credibilidad de una observación OVNI adelantados por Hynek, Olsen, Poher y otros, los investigadores españoles V.J. Ballester Olmos y M. Guasp Carrascosa (Ballester y Guasp, 1981 y 1989), desarrollaron un triple sistema de valoración objetiva de la información OVNI que confluyó en un índice total que agrupaba los valores de los tres y que se definía como índice de certidumbre. Se trata de una avanzada forma de cuantificar los informes OVNI con el fin de homologar y homogeneizar su expresión en valores numéricos. Disponiendo de los tres valores antedichos se construye un índice de certidumbre, que es un algoritmo que los interrelaciona mediante la fórmula: Δ = (Q x Σ x π ) Siendo delta un grado de certidumbre que se indica en forma de porcentaje (por ejemplo, 0,15 señala que existe una certeza sobre la veracidad de unos hechos anormales equivalente al 15%). Índice de calidad de la información OVNI (Q) es la validez en términos matemáticos que el relato de un caso ufológico tiene para el analista. Basado en el conjunto de datos disponibles y en el examen de la fuente de información, Q adopta valores entre 0 y 1. La cantidad de datos acumulados y la probabilidad de que éstos sean fidedignos varía según se trate de material procedente de una fuente de primera mano, una encuesta indirecta, datos de prensa, rumores, etc. El índice de certidumbre Ballester-Guasp ha sido adoptado por el Mufon norteamericano como el método oficial de tasación objetiva de los informes OVNI y como tal se ha incorporado con todos los honores en su Manual del investigador de campo (Andrus Jr., Walter H. (ed.): MUFON Field Investigator’s Manual, MUFON, Seguin, 4ª ed., 1995).[31][VB] Para el índice de extrañeza, Ballester y Guasp proponen una escala aditiva que consigne la experiencia anómala, existencia 127 Indiscernibilidad INDISCERNIBILIDAD través del informe se ofrece a los interesados y a otros estudiosos un relato del incidente, de la investigación llevada a cabo y de las conclusiones a que ha llegado su autor, de tal forma que se convierte en algo autónomo: idealmente un informe correctamente realizado debe suministrar toda la información relevante para una adecuada comprensión del episodio por parte de otros investigadores o para su posterior reencuesta. Para ello deberá incluir todos los detalles significativos del incidente y del relato del testigo: expresiones comunes usadas por éste, datos espacio-temporales, cronología de la observación, etc., y una conclusión donde se apunten posibles explicaciones (si caben) para la visión inicialmente misteriosa, consultas evacuadas a todos aquellos organismos que pudieran aportar informes o datos técnicos relacionados con el caso o que permitiesen deducir conclusiones importantes (v. resumen modelo). El informe OVNI muestra la habilidad y capacidad del investigador o simple aficionado para transmitir unos hechos a otros interesados de manera imparcial, dentro de los cánones habituales de rigor científico, objetividad, conocimiento público, desinterés, etc. La fidelidad a tales normas permite introducir juicios éticos o de valor respecto a la validez como investigador ufológico de un individuo concreto. [RC] Véase Casos positivos y negativos. INFORESPACE Publicación semestral del grupo belga Sobeps. El último de sus números apareció en octubre de 2007. Pueden consultarse en línea en: http://files.afu.se/Downloads/ ?dir=Magazines/Belgium/Inforespace INFORME OVNI Entre otras muchas facetas, una de las que más claramente diferencian a la ufología periodística de la de inspiración crítica es el contenido habitual de los informes de investigación de un caso concreto. Ha sido frecuente ofrecer al interesado una simple transcripción de las palabras del observador interpretándolas invariablemente como prueba de la presencia de algún fenómeno extraño o de la manifestación de un poder ajeno a la Tierra. No otro podía ser el resultado de tal valoración porque puede decirse que ésta estaba prefijada de antemano: en un contexto de supuestos misterios, sensacionales “revelaciones” y creencias en visitas extraterrestres, ciertos informadores, inmersos en tal mundo y cultivadores del mismo, se limitaban -y aún se limitan- a extraer en sus informes conclusiones que no se derivan legítimamente de las premisas que han barajado. INGENIOS ASTRONÁUTICOS Prácticamente desde sus inicios la astronáutica terrestre ha dado lugar a confusiones que muchas personas han atribuido a los OVNIs. Es seguro que bastantes casos de los llamados cohetes fantasmas vistos en Escandinavia en 1946 y de los supuestos platillos estrellados Un buen informe ufológico debe ser deudor de una buena investigación particular. Máxima objetividad es la condición fundamental exigible a toda investigación, y no menos en este terreno. A 128 Ingenios astronáuticos en los desiertos norteamericanos se deben a las primeras experiencias con cohetes de las grandes potencias. se ha encontrado siempre una explicación convincente para los mismos (Oberg 1982). Finalmente la reentrada en la atmósfera de algún elemento en órbita, desde satélites a mera "chatarra espacial", con ese gran despliegue luminoso provocado por la fricción, ha generado a lo largo de los años muchos casos de avistamientos masivos de supuestos OVNIs. La lista sería interminable, así que sólo mencionaremos varios: 19/9/67 en Kazakhstan (pruebas de misiles balísticos); 23/02/71 sobre la mitad norte de la Península Ibérica (cohete Tibère); 14/06/80 y 31/10/81 sobre Chile y Argentina (Cosmos 1188 y Cosmos 1317), 5/11/90 sobre Europa (reentrada de la tercera etapa del cohete Protón). Todas las fases de una trayectoria orbital ofrecen estímulos capaces de ser malinterpretados: El lanzamiento, especialmente si es nocturno, puede ser visto a grandes distancias. Y si se trata de pruebas secretas de misiles resultan muy díficiles de confirmar con la suficiente rapidez. Como ejemplos de supuestos OVNIs recordaríamos el OVNImedusa de Petrozavodsk el 20/9/77 debido al lanzamiento del satélite soviético Cosmos 955, y el famoso OVNI de Canarias el 22/6/76 que un médico de Galdar llegó a describir como una nave con gigantescos seres en su interior y que consistió realmente en el lanzamiento de un misil desde un submarino. El problema con este tipo de avistamientos masivos es que los distintos informes que se van acumulando ofrecen una cierta disparidad respecto a la hora de lo sucedido, la dirección del fenómeno y su duración o proximidad al testigo, lo cual permite a los creyentes (haciendo caso omiso a la poca fiabilidad del testimonio humano) llegar a argumentar que los OVNIs aprovechan dichos espectáculos atmosféricos para intentar pasar desapercibidos, supeditando por tanto sus propios planes a la difícil coincidencia con un bólido o un lanzamiento. Así el periodista J.J. Benítez (1993) afirma: "toda una colección de naves no humanas (...) se precipitó sobre buena parte de Europa. Y lo hizo, justamente, de forma y manera que coincidiera con la reentrada de los restos de un cohete espacial humano" (pág. 399) En ocasiones se trata de descargar en la alta atmósfera nubes de bario, sodio y otros elementos químicos para su estudio. Ello da lugar a que se hable de OVNIs que se desintegran o son destruidos. Ejemplo paradigmático fue el fenómeno visto y fotografiado sobre Barcelona la noche del 12/6/74 cuyo origen estuvo en un cohete meteorológico francés. Algo similar ocurrió con otros rastros luminosos aparecidos en la misma zona el 21/3/89, o el 21/1/76 sobre Andalucía y Extremadura. Si se trata de vuelos tripulados, los propios astronautas (supuestamente testigos de la máxima fiabilidad) pueden sufrir confusiones o ser malinterpretados. Y cualquier mancha luminosa en alguna de las miles de fotos publicadas ofrece al creyente la oportunidad de hablar de OVNIs. Pese a que en su momento el propio Dr. Hynek defendió la validez de algunos de estos casos, Aunque existen organismos como el NORAD que realizan el seguimiento de algunos materiales en órbita, no siempre es posible predecir dónde y cuándo se producirá su reingreso en la atmósfera ni 129 Internet, Ufología en para la Ufología y para las paraciencias en general, por lo que no es de extrañar dada la coincidencia temporal que el boom de Internet fuera parejo a la polémica del caso Roswell: el informe GAO, la película de Santilli, desmentidos y contradesmentidos, todo fue transmitido por las redes, volcándose creyentes y escépticos en un alarde informativo inusitado. confirmar a posteriori que sea ésta la explicación de unas determinadas observaciones. [35, 41, 170][LG] INTERNET, Ufología en Internet (literalmente "entre redes", "una red de redes") es el sistema de comunicación por excelencia entre redes de ordenadores. Aunque su origen es bastante anterior, no fue hasta 1992 cuando comenzó a popularizarse, debido en gran parte a la accesibilidad y comodidad del entorno gráfico hipermedia (WWW, World Wide Web, la telaraña que envuelve el mundo). El año 1995 fue testigo de la reaparición de los alienígenas que producen mutilaciones de ganado, al menos eso creyeron ver algunos en los supuestos ataques del “chupacabras”, para otros una extraña bestia que surgiendo de Puerto Rico saltó a Florida, México e incluso Nueva York. La creencia en este ser está sospechosamente vinculada a las informaciones poco veraces transmitidas a través de Internet. Es curioso como la barrera del idioma (inglés-español) forjó en cierta medida dos corrientes propias con características particulares. La facilidad y precio asequible de acceso desde cualquier lugar del planeta con un simple ordenador, un módem y una línea telefónica motivaron la voluminosa aparición de páginas informativas en las webs de organizaciones y particulares muy alejadas en lo general del interés científicomilitar para el que fue concebido. Pese a esto ya existían con anterioridad boletines accesibles por ordenador y conocidos comúnmente por BBS (Bulletin Board Service), pero había que disponer del número de teléfono de acceso concreto y realizar una llamada telefónica a ese en particular. Al margen de las modas temporales son numerosas las organizaciones y particulares relacionados con los OVNIs que han establecido su web informativo más o menos extenso. En el orden internacional entre las primeras citemos sin ánimo extensivo al Bufora, al Mufon, al Cisu, al Csicop o al AUFORA, que se distinguen además por poseer una lista de correos muy concurrida. Un boletín electrónico muy curioso es el Groom Lake Desert Rat referido al Área 51 y a la base secreta de Groom Lake, así como el Area 51 Research Center. También han nacido organizaciones con la propia red como el Internet Ufo Group. Entre los particulares destacar a Willy Smith o Stanton Friedman. También revistas como Flying Saucer Review o Fortean Times han presentado sus páginas electrónicas. En Internet, junto a los kioscos informativos -las webs- han prodigado las listas de correo que no son otra cosa que centros de recogida de correo electrónico que a su vez los remiten a todos los afiliados. También se han popularizado los newsgroups, fórums de debate público en que los escritos se cuelgan de un tablero virtual y son accesibles a cualquiera a través de la red. Un medio tan ágil y que llega a todos los rincones del planeta era campo abonado 130 Intraterrestres En el panorama español la primera página web se debe al investigador de temas paranormales Juan Carlos Valero (OVNIS en INTERNET), aparecida a principios del verano de 1996, poco antes de la de Josep Guijarro (Punto de encuentro OVNI). A ella se han sumado rápidamente otras como la del Cei, el LACIP (Laboratorio Científico de Investigaciones Paranormales) y UFOES, una página de enlaces a otros webs en español e información sobre la lista del mismo nombre, lugar de reunión de ufólogos hispanohablantes de uno y otro lado del Atlántico. Es de esperar que otras organizaciones y particulares vayan sumándose a las presentes. [87][ JAB] datos procedentes de los medios de comunicación, en gran número de casos sin mayor intervención del investigador, dando como resultado una información deficiente y meramente especulativa. Las primeras encuestas serias fueron realizadas en el seno de las comisiones de estudio desarrolladas en EE.UU. por escaso personal militar (v. Sign y Grudge, proyectos). Los grupos privados intentaron normalizar esta actividad mediante la protocolización de cuestionarios, que en gran medida eran malas copias de los utilizados por los estamentos militares o las comisiones de estudio oficiales. En España el primer intento de normalizar esta actividad corrió por parte del Cei con la publicación de circulares normativas para cada caso específico, seguido después por la Rnc y su Guía del corresponsal (1971), inspirada en la Aide-Memoire de l’Enquéteur de la entidad francesa Lumières Dans La Nuit. En 1979 Alberto Adell Sabates, ingeniero técnico y miembro del CEI, publicó Manual del Ufólogo (Ed. 7 y 1/2, Barcelona, 1979), un compendio pormenorizado de normas y herramientas de enorme utilidad para el investigador, aunque también plagado de notables carencias. Siguiendo en España, sería más tarde V.J. Ballester Olmos quien con su libro Investigación OVNI (Plaza & Janés, Barcelona, 1984) profundizara en la educación del encuestador dando pautas en el capítulo dedicado a la infraestructura de la investigación ufológica, la “Guía para el encuestador”. Fuera de nuestro país, son también reseñables las obras Guía de Procedimientos para Recopilar Datos de Experiencias OVNI de Heriberto Janosch y Alejandro Agostinelli (CIU, 1988), y el MUFON Field Investigator’s Manual coordinado por Walter Andrus Jr. (MUFON, 4ª edición, 1995). INTRATERRESTRES Supuestos habitantes del interior de la Tierra cuya avanzada tecnología sería la causa de los avistamientos de OVNIs. Véase Hipótesis intraterrestre. INVERSIÓN TÉRMICA Véanse Fenómenos ópticos y Radar y OVNIs. INVESTIGACIÓN DE CAMPO Actividad encaminada al descubrimiento de nuevos conocimientos en el campo de las ciencias mediante un conjunto de diligencias realizadas por los investigadores en el lugar de los hechos y mediante la consulta de las fuentes documentales que lo dieron a conocer públicamente. En Ufología la investigación de campo ha estado deformada por la acumulación de 131 Investigación de gabinete o analítica En los últimos años, por actitudes manifiestamente sesgadas, se ha pretendido magnificar la labor del encuestador, a todas luces insuficiente, propiciando no la encuesta sino el reportaje periodístico, en clara contraposición a lo que debe ser una actividad encaminada a obtener datos fidedignos y cuantificables por la ciencia. Un encuestador no puede limitarse a realizar una entrevista que luego dará a conocer anteponiendo sus personales convicciones, sino que habrá de tratarse de persona cualificada, con amplios conocimientos, y poseedor de una esmerada técnica de encuesta que permita dilucidar errores o cuando menos facilitar datos no contaminados para que los analistas puedan llegar a determinar la naturaleza del fenómeno reportado. La investigación de campo -y esto es importante subrayarlo- no puede suponer la única base para el estudio de un fenómeno no reproducible en laboratorio, sino el primer eslabón importantísimo en una cadena de sucesivos especialistas. [ JR] información sobre los hechos sea factible reunir, la investigación de gabinete busca insertar esos datos en la premisa mayor que suponen el conjunto de conocimientos establecidos por la ciencia, con el objetivo de obtener como conclusión un pronunciamiento lo más riguroso posible sobre la naturaleza de la experiencia denunciada. Naturalmente, en la mayoría de las ocasiones ello supone, por un lado, encontrar una explicación banal a los hechos y, por otro, rebatir en consecuencia la tesis extraterrestre que puede estar implícita en el informe elaborado por el investigador de campo. Por estos motivos, dicho análisis a posteriori no resulta del agrado de los sectores más apegados a la HET, que interpretan cualquier explicación alternativa como una “maniobra de desinformación” o una “intrusión ilegítima” en el trabajo del ufólogo que primeramente encuestó el incidente, olvidando que investigación de campo y de gabinete no son términos contrapuestos sino complementarios. Prácticamente todos los autores que practican esta última son o han sido investigadores sobre el terreno, cosa que no puede decirse a la inversa. INVESTIGACIÓN DE GABINETE O ANALÍTICA El máximo exponente de la investigación analítica a escala internacional ha sido sin duda J.A. Hynek, quien tras su implicación con las comisiones de estudio oficiales en EE.UU. defendió la realidad del fenómeno OVNI como objeto de estudio científico. En España fue pionero de esta orientación el Cei, destacando entre sus miembros la figura de Félix Ares de Blas, autor del Estudio de la Oleada 1968-1969 (1970). Posteriormente ha descollado en este campo la labor de Vicente-Juan Ballester Olmos (con Investigación OVNI, 1984, y Los OVNIS y la Ciencia, 1989) y, más recientemente, la de Manuel Borraz Metodología investigativa basada en el análisis a la luz de diversas disciplinas y procedimientos de los datos previamente obtenidos a través de las encuestas sobre el terreno. En Ufología fue bautizada peyorativamente por el periodista J.J. Benítez como “investigación de salón”, al considerar la no implicación directa con el caso y los testigos como intrusismo en la labor del investigador de campo. Así como el objetivo de la investigación de campo es fundamentalmente la recogida de cuanta 132 IUR Aymerich. [ JR, MM] IUR Siglas de International UFO Reporter, publicación trimestral (semestral desde 1996) del Cufos. Dejó de editarse en 2012. DTU 133 Journal of Scientific Exploration J JOURNAL OF SCIENTIFIC EXPLORATION rigor y documentación y que la convierten en una fuente ineludible de referencias científicas sobre OVNIs y fenómenos paranormales. Dirección: P.O. Box 5848, Stanford, California 94309-5848, EE.UU. En Internet: https:// www.scientificexploration.org [VB] Revista trimestral de contenido y procedimiento académico sobre temas en los límites de la ciencia publicada por la Society for Scientific Exploration (SSE). Fundada en 1981 por el profesor Peter Sturrock de la Universidad de Stanford, en 1987 comenzó a editarse por Pergamon Press, entidad especializada en literatura científica, con Donald Howard como director. A partir de 1992 la publicación pasó a ser producida por la propia SSE. En sus diez primeros años ha constituido la más destacada tribuna científica multidisciplinar para temas como OVNIs, parapsicología y anomalías en general. Ciñéndonos a aspectos ufológicos, de entre los numerosísimos trabajos aparecidos en ella destacan los de Sturrock (informe Condon), Haines, Maccabee, Vallée, Ballester Olmos, Henry, Bounias, Velasco, Derr, Persinger, Wood, Wieder, Guérin, Grigor’ev (rayos globulares), etc. JOURNAL OF UFO STUDIES Publicación anual editada por el grupo estadounidense Cufos hasta 2006. JUST CAUSE Publicación trimestral órgano oficial de Citizens Against UFO Secrecy (CAUS, Ciudadanos contra el secreto OVNI). En sus comienzos en 1978 el equipo editorial estaba integrado por W. Todd Zechel, a su vez director de investigaciones del Ground Saucer Watch (GSW), Steve Stoikes y Brad Sparks, con el asesoramiento legal de Peter Gersten (abogado del GSW). El objetivo inicial de CAUS fue principalmente plantear pleitos contra la administración norteamericana, amparándose en la Ley de Libertad de Información (FOIA), para conseguir documentos sobre OVNIs de los archivos de la CIA, la NSA, las Fuerzas Armadas y otros organismos oficiales. En 1979, Larry Bryant se hizo cargo de la revista, que dejó de publicarse poco después, al tiempo que Zechel se decantaba por extremistas lecturas paranoico- En la actualidad la revista está dirigida por el Dr. Berhard Hadisch con el apoyo de un consejo editorial integrado por un selecto grupo de eruditos entre los que se cuentan los profesores Rémy Chauvin, O. Costa de Beauregard, Richard Henry, Kunitomo Sakurai y Yervan Terzian. Un científico, naturalmente, puede errar tanto como un profano; tiene creencias que pueden dominar su lado racional y, en suma, dejarse llevar por la ceguera. Pero esta publicación aporta trabajos que han superado controles de calidad que aseguran un alto nivel de 134 Just Cause XXX conspiracionistas sobre la implicación del gobierno estadounidense en este asunto. sobre Majestic-12, Roswell, el clásico caso Mantell, los foo-fighters, los cohetes fantasma escandinavos, el proyecto Mogul y un largo etcétera que convierten esta publicación en una referencia imprescindible para el estudioso no dogmático. Dirección: CAUS, Box 176, Stoneham, Massachusetts 02180, EE.UU. [VB] DTU En 1984, Lawrence Fawcett y Barry Greenwood, miembros de CAUS y autores del célebre libro Clear Intent, decidieron reactivar esta publicación bajo una perspectiva sensata y documentada. Fawcett se jubiló en 1994 y desde entonces Barry Greenwood es el responsable de este boletín, que ha ido evolucionando desde sus orígenes hasta convertirse hoy en “un órgano que informa, analiza y frecuentemente critica hechos relacionados con el interés gubernamental por los OVNIs, así como actitudes de investigadores civiles relacionadas con lo mismo... Más que representar a machamartillo la tesis de las conspiraciones gubernamentales ejecutadas con una perfección que no se ve en otros campos, quisimos ser más realistas... creíamos que el gobierno tenía la respuesta al fenómeno. Muy frecuentemente maniobras gubernamentales apoyaban la idea de que se escondían respuestas. Ya no pensamos que el gobierno, o cualquier otro gobierno, tenga una respuesta. Creemos que todavía se mantiene apartada de la vista del público mucha información. Lo que importa no son las creencias sino las pruebas. Para eso se creó esta organización: para escudriñar en los registros gubernamentales y hacer una valoración rigurosa. Hay todavía huecos en la historia del fenómeno OVNI y hemos aprendido a tratar también con este aspecto. El presente sólo se entiende en el contexto del pasado” (Barry Greenwood, Just Cause, 39, marzo de 1994, p. 8). Just Cause ha aportado a la comunidad ufológica información novedosa y destacada sobre desconocidos aspectos militares relacionados con el fenómeno OVNI, tanto de EE.UU. como del resto del mundo, brindando competentes ensayos críticos 135 Keel, John A. K KEEL, John A complicado sistema de nueva física relacionado con teorías del continuum espaciotemporal. Es posible, incluso altamente probable, que un sutil sistema de control cosmológico haya funcionado desde los albores de la humanidad y que los OVNIs sean parte de dicho sistema”. (Nueva York, 1930-2009). Pseudónimo de Alva John Kiehle, prolífico escritor, periodista y guionista de radio y televisión norteamericano considerado uno de los principales partidarios de las tesis paraufológicas. Según Keel, los OVNIs son ejemplos actuales de un fenómeno ya conocido antiguamente: las transmogrificaciones, manifiestaciones de una energía inteligente que poseería la capacidad de hacerse invisible a voluntad variando la frecuencia de la luz que refleja. Igualmente, ha dedicado una especial atención en sus obras a la fauna fantástica y sus relaciones con los OVNIs. Autor, entre otros, de Operation Trojan Horse (1970) y The Mothman Prophecies (1975), en España únicamente ha sido traducido uno de sus libros: El enigma de las extrañas criaturas (ATE, Barcelona, 1981). KEELINIANO Dícese del epifenómeno de naturaleza aparentemente parafísica que puede acompañar el avistamiento de un OVNI o suceder con posterioridad al mismo (p. ej.: llamadas telefónicas de hombres de negro, visitantes de dormitorio, incoherencias físicas). Toma su nombre de las ideas de John A. Keel. KEYHOE, Donald E(dward) Opinión sobre el fenómeno (tomada de Ronald D. Story (ed.), 1980: The Encyclopedia of UFOs): “Fundamentalmente, una gran parte de los relatos OVNI son subjetivos y muchos incidentes son en realidad el producto de un complejo proceso alucinatorio, especialmente los informes sobre EC-3 y contactados. El mismo proceso estimuló en otros siglos las creencias religiosas, las historias sobre hadas y las tradiciones ocultistas (...). Mientras no podamos explicar satisfactoriamente todos los sucesos OVNI en términos de conocimientos y tecnologías actuales creo que la solución definitiva implicará un Véanse Hipótesis extraterrestre y Nicap. KLASS, Philip J(ulian) (Des Moines, Iowa, 1919-Cocoa, Florida, 2005). Philip Klass es considerado como el más importante debunker ufológico en la actualidad, tras el fallecimiento del profesor Donald H. Menzel. Es miembro fundador del Csicop y preside su subcomité OVNI. Desde 1966 ha investigado personalmente algunos de los 136 Klass, Philip XXX J. más famosos y desafiantes casos OVNI explicando la mayoría de ellos en términos prosaicos y terrestres. Tras graduarse en la Universidad del Estado de Iowa en 1941 en ingeniería eléctrica, Klass trabajó para la empresa General Electric (1941-51) en su división de electrónica aeronáutica. De allí se incorporó a la revista Aviation Week & Space Technology, donde pronto alcanzó el puesto de editor jefe que mantuvo hasta su jubilación. Desde 1990 publica un boletín aperiódico titulado Skeptics UFO Newsletter. Dirección: 404 "N" St. SW. Washington DC 20024, USA. Su bibliografía sobre OVNIs consiste en decenas de artículos y cuatro libros: UFOsIdentified (1968); UFOs Explained (1974); UFOs.The public deceived (1983) y UFO Abductions, a dangerous game (1989), ninguno de ellos traducido al castellano. Opinión sobre el fenómeno (extraída del prólogo de su último libro, UFO Abductions, a dangerous game): “Después de 22 años investigando informes de OVNIs y abducciones puedo asegurar que no existe ninguna evidencia física creíble que indique que la Tierra está siendo visitada por seres extraterrestres (...). (Las abducciones) han pasado de ser un mito OVNI relativamente inofensivo a convertirse en un culto peligroso (...) que amenaza la salud mental, e incluso quizá la vida, de aquellos que involuntariamente se convierten en partícipes”. DTU 137 La Alternativa Racional L LA ALTERNATIVA RACIONAL horas, y volvían a ser más frecuentes al amanecer. Pese a que el escaso número de casos que cotejó Vallée (unos 200) y la contraencuesta negativa de muchos de ellos obligan en la actualidad a matizar mucho la validez de sus apreciaciones, lo cierto es que la nocturnidad de la casuística ha sido puesta de relieve en muchos otros estudios. Las causas de este comportamiento, no obstante, se han de buscar más en los testigos que en el fenómeno en sí, pues es durante la noche cuando las confusiones son más comunes y cuando más relajados se encuentran los sentidos. De hecho, con el paso del tiempo parece también manifestarse una tendencia general hacia la progresiva nocturnidad de los avistamientos, en parte debida a que los ingenios humanos que en 1954 podían aún causar extrañeza a plena luz del día (globos, helicópteros, cohetes) son en la actualidad más conocidos y fácilmente identificables, con lo que sólo permanecen “no identificados” por los testigos aquellos objetos nocturnos de los que se perciben escasos rasgos. [27, 251][MM] (LAR). Publicación trimestral editada por el grupo español Arp (Alternativa Racional a las Pseudociencias). hasta 1998, cuando tomó su relevo la revista El Escéptico: https://www.escepticos.es/revistas LAWSON, Alvin H. Véase Hipótesis del trauma natal. LAZAR, Robert Scott Véase Área 51. LEY HORARIA Tendencia a la nocturnidad de los avistamientos OVNI constatada estadísticamente por Jacques Vallée en los casos de aterrizaje de la oleada francesa de 1954. Sobre los datos extraídos en esa ocasión, Vallée formuló en 1962 una “ley negativa” (la densidad de casuística es inversamente proporcional a la densidad de población) y dos “leyes positivas”: los objetos denunciados eran figuras de revolución (discos) de pequeño tamaño, y la famosa “ley horaria”. Según ésta, las observaciones de tipo-I eran muy raras durante el día, alcanzaban su máximo entre las 20 y las 0 horas, disminuían desde entonces hasta las 2 LEYENDAS URBANAS Se definen como aquellas historias que la mayoría de la gente escucha (y repite) como relatos verídicos con rasgos modernos y siempre referidos a personas no identificadas (“el amigo de un amigo”); pero aunque los detalles puedan ser actuales, las estructuras e ideas subyacentes son muy antiguas. Algunas de estas leyendas urbanas tienen su origen 138 Lleget Colomer, Màrius sin duda en incidentes reales, pero el paso del tiempo y las sucesivas repeticiones las han ido adornando y redondeando hasta dejarlas demasiado perfectas para ser ciertas. Además, todas estas narrativas orales comparten una formulación común, explotando la paranoia y los temores irracionales, especialmente en los rituales de la vida diaria, exponiendo a la luz prejuicios y avisos morales o sociales. Periodista y escritor, pionero en la divulgación de la Ufología en España. Interesado desde muy joven por la ciencia en general y por la Astronomía en particular, se sintió atraído por los OVNIs a raíz de las noticias de prensa que vio publicadas sobre cohetes fantasmas escandinavos (1946). Fue cofundador en 1947 de la Asociación Astronómica Aster y, en 1958, del Cei de Barcelona. Especialista en el planeta Marte (Marte, la vida en el cosmos, 1971), en 1972 abandonó prácticamente la Ufología a causa del suicidio de dos jóvenes contactados de Terrassa que habían acudido a una de sus charlas. Ameno conferenciante, colaborador de numerosos medios escritos y radiofónicos, fue autor de una treintena de títulos sobre diversos temas, en especial de Astronáutica y Astronomía. De su producción estrictamente ufológica destacan el clásico Mito y realidad de los platillos volantes (Telstar, Barcelona, 1967), Los OVNI en 25.000 palabras (Bruguera, Barcelona, 1974), Nosotros los extraterrestres (Karma-7, Barcelona, 1978), OVNIs: Enigmas del más allá (Producciones Editoriales, Barcelona, 1979) y OVNIs y agujeros negros (Plaza & Janés, Barcelona, 1981). Como ejemplos más conocidos y remotamente vinculados al campo de esta obra podríamos citar las abundantes versiones sobre el autoestopista fantasma sólo en contadas ocasiones es posible localizar a un supuesto testigo (Goss, 1984), o los rumores sobre secuestros de niños o jovencitas en grandes almacenes con destino a la trata de blancas o al tráfico de órganos. Muchos consideran los rumores sobre felinos misteriosos en Gran Bretaña, el Demonio de Jersey o las famosas apariciones del Mothman (investigadas por John Keel) como leyendas urbanas, cuyo más moderno exponente sería la histeria desatada en los últimos años en Centroamérica por el Chupacabras. Ya dentro del campo estrictamente ufológico, podrían mencionarse los rumores sobre platillos estrellados o sobre los famosos Hombres de Negro. Y no hay tanta distancia entre esos helicópteros que, al parecer, sueltan serpientes en paracaídas en los bosques asturianos y aquellos otros mucho más siniestros y totalmente pintados de negro que parecen acosar a algunos abducidos (v. también Antropología de los ovnis). [49, 101, 247][LG] LUCES DE TERREMOTO Véase Hipótesis tectónicas LUMIÈRES DANS LA NUIT (LDLN). Revista bimestral francesa fundada en febrero de 1958 por Raymond Veillith, quien en el editorial de su primer número afirmaba que “muchas cuestiones pueden observarse con nueva luz, a condición de que los elementos decisivos, LLEGET COLOMER, Màrius (Granollers, 1917-Barcelona, 1988). 139 Luz nocturna LUZ SÓLIDA específicos para captarlas, resulten bien evidenciados”. Resumía así la filosofía de la publicación, partidaria siempre de la naturaleza física del fenómeno OVNI y de su origen inteligente, así como de la existencia de una “odiosa conspiración de silencio y su necesidad personal de servir a la verdad”. (luz tubular). Epifenómeno luminoso reportado en algunos avistamientos de OVNIs que consiste en la emisión de haces de luz compactos y coherentes que muestran un comportamiento similar al de los cuerpos sólidos, retrayéndose o doblándose sin dispersión del foco. Dichos tubos luminosos tienen su origen en los OVNIs, desde los que se proyectan generalmente sobre el suelo a modo de soportes. En otras ocasiones aparentan ser orientados con finalidades exploratorias, pudiendo llegar a interactuar con los testigos, como en el suceso acaecido el 28/07/1975 en Sierra Aitana (Alicante), donde un haz luminoso que describió una curva en el aire impactó en los ojos del testigo evitando que cayera por una terraza. El tamaño de estos tubos de luz suele ser pequeño, aunque a veces, como en el famoso caso Trancas (Tucumán, Argentina, 21/10/1963), se han descrito de gran tamaño. Esta última categoría está también presente en diversos casos de encuentros cercanos de tercer tipo (Imjärvi, Finlandia, 07/01/1970) y, sobre todo, en los relatos de episodios de abducción, donde es común ascender a la “nave” a través de un haz de luz. La explicación de hechos tan hetereogéneos y desacostumbrados no puede hacerse de manera generalizada, así que cabe referirla a cada uno de los casos concretos; si bien parece que juega aquí un papel importante la interpretación según los patrones culturales contemporáneos (rayos láser, ciencia ficción) de destellos o ráfagas de luz más bien comunes. [MM] DTU LDLN pasó por diversas situaciones desde su primera época, en la cual la mitad de sus ediciones eran ciclostiladas y escasamente difundidas, hasta su definitivo asentamiento con periodicidad bimestral y calidad offset en los años setenta, cuando contó con unos 5.000 suscriptores. Al amparo de la revista surgió una estructura de organizaciones regionales, de una docena o más de componentes, que en su fase de mayor desarrollo reunió a 1.200 asociados. Gracias a ella se desarrollaron una serie de muy diversas actividades en los campos de la detección de los efectos electromagnéticos de los OVNIs, sesiones de observación del cielo, recogida sistemática de casuística, análisis fotográfico, etcétera. La revista y su grupo homónimo siguen activos, pese a la desaparición de su fundador, tras casi cuatro décadas de actividad, perteneciendo a lo que podríamos denominar la “ufología clásica francesa”, que dispuso en su momento de colaboradores tan conocidos como Aimé Michel e incluso Michel Monnerie, promotor tiempo después de la hipótesis psicosocial. Dirección: Lumières dans la Nuit BP3, 77123 Le Vaudoué, Francia. En Internet: http://www.ldlnufologie.com/ [ JA] LUZ NOCTURNA Véase Clasificación de Hynek. 140 Magnet, proyecto M MAGNET, proyecto Véanse detección de OVNIs propulsión de los OVNIs. Sandell, Nigel Watson, Peter Rogerson y el editor John Rimmer, este boletín se ha convertido casi en el único exponente del mundo anglosajón en defender desde dentro de la propia comunidad ufológica la llamada hipótesis psicosocial. Mantiene un reconocido prestigio y en sus páginas han tenido lugar algunos de los más clarificadores debates sobre abducciones, naves aéreas, platillos estrellados, luces de terremoto, etc. Mención aparte merecen los artículos sobre el nacimiento y auge de la ufología americana desarrollados por Martin Kottmeyer. También es de destacar su empeño en difundir dentro de la comunidad de habla inglesa los trabajos de los ufólogos europeos (fundamentalmente franceses e italianos). En mayo de 1988, conmemorando su vigésimo aniversario, Magonia organizó su primera y única (hasta el momento) Conferencia Internacional. y MAGONIA Boletín trimestral publicado en Gran Bretaña sin vinculación actual con ninguna organización o grupo ufológico. Su publicación se inicio en 1968 como el Merseyside UFO Bulletin (MUFOB), y no fue hasta el otoño de 1979 cuando el número 50 fue rebautizado como Magonia. Este era el nombre utilizado en la Europa medieval para describir un fabuloso reino fuente de todo tipo de visiones y maravillas; se hizo conocido en el contexto ufológico cuando fue empleado por Jacques Vallée en su crucial libro Pasaporte a Magonia. Mediante dicho cambio los editores trataban de destacar algo que ya había ido reflejándose en el boletín con el paso del tiempo: "el misterio OVNI es sólo parte de un enigma mayor donde nos enfrentamos a visiones, rumores, creencias y experiencias humanas" (MUFOB 49, editorial). Desde este momento se incluyen cada vez más artículos sobre otros aspectos aparentemente alejados de la ufología, como satanismo, hipnosis, leyendas urbanas, etc., lo que ha llevado a añadir el siguiente subtítulo desde 1994: "Interpretando las creencias y visiones contemporáneas". Dejó de publicarse en abril de 2009. Los números de la revista pueden consultarse en: http://fi les.afu.se/Downloads/?dir=./ Magazines/United Kingdom/Magonia y en el blog https://pelicanist.blogspot.com/ MAGONIA, catálogo Catálogo mundial de casos Tipo-I (aterrizajes, v. Clasificación de Vallée) registrados entre 1868 y 1968. Recibe este nombre por aparecer como apéndice de la obra de Jacques Vallée Pasaporte a Magonia (1972). Fue elaborado por este Conforme pasaron los años y gracias a la labor de gente como John Harney, Roger 141 Majestic-12 mismo autor y otros colaboradores entre 1961 y 1969, a partir de referencias tomadas de periódicos, revistas ufológicas y libros sobre la materia. Reúne un total de 923 incidentes de los que se expresa su número de orden, fecha, hora, lugar y fuente, seguidos de un breve resumen con los datos de los testigos y la descripción del objeto y los seres asociados al mismo. Pese a haber sido muy criticado por la escasa credibilidad de muchas de sus fuentes, en su momento constituyó un hito histórico que dio lugar a iniciativas semejantes de otros autores.[252][MM] septiembre de 1947 por orden ejecutiva del Presidente Truman, siguiendo la recomendación del Dr. Vannevar Bush y del Secretario de Estado de Defensa, James Forrestal. Se trataba de las siguientes personalidades: Almirante Roscoe H. Hillenkoetter, Dr. Vannevar Bush, Secretario de Estado James Forrestal, General Nathan F. Twining, General Hoyt S. Vandenberg, Dr. Detlev Bronk, Dr. Jerome Hunsaker, Sidney W. Souers, Gordon Gray, Dr. Donald Menzel, General Robert M. Montague y Dr. Lloyd V. Berkner. Todos ellos eran individuos muy conocidos en la época, importantes científicos, miembros de los Servicios de Inteligencia, o responsables políticos de altísimo nivel. MAJESTIC-12 Nombre dado a un supuesto grupo de doce personalidades norteamericanas (científicos, políticos y militares) que pertenecerían a un comité secreto creado a partir de septiembre de 1947 en respuesta a los problemas originados en las esferas gubernamentales por la presunta caída en las proximidades de Roswell (Nuevo México) de una nave extraterrestre tripulada y su posterior recuperación. La primera parte del documento recoge los elementos sustanciales que preocupaban al Presidente de los EE.UU. y a sus colaboradores, centrados en los platillos volantes de la época. Curiosamente, se alude a la observación del 24/6/1947 cuyo testigo, Kenneth Arnold, alcanzaría gran protagonismo en toda la literatura especializada posterior. Se menciona también la falta inicial de pruebas específicas sobre el fenómeno, hasta que se descubren en una zona de Nuevo México los restos de un objeto en forma de disco. Se afirma que el 7 de julio de 1947 se puso en marcha una operación militar secreta para recuperar esos restos y asegurar su estudio en condiciones adecuadas. Igualmente, que se hallaron cuatro pequeños seres humanoides muertos a consecuencia del accidente y cuyos cuerpos fueron también recogidos. Los testigos, tanto militares como civiles, fueron conminados a guardar el secreto y se ofreció a la prensa una explicación tranquilizadora basada en la recuperación de los restos de un gran globo meteorológico. En junio de 1987, durante el congreso anual del Mufon, el ufólogo Bill Moore dio a conocer públicamente los documentos top secret que hacían referencia a la creación del comité Majestic-12. Tres años antes, uno de sus colaboradores, Jaime Shandera, había recibido en su domicilio una serie de negativos fotográficos que contenían reproducciones de tales documentos, consistentes en ocho páginas mecanografiadas en las que aparecía con profusión el sello Top Secret/Majic/Eyes Only. En la segunda página se relacionaban los distintos miembros del Majestic-12, creado -según dichas fuentes- el 24 de 142 Majestic-12 El documento explica que el 19 de septiembre de 1947 se concluyó que el objeto discoidal era probablemente un artefacto de reconocimiento con una área de actuación reducida. El análisis de los cuerpos humanoides, comunicado el 30 de noviembre de 1947 por el Dr. Detlev Bronk, revela notables diferencias en los procesos de evolución biológica respecto a los seres humanos. Se alude a los cuerpos como “EBEs”, “Entidades Biológicas Extraterrestres”. Entre sus conclusiones existe también la sugerencia de que el origen de tales seres probablemente sea externo a nuestro propio sistema solar. por sus continuas actividades en pro de la desclasificación de los documentos oficiales ufológicos, fue el primero en publicar una refutación muy puntual de Majestic-12. Basándose en datos como fechas, estilo de las diversas fórmulas oficiales empleadas, tipo de máquina de escribir, relación de nombres que cita el documento como integrantes del comité y otros muchos, afirmó con rotundidad que se trataba de un montaje. Tiempo después, su conclusión se vio reforzada por el descubrimiento de Philip Klass referente a que la firma del Presidente Truman, estampada al final del memorándum, no era original. Klass afirma, sin la menor duda, que se trata de una reproducción extraída de una carta que el Presidente dirigió a su consejero científico Vannevar Bush, conservada en la Biblioteca del Congreso. Como consecuencia de las dificultades para explotar en profundidad los materiales recuperados en Nuevo México, se promovió un programa específico para el estudio de los platillos volantes, supuesto origen del proyecto Sign de la USAF. Junto a esta polémica, muy aireada en las publicaciones y congresos especilizados americanos, estalló el definitivo escándalo cuando Bill Moore, primer difusor público del memorándum Majestic-12, confesó en el congreso del MUFON de 1989 que desde hacía ocho años colaboraba con algunas agencias del gobierno para difundir determinadas informaciones manipuladas entre la comunidad ufológica americana. La credibilidad del “portavoz público” del comité Majestic se vino entonces abajo, haciendo recaer sobre sí todas las sospechas de ser el autor necesario para la elaboración del informe. El informe Majestic recoge también otros presuntos casos de platillos estrellados que tuvieron lugar dos años después, en 1950, cerca de la frontera mexicana, en el estado de Texas. Finalmente, la documentación explica que razones tecnológicas e internacionales, así como la necesidad de evitar a toda costa un pánico colectivo, obligaban a adoptar estrictas medidas de seguridad para cualquier administración norteamericana, consagrando el dispositivo secreto MJ-194904P/78 (Top Secret/Majic/Eyes Only) como medio para controlar públicamente este tipo de información. La polémica, evidentemente, no ha quedado cerrada con estas revelaciones. Determinados sectores de la ufología americana, encabezados por Stanton Friedman o el propio Bruce Maccabee (autores ambos de muchas páginas dedicadas al Majestic-12), estiman que pudo existir manipulación por intereses políticos. Tras la publicidad proporcionada al supuesto documento secreto por Bill Moore, surgió una controversia centrada principalmente en la autenticidad del mismo, origen y consecuencias sociales. Su compatriota Barry Greenwood, conocido 143 Manises, caso No obstante, opinan que el memorándum, a pesar de tratarse de un documento falso, contiene información auténtica sobre lo ocurrido en los años cuarenta y la línea política adoptada por las sucesivas administraciones americanas para ocultar la verdadera naturaleza de las observaciones OVNI. tres luces rojas con destellos de diversos colores y de un tamaño cuatro veces superior al de Venus que se elevaron lentamente en dirección al sur. Mientras tanto, un caza Mirage F-1 despegó de la base de Los Llanos (Albacete) con la misión de interceptar los objetos reportados. El piloto militar comunicó que, pese a tener las luces de colores a la vista, cada vez que intentaba acercárseles éstas se alejaban, se producían interferencias en la radio y no funcionaba la cámara fotográfica, por lo que abandonó la persecución poco después de una hora y 45 minutos. Con posterioridad han seguido apareciendo documentos relacionados con este supuesto grupo (p. ej.: un manual de operaciones), siempre mediante el mismo procedimiento: un rollo de negativos fotográficos de supuesta documentación oficial. [90, 103, 130, 144][ JA, LG] Días después, el 16 de noviembre, el diario palmesano Última Hora publicó una fotografía del supuesto OVNI de Manises como empezó ya a conocerse el caso- tomada por el contactado mallorquín Pep Climent. En ella aparecía una luminosidad triangular entre las montañas de la que se afirmaba que había sido vista a las 2:30 horas del día 12. Según se dijo más tarde, los negativos de la toma habían sido confiscados por el Ejército del Aire, pero lo cierto es que no hay constancia de tal hecho y no figuran en el expediente del caso desclasificado en agosto de 1994. No obstante, diversos análisis efectuados sobre la imagen por el Gsw, así como la investigación sobre el terreno del periodista Mario Alberto Morales (Ballester, 1984), han permitido determinar sin lugar a dudas el carácter fraudulento de esta fotografía, que no mostraría más que un reflejo del sol en la lente de la cámara. MANISES, caso El 11 de noviembre de 1979, un avión Super-Caravelle de la compañía aérea española TAE que efectuaba el vuelo chárter Salzburgo-Palma-Tenerife contactó pasadas las 22:00 horas con el Control Aéreo de Barcelona para conocer si existía algún tráfico (otra aeronave) en sus cercanías. El motivo de la consulta obedecía a que en esos momentos, cuando después de haber despegado de Mallorca se sobrevolaba Ibiza, la tripulación estaba divisando dos luces rojas a su misma altura y a una distancia estimada de tres millas del aparato. Desde Barcelona se informó que no había ningún otro avión en las proximidades, pero el comandante insistió en que el tráfico desconocido se estaba aproximando cada vez más, casi a media milla, y que prefería aterrizar en Valencia antes de comprometer la seguridad del pasaje. Tras otorgársele autorización para la maniobra, desde el aeropuerto de Manises diversas personas, entre ellas el propio director de la instalación, avistaron durante unas dos horas El suceso recibió un tratamiento desmesurado por parte de los medios de comunicación, hasta el punto que el entonces diputado socialista Enrique Múgica Herzog elevó el 26 de septiembre de 1980 una pregunta parlamentaria al 144 Marciano Gobierno en relación a estos hechos, que nunca fue contestada. MARTE En la actualidad parece claro, según las investigaciones de V.J. Ballester Olmos y J.A. Fernández Peris, que lo divisado desde el aeropuerto de Manises responde a estímulos astronómicos, en tanto que para el resto del episodio se han señalado las luces de la refinería de Escombreras (Murcia) como causa del avistamiento de la tripulación (v. J.A. Fernández Peris: El expediente Manises, Fundación Anomalía, 2000). Es de destacar que el pasaje del avión, unos 109 turistas austríacos y alemanes, no advirtió nada del incidente y nada más aterrizar formuló diversas reclamaciones por lo que consideraban una avería del aparato. Se ha apuntado igualmente a los serios problemas psicológicos del comandante como causa concurrente de los hechos. [29, 232][MM] En toda la historia de la humanidad nunca ha habido un astro de nuestro sistema solar que despertase tantos ecos en la imaginación literaria como el denominado “planeta rojo”. Durante más de un siglo, Marte ha sido el hogar privilegiado para albergar los primeros seres extraterrestres, nuestros vecinos más próximos. Los escasísimos pioneros, exploradores del cielo, que dispusieron de medios técnicos adecuados, telescopios con la suficiente capacidad de ampliación y definición, ya establecieron en el siglo XVIII sus rasgos principales. Descubrieron un planeta cambiante según las estaciones, con nubes en movimiento e inverosímiles campos de hielo que recordaban algunas regiones de nuestro propio mundo. La identificación con la teoría sobre la “pluralidad de mundos habitados” impulsó la inclinación de aquellos primeros investigadores de Marte a identificar las manchas oscuras visibles en sus telescopios con oceanos, y las de apariencia rojo/anaranjada con desiertos. MARCIANO 1. Supuesto habitante del planeta Marte. 2. Nombre con el que popularmente se conocen los hipotéticos seres extraterrestres inteligentes. 3. Lengua con la cual la médium Hélène Smith (pseudónimo de Catherine Elise Müller) se expresaba durante sus supuestos contactos telepáticos con habitantes de Marte a finales del s. XIX. Llegó de este modo a describir la vida marciana, sus características, flora, fauna, etc., creando gran expectación en el mundo científico de la época. Théodore Flournoy, profesor de Psicología de la Universidad de Ginebra, estudió el caso y concluyó que se trataba de una mera creación inconsciente de la médium, pues cada letra marciana tenía su equivalencia exacta en el francés, lengua materna de Smith. [202][MM] Es en Italia en 1869, cuando Pietro Angelo Secchi utiliza el término canali, para referirse a determinadas líneas finas y no bien definidas que unían aparentemente las zonas más oscuras de grandes dimensiones, distribuidas un poco al azar por la superfice del “planeta rojo”. Tiempo después, su compatriota Giovanni Schiaparelli, pudo realizar una serie de observaciones mucho más detalladas. De ese modo, identificó los canali con una completa red que cubría Marte, algunos de ellos de líneas pares y paralelas. Casi a finales del pasado siglo, el astrónomo norteamericano Percival Lowell identificó aquellos canali con un completo 145 Marte sistema de irrigación. En su interpretación, enormes canales de centenares de kilómetros servirían para mantener la vegetación estacional del suelo marciano, transportando agua desde las regiones polares hacia el ecuador. Había nacido así la avanzada civilización del planeta Marte, cuyos ecos nos han llegado hasta las postrimerías del siglo XX con la misma intensidad que hace un siglo, si no compruébese la agitación provocada en todos los medios de información tras el descubrimiento, en agosto de 1996, de posibles restos de vida marciana en un meteorito hallado en la Antártida (v. Exobiología). superficie por medio de los Mariner, proporcionó nuevas evidencias sobre el verdadero aspecto y evolución del mundo marciano. Junto a gigantescas montañas como el Olimpus Mons, las mayores del sistema solar conocidas, se abren vastos cañones que bien pudieron ser confundidos por Lowell con sus canales de irrigación, cráteres aparentemente de origen volcánico y meteorítico, así como formaciones sedimentarias y enormes depósitos de polvo, movidos estacionalmente en forma de tormentas de arena, causantes de las manchas oscuras descritas por Schiaparelli. Su aspecto desértico anula muchas de las iniciales posibilidades de descubrir la ansiada “civilización del planeta Marte”, descrita por Burroughs, Wells, Asimov o el mismísimo Ray Bradbury en sus amplios frescos de ciencia ficción. No obstante, y a pesar de la impresión inicial negativa, existe agua en diversas formas y suficiente cantidad en las arenas marcianas. Los científicos afirman que son tres los modos posibles de conservación de tan preciado elemento. El primero, en forma de hielo, mezclado con nieve carbónica en los polos del planeta. También mezclado con el suelo, en forma de permafrost, similar al que compone el continente Antártico. Su profundidad podría alcanzar un kilómetro en el ecuador y unos ocho en la próximidad de los polos y el tercer medio, en forma molecular enlazado químicamente en las redes cristalinas de los minerales del suelo, lo que se denomina agua de hidración. Conocer las auténticas posibilidades del planeta Marte para albergar algún tipo de vida ha estado en la mente de los científicos desde las primeras observaciones astronómicas. La capacidad para mantener una cierta atmósfera similar en sus componentes a la de la Tierra, aunque mucho más leve, propició toda suerte de cálculos de aproximación, hasta llegar a las primeras mediciones realizadas por los satélites lanzados hacia la órbita del planeta, los Mariner, que entre 1964 y 1972 facilitaron los primeros datos bastante fiables y enfriaron totalmente el ambiente. La presión atmósferica en la superficie marciana oscila entre 5 y 10 milibares, apenas una fracción mínima de la terrestre, que es de 1.000 mb a nivel del mar. La temperatura del suelo oscila entre -112º C en la zona nocturna a 25º C por el día en la estación veraniega, son por tanto condiciones muy penosas para poder albergar una vida altamente desarrollada, al menos en la forma que se conoce en la Tierra. Existiría pues agua en Marte, quizás suficiente para la pervivencia de una vida muy elemental oculta a la radiación ultravioleta letal y esterilizante en profundas grietas o cavidades subterráneas. En 1976, el Viking 1, después de un largo periplo de La exploración del planeta Marte, iniciada mediante la cartografía de su 146 Marte decenas de millones de kilómetros por el espacio, llega a la superficie de Marte con el propósito de establecer por medio de diversos análisis químicos la existencia o no de vida, al menos en ese pequeñísimo espacio objeto de su exploración. Las imágenes emitidas hacia la Tierra mostraban un lugar caótico, cubierto de piedras, dunas y polvo rojizo de óxido de hierro. Ninguna de estas sondas ni los experimentos prebióticos preparados por los científicos pudieron descubrir las menores señales del tan esperado espécimen marciano. Ningún enano verde acudió al encuentro, Marte permanecía mudo. iluminación situada en el ángulo apropiado, tal como dejaron patente en abril de 1998 las imágenes captadas por la sonda Mars Global Surveyor. El enigma de las presuntas pirámides y la “esfinge” marciana no ha contribuido a clarificar ninguno de los problemas planteados, simplemente es un rasgo más de la enorme expectación que se cierne en torno al “planeta rojo”. Lo que parece indiscutible hasta este momento, es la ausencia total de vida en los restos analizados por el último satélite Viking que logró alcanzar suavemente su superficie. Se apuesta por la posibilidad de que durante los períodos en los cuales el agua de los polos alcanza su fase líquida, y en zonas profundas a resguardo de la implacable radiación ultravioleta que día tras día bombardea el suelo marciano, se manifiesten las condiciones necesarias para que sobrevivan determinados microorganismos, aunque puede pasar mucho tiempo antes de que estemos en condiciones de comprobarlo. Pero frente a estos datos proporcionados por los satélites, Marte no dejaba de sorprender y los amantes de lo insólito también recibieron su recompensa. Entre las numerosas imágenes enviadas a las estaciones terrestres por el Viking 1 y 2, aparece una sección del planeta Marte, captada desde 1870 Kms. de altura, próxima a la región denominada Mare Acidalium, que presenta varias formaciones de aspecto regular y bases cuadradas semejantes a enormes pirámides egipcias y de aproximadamente tres kilómetros de diagonal. Próximo a ellas se observa en otra imagen un enorme rostro de apariencia humana, aparentemente esculpido en la piedra de Marte, que muchos compararon con una inmensa esfinge que mira ciegamente hacia el espacio. Como es lógico, estas fotografías desataron una enorme polémica entre los que sustentaban la existencia de una antigua civilización en el planeta rojo, aunque los planetólogos manifestaron su clara opinión de que la explicación más probable era que se trataba de formaciones geológicas elaboradas por la erosión, provocada por las tempestades de arena en el suelo marciano más el azar y una En el verano de 1877, el astrónomo americano Asaph Hall descubrió, explorando con su telescopio el espacio próximo a Marte, una débil luz, su luna exterior Deimos. Poco tiempo después detectaría la luna más próxima y brillante, que se conocería a partir de ese momento como Fobos. Ambos, son cuerpos de pequeñas dimensiones, con forma de patata, y numerosos cráteres de superficie negrísima recubierta de polvo. Tanto los satélites Mariner como los Viking, fotografiaron y dirigieron sus instrumentos hacia las dos lunas marcianas, descubriendo que por su composición podrían ser asteroides de tipo carbonáceo procedentes del llamado cinturón de asteroides ubicado más allá de la órbita de Júpiter. Las órbitas de estas lunas son muy curiosas. Deimos tarda treinta 147 Matrix, informe horas y un cuarto en orbitar en torno al planeta, y Fobos lo hace en siete horas y media. Teniendo en cuenta que Marte posee un período de rotación ligeramente superior a 24 horas, Deimos se desplaza muy lento en su cielo, demorándose sesenta horas desde su aurora hasta el ocaso. Fobos por su veloz período de traslación, solamente emplea cuatro horas y media para cruzar el cielo, tiempo en el que un observador ubicado en Marte contemplaría los distintos cambios de fase de esa pequeña luna. Estas características hicieron especular a algunos astrónomos sobre la naturaleza física de Fobos, apuntando la posibilidad de que estuviera hueco y se tratase realmente de un objeto artificial (v. Tierra hueca, teoría de la). Las pruebas acumuladas por los distintos satélites durante dos décadas demuestran que se trata de una roca de 19 x 27 kms., cuya seña de identidad más importante es el cráter Stickney, de 8 kms. de diámetro. Book Service, y en él se recopilaban artículos de prensa, cartas privadas, entrevistas y fotografías de diversas fuentes, destacando unas declaraciones del piloto John Lear al Sun de Las Vegas del 22/05/1988 y el testimonio del informático Paul Bennewitz. La tesis propuesta por The Matrix era la existencia de un supuesto pacto entre el gobierno norteamericano y una civilización alienígena procedente de la estrella Zeta Retículi. Las prestaciones serían tecnología extraterrestre a cambio de permitir a los grises realizar abducciones, mutilaciones de ganado y experimentos genéticos sobre suelo estadounidense. El primer contacto habría tenido lugar en la Base de la Fuerza Aérea de Holloman en 1964, y desde entonces esa colaboración alieno-terrestre habría ido en aumento hasta el punto de acondicionarse bases subterráneas para la construcción de aeronaves inspiradas en diseños extraterrestres. Uno de esos lugares sería el conocido como Groom Lake, o Área 51. Sin duda la futura exploración del planeta Marte, que se ha visto retrasada tanto por el recorte de proyectos como por el fracaso de algunos satélites, proporcionará las respuestas definitivas, a la espera de que el hombre pueda dar sus primeros pasos en las arenas rojizas de un mundo que siempre ha sido la residencia de los míticos marcianos, los primeros extraterrestres del sistema solar. Más información en Internet: https:// mars.nasa.gov/ [24][ JA] En resumen, lo que Bennewitz y Lear aseguraban era que a los abducidos se les implantaba una especie de microchip en el cerebro a través de las fosas nasales, con objeto de controlar su comportamiento y enviarles instrucciones desde sus naves (o bases terrestres) mediante ondas de baja frecuencia, como las que Bennewitz creyó interceptar mientras estudiaba un caso de abducción ocurrido en 1980 (Cimarrón, Nuevo México). Otros objetivos que perseguirían los “grises” con las abducciones serían los de crear una raza híbrida para dominar la Tierra, usando técnicas de fecundación artificial con mujeres humanas a las que extraerían los fetos antes de nacer. Otra denominación que popularizó The Matrix para referirse a los “grises” fueron las siglas Ebe, aparecidas por primera vez en uno MATRIX, informe Obra en varios volúmenes atribuida a Valdemar Valerian, pseudónimo tras el que se ocultaría, al parecer, un capitán de AFOSI (Air Force Office of Special Investigations) llamado John Grace. El primero, de 361 páginas, fue publicado en 1988 por Arcturus 148 Maugé, Claude de los documentos Majestic-12. que componen aplicación no reduccionista de la HPS. En 1989 presentó un ensayo, luego devenido en clásico, donde proponía una definición “dialéctica” del estímulo OVNI y una nueva clasificación de las experiencias según la calidad de los informes. Ha reunido la primera “bibliografía comentada de la literatura ufológica en lengua francesa”, donde reseña 252 títulos, recogida en la obra de Pinvidic (ed.) OVNI: vers une anthropologie d’un mythe contemporain (Ed. Heimdal, Bayeux, 1993, pp. 515-550.) Dos años después, William L. Moore confesó haber colaborado con elementos de AFOSI destinados en Kirtland AFB, base desde la que Bennewitz creía que se “monitorizaba” a distintos abducidos. Moore reconoció que había participado en una campaña desinformativa contra Bennewitz, que, entre otros apartados, incluyó todo lo relativo al acuerdo entre EE.UU. y los EBEs. De este modo la credibilidad de The Matrix, de por sí precaria, se vio todavía más mermada, ya que uno de sus pilares eran precisamente las declaraciones de Paul Bennewitz. Pese a todo, en 1990 se publicó la segunda entrega, nuevamente firmada por Valdemar Valerian y distribuida por el grupo Nevada Aerial Research de Las Vegas, constituido en torno a las ideas de John Lear. Es aquí donde rizando el rizo de lo absurdo se menciona (págs. 85 y ss.) uno de los platos favoritos de los “grises”: absorber con sus manos un caldo elaborado con sangre humana extraída a algunos abducidos, pobres despojos a los que conservarían vivos en enormes cápsulas e inmersos en un líquido ámbar. [89, 226] [ JM] Opinión sobre el fenómeno (tomada de su artículo “Le phénomène OVNI: un bref état de la question”, en Pinvidic, op. cit.): “A pesar de algunas objeciones perfectamente legítimas, parece que la hipótesis socio-psicológica ha comenzado a tomar cierta consistencia, volviendo cada vez más inútil invocar a extraterrestres o a fenómenos psi. De todos modos, para tener acceso a un eventual fenómeno OVNI en sentido estricto, es necesario descortezar la escoria sociopsicológica con la que los medios y los ufólogos nos han embetunado durante más de 40 años. Puede ser que la pepita, en definitiva, esté hecha del mismo material que la ganga, algo que temo bastante, pero también puede que sea de un metal brillante y precioso. La paradoja sería entonces que los ufólogos hubiesen impuesto un ocultamiento del fenómeno verdadero de una manera bastante más eficaz que la de los medios oficiales o de los científicos racionalistas que tanto deploran....” MAUGÉ, Claude (n. 1946). Ufólogo francés, profesor de enseñanza secundaria de Matemáticas y Física, y destacado sistematizador de las ideas psicosociológicas. Autor de una demoledora revisión del catálogo de Claude Poher y compilador de un completo “listado de casos OVNI/OVI clásicos” (con 525 entradas en 1988), Maugé fue uno de los primeros ufólogos de la generación de Michel Monnerie que se enrolaron en una MEDIOS DE COMUNICACIÓN Ver Tabloides. 149 Menzel, Donald H. MÉHEUST, Bertrand Esta es la única que permite resolver la contradicción (según la cual) es posible estar sano, obrar de buena fe y pretender, sin embargo, haber sido abducido por extraterrestres (...) Es una propiedad paradójica pero esencial del espíritu humano elaborar como reales, y también como excesivamente reales, los seres fantásticos de la cultura. Si esta hipótesis es correcta, los relatos de abducción demostrarían que la facultad de recrear por medio del trance no sólo está reservada a los «primitivos» sino que sobrevive en el occidente contemporáneo.” (n. 1947). Ufólogo francés y profesor de filosofía de enseñanza secundaria, profesión que ha desempeñado en Argelia, Gabón y Francia. Aunque más conocido por sus aportaciones teóricas, ha desarrollado una intensa labor de campo especialmente en Borgoña y Gabón, donde estudió el conocimiento de los estererotipos ufológicos en poblaciones no expuestas al mito de los platillos volantes (“Proyecto Nabokok”). Es autor de dos influyentes libros, Science Fiction et soucoupes volantes (Mercure de France, París, 1978) y Soucoupes volantes et folklore (Mercure de France, París, 1985), donde aborda la dimensión folklórica del fenómeno OVNI, que para Mehéust debe entenderse como una reactualización de antiguos patrones culturales ya presentes en relatos medievales y en las obras de cienciaficción de principios del siglo XX (v. Ciencia ficción, ufología y). En trabajos posteriores abandonó la hipótesis paraufológica, que lo emparentaba con la escuela de Vallée, para enfrentarse con los partidarios de que los relatos de abducciones se basan en experiencias reales, como puso de manifiesto en las actas de una polémica que mantuvo con el etnólogo pro-OVNI norteamericano Thomas “Eddie” Bullard (Sheffield, Inglaterra, 1991). MENZEL, Donald H(oward) (1901-1976). Considerado como el más destacado debunker en la primera época de la Ufología, Donald Menzel, un astrónomo americano bien conocido, fue autor de tres libros y numerosos artículos que intentaban explicar el fenómeno OVNI como una combinación de sucesos naturales y otros originados por el hombre. Pese a que la mayoría de los defensores de los OVNIs lo consideraban su "archienemigo", sus explicaciones eran por lo general bastante razonables, y desde luego el Dr. Menzel poseía la suficiente preparación técnica para evaluar tal tipo de casos. Aunque también es cierto que sólo muy raramente hacía comprobaciones sobre casos concretos, prefiriendo dar explicaciones más teóricas y generalistas. Sin embargo, una vez llegó a afirmar: “en nuestra propia Vía Láctea pueden existir hasta un millón de planetas, todos habitados por vida inteligente”. Opinión sobre el fenómeno (tomada de su artículo “Los OVNIs, el mito y el folklore”, en Más Allá, número monográfico, 1991.): “Si tenemos en cuenta que no existen pruebas concretas susceptibles de apoyar la hipótesis suprahumana (para explicar los casos de abducción), debemos concentrar toda la atención sobre la hipótesis de que se trata de vivencias alucinatorias no patológicas, es decir, estados de trance espontáneos. (...) Tras obtener su doctorado en astrofísica por la Universidad de Princeton en 1924, Menzel enseñó astronomía en varias universidades y trabajó en el observatorio 150 MIB Lick de la Universidad de California hasta 1932. Entonces entró en la facultad de Harvard donde llegó a ser profesor emérito del Harvard College Observatory hasta su muerte a finales de 1976. Menzel fue miembro de diversas sociedades científicas y presidente de la Sociedad Astronómica Americana (1954-56) y recibió muchos premios y distinciones. Sus principales actividades de investigación se centraban en la astronomía solar y estelar, atmósferas planetarias, propagación de radio y problemas ionosféricos. Ubaye-Serre-Ponçon, 1919-1992). Investigador francés, pionero en Europa del estudio del fenómeno OVNI con pretensiones científicas. Licenciado en Filosofía y Letras, matemático e ingeniero, compensaba con una vasta erudición las deformidades físicas que padecía. Comenzó a interesarse por los OVNIs a raíz del seguimiento que hizo de las observaciones de cohetes fantasmas escandinavos (1946), y en 1954 publicó su primera obra sobre el tema: Lueurs sur les Soucoupes Volantes. Ese mismo año se produjo en Francia una intensa oleada de avistamientos que Michel fue recopilando gracias a los corresponsales que había reunido entre los lectores de su libro. El análisis de la casuística denunciada le condujo a formular su hipótesis de las ortotenias, expuesta en su obra Mysterieux objects celestes (1958; trad. española: Los misteriosos platillos volantes, 1963). Convertido desde entonces en la máxima figura de la ufología gala, su pensamiento racionalista y pro-HET ejerció una marcada influencia en Francia hasta el advenimiento de las concepciones psicosociológicas. Retirado en 1975 de su trabajo en la RadioTelevisión francesa, en sus últimos tiempos se inclinó hacia los aspectos psíquicos y místicos del fenómeno, hasta que en 1980 abandonó por completo el cultivo de la Ufología. Bibliografía OVNI: Flying Saucers (1953); The World of Flying Saucers, con Lyle Boyd (1964); y The UFO Enigma: A Definitive Explanation of the UFO Phenomenon, con Ernest Taves (1977). Opinión sobre el fenómeno (extraída y adaptada de “UFOs: The Modern Myth”, artículo de Donald Menzel publicado en Carl Sagan y Thornton Page (eds.), UFOs-A Scientific Debate, 1972): “En mi opinión, la cuestión de si existe vida inteligente en nuestro sistema solar o más allá es irrelevante para esta discusión. Tampoco niego la posibilidad de que algún día nos visiten realmente seres de otros planetas. Sólo afirmo que los informes de OVNIs conocidos hasta la fecha no representan ningún tipo de actividad extraterrestre.” Opinión sobre el fenómeno (tomada de Ronald D. Story (ed.), 1980: The Encyclopedia of UFOs): “Si la consecución del nivel de complejidad psicológica de los seres humanos es un fenómeno normal y no milagroso, se debe admitir entonces la existencia de un medio psíquico cósmico que habría sobrepasado el nivel de complejidad humano hace algunos millones de años. (...) Según mi entender, basado en premisas demasiado complicadas para exponerlas MIB Siglas de Men In Black, en inglés hombres de negro. MICHEL, Aimé (Saint Vicent-les-Forts, actualmente 151 Mito aquí, los OVNIs son una manifestación de ese medio, presente, de alguna ignota manera, desde el origen del sistema solar. Creo que sería un gran misterio que algo como los OVNIs no existiera, ya que estaría en contradicción con todo lo que sabemos sobre astrofísica, biología, etcétera; pues la existencia de los OVNIs con toda su insondable extrañeza se adecúa a lo que la ciencia nos permite esperar. ” caso lo que hacemos es interpretar el estímulo simbólicamente, creando nuevas categorías para las experiencias anómalas, a las que damos los nombres de milagros, fenómenos paranormales, ovnis, etc., de manera que nunca quede un estímulo en la indefinición”. Dentro del gran mito de los extraterrestres se han desarrollado otros mitos como resultado de la inaprensibilidad e indefinición permanente de toda la Ufología. Ello ha propiciado una progresiva complejización que permite su supervivencia en el seno de las sociedades occidentales. La industria cinematográfica ha tenido gran influencia en este sentido. Desde las películas de serie B de los años 50 con malvados alienígenas reflejo de la guerra fría y el peligro comunista, a los años 80 con los filmes de Spielberg que estereotiparon física y moralmente al extraterrestre bueno. Más recientemente Independence Day (1996) recupera a los extraterrestres malvados, aunque no parece que vaya a influir en la evolución del mito ovni; más bien se trata de un filme con claras implicaciones ideológicas y propagandísticas. Entre los mitos de la cultura ufológica destaca el de las abducciones, rapto de seres humanos por parte de extraterrestres para fines experimentales, con amplia incidencia en Estados Unidos aunque escasa en España. Los OVNIs accidentados (v. platillos estrellados), o ufo crash, también forman parte del folklore ovni. El ejemplo clásico es el supuesto accidente de un platillo volante en Roswell, Nuevo México, en julio de 1947, en donde se habrían descubierto los cadáveres de varios alienígenas. Otro importante mito es el del encubrimiento oficial de información relativa a los OVNIs, recientemente propalado gracias a MITO Fábulas o ficciones alegóricas, especialmente en materia religiosa. Según el historiador de las religiones Mircea Eliade “el mito cuenta una historia sagrada, un acontecimiento que ha tenido lugar en el tiempo primordial, en el tiempo fabuloso de los comienzos”. En nuestras sociedades occidentales desacralizadas el mito juega un papel de menor importancia; no es vivido como lo Real por excelencia, sino como lo misterioso y enigmático, aquello que la ciencia (para algunos el mito de la Modernidad junto con sus aplicaciones tecnológicas) no ha podido explicar adecuadamente. En el terreno de la Ufología el principal mito y el que ha dado origen en gran medida a todas sus ramificaciones, es la venida de seres extraterrestres al planeta Tierra a bordo de naves interplanetarias. Como señala Cabria (1993; 206), “cuando visualizamos algo que no podemos clasificar en nuestro sistema de categorías podemos ignorarlo por incómodo, aislarlo como una anomalía científica o distorsionarlo para hacerlo encajar en nuestro esquema mental” (fenómeno que se produciría en el caso del síndrome de transgresión de la realidad). Según Cabria, “en este último 152 MOC series como Expediente X. Estados Unidos sería el paradigma de tal labor de desinformación ufológica; por ejemplo los documentos del Majestic-12, una supuesta comisión de científicos americanos para estudiar los restos de Roswell y que fue un monumental fraude. En España el secretismo oficial se centraba en el Ejército del Aire, que custodiaba numerosos informes OVNI con categoría de información confidencial hasta que en 1992 se inició el proceso de desclasificación. Con él caía un viejo mito; quizá por eso cierto sector de investigadores no han visto con buenos ojos su puesta en marcha. MOC Siglas de Misterieux Objets Célestes (misteriosos objetos celestes, en francés), denominación dada por la aviación francesa y suiza a los platillos volantes. Según Jacques Vallée (1966), tal nombre no era sino un intento de despojar al fenómeno de una terminología desagradable que no podía ser empleada en el campo científico. Estas siglas tuvieron escaso éxito por imponerse UFO u OVNI, y su utilización se limitó a la terminología del GEPAN en tiempos de Claude Poher. [250][ JR] Otros mitos o simples ideas apriorísticas hacen referencia, en el entorno de interesados menos críticos, a la exactitud del testigo en el momento de informar de su observación, y por lo mismo, a que las explicaciones convencionales sólo son pertinentes cuando estas se ajustan exactamente a la descripción proporcionada por el testigo. MONNERIE, Michel (n. 1940) Ufólogo francés, restaurador de obras de arte en los Archivos Nacionales de París y pionero de la hipótesis psicosociológica. Poseedor de una reconocida trayectoria dentro del movimiento ufológico francés, fue coordinador desde finales de los sesenta del RESUFO, la red de detección fotográfica de Lumières dans la Nuit, y director entre 1973 y 1978 de la desaparecida SPEPSE (Societé Parisienne d’Etudes des Phénomènes Spatiaux et Etranges). En su primer libro, Et si les OVNI n’existaient pas?, (Editions Les Humanoïdes Associés, París, 1977) bosqueja su Hipótesis Psicosociológica, según la cual los informes OVNI son objetos o fenómenos explicables no reconocidos por el testigo. Más tarde, en Le naufrage des extraterrestres (Nouvelles Editions Rationalistes, París, 1979), sistematiza la HPS en un contexto histórico, planteando que el fenómeno OVNI es un mito surgido en la era de la conquista espacial. Aunque actualmente ha reducido su actividad a alguna colaboración esporádica en medios Lo que parece indudable es en que las sociedades modernas, a pesar de su profunda desacralización, siguen perviviendo algunos mitos, y de forma destacada el de la existencia de extraterrestres que nos visitan. Decía Eliade que a través de los mitos se manifestaba lo sagrado; en el caso de los ovnis, gracias a su significado connotativo (naves extraterrestres), estos se habrían convertido, al menos para algunos grupos de culto como los contactados y las sectas platillistas, en una manifestación de seres superiores, en una hierofanía en la que confluirían el sentido por lo maravilloso bajo la inspiración de la técnica y el control de la información, como manifestación de poder en un mundo secularizado en el que reviven viejos patrones creenciales (v. también Antropología de los ovnis). [54][RC] 153 Monteiro, Cassiano José especializados, sigue siendo considerado como uno de los autores fundamentales de la ufología contemporánea. creación de la Cnifo. Mantiene desde hace años contactos regulares con los ufólogos españoles, siendo representante en Portugal de Cuadernos de Ufología y activo colaborador de V.J. Ballester Olmos y J. A. Fernández Peris en la confección de sus catálogos sobre aterrizajes ibéricos. Por su parte, es autor (junto a Luis R. González en su fase inicial) de un Catálogo de Observaciones de Humanoides en la Península Ibérica, publicado por la CNIFO en 1983 y en cuya actualización sigue trabajando. Interesado en los casos OVNI de mayor complejidad y con aspectos ligados al folklore local, defiende una mayor aproximación del fenómeno a la ciencia, tanto en el aspecto de la aplicación de métodos objetivos de investigación como en el del mayor compromiso de los científicos en el estudio del problema. Opinión sobre el fenómeno (extraída del prefacio de su libro Le naufrage des extraterrestres, op. cit.): “A. El mito extraterrestre, perfectamente creíble, tecnológicamente posible, forma un cuadro universalmente aceptado. - su existencia autoriza a explicar ciertas observaciones que lo amplifican en un infernal círculo vicioso. B. Una observación describe (casi) siempre una escena o un objeto real, vulgar o extraño, no reconocido, no identificado. -influenciado por el mito OVNI el testigo transmite su observación y detalles según su conocimiento conciente o no del fenómeno, MONTEJO AGUILERA, José Juan - a partir de un cierto nivel de extrañeza, de emoción, de angustia, el observador se desliza en un segundo estado donde el inconsciente va a ser el maestro que procede a la elaboración de una escena OVNI más o menos alejada de la realidad. Llevado esto al extremo, nos encontramos con las alucionaciones y las visiones.” (n. Madrid, 1967). Licenciado en Derecho, empezó a interesarse por el fenómeno OVNI en 1980, y de modo activo a partir de 1987. Miembro del Colectivo Cuadernos desde sus inicios, fundó en 1990 con Enrique Padial el Proyecto FAECOM (Fenómenos Aéreos Extraños de la Comunidad de Madrid) para la catalogación de la casuística madrileña. Su labor investigadora se ha centrado en una encuesta metódica del asunto Ummo y sus diversas ramificaciones (como el caso de la “mano cortada”, en el que fue el primer investigador que accedió al expediente judicial), sobre el que ha publicado esclarecedores artículos en Cuadernos de Ufología (nº 16-17, 1994) y otras revistas. Es también autor de otras investigaciones inéditas, como la de las Islas Hespérides (junto a Vicente Pérez Baeza), un MONTEIRO, Cassiano José (n. Maia, 1961). Ufólogo portugués, licenciado en ingeniería química. Estudioso del fenómeno OVNI desde 1976, ha destacado como investigador de campo de algunos de los más conocidos casos portugueses. Tras los intentos de organizar una red nacional de corresponsales a semejanza de la Rnc española, se afilió en 1979 a CEFAI, desde donde participó en la 154 Morón, caso archipiélago “fantasma” de los Mares del Sur. ABC de Andalucía. Opinión sobre el fenómeno: “La información sobre los OVNIs es eminentemente testimonial, es decir, de personas que afirman haber visto objetos volantes no identificados que, tras la correspondiente investigación, pueden quedar explicados como errores de percepción por parte de los testigos, confusiones con aeronaves convencionales y fenómenos naturales, sin olvidar los fraudes. Opino que los casos restantes, que algunos cifran en un 5% (aunque este porcentaje pudiera ser mayor), pueden tener un origen diverso, incluido el foráneo. Alrededor del OVNI «puro y duro» se ha ido perfilando un mito contemporáneo cuyo análisis y estudio pienso que cada vez será más importante, dado su notable interés para la antropología social.” La investigación del caso corrió a cargo de un nutrido grupo de estudiosos entre los que se encontraban Manuel Osuna, Ignacio Darnaude, Felipe Laffite, Juan Salas, Rafael Llamas y José Ruesga, si bien las investigaciones más profundas fueron realizadas por Laffite y Salas, quienes visitaron el lugar en repetidas ocasiones. La intervención de la Guardia Civil, de la Jefatura Agronómica y del propio Ejército añadió al caso connotaciones de especial interés. En los años ochenta Juan Antonio Fernández Peris redactó un voluminoso trabajo (inédito) en el que concluía que las huellas se debían a la caída fortuita de un rayo. Como clásico de la casuística española, el caso Morón permanece abierto al no haber hecho públicas sus conclusiones el ingeniero Felipe Laffite, que disiente de las de Fernández Peris.[69, 142][ JR] MORÓN, caso MUFON Sobre las tres de la madrugada del 11 de mayo de 1970, un solitario campesino oyó un trueno ensordecedor en el Rancho del Maestro Oliva del término de Morón de la Frontera (Sevilla). La noche era lluviosa, y a la mañana siguiente, al ir a visitar su sembrado de girasoles, descubrió un gran lunar de plantas en el suelo junto a un arroyo. Se trataba de dos círculos separados entre sí por unos 25 metros que tenían en su centro unos agujeros de aparente forma geométrica. La primera impresión del descubridor de las huellas fue que se trataba de una bomba o de un rayo, sin embargo, con posterioridad la hipótesis de que las huellas habían sido producidas por un OVNI cobra voz y en pocas fechas el caso es conocido internacionalmente, gracias sobre todo a la cobertura informativa que le prestó el diario (Mutual UFO Network). La mayor organización ufológica del mundo en la actualidad, fundada el 31 de mayo de 1969 por Walter H. Andrus, Jr. en Quincy, Illinois (EE.UU.). En origen fue una escisión del Apro llamada Midwest UFO Network, pero cambió de nombre al ampliarse su ámbito territorial. Siempre bajo la dirección de Andrus, desde 1975 ha venido operando desde Seguin, Texas. Organización de estructura descentralizada, cuenta con miembros y delegaciones en diversos países. Edita la revista mensual MUFON Journal, que hasta junio de 1976 se tituló Skylook, y desde entonces hasta 2016 MUFON UFO Journal. Desde la misma, y en consonancia con la filosofía del grupo, se ha seguido una línea editorial hasta hace poco claramente 155 MUFON UFO Journal orientada hacia la HET, fiel reflejo por otro lado de las ideas imperantes en los EE.UU. Desde 1970, el MUFON organiza cada verano en una diferente ciudad norteamericana un simposio, cuyas conferencias -pronunciadas por prominentes figuras de la ufología internacional- se recogen en el anuario MUFON UFO Symposium Proceedings. Dirección: 103 Oldtowne Road; Seguin, Texas 78155. En Internet: http://www. mufon.com [MM] realizado con precisión quirúrgica y de forma muy selectiva. La amplia publicidad dispensada a la muerte de Snippy y su eco en los medios ufológicos propiciaron la asociación de estos hechos con el fenómeno OVNI, y así se mencionan las mutilaciones de animales en el contexto de observaciones de luces nocturnas o de misteriosos helicópteros de color negro (a veces ni siquiera visibles) que a menudo se han denunciado en las cercanías de las localidades afectadas, y que algunos vinculan con proyectos militares secretos. MUFON UFO JOURNAL Sin embargo, la relación de esta clase de incidentes con los OVNIs dista de estar clara. La campaña de mutilaciones de 1974-76 fue al menos en parte causada por un grupo satánico, que operó primero en Minnesota y más tarde en Texas, y que empleaba las vísceras extraídas para sus ritos. En 1980, una investigación del FBI concluyó que todo era debido a la acción de animales depredadores, y de hecho las comprobaciones in situ de este tipo de historias han revelado no pocas inexactitudes y exageraciones de hechos perfectamente naturales. La periodista estadounidense Linda Milton Howe, autora del libro An Alien Harvest (1989), ha sido quien más ha intentado en los últimos tiempos establecer la conexión entre ambos fenómenos, partiendo de relatos de testigos de supuestas actuaciones de alienígenas sobre el ganado y de informes oficiales secretos que los explicarían en términos de las necesidades alimenticias de los grises. Mas la probada falsedad de los documentos de Majestic 12 y el hecho de que los testigos incluyeran las mutilaciones dentro de un relato de abducción privan de toda base a estas especulaciones. Revista mensual editada por el grupo americano Mufon. Fundada en 1967, hasta junio de 1976 se denominaba Skylook, y, desde 2016, simplemente MUFON Journal. MUTILACIONES DE GANADO El 8 de septiembre de 1967 fue hallado en Alamosa, Colorado (EE.UU.), el cuerpo sin vida del caballo Snippy, al que le faltaban la cabeza y diversos órganos, sin que ningún indicio explicase la causa de la muerte. Poco tiempo después, una oleada de incidentes similares azotó los estados de Pennsylvania y Kansas, la primera de una serie completada por la de 1974-76 en el Medio Oeste, la de principios de los años 80 en Colorado, y la de los primeros 90 en el Profundo Sur (especialmente en Alabama). En todas estas ocasiones se reiteraron las circunstancias de animales domésticos (caballos, toros, ovejas, gallinas o patos) misteriosamente fallecidos, con pérdidas de vísceras internas o completamente desangrados, y sin huellas de depredadores en las proximidades. Aparentemente, en algunos casos la extirpación de los órganos parecía haberse Aunque se trata de una fenomenología 156 Mutilaciones de ganado típicamente norteamericana, el misterio de las agresiones a animales se ha extendido por diversos países, donde se ha relacionado no directamente con OVNIs sino con criaturas extrañas como el “chupacabras” portorriqueño, monstruoso ser bípedo que vacía de sangre a sus víctimas por una única incisión en el cuello. Esta especie de vampiro, al que nadie ha podido jamás capturar pese a sus frecuentes ataques incluso a personas, a partir de su aparición en 1995 ha ido ampliando progresivamente su radio de acción desde la isla caribeña hasta Centroamérica, México, los EE.UU. y Europa, en lo que parece ser una expansión mediática de meros rumores e histerias sin fundamento. Inevitablemente, se ofrecen tantas hipótesis sobre su naturaleza como escenarios de sus fechorías, entre las que se encuentra la de que sea un ser extraterrestre o interdimensional que experimentaría con sangre humana para infectarla con virus como el del SIDA. [21][MM] DTU 157 Nave aérea de 1896-1897 N NAVE AÉREA DE 1896-97 época flotaba la idea desde hacía años, y así ya en 1886 Julio Verne había escrito Robur el Conquistador (traducida en EE.UU. justo en 1897) donde se describe una nave aérea similar cuyas maniobras fueron fielmente reflejadas hasta en sus menores detalles en los distintos casos conocidos de esta oleada. También es curioso como dicha oleada prefigura el posterior fenómeno de los platillos volantes, incluyendo fotografías (falsas por lo general), raptos de animales, visiones de los tripulantes (generalmente humanos), naves estrelladas, visitas al interior del artefacto, etc. La primera gran oleada de extrañas naves en los cielos de Norteamérica fue descubierta en los años sesenta por algunos ufólogos curiosos que buscando precedentes históricos a los platillos volantes revisaron las hemerotecas. Para entonces resultaba ya imposible entrevistar a los testigos originales, y en los pocos casos en que se pudo localizar a familiares o contemporáneos (p. ej.: la nave estrellada en Aurora, el rapto de un ternero en Leroy, Kansas) estos afirmaron que se trataba de bromas. Para los partidarios de la HET, esta oleada sería una muestra del carácter mimético del fenómeno, que adoptaría tecnologías sólo ligeramente por delante de las humanas, con no se sabe qué intenciones. Para los escépticos se trata de confusiones astronómicas (para las luces vistas de noche) junto con una gran mayoría de fraudes y/o bromas periodísticas o de los radiotelegrafistas. [52, 62, 68, 258][LG] La nave aérea (sólo se conocen un par de casos en que se viese más de una al mismo tiempo) era descrita como con forma de cigarro, aparentemente metálica, y provista de alas, hélices, aletas u otros apéndices. De noche aparecía como luces brillantes que a veces dejaban vislumbrar una estructura oscura detrás. Su velocidad era muy reducida, pero a veces se desplazaba contra el viento. Lo asombroso del asunto es que, según la historia oficial de la aeronáutica terrestre, en aquella época no se disponía en Norteamérica de la tecnología necesaria para producir un vehículo de esas características: el primer dirigible exitoso fue desarrollado por Alberto Santos Dumont en 1901, y el vuelo de los hermanos Wright no tendría lugar hasta 1903. Ello no fue obstáculo para que distintos aprovechados asegurasen ser los inventores de la nave aérea, llegando incluso a solicitar patentes. Pero aunque la tecnología no existiese, en el ambiente de la NAVE NODRIZA Dentro del folklore ufológico se han ido asentando creencias que encajarían dentro de la lógica tecnológica de los visitantes espaciales. Entre ellas se encuentra la visión de grandes naves, normalmente con forma de cigarro, que servirían para transportar naves de pequeño tamaño y más operativas de cara al reconocimiento del planeta; idea que parece tener su origen en la primera 158 NICAP tipología de OVNIs que confeccionó Donald Keyhoe en su libro The Flying Saucers Are Real (1950). Existen numerosos testimonios de observaciones de esta clase de “naves”, aunque tan dudosos como pueda ser cualquier otro. Diversos libros de corte sensacionalista están plagados de visiones de este tipo, en los que se las presenta como objetos totalmente reales, entrando a formar parte de la especulación platillista como una pieza más del engranaje de las creencias ufológicas. El propio Jacques Vallée incluyó en su tipología OVNI de 1966 un apartado dedicado a estas naves en forma de cigarro o huso (v. clasificación de Vallée). [RC] de filosofía muy conservadora, sostuvo desde sus inicios la tesis del encubrimiento gubernamental de las supuestas pruebas de los OVNIs y una beligerante actitud respecto a los contactados. Del mismo modo, tardó mucho en aceptar los casos de encuentros cercanos de tercer tipo (v. clasificación de Hynek), que apenas sí se mencionan en la obra clásica del NICAP, The UFO Evidence (Hall, 1964), especie de summa ufológica para muchos aún no superada, y que sólo serían reconocidos como experiencias OVNI válidas en UFOs: A New Look (1969), monografía que junto a Strange Effects from UFOs (1969) fue el último fruto bibliográfico del centro. NICAP La reactivación del interés por los OVNIs a mediados de los sesenta alivió los problemas económicos del NICAP, que en 1966 alcanzó la cifra máxima de 14.000 afiliados. Inicialmente el grupo prestó su apoyo al proyecto de la Universidad de Colorado (v. Condon, informe), mas en septiembre de 1967, cuando se hizo evidente la orientación negativa que iba tomando, rompió con él y anunció una especie de contra-proyecto que nunca llegaría a materializarse. Cerrado el Proyecto Blue Book en 1969, decayó en los EE.UU. el interés por la Ufología y el NICAP se enfrentó a su peor crisis financiera. Ello provocó en 1970 la destitución de Keyhoe y el nombramiento de John L. Acuff como nuevo director, cargo que abandonaría en 1978 -en medio de acusaciones contra la CIA por haber saboteado la organizaciónen favor de Alan N. Hall. En 1980 apareció el último número de The U.F.O. Investigator, lo que marcó la desaparición del centro y el traspaso de sus archivos al Cufos. [110] [MM] (National Investigations Committee on Aerial Phenomena). La mayor entidad privada a nivel mundial creada exclusivamente para el estudio del fenómeno OVNI. Fue fundada en Washington D.C. el 24 de octubre de 1956 a instancias de Thomas Townsend Brown, un físico conocido por sus especulaciones sobre la antigravedad, y un grupo de científicos y militares en la reserva. La megalomanía de este sujeto le descabalgó pronto de la dirección, que fue asumida en enero de 1957 por Donald E. Keyhoe, articulista aeronáutico y principal proponente de la hipótesis extraterrestre. En julio de ese mismo año el grupo empezó a editar The U.F.O. Investigator, un boletín de variable periodicidad y número de páginas que sirvió para mantener conectada la red de corresponsales que se fue creando a lo ancho de todos los EE.UU. Pese a que en 1958 ya contaba con 5.000 miembros, el NICAP arrastró siempre una crónica crisis financiera sólo soportable gracias a las aportaciones que hacía Keyhoe de su propio bolsillo. Entidad 159 Nidos OVNI NIDOS OVNI El avistamiento del granjero australiano George Pedley el 19 de Enero de 1966 en Tully (Queensland) introdujo el concepto "nido OVNI" en la terminología ufológica. Se denominaron así las supuestas huellas de aterrizaje de OVNIs que consistían en depresiones circulares en la hierba, dispuestas en el sentido de la agujas del reloj (o viceversa). Casos similares volvieron a repetirse en Nueva Gales del Sur (Australia) en 1969, y esporádicamente en distintas zonas del mundo. Con la difusión pública del fenómeno de los círculos en campos de cereales en la década de los 90, algunos ufólogos quisieron encontrar paralelismos entre ambos tipos de sucesos, pero sin mucha validez. Lo cierto es que aquel granjero australiano sólo tuvo una visión fugaz del OVNI ya situado a unos 10 mts de altura, por lo que su conexión con la huella es dudosa. Además los habitantes de la zona aseguraron que por esa época del año no era inusitado encontrar huellas similares, atribuyéndolas a tornados, precedente de la posterior teoría del Dr. Meaden y su "vórtice de plasma". [61] [LG] NO-CONTACTO, problema del Véase Ceti. NUEVA ERA (New Age). Véase Religión y ovnis. DTU 160 Observación radar-visual O OBSERVACIÓN RADAR-VISUAL que de haber hallado alguna ley cíclica que gobernase las mismas se habría logrado su predicción; de ahí a la explicación definitiva del enigma OVNI habría un corto espacio temporal. Pero, como han señalado Ballester y Guasp (1989) respecto a la concentración de casuística en períodos de tiempo delimitados, el problema fundamental “consiste en averiguar si la oleada es en realidad una propiedad inherente al fenómeno OVNI, o bien una parte accidental del mismo; es decir, se plantea dilucidar si ocurren más apariciones de OVNIs durante los períodos de oleadas o si éstas sólo reflejan una convergencia de denuncias que es independiente del nivel de las manifestaciones OVNI”. Los exinvestigadores Félix Ares de Blas y David G. López se decantan por la influencia exclusivamente externa sobre el origen de las mismas, después de realizar diversos estudios de distribución anual, mensual y semanal; simultáneamente indicaron que la presencia OVNI “debe ser” uniforme en el tiempo sin el concurso de estos factores extrínsecos, relacionados con la influencia de los medios de comunicación y la sociología de los testigos. Véase Clasificación de Hynek. OCULTACIÓN OFICIAL Véase Encubrimiento gubernamental. OLEADA Acumulación de denuncias ufológicas independientemente de que sean casos explicados o no- en un período de tiempo bien determinado, sobrepasando en mucho el nivel o frecuencia habitual de la casuística en una zona geográfica localizada. Son diversas las oleadas que se han producido en diferentes partes del mundo desde 1947. Destacan las de 1952 en EE.UU., 1954 en Francia, 1968-69 en todo Occidente y la de 1974-75 en algunos países. España ha sufrido diversas oleadas OVNI prácticamente desde el inicio del fenómeno. Las más destacadas son las de 1950, 1968-69, 1974-79 y una muy reciente iniciada en 1995, ampliamente divulgada en los medios de comunicación comerciales y respecto a la cual es todavía pronto para pronunciarse sobre su verdadera naturaleza. No sabemos si realmente existe algún factor intrínseco en la fenomenología OVNI que provoque su aumento y acumulación; muy probablemente no, como ya indicaron Ares y López. Es posible que operen factores como el contagio social de falsas experiencias, histeria de masas, etc., de modo similar a como ocurre en las alertas Tradicionalmente los estudiosos de talante más racionalista han dedicado grandes esfuerzos a analizar estos períodos de acumulación de casuística ufológica, ya 161 Oleada comunicación siguen provocando la denuncia de nuevos incidentes, creando una oleada, hasta que, por saturación de la opinión pública y desinterés de los medios, se diluye. Alcíbar destaca que este proceso actúa inconscientemente en la mente de futuros testigos de un hecho con apariencia anómala, condicionándolos a confundir con hechos extraños fenómenos triviales, al menos en ese intervalo temporal en que el proceso descrito es operativo. El problema radica en cómo distinguir los genuinos OVNIs de aquellos que tienen un “origen social”, y, en primer lugar, si realmente hay casos que no tengan un origen social. Éste es el terreno de la Ufología donde se produce una mayor incertidumbre: cuanto más profundizamos en el estudio de las oleadas menos sabemos de los casos particulares que las componen y viceversa. La teoría de la resonancia social (esta influencia de los medios de comunicación sobre las denuncias y viceversa) es la que parece adaptarse mejor a la dinámica de las oeladas, pero no existe consenso internacional respecto a la misma entre la ufología racionalista. ovni. Mutatis mutandi, concurrirían factores similares a los que entran en juego cuando se producen algunos casos de una determinada enfermedad y una gran cantidad de personas desean vacunarse sin haber necesidad, ya que se trata de un simple repunte en la incidencia normal del mal, como hubo oportunidad de comprobar en los casos españoles de meningitis de 1997. De lo que sí parece haber constancia es de la influencia de los medios de comunicación en la génesis de estas oleadas. De hecho, es muy probable que en ciertas ocasiones se hayan valido de esta capacidad para generar oleadas ficticias con las que aumentar sus ventas, sabedores del interés social que despiertan tales relatos, convenientemente aderezados y publicitados. Es también factible que el público se sienta sensibilizado ante las numerosas referencias periodísitcas, televisivas, etc., y preste mayor atención a estímulos luminosos que anteriormente pasaban desapercibidos, aumentando, de esta forma, la posibilidad de confusión. De igual manera, testigos de observaciones no identificadas que sienten vergüenza o timidez a notificar su visión pueden sentirse respaldados ante la avalancha de casos en los medios a hacer partícipes a los mismos de su experiencia. Recientemente la revista especializada MUFON UFO Journal ha recogido en sus páginas una polémica en torno a las oleadas. Se trata de la propuesta del investigador norteamericano Martin Kottmeyer (en The Anomalist nº 3), para quien esta concentración de casuística puede tener su origen en algún tipo de respuesta histérica ante graves acontecimientos sociales. La teoría de la “paranoia” ha despertado aceradas críticas tachándola de contener numerosas suposiciones muy dudosas y fallos metodológicos, y que, en realidad, tal hipótesis no es un intento de explicar las oleadas, sino el fenómeno OVNI globalmente. [13, 31][RC] El investigador sevillano José Miguel Alcíbar Cuello (1990) propuso un esquema en el que sistematizaba la influencia social en el aumento de la casuística ufológica. Según éste, un incidente OVNI espectacular, ya sea genuino o falso, es recogido en los medios de comunicación, provocando una avalancha de noticias y casuística, fundamentalmente simples confusiones con fenómenos naturales, que pueden estar referidas incluso a fechas anteriores a las que se produjo el incidente que actuó como detonante. Al ser éstas reproducidas en los medios de 162 ONI ONI Pero ya en 1966 Jacques Valleé, en una de las primeras aplicaciones del ordenador a la Ufología, demostró que todas las alineaciones de 3, 4 y hasta 5 puntos podían ser explicadas por el azar. Por ello, todas las discusiones posteriores (Toulet, Menzel, Saunders, Scornaux, etc.) se han centrado en la línea Bavic (de 6 puntos) y su extensión en el espacio (continuando el gran círculo terrestre hasta España, Brasil y Argentina) y en el tiempo; así como en la importancia de una mayor precisión en las coordenadas geográficas y en el ancho admitido para las ortotenias. En la actualidad, incluso la línea BAVIC se atribuye al mero azar. Siglas de Objeto No Identificado, denominación propuesta por Eduardo Buelta para designar a los platillos volantes. Escasamente utilizada, sólo se empleó por parte del Ceoni y por el Cei en determinadas épocas. ORTOTENIA Del griego "tendido en línea recta", término creado por el ufólogo francés Aimé Michel para denominar su descubrimiento de que los casos OVNI divulgados durante la oleada francesa de 1954 se disponían sobre "rectas de la proyección Lambert" (no geodésicas u ortodrómicas) pero dejando bien claro que "no se había visto el mismo objeto en los distintos lugares ni la alineación se corresponde en absoluto con una trayectoria". Además dichas alineaciones sólo se mantenían durante un día solar, eran efímeras. Una dificultad mayor era que por lo general se trataba de alineaciones de 3 puntos, siendo escasas las de 4 y 5 puntos. Muchos han sido los ufólogos que han extendido estos trabajos a sus países respectivos (Antonio Ribera para la oleada española de 1950, Olavo T. Fontes para Brasil en 1960, etc.) suavizando las restricciones iniciales hasta el extremo de que el capitán Bruce Cathie llegó a describir (en varios libros a partir de 1968) una supuesta red mundial de líneas electromagnéticas por la que se desplazarían los OVNIs. Históricamente puede considerarse como una de las primeras "pruebas objetivas" esgrimidas por los ufólogos para demostrar la existencia real del fenómeno. En palabras de Michel, "los fenómenos psicológicos -alucinaciones o fraudes- no se dan en líneas rectas". Pero la atracción por trazar líneas en un mapa y buscar una coherencia oculta tras los informes OVNI es irresistible para algunos, y con el paso de los años han reaparecido periódicamente propuestas similares. Entre ellas destaca la realizada en 1978 por J.C. Fumoux y J.F. Gille denominada "isocelia" y que pretendía que los avistamientos OVNI se agrupaban en triángulos isósceles. Miembros del Gepan coordinados por Esterlé pudieron demostrar poco después que se trataba de otra ilusión atribuible al mero azar (AA.VV., 1981). Este tipo de hipótesis se han aplicado también a otros fenómenos anómalos como las llamadas líneas de leys (alineaciones de monumentos megalíticos) o los casos de combustión humana espontánea. [3][LG] OSUNA LLORENTE, Manuel (Umbrete, 1914-1982) Pionero de la investigación de los OVNIs en Andalucía. Su vida profesional estuvo dedicada a la enseñanza como docente y director de varias escuelas. Intelectual nato, fue autor de diversas obras de una interesante prosa 163 Oui-ja, tabla OVI literaria. Se inició en la investigación de campo a principios de los años sesenta, actividad en la que se mostró prolífico (José Ruesga ha catalogado más de 350 informes salidos de su pluma), si bien no consiguió publicar ninguno de los libros que escribió sobre esta materia. Defensor de la HET más clásica, se decantó en sus últimos años por una explicación ultraterrena del fenómeno a raíz de las experiencias vividas por él y sus colaboradores en la finca de El Condesito, en Rociana (Huelva). Allí practicó, entre 1974 y 1975, repetidos intentos de contacto con extraterrestres en sesiones de observación nocturna y mediante técnicas de psicofonía. A su muerte le fue dedicada la calle donde residía. Siglas de Objeto Volante Identificado, traducción del inglés IFO (Identified Flying Object) empleada en relación a todos los estímulos de una observación OVNI que han sido, o pueden ser, explicados convencionalmente. Este concepto, creación de Josef A. Hynek (v. “UFOs Merit Scientific Study,” en Science, vol. 154:329, 1966), suele denotar en quien lo usa una opinión favorable a la existencia de un genuino fenómeno OVNI y a la posibilidad de discernir entre éste y el conjunto de fraudes, errores de observación y psicopatologías que causarían los OVIs. (v. casos positivos y negativos, depuración, falacia del residuo). [MM] Opinión sobre el fenómeno (tomada y adaptada de V.J. Ballester Olmos, Investigación OVNI, Plaza&Janés, Barcelona, 1984): "El fenómeno OVNI puede corresponder a la presencia de seres extraterrestres llegados a nuestro planeta y abandonados a su suerte en zonas que pueden constituir un cosmódromo excepcional por sus condiciones naturales. Por otra parte, mi convencimiento descansa en la certidumbre moral de millares de casos testificados por millones de criaturas racionales durante los últimos veinte años, pertenecientes a todas las razas, continentes, estratos sociales, y a los más variados niveles de formación. La persistencia del fenómeno asegura, ya de por sí, un mínimo porcentaje de veracidad. Jamás una fantasía tuvo tamaña consistencia ni tanto alongamiento en el espacio y en el tiempo". OVNI Siglas de Objeto Volante No Identificado, el propugnado objeto de estudio de la Ufología. El término es traducción literal del inglés UFO (Unidentified Flying Object), acrónimo acuñado por Edward J. Ruppelt, el primer director del proyecto Blue Book. Este vocablo carecía originariamente de las connotaciones extraterrestristas que con posterioridad tanto le han caracterizado, y se limitaba a designar los fenómenos reportados por el personal de las Fuerzas Aéreas estadounidenses que no habían podido ser adecuadamente identificados. No obstante, la influencia de las tesis de Keyhoe y otros condujo a su equiparación con platillo volante y con su significado de nave extraterrestre, término al que sustituyó en los años sesenta en un frustrado intento de los ufólogos por operar con un concepto más aséptico y científico. La necesidad de OUI-JA, tabla Véase Contactismo. 164 Ovni matizar estos aspectos tan subjetivos ha propiciado el uso de "fenómeno OVNI", expresión aparentemente más amplia y menos comprometida, pero que aún conserva los mismos defectos. En la actualidad, el término "fenómeno aéreo anómalo" parece promoverse con fuerza como sustituto de OVNI. de lo que se entiende por "objeto", por "volante" y por "no identificado". Lo fundamental en esta última clase es precisar esa no identificación y sus responsables, lo que permite dos posibilidades: considerar al propio testigo (tipo Condon) o a los expertos (tipo Hynek) como autores de la misma. Es precisamente la definición de Hynek la que ha cosechado más popularidad. Según ésta, puede definirse el OVNI como el informe de la percepción en el cielo o sobre el suelo de un objeto o una luz cuya apariencia, trayectoria, comportamiento dinámico y aspectos luminosos no sólo no sugieren una explicación convencional o lógica a los testigos, sino que permanece sin identificar cuando individuos técnicamente idóneos examinan toda la información disponible. El intento más logrado de perfeccionarla ha corrido a cargo de Claude Maugé, quien distingue entre "pre-ovni" (antes de la peritación de los expertos: el fenómeno OVNI en sentido amplio), "cuasi-ovni" (después de aquélla: fenómeno OVNI en sentido restringido) y verdadero OVNI (fenómeno OVNI en sentido estricto), cuando su naturaleza permanece sin ser aclarada pese a los avances científicos y en la reinvestigación del caso.[120][LG, MM] Nominalismos aparte, cabe destacar que, por motivos evidentes vinculados a la transitoriedad de los avistamientos y a la casi nula evidencia que dejan tras de sí, la consideración del OVNI como el objeto de estudio de la Ufología debe matizarse en el sentido de reconocer que lo que realmente pueden llegar a investigar los ufólogos no son sino los informes sobre los casos denunciados. Esta circunstancia, que pese a ser bien conocida no suele ser consecuentemente considerada, ha llevado a algunos a postular como objeto de estudio de la disciplina el llamado incidente o suceso ufológico, una estructura en la que el testigo participaría en igualdad de condiciones con lo observado, junto a otros elementos (entorno, percepción, entidades) cuya inclusión es más discutida. Sin embargo, las propuestas de este tipo, aunque útiles a otros efectos, mantienen la indefinición sobre el concreto significado que encubren las siglas OVNI. Otras posturas más reduccionistas han concentrado los esfuerzos en elaborar definiciones de OVNI capaces de garantizar unos contenidos operativos mínimos. En este sentido, la inexistencia de consenso sobre su naturaleza ha obligado a decantarse por dos modelos definitorios: a) el fenomenológico, que explica el OVNI en función de sus propiedades características (si se admite que las posee), y b) el del "grado de investigación" del caso, esto es, definiendo el OVNI negativamente mediante la exégesis Ovni En minúsculas, el fenómeno OVNI entendido como referente social y al margen de su supuesta materialidad. A diferencia de OVNI y su pretendida asepsia, el ovni es el término empleado por los autores -incluidos los de la presente obra- para denotar la carga mítica añadida al concepto en forma de creencia popular en su origen extraterrestre. Así como el OVNI, en mayúsculas, aspiraría a ser tenido en cuenta desde la perspectiva de 165 OVNI Présence las ciencias naturales, el ovni se propone como objeto de estudio para las ciencias sociales (v. Antropología de los ovnis, mito). [MM] OVNI PRÉSENCE Revista trimestral, desaparecida en 1996, del grupo francés SOS-Ovni. OVNILOGÍA Véase Ufología. OZMA, proyecto Véase SETI, proyectos. DTU 166 Palmer, Ray P PALMER, Ray la Ufología. (1911-1977). Véase Ciencia ficción, Ufología y y Tierra hueca, teoría de la. Estas actividades parten de la creencia en que hay otro mundo cualitativamente diferente del que estudian las ciencias físicas y al que no es posible acceder mediante la lógica y la razón. El sentido de lo maravilloso siempre presente en épocas de crisis y todas estas actividades se presentan como capaces de aportar conocimientos seguros o poseedoras de técnicas para mejorar el bienestar físico y espiritual. Profundas creencias culturales asentadas en la mente de la mayoría de los contemporáneos, son las responsables de que estas “ciencias alternativas” resuciten periódicamente. PAPERS D'OVNIS Revista bimestral del grupo barcelonés Cei, fundada por Joan Crexell. Ha conocido dos etapas: entre enero de 1994 y diciembre de 1996 (mensual, aunque desde abril de 1995 sólo se publicaron números dobles cada dos meses), y a partir de enero de 1997 (inicialmente bimestral y trimestral desde enero de 2000). Dejó de editarse en 2004, aunque tiempo después dio pie a una nueva publicación: Nous Papers d'OVNIs (desde 2014). Puede consultarse en http:// files.afu.se/Downloads/?dir=Magazines/ Spain and Catalonia/Papers d'Ovnis (CEI, eISSN 2604-1391) En cierto modo el enfrentamiento ciencia-paraciencia actualiza el viejo conflicto entre ciencia y religión. Actualmente las religiones tradicionales han perdido capacidad de influencia y han sido sustituidas en buena medida por un maremágnum de creencias y prácticas a menudo sin el más mínimo fundamento, pero que cumplen el papel asignado tradicionalmente a las explicaciones irracionales: dar cuenta de lo que la ciencia parece no poder explicar y ofrecer seguridades al ser humano ante los desconocidos peligros del exterior. No es nuevo el enfrentamiento y la crítica de lo que actualmente se entiende por paraciencias y que en ámbitos escépticos se denomina más claramente pseudociencias. Desde principios de siglo se encuentran referencias a estas manifestaciones acientíficas. Por ejemplo el filósofo de la historia Oswald PARACIENCIAS El prefijo para habitualmente significa al margen de o contra. En este caso haría referencia a todos aquellos saberes y conjunto de disciplinas supuestamente situadas más allá de los conocimientos científicos ordinarios, como por ejemplo la astrología, las mancias, la sanación por métodos no convencionales (como la imposición de manos), los fenómenos que estudia la Parapsicología, y, en cierta forma, 167 Paraufología Spengler se refirió al surgimiento de una segunda religiosidad en épocas de decadencia, cuando las grandes civilizaciones pierden fe en los ideales tradicionales. También el tradicionalista René Guénon criticó el espiritismo por confundir simples residuos psíquicos (como manifestaciones que hoy denominaríamos “viajes astrales”, “canalizaciones”, etc.), y lo auténticamente espiritual. controladas e independientes. Los medios de comunicación son en gran medida responsables del auge de las paraciencias porque al ser un producto que vende es potenciado sin medida. Se entra aquí en un terreno muy importante: el de la deontología periodística y las políticas editoriales; y en definitiva, el de la responsabilidad de todo informador ante sus oyentes. En su inmensa mayoría el periodismo carece del menor sentido crítico y en sus medios de expresión suelen aparecer toda suerte de charlatanes y embaucadores, ante lo que se apela a un abusivo “derecho de expresión”. [RC] La Ufología es con demasiada frecuencia asimilada a las paraciencias. A menudo vemos cómo en programas radiofónicos se pasa sin solución de continuidad de las experiencias psíquicas de un médium o de la echada de cartas de un tarotista al “experto” de turno en los ovnis. En muchos casos es realmente justa tal indiferenciación, puesto que el discurso habitual de todos ellos no se diferencia por una posibilidad mayor de comprobación y contrastación independiente. Las paraciencias en el terreno ufológico podemos encontrarlas entre los miembros de sectas ufológicas que realizan prácticas de relajación y concentración mediante exóticas técnicas, o entre los abducidos y contactados que supuestamente han desarrollado poderes extrasensoriales. Desde el punto de vista metodológico, la Ufología sería igualmente una pseudociencia ya que no es posible llevar a cabo estudios de características científicas por la propia “naturaleza” del fenómeno, y tradicionalmente no se han propuesto hipótesis realmente falsables ni predictivas, características ineludibles del conocimiento científico. PARAUFOLOGÍA Véase Hipótesis paraufológica. PEDRAJO GÓMEZ, Manuel (Santander, 1925 - 2001). Su padre, culto autodidacta, y su madre, maestra, son sus profesores. A los quince años continúa sus estudios en París, donde reside varios años. A su vuelta a España estudia en la Escuela Normal de San Sebastián y, posteriormente, en la Universidad Central de Madrid. Termina allí sus estudios de Filosofía e Historia Antigua (Filología Semítica). Desde los inicios del fenómeno en la década de los cuarenta, se interesa por su evolución principalmente en Norteamérica. Tras un período amplio de recopilación de datos, escribe en 1954 Los platillos volantes y la evidencia (ed. del autor). Se trata del primer libro en su género editado en España, haciéndose eco de observaciones originales de la época, como la producida en Villares del Saz (Cuenca) en julio de 1953. En último término, las paraciencias renuncian a la práctica del método científico, a la comprobación de sus predicciones y conocimientos mediante experiencias 168 Percepción Pronto el autor desconfía de sus primeras hipótesis, relacionadas con el planeta Marte, en consonancia con aquel momento histórico y comienza una dilatada época de conferencias y colaboraciones en revistas y periódicos, evolucionando claramente desde sus primeras interpretaciones. de realidad” (posteriormente, su evocación por la memoria la transformará, por influjo del raciocinio, de certeza inmediata a “juicio de realidad”), y el Objeto, que es lo que percibimos. Opinión sobre el fenómeno: “El denominado fenómeno OVNI está dirigido por un Poder desconocido, cuya finalidad aparente demuestra un especial interés por la Biosfera, hombre incluido. Tan misterioso como el origen en sí, es la manipulación mundial de la información, bien presentándose casos absolutamente falsos, cuidadosamente orquestados, bien mediante los evidentes esfuerzos de ciertos estamentos por silenciar y desmentir categóricamente los hechos.” • A igualdad de circunstancias, estímulos más próximos tienden a agruparse como formando parte de un mismo objeto. PERCEPCIÓN a) El ser humano no percibe las cosas tal y como son para él. Los sentidos entregan al cerebro la materia prima y éste la organiza e interpreta. La interpretación de la realidad no siempre es fidedigna. Las leyes básicas de la percepción son: • A igualdad de circunstancias, estímulos semejantes tienden a percibirse como formando parte de un mismo objeto. • Tendemos a buscar continuidad en los estímulos. • Tendemos a completar las figuras. Dicho esto, habría que considerar dos cuestiones: La percepción es un proceso extremadamente complejo en el cual diversos datos sensoriales se integran, con ayuda de la memoria y de mecanismos de relación, para crear en el sistema nervioso un esquema desencadenante de un reconocimiento, una identificación o, si es necesario, una reacción apropiada (Haines, 1980). b) El mecanismo de integración e interpretación de la percepción está influido por factores socioculturales y por la experiencia (aprendizaje), así como por tendencias de carácter afectivo (tenemos tendencia a percibir aquello que deseamos) o de sugestión. En la psicología de la percepción clásica se considera que el ser humano percibe el mundo que le rodea simultáneamente a través de todos sus sentidos, aunque, en función de la actividad que se encuentre desarrollando en el momento de la percepción, unos sentidos participarán en mayor proporción que otros. En los procesos perceptivos hay que distinguir entre el Sujeto (perceptor), que vive la sensopercepción con la “certeza inmediata A la vista de los datos anteriores, se puede concluir que no percibimos solamente lo que tenemos enfrente, sino también lo que llevamos dentro, y percibimos “la mejor figura” que es coherente con una imagen de referencia dada. La psicología cognitiva ha establecido claramente que nuestros intereses, actitudes y contenidos mentales y culturales tienen una influencia directa sobre 169 Pereira, Jader U. PESO el proceso perceptivo y determinan su contenido, de manera que se puede decir que una gran parte del mismo es “deducido”. En los casos de alerta, tensión extrema y ansiedad por parte del observador aumenta la tendencia a la deformación o mala interpretación de los estímulos percibidos. Es decir, los testigos generalmente sí aciertan cuando manifiestan haber percibido algo, en determinadas circunstancias, pero pueden equivocarse (y lo hacen muy frecuentemente) sobre la naturaleza de lo percibido. [MG] Para algunos autores, medida del valor de un caso OVNI. Véase Credibilidad e Índices de Ballester-Guasp. PHÉNOMÈNA Revista bimestral del grupo francés SOSOvni. Dejó de publicarse en el año 2002. PHÉNOMÈNES SPATIAUX Publicación trimestral, ya desaparecida, del Gepa francés. PEREIRA, Jader U. Secretario del Grupo Gaucho de Investigação de Objetos Aéreos Não Identificados (GGIOANI) de Porto Alegre (Brasil), pasó a la historia de la ufología internacional por un excelente estudio sobre 333 casos de supuestos humanoides relacionados con el OVNIs; trabajo de sistematización que, aunque hoy ha quedado obsoleto, fue de gran importancia en su tiempo al abordar con valentía uno de los aspectos más controvertidos del fenómeno (v. tipologías de humanoides). Traducido a varios idiomas, el Gepa francés le dedicó una esmerada edición en 1974 en el segundo número especial de su publicación Phénomènes Spatiaux bajo el título de Les Extra-terrestres. En España fue publicado en los números 5 a 10 de Stendek. [172] PINVIDIC, Thierry Ufólogo francés. La influencia de Carl Gustav Jung y Jacques Vallée se nota en su primer libro, Le noeud gordien (France Empire, París, 1979), donde postula que “los OVNIs son artefactos físicos asociados a un componente psíquico que manipula el simbolismo arquetípico y los fundamentos antropológicos del mito”. En su transición a la ufología escéptica, en 1982 manifiesta su “apoyo crítico” al modelo psicosociológico de Michel Monnerie y se convierte en un dinámico referente de la llamada “nueva ufología” francesa que encontró su foro en la revista OVNI Présence (convertida, desde 1996, en Anomalies). El estudio que realizó en Argelia sobre la dispersión del estereotipo platillista en una nación islámica (Ed. F.F.U., 1983), su viaje de investigación sobre los círculos en campos de cereales en Inglaterra para la revista Science et Vie (Nº 878, noviembre 1990) y la compilación OVNI: vers une anthropologie d’un mythe contemporain (Editions Heimdal, Bayeux, PERSPECTIVAS UFOLÓGICAS Revista cuatrimestral, ya desaparecida, del grupo mexicano Somie. 170 Plana Crivillén, Joan 1993) son sus tres contribuciones más valoradas. En sus últimas aportaciones apunta a sentar las bases de una epistemología del dominio ufológico y, por extensión, de los sistemas de conocimientos interesados en las anomalías científicas. el análisis de la casuística con implicaciones militares (v. desclasificación). Colaborador y corresponsal de revistas como Lumières dans la Nuit y Flying Saucer Review, destacan entre sus principales trabajos “Actitud de las autoridades españolas sobre el fenómeno OVNI” (con Ballester y Fernández Peris, 1987; incluido en [30]), “OVNIS: Materia reservada” (con Ballester, en Cuadernos de Ufología nº 13, 1992) y “Stealth: los fantasmas aéreos modernos” (en Cuadernos de Ufología nº 18, 1995). Opinión sobre el fenómeno (tomada de su artículo “Un gran classique qui a la vie dure”, en Pinvidic, 1993, op. cit.): “He aquí una historia que no hemos explicado o, más aún, ¡que nunca llegaremos a explicar! Si todas las hipótesis parecen aceptables, ninguna parece imponerse actualmente, y no deseamos privilegiar la que nos caiga mejor sin tener los medios para demostrar que está bien fundamentada. El OVNI es una noción compleja. Sabemos unas pocas cosas sobre él. Escribir su historia devendrá, sin duda, reeditar la historia misma del hombre, la de sus temores, de sus esperanzas, de sus creencias, de sus certezas. Esto es de un interés inmenso. Pero, en consecuencia, la solución no llegará de hoy para mañana...” PLATILLISTA Adjetivo, generalmente despectivo, que se aplica a la persona, grupo o tendencia que sustenta el origen extraterrestre de los OVNIs de una manera ingenua, dogmática o irreflexiva. PLATILLO VOLANTE (plato volador). Término mayoritariamente empleado hasta bien entrada la década de los sesenta para referirse a lo que hoy conocemos como Ovnis, independientemente de la forma concreta de lo observado. Es una traducción literal de la expresión inglesa flying saucer, denominación fruto de un equívoco histórico a la hora de interpretar los periodistas la descripción de Kenneth Arnold, el primer testigo moderno de un avistamiento ufológico. Éste, en sus declaraciones al periódico East Oregonian al día siguiente de su observación, explicó el movimiento de los objetos en términos de “like a saucer if you skipped it across the water” (como un platillo saltando sobre el agua), cosa que en el despacho remitido por el PLANA CRIVILLÉN, Joan (n. Sabadell, 1956). Ufólogo de la llamada tercera generación, especialista en observaciones OVNI por parte de pilotos civiles y militares y, en general, de miembros de las Fuerzas Armadas y Cuerpos de Seguridad del Estado. Interesado por el fenómeno OVNI desde 1976, ingresó en el Cei en 1982, de cuya directiva entró a formar parte en 1985 y en el que ostenta desde 1995 el cargo de vicepresidente. Hacia 1985 inició la compilación del catálogo AEROCAT sobre avistamientos desde aeronaves o aeropuertos españoles (con más de 250 entradas en 1995), y desde 1989 ha venido colaborando con V.J. Ballester Olmos en 171 Platillos estrellados periodista Bill Bequette a la Associated Press se convirtió en “saucer-like objects flying at incredible speed” (objetos en forma de platillo volando a una velocidad increíble), y que finalmente apareció en los titulares de prensa como flying saucers o flying disks. objeto, generalmente un disco, es trasladado hasta una base aérea donde se almacena en una zona de máxima seguridad. Como elementos adicionales podemos encontrar el hallazgo de seres alienígenas (normalmente muertos, aunque no siempre), la realización de autopsias, la visita de personalidades importantes como el Presidente, etc. No obstante, no era ésta la primera vez que se mencionaba la palabra “platillo” en relación a un suceso semejante, pues el 25 de enero de 1878 el Daily News de Denison (Texas, EE.UU.) publicó la historia de un tal John Martin que decía haber visto un objeto con forma de plato sobrevolar su casa. Por otro lado, el número de marzo de 1918 de la revista de ciencia ficción Electrical Experimenter mostró en portada una nave con la misma geometría; y la más conocida Amazing Stories ilustró su contraportada, poco antes de la oleada de 1947, con una imagen de un grupo de “flying saucer spaceships” (astronaves discoidales volantes) desplazándose en formación. [135, 143, 240][MM] De hecho, este tipo de historias son ya "oficialmente" folklore. El destacado folklorista Jan Harold Brunvand encuadra el motivo de los platillos estrellados en la categoria de leyendas urbanas sobre descubrimientos maravillosos suprimidos por el gobierno, junto con el motor de agua y la bombilla de eterna duración. Ello no ha sido óbice para que los creyentes hayan ido recopilando documentos oficiales, declaraciones juradas y confesiones en el lecho de muerte, apoyando la realidad de lo ocurrido. En cuadro aparte se comentan algunos casos más documentados. El primer informe conocido sobre cadáveres extraterrestres aparece (con foto incluida) en Un habitant de la planète Mars de Henri de Parville (1865) que habría llegado a la Tierra dentro de un aerolito. Poco después se menciona un incidente similar ocurrido en Carcarañá (Argentina) en 1877. PLATILLOS ESTRELLADOS Desde el momento en que se considera a los OVNIs como naves extraterrestres tripuladas, y a falta del tópico aterrizaje frente a la Casa Blanca, la prueba definitiva de su existencia sería disponer de uno de estos platillos y de sus tripulantes. Ya desde la prehistoria del fenómeno OVNI han aparecido rumores al respecto. El relato básico a partir del cual se desarrollan las ya casi infinitas variantes sería el siguiente: Una nave espacial desconocida es vista estrellarse, o encontrada accidentada ya en tierra, invariablemente en un lugar remoto de cualquier país (primordialmente los Estados Unidos). Si existen testigos civiles estos son rápidamente silenciados por los militares y el Aunque las noticias sobre OVNIs estrellados resurgirían cada cierto tiempo desde entonces, los ufólogos más serios en su lucha por lograr un status científico para la Ufología no les prestaban demasiada atención (recordemos que ni siquiera los avistamientos de tripulantes llegaron a ser plenamente aceptados hasta finales de los años 60). Además, el publicitado fraude de Frank Scully en su libro Behind the Flying Saucers (1950) sirvió para aumentar el rechazo a este tipo de relatos. Por ello no 172 Platillos estrellados sería hasta 1977, quizá no casualmente en medio de una gran escasez de informes OVNI, que Leonard Stringfield volvió a sacar el tema a la palestra mediante su libro Situation Red, the UFO Siege, al que siguieron con los años hasta cuatro Status Reports: The crash/retrieval syndrome. Estos trabajos, llenos de evidencias provocativas pero generalmente anónimas (los testigos nunca se identifican por temor a represalias otra constante-), prendieron la llama del actual incendio. Según este autor, ¡hasta 24 incidentes de este tipo se habrían sucedido desde 1947! No sabe uno qué es más increíble, si la alta tasa de accidentes de estas aeronaves (siempre sobre zonas despobladas) y la ausencia de cualquier intento de salvamento por parte de los propios extraterrestres; o la extraordinaria eficiencia de los militares y científicos para llegar con inusitada rapidez al lugar de los hechos, recoger apresuradamente hasta el más mínimo fragmento revelador, y lograr guardar el secreto de los mismos hasta la fecha (pese a los miles de personas que han debido verse involucradas en casi cincuenta años). De todas formas, como ocurre con el caso de Roswell, algunas historias pueden tener cierta base real, pero referidas a posibles prototipos secretos. Los militares son perfectamente conscientes del gran interés que el fenómeno OVNI genera en la gente, por lo que no sería de extrañar que en ocasiones hayan llegado a manipular tanto a los testigos como a los medios de comunicación para hacer creer que lo que se CIEN AÑOS DE PLATILLOS ESTRELLADOS • Aurora (Texas),17 de abril de 1897.- Una extraña nave aérea se estrelló supuestamente contra un molino de viento en este pequeño poblado de Texas, estallando y esparciendo los restos en una amplia zona. Pudo recuperarse el cuerpo del piloto, pero aunque muy quemado era evidente que "no era habitante de este mundo", siendo enterrado en el cementerio de la localidad. La nave estaba construida en un metal desconocido semejante al aluminio y también aparecieron unos "extraños jeroglíficos". La noticia no tuvo gran repercusión en la época, y durante aquella oleada se informó de otros algunos incidentes similares. No fue hasta mediados de los años sesenta cuando la noticia cayó en manos de los ufólogos, que pronto visitaron la zona encontrando testigos ya ancianos y llegando incluso a excavar la supuesta tumba con detectores de metales. Pero la opinión mayoritaria actual es que todo fue un engaño. (Chariton, 1991) •Aztec (Nuevo México), marzo de 1950.- Dos platillos volantes con un diámetro de 30 metros y construidos de una aleación desconocida, se habrían posado cerca de esta población del desierto americano, recuperándose hasta 34 cadáveres de unos seres de baja estatura (inferior a 1 metro), totalmente quemados, que según pudo determinarse posteriormente procedían de Venus. Cuando el periodista Frank Scully publicó esta historia en su libro Behind the Flying Saucers (1950) llegó a vender más de 60.000 ejemplares. Desgraciadamente tras una prolongada investigación todo el incidente fue denunciado en la revista True (Septiembre de 1952) como un fraude organizado por dos timadores profesionales (Newton y GeBauer). No obstante, incluso en la actualidad siguen apareciendo investigadores que defienden su realidad. Este caso tiene el privilegio de haberse convertido en el eje central de uno de los primeros libros españoles sobre OVNIs, Vienen los platillos volantes, de Enrique Miguel Borjas (1957). 173 Platillos estrellados • Isla Spitzbergen (Atlántico Norte), mediados de junio de 1952.- En plena oleada OVNI sobre Estados Unidos aparece en un periódico alemán la noticia de que una escuadrilla de reactores noruegos, tras sufrir algunas interferencias electromagnéticas, descubrió en las nevadas soledades de esta isla ártica un disco circular de unos 40 a 50 metros de diámetro, accidentado. El examen posterior permite comprobar que no está tripulado y que los instrumentos de a bordo están en ruso. Se comenta incluso que pudiera tratarse del desarrollo de alguna arma secreta nazi capturada tras el final de la guerra. Aunque la noticia llegó a manos de la CIA no causó gran revuelo hasta 1954 cuando vuelve a resurgir ¡con 4 diferentes versiones! que potencian el origen extraterrestre del aparato. Todos los investigadores serios que han analizado los datos, coinciden en que se trató de un fraude periodístico. (UFO-Norway. Ole Jonny Brænne. INTERNET 30/06/1995). •Varginha (Brasil), 20 de enero de 1996.- La última historia que está causando sensación al tratarse de un suceso reciente y en un lugar poblado. Todo empezó cuando tres niñas dijeron ver una extraña criatura (humanoide y desnuda, de color marrón, con tres bultos en la cabeza y grandes ojos rojizos) en plena tarde y salieron huyendo. Cuando los ufólogos llegaron a investigar encontraron diversos testigos que hablaban de al menos dos seres, los cuales habrían sido capturados por los bomberos o los militares. Habrían sido llevados a un hospital donde fallecieron, desde donde serían trasladados después en dirección desconocida para su autopsia. Parece que la noche anterior se habría visto un OVNI con forma de submarino que dejaba tras de sí una estela de humo. Sin embargo, ha sido imposible obtener un relato coherente; se trata de una confusa mezcla de testimonios directos, rumores y especulaciones. Naturalmente tanto las autoridades como los médicos supuestamente involucrados lo niegan todo. PLATÍVOLO había estrellado y recuperado era precisamente una nave extraterrestre. Un ejemplo claro parece ser el famoso caso del bosque de Rendlesham (Gran Bretaña) en Diciembre de 1980 donde supuestamente un OVNI aterrizó en medio de un bosque cercano a una base aérea. Sinónimo de OVNI, escasamente usado, formado por la contracción de "platillo" y "volante". PROPULSIÓN DE LOS OVNIS A la espera de la exhibición pública de estas supuestas pruebas ocultas, los ufólogos partidarios de la HET han tenido que conformarse con el análisis de algunos supuestos restos que han logrado caer en manos civiles, y con una serie de fotografías y películas de extraterrestres que han ido saliendo a la luz. [243, 244, 245, 258][LG] La teorización sobre los posibles medios de propulsión de los OVNIs resulta inevitable desde el punto de vista de la hipótesis extraterrestre, que considera a estos como vehículos de exploración de civilizaciones extraplanetarias, pues deben explicarse dos aspectos: a) de qué forma logran viajar estas naves desde su mundo de origen hasta la Tierra, y b) cómo pueden realizar las proezas aeronáuticas que se les atribuyen. 174 Propulsión de los OVNIs La primera cuestión resultó ser especialmente delicada desde que los astrónomos y las sondas espaciales descartaron Marte y los restantes planetas del Sistema Solar como posible origen de los platillos volantes, pues no quedó más remedio que hacerlos venir de otras estrellas. El viaje interestelar supone dificultades de todo orden, principalmente por las enormes distancias que se deben cubrir, respetando además el límite de la velocidad de la luz. Aunque es teóricamente posible efectuar viajes “lentos” (de cientos o miles de años de duración) esta solución no podría explicar el alto volumen de casuística registrado, así que los diversos autores han apostado por desplazamientos translumínicos, aunque sin precisar muy bien el medio empleado. De hecho, los ufólogos han sido más bien conservadores y poco imaginativos en este terreno, limitándose a importar las propuestas de la ciencia ficción (hiperespacio) o de algunos físicos teóricos (agujeros de gusano, plegamientos del espacio). Ciertamente nadie ha anticipado las tesis de Alcubierre (1994), que -al menos sobre el papel- permitirían alcanzar velocidades mayores que la de la luz sin violar las leyes físicas establecidas. gravedad y electromagnetismo (“electrogravitación”) o por otros medios. Fue el proyecto Magnet canadiense (1950-1954), destinado a la observación de los platillos volantes y al estudio de la tecnología que exhibían, el que por medio de su director Wilbert B. Smith avanzó en 1952 la hipótesis del electromagnetismo como medio de propulsión de las naves extraterrestres (Ribera, 1966), idea que al parecer le fue sugerida por los propios alienígenas. Posteriormente, Thomas Towsend Brown (v. nicap), Jean-Pierre Petit y algunos otros científicos -citados por Schneider (1977)- han sostenido la posibilidad de oponer a la gravedad campos electromagnéticos capaces de anularla o de propulsar vehículos con su ayuda. Pese a que incluso se han presentado desarrollos prácticos de estas ideas, nunca se ha podido contrastar la viabilidad de las mismas y nada más se ha vuelto a saber de ellas. Por su parte, los restantes autores se han aferrado a conceptos aún más especulativos. El capitán Jean Plantier destacó en 1955 con su hipótesis sobre la aplicación a cada uno de los átomos del OVNI de una fuerza proporcional a su masa, que tendría su fuente en una misteriosa “energía del espacio”. El profesor Marcel Pagès (Ribera, 1983) reducía la gravitación a un problema de “presión de fotones”, con lo cual un objeto podría flotar simplemente por aplicación del principio de Arquímedes si se realizaba el “vacío fotónico” en su interior. En España, en cambio, Francisco Aréjula (1973) resucitó las tesis de Plantier substituyendo la “energía del espacio” por unas “cargas gigantes” de origen cosmológico, que se encontrarían en unos “bulbos” probablemente existentes en Fobos. Respecto al segundo punto, a menudo los informes OVNI refieren aceleraciones prodigiosas, detenciones bruscas, cambios de dirección de 90 grados, descensos en “hoja muerta” y otras anomalías dinámicas que violan los principios físicos del movimiento de cuerpos materiales. A excepción de Eduardo Buelta, que postuló la utilización de motores iónicos, el resto de los autores resolvió la cuestión atribuyendo al OVNI una especie de campo gravitatorio propio que le sustraería de la acción de fuerzas externas. La manera de conseguir esto puede ser mediante la conexión entre Al margen del lastre de las evidentes 175 Próspera Muñoz, caso dificultades teóricas de todas estas concepciones, el propulsionismo desapareció a finales de los años setenta cuando la materialidad de los OVNIs empezó a ser cuestionada por la paraufología primero y por las hipótesis psicosociológicas después. [14, 22, 191, 196, 220][MM] hermana de la primera parte del contacto. Nunca se ha encontrado el objeto escondido. Del estudio realizado con el concurso de varios médicos, Ruesga acepta la no implicación de Próspera en un fraude, si bien asigna una naturaleza puramente psíquica a todo el caso como puro mecanismo de defensa de la testigo, pues encuentra en sus relatos grandes inconsistencias. Próspera es una gran interesada en temas paranormales y ha conseguido un amplio reconocimiento en medios ufológicos a raíz de su testimonio profusamente divulgado en radio, prensa y televisión-, mostrándose siempre abierta al análisis de su experiencia. [4, 197, 205][ JR] PRÓSPERA MUÑOZ, caso En 1983, y a raíz de la lectura de un libro de Antonio Ribera sobre secuestros por extraterrestres, Próspera Muñoz, una trabajadora de Telefónica residente en Gerona, comienza a recordar una experiencia personal de contacto extraterrestre. Acude al propio Ribera y es sometida a hipnosis regresiva, de la que se extrae que en 1947, siendo ella una niña que vivía con su familia en Jumilla (Murcia), sufrió una abducción. El suceso se habría producido así: encontrándose con su hermana en pleno día, observan ambas el aterrizaje de un objeto luminoso a la puerta de su casa. Dos individuos de pequeña estatura y voluminosas cabezas se dirigen a ellas haciéndoles preguntas absurdas y entregan a Próspera un artefacto que ésta esconde en alguna parte de la casa. Aquella misma noche Próspera es visitada de nuevo por los mismos sujetos, que la transportan al interior de un platillo, la examinan y le implantan un diminuto artilugio en la base del cráneo. En la regresión hipnótica aparecen también recuerdos de otros contactos que Próspera Muñoz habría tenido a lo largo de su vida con seres idénticos. PROTOTIPOS EXPERIMENTALES Se entienden por tales aquellas aeronaves desarrolladas en diferentes naciones, ya sea teórica, conceptual o industrialmente, y concebidas para desplazarse en un medio aéreo mediante procedimientos convencionales o no convencionales que modifican notablemente su aspecto habitual, pretendiendo con ello conseguir mejores características y prestaciones de vuelo, introduciendo cambios en su grupo propulsor (motor o combustible), mejora de la sustentación o de la gobernabilidad, que se acompañan de cambios de diseño cuya apariencia puede ser incluso la de un plato o disco biconvexo, similar a la forma vulgarmente aceptada como la de un OVNI. Grosso modo podemos distinguir tres tipos de aeronaves que responden a estos criterios: • Aviones de ala circular o redonda. José Ruesga ha realizado un seguimiento del caso durante cinco años, obteniendo una dudosa confirmación de la • Prototipos de ala circular giratoria. • Proyectos de artefactos con forma de 176 Prototipos experimentales disco biconvexo. voladora” (fliegender Bierdeckel) y que sirvió para comprobar la inestabilidad y escasa maniobrabilidad que proporcionaba este tipo de configuración. Capturado este prototipo por los rusos, permitió el desarrollo en la URSS del Shukanov Diskoplan 1 (1958) y del Diskoplan 2 (1962). “Platillos volantes” y prototipos experimentales en la Alemania del III Reich. Tras la Primera Guerra Mundial, el Tratado de Versalles impuso al Estado Alemán una serie de limitaciones en armamento militar que se intentaron burlar, subrepticiamente al principio, y después con menor cautela conforme se fueron suavizando las condiciones del Armisticio de la Gran Guerra, hasta prescindir de todo disimulo, particularmente desde el acceso al poder del Partido Nacionalsocialista. Los nazis favorecieron el reequipamiento de las Fuerzas Armadas e impulsaron la búsqueda de armas nuevas no recogidas o contempladas en el Tratado, permitiendo de una parte disponer de los medios para el desarrollo técnico de conceptos en experimentación (como la aplicación del motor cohete como medio de propulsión) y, de otra, intentar adaptar a la ingeniería aeronáutica criterios teóricos que en aquellos días de grandes cambios (ideológicos, técnicos, sociales, etc.) alcanzaban gran predicamento, incluyendo los defendidos por sociedades de índole teosófica, metapsíquica, espiritistas u ocultistas, dando estas últimas origen a una serie de ideas propugnadas en aquel entonces, de las que – como hemos indicado– tenemos noticia, pero no constancia fehaciente de que se llevaran a buen término, aunque sí es evidente que la ferocidad de los bombardeos Aliados sobre Alemania en las postrimerías de la guerra exacerbó la búsqueda de armas que permitieran detenerlos. B) Mención aparte requiere el Triebflügel (“ala propulsada”) basado en la patente solicitada por el ingeniero alemán Otto Muck (10 de septiembre de 1938) para un avión de despegue vertical (Vertical Take Off and Landing: V.T.O.L. –despegue y aterrizaje vertical). Basándose en este principio, la Focke Wulf lo llevó a la práctica, con el diseño de un avión de fuselaje ahusado que se apoyaba en el suelo sobre su cola, hacia la mitad de su sección estaba rodeado por tres largas palas instaladas en un anillo que podía girar libremente, y que tenía incorporadas cada una en su estremo distal un motor a reacción; al funcionar los motores, los brazos rotatorios actuaban como las palas de un helicóptero, permitiendo el despegue vertical. No superó la fase de prototipo. C) Otra aplicación de este principio fueron las minas antiaéreas (Flakmine) Flugschnittel, las V-7 Feuerball (“bola de fuego”) y Kugelblitz (“rayo en bola”), capaces de elevar entre doscientos y mil kilogramos de explosivos. Tenían aspecto esferoidal, rodeadas por un anillo móvil donde se encastraban varias palas, un motor cohete situado verticalmente en el Feuerball, o en los extremos de cuatro de las palas en el Kugelblitz, impelía al ingenio hacia arriba, incrementando las palas al girar la fuerza ascensional, haciéndose deflagar por radio entre las formaciones de bombarderos cuando llegaban a la misma altitud. Se conserva muy poca información sobre estos A) Aviones de ala circular : se desarrolló uno en Alemania durante la guerra por Arthur Sack, el AS5, prácticamente una maqueta a escala para evaluación, que dio origen al AS6/V1 (1944), un prototipo que por su forma fue denominado “torta 177 Prototipos experimentales proyectos. Otros estudios independientes de los anteriores son: D) Durante la Segunda Guerra Mundial, surgen también en Alemania varios modelos con forma de disco, basados en los principios del “Autogiro” desarrollado en la década de los años treinta por el ingeniero español Juan de la Cierva, aunque con aplicaciones distintas. E) El Aerodino de Henri Coanda, ingeniero rumano que estudió el efecto que lleva su nombre, consistente en el empuje que ejerce sobre una superficie el fluido que incide sobre la misma y el aprovechamiento de dicho empuje para desplazar un cuerpo físico. Los alemanes diseñaron un modelo de aeronave lenticular que lanzaba un flujo de doce reactores dispuestos radialmente sobre el borde del ingenio; se atribuye a la escasez de motores y de combustible el retraso del proyecto, que se considera que influyó en el desarrollo del AVRO VZ-9 Saucer. Se ha publicado que se patentó el 15 de febrero de 1938 con el número 2.108.652 (sin especificar el país). El Dr. Miethe en las instalaciones de la BMW próximas a Praga diseñó un modelo de autogiro denominado Flügelrad, donde las palas rotatorias se encontraban en torno al habitáculo del tripulante, el cual tenía forma de cúpula; la eyección de gases procedentes de un motor a reacción situado horizontalmente bajo las palas del rotor, que al ser orientables podían modificar la incidencia del flujo de gases permitiendo maniobrar el aparato; el giro de las palas del rotor proporcionaba la estabilidad de la que carecía el AS6/V1. • Flügelrad III, del que se diseñó un modelo, siendo éste –el de mayor tamaño– el conocido en la ufología como “disco de Miethe”. F) El Disco Omega : su proyecto se atribuye al ingeniero Andreas Epp, y vendría a ser la quintaesencia del Triebflügel, el Flügelrad y la aplicación del “efecto Coanda”. Básicamente sería una cabina con forma de cúpula situada sobre un disco biconvexo, ocho hélices cuatripalas distribuidas horizontalmente en el cuerpo del disco proporcionarían sustentación como en los actuales “hovercraft” y dos palas dotadas cada una de un reactor en su extremo distal girarían sobre el borde del disco generando un “efecto Coanda”. Se patentó en Alemania Occidental el 22 de abril de 1956 y se ha afirmado que se ofreció a la Fuerza Aérea de los Estados Unidos. No es fácil conseguir información sobre estos proyectos de la BMW, dado que Praga fue ocupada por tropas soviéticas bajo el mando del Mariscal Koniev el último día con movimiento de tropas del conflicto (10 de mayo de 1945, cuando Berlín había sucumbido el 30 de abril y el Armisticio se había firmado el 7 de mayo). Las posibilidades de estudiar la veracidad de los dos últimos proyectos son escasas, aunque no es imposible avanzar en su investigación, pero se conocen otros que se consideran evidentemente como una superchería, tales como las peonzas voladoras atribuidas a Viktor Schauberger, conocidas como Haunebu y supuestamente Se proyectaron varios modelos: • Flügelrad I, del que se estudiaron dos variantes, la V1 (monoplaza) y la V2 (biplaza). • Flügelrad II, con tres variantes, la V1, la V2 y la V3. 178 Prototipos experimentales movidas por “energía diamagnética”, o las de la “serie VRIL” de un denominado “grupo Schumann”. Otras historias como la del “Sonderbüro nº 13” (Operación Urano, dirigida por el profesor Kamper, v. Foofighter) ya fueron desenmascaradas por el investigador Thierry Pinvidic como una invención periodística, o la aparecida en 1978 en el único número de la revista Brissant, donde en un artículo sobre Schriever (coartífice del Flügelrad, cuya existencia dio a conocer en 1950), aparecen los planos de un disco con turbinas electronagnéticas, radares láser y computadoras, que recuerdan a la patente inglesa que se menciona más adelante. para un “vehículo espacial” donde se explicaba cómo construir un “platillo volante” con capacidad para veintidós pasajeros. Se inspiraba en la aplicación de la energía nuclear como medio de propulsión y basaba su utilidad en la posibilidad teórica de rápidos viajes intercontinentales. La patente expiró en 1976 sin que prosperase la idea. Fuera del contexto de “prototipos experimentales” que se expone (dado que se trata de aeronaves no tripuladas de reconocimiento por control remoto), existen dos modelos británicos susceptibles de confundirse con OVNIs, ambos de la firma Westland, que en 1968 comenzó el desarrollo de un helicóptero de control remoto (RHP) que condujo a la evaluación en 1976 del pequeño y rechoncho Wisp, lo cual permitió a su vez desarrollar el más complejo Wideye (asociable con algunos casos OVNI). El programa no obstante se canceló oficialmente en 1979, aunque la Westland mantuvo los estudios sobre el mismo. Diseño de un “platillo volante” en España. El español José Infante Lozano, profesor de Física y Matemáticas del Instituto de Bachillerato de Jumilla (Murcia), patentó en Madrid en octubre de 1955 una turbina aérea con forma de “disco volante”. El ingenio constaba de dos cuerpos, uno central para los tripulantes y otro exterior que en la patente se describe en los siguientes términos: “El funcionamiento de la turbina depende de leyes físicas, a base de consumo de carburantes y elementos de ignición, cuyos gases originarían un movimiento circular, lo que provocaría la absorción de grandes masas de aire que, al ser comprimidas en el interior, salen con fuerza por los extremos donde se produce la ignición”. El diseño no superó la fase de tablero de dibujo, pero conceptualmente aplicaba los mismos planteamientos del Focke Wulf Triebflügel y los Flügelrad de la BMW. “Platillos volantes” soviéticos. Tras la desaparición de la Unión Soviética, algunos proyectos exclusivamente militares han accedido a los medios de difusión; sin aludir a los proyectos de aviones de ala circular ya citados de Shukanov, se conoce al menos la existencia de dos ingenios similares a objetos discoidales. Se trata del Thermoplano (Russia 4001), un prototipo de dirigible con forma de disco biconvexo que volaría a alturas no superiores a 8.000 metros a una velocidad de 150 a 200 km/h (lento), y del Ekip L3 (proyecto Tarielka), cuyo jefe de diseño es Lev Shukin (ingeniero responsable de la misión “ApoloSoyuz” de 1974) y en su concepción parecen integrarse todos los conceptos vertidos en el Patente inglesa para un “platillo volante”. En 1973 se concedió a la Compañía Inglesa de Ferrocarriles la patente 179 Prototipos experimentales disco Omega de Andreas Epp: reactores integrados en el fuselaje, que proyectan el flujo sobre los bordes del mismo permitiéndole desplazarse, y que despega y aterriza sobre un colchón de aire en cualquier lugar. Se ha construido un modelo a escala, y un prototipo de quince metros de ancho por once de largo, con nueve toneladas de peso, capaz de transportar a dieciocho personas a 650 km/h. Si el proyecto progresa, se desarrollaría un modelo mayor. 810 a 880 km/h, despertando la admiración de quienes lo contemplaron volar. Pero la Chance Vought ya tenía un pedido oficial (25 de junio de 1946) para otro avión desarrollado tras el conflicto mundial a partir del botín de guerra, cuando llegaron a conocimiento de los proyectistas estadounidenses estudios como el de la Arado germánica sobre el Arado Ar II-1 Nachtjäger, que dieron paso al Vought F7U Cutlass, que alcanzaba 1094 km/h, dando al traste con el futuro del Flap Jack. Prototipos experimentales en Norteamérica. Existen en Norteamérica bastantes aeronaves asociadas o susceptibles de asociarse con OVNIs o casuística ufológica. Ya en el libro de Kenneth Arnold y Ray Palmer The Coming of the Saucers (1952) se menciona un “platillo” por el hombre, el del Dr. E.W. Kay de Glendale, California, quien construyó un modelo reducido que debería mantenerse en vuelo como un helicóptero gracias a las palas de un rotor situado sobre el disco. Un “aerodino” de ala circular giratoria fue construido por Jonathan E. Cadwell, carpintero iniciado en aeronáutica que a finales de los años treinta construyó dos prototipos: uno, con fuselaje convencional sobre el que montaba un disco del que sobresalían unas palas como las de un autogiro; el otro, que parecía un carrete de cables de teléfono, con dos grandes discos paralelos de los que sobresalían las palas de sendos rotores que giraban en sentido opuesto. Cadwell había creado una sociedad mercantil denominada “Grey Goose Corporation” (Sociedad Ganso Gris), vendiendo indiscriminadamente acciones de su compañía cada vez que necesitaba fondos para su proyecto. Hacia 1940 la Justicia empezó a indagar sobre las finanzas de Cadwell, y éste desapareció sin dejar otro rastro que sus prototipos abandonados en el cobertizo de un secadero de tabaco, en una granja de Glen Burnie cerca de Baltimore, en Maryland, donde los encontraron en 1949 investigadores de la U.S. Air Force (USAF) alertados por un antiguo accionista que relacionó su malograda inversión con los OVNIs. No obstante, quizá el más famoso (por haber sido asociado precisamente con la observación de Kenneth Arnold) sea el Chance Vought XF5U, conocido también por Flap Jack o Flying Pancake entre otras denominaciones. Este avión data de un proyecto de 1939, que dio origen a un modelo de evaluación, el V-173, a partir del cual se construyó el primer prototipo del XF5U, que no estuvo concluido hasta el 25 de junio de 1945. Caracterizado por su aspecto de “hamburguesa”, realizó su primer vuelo al año siguiente. La velocidad de 680 km/h que le conferían sus motores era insuficiente para permitirle competir con los nuevos aviones a reacción, por lo que el grupo propulsor se cambió por dos turbohélices que le permitieron alcanzar de Otro avión de formas inusuales fue el diseñado en los años cuarenta por Michael Gluhareff, exiliado ruso afincado en los 180 Psicología del testigo OVNI Estados Unidos. Era un ala volante en delta, propulsado por hélices en posición “canard” (en la cola del avión), que guarda un notable parecido con el fotografiado por William A. Rhodes el 10 de julio de 1947 en Phoenix, Arizona (v. Hipótesis del arma secreta), aunque no se ha podido demostrar que superara la fase del tablero de dibujo. “platillo volante” que denominó M 200 X. Para atraer publicidad e inversiones, Moller pilotó el vuelo de su prototipo, el cual tuvo lugar por exigencias de la compañía aseguradora con el vehículo suspendido de un cable por una grúa. Fuera del contexto de los prototipos experimentales quedan (como se indicó anteriormente en relación con los prototipos británicos) los vehículos de control remoto “RPV” (Remotely Piloted Vehicles) con todo tipo de formas, ya sean convencionales o extrañas, como el canadiense CL 227, con aspecto de cacahuete, el construido por la NASA para evaluación de instrumentos, o el Cipher de Sikorsky, un helicóptero no tripulado con forma de rosquilla. Uno de los prototipos experimentales más conocidos de “platillo volante” es el AVRO (de la compañía A.V. Roe Canada Ltd.), también conocido como AVRO Saucer o AVROCAR. Se trata de un modelo de aeronave con forma de “platillo” (o disco biconvexo) que dicha empresa desarrolló durante la década de los años cincuenta, aunque sin conseguir alcanzar las expectativas que del mismo se esperaban, por lo que el proyecto se demoró, proponiéndose su compra por los Estados Unidos como solución que permitiera su continuidad. El Ejército (U.S. Army) y la Fuerza Aérea (USAF) adquirieron el prototipo, que efectuó vuelos de prueba en 1959 prosiguiendo los ensayos. Finalmente, el proyecto se abandonó a principios de los sesenta y el prototipo se encuentra hoy en el Museo de las Fuerzas Aéreas estadounidenses en Fort Eustis, Virginia. Recientemente (junio de 1995) se hizo pública en Estados Unidos la existencia del U.A.V. Tier 3 (Unmanned Air Vehicle, conocidos comúnmente como drone). Denominado Dark Star (Estrella Oscura), fue desarrollado en once meses por la Lockheed Martin y la Boeing, quienes aplicaron su tecnología stealth (furtivo) en el diseño de un avión espía no tripulado con forma de “platillo” sustentado por las alas de un velero (21 metros de envergadura). [2, 111, 149, 164, 237, 238][AP] Paul Moller es un ingeniero californiano cuyo sueño de la niñez era poder acudir al trabajo en su propio avión utilitario. Llevando a la realidad su sueño, hizo un avión a su medida, creando previamente la compañía Moller International, diseñando un vehículo de líneas aerodinámicas y del tamaño de un automóvil, con características de despegue y aterrizaje vertical (V.T.O.L.), capaz de desplazarse a 600 km/h. Para verificar el sistema informático que controlaba la estabilidad del vehículo, Moller construyó una plataforma experimental no operativa con forma de PSICOLOGÍA DEL TESTIGO OVNI En los diversos estudios sobre grupos y poblaciones tratando de hallar unas características psicológicas que pudieran aplicarse al testigo típico de avistamientos o fenómenos OVNI, no parece haberse encontrado ningún rasgo especial que los distinga de la población general. Se les ha catalogado como personas normales (y habitualmente sinceras, en lo que se refiere a su observación), sin patologías psíquicas 181 Psicología del testigo OVNI específicas. Tampoco parece haber diferencias cuantificables en cuanto al posible exceso de imaginación. Sin embargo, sí puede ser determinante el factor “creencia” en los OVNIs y en los alienígenas para establecer diferencias en la psicología del testigo OVNI, sobre todo en su vertiente de interpretación de sucesos externos, esto es, cuando el testigo percibe una visión o estímulo del entorno, de naturaleza desconocida para él, y trata de explicarla de acuerdo con sus patrones sociales, culturales y personales (v. componente psíquico y transgresión de la realidad, síndrome de). Si la persona cree en la existencia de entidades extraterrestres, tenderá a interpretar el suceso como la manifestación de una de éstas. Esto es así particularmente cuando el testigo no es un mero espectador de algo externo a él (por ejemplo, una observación de un objeto luminoso en el cielo), sino cuando es protagonista del suceso (casos de encuentros cercanos o de abducciones), pues cabe una mayor elaboración psíquica. A este respecto, el acontecimiento puede explicarse como una producción inconsciente que responde a necesidades emocionales (angustia vital) o, incluso, y refiriéndose concretamente a la experiencia de los visitantes de dormitorio, algunos estudiosos la atribuyen a descargas en los lóbulos temporales del cerebro, motivadas por cambios en los campos magnéticos, aunque combinado todo ello con los datos personales y culturales del individuo (M. Persinger, 1983). [175][MG] DTU 182 Radar-visual R RADAR-VISUAL (radar-óptico). Véase Clasificación de Hynek. RADAR Y OVNIS La relación entre radar y OVNIs viene ya desde muy antiguo, pues el primer caso en el que se cita la detección de un objeto extraño por este medio fue el ocurrido en verano de 1948 en Goose Bay (Labrador, Canadá). Indudablemente, desde la perspectiva que considera los OVNIs como naves o cuerpos físicos de indiscutible materialidad está claro que el radar debe jugar un importante papel en la detección de tales objetos, pero también es cierto que la fiabilidad de este dispositivo no permite asegurar que en el 100% de las ocasiones lo reflejado en la pantalla se corresponda con un cuerpo sólido y real. De hecho, en Ufología generalmente se han venido considerando únicamente válidos aquellos sucesos de detección por radar simultáneamente acompañados por la visión del OVNI por testigos; son los llamados casos radar-visual, la única de las categorías de la clasificación de Hynek que había sido previamente definida. Cuando esto no se da, y en general respecto a toda clase de anomalías de tipo técnico, se suele hablar de ángeles de radar, esto es, trazas virtuales sin correlación con blancos reales. La aparición de dichos “ángeles” obedece a una multiplicidad de causas de larga y compleja explicación, así que expondremos ahora sólo las más comunes. Reflejos. La captación de las ondas de radio emitidas por el radar y reflejadas por los objetos que encuentran constituye la base del funcionamiento de este dispositivo. Los problemas surgen cuando esos reflejos muestran discordancias con la realidad, supuesto que puede deberse a que las señales reflejadas proceden de un lóbulo lateral del haz principal que ha sido generado por difracción, fenómeno que simulará la presencia de un objeto donde no lo hay. No es una disfunción muy común ni grave debido a la escasa potencia de estos haces secundarios, pero que se complica cuando un blanco seguido por el tren de ondas principal devuelve esa señal secundaria a través del reflejo de un objeto en tierra. Como el recorrido de propagación es entonces mayor que la distancia real, el eco fantasma parecerá perseguir al eco regular. Además, al ser su señal más débil, será interpretada como la de un objeto más pequeño, dificultando aún más su identificación. Propagación anómala. El más importante de estos efectos es la superrefracción, provocada por situaciones de inversión térmica (un incremento de la temperatura con la altitud) en las capas bajas de la atmósfera que causan la desviación de los haces de radar. En tales condiciones el radar es capaz de detectar blancos que están situados por debajo del horizonte normal al refractarse las ondas sobre la curvatura de la 183 183 RAMA, misión Tierra. Cuando esta superrefracción es muy alta, un haz apuntado horizontalmente puede doblarse en un arco con la misma o mayor curvatura que la superficie terrestre y propagarse a grandes distancias más allá del horizonte (lo que se conoce como ducting). En estos casos puede que un blanco móvil, como un avión a baja altura tras el horizonte, aparezca en pantalla a una distancia que normalmente indicaría la presencia del objeto a gran altitud. No obstante, dado que en condiciones así se observan extensas zonas de confusión radárica, los operadores ajustan sus interpretaciones corrigiendo tales circunstancias. que se acercan o, por el contrario, a unirse en una gran señal cuando se alejan. Por su parte, el fenómeno opuesto de la subrefracción se produce al refractarse hacia arriba las ondas del radar y reducirse grandemente el horizonte de radio. Puede ocurrir tanto por una inversión de la humedad (un incremento de ésta con la altura) como cuando, en ambientes desérticos, la densidad del aire en las capas bajas es, o bien constante, o bien aumenta con la altura (una inversión de densidad). La subrefracción puede ser relevante cuando provoca reflejos de radar sobre terrenos que de otra manera no son buenos reflectores de las ondas de radio. Los efectos de capas refractivas a gran altura son más insidiosos, puesto que producen una reflexión parcial de las ondas de radio. En un radar orientado verticalmente estos efectos apenas se perciben, pero a medida que disminuye su ángulo de elevación la reflexión aumenta rápidamente, aproximándose al 100% cuando el ángulo tiende a cero. Los retornos de objetos situados sobre el suelo reflejados desde capas elevadas son ocasionalmente lo suficientemente intensos como para aparecer en las pantallas, siempre que la antena esté orientada con ángulos de elevación menores de 10 o 15 grados. Dichos retornos son difíciles de diagnosticar porque rara vez se acompañan de otras evidencias de propagación anómala. Sin embargo, hay tres características que permiten identificarlos: 1) los bancos reflectantes son arrastrados por el viento, por lo que los “ángeles” simulan moverse en la dirección en que éste sopla; 2) estos falsos blancos se desvanecen si se aproximan a menos de 15-30 km. del radar, debido a la reducción de la señal reflejada al incrementarse el ángulo de elevación; 3) tienen, por lo mismo, tendencia a dividirse en numerosos blancos menores a medida Igualmente son significativos para los radares equipados con MTI (Moving Target Indicator, indicador de blancos móviles) el “centelleo” y los efectos de multipath característicos de la propagación de las ondas de radio bajo condiciones excesivamente turbulentas, ya que las rápidas fluctuaciones en las señales recibidas simulan el eco de un blanco “vibrante” que trastorna los circuitos MTI y causa la representación de un objeto aparentemente estacionario. Tanto en estos supuestos como en otros, una correcta identificación de los “ángeles” pasa muchas veces por la consulta a los propios operadores del radar, que son quienes, con su experiencia en el manejo del mismo y con el conocimiento de las condiciones atmosféricas en que se desenvuelve, mejor pueden determinar la naturaleza de la señal anómala. [240][MM] RAMA, misión Grupo contactista peruano, con numerosas delegaciones internacionales, 184 Rayo en bola fundado en 1974 por Sixto Paz Wells y disuelto en 1991. Alcanzó gran difusión en España gracias a los artículos y al primer libro de J.J. Benítez. Véase sectas ufológicas. segundos, sin que parezca exceder de los 15 minutos. Cuando desaparecen pueden hacerlo de dos formas: a) mediante explosión, que contadas veces genera un residuo de humo u hollín, y b) en silencio, disminuyendo progresivamente de tamaño y luminosidad. Hay casos en los cuales las centellas han provocado muertes de hombres y animales o desperfectos, junto a otros más curiosos en los que han transportado objetos frágiles sin dañarlos, vaciado tinteros, desnudado personas o quemado el vello púbico a una muchacha sin otro mayor trastorno. Pese a que pueda parecer un fenómeno muy infrecuente, algunos estudios apuntan a que podrían ser tan comunes como los relámpagos normales. Igualmente, no está del todo clara su relación con estos últimos, pues se han observado con independencia de la existencia de una tormenta eléctrica y durante terremotos, erupciones volcánicas, ventiscas, tornados, ciclones y temporales. RAYO EN BOLA (rayo globular, centella). Fenómeno atmosférico con apariencia de esferoide luminoso, de naturaleza aún no bien determinada pero presuntamente asociada al relámpago común. De forma generalmente esférica (aunque se han visto cilíndricos o en forma de anillos o halos) los rayos globulares poseen un diámetro promedio de 35 cm., que en raras ocasiones alcanza hasta los 10 m., y tres posibles tipos de estructura: 1) sólida, con superficie mate o un núcleo sólido con envoltura traslúcida, 2) rotante y 3) con estructura de llama. No existe consenso entre los autores sobre los colores predominantes, que por lo general abarcarían una gama de rojos, naranjas, amarillos, verdes y violetas. El rayo en bola puede producir un sonido parecido al zumbido de las abejas y un olor sulfuroso o de ozono. Se ha estimado su temperatura entre los 4.000 y los 6.000 grados Kelvin, sin descartar otras mucho menores. En cuanto a sus movimientos, existen diversas posibilidades: entre las nubes o entre éstas y el suelo, en horizontal sobre la tierra, errático o incluso estacionario. Aparentemente puede desplazarse contra el viento o estar detenido sin que éste le afecte, y se le ha visto atravesar ventanas o paredes sin dificultad. Su velocidad ronda los 2 m/s, si bien existen ejemplos de traslaciones a cientos de kilómetros por hora. La duración de este fenómeno es de un promedio de 15 A lo largo de los años se han formulado infinidad de hipótesis explicativas de los rayos globulares sin que se haya conseguido describir a plena satisfacción de todos los mecanismos que lo originan. Se ha recurrido a tesis electromagnéticas, químicas, radioactivas e incluso psicológicas (alucinaciones, ilusiones ópticas o fosfenos), por más que en la actualidad su realidad física esté ya establecida. La última propuesta ha corrido a cargo del catedrático de la madrileña Universidad Complutense Antonio Fernández Rañada y del doctorando José Luis Trueba, quienes en la revista Nature de 05/09/1996 expusieron un modelo basado en la formación durante las tormentas de nudos electromagnéticos que confinarían pequeñas bolas de plasma ionizado. A efectos ufológicos, la importancia de 185 Redón Trabal, Pere los rayos en bola parece evidente, pues muchas de sus características coinciden con las que tienen atribuidas los OVNIs. No obstante, el desconocimiento de la génesis de este fenómeno no favorece precisamente su utilidad a la hora de explicar los avistamientos. El primero en conectar el fenómeno OVNI con las centellas fue el astrónomo americano A.F. Jenzano, idea que fue recogida por Philip J. Klass en 1961 y que constituyó una de las bases de su obra UFO’s: Identified (1968), en la que aplicaba con carácter general esta explicación a todo tipo de sucesos ufológicos. Posteriormente, tanto él como otros autores han moderado mucho su postura, y hoy sólo se propone la identificación de OVNIs con rayos globulares para casos muy concretos. [129, 206][MM] secretaria de CEI-Madrid, que se integraría desde entonces en las tareas de la sede barcelonesa. El 30 de junio de 1980 Redón asumió la presidencia del CEI, cargo que aún ostenta. REENCUESTA Como es sabido existen casos OVNI de diferente complejidad. Por tal motivo, el tiempo y el esfuerzo necesarios para explicar un caso pueden ser muy diferentes de los requeridos por otro caso más sencillo, por ejemplo una observación nocturna y lejana. Los casos complejos, mal investigados o antiguos para los que no se pudo hallar explicación, requieren ser reinvestigados, reencuestados, de acuerdo con la importancia que para un estudio concreto tengan. A menudo el reencuestador se vale de los informes previos realizados por otro investigador, o de una simple información periodística, escasa y dudosa por principio. Para compensar tales defectos se hace necesario repetir la investigación llevada a cabo por el anterior encuestador o completarla significativamente. En tal proceso se intentará contrastar la versión antigua con los nuevos hallazgos, por esta razón también se ha denominado esta labor con el término contraencuesta, utilizado por primera vez por el investigador francés Aimé Michel en 1970 para referirse a la necesaria reinvestigación de la conocida oleada francesa de 1954. REDÓN TRABAL, PERE (n. Barcelona, 1941). Interesado por los no identificados desde finales de los años cuarenta a consecuencia de su pasión por la aviación, su pequeña colección de recortes fue la base del archivo del Cei cuando junto a otros jóvenes se incorporó a la entidad en 1967. Miembro de la Junta Directiva desde la reactivación del grupo, ocupó algo más tarde el puesto de Secretario General, desde donde atendió las labores de creación del archivo y la biblioteca. En el marco de esa función estableció contactos con investigadores de toda España e Hispanoamérica, a los que atrajo al centro y con los que creó una red de corresponsales que cubrió toda la geografía nacional. Junto a Joan Crexells y Josep Serra impulsó en 1970 la aparición de Stendek, de la que a partir de 1974 sería su director. En 1975 contrajo matrimonio con María del Carmen Tamayo Ballesteros, hasta aquel momento Las dificultades de la labor de reencuesta son obvias: debido a que el tiempo transcurrido desde la observación puede abarcar décadas, el testigo quizá haya fallecido o sea imposible localizarlo. A pesar de ello la reencuesta es necesaria, pues permite aclarar puntos oscuros, reforzar o 186 Religión y ovnis rebajar la extrañeza del caso o que el autor de un fraude confiese, ya que no tiene sentido mantener el engaño durante tanto tiempo. En resumen, se trata de una labor de análisis contraria por principio a la que es habitual en los medios comerciales, donde la permanente búsqueda de novedades no permite la reevaluación de los episodios de cierta antigüedad.[RC] círculos como el líder extraterrestre de una civilización hiperevolucionada, transmisor un mensaje de corte salvacionista semejante al de la religión judeocristiana. En el escalafón inferior se encontrarían los ángeles, igualmente extraterrestres. Pero ya sea esta figura religiosa la que en último término inspira el contacto o los propios extraterrestres, siempre se presenta como vehículo para cambios substanciales en la sociedad actual, que es señalada como perniciosa e injusta (véase Cabria, 1993; 143-62). Como portavoces de los seres superiores -en este caso extraterrestresestarían los contactados, versión actualizada de los viejos profetas judíos. REENTRADA DE SATÉLITE Véase Ingenios astronáuticos. RELIGIÓN Y OVNIS Entre los paralelismos que se han advertido entre las religiones tradicionales y el movimiento ufológico destacan las abducciones, que se asemejan a las ceremonias de iniciación. A través de ellas surge un hombre nuevo, liberado de la cáscara del hombre antiguo, evolucionado, similar a la conversión de Saulo. Algunos supuestos abducidos han afirmado que su vida cambió drásticamente después de sufrir su experiencia. Desde finales de los años 60 se ha ido extendiendo por todo el mundo occidental una forma pretendidamente novedosa de entender la sociedad humana y todos los problemas que la aquejan, movimiento que ha sido denominado Nueva Era. Entre otras facetas de la vida a las que ha influido destaca el abandono o deserción de la práctica de las religiones tradicionales, concretamente del cristianismo en Occidente. Los ovnis, asimilados en la mitología popular a la presencia de naves extraterrestres, fueron captados por esa corriente ideológica trasformándolos en mensajeros o intermediarios de lo alto; como tales, se habrían comunicado con algunos elegidos transmitiéndoles una serie de principios semejantes en su forma a los de las religiones tradicionales, como ha ocurrido siempre en momentos de crisis y desasosiego. Es decir, el contacto extraterrestre se habría transformado en un culto mesiánico, a modo de reelaboración de ciertas religiones tradicionales, como el cristianismo, al nuevo contexto. Por ejemplo, la figura mesiánica de Jesucristo es reinterpretada en algunos A medida que los aspectos más claramente religiosos del culto ovni se desarrollaban entraban en conflicto con las religiones tradicionales y en particular con el catolicismo. En este sentido algunos divulgadores pseudocientíficos como Erich von Däniken han afirmado que lo que en la religión son considerados ángeles no eran más que astronautas con afán civilizador, como en el caso de la Biblia, o en el caso del propio Dios de la religión judeocristiana, considerado por autores similares como un ser no divino pero tecnológicamente superior y que habría creado la religión por motivos desconocidos. Otros han querido ver manifestaciones demoníacas en la 187 Repeater presencia de extraterrestres en la Tierra, que estarían en connivencia con algunos gobiernos para intercambiar tecnología alienígena por seres humanos con los que realizar experiencias para mejorar su raza. puede ser considerada como absurda". Pero la realidad es muy tozuda. Incluso el propio Kenneth Arnold aseguró haber visto OVNIS en 6 ocasiones más. Y con la expansión acelerada del fenómeno abducción esa posición cautelosa ha sido completamente olvidada y el término ha caído en desuso. En la actualidad casi se ha cerrado el círculo y muchos creyentes aceptan los encuentros repetidos hablando de "elegidos".[LG] Como todo acontecimiento con apariencia misteriosa, la casuística ufológica tiene cabida en la Nueva Era, ese movimiento difuso con claras implicaciones comerciales. Desde este punto de vista el fenómeno ovni tendría una función similar a la de otros fenómenos paranormales: la evolución de la conciencia, quid de la cuestión Nueva Era. Es igual que sean fenomenologías completamente distintas; lo importante es que serán capaces de producir en el adepto un cambio cualitativo, hasta transformar su percepción de manera holista y transcendente. Como todo acontecimiento social, la religiosidad ufológica está plenamente influenciada por los tiempos. Basta releer los mensajes transmitidos por George Adamski, en plena guerra fría, y los que actualmente dan a conocer los nuevos contactados, en los que predomina el intento de cambio interior y evolución espiritual; la posmodernidad ha llegado a los ovnis y su principal actor, el yo, entra en escena. [55][RC] RESIDUO Remanente de casos de una determinada colección de casuística que permanece inexplicado tras la eliminación de los OVIs que ésta pudiera contener, y que comúnmente es tenido como representación genuina del verdadero fenómeno OVNI. Véase Depuración de casuística y Falacia del residuo. RESUMEN MODELO La Ufología, en el intento por normalizar sus métodos y la presentación de los informes OVNI a los interesados, ha diseñado formas más o menos estandarizadas de recopilar y dar a conocer la información de cada caso OVNI. Al ser el volumen de ésta elevado en numerosas ocasiones, se ha tendido a presentar de manera resumida los detalles más relevantes, intentando construir un relato objetivo, coherente y que aportase la mayor cantidad de datos útiles, tanto de tipo físico como sociológico. Un sumario o resumen de un incidente ufológico, si quiere contar con la confianza de la comunidad de investigadores, debería incluir (en poco más REPEATER Término anglosajón que podríamos traducir por "reincidente". Se refiere a aquellas personas que aseguran haber tenido dos o más experiencias OVNI. En los años setenta, y frente al fenómeno de los contactados, el propio Dr. Hynek defendía que... "la idea de que una persona pueda tener docenas de avistamientos OVNI, mientras que la gran mayoría de las personas no hayan visto nunca un OVNI, 188 Rey Brea, Óscar de una página) los siguientes datos: fecha completa del incidente, datos personales del testigo o testigos principales (siempre que no lo hayan prohibido expresamente), lugar exacto del incidente, cercanía de cualquier tipo de instalación al citado lugar, descripción minuciosa de la observación y de las características del fenómeno u objeto observado, dirección de aparición y desaparición, etc. En el caso de tratarse de un caso de aterrizaje la precisión de los datos obtenidos deberá ser mayor, sumándose a ellos todo tipo de datos biométricos en caso de haberse observado algún tipo de criatura o humanoide. de fenómenos naturales y otros hechos banales. Autor poco prolífico y centrado en la correspondencia con otros investigadores, cabe destacar en su producción los artículos “Falso OVNI en Galicia” (en Stendek nº 14, 1973) y, especialmente, “Algo sobre las fotografías del supuesto OVNI de San José de Valderas” (en Stendek nº 9, 1972), donde puso al descubierto el fraude de las fotos de la nave “ummita” del caso de San José de Valderas. RIBERA I JORDÀ, Antoni (Barcelona, 1920 – Sant Feliu de Codines, 2001). Escritor y traductor en varios idiomas (más de 300 obras), con estudios de Filosofía y Letras interrumpidos por la guerra civil, y considerado como el padre y el decano de la ufología española. Fue cofundador del CRIS (Centro de Recuperación e Investigaciones Submarinas) y del Cei (1958), del que fue Presidente de Honor en 1968. Director de la revista Horizonte entre 1968 y 1971, organizó en 1975 una célebre expedición a la Isla de Pascua, y en 1977, el I Congreso Nacional de Ufología en Barcelona. Dos años más tarde fue invitado a disertar ante el Grupo de Estudios OVNI (UFO Study Group) de la Cámara de los Lores británica. En 1989 recibió la Creu de Sant Jordi de la Generalitat de Catalunya en reconocimiento por sus aportaciones a la cultura catalana. Fue autor de una veintena de títulos sobre OVNIs, entre los que destacan El gran enigma de los platillos volantes (1966), auténtica Biblia del fenómeno para toda una generación de ufólogos, Un caso perfecto (con Rafael Farriols, 1969) y El misterio de Ummo (1979), que popularizaron este fraude, así como de Secuestrados por extraterrestres (1981), Por último es necesario hacer constar las señas del investigador o del centro que ha llevado a cabo la investigación cuyo producto es el informe publicado, indicando si se trata de una encuesta de primera mano (original) o basada en gestiones previas de otros encuestadores. [31][RC] REY BREA, Óscar (A Coruña, c. 1923-A Coruña, 1977). Uno de los pioneros de la ufología española, combatiente en la División Azul (1941-43) y auxiliar de meteorología. Con formación autodidacta en ciencias y aficionado a la astronomía, empezó a interesarse por los OVNIs a raíz de un avistamiento del que fue testigo el año 1943 en Puskin (URSS). En 1950 avanzó en el diario El Ideal Gallego el origen marciano de los platillos volantes, y el 9/4/1954 publicó en el periódico Pueblo la primera referencia conocida de la teoría del ciclo bienal. Fue también uno de los primeros proponentes de los sistemas de propulsión de los OVNIs y de la necesidad de seguir una metodología en la elaboración de los censos de casuística que los depurara 189 RNC primera exposición monográfica de las abducciones en lengua española. Coordinadora de Estudios sobre el Fenómeno OVNI en 1981. Su trabajo se concentró en el área de la compilación de catálogos de casuística de cobertura regional (Proyecto CATARES). En la actualidad sus elementos humanos y materiales se hallan comprometidos en el proyecto que representa Cuadernos de Ufología.[ JR] Opinión sobre el fenómeno (extraída de una entrevista en Expedientes Secretos, nº 4, 26 octubre 1996): ”En 1979 tuve el gran honor de ser invitado a hablar ante la Cámara de los Lores de Inglaterra, donde había un grupo de estudio OVNI, y les dije dos cosas que repetiré ahora. Primero, que el fenómeno es real. Hay miles de observaciones y de éstas quedan por lo menos un 5 por ciento que son completamente inexplicables. Muchas de estas observaciones son realizadas por pilotos militares o civiles, gente muy solvente. En segundo lugar, la hipótesis más plausible es la extraterrestre. Todavía no tenemos certeza, pero es lo más probable (...). Estos seres vienen de nuestra propia galaxia. Determinados datos indican que pueden proceder de una estrella que se encuentra a 38 años luz de la Tierra. Es posible que algunos de ellos vengan de allí, pero no todos. Porque no hay sólo un tipo de visitantes. Por lo menos existen cuatro o cinco diferentes.” ROBERTSON, panel A comienzos de 1953, y como consecuencia de las tensiones de la “guerra fría”, de las presiones que por parte de los medios de información y opinión pública someten de continuo a la Fuerza Aérea, y de las inquisitivas indagaciones de algunos investigadores privados como Donald Keyhoe, que denuncian permanentemente una política de ocultación y secreto del gobierno, se reúne en Washington del 14 al 18 de enero, bajo la presidencia del doctor H.P. Robertson, profesor del Californian Institute Technology (CALTECH), una comisión oficial de expertos científicos, militares y oficiales de inteligencia, con el propósito de dilucidar la importancia de las informaciones OVNI conocidas hasta ese momento por los medios oficiales norteamericanos. Componen también la comisión otros científicos de gran reputación como el Dr. Luis W. Álvarez, premio Nobel de física de 1968, el astrónomo Thornton Page, Lloyd V. Berkner, Samuel Goldsmit, el general W.M. Garland, director del ATIC (Air Technical Intelligence Center), Josef Allen Hynek, consejero científico del proyecto Blue Book, Marshall Chadwell, Ralph Clark y Philip Strong (los tres agentes de la CIA). Durante ese tiempo, examinan determinados casos previamente seleccionados por los investigadores del Blue RNC (Red Nacional de Corresponsales). Entidad decana en Andalucía en la recogida de casuística OVNI y su investigación. Fue creada en Sevilla por José Ruesga Montiel el 27 de febrero de 1969 y constituyó la mayor red de corresponsales de la época, llegando a contar con más de 200 socios y una delegación en Barcelona. Sus órganos de difusión escrita fueron Hojas informativas y OVNI: Ufología Informativa. La RNC tuvo una clara vocación organizativa, aglutinando la ufología andaluza a principios de los setenta y desempeñando la secretaría de la 190 Roswell, caso ROSWELL, caso Book, así como otros procedentes de las comisiones oficiales creadas con anterioridad y diverso material gráfico (algunas filmaciones), que muestran las evoluciones de tales fenómenos. La verdadera investigación sobre este supuesto platillo estrellado, no comenzó hasta 1978, pero al menos todos (creyentes y escépticos) admiten que algo se estrelló en Roswell y fue recuperado por los militares. La cuestión es que después de 4 libros (El Incidente, Charles Berlitz y William Moore, 1980; UFO crash at Roswell, Kevin Randle y Don Schmitt, 1991; Crash at Corona, Stanton Friedman y Don Berliner, 1992; The truth about the UFO crash at Roswell, Kevin Randle y Don Schmitt, 1994) y centenares de artículos, todo está mucho más complicado que al principio. En sus conclusiones, el informe Robertson, estima que los OVNIs no constituyen una amenaza para la seguridad nacional, ni representan manifestaciones que obliguen a revisar los conceptos científicos ya establecidos. Principalmente, se hace hincapié en la necesidad de eliminar el “ruido” que provocan los cada vez más numerosos informes OVNI, que podrían ocultar actividades de carácter ofensivo procedentes del denominado “telón de acero” cuyas señales no serían detectables para la Defensa nacional. Se recomienda, pues, atemperar la creciente influencia que estos fenómenos tienen para la opinión pública del país, eliminando el aura de misterio que los rodea y educando al público para que identifique correctamente tales estímulos. El dos de julio de 1947, tras una tormenta sobre la zona de Roswell, un granjero descubrió gran cantidad de restos en un rancho cercano. Se describen como láminas muy finas y ligeras junto a pequeñas barras metálicas cubiertas de caracteres jeroglíficos. Todos los restos fueron inmediatamente recogidos por la Fuerza Aérea que los trasladó a la base de Wright Patterson, no sin antes difundir un comunicado ¡reconociendo la captura de uno de esos discos voladores! Pocas horas más tarde habría un desmentido oficial atribuyendo los restos a un globo meteorológico. Y esto sería todo hasta más de 30 años después. Cierta historiografía ufológica considera que estas sugerencias, plenamente apoyadas por los servicios de inteligencia, inauguran el período de debunking (desprestigio), que presidiría, a partir de entonces, las declaraciones oficiales sobre cualquier observación OVNI producida en el territorio de los EE.UU., relacionándolo con la promulgación ese mismo año de la Air Force Regulation AFR 200-2, orden dictada por el Secretario de Estado Henry Talbot que regulaba los procedimientos para controlar y minimizar los efectos de las observaciones frente a la opinión pública, fundamentalmente, de las generadas en el seno de la propia Fuerza Aérea.[ JA] Tras las sucesivas visitas de distintos ufólogos a partir de 1978, han ido apareciendo más y más testigos (90 según la última estimación) que aseguran tener datos de primera mano sobre lo ocurrido. Por desgracia, estos datos casi nunca son compatibles. De hecho, cada uno de los autores citados anteriormente, defiende unas fechas, un lugar de caída, un tipo de nave y unos tripulantes distintos de los demás. Las últimas aportaciones a esta controversia han 191 Ruesga Montiel, José sido dos informes oficiales, uno de la Fuerza Aérea (Septiembre 1994) y otro de la Oficina Auditora del Congreso ( Julio 1995) manifestando que los restos recuperados en Roswell pertenecían a unos experimentos de alto secreto con globos denominados "Proyecto MOGUL", negando cualquier informe sobre cadáveres extraterrestres. 1978. Dedicado a la investigación de campo durante casi veinticinco años, es autor del Proyecto CATARES (catálogo de 1.300 casos OVNI de Andalucía, 1979) y de diversos artículos sobre casuística. En 1983 creó la publicación Cuadernos de Ufología, de la que es actualmente coeditor. Es igualmente coordinador del Colectivo Cuadernos y vicepresidente primero de la Fundación Anomalía. Por el lado contrario, tenemos la creación del Centro de Investigación y Museo Internacional OVNI de Roswell, Box 2221, NM 88202, USA, (400 North Main, Roswell), Tel: 505 625-9495 y la aparición de dos películas. Una titulada Roswell y protagonizada por Martin Sheen y Kyle MacLachlan no ha alcanzado gran difusión; pero la segunda, una supuesta filmación real de la autopsia de un extraterrestre adquirida al anciano cámara que la realizó en 1947, ha llegado a todos los rincones del mundo, generando enormes ingresos para su propietario actual, el Sr. Ray Santilli. Un año después de que salieran a la luz las primeras imágenes ha podido demostrarse más allá de toda duda razonable que se trata de un nuevo fraude. También algunos supuestos fragmentos han resultado ser falsos una vez analizados. [45, 228][LG] Opinión sobre el fenómeno (extraída de Papers d'Ovnis nº31-32, julioagosto 1996): "A estas alturas de mi vida biológica y ufológica veo el fenómeno como una inevitable mezcla de muchas consecuencias, que no orígenes. Hay un substrato importante de un fenómeno psicosocial, generado fundamentalmente por los medios de comunicación y los intereses de las grandes potencias, principalmente Estados Unidos. La buena voluntad pero escasa preparación intelectual de la mayoría de los ufólogos ha generado estereotipos que han calado fuertemente también en nuestras filas. Por último estoy convencido de que un número más o menos importante de casuística responde a un fenómeno físico, cuyas características básicas se confunden con otros factores o estereotipos, pero que terminarán por definir un origen físico y terrestre de lo que venimos llamando fenómeno Ovni". RUESGA MONTIEL, José (n. Sevilla, 1947). Investigador y, sobre todo, organizador nato, se le puede considerar un "corredor de fondo de la Ufología". Se formó en Arquitectura Técnica e Historia Antigua (Arqueología) en la Universidad de Sevilla y UNED, respectivamente. Presidente y fundador en 1969 de la Rnc, dirigió el intento de integración de la ufología andaluza de los primeros años setenta y tuvo un papel fundamental en la creación de la Cefo en RUIZ NOGUEZ, Luis (n. Ciudad de México, 1957). Ingeniero Químico agregado de la Facultad de Química de la Universidad Nacional Autónoma de México. Especialista en Estadística Aplicada por el Instituto de Matemáticas y Sistemas de la misma universidad. Desde comienzos de los años 192 Ruiz Noguez, Luis setenta se interesó por el estudio de los OVNIs, siendo en México delegado de la CIU (Comisión de Investigaciones Ufológicas) de Buenos Aires (1983) y representante de Cuadernos de Ufología. En 1994 creó con otros estudiosos de la Somie la revista Perspectivas Ufológicas, de cuyo consejo editorial forma parte. Estudioso de los aspectos físicos del fenómeno OVNI, destacan entre sus trabajos (a veces firmados con el pseudónimo “Roberto López”) “Centellas o rayos en bola” (en Cuadernos de Ufología nº 7, 2ª época, 1990), “Difracción de la luz” (CdU. nº 8, 2ª época, 1990), “Meteoritos” (CdU. nº 12, 2ª época, 1992), “La historia gráfica de los Ovnis en México” (en Perspectivas Ufológicas nº 3, 1994) y “Dossier Astroarqueología” (Perspectivas Ufológicas nº 5, 1995). DTU 193 San José de Valderas, caso S SAN JOSÉ DE VALDERAS, caso coordenadas geográficas que limitaban la zona del probable aterrizaje (afueras de Madrid). Esta circunstancia animó a varios conocedores de la carta a recorrerse dicha zona armados de cámaras fotográficas y tomavistas, que cesaron en su empeño (y sin éxito) tras ver la portada del periódico Informaciones. Supuesta observación de un OVNI ocurrida entre las 20:00 y las 21:00 h. del primero de junio de 1967. Ese día, diversos vecinos y paseantes que se hallaban cerca de los castillos de San José de Valderas (Alcorcón, Madrid) vieron aproximarse un objeto con luz anaranjada que durante unos minutos evolucionó sobre los castillos para alejarse en dirección NE hacia Madrid. Poco después, el mismo objeto -descrito como dos palanganas encaradas y, de acuerdo a algunos testimonios, con un signo en su parte inferios- habría aterrizado al lado del restaurante La Ponderosa, en el km. 3 de la carretera a Boadilla del Monte. Según las primeras noticias de la prensa madrileña (diarios Informaciones y Pueblo del 2 de junio) los testigos se contaban por docenas, e incluso el primero de ellos publicaba en primera plana dos fotografías del OVNI en San José de Valderas, apreciándose en una de ellas una especie de letra H cruzada por una barra vertical más corta en su centro. Se trataba del mismo símbolo que unos autodenominados expedicionarios del planeta Ummo habían estampado en cartas mecanografiadas que un grupo de Madrid (Fernando Sesma y varios contertulios de la Ballena Alegre) venía recibiendo desde comienzos de 1966. A Sesma y a otros contactados con Ummo les había llegado el 30 de mayo de 1967 una misiva ummita anunciándoles que en los días siguientes tres de sus naves aterrizarían en Bolivia, Brasil y España, indicándose en este último caso las Las dos fotografías que publicó no fueron las únicas que alguien tomó esa tarde. Casi tres meses después, un tal Antonio Pardo escribió a Màrius Lleget contándole que él y su familia estaban en San José de Valderas y que no sólo vieron el platillo, sino que él incluso lo fotografió. Envió dos copias a Lleget, junto a un material plástico y metálico que recuperó en el barrio de Santa Mónica (colindante con la carretera a Boadilla), al parecer relacionado con el aterrizaje. La investigación de este caso, al igual que el de su antecedente registrado la tarde del 6/02/1966 en el barrio madrileño de Aluche (donde se habría posado un platillo ummita que dejó tres huellas en triángulo), fue recogida en el libro Un caso perfecto (eds. de 1969 y 1973), escrito por Antonio Ribera y Rafael Farriols. Ambos contaron con la colaboración del testigo principal de Aluche, José Luis Jordán Peña, quien había recogido la mayor parte de los testimonios de San José de Valderas y Santa Mónica. Tal circunstancia, así como que la mayoría de los testigos fueran anónimos o con apodos, y unos curiosos paralelismos entre su encuesta y la efectuada por el presunto Antonio Pardo (a quien nunca se 194 Sectas ufológicas pudo localizar), sirvieron para que desde el primer momento la duda planeara sobre el “caso perfecto”. Además, en el libro se aseguraba que los restos remitidos por Pardo a Lleget estaban al alcance de la técnica terrestre, según se desprendía del análisis realizado por el Instituto Nacional de Técnica Aeroespacial. contactado con entidades extraterrestres o incluso encarnación de una entidad ajena a nuestro planeta. Poseen un cuerpo doctrinal a menudo dogmático y forman un grupo cerrado sobre sí mismo, regido por un líder carismático. Se caracterizan por seguir una doctrina de tipo milenarista, según la cual el fin del mundo está más o menos próximo y sólo sobrevivirán aquellos que se hayan convertido en adeptos seguidores. El primer contacto mesiánico, no divulgado hasta 1953 en los medios de comunicación, habría tenido lugar en noviembre de 1952 en el desierto de California, cuando George Adamsky mantuvo una conversación con un ser procedente de Venus que le empleó como portavoz de un mensaje de amonestación a la humanidad por la carrera armamentista. A pesar de que su historia está totalmente desacreditada, su influencia en posteriores relatos similares ha sido decisiva. En este tipo de tipo de grupos sectarios existen muchas formas de "contacto", aunque la más tradicional ha sido el contacto visual con previa cita. Los ejemplos máximos de sectas ufológicas son la Misión RAMA, iniciada en 1974 por el contactado peruano Sixto Paz y reconvertida en Misión Humanidad en 1991, Fraternidad Cósmica (Fratellanza Cosmica) del italiano Eugenio Siragusa y la Aetherius Society inglesa, fundada en 1955 por George King. En numerosas ocasiones la creencia de ser elegido por los extraterrestres se manifiesta como lo que en psiquiatría se denomina delirio pranoide, aunque es necesario que los adeptos participen en un sistema de creencias que permita que la secta prospere. Diferentes sectas platillistas han derivado hacia el sectarismo destructivo. Como ejemplo extremo de ello, el 26 de marzo de 1997 fueron hallados en San Diego (California, EE.UU.) los cuerpos sin vida de 39 adeptos del grupo Heaven’s Gate, que se La evidencia fotográfica (cinco negativos del fotógrafo anónimo y dos copias de Antonio Pardo) fue posteriormente examinada por estudiosos españoles y extranjeros, entre los que cabe destacar a Óscar Rey Brea, Claude Poher (v. Gepan) y los analistas del Gsw americano. Todos ellos coincidieron en afirmar que se trataba de un montaje realizado con una maqueta de no más de medio metro a la que se había pintado el signo ummita. Tanto Poher como el GSW utilizaron computadoras para su peritaje (el primero, sobre los negativos originales), y sus conclusiones fueron confirmadas por otro análisis hecho por Carles Berché en 1994. Aunque hay personas que hoy en día aseguran haber visto entonces un OVNI (sin la H), como Eugenia Arbiol y Anselmo de Virto, la teoría más plausible es la de un complejo fraude perfectamente escenificado. En fecha reciente, Vicente Ortuño -amigo de Jordán Peña y también testigo de Aluche- ha reconocido que ambos fueron los autores del trucaje fotográfico. [7, 192][ JM] SECTAS UFOLÓGICAS Las sectas son conjuntos de seguidores de una ideología o creyentes en una doctrina religiosa que se ha independizado de otra. En el caso de las sectas ufológicas, suelen organizarse alrededor de un líder carismático que a menudo se presenta como 195 SEPRA habían suicidado ritualmente con la esperanza de reunirse con una supuesta nave extraterrestre que acompañaba al cometa Hale-Bopp. [11, 42, 55][RC] un retorno a sus orígenes astrológicos y esotéricos. Entre su producción bibliográfica destacan Lorsomoromba-El mensaje filosófico del hombre del espacio (1955), Yo, confidente de los hombres del espacio (1965), La lógica del visitante del espacio (1969), y El Universo de perfil (1976). SEPRA Opinión sobre el fenómeno (extraída del nº 20 de 2001, Buenos Aires, 1970, y del nº 4 de Telepsiquia, Barcelona, 1977): “ Los investigadores científicos de los OVNIs no hacen otra cosa que una especie de coleccionismo de casos (...) y escamoteando siempre el hecho más desconcertante y fundamental de que LOS EXTRATERRESTRES SE CONTRADICEN (...). Hoy puedo asegurar una cosa: LOS MENSAJES Y CONDUCTAS DEL VISITANTE DEL ESPACIO SE REALIZAN EN UN LENGUAJE COSMOSIMBÓLICO (que) cuando pretenden traducirse en nuestra gramática lógica resultan incongruentes.” (1970) Véase Gepan. SESMA MANZANO, Fernando (Ceuta, 1908-Madrid, 1982). Escritor y funcionario de Telégrafos, pionero del asociacionismo ufológico y del contactismo en España. En otoño de 1954 publicó en el diario Madrid una serie titulada “El origen extraterrestre de los platillos volantes” cuya repercusión motivó la creación en diciembre del mismo año de la Sociedad de Amigos de los Visitantes del Espacio BURU, la primera de tal carácter en España, que celebraba sus reuniones en “La Ballena Alegre” (el sótano del madrileño Café Lion). A partir de 1962 Sesma empezó a ser receptor postal y telefónico de supuestos mensajes extraterrestres (sobre todo de un tal “Saliano”), de contenido entre hilarante y absurdo. En 1965 Sesma disolvió la Sociedad BURU, pero prosiguieron las tertulias en “La Ballena Alegre”, donde desde 1966 se fueron recibiendo una serie de informes cuya autoría reclamaban los expedicionarios del planeta Ummo en la Tierra. A finales de los años 60 la popularidad de Sesma en los medios alcanzó sus más altas cotas, aunque desaparecieron por entonces las reuniones del sótano del Café Lion. La década de los 70 marcó un punto de inflexión en su pensamiento, al reconocer el carácter fraudulento de algunas de las experiencias que, engañado, había protagonizado. Sus últimos años supusieron (Sobre el asunto de Ummo): “Si difícil es que las personas crean al principio en algo insólito, más difícil resulta que dejen de creerlo cuando ya lo han aceptado (...) Tampoco creo que pueda esperarse, en mucho tiempo, ningún contacto público con los visitantes espaciales. Nuestra sintonía es demasiado estrecha para que exista ningún diálogo colectivo. Sus palabras, o sus símbolos, no serían comprendidos (...) y Ellos no son conquistadores, como nosotros, de ningún Espejismo Exterior.” (1977) SETI, proyectos SETI son las siglas de Search for Extra196 SETI, proyectos NASA el 12 de octubre de 1992 y suspendido por motivos presupuestarios un año más tarde. Consistió en un rastreo generalizado del espectro electromagnético en la zona comprendida entre 1 y 10 Ghz., en la que el ruido de fondo interestelar es especialmente reducido, complementado con una búsqueda más sensible en la dirección de unas 800 estrellas de tipo similar al Sol. Terrestrial Intelligence, búsqueda de inteligencia extraterrestre. Supone adoptar una posición pasiva en la captación de señales de seres inteligentes de otros mundos. El primero de los múltiples intentos de este tipo desarrollados en diversas partes del planeta fue el llamado Proyecto Ozma (nombre del rey de la imaginaria tierra de Oz), dirigido por Frank Donald Drake, que enfocó las estrellas Tau Ceti y Epsilon Eridani con un radiotelescopio paraboloide de 25'5 m. de diámetro del National Radio Astronomy Observatory (NRAO), en Green Bank (Virginia Occidental, EE.UU.). Durante 150 horas hábiles, desde el 8 de abril de 1960 hasta julio de ese mismo año, se escuchó la frecuencia de 1.420 Mhz., correspondiente a una longitud de onda de 21'1 cm., en la creencia de que debería poseer carácter universal al ser la propia del átomo de hidrógeno. No se cosechó ningún resultado positivo, pero se sentaron las bases para posteriores intentos. Desde 1972 a 1976, también en el NRAO, Zuckerman y Palmer emprendieron el Proyecto Ozma II, con radiotelescopios de hasta 90 m. de diámetro y 384 receptores de radio, frente al único con que contó Drake. Se examinaron esta vez 700 estrellas, pero sin que se captara nada anormal. De los proyectos que en la actualidad siguen en marcha, el más veterano es el que desde diciembre de 1973 lleva a cabo el observatorio de la Universidad de Ohio con un radiotelescopio de 52'5 m. sintonizado en la banda de 1'4 a 1'7 Ghz. En 1977 este instrumento captó una intensa señal de origen desconocido que no volvió a repetirse, por lo que quedó sin explicación. La Sociedad Planetaria, que cuenta ya con experiencias anteriores en este campo (proyecto Sentinel), auspicia desde 1985 el META (Megachannel ET Assay), que se desarrolla desde el radiotelescopio Cassegrain de la Universidad de Harvard, y que habría descubierto señales no repetitivas de banda estrecha en el plano de la Vía Láctea. Otros proyectos aún en desarrollo son el Phoenix, que emplea el radiotelescopio australiano Parkes, y el Serendip del Laboratorio de Ciencias Espaciales de la Universidad de Berkeley. En mayo de 1999 se puso en marcha el proyecto Seti@Home, auspiciado por esta misma universidad y diversas empresas comerciales, que cuenta con la particularidad de que las señales obtenidas (procedentes del radiotelescopio de Arecibo) son analizadas por los ordenadores de cerca de un millón de internautas de todo el mundo mediante un programa “salvapantallas” especialmente diseñado para esta función. Sin contar algunos programas particulares desarrollados por aficionados (cuya labor puede ser exitosa en un campo poco atendido por los grandes radiotelescopios, con largas listas de espera para su utilización), desde estos primeros intentos se han sucedido más de cuarenta proyectos similares con financiación pública en EE.UU., la antigua URSS, Canadá, Francia, Australia y Argentina. El más ambicioso de todo ellos fue el MCSA (Multi Channel Spectrum Analyzer), iniciado por la Algunos han criticado la preferencia 197 Sierra Albert, Javier mostrada por todas estas iniciativas por las ondas de radio como medio de comunicación interestelar frente a otras opciones como los láseres, los rayos gamma o las ondas gravitatorias. En realidad, dicha elección viene determinada claramente por motivos tecnológicos, pues los radiotelescopios son los únicos instrumentos disponibles en abundancia para escudriñar el cielo. Esto pone de relevancia como la enorme escala temporal del Universo es contraria al encuentro de civilizaciones con el mismo nivel de desarrollo.[75, 214][ JR, LG, MM] Amigos de Expediente-X. SIGN Y GRUDGE, proyectos Primeros proyectos de la Fuerza Aérea estadounidense encaminados a determinar si los OVNIs podían constituir una amenaza para la seguridad nacional. Tras los numerosos avistamientos registrados en 1947, la USAF decidió en septiembre de ese mismo año iniciar un proyecto que analizase los diversos incidentes que se reportaban. El 22 de enero de 1948 arrancó así el Proyecto Sign (en inglés, signo; popularmente conocido como Saucer, platillo) en el seno del Air Technical Intelligence Center (ATIC), con sede en la actual base de Wright-Patterson (Ohio). Sus objetivos se centraban en la recolección y evaluación de toda la información relativa a avistamientos y fenómenos atmosféricos que pudieran presumirse de interés para la seguridad nacional. La inexperiencia de los militares en estas labores hizo que, pese a haberse percatado ya del problema de distinguir la señal entre el ingente ruido provocado por objetos astronómicos y aeronáuticos, se desperdiciaran muchos esfuerzos en casos de escasa extrañeza o investigando inútilmente la vida privada de los testigos, fundamentalmente por carecer de métodos de trabajo estandarizados. Por otro lado, la diversidad de opiniones sobre la naturaleza de los OVNIs provocó fuertes controversias dentro del proyecto. Durante los primeros meses fueron los partidarios de la HET quienes llevaron la iniciativa, hasta que después del caso de los pilotos Chiles y Whitted (23/07/1948) el ATIC elaboró un informe sobre el estado de la cuestión en el que se concluía que los OVNIs eran naves de otros mundos (agosto de 1948). El jefe del SIERRA ALBERT, Javier (n. Teruel, 1972). Periodista e investigador ufológico, miembro de la llamada cuarta (o tercera) generación. Especializado en el tema de los contactados, fue promotor del frustrado CATCE (catálogo de contactados españoles, 1988) y es autor de los libros Extraterrestres: Dioses de una nueva religión (C.M. Chaminade, Madrid, 1990), Técnicas del contacto extraterrestre (Héptada, Madrid, 1992) y Los guías del Cosmos (Grijalbo, Madrid, 1996). Ha escrito igualmente Roswell. Secreto de estado (EDAF, Madrid, 1995) y, con Enrique de Vicente, ¿Qué se oculta tras los expedientes X? (Grijalbo, Madrid, 1996). Junto a Josep Guijarro emprendió el proyecto diana (1990), cuyos resultados aún no se han dado a conocer. En 1992 fue el coordinador de la enciclopedia Más Allá de los OVNIs (Héptada, Madrid), y poco después compilaría la bibliografía ufológica que aparece en la obra de Ignacio Cabria Entre ufólogos, creyentes y contactados (1993). Actualmente desempeña los cargos de subdirector de la revista Más Allá de la ciencia y de presidente del llamado Club de 198 Smith, Willy Estado Mayor de la USAF rechazó dichas conclusiones por falta de pruebas que las apoyaran, lo que facilitó el acceso al control del proyecto de quienes apostaban por explicaciones convencionales. fue el nacimiento de un nuevo proyecto oficial, el Blue Book, en marzo de 1952. [110, 191, 240][MM] SMITH, Willy El 11 de febrero de 1949, la Fuerza Aérea cambió el nombre del programa por Grudge (rencor, en inglés), alegando que el anterior había quedado comprometido, y el 27 de abril el Proyecto Sign presentaba su informe final, según el cual el 30% de los casos habían sido explicados satisfactoriamente y otro 30% podrían haberlo sido de haberse contado con más datos. El cambio de nombre conllevó también un giro en la política del ATIC, pues con los escépticos al mando el Proyecto Grudge persiguió dar respuestas convencionales a todos los incidentes que le llegaban. A tal efecto, permitió que el periodista del Saturday Evening Post Sidney Shallet tuviera acceso a sus archivos para que redactara un artículo que presentase los OVNIs como una pérdida de tiempo. El trabajo, publicado el 30 de abril de 1949, tuvo un efecto contrario al deseado, pues inundó el ATIC con nuevos casos y suscitó la reacción de defensores de la HET como el mayor Keyhoe, que con sus artículos de réplica presentaron los OVNIs por primera vez ante el gran público como naves de procedencia extraterrestre. El 27 de diciembre de ese mismo año el Proyecto Grudge dio a conocer su informe final, que arrojaba un 23% de sucesos no identificados para los que se sugería una explicación de tipo psicológico. Aunque a menudo se considera que el proyecto se cerró con ese informe, lo cierto es que se mantuvo de forma precaria durante otros dos años. En septiembre de 1951 el capitán Edward J. Ruppelt se hizo cargo del mismo y emprendió una serie de reformas destinadas a dotarlo de nueva vida. Fruto de las mismas (1921-2006). Doctor en Física por la Michigan University, dedicó la mayor parte de su trayectoria profesional a la enseñanza de esta materia en diversas universidades, entre ellas la Politécnica de Valencia, hasta que en 1980 abandonó esta actividad para dedicarse exclusivamente a la investigación ufológica. Interesado por los OVNIS desde los años cincuenta y profundo conocedor de la ufología hispanoamericana, fue un año sabático empleado en contactar con los investigadores españoles y franceses lo que le llevó a adoptar esa decisión. A finales de 1983 emprendió junto a J.A. Hynek el proyecto Unicat, cuya dirección asumió tras la muerte de éste en 1986, al que consagra hoy todos sus esfuerzos. En el terreno profesional ha publicado el libro Problems in Modern Physics, mientras que en el plano ufológico ha contribuido con diversos trabajos en revistas como Mufon UFO Journal, IUR, Stendek, Cuadernos de Ufología, Magonia, UFO Press, etcétera. Opinión sobre el fenómeno (extraída de su artículo “Panorama sobre el fenómeno OVNI”, en Cuadernos de Ufología nº 11, 2ª época, 1991): “El desarrollo de la ufología ha sido mutilado seriamente por la falta de unidad y metas comunes de los practicantes, y el descuido sistemático del método científico. La cantidad de pruebas recogidas es suficiente para establecer la existencia de un problema, de que algo fuera del reino de la ciencia convencional existe realmente, y que es suficiente para justificar la recogida sistemática de información al 199 SOBEPS respecto. Esto es precisamente lo que no se ha hecho. Aunque se ha recogido información durante más de cuarenta años, la calidad de ésta es tan variada que resulta inútil cualquier intento de interpretación estadística.” que duró más de dos años. En noviembre de 1988 la SOBEPS organizó el Premier Congrès Europèen sur les Phénomènes Aériens Anormaux (Primer Congreso Europeo sobre Fenómenos Aéreos Anormales), que reunió a destacados estudiosos mundiales: Walter Andrus (MUFON, EE.UU.), Richard Haines, Jacques Vallée, Vladimir Rubtsov (URSS), Pierre Lagrange, etc. SOBEPS (Société Belge d’Etude des Phénomènes Spatiaux). Entidad belga de investigación ufológica fundada el 20 de mayo de 1971 por Lucien Clerebaut. Con sede en Bruselas, nació con la voluntad de estructurar un grupo de estudio cuya base, compuesta por jóvenes universitarios, mantenía un enfoque científico del problema. Durante el invierno de 1972 publicó el primer número de Inforespace, todavía hoy órgano de difusión de la asociación, cuya periodicidad ha pasado de seis números anuales a dos, siguiendo los vaivenes de su afiliación. Ésta alcanzó su cénit en 1976 con 1750 asociados, que serían menos de 500 diez años después. Durante sus veinticinco años de existencia ha realizado miles de investigaciones, decenas de conferencias, emisiones de radio y televisión, así como múltiples gestiones con autoridades políticas, militares y científicas en un notable intento por hacer progresar la idea de la necesidad de investigar racionalmente el fenómeno OVNI. La organización se disolvió en junio de 2007, y dio paso a una estructura más informal conocida como COBEPS: http://www.cobeps.org [ JA] SOCIETY FOR SCIENTIFIC EXPLORATION En su vertiente de información pública, destaca la edición en 1977 de su primera monografía, Des soucoupes volantes aux OVNI. La chronique des OVNI (De los platillos volantes a los OVNI. Crónica de los OVNI), dedicada a trazar un panorama histórico de la casuística desde la antigüedad hasta 1947. Su mayor aportación se recoge en dos extensos libros, Vague d’OVNI sur la Belgique. Un dossier exceptionnel (Oleada OVNI en Bélgica. Un dossier excepcional, 1991) y Vague d’OVNI sur la Belgique 2. Une énigme non résolue (Oleada OVNI en Bélgica 2. Un enigma sin resolver, 1994), en los que se analiza el enorme aluvión de casuística que se produjo sobre el territorio belga desde el mes de noviembre de 1989 y Véase Journal of Scientific Exploration. SOCIOLOGÍA DE LOS OVNIS Una sociología de los ovnis debe estudiar las diversas facetas del fenómeno que tienen una incidencia social, desde la dinámica de difusión de la noticia de una observación OVNI hasta el análisis de la opinión pública al respecto, pasando por la estructura social de los grupos de seguidores y aficionados a los ovnis. Un estudio desde el punto de vista de la 200 Sociología de los ovnis dinámica de la comunicación social debe analizar la constitución del fenómeno de los ovnis como rumor, en el sentido de que se trata de información no contrastada que circula en un proceso de transformación y recreación continuas. Es importante a este respecto analizar el flujo de la comunicación entre los siguientes polos: el observador de un ovni, que emite una primera interpretación de lo que ha visto, el investigador que realiza el informe del caso con un juicio sobre su naturaleza extraterrestre o similar, los medios de comunicación que lo difunden proyectando una idea más o menos fantástica del fenómeno, los científicos y escépticos emitiendo su versión negativa, y el público, que mantiene una determinada creencia y un interés por ciertos aspectos de la narración. Todos estos polos actúan en una dinámica de conflicto social, influyendo unos factores sobre los otros en la creación del rumor. norteamericana, y un poco menos la europea, piensan que los ovnis son “reales”, y este concepto de “real” ha venido íntimamente asociado con la creencia en su origen extraterrestre. En una encuesta realizada en 1973 por Gallup en EE.UU. en plena fiebre de apariciones de humanoides, nada menos que un 11% de la población encuestada declaró haber sido testigo de un OVNI, lo que representaba 15 millones de personas. Esta tendencia se ha desplazado hacia el tema estrella de la última década: las llamadas abducciones o secuestros en el interior de ovnis. Gracias a la publicidad conseguida por los libros del escritor Budd Hopkins, numerosas personas han declarado sentir los síntomas asociados por éste a los casos de abducción, creyendo haber sido objeto de una experiencia no consciente de secuestro por extraterrestres. Al ser los ovnis uno de los fenómenos de mayor atractivo popular en las últimas décadas, se ha formado en torno al tema una comunidad de seguidores, críticos, etc., que ha adquirido características diferenciadas de un país a otro. En el caso de España podemos clasificar este colectivo de la siguiente manera: Otra perspectiva a adoptar por el sociólogo es la del análisis de la opinión pública a través de las encuestas de opinión. La primera la realizó en 1947 la agencia Gallup en EE.UU. con motivo de la oleada de observaciones que siguió al caso Arnold. Los resultados reflejaban que apenas bautizado el fenómeno, nada menos que el 90% de los norteamericanos habían oído hablar de “platillos volantes”. Pero, a diferencia de hoy, estos no eran considerados inicialmente objetos no convencionales, sino más bien vehículos experimentales de alguna potencia extranjera. Fue en 1950 cuando algunos investigadores y periodistas introdujeron en la opinión pública, primero en EE.UU. y luego en todo el mundo occidental, la idea de que eran “algo de otro planeta”. A partir de los años sesenta todas las encuestas han revelado que aproximadamente la mitad de la población a) Grupos ufológicos. Por lo general están constituidos legalmente como asociaciones con una denominación del tipo “centro investigador” y compuestos por un reducido grupo de personas, en su mayoría jóvenes. Estudian el tema ovni desde actitudes favorables a la existencia de un fenómeno inexplicado, al que asocian preferentemente con un origen extraterrestre. La actividad principal de estos grupos suele ser la investigación de campo, entendida como recogida de testimonios ufológicos y la divulgación de sus investigaciones particulares en boletines amateurs y programas de radio. Gran parte del trabajo 201 Sociología de los ovnis desarrollado se invierte en labores administrativas y burocráticas, y de hecho se presentan de forma pública como grupos con un alto grado de organización, que suele ser más teórica que real. Estos grupos se han unido en algunos períodos en organizaciones de escala regional o nacional, pero en los últimos tiempos han perdido la categoría de vanguardia en el estudio de los ovnis que tuvieron en las décadas de los 60 y 70, manteniendo posiciones teóricas poco innovadoras. Estos grupos estuvieron en otra época implicados en la organización de eventos divulgativos de tipo congreso, pero en los últimos tiempos esta clase de actos han pasado a ser organizados por editoriales o empresarios de temas mágicos con objetivos comerciales. disperso en multitud de tendencias individuales y de grupo, y que siguen doctrinas eclécticas de tipo mesiánico sobre el contacto con extraterrestres. Por lo general forman grupos pequeños no jerárquicos, laxos en sus vínculos, no proselitistas y que unen a sus miembros en relaciones más emotivas y espiritualistas que intelectuales. d) Investigadores críticos. Generalmente son investigadores veteranos que han evolucionado desde la ufología clásica organizada en centros investigadores a una actividad más individualizada y orientada de forma racionalista y crítica. La ufología racionalista se ha organizado en España en torno a la revista Cuadernos de Ufología, cuyo Colectivo ha dado lugar recientemente a la Fundación Anomalía. Entre sus objetivos están la investigación científica del fenómeno OVNI, la difusión de una visión crítica sobre el mismo, el fomento de los estudios en esa línea y la preservación de la documentación sobre la subcultura ovni. b) Ufólogos periodistas. Algunos ufólogos han tendido a la profesionalización como divulgadores del tema ovni en las revistas mágicas de distribución comercial, como autores de libros y difusores en radio y televisión, en una línea continuadora de la ufología clásica de orientación proextraterrestre. Los jóvenes ufólogos que se han iniciado en la profesión periodística en los últimos años se han especializado en aspectos altamente controvertidos del fenómeno ovni y son los promotores ante la opinión pública de las afirmaciones más extravagantes de la moderna ufología internacional. Trabajan de forma individualista y son las voces más influyentes en los congresos de Ufología. e) Escépticos. Son detractores de las pretensiones de la Parapsicología, la Ufología y las magias. Procedentes algunos de las líneas de la ufología más racionalista, se han volcado en una labor desmitificadora descalificando aquellos fenómenos desde el punto de vista de la ciencia y desenmascarando el fraude donde lo hay. Se puede englobar a todos estos colectivos, salvo el último, bajo el nombre genérico de “movimiento ufológico”, aunque las diferencias entre ellos sean muy considerables. Podríamos decir que existe una conexión más cercana entre los dos primeros sectores -ufólogos y divulgadoresy los dos últimos -críticos y escépticos-, quedando los contactados como un sector al margen al no considerarse ellos mismos investigadores, por enfocar su actividad c) Contactados. Los contactados buscan la comunicación física o psíquica con los extraterrestres, y en ocasiones se sienten elegidos para algún tipo de misión por los seres del espacio. El contactismo supone una experiencia religiosa de tipo místico. Se trata de un colectivo no homogéneo, 202 Socorro, caso como un culto. mirarle sobresaltada. El patrullero detuvo su vehículo, bajó del mismo, informó por radio que se disponía a examinar el coche volcado y empezó a dirigirse hacia donde estaba. En ese momento escuchó un nuevo bramido que fue aumentando tanto de intensidad como de frecuencia, cada vez más agudo, al tiempo que una llama azulada aparecía bajo el objeto y éste comenzaba a elevarse lentamente. Temiendo que pudiera estallar, Zamora corrió a refugiarse tras el automóvil de forma tan atropellada que se golpeó con él y le cayeron las gafas, aunque eso no detuvo su carrera. Entretanto, el objeto (ovalado, de color blanco-aluminio y con una insignia roja en un costado) había ascendido hasta la altura en que se hallaba el coche. Cuando cesó el bramido, el policía dejó de correr, se volvió hacia el OVNI y lo vio alejarse en dirección sudoeste a gran velocidad. Justo entonces oyó durante un segundo un agudo chirrido que fue haciéndose más grave hasta desaparecer. Mientras veía aún el objeto perderse en la distancia, contactó por radio con el sargento Chávez, con quien después examinaría el lugar del aterrizaje. Los sociólogos no se han ocupado de estudiar a los testigos de objetos voladores no identificados, pues en principio no constituyen un grupo con características peculiares ni la observación de ovnis constituye un fenómeno de una incidencia social notable. Ocupando el terreno de aquellos, unos pocos ufólogos críticos han cuestionado en los últimos tiempos el axioma según el cual los testigos de ovnis son personas normales que representan una media de la sociedad, preguntándose en cambio si no se da en la mayoría de ellos el “síndrome de la personalidad propensa a la fantasía” (v. Hipótesis explicativas). Un apartado muy especial es el estudio de los cultos extraterrestres desde el punto de vista de la sociología de la religión, campo que los sociólogos comparten con los antropólogos. [59, 86, 150] [IC]. SOCORRO, caso A las 17:45 h. del 24 de abril de 1964, el inspector de policía Lonnie Zamora perseguía en Socorro (Nuevo México, EE.UU.) a un automóvil que había sobrepasado el limite de velocidad permitido. De repente oyó una especie de bramido y vio una llama azulada en el cielo que le hizo pensar que tal vez hubiese explotado un polvorín que se hallaba en las inmediaciones. Abandonó la persecución y se dirigió hacia el lugar desde donde parecía proceder la llamarada, dejando la carretera y avanzando con el coche campo a través. Al alcanzar una colina observó a unos 150-200 m. un objeto brillante que a primera vista le pareció un automóvil volcado. Junto a éste se encontraban dos personas vestidas de blanco, una de las cuales pareció girarse y En el mismo se descubrieron cuatro huellas cuadrangulares dispuestas trapezoidalmente y cuatro zonas chamuscadas, tres de ellas coincidentes con las anteriores marcas, junto a varias pequeñas indentaciones circulares de carácter superficial. Zamora creyó recordar que el objeto se sostenía sobre cuatro patas que se corresponderían con las huellas cuadradas, así como que antes de sobresaltarse por el bramido había escuchado dos o tres golpes secos como los de una puerta pesada al cerrarse. Aunque se efectuaron algunos análisis de muestras del suelo y de las plantas del lugar del aterrizaje, ninguno arrojó resultados anormales. Aunque no fueron entrevistados hasta mayo de 1978, fue 203 SOMIE posible localizar a otros dos testigos, conductores que se encontraban en las cercanías, quienes recordaron haber visto una nube de humo negro y, al menos uno de ellos, un objeto con forma de huevo que ascendió verticalmente a través de la humareda. Phénomèna (hasta 2002) y editó hasta 1996 la publicación trimestral OVNI-Présence, que a lo largo de sus casi dos décadas de existencia manifestó un patente interés por el tratamiento objetivo de la información ufológica. En su primera época OVNIPrésence se editaba en xerocopia, pasando a partir de 1986 a hacerlo en offset, con un notable aumento de la calidad tanto de la impresión como de sus contenidos. Sus dossieres sobre la ufología británica, Ummo y el pánico de 1938 por la popular emisión de Orson Welles de La guerra de los mundos (v. extraterrestre) obtuvieron el reconocimiento de la comunidad ufológica internacional. Al frente de la redacción estuvo el investigador Yves Bosson, quien más tarde sería también director de la sucesora de OVNI-Présence: la publicación trimestral Anomalíes, editada entre 1996 y 1999, y más volcada hacia otras temáticas como el folklore contemporáneo y las leyendas urbanas. SOS OVNI cesó su actividad en 2002, aunque sus publicaciones pueden consultarse en los archivos del AFU sueco: http://files.afu.se/Downloads/ ?dir=./Magazines/France [ JA] La aparente materialidad de este suceso, que sólo parece explicable en términos de un prototipo experimental desconocido o de una nave extraterrestre, fue históricamente determinante para que los ufólogos estadounidenses empezaran a admitir la veracidad de los casos de aterrizajes, que hasta entonces se despreciaban por su relación con el contactismo. La investigación de las Fuerzas Aéreas, en la que participó J.A. Hynek, no consiguió aclarar el incidente, pese a que se intentó seguir la pista de algún artefacto militar secreto que hubiera podido operar en la zona. [MM] SOMIE (Sociedad Mexicana de Investigación Escéptica). Entidad creada en 1989, activa hasta principios de 2000, y que fue editora, entre 1993 y 1997, de la revista cuatrimestral Perspectivas Ufológicas, la cual puede consultarse en http://files.afu.se/ Downloads/?dir=Magazines/Mexico/ Perspectivas SPEC Siglas de la Sociedade Portuguesa de Exploração Científica. Véase Cnifo. STARLIGHT INTERNATIONAL, proyecto SOS OVNI Grupo privado francés con origen en la AESV (Association d’Etude sur les Soucoupes Volantes) fundada en 1974 y dirigida por Perry Petrakis. Con ramificaciones en la Suiza francófona y en el Québec, publicó la revista bimestral Véase Detección de OVNIs. STENDEK Revista trimestral editada por el Cei 204 Stoliarov, comité STOLIAROV, comité entre 1970 y 1981 (para conocer el porqué de su nombre, véase Desapariciones misteriosas). Sucesora del antiguo Boletín del CEI (enero 1959-diciembre 1961), culminó la idea de crear un servicio informativo serio y veraz que fuera el portavoz de los investigadores asociados al CEI y otros elementos afines. Joan Crexell, Josep Serra y Pere Redón plantearon el proyecto a la Junta del CEI a principios de 1970, y su primer número apareció el mes de junio siguiente. La aportación económica inicial corrió a cargo de Crexell, quien con su experiencia se convirtió en el primer director. En julio de 1971 se editó un número monográfico para dar a conocer el catálogo de aterrizajes de Ballester Olmos. A sus páginas fueron acudiendo aquellos autores que con espíritu científico deseaban exponer sus trabajos de campo y de gabinete. En 1979 llegó a tener 1.400 suscriptores, cifra que permitió recoger en dos volúmenes (Sí, están. Aproximación científica a los OVNIS, ed. 7 1/2, Barcelona, 1978) los mejores trabajos publicados hasta ese momento. El declive del interés serio por el tema también afectó a Stendek, que vio reducido su número de suscriptores hasta el punto de decidirse suspender su edición ante la posibilidad de que su crisis afectara a la economía del CEI. El último ejemplar apareció en diciembre de 1981, tras haberse publicado 45 números y un extra. Por sus páginas pasaron todos los investigadores serios de habla española. En los últimos años la revista contó con un grupo de titulados que actuó como Consejo de Consultores. Hoy en día, Stendek sigue siendo una publicación de referencia obligada para los investigadores españoles. Se puede consultar en Internet en: https:// bit.ly/stendek[PR] El desconocimiento que durante décadas afectó a todos los asuntos internos soviéticos es también trasladable a los aspectos ufológicos. Durante mucho tiempo, extremos fundamentales como la intervención oficial en los estudios sobre estos temas han sido desconocidos para el gran público, aunque algunos autores, como recientemente Boris Churinov y hace años Hobana y Weverberg, hayan presentado mucha de la documentación reunida por los estudiosos privados que también existían tras el llamado “telón de acero”. En 1967 causó verdadera sensación el anuncio que la prensa mundial hizo de la creación de un Comité oficial de investigación denominado “Stoliarov”. Lo que en principio parecía ser la versión soviética del Comité Condon norteamericano se reveló como un voluntarioso intento de algunos veteranos investigadores para centralizar la información de los casos OVNI dispersos por su inmenso territorio. A iniciativa del físico Felix Ziegel, el 17 de mayo de ese año se reunieron unos cuarenta interesados bajo la cobertura de la Casa Central de la Aviación y Cosmonáutica, dependiente de las Fuerzas Armadas. Ziegel propuso entonces la creación de un Comité público que estudiara el fenómeno, tras presentar algunos casos ocurridos en la URSS y otros países. Porfiri Stoliarov, a la sazón general de Aviación, sugirió entonces formar un comité preparatorio para la creación posterior de un específico comité de investigación sobre los OVNIs, en el que se inscribieron un total de diecisiete personas. Con la alegría del momento, fueron elegidos provisionalmente como presidente Stoliarov, como secretario el periodista Arkady Tikhonov, y como 205 Stoliarov, comité ayudantes el físico Felix Ziegel y Alexander Kazantsev, conocido por sus hipótesis astroarqueológicas. En octubre de 1967, después de varios meses de preparación, se creó la Sección OVNI dentro del Comité de Cosmonáutica de la Sociedad de Apoyo al Ejército, la Aviación y la Marina. Su primera asamblea tuvo lugar el 18 de octubre en presencia de 350 asistentes. El 10 de noviembre, a las 20 horas, el general Stoliarov y Ziguel presentaron en la primera cadena de la televisión estatal algunos casos de observaciones, anunciando la creación de la Sección OVNI, cuya finalidad sería combatir las interpretaciones seudocientíficas de la cuestión. El general Stoliarov invitó también a quienes estuvieran interesados a afiliarse a su asociación. El impacto público causado por estas declaraciones fue inesperado, pues al día siguiente todos los corresponsales extranjeros entrevistaron a Félix Ziguel y la prensa mundial se hizo eco del acontecimiento. Las noticias publicadas no pudieron ser más erróneas, pues abonaban la impresión de que las autoridades del estado soviético pretendían estudiar el tema. El efecto de estas informaciones fue bastante mal recibido en los círculos del gobierno, que, en la lógica soviética, ordenaron la disolución de la citada Sección OVNI sin proporcionar ningún tipo de explicaciones a los interesados. El propio general Stoliarov se apartó definitivamente de cualquier actividad ufológica. Con ello, el primer grupo ufológico privado soviético había durado escasamente cuarenta y cinco días. [63, 115, 183, 264][ JA] DTU 206 Tabloides T TABLOIDES El primer periódico de estas características que comenzó a publicar reportajes sobre OVNIs, fue el National Enquirer en los años setenta, inicialmente con cierta corrección, pero esta fue deteriorándose con el transcurso del tiempo, dando origen a las denominadas “noticias basura”. En décadas posteriores comenzó a ser imitado por otras publicaciones similares (Weekly World News, Sun, y otros), que encontraron en la Ufología un filón que explotar económicamente y que comenzaron a subir progresivamente el tono portentoso de las “noticias basura”, cada vez más peregrinas, basadas en fuentes inexistentes o difíciles de comprobar, y con credibilidad y fiabilidad mas que dudosa las relacionadas específicamente con OVNIs. Se conoce comúnmente por “tabloides” a aquellas publicaciones de carácter sensacionalista que incluyen entre las noticias que divulgan, todo aquello que capte la atención del público al que van dirigidas, por disparatadas que dichas noticias sean, y entre las cuales se encuentran también noticias sobre OVNIs. El vocablo tabloide corresponde a una forma de presentación farmacológica de medicamentos, el cual se comercializa comprimido en forma de tabletas. Alfred Harmsworth, fundador del londinense Daily Mirror (1903), llamó “tabloides” (tabloids) a los artículos periodísticos breves, de prosa ágil e historias cortas que caracterizaban a su diario, intentando sugerir noticias o reportajes “comprimidos” y densos en contenido. El estilo periodístico propiciado por el Daily Mirror fue difundido en Estados Unidos por el Daily News de Nueva York (1919), llevándolo a extremos sensacionalistas. Aunque la evidencia de la falsedad es bien patente en las “noticias basura” por los dislates que refieren, de hecho tienen gran aceptación entre el público, generalmente personas con bajo nivel cultural. En la práctica, los tabloides constituyen un excelente medio donde propalar cualquier infundio, creando a fuerza de necedades unas expectativas, que hacen imposible cualquier intento serio de mostrar la verdad, erigiéndose en un excelente medio de expresión para cualquier trastornado necesitado de protagonismo o afán de notoriedad, sustentado por un periodismo sin escrúpulos. [227][AP] En la década de los ochenta y los noventa, los tabloides se decantaron por noticias de sexo, escándalos y cotilleos, por lo que actualmente la acepción de tabloide se ha convertido en sinónimo de prensa amarilla y sensacionalismo. Esta prensa sensacionalista se vende en Norteamérica en centros comerciales además de en establecimientos o puntos de venta de letra impresa, por lo que tienen gran difusión. 207 Teleportación TELEPORTACIÓN valor de un caso OVNI en función de los grados de extrañeza y credibilidad que exhibe. La idea original procede de Hynek, quien propuso una “matriz de extrañeza y probabilidad” como sistema para discernir cómoda y rápidamente entre OVNIs y OVIs, según la distancia que separaba sus coordenadas del origen de la gráfica. Saunders (1968) empleó esta técnica para la evaluación de la casuística registrada por el comité Condon primero y por el Ufocat después, si bien distinguiendo un mayor número de posibles valores en la objetividad que en la extrañeza. Algunos autores emplean este término (creado por Charles Fort como sinónimo de psicokinesis) para referirse a la supuesta habilidad manifestada por los OVNIs o sus tripulantes para mover objetos e incluso personas de un lugar a otro supuestamente mediante transmisión de materia. Al principio a este tipo de informes no se les daba una lectura ufológica, sino que se les trataba como hechos forteanos o como desapariciones misteriosas. El más destacado, con libro y película propios, sería el llamado "misterio de Filadelfia" donde un barco de la Armada americana habría sido teletransportado. Recientemente se ha demostrado (Valleé 1994) la falsedad del incidente. En España, fue Albert Adell (1974 y 1979) quien rediseñó el mismo método con el nombre de test de extrañeza-credibilidad. Para ello situó en el eje de abcisas los 10 factores que para él representaban el grado de credibilidad (π) del testigo (profesión, objetividad, salud, reputación, notoriedad, habilidad, número, quehacer, agudeza visual y cultura), y en el de ordenadas otros tantos correspondientes a la extrañeza (Σ) de la observación (sonido, luz, forma, velocidad, evolución, tiempo, cantidad, ambiente, luces secundarias y efectos secundarios). La conjunción de estos índices dibujaba un cuadro dividido en cuatro zonas delimitadas por el valor cuatro. De esta manera, sólo los casos con un grado superior a cinco en ambas escalas se consideraban aptos para una ulterior atención analítica. Por debajo quedaban los supuestos de testigo poco fiable (π < 5), fenómeno identificable (Σ < 5) e inaceptable (π < 5 y Σ < 5). Aunque este sistema poseía un gran atractivo por su sencillez, las subjetividades presentes en la consideración de sus factores mermaron su difusión en favor de métodos de valoración más objetivos, como el representado por los índices de Ballester-Guasp. [9, 10] [MM] La interpretación ufológica de algunos casos se potenció a través de varios artículos publicados en la prestigiosa Flying Saucer Review durante los años setenta, y con la proliferación posterior de abducciones ha pasado a convertirse en un cliché habitual. La teleportación del matrimonio Vidal y su automóvil desde Chascomús (Argentina) hasta México en 1968 se consideró inicialmente como la versión argentina del caso Hill, con tiempo perdido inclusive... luego todo ha resultado ser un montaje publicitario. [255][LG] TEORÍA DE PROCESOS DE LOS OVNIS Véase ufología procesal. TEST DE EXTRAÑEZACREDIBILIDAD Método de representación gráfica del 208 Testigo TESTIGO bromas y fabulaciones, consideración social en general, etc. Perceptor de un avistamiento o de una observación asociada en primera instancia a un fenómeno u objeto anómalo. En el curso de la encuesta pediremos al testigo un relato completo de su observación, cuidando de no realizar preguntas que puedan influir en el mismo, y de valorar, de forma objetiva, su fiabilidad como testigo. Esta ha sido una preocupación metodológica habitual entre los estudiosos de clara vocación científica, como es el caso del intento de medida de la subjetividad del testimonio propuesta por V.J. Ballester Olmos (1984). Parte el estudioso valenciano de la constatación de que, en numerosas ocasiones, los testigos interpretan erróneamente la visión correctamente percibida por los órganos sensoriales y, en otras, la versión que nos ofrecen de lo observado no se corresponde con la idea que tienen en su mente, debido a la dificultad para transmitir verbalmente esa información; de ahí la nefasta influencia de las preguntas tendenciosas sobre el testigo. Ballester propone un modelo que incluye diversos parámetros: concordancia y consistencia de los testimonios, personalidad y comportamiento del sujeto con relación a los “temas extraños”, impacto del caso en las creencias del testigo, y aspectos psicofisiológicos del testigo. Cada uno de estos parámetros se compone a su vez de diversos elementos cuya presencia o ausencia en el caso que está siendo analizado determinará una mayor o menor subjetividad en el testimonio oral, lo cual nos permitirá desecharlo como fenómeno presuntamente anómalo o mantener su extrañeza y grado de confiabilidad. [29][RC] Al no poder la Ufología convertirse, por su propia naturaleza, en ciencia empírica, será el estudio del testigo y del relato proporcionado por el mismo, tomados conjuntamente, la materia de estudio para el investigador OVNI. La Ufología estudia predominantemente relatos orales de personas que dicen haber contemplado un suceso aparentemente enigmático. El testimonio del perceptor ha sido tradicionalmente sobrevalorado, presentándolo como la realidad fidedigna de lo observado, obviando que el testigo es un transmisor cuya versión final de su visión puede variar mucho de la realidad original. Según se puede leer en determinados informes, da la impresión de que nos encontramos ante un oráculo que nos está transmitiendo las verdades ocultas, como implicado directo, de los misterios celestes. La Ufología de inspiración científica asume que gran parte del relato del testigo puede ser erróneo, que no falso. Al menos habrá expresado honradamente una verdad suya, sin deseo de engañar al encuestador (aunque también puede darse el caso). Al ser el testigo el principal “objeto de estudio” ufológico, debemos informarnos de cualquier detalle que nos permita extraer alguna conclusión sobre su fiabilidad como perceptor de un hecho anómalo: sus aficiones, interés por los temas extraños, familiaridad y conocimientos de los fenómenos astronómicos y atmosféricos convencionales, etc. Es conveniente también que nos informemos en su comunidad de residencia de su personalidad, propensión a 209 Tiempo perdido TIEMPO PERDIDO divulgar su teoría de una Tierra hueca formada por cinco esferas concéntricas. Su influencia se descubre en algunos relatos de Edgar Allan Poe, y especialmente en la colección de novelas de Edgar Rice Burroughs sobre Pellucidar (1914-15). Muchos otros autores han utilizado la misma idea para los más variados propósitos (generalmente la localización de sociedades utópicas), pero sin tomarla en serio por razones evidentes. (missing time). Pérdida parcial de memoria del testigo de un avistamiento que suele coincidir con lo que él mismo considera que es el momento de máxima cercanía al OVNI. Este fenómeno es casi una constante en los sucesos de abducción, en los que el testigo pierde la memoria consciente de haber estado dentro del OVNI y sus recuerdos sólo afloran mediante el empleo de técnicas de hipnosis regresiva. El término comenzó a utilizarse a raíz de la publicación del libro de Budd Hopkins Missing Time (New York, Marek Publishers, 1981).[ JR] En la actualidad se dispone de una abrumadora cantidad de pruebas físicas (gracias a los estudios sismológicos de las ondas producidas por los terremotos y las propias oscilaciones terrestres) que demuestran no sólo que la Tierra es esférica y sólida, sino que su masa se concentra hacia el centro y por tanto no puede ser hueca. TIERRA HUECA, teoría de la El concepto de la Tierra como una cáscara hueca y esférica, con una superficie interior cóncava y habitable a la que pudiera accederse mediante aberturas en los Polos, cavernas, o máquinas excavadoras ha sido uno de los motivos más empleados por la ficción científica de los siglos XVIII y XIX. Su origen dual potenciaba su atractivo. Tradicionalmente el infierno de todas las religiones se ha ubicado en el interior de la Tierra. Y desde la naciente perspectiva científica fue nada menos que el propio astrónomo Edmond Halley (1656-1742) quien, para explicar algunos fenómenos magnéticos, sugirió que la Tierra (y otros planetas) consistía en una serie de esferas concéntricas unas dentro de otras y rodeando un pequeño sol central, cada una de ellas con posibles aperturas polares. Sin embargo, en el peculiar mundillo de la Ufología no podían faltar autores como el americano Raymond Bernard, que todavía en 1964 defendía esta teoría en su libro The Hollow Earth. De hecho, el verdadero responsable de su difusión actual fue Ray Palmer, primer impulsor del fenómeno de los platillos volantes (v. Ciencia ficción, Ufología y), quien ya en 1946 había defendido ideas paranoicas en torno a cavernas subterráneas habitadas por robots (el llamado “misterio Shaver”) y que en 1970 hizo circular, con el título de “primera foto del agujero del polo”, una imagen tomada por un satélite de la NASA en la que aparecía una zona negra en el Polo Norte. El efecto se debía simplemente a que no se trataba de una foto única sino de un mosaico compuesto de decenas de fotos tomadas a mediodía a lo largo de un invierno; el círculo oscuro mostraba las latitudes no alcanzadas por el sol durante la larga “noche” polar. Esta explicación lógica y sencilla no fue óbice El mayor empuje a la difusión de esta idea lo aportó un persuasivo oficial norteamericano, John Cleves Symmes (1780-1829), quien dedicó toda su vida a 210 Tipologías de humanoides para que las revistas pseudocientíficas de diversos países occidentales lanzaran este absurdo como una noticia sensacional. Con razón Antonio Ribera ha definido la teoría de la Tierra hueca como una teoría hueca. improbable que, de existir otros seres inteligentes, tengan una forma similar a la nuestra. Sin embargo, algunos defienden apelar a la llamada evolución convergente, proceso mediante el cual seres vivos enfrentados a los mismos problemas desarrollan de forma independiente soluciones análogas, adquiriendo una apariencia externa muy similar. Por ejemplo, la simetría bilateral, la concentración del cerebro, boca y principales órganos sensoriales en la misma zona del cuerpo, así como la locomoción en base a un número reducido de extremidades, son estructuras desarrolladas en todos los animales superiores. Más explotada ha sido la idea de los satélites huecos. En los años sesenta el ufólogo ruso Iosif Shklovsky sugirió que las lunas de Marte (Fobos y Deimos) eran naves espaciales huecas de tamaño gigantesco. Tras las fotos de la nave Viking en 1977 resultó evidente su error, aunque no es raro que vuelva a citarse de vez en cuando. Pero Don Wilson fue más allá en su libro Our Mysterious Spaceship Moon (1975), donde defendía que nuestro propio satélite, la Luna, era hueco, pese a que en esos mismos momentos había astronautas americanos allí depositando instrumentos y tomando muestras que demuestran claramente lo contrario. [82, 169, 170, 224, 240][IC, LG] Enfrentados a un montón de datos inexplicados, resulta tentador tratar de hacerlos encajar en unas tipologías claramente definidas, especialmente cuando tales clasificaciones aportan una apariencia de "respetabilidad científica" a un asunto por demás herético. En 1964 apareció el primer trabajo estadístico sobre este tipo de casos (Vallée 1964) donde se establecían ya tres grupos de ocupantes bien diferenciados (y que siguen vigentes en la actualidad): TIPOLOGÍAS DE HUMANOIDES La gran paradoja es que los seres descritos como asociados a los OVNIS se nos parecen mucho y al mismo tiempo demasiado poco. Es cierto que prolifera la morfología similar a la humana (de ahí el término humanoide), aunque nunca podremos saber si ello se debe a una autocensura previa por parte de los propios testigos o de los investigadores hacia aquellas formas que se apartan de lo habitual. • El grupo más numeroso, de seres de pequeña talla, de altura inferior a la humana media. • Aquellos seres cuya estatura puede tomarse como normal o moderadamente alta, y aspecto muy terrestre. • Y los seres de estatura gigantesca, superior a los 2 metros. De todas formas, si vamos más allá del aspecto general (forma humanoide) seguimos encontrando una gran diversidad, una increíble fauna, locura de cualquier taxonomista. Dada la gran cantidad de elementos aleatorios que intervienen en la evolución de los seres vivos, es muy Las tipologías más detalladas y divulgadas (por su atractivo iconográfico) han sido las de Pereira (1974) que establecía hasta 12 tipos principales subdividos en 23 variantes; y la de Zurcher 211 Tipologías de OVNIs (1979) con 8 grupos y 16 variantes. Otros estudios estadísticos importantes son los del profesor James M. McCampbell en EE.UU. y de Alain Gamard en Francia. El último trabajo conocido en esta línea es el de Patrick Huyghe (1996). D) Relación circunstancial: los seres son vistos en una zona con actividad OVNI. E) Sin relación: los seres aparecen aislados y sin relación con actividad OVNI. F) Asociación implícita: proximidad con un OVNI y se reciben mensajes sin ver ningún ser. Algunos estudiosos han evitado la morfología centrándose más en el comportamiento. Así, J.B. Musgrave (1979) distinguía 8 formas de interacción con los testigos, y Jenny Randles (1981) establecía estos otros tipos: G) Abducciones. Más allá de las simples tipologías morfológicas o de comportamiento se han intentado sistematizar otros aspectos como la fonética de estos seres (Edwards 1970) e incluso su inteligencia ( Jamaludin 1981). Por último, no podemos dejar de señalar la propuesta de Lawson (1979) señalando 6 tipos principales de entidades (humanos, humanoides, animales, robots, exóticos y aparicionales) y sus posibles ancestros terrestres en los campos de la mitología, el folklore, la ciencia ficción y hasta ¡los cómics de superhéroes! • Observadores: entidades que simplemente observan o permanecen dentro del OVNI. • Catadores: aquellos que recogen muestras del entorno. • Técnicos: los que emplean o reparan aparatos. • Examinadores: aquellos que hacen preguntan o realizan exámenes. El riesgo de cualquier tipología es que supone rechazar (consciente o inconscientemente) todos aquellos casos que no se adaptan a los tipos establecidos. Se conocen casos de seres descritos sin cabeza, sin extremidades, transparentes parcialmente, con alas, de formas geométricas, etc. etc.. Y al mismo tiempo, el disponer de unos dibujos atrayentes (o de unas categorías estrictas) reduce la diversidad al encauzar el testimonio dentro de unos modelos ya establecidos. [26, 78, 119, 122, 145, 168, 172, 249, 267][LG] • Mensajeros: los que imparten mensajes. • Agresores: aquellos que se muestran agresivos, aunque sea como autodefensa. Y el MUFON en su HUMCAT (CATálogo de casos de HUManoides) establecía una tipología según el grado de proximidad entre el OVNI y los seres. Resultaban 5 tipos (a los que luego añadieron 2 más): A) Relación explícita: los seres se encuentran en el interior del OVNI. B) Asociación directa: los seres entran o salen del OVNI. TIPOLOGÍAS DE OVNIS C) Asociación deducida: los seres se encuentran en las cercanías del OVNI. Las tipologías contraposición con 212 están en clara las clasificaciones Tipologías de OVNIs clásicas establecidas por Vallée y Hynek referidas a categorías de sucesos, en principio-, si bien la primera de ellas está fuertemente influenciada por la idea básica de los tipos que en 1958 estableció Aimé Michel al analizar la oleada francesa de 1954. Esta clasificación tipológica tuvo en su momento una razón de ser al estudiar la afinidad de los fenómenos que parecían producirse a lo largo de líneas rectas llamadas ortotenias, pero con posterioridad su justificación viene de la mano de la búsqueda de señas de identidad para lo que podía ser un fenómeno original. • Objetos elípticos: 66,5% • Objetos en forma de cohete / avión: 8,6% • Objetos en forma de meteoro o cometa: 1,6% • Objetos lenticulares, cónicos o con forma de lágrima: 6,4% • Objetos con forma de llama: 3,6% • Otras formas: 13,3% Por su parte, el clásico estudio del NICAP The UFO Evidence (1964) estableció estas cifras sobre 575 casos (19421963): Las establecidas entonces fueron las siguientes: 1) forma imprecisa, 2) disco, globo o esfera, 3) platillo boca abajo, 4) ovoide, 5) elipsoide, 6) cigarro, 7) cigarro premunido de una cúpula en su mitad, 8) medialuna, 9) cúpula, 10) cúpula con tallos (disco-medusa), 11) objeto circular coronado por una cúpula, 12) obús, 13) objeto circular partido en dos, 14) objeto compuesto cuyas dos partes permanecen unidas según el aspecto que presenten, 15) objeto en formación numerosa, 16) objeto en forma de llama. Estas variantes originarias se vieron modificadas sucesivamente al correr de los años, aunque sin duda fueron la base para diferentes estudios sobre el particular. La abundancia de formas contrasta aquí con la que históricamente fue la primera de las tipologías, elaborada por Keyhoe en 1950, y que sólo poseía tres categorías: objetos de pequeño tamaño, discos y grandes cigarros-puro. • Discos: 26% • Círculos: 17% • Elipses / óvalos: 13% • Triángulos: 2% • Cigarros: 8,3% • Luces: 24,3% • Otros y radar (no visuales): 9,4% En España, Ares de Blas analizó una muestra de 2.514 casos registrados entre 1950 y 1978, dando como resultado el siguiente cuadro: • Objetos discoidales: 25% • Objetos fusiformes u ovoides: 22% Dentro del desarrollo del proyecto Blue Book, el llamado Special Report 14 (1955) estudió 3.201 casos entre 1947 y 1952 que arrojaron los siguientes porcentajes (sobre 498 incidentes con datos al respecto): • Objetos esféricos: 16% • Objetos triangulares: 4% • Objetos puntuales: 15% • Otras formas: 18% 213 Tosselli, Paolo La gran variedad de categorías empleadas por los distintos autores y la disparidad de resultados obtenidos apunta las dificultades de los estudios de esta clase. Por si esto no fuera ya problema, en los últimos tiempos la casuística parece registrar una tendencia hacia la desaparición de las formas definidas y en favor de los cuerpos difusos o de difícil descripción, cosa que se ha interpretado como un indicio de la “espiritualización” del fenómeno y del abandono de los clichés “mecanicistas” clásicos. [110, 162][ JR, MM] dell’Italia d'oggi (Sonzogno, Milano, 1994). Socio fundador del Cisu, es uno de los principales redactores de la revista UFO y un activo investigador de los casos de su zona de influencia. En 1996 publicó FBI, dossier UFO - I veri X-File (Armenia Editore, Milano), donde se ocupa de los informes ufológicos del FBI recuperados a través del Acta de Libertad de Información (FOIA). Opinión sobre el fenómeno (tomada de la citada conferencia): “Se da una especial situación interna que conduce a un individuo a «vivir» una experiencia OVNI. En general es cosa de personas que ya son propensas a este tipo de experiencia. Cada experiencia particular resulta de una significativa concomitancia de factores psicofisiológicos, físicos y psicológicos cuya interacción genera el nivel de transposición de los casos OVI, es decir, una confusión, una transformación proyectiva o una elaboración proyectiva (...) Sucedería, pues, una especie de transposición ufológica de un estímulo corriente identificado distintamente por el testigo, algo parecido a lo que ocurre con las manchas de tinta del famoso test de Rorschach. El estímulo inicial sólo constituye la oportunidad para que se manifieste el síntoma (el fenómeno). Nada excesivamente patológico. Al contrario, dado que existen miles de casos OVI, parece que el proceso es bastante normal, probablemente demasiado normal, hasta el punto de que quizá no se limite a ser únicamente aplicable al fenómeno OVI (...) No tengo ninguna certeza sobre la real naturaleza del fenómeno OVNI. Como los casos OVI son producto de numerosos fenómenos diferentes (estrellas, aviones, meteoros, faros, lanzamientos de misiles, reflejos, globos sonda, etc.), quizás los casos OVNI no son causados por un único fenómeno, aunque sí por más de un TOSELLI, Paolo (n. Alessandria, 1960). Ufólogo italiano, directivo de la Oficina Nacional de Distribución de Gas. Comenzó a interesarse por los OVNI en 1973, cuando fundó el grupo A.C.O.M. Desde 1978 es atraído por los aspectos psicológicos, perceptivos y sociológicos de las experiencias OVNI, desarrollando un creciente interés por los llamados OVIs, tema en el que pronto se convierte en uno de los mayores referentes mundiales. Su conferencia Examinando los casos O.V.I.: El Factor Humano (presentada en el International UPIAR Colloquium on Human Sciences and UFO Phenomena, Salzburgo, Julio de 1982) es ya un clásico de la literatura especializada. En la década de los ochenta contribuyó a la fundación de la Cooperativa Upiar, al tiempo que sus artículos se publicaban en numerosas revistas internacionales. Desde los años 90 se dedica activamente a la recopilación de leyendas urbanas. Fue redactor jefe de la revista Tutto Storie y hoy dirige la asociación Centro per la Raccolta delle Voci e Leggende Contemporanee (CeRaVoLC), tema sobre el cual escribió La famosa invasione delle vipere volanti e altre leggende contemporanee 214 Transgresión de la realidad, síndrome de fenómeno entre muchos otros.” puro; raramente se da la primera sin la segunda y es muy difícil separar ésta de la tercera. [30][MM] TRANSGRESIÓN DE LA REALIDAD, síndrome de TRIÁNGULOS MORTALES Modificación sustancial de una percepción óptica cuando ésta se acomoda al arquetipo popularizado del fenómeno OVNI, incorporando sus detalles más característicos de forma, estructura, dinámica u otras propiedades. Nombre que reciben ciertas áreas geográficas, normalmente delimitadas por triángulos, donde se dice que han tenido lugar desapariciones de barcos y aviones por motivos desconocidos, comúnmente asociados a la presencia en dichas zonas de anomalías físicas o bases de OVNIs. Tal síndrome aparece cuando el hecho visual primario se presenta ante el observador de manera sorpresiva y éste, incapaz de identificarlo adecuadamente, lo complementa con símbolos y atributos extraídos de sus concepciones personales sobre los OVNIs. No se trata de un trastorno mental ni de una alucinación, sino meramente de un subproducto de los complejos procesos fisiológicos, psicológicos y sociales que intervienen en el registro, retención, recuerdo y reproducción de la información visual. Según Paolo Toselli, las modificaciones introducidas por el testigo pueden ser de tres clases: a) falsa interpretación, cuando el observador hace una descripción exacta de lo ocurrido, pero lo interpreta equivocadamente; b) transformación proyectiva, cuando éste proyecta su conocimiento consciente o inconsciente del fenómeno OVNI en el objeto observado, transformándolo en algo más cercano a sus expectativas ufológicas; c) elaboración proyectiva, cuando el testigo asigna a su visión considerables elementos subjetivos y adiciona patrones y propiedades irreales (la Luna que “persigue” a un automóvil, el televisor que muestra interferencias a causa del ocaso de una estrella, etc.). En la práctica, estas divisiones teóricas pocas veces se presentan en estado El origen de este tipo de especulaciones se remonta a Charles Fort y a los pioneros de la ufología americana Morris K. Jessup e Ivan T. Sanderson, quienes relacionaron la supuesta existencia de bases submarinas alienígenas con las desapariciones de barcos o de sus tripulaciones, integrando en el cuerpo de las viejas leyendas marinas a toda una serie de pérdidas recientes de embarcaciones y aeronaves. No obstante, la indudable popularidad alcanzada por estas ideas se debe a Charles Berlitz, quien con su obra El triángulo de las Bermudas (1974) dio pie para que diversos autores descubrieran zonas semejantes en diferentes partes del mundo. Lawrence David Kusche (1977) pronto desmontó los fraudes y mistificaciones que se escondían tras tantas supuestas desapariciones simplemente acudiendo a las fuentes originales y a los registros de las navieras y las compañías de seguros, que dejaron clara la falsedad de los casos invocados por Berlitz. Por si fuera poco, en mayo de 1991 fueron supuestamente encontrados bajo el mar los restos del vuelo 19, el escuadrón de cinco aeronaves cuyo desvanecimiento cerca de Fort Lauderdale (Florida) en diciembre de 1945 se tenía como el caso fundador del 215 TST misterio del triángulo de las Bermudas. Central, y explotó sobre la tundra provocando una columna de fuego que fue vista desde Kirensk, a 400 km. de distancia, y una terrible explosión cuya onda expansiva provocó ráfagas de viento que azotaron la estepa con tal fuerza que derribaron caballos que se encontraban a más de 600 kms. El choque fue registrado por los sismógrafos de Washington y Java, y las ondas expansivas en la atmósfera rodearon la Tierra en todas direcciones. En las noches que siguieron a la explosión, el cielo mostró una extraña luminosidad debida a las partículas en suspensión en la alta atmósfera. El lugar del impacto, situado en una zona deshabitada, no fue visitado hasta el año 1921. La expedición científica que accedió al lugar localizó una área de unos 30 km. en la que los árboles habían sido talados en todas direcciones. Más allá, los árboles presentaban las copas segadas o planas, y más al centro se encontraban carbonizados. No se encontró cráter alguno. En España, Antonio Ribera, quien ya había prestado una especial atención a las bases submarinas en obras anteriores, escribió en 1976 un libro en el que identificaba hasta doce diferentes triángulos repartidos simétricamente por la geografía mundial, incluyendo los de Canarias y el del Mediterráneo, y aparentemente vinculados a zonas calientes ufológicas. El triángulo de las Baleares (o de Ibiza, como también se le ha llamado) ha sido el que, siguiendo las tesis de Ribera, ha suscitado una mayor atención por parte de los ufólogos hispanos. Especialmente discutida ha sido su concreta delimitación, que cuenta con hasta cuatro incompatibles versiones muy débilmente argumentadas. Las discrepancias surgen según se pretenda ubicar en las Pitiusas o cerca de la costa norte mallorquina la oculta base submarina responsable de los supuestamente- frecuentes avistamientos. La reciente catalogación de la casuística balear (CASUIB) permite desechar la gratuita consideración del archipiélago como área de abundante fenomenología OVNI, con lo que desaparece por completo la necesidad de trazar polígonos para acotar lo que no son sino especulaciones sin fundamento alguno.[19, 44, 138, 193][MM] Aunque hoy en día está ya claro el origen cósmico de tal devastación (en función del hallazgo de altas concentraciones de cobre, oro y níquel en las resinas de los árboles del lugar, con valores anómalos respecto a los terrestres), albergándose dudas únicamente sobre si se trató de un meteorito o de un cometa, el propio misterio de este incidente y el hecho de que no se hallasen fragmentos de ningún tipo ha hecho especular a más de uno acerca de la naturaleza ufológica del fenómeno. El primero de ellos fue el investigador ruso A. Kazantsev, quien en 1947 formuló ya la tesis de que en realidad se había tratado de la desintegración de una astronave extraterrestre a consecuencia de una explosión nuclear. Se pretendía explicar así el hallazgo de niveles radiactivos ligeramente altos en la zona devastada y la apariencia cilíndrica que algunos testigos TST Siglas de Tectonic Strain Theory (a veces Tectonic Stress Theory), teoría de la tensión tectónica. Véase Hipótesis tectónicas. TUNGUSKA El 30 de junio de 1908, una bola ígnea cruzó por encima del Yenisei, en la Siberia 216 Twinkle, proyecto habían otorgado al cuerpo estrellado. Más tarde, ideas semejantes serían expuestas en los libros The Fire Came by (Baxter y Atkins, 1976) y Cauldron of Hell (Stoneley, 1977), que durante cierto tiempo volvieron a poner de moda, a nivel popular, la hipótesis de la nave alienígena. imposible encontrar restos de ningún tipo pese a contar con descripciones bastante detalladas de las trayectorias seguidas por aquéllas. Esta circunstancia y el aumento de las observaciones propiciaron una reunión de expertos en el laboratorio de Los Álamos a mediados de febrero de 1949. En ella expuso La Paz su tesis de que no se trataba de meteoros ordinarios sino de objetos artificiales, pues su trayectoria era horizontal, volaban a relativamente baja velocidad y no producían ningún sonido ni dejaban trazas de ionización. Estas conclusiones indujeron a la Fuerza Aérea a clasificar como secretas las investigaciones sobre las bolas de fuego verde, ante el temor de que pudieran tratarse de cohetes soviéticos desarrollados a partir de modelos de V-2 capturados al final de la Segunda Guerra Mundial. Posteriormente, uno de los participantes de esa conferencia, el Dr. Joseph Kaplan de la UCLA, presentó en noviembre de 1949 un estudio realizado sobre 46 informes de avistamientos que sugería que los objetos reportados eran un extraño tipo de meteoros, observado ya en otros lugares de América, pero en trayectorias verticales. Para explicar por qué se divisaban principalmente sobre Nuevo México, Kaplan apuntaba que la extraordinaria visibilidad de la región junto al gran número de centinelas destacados en las instalaciones militares y nucleares del estado propiciaban este tipo de denuncias. Sin embargo, las escasas pruebas argüidas por los partidarios de ésta son fácilmente rebatibles: la radiación registrada tiene su explicación en el intenso calor que generó el impacto, y los testimonios que hablaban de una forma cilíndrica (recogidos veinte años después de los hechos) deben concordarse con los que mencionan una bola brillante con una cola fulgente, posible fuente del error de tales relatos. [240][CL, MM] TWINKLE, proyecto A finales de 1948, el estado de Nuevo México (EE.UU.) experimentó una oleada de observaciones de lo que parecían ser unas extrañas bolas de fuego verde que inicialmente se creyeron de origen meteorítico. No obstante, la gran cantidad de avistamientos y las estratégicas localizaciones desde donde se efectuaban alertaron a los militares sobre la necesidad de proceder a determinar su origen. Dado que estos objetos se comportaban como bólidos, la Fuerza Aérea consultó el tema con el Dr. Lincoln La Paz, director del Instituto de Meteoritos de la Universidad de Nuevo México y reputado experto en la materia. En diciembre de ese mismo año, La Paz inició sus investigaciones, centradas en localizar posibles fragmentos en los lugares de impacto de una de esas bolas, según la metodología que tan buenos resultados le había proporcionado en sus estudios sobre meteoritos. Sin embargo, le resultó Ese mismo mes las autoridades militares, siguiendo las recomendaciones de la reunión de Los Álamos, emprendieron el proyecto Twinkle para despejar las incógnitas existentes. Éste consistió en la instalación de tres estaciones de seguimiento fotográfico en Nuevo México capaces de determinar la velocidad y altura de las bolas de fuego. Desafortunadamente, la escasez de medios 217 Twinkle, proyecto materiales y humanos (sólo se consiguió una de las tres cámaras previstas) limitaron la eficacia del proyecto, que no logró registrar ningún objeto extraño. El estallido de la Guerra de Corea en 1950 y las nuevas prioridades que impuso marcaron el final de todos estos intentos. No obstante, los avistamientos continuaron sucediéndose hasta 1952, en que cesaron bruscamente. Más tarde, en septiembre de 1954 se dio aún un breve flap de este tipo, tras el cual las bolas de fuego verde no volvieron ya a ser vistas. [240][MM] DTU 218 UFO U UFO avistamiento, duración, latitud/longitud, y otros campos. Su autor continuó con la labor de recopilación aun después de abandonar en 1968 el equipo de Condon, incorporando casos procedentes de los archivos del proyecto Blue Book y de otras naciones. En 1975 Saunders donó el catálogo al CUFOS, que lo continuó desarrollando a través de Fred Merritt. Cuando en 1980 fue finalmente abandonado, UFOCAT contaba con unas 106.000 entradas, muchas de ellas repetidas. Por su condición de repertorio no sometido a depuración, y pese a que en ocasiones se ha presentado como prueba de la existencia de un fenómeno desconocido, su valor a estos efectos es escaso, pues se considera que no más de un 1 ó 2% de sus incidentes pueden tener algún valor científico. En 1990, Donald A. Johnson (psicólogo asociado al CUFOS) emprendió las labores de actualización de su contenido. [66, 233][MM] 1.Siglas inglesas de Unidentified Flying Object, objeto volante no identificado. Este acrónimo fue creado por el capitán Edward J. Ruppelt, primer director del proyecto Blue Book. Véase OVNI. 2. Revista semestral italiana del grupo Cisu. Dirección en Internet: https://ufo.it UFO-CRASH Véase Platillos estrellados. UFO TIMES Publicación del grupo británico Bufora. Dejó de publicarse en octubre de 1997. UFOCAT, catálogo (UFO CATalogue). Primer catálogo ufológico de cierta extensión gestionado informáticamente. Fue creado en 1967 por David R. Saunders, entonces empleado en el estudio que elaboraba la Universidad de Colorado (v. Condon, informe), a partir de 3.000 casos que Jacques Vallée donó al proyecto. Saunders descompuso los incidentes en parámetros relativos a la fuente, fecha, hora, localidad, estado, condado (o país), nombres, edades y sexo de los testigos, circunstancias especiales del UFOLOGÍA En sentido estricto, actividad de estudio del fenómeno OVNI desarrollada por grupos de investigadores aficionados. En sentido amplio, conjunto de creencias populares y actividades sociales relacionadas con los ovnis. El término fue acuñado en 1955 por el escritor Morris K. Jessup a partir de la palabra inglesa para OVNI, aunque su uso no se extendió hasta los años setenta. En algunos países iberoamericanos es más 219 Ufología utilizada la expresión "ovnilogía". de ganado o los círculos en campos de cereales, cuya conexión con OVNIs es más bien dudosa. Igualmente, se tiene por ufólogos a personas que elaboran trabajos de divulgación más próximos al sensacionalismo periodístico que a la reflexión científica, o a una serie de personajes de dudosa credibilidad dedicados a propagar de manera incuestionable la presencia alienígena en la Tierra. De hecho, lo poco que parecen tener en común todas estas manifestaciones es su adhesión dogmática a la hipótesis extraterrestre, ligada mayoritariamente a un escaso rigor metodológico y a unas finalidades sectarias o comerciales. En su primera acepción, la Ufología ha pretendido siempre ser presentada como una ciencia natural más, cuyo campo de estudio se centraría en objetos físicos de naturaleza desconocida pero independiente del testigo. No obstante, los escasos resultados conseguidos con esta orientación, disfrazados de disputas estériles sobre la metodología aplicable, hizo ver a muchos la imposibilidad de hacer ciencia partiendo de un concepto tan vago del objeto a estudiar. La inconcreción de lo que era un OVNI impedía, según estos, obtener datos o metodologías contrastables que dotasen a la Ufología de contenido científico. Ahora bien, la negación por esta vía de la Ufología como ciencia peca de una visión exclusivamente fisicalista de lo científico, pues el problema de la laxitud del objeto de estudio está igualmente presente en las ciencias sociales, cuyo carácter académico nadie discute. De hecho, la orientación humanística de los partidarios de la "nueva ufología" (despreocupados por la naturaleza física o no de los OVNIs) permitiría la consideración de la Ufología como ciencia social ocupada en el estudio de los relatos de los testigos. Sin embargo, la fuerte carga fisicalista de la ufología clásica y de parte de la casuística, unido al descrédito generalizado de la disciplina y a una cierta infructuosidad de las tesis psicosociales, han impedido el asentamiento de la Ufología como ciencia humanística. Escuelas ufológicas. Hablar de escuelas en Ufología resulta arriesgado, dada la gran diversidad de ideas y actitudes que es posible encontrar, incluso en relación a un mismo autor. Además, a menudo ello se confunde con el seguimiento de una concreta hipótesis explicativa, pese a que hay teorías que no han originado ninguna escuela. Si por escuela de pensamiento entendemos una identidad de métodos y conceptos sobre la Ufología en sí y su objeto de estudio, la primera clasificación que podemos establecer es la que separa a los que se detienen a reflexionar sobre estas cuestiones de quienes no lo hacen, es decir, la mal llamada ufología "de campo", caracterizada por su carácter asistemático y antimetodológico y por el apego ciego a la HET. Una segunda escuela, a la que podemos llamar clásica, postularía la objetividad del concepto de OVNI y su estudio mediante técnicas propias de las ciencias naturales. Se incluirían aquí todos los seguidores de la HET, los de las hipótesis tectónicas y naturalistas, y quienes sin apoyar ninguna teoría creen sin embargo en la En sentido amplio, la Ufología engloba una extensa variedad de conceptos y prácticas no siempre directamente vinculados a los OVNIs, constituyendo más un referente cultural que científico. Así, se emplea en relación a cosas tan dispares como la astroarqueología, las mutilaciones 220 Ufología independencia del OVNI respecto al testigo. La escuela psicosociológica, en cambio, es partidaria de la subjetividad del OVNI y apuesta por una metodología semejante a la de las ciencias sociales. El centro de atención recae, por tanto, en el testigo y en sus circunstancias o motivaciones. verá respaldada por la coincidencia entre las oleadas de avistamientos y las oposiciones de Marte, advertida ya por Óscar Rey Brea (1954) y luego retomada por Eduardo Buelta, Aimé Michel y otros (v. Ciclo bienal). Precisamente la oleada de 1954 sobre Francia proporcionó a Michel los datos necesarios para elaborar su teoría de las ortotenias (1958), posteriormente aplicada en diversos países. Sobre el mismo repertorio de observaciones, Jacques Vallée (1965) deducirá sus tres clásicas leyes de los aterrizajes. Cabría incluso considerar una cuarta, la escuela escéptica, que no creería tampoco en la sustantividad de los OVNIs, pero mostrándose indiferente por los aspectos humanísticos del problema. De hecho el escepticismo tiene un campo de acción más amplio que el estrictamente ufológico, pero podría tenerse por escuela en la medida en que sus representantes provienen de la Ufología o se ocupan de ella. Pese a que no consideran a ésta ni siquiera disciplina independiente, los escépticos principalmente se acercan a la Ufología para rebatir sus postulados mediante análisis de tipo fisicalista. En 1969, el controvertido informe Condon concluyó que no había evidencia suficiente que justificase la investigación de los OVNIs, lo que supuso el abandono del proyecto Blue Book. Ese mismo año, Vallée publica Pasaporte a Magonia, primera muestra de las tesis folkloristas y paraufológicas que abundarían en la década de los setenta. En 1976, Persinger avanza lo que luego sería la hipótesis tectónica; en tanto que al año siguiente Monnerie se atreve a cuestionar la validez de la HET desde dentro de la propia Ufología, actitud que originará la escuela psicosocial. Allan Hendry alcanzará poco después (1979) las mismas conclusiones partiendo de bases distintas. Desarrollo histórico. Ciñéndonos al concepto estricto de Ufología, ésta nace con el primer avistamiento de Kenneth Arnold en 1947, que junto a otros motivará una serie de proyectos militares estadounidenses (Twinkle, Sign y Grudge, Blue Book) destinados a determinar si detrás de los OVNIs se escondía alguna amenaza para la seguridad nacional. En 1949, el mayor Donald E. Keyhoe plantea por primera vez públicamente la HET, al proponer un origen marciano para los platillos volantes. En 1952, con el proyecto Magnet, comienza la especulación sobre la propulsión de los OVNIs, que gozaría de un cierto cultivo en diversas épocas. Al año siguiente, Donald H. Menzel publica el primer libro en que se ofrecen explicaciones convencionales para la creciente casuística. No obstante, la HET se Los años 80 y 90 serán testigos de dos hechos cruciales. Por un lado, de una sequía de casos como nunca se había conocido, y por otro, del progresivo distanciamiento entre la ufología europea y la americana. Mientras en el viejo continente el revisionismo de incidentes e ideas es la nota predominante, en los EE.UU. continúa la adhesión a la HET como en décadas anteriores, incrementada por una serie de nuevos tópicos que en realidad retrotraen la Ufología a su infancia: abducciones, platillos estrellados, conspira221 Ufología española cionismo gubernamental, etcétera. Además, a nivel mundial se constata una tendencia a la comercialización y a la popularización banal de los conceptos ufológicos que amenaza peligrosamente con disolverlos en la llamada "New Age".[IC, MM] posteriormente sería conocido como la “segunda generación” de ufólogos españoles. Eran por lo general estudiantes muy jóvenes que se organizaban en grupos de ámbito local, con preferencias muy claras por la investigación de campo y la edición de modestos boletines. Aunque partían de la convicción en el origen extraterrestre de los OVNIs, pretendían una investigación objetiva. Algunos jóvenes universitarios tomaron una orientación científica a través del estudio estadístico de los casos. La revista Stendek, editada por el CEI de Barcelona, fue el medio de difusión de este sector y la tribuna más importante para el debate y el avance de la Ufología rigurosa del momento. Este llamado “sector científico” se constituyó en 1978 en Consejo de Consultores de Stendek. Otro sector, el de los grupos investigadores, más orientados a la investigación de campo y a la divulgación, se organizó el mismo año como Coordinadora de Estudios sobre el Fenómeno OVNI (Cefo), teniendo por órgano de expresión la revista Vimana, editada por el Ciove de Santander. UFOLOGÍA ESPAÑOLA Los primeros estudiosos españoles del fenómeno OVNI empezaron su actividad a partir de la aparición en nuestro país de los platillos volantes en 1950 y de su popularización en la prensa. Estos pioneros desempeñaron su labor de manera generalmente individual. Óscar Rey Brea realizó algunas colaboraciones en la prensa de La Coruña, Manuel Pedrajo escribió desde Santander el primer libro español sobre la materia (1954), y Eduardo Buelta dirigió en Barcelona una agrupación de aficionados al tema, en el que estaban Màrius Lleget y Antonio Ribera. Este grupo dio lugar en 1958 al Centro de Estudios Interplanetarios (Cei). En aquellos pioneros animaba el afán por propagar su teoría de la presencia de astronaves de origen marciano en nuestro planeta. Tras el furor popular y de los medios de comunicación por los extraterrestres durante los años 1978 y 1979, el tema decayó en el interés del público, reduciéndose considerablemente el número de casos OVNI en los años ochenta, lo que condujo a una profunda crisis de la ufología española. A partir de 1982, un sector superviviente de ufólogos con orientación crítica hacia la HET se agrupó en torno del boletín Cuadernos de Ufología (CdU.), editado desde Sevilla por José Ruesga. En 1987 pasaría a publicarse en formato libro, coeditado por Julio Arcas, y en 1994 aglutinaría a profesionales de distintas ramas en el Colectivo Cuadernos, precedente inmediato de la Fundación Anomalía. En En 1966 apareció en las librerías El gran enigma de los platillos volantes, obra de gran influencia en la naciente generación de investigadores del tema y que convirtió a su autor, Antonio Ribera, en la personalidad más reconocida en el estudio de los OVNIs, como se empezaba a denominar el fenómeno. Fue a partir de 1967, al calor de la carrera espacial y el furor por el espacio, cuando surgiría en España un movimiento de estudiosos de los OVNIs que 222 Ufología procesal este grupo destaca la labor de Vicente-Juan Ballester Olmos como inspirador de toda una escuela de investigadores y autor de cinco libros con un enfoque científico del fenómeno. matemática, deductiva y estadística, que fue desarrollado inicialmente por Guasp en su obra Teoría de Procesos de los OVNIs (1973), así como en un conjunto de publicaciones posteriores, y que en 1989 fue actualizado por Ballester y Guasp en el libro Los OVNIS y la Ciencia (2ª ed.). Otra perspectiva diferente del tema es la de las agrupaciones que tienen por actividad principal la recogida de testimonios OVNI y su divulgación por medio de boletines y programas de radio, de forma amateur y partiendo siempre de una postura favorable al origen extraterrestre del fenómeno. Por otro lado, Juan José Benítez estrenó en España la Ufología como actividad profesional a través de su trabajo periodístico sobre casuística OVNI e insistiendo siempre en un decidido partidismo por la HET. En los últimos años se ha ido generalizando la dedicación de los jóvenes ufólogos a la divulgación profesional en revistas de gran tirada llamadas “paracientíficas” por unos y “pseudocientíficas” por otros. Los sectores más escépticos han calificado a este sector como sensacionalista y carente de rigor por su dependencia del mercado. La ufología procesal supuso el primer intento serio hacia la predicción de la fenomenología OVNI y, en cierto modo, puede considerarse en línea con aquellos otros enfoques previos más elementales, como la teoría del ciclo bienal de Buelta, las ortotenias de Michel o la hipótesis del desplazamiento de las oleadas de Saunders, que habían perseguido con idéntica filosofía la predicción de los acontecimientos ufológicos y que, con toda independencia de su exactitud o acierto, han escrito uno de los capítulos más importantes en el desarrollo de la Ufología; de ahí el hecho de que la teoría procesal estuviese inicialmente vinculada al análisis de la geometría de las correlaciones Tierra/Marte, cuya consideración tuvo su justificación en la necesidad de crear un modelo teórico sobre el fenómeno OVNI, una excusa formal, pues, para poder llegar a proponer una nueva metodología mucho más general que permitiese someter la hipótesis extraterrestre a prueba, aunque dicha vinculación le valiera que algunas de sus conclusiones puedan resultar demasiado comprometidas. Desde una posición de defensa de la ciencia, al margen de la Ufología y enfrentada a ella, la asociación Alternativa Racional a las Pseudociencias (Arp), fundada por Félix Ares y dirigida actualmente por Javier Armentia, estudia el fenómeno OVNI desde un escepticismo militante, criticando duramente las investigaciones de los ufólogos más creyentes y denunciando las actitudes mercantilistas del periodismo especializado en estos temas.[55, 171, 191][IC] Partía la ufología procesal de la premisa de que era posible construir una geometría con potencial predictivo sobre la base de los parámetros espacio-temporales de la observación del OVNI (lugar, hora, duración de la observación, dirección de vuelo, etc.). Se trataba de conseguir un paradigma de naturaleza matemática que permitiera someter a prueba la hipótesis de la realidad de los OVNIs, aislando del UFOLOGÍA PROCESAL Enfoque ufológico, de naturaleza 223 Ufología racional conjunto de indicios que podían apuntar hacia su naturaleza física solamente aquellos parámetros que podían contener información sobre el cómo, dónde y cuándo aparecen estos, no entrando en absoluto a considerar el contenido propio de los sucesos. Entre los diferentes aspectos tratados por la ufología procesal cabe resaltar el estudio de las direcciones de vuelo de los OVNIs y su relación con los lugares de observación. Se diseñó un método de evaluación numérica para la predicción de las mismas, pronosticando con sorprendente acierto la estadística de las direcciones de vuelo de muestras casuísticas de países como España, Argentina y EE.UU. científico como para admitir su desarrollo teórico -objetivo fundamental de los estudios procesales-, lo que finalmente condicionó su desarrollo. [31, 106] [MGC] UFOLOGÍA RACIONAL Revista cuatrimestral del grupo argentino CIFO (Círculo de Investigadores del Fenómeno OVNI) de Rosario, desaparecida en 1999. UFÓLOGO 1. Persona dedicada al estudio o cultivo de la Ufología. 2. Impropiamente, aficionado al tema o, en general, cualquier sujeto que se atribuya una especial relación con los ovnis o con extraterrestres.[MM] Otro aspecto notable de la teoría procesal fue el llamado problema fundamental. Se trataba de conocer con antelación el lugar geográfico de la aparición del OVNI, previo conocimiento de otros parámetros relevantes de la observación, problema considerado como una questio crucis estrechamente vinculada a la posibilidad de aislar parámetros racionalizables del fenómeno OVNI y de la existencia del paradigma procesal de la Ufología. A pesar de los infructuosos esfuerzos dirigidos en esa dirección, su tratamiento detallado constituye una de las aportaciones intelectuales más notables de la ufología teórica. UFONAUTA Tripulante de un OVNI, entendido éste como nave extraterrestre. Véase Entidades. UMMO Supuesto planeta situado a 14,6 años-luz del sistema solar y que orbitaría la estrella Iumma, identificada por algunos como la Wolf 424 de los catálogos terrestres. Los estudios procesales fueron ampliamente aclamados en su día, tanto por investigadores españoles como extranjeros, llegando a despertar el entusiasmo crítico de personalidades como Aimé Michel; sin embargo, a pesar de los alentadores resultados obtenidos, la teoría se resintió pronto del hecho de que la fenomenología OVNI no está lo suficientemente consolidada desde el punto de vista En torno a Ummo se ha tejido en los últimos treinta años una historia de contactismo caracterizada por la recepción de mensajes postales y telefónicos sobre diversos temas. Aunque las primeras comunicaciones datan de 1966, los presuntos ummitas afirmaban estar en la Tierra desde el 28/03/1950, día en que 224 Ummo aterrizaron con varias de sus naves en el Cheval Blanc, cerca de La Javie (Alpes franceses). El viaje fue motivado (siempre según estas mismas fuentes) por la descodificación de una señal radioeléctrica emitida desde un buque noruego en 1934, que logró atravesar las altas capas de la atmósfera y fue captada en Ummo 14 años después. Tras familiarizarse con la lengua y culturas francesas (mediante el robo incluso de objetos de uso cotidiano en una granja de la zona), se distribuyeron por otros países europeos. Llegaron a España en 1952, donde utilizaron como tapadera de sus actividades a Margarita Ruiz de Lihory. Los ummitas responden al tipo nórdico, por lo que podían pasar desapercibidos. que los informes iban elevando su nivel científico y, sobre todo, con las circunstancias que rodearon el caso de San José de Valderas. Tras este año y medio en que se concentró el mayor volumen de correspondencia ummita (unos 500 folios), siguieron unas etapas sucesivas que se caracterizaron por unos mensajes más esporádicos y de menor calidad -salvo excepciones- y por la pérdida de la hegemonía madrileña, con nuevos corresponsales en otras zonas, como los mismos autores de Un caso perfecto (1969), Antonio Ribera y Rafael Farriols. En esta época cabe destacar las declaraciones en 1968 del Padre Enrique López Guerrero sobre una colonia ummita, y la separación del grupo Ummo de Madrid de las reuniones de Sesma para participar en la fundación de Eridani AEC. Aunque Eridani se disolvió en 1975, el grupo madrileño (al que se habían incorporado Juan Aguirre, Jorge Barrenechea y Juan Domínguez, entre otros) continuó recibiendo cartas tras un paréntesis de tres años al ritmo de una por año desde 1979 hasta 1984; para llegar a la última etapa importante de 1987-88, en la que se enviaron varios informes que por su contenido y extensión parecían una reedición de la “era clásica” (con un total de unas 250 hojas). Pero esta vez los temas tratados acusaban una mayor tendencia hacia la Cosmología y la Teología (alma colectiva, generación de los oemmiwoas, etc.). En 1990-91 y 1993 se recibieron varias cartas, y en 1996 otras dos sobre una posible reactivación del grupo Ummo en España. Todos estos detalles sólo fueron conocidos a partir de 1966, año en que Fernando Sesma y otros asistentes a las reuniones de La Ballena Alegre (como Dionisio Garrido, Enrique Villagrasa, Alicia Araujo) empezaron a recibir cartas mecanografiadas y llamadas telefónicas de los autodenominados “expedicionarios de Ummo”. Los contenidos variaban desde la exposición de diversas disciplinas (Biología, Física, Astronomía, Cosmología, Teología) hasta lo meramente anecdótico (instrucciones sobre las tertulias, citas frustradas con los ummitas, su vida cotidiana). La recepción de los primeros informes coincidió con el supuesto aterrizaje de Aluche (6/02/1966), donde uno de los testigos ( José Luis Jordán) dijo haber visto en la parte inferior del OVNI una raya vertical entre dos paréntesis opuestos, cosa que se “confirmaría” en una carta ummita posterior que aclararía que se trataba de una de sus naves. Esta prueba sirvió para que algunos de los contactados -y otros curiososempezaran a tomarse en serio el asunto de Ummo, creencia que se acentuó a medida El estudio de la cuestión ummita (denominado ummología) ha presentado dos facetas principales, el contenido de los informes y la verificación de las contadas pruebas que los han apoyado. Las primeras 225 Ummocat investigaciones se realizaron por los propios receptores, pero casi siempre quedaban inconclusas porque la consigna ummita era información contra silencio, en el que se incluía la prohibición de cualquier indagación so pena de cesar el contacto. Estudios posteriores han puesto de manifiesto numerosos errores y contradicciones en las cartas técnicas, así como el carácter fraudulento o inexistente de algunas de las evidencias en favor de la autenticidad de Ummo. En 1993, José Luis Jordán Peña (n. Alicante, 1931) confesó haber sido el creador del mito llamado Ummo, versión corroborada por Vicente Ortuño (segundo testigo de Aluche y amigo suyo desde hacía tiempo, extremo siempre negado por ambos hasta 1989). Pese a ser numerosas las sospechas que desde antiguo recaían en Jordán, esta explicación no ha satisfecho a varios ummólogos, que aún creen en el origen extraterrestre de Ummo, o bien que se trate de un experimento sociológico terrícola donde Jordán se habría limitado a jugar un papel secundario (p. ej., como autor de algunas cartas que coinciden con sus aficiones, y de los dibujos que ilustran una parte de los informes). Aunque la investigación de las dos facetas indicadas no ha finalizado, lo obtenido hasta ahora apunta claramente al origen terrestre y español del asunto Ummo, una leyenda contemporánea que se resiste a desaparecer. [7, 12, 53, 194, 198] [ JM] amplió a todo tipo de referencias de libros, artículos, comentarios y rumores relacionados directa o indirectamente con el asunto Ummo. En marzo de 1997 el Ummocat estaba compuesto por 964 páginas y constaba de 3.919 referencias. En palabras de su autor, este catálogo “pretende sistematizar el material informativo elaborado por los responsables de Ummo, y consiste en la recopilación bibliográfica de las unidades documentales a las que hemos tenido acceso”. Darnaude prosigue su tarea recopiladora, incluyendo aportaciones de diversos investigadores, y en octubre de 1994 preparó un “Catálogo Resumido de la Literatura Ummita” donde por primera vez se recoge la cifra de 1.148 páginas como total de las cartas e informes de Ummo conocidos (entre 1966 y 1993). El Ummocat fue publicado parcialmente (hasta la referencia nº 232) en la obra de Antonio Ribera Ummo informa a la Tierra (Plaza & Janés, Barcelona, 1987). [ JM] UNICAT, catálogo Base de datos de ámbito mundial (UNIversal CATalogue) sobre casuística ufológica de alta calidad, ideada en 1984 por Josef A. Hynek y Willy Smith y continuada por este último en la actualidad. Su objetivo era superar los defectos que presentaba el Ufocat mediante la creación de un catálogo que permitiera analizar el verdadero fenómeno OVNI separándolo de todos los OVIs que habitualmente también se incluyen en repertorios semejantes. Para ello se establecieron cuatro condiciones mínimas que debían cumplir todos los incidentes considerados: 1) contar con al menos dos testigos, 2) haber sido investigados por ufólogos conocidos, 3) recogerse en un informe detallado, y 4) UMMOCAT Catálogo de documentación sobre el asunto Ummo iniciado por Ignacio Darnaude Rojas-Marcos en 1980. Su primer objetivo fue recopilar en breves notas las cartas e informes ummitas recibidos desde 1966, aunque posteriormente se 226 UPIAR contener al menos 10 parámetros básicos. Los casos que superan estos requisitos y la constante depuración a la que están sometidos pasan a integrar el UNICAT, en tanto que los restantes (los OVIs, los sucesos en los que falta información básica o poseen un único testigo, y aquellos con información insuficiente para su evaluación) conforman el MAYBECAT, a la espera de que el progreso de su reinvestigación permita incluirlos en el primer grupo o mantenerlos como muestra para estudios comparativos. El catálogo UNICAT, de carácter dinámico, poseía en 1991 unos 800 registros desglosados en unos 255 campos que se agrupan en seis categorías generales: a) testigo, b) investigador/investigación, c) información (básica y específica), d) extrañeza, y e) peso. anuales (1976, 1977 y 1978-79) fueron publicados por EDITECS, una empresa editorial de Bolonia (Italia), y el cuarto y último (1980-81) por la sociedad cooperativa UPIAR. En 1981 recibió el prestigioso premio “International Achievement Award” del Fund for UFO Research. Su objetivo era la publicación anual (en inglés) de extensos trabajos en profundidad sobre aspectos científicos del fenómeno OVNI, procedentes de todo el mundo y siguiendo los más estrictos criterios académicos. Se clasificaban en cinco secciones: Aspectos Físicos, Recogida y procesamiento de datos, Encuentros cercanos, Aspectos psicológicos y perceptivos, Epistemología. En 1978 publicó además una monografía de Maurice G. de San titulada Hipótesis sobre el origen de los OVNIs. Y como canto de cisne final, en 1982 aparecieron las Actas del Coloquio Internacional UPIAR sobre Ciencias Humanas y Fenómeno OVNI, celebrado en Salzburgo del 26 al 29 de julio de 1982 con la asistencia de investigadores de gran valía como Hillary Evans, Alvin H. Lawson, Claude Maugé, Paolo Toselli y muchos otros. Pese a su aparente objetividad, la metodología del proyecto ha sido criticada (Fuller y van Utrecht, 1989) por ser proclive hacia la concepción del OVNI como fenómeno tecnológico y por introducir sesgos distorsionantes a la hora de seleccionar los casos. Después de que Smith abandonara la Ufología en 2004, cedió los archivos de UNICAT a Jan Aldrich (1947 Project: http://www.project1947.com). [93, 231] [ JR, MM] Considerando que la periodicidad anual limitaba la actualidad de lo publicado, bajo la dirección de Vicente-Juan Ballester Olmos nació un boletín más modesto en páginas pero con una cadencia semestral, UPIAR Research in Progress, cuyo último número (vol. II nº 2) aparecería en 1983, señalando el final de todo este apasionante proyecto adelantado a su tiempo (el Journal of UFO Studies del CUFOS no vería la luz hasta 1979) y con un carácter europeo/ mediterráneo digno de mejor éxito frente a la preponderancia anglosajona. UPIAR Siglas de UFO Phenomena International Annual Review, la primera revista ufológica internacional editada con las características y criterios de selección de las publicaciones científicas. Nació en 1976 bajo los auspicios del Centro Unico Nazionale (CUN) y la dirección de dos italianos: el Dr. Roberto Farabone (físico) y el Dr. Francesco Izzo (bioquímico). Los tres primeros volúmenes 227 Uránida Una vez desaparecidas ambas revistas, la sociedad UPIAR, constituida en cooperativa editorial exclusiva de textos ufológicos, ha seguido publicando esporádicamente interesantes monografías, por ejemplo, sobre los casos OVNI y OVI de 1977 en Italia y Francia. Tras la constitución del Cisu (1985) publica semestralmente su revista UFO. En Internet: http:// www.upiar.com/[LG] URÁNIDA Dícese del nativo del espacio. Término empleado por Hermann Oberth para designar a los Ufonautas. DTU 228 Vallée, Jacques Francis V VALLÉE, Jacques Francis (tomada de Ronald D. Story (ed.), 1980: The Encyclopedia of UFOs): “Propongo la hipótesis de la existencia de un sistema de control sobre la conciencia humana. No he podido determinar si es natural o espontáneo, si es explicable en términos de genética o de psicología social, ni si es un fenómeno ordinario o si su naturaleza es artificial y está sometido a una voluntad sobrehumana. Podría estar completamente determinado por leyes que todavía no hemos descubierto. Sugiero que lo que se da en los encuentros cercanos con OVNIs es el control de las creencias humanas, el control de la relación entre nuestra conciencia y la realidad física; que dicho control ha sido constante a lo largo de la historia y que es secundario que asuma ahora la forma de avistamientos de visitantes del espacio.” (n. 1939). Ufólogo francés, licenciado en Astrofísica y doctor en informática, escritor de ciencia ficción y una de las personalidades más influyentes de la Ufología de los años sesenta y setenta. Según él mismo cuenta, su interés por el fenómeno nació a raíz de ser testigo en 1961 de cómo se borraba deliberadamente en el Observatorio de París una cinta que contenía el registro de un OVNI. En 1962 emigró a los EE.UU. para proseguir sus estudios en la Northwestern University, donde conoció a Josef A. Hynek, quien le inició en el tema y con quien colaboró estrechamente hasta que en 1967 Vallée se mudó a San Francisco. Creador del catálogo Magonia, es autor de diversos libros sobre el fenómeno, entre los que destacan dos clásicos de la ufología cientifista como Anatomy of a Phenomenon (1965) y Fenómenos insólitos del espacio (1966, trad. española de 1967), junto a una de las obras claves para la historia del movimiento ufológico: Pasaporte a Magonia (1969, trad. de 1972), donde se expuso por primera vez en muchos años una hipótesis alternativa a la HET (v. hipótesis del sistema de control) que está en el origen de los ulteriores desarrollos teóricos de las concepciones paraufológicas y psicosociológicas. Sus obras posteriores a ésta (El colegio invisible, 1975; Messengers of Deception, 1979; Dimensions, 1988) pueden considerarse de tono menor, con la excepción quizás de Confrontations (1990). VALLÈS, caso del Caso radar-visual aún inexplicado ocurrido en la comarca catalana del Vallès e investigado por Joan Plana. A las 21.30 h del 29 de noviembre de 1985, miembros de la policía municipal de Terrassa (Barcelona) y numerosos testigos observaron una luz extraña de apreciable tamaño estática en el cielo. A las 22.30 la policía municipal comunica el suceso a la Guardia Urbana de Barcelona, que hacia las 22.45 lo notifica al Centro de Control Aéreo (CCA) del Prat, desde donde se detecta (tras apagar el dispositivo MTI de cancelación de ecos fijos) un eco fijo al norte de Terrassa con Opinión sobre el fenómeno 229 VDD forma de mancha amplia y de un tamaño superior a los 200 m. La Guardia Urbana informó igualmente a la emisora autonómica TV-3, que desplaza hasta Terrassa un equipo móvil que filma en vídeo una luz de dimensiones tres o cuatro veces superiores a las estrellas normales. antes descritos. Cuando el avión sobrevuela Castelldefels los pilotos observan en el área de aquellas dos poblaciones un resplandor luminoso blanco muy potente en el cielo que lanza destellos. Este objeto, con eco fijo en el radar, permaneció en la zona hasta aproximadamente las 06.00 h. A las 23.30, los controladores del CCA de Barcelona dirigen varios aviones comerciales hacia la zona de Sabadell y Terrassa, pero no se observó desde ellos nada extraño. No obstante, a las 01.30 h del 30 de noviembre, un Boeing 727 de Iberia procedente de Tenerife distingue, encontrándose cerca de Sitges, una luz azulblanca suave en forma de línea y con ventanas (“como una granja de pollos”) al noroeste de Sabadell que coincide con la posición del eco del radar. Los equipos de radar del CCA de Barcelona fueron verificados y se descartaron anomalías técnicas. Al parecer, desde el EVA-4 de Roses (Girona) también se detectaron los tres objetos, estimándose en 8.000 m la altura del eco fijo. [185][MM] VDD Siglas de Visitante De Dormitorio. Acrónimo acuñado por el ufólogo Pedro Canto (1994) para referirse a una de las categorías de ebds: "1,3 m de altura, macrocéfalos, ojos negros sin pupila, almendrados y extremadamente grandes, con complexión raquítica y desagradable". [58][LG] Más tarde, a las 04’20 h, en las pantallas de radar del CCA de Barcelona aparece un eco primario móvil, a unos 7,4 km al norte de Sabadell, de 9 km de longitud pero de inapreciable anchura, que se mueve con rumbo sur a unos 90 km/h. A los 50 segundos, del gran eco se desprende por su extremo derecho otro de unos 20 m que se desplaza con rumbo sudeste durante 15 segundos (unos 4 km) y desaparece. El eco mayor, orientado de este a oeste y moviéndose con su cara más amplia hacia el sur, también se desvanece de repente al minuto y medio de su detección, después de recorrer 2.250 m. No pudo determinarse la altura de ninguno de los ecos, que no fueron observados por testigos en tierra. VED Acrónimo de Vehículo Extraterrestre Dirigido, término propuesto por Antonio Ribera y Rafael Farriols en su obra Un caso perfecto (Pomaire, Barcelona, 1969) para designar a los OVNIs, entendidos categórica e indudablemente como naves de otros mundos. Poco después, un Aviocar C-212 del servicio postal despega a las 04.31 h de Barcelona hacia Madrid. Los controladores le hacen seguir una ruta diferente a la habitual (sobre Sabadell y Terrassa) para prevenir una posible colisión con los ecos VENUS Véase Fenómenos astronómicos. 230 Verga, Maurizio VERGA, Maurizio aunque los OVNIs existieran como fenómeno físico, la relación entre ellos y las huellas debería establecerse de una manera mucho más concluyente que en la actualidad.” (n. Cermenate, 1963). Técnico textil italiano dedicado a la investigación ufológica desde 1977. Especializado en encuentros cercanos y en casos con huellas, ha destacado especialmente por su labor en el campo de las aplicaciones ufológicas de la informática, donde ha desarrollado diversos programas de bases de datos (COMPICAT) y servicios de BBS, amén de editar The Computer UFO Newsletter, la única revista del mundo exclusivamente dedicada a la utilización de los ordenadores en Ufología. Es autor de ITACAT, catálogo de encuentros cercanos en Italia, TRACAT, catálogo de incidentes italianos que presentan huellas, y PHOTOCAT, catálogo fotográfico. En 1985 abandonó el Centro Ufologico Nazionale para fundar, junto a otros investigadores, el cisu, entidad de inspiración psicosociológica de la que actualmene es dirigente. VIDAL, caso 1. Véase teleportación. 2. Hacia las 00.30 h. del 30 de julio de 1975, el labrador Feliciano Vidal Chorent, de 69 años de edad, fue testigo de un peculiar incidente en el paraje conocido como Fuente de la Virgen, en el término municipal de Alborache (Valencia). Esa noche fue despertado por el bufar de su burra, que se encontraba atada a un árbol a unos 10 o 15 m. de su ventana. Sobresaltado por la posible presencia de intrusos y por los ladridos de su perro, salió al exterior armado con un cuchillo, instante en el que un impresionante fogonazo de luz iluminó toda la casa durante breves segundos. Después de recuperarse de la momentánea parálisis y del deslumbramiento, Feliciano Fuster examinó los alrededores sin encontrar rastros de ningún tipo. Opinión sobre el fenómeno (extraída de “Physical traces: real evidence?”, artículo de Evans, Hillary y Spencer, John (eds.): UFOS 1947-1987. The 40 years search for an explanation, Fortean Tomes, London, 1987): “Si queremos desarrollar la investigación ufológica como un campo de estudio serio, debemos estar preparados para abandonar dogmas profundamente asentados e ilusiones ampliamente sostenidas (...). Particularmente, no nos deberíamos aferrar a la creencia de que las huellas físicas son evidencia de un fenómeno OVNI físico, a menos que contemos con razones más poderosas que las que actualmente poseemos. Pese a lo que comúnmente se cree -incluso por muchos ufólogos- no existe certeza de ninguna clase respecto a que el fenómeno OVNI tenga una base en la realidad física (...). E incluso Unos 15 días después de estos hechos, el testigo advirtió que padecía visión duplicada de ciertos objetos lejanos (veía “una Luna de cuatro puntas”); y, pasados dos meses, experimentó una notable mejoría en la artrosis que presentaba. A los 6 o 7 meses, en febrero o marzo de 1976, su perro perdió paulatinamente la vitalidad y el apetito al tiempo que quedaba impregnado de bultos por todo el cuerpo, lo que le condujo a un estado lastimoso que obligó a sacrificarlo ese mismo mes de abril. Hacia mayo, la burra empezó a mostrar los mismos síntomas, acompañados además de una progresiva rigidez, que también condujeron a su 231 VImana sacrificio dos o tres meses después. A juicio de Miguel Guasp, investigador del caso junto a V. J. Ballester Olmos, la diplopía de Feliciano Fuster podía ser causada por una catarata provocada por la exposición a una intensa luz, en tanto que señala que el alivio de la artrosis es propio de los tratamientos antiinflamatorios con radioterapia. Por lo que respecta al proceso canceroso que sobrevino a los animales, los síntomas que presentaron son típicos de un carcinoma de piel inducido mediante radiaciones. En consecuencia, Guasp concluye que probablemente los afectados se vieron expuestos a una radiación electromagnética de amplio espectro (incluyendo desde luz infrarroja hasta rayos X o gamma) en una dosis próxima a los 100 rad; que, dada la breve duración del suceso, supondría una tasa de dosis de 36.000 rad/h, tasa extraordinariamente alta. [31, 107][MM] que una de cada cinco personas las ha sufrido alguna vez en su vida. Consisten en la visualización, inesperada e involuntaria de una cara, escena o paisaje, generalmente desconocidos y sin un significado evidente pero perfectamente claros y "totalmente reales". No obstante, se dan muchas variaciones de esta norma básica (las imágenes parecen reaccionar ante el testigo, se escuchan voces, etc.). Dado su carácter inesperado e involuntario muchos testigos apuntan un origen externo, aunque los científicos defienden causas meramente psicológicas. VIMANA VISITANTES DE DORMITORIO 1. Nombre que recibían en la mitología hindú los vehículos de los dioses. 2. Revista, ya desaparecida, editada por el grupo santanderino Ciove. Tuvo dos etapas: entre 1975 y 1977 (periodicidad variable) y entre 1978 y 1981 (trimestral), época durante la cual fue también el órgano de expresión de la Cefo. Está disponible en Internet en: https:/ /bit.ly/3vp76Tl Desde tiempo inmemorial muchas personas han descrito la presencia de seres en sus dormitorios durante la noche. A veces, sólo notan una presencia extraña sobre ellos como aplastándolos, mientras se sienten paralizados (Hufford 1982). Otro tipo de visitante nocturno son los famosos íncubos y súcubos de la Edad Media, con unas actividades más centradas en el sexo. Con la divulgación del fenómeno OVNI en las últimas décadas empezaron a conocerse casos donde las entidades observadas aparecían como luminosas, y naturalmente algunos ufólogos dieron una interpretación extraterrestre a este tipo de encuentros, pese a no existir avistamiento de ningún OVNI. Y en la actualidad, cuando la opinión pública ya acepta como hecho demostrado el secuestro repetitivo y metódico de humanos Este tipo de visiones se han apuntado como una posible explicación de los llamados visitantes de dormitorio, e incluso ufólogos como Keith Basterfield extienden su validez a las propias Abducciones. [148][LG] VISIONES HIPNAGÓGICAS E HIPNOPÓMPICAS Se refieren a las extraordinarias experiencias visuales que muchas personas dicen haber sufrido al dormirse (hipnagógicas) o al despertarse (hipnopómpicas). Se ha llegado a estimar 232 Voronezh, caso en sus propias casas por parte de seres de origen y propósitos desconocidos, este tipo de experiencias, cuya raíz psicológica y onírica nunca ha podido descartarse, constituyen la mayoría de informes de abducción, existiendo una retroalimentación constante entre ambos fenómenos. [58, 81, 83, 118][LG] de escoria de color rojo oscuro, cuyo análisis mineralógico preliminar mostró que no reaccionaba normalmente a los ácidos y que no existía ninguna parecida en la Tierra. Investigaciones posteriores desmintieron las informaciones sobre la piedra, sobre la pistola desintegradora, y sobre un signo parecido al de Ummo que se dijo que habían visto algunos testigos, que en realidad -según B. Chourinov- no habían estado presentes y lo habían afirmado a instancias de un ufólogo local que había mostrado una foto del falso OVNI de San José de Valderas. Se supo también que, pese a que se había sostenido que gran cantidad de viandantes había presenciado el aterrizaje, no hubo más testigos que los escolares. En definitiva, a medida que se conocían nuevos datos se iba perfilando más la posibilidad de que todo quedase en un fraude o broma pergeñado por los tres niños, que se habían servido de elementos de la ciencia ficción más pedrestre para confeccionar un fantasioso relato que quizás pudo tener como desencadenante algún estímulo astronómico. VORONEZH, caso Presunto aterrizaje de un OVNI ocurrido el 27 de septiembre de 1989 en un parque de dicha ciudad, sita a 500 km. al SE de Moscú. La primera versión del suceso fue divulgada por la Agencia TASS a la prensa de todo el mundo, que se hizo eco del mismo con titulares tan espectaculares como “Científicos soviéticos confirman el aterrizaje en la URSS de un «ovni» tripulado por gigantes” (ABC, Madrid, 10/10/1989). En ella se decía que un objeto de color rojo y esférico se había posado en el parque y que de él bajaron por una escalerilla tres seres gigantescos y una especie de robot. Aquéllos tenían cabezas muy pequeñas en relación con su estatura, con tres ojos, y vestían un mono plateado. Uno de ellos disparó con una especie de pistola a un niño y un rayo le hizo desaparecer momentáneamente. Poco después la nave despegó, dejando una huella circular de 20 m. de diámetro en cuyo interior había cuatro hendiduras de 4 o 5 cm. de profundidad, situadas en los vértices de un rombo. Los testigos inicialmente conocidos fueron los niños de 12 a 13 años Julia Sholojova, Vasia Surin y Zhena Blinov, alumnos de la Escuela Secundaria nº 33 de Voronezh. Los primeros investigadores (coordinados por Genrij Silanov, director del Laboratorio de Geofísica de Voronezh) habrían encontrado en la zona del aterrizaje una piedra o trozos La importancia de este caso no reside tanto en los hechos en sí, como en la extraordinaria difusión que recibieron en Occidente, fruto de la expectación que el proceso político de la perestroika había suscitado respecto a todo lo procedente de la URSS. La reproducción de la noticia por la Agencia oficial TASS y la mención de los científicos implicados indujo a los medios a creer que se trataba de una confirmación oficial del suceso, cuando lo cierto es que en la Unión Soviética apenas sí tuvo repercusión. Desde la perspectiva actual, el incidente no deja de poseer su interés como muestra de la propagación de la mitología ufológica desde el mundo occidental a otras culturas, y como ejemplo de la retroalimentación entre la prensa y los 233 Voronezh, caso testigos de avistamientos, pues poco después fueron varios los casos denunciados (el más conocido, el de Conil, Huelva de 29/09/1989) que presentaron elementos coincidentes con el de Voronezh.[32][ JM, MM] DTU 234 Zeta Reticuli Z ZETA RETÍCULI supuestamente abundantes en las zonas de fallas tectónicas (v. hipótesis tectónica). Sistema estelar de origen de los grises, según sostienen los defensores de las abducciones. Esta convicción surge de la interpretación realizada por Marjorie Fish del mapa estelar que Betty Hill dijo contemplar en la nave de sus raptores, donde aparecerían estas dos estrellas (Z-1 y Z-2, que conforman un par óptico) de clase G (v. Hill, caso). Posteriormente, William J. Herrmann, supuestamente abducido varias veces entre 1977 y 1979, ha declarado que sus raptores también le señalaron estas estrellas como su lugar de origen, lo que parece una evidente mistificación urdida a partir del relato de Betty Hill.[196] [MM] La existencia de zonas con mayor presencia de OVNIs parece estar en relación, directa o inversa, con la densidad de población. Son clásicos los hallazgos de los investigadores Claude Poher y Jacques Vallée, que en 1975 afirmaban la semejanza de la casuística francesa y mundial en relación a una mayor presencia del fenómeno en zonas deshabitadas (Ballester, 1978). Sin embargo, David Saunders llegó el mismo año a la conclusión opuesta respecto a la casuística norteamericana, afirmando que “el número de informes es una función positivamente acelerada de la población”: donde hay más testigos potenciales se producirán más casos OVNI. ZONA CALIENTE Otro factor que en cierta medida ha influido para que en algunas zonas exista mayor cantidad proporcional y absoluta de casuística ufológica es el denominado “peso del encuestador local”. La presencia de investigadores activos en ciertas localidades, recogiendo y divulgando los casos de los que tienen conocimiento, puede provocar la impresión de que se están produciendo más episodios que en zonas o provincias donde no existen agrupaciones ufológicas. Área geográfica respecto a la que existe la creencia de que el fenómeno OVNI se manifiesta con mayor frecuencia que en otras. A menudo estos lugares suelen coincidir con zonas de gran actividad tectónica, lo cual ha llevado a proponer teorías que explican la aparición de luces misteriosas originadas por la actividad sísmica, que provocaría ciertos fenómenos luminosos debido al efecto piezoeléctrico, según el cual enormes presiones en la corteza terrestre producirían la acumulación de grandes voltajes eléctricos. Se generarían de esta forma ionizaciones del aire que saldrían al exterior con formas luminosas, Por último, no es desdeñable tampoco el componente mágico o mítico atribuido a algunas áreas geográficas más localizadas (montañas, islas, yacimientos arqueológicos, etc.), que puede generar verdaderas “leyendas ufológicas” a partir de muy escasas 235 Zona caliente observaciones. En España sería el caso de los archipiélagos balear y canario, tenidos tradicionalmente como zonas calientes, pero sin que las compilaciones regionales de casuística (CASUIB y CATCAN, respectivamente) avalen para nada dicha fama, sino más bien la contradigan. [28][RC, MM] DTU 236 Apéndice bibliogràfico APÉNDICE BIBLIOGRÁFICO [1]: AA.VV.(1967): Los humanoides, Pomaire, Barcelona. Trad. de AA.VV. (1966): “The Humanoids”, en Flying Saucer Review nº especial octubre/noviembre. [2]: AA.VV. (1978): Crónica militar y Política de la Segunda Guerra Mundial, SARPE, Madrid.. [3]: AA.VV. (1981): Note Technique nº 3: Methodologie d’un probleme, GEPANCNES, Toulouse. [4]: AA.VV. (1985): “Las abducciones a debate II”, en Cuadernos de Ufología nº 13-14, 1ª época. [5]: AA.VV. (1990): “Forum: Tectonic Stress Theory,” en Journal of UFO Studies, vol. 2. [6]: AA.VV. (1994): “Ciencia Ficción USA años 50”, en Nosferatu-revista de cine, nº monográfico 14/15. [7]: AA.VV. (1994): “Ummo: la historia interminable”, en Cuadernos de Ufología, nº 16-17, 2ª época. [8]: AA.VV. (1994): “Vida en el universo”, en Investigación y Ciencia nº 219. [9]: Adell, Alberto (1974): “Test de extrañeza-credibilidad”, en Stendek nº 16. creencia OVNI”, en Cuadernos de Ufología, nº 13, 2ª época. [12]: Aguirre Ceberio, Juan Miguel (1985): Escritos de Ummo, Santos Reprografía, Madrid. [13]: Alcíbar Cuello, José Miguel (1990): “Reflexiones en torno a la evolución de las manifestaciones OVNI”, en Cuadernos de Ufología nº 7, 2ª época. [14]: Alcubierre, Miguel (1994): “The warp drive: hyper-fast travel within general relativity”, en Class. Quantum Grav., 11, L73. [15]: Alés, Antonio José y Madrid, Andrés (1979): Alerta OVNI, Ed. Uve, Madrid. [16]: Alfonseca, Manuel (1993): La vida en otros mundos, McGraw-Hill, Madrid. [17]: Allen, Robin (1994): “Crop circles: The Full Story”, en The Skeptic, vol. 8-1 y vol. 8-2. [18]: Angel, J. Roger P. y Woolf, Neville J.(1996): “La búsqueda de vida en otros planetas”, en Investigación y Ciencia nº 237. [19]: d’Arbó, Sebastià (1992): “El triángulo de las Baleares”, en Año Cero nº 25 [10]: Adell, Alberto (1979): Manual del ufólogo, Ed. 7 1/2, Barcelona. [20]: Ardanuy Baró, Jordi (1996): “El Área 51: bases aéreas y tecnología alienígena”, en Papers d’OVNIS nº 29-30. [11]: Agostinelli, Alejandro C. (1992): “Las sectas platillistas. Hacia una sociología de la [21]: Ardanuy Baró, Jordi (1996): “El ‘Chupacabras’:¿un alienígena?”, en Papers 237 Apéndice bibliogràfico d’OVNIS nº 33-34. Ufología nº 7, 2ª época. [22]: Aréjula, Francisco (1973): Hacia una física de los OVNI, ed. del autor, Barcelona. [33]: Ballester Olmos, Vicente Juan (1990): “Ecología de los aterrizajes OVNI”, en Cuadernos de Ufología nº 8, 2ª época. [23]: Asimov, Isaac (1981): Civilizaciones extraterrestres, Bruguera, Barcelona. Trad. de Asimov (1979): Extraterrestrial Civilizations, Crown Publishers, New York. [34]: Ballester Olmos, Vicente Juan y Plana Crivillén, Joan (1992): “OVNIS: Materia reservada”, en Cuadernos de Ufología nº 13, 2ª época. [24]: Asimov, Isaac (1986): Marte el planeta rojo, Alianza editorial, Madrid. Trad. de Asimov (1977): Mars, the Red Planet. [35]: Ballester Olmos, Vicente Juan (1995): Expedientes insólitos, Temas de Hoy, Madrid. [25]: Baker, Robert A. (1988): “Los alienígenas entre nosotros: De nuevo la regresión hipnótica”, en Cuadernos de Ufología nº 4, 2ª época. [36]: Banchs, Roberto E. (1980): Los OVNIs y sus ocupantes, Ediciones Tres Tiempos, Buenos Aires. [37]: Barlowe, Wayne Douglas et al. (1979): Barlowe’s Guide to Extraterrestrials, Workman Publishing, New York. [26]: Ballester Olmos, Vicente-Juan (1973): “Biometric data in 19 UFO occupant cases”, en Flying Saucer Review XIX, 3. [38]: Barthel, Gèrard y Brucker, Jacques (1979): La grande peur martienne, Nouvelles Editions Rationalistes, París. [27]: Ballester Olmos, Vicente Juan y Guasp Carrascosa, Miguel (1973): “Cuantización de la ley horaria”, en Stendek nº 14. [39]: Bassa, J. y Freixas, R. (1993): El cine de ciencia-ficción, Paidós, Barcelona [28]: Ballester Olmos, Vicente Juan (1978): OVNIS: el fenómeno aterrizaje, Plaza & Janés, Barcelona. [40]: Bastida Freijedo, Ángel (1992): “Los OVNIs y el Ejército del Aire”, en Revista de Aeronáutica y Astronáutica nº 615. [29]: Ballester Olmos, Vicente Juan (1984): Investigación OVNI, Plaza & Janés, Barcelona. [41]: Benítez, J.J. (1993): Reservada, Planeta, Barcelona. [30]: Ballester Olmos, Vicente Juan y Fernández Peris, Juan Antonio (1987): Enciclopedia de los encuentros cercanos con OVNIS, Plaza & Janés, Barcelona. Materia [42]: Berché i Cruz, Carles (1992): “Sectas y contactismo”, en Cuadernos de Ufología nº 12, 2ª época. [43]: Berché i Cruz, Carles (1994): “No perdamos el hilo... de San José de Valderas”, en Cuadernos de Ufología nº 16-17, 2ª época. [31]: Ballester Olmos, Vicente Juan y Guasp Carrascosa, Miguel (1989): Los OVNIS y la Ciencia, 2ª ed., Plaza & Janés, Barcelona. (Primera edición de 1981) [44]: Berlitz, Charles (1975): El triángulo de las Bermudas, Pomaire, Barcelona. Trad. de Berlitz (1974): The Bermuda Triangle. [32]: Ballester Olmos, Vicente Juan (1990): “Voronezh (CCCP)”, en Cuadernos de 238 Apéndice bibliogràfico [45]: Berlitz, Charles y Moore, William L. (1981): El incidente, Plaza & Janés, Barcelona. Trad. de Berlitz y Moore (1980): The Roswell Incident, Grosset & Dunlap, New York. [56]: Campo Pérez, Ricardo (1996): “El fenómeno OVNI en Canarias”, en Cuadernos de Ufología nº 19-20, 2ª época. [57]: Candel Vila, Rafael (1976): Atlas de Meteorología, Ediciones Jover, Barcelona. [46]: Borraz Aymerich, Manuel (1992): “Los gigantes de Gáldar y los avistamientos canarios”, monografía nº 1 de Cuadernos de Ufología, Santander. [58]: Cantó, Pedro P. (1994): Visitantes de dormitorio: el contacto sexual con extraterrestres, Temas de Hoy, Madrid. [59]: Cantril, Harley (1941): “La invasión desde Marte”, en Revista de Occidente. [47]: Borraz Aymerich, Manuel (1996): OVNIs: historias increíbles con explicaciones creíbles, Centro de Estudios Interplanetarios, Barcelona. [60]: CEI (1995): Fenómenos celestes en el pasado, Centro de Estudios Interplanetarios, Barcelona. [48]: Boyce, Chris (1979): Extraterrestrial Encounter: A Personal Perspective, New English Library, London. [61]: Chalker, Bill (1997): “Las evidencias de OVNIs con efectos físicos en Australia”, en Cuadernos de Ufología nº 1, 3ª época. [49]: Brunvand, Jan Harold (1981): The vanishing hitchhiker.Urban legends and their Meanings, W.W. Norton & Co., New York. [62]: Chariton, Wallace O. (1991): The Great Texas Airship Mystery, Wordware Publishing, Texas. [50]: Budden, Albert (1994): Allergies and Aliens, Discovery Times Press, London. [63]: Chourinov, Boris (1995): OVNIs en Russie, Guy Trédaniel Editeur, París. [51]: Budden, Albert (1995): UFO The Psychic Solution, Blandford, London. [64]: Christin, Pierre y Mezieres, JeanClaude (1992): Los Habitantes del Cielo: atlas cósmico de Valerian y Laureline, Grijalbo/Dargaud, Barcelona. Trad. de Christin y Mezieres (1991): Les habitants du ciel, Dargaud Editeur, París. [52]: Bullard, Thomas E. (1982): The Airship File, ed. del autor, Bloomington. [53]: Cabria García, Ignacio y Berché, Carles (1988): “Dossier: historia del contactismo español”, en Cuadernos de Ufología nº 3, 2ª época. [65]: Clark, Jerome y Coleman, Loren (1975): The Unidentified, Warner, New York. [54]: Cabria García, Ignacio (1991): “El mito de la venida de los extraterrestres: génesis y estructura”, en Cuadernos de Ufología nº 11, 2ª época. [66]: Clark, Jerome (1996): High Strangeness:UFOs from 1960 through 1979, Omnigraphics, Detroit. [55]: Cabria García, Ignacio (1993): Entre ufólogos, creyentes y contactados. Una historia social de los OVNIS en España, Cuadernos de Ufología, Santander. [67]: Clute, John (1996): Ciencia ficción: enciclopedia ilustrada, Ediciones B, Barcelona. Trad. de Clute (1995): Science 239 Apéndice bibliogràfico XVI, 2. Fiction: The Illustrated Encyclopedia, Dorling Kindersley, London. [79]: Eeben, Heinrich K. (1985): Estamos solos en el Cosmos, Planeta, Barcelona. Trad. de Erben (1984): Intelligenzen im Kosmos?, P. Piper GmbH & Co KG, Münich. [68]: Cohen, Daniel (1981): The Great Airship Mystery, Dodd & Mead, New York. [69]: Crexell, Joan (1970): “Las huellas de Morón de la Frontera”, en Stendek nº 2. [80]: Evans, Hillary (1983): The Evidence for UFOs, The Aquarian Press, Wellingborough. [70]: Däniken, Erich von (1970): Recuerdos del futuro, Plaza & Janés, Barcelona. [81]: Evans, Hillary (1984): Visiones, apariciones, visitantes del espacio, Kier, Buenos Aires. Trad. de Evans (1984): Visions, Apparitions, Alien visitors, The Aquarian Press, Wellingborough. [71]: Däniken, Erich von (1971): Regreso a las estrellas, Plaza & Janés, Barcelona. [72]: Däniken, Erich von (1975): Las apariciones, Martínez Roca, Barcelona. Trad. de Däniken (1974): Erscheinungen, EconVerlag, Düsseldorf. [82]: Evans, Hillary y Spencer, John (eds.) (1987): UFOS 1947-1987. The 40 years search for an explanation, Fortean Tomes, London. [73]: Delgado, Pat y Andrews, Colin (1994): Testimonios circulares, Tikal, Madrid. Trad. de Delgado y Andrews (1989): Circular Evidence, Bloomsbury, London. [83]: Evans, Hillary (1989): Alterate States of Consciousness, The Aquarian Press, Wellingborough. [84]: Evans, Hillary y Stacy, Dennis (eds.) (1997): 1947-1997. Fifty years of flying saucers, John Brown Publishing, London. [74]: Devereux, Paul (1982): Earth Lights, Turnstone Press, Wellingborough. [75]: Drake, Frank y Sobel, Dava (1993): ¿Hay alguien más en el universo?, Argos Vergara, Buenos Aires. Trad. de Drake y Sobel (1992): Is anyone out there?, Delacorte Press, New York. [85]: Fernandes, Joaquim y D’Armada, Fina (1981): Intervenção Extraterrestre em Fátima, Livraria Bertrand, Lisboa. [86]: Festinger, Leon, Riecken, Henry y Schachter, Stanley (1956): When Profecy Fails, Harper and Row Publishers, New York. [76]: Durant, Henry (1972): OVNIs: realidad o ficción, Daimon, Barcelona. Trad. de Durant (1970): Le livre noir des soucopes volantes, Robert Laffont, París. [87]: Flò, Martí y Ardanuy, Jordi (1996): “OVNIS y Webs. Viaje ufológico por las autopistas de la información”, en Papers d’OVNIS nº 27-28. [77]: Edwards, Frank (1966): Platillos volantes, asunto serio, Diana, Barcelona. Trad. de Edwards (1966): Flying Saucers, Serious Business. [88]: Fort, Charles (1969): El libro de los Condenados, Rumeu Editor, Barcelona. Trad. de Fort (1919): The Book of the Damned. Última edición: Fort (1995): [78]: Edwards, P. M. H. (1970): “Speech of aliens”, en Flying Saucer Review XVI, 1 y 240 Apéndice bibliogràfico Charles Fort’s Book of the Damned, Fortean Times Library, London. OVNIs en Canarias, Centro de la Cultura Popular Canaria, Tenerife. [89]: Freixedo, Salvador (1990): La amenaza extraterrestre, Bitácora, Madrid. [100]: González, Luis R. (1983): “Los casos negativos de humanoides ibéricos. Un análisis preliminar”, en Cuadernos de Ufología, nº 3, 1ª época. [90]: Friedman, Stanton (1990): Final Report on Operation Majestic 12, Fund for UFO Research, Mount Rainier. [101]: Goss, Michael (1984): The evidence for phantom hitch-hikers, The Aquarian Press, Wellingborough. [91]: Fuller, Curtis G. (ed.) (1980): Proceedings of the first international UFO Congress, Warner Books, New York. [102]: Green Bukley, Timothy (ed.) (1990): The UFO Silencers, Inner Light Publications, Brunswick. [92]: Fuller, John G. (1970): El viaje interrumpido, Plaza & Janés, Barcelona. Trad. de Fuller (1966): The Interrupted Journey, Dial Press, New York. [103]: Greenwood, Barry (1990): “MJ-12: Una valoración negativa”, en Cuadernos de Ufología nº 9-10, 2ª época. [93]: Fuller, Paul y van Utrecht, Wim (1989): “The Trouble with UNICAT,” en International UFO Reporter, julio/agosto (14,4) y septiembre/octubre (14,5). [104]: Greenwood, Barry (1996): “El informe de la Fuerza Aérea sobre Roswell”, en Cuadernos de Ufología nº 19-20, 2ª época. [94]: Gardner, Martin (1993): “The False Memory Syndrome”, en The Skeptical Inquirer 17-4. [105]: Grupo P.S.I. (1975): “El proyecto Starlight”, en Stendek nº 20. [106]: Guasp, Miguel (1973): Teoría de procesos de los OVNI, ed. del autor, Valencia. [95]: Gardner, Martin (1994): “The Tragedies of False Memories: The Accused Are Striking Back, But Grave Injustices Have Been Done”, en The Skeptical Inquirer 18-5. [107]: Guasp, Miguel (1992): “Efectos radiofisiológicos presuntamente vinculados a observaciones de OVNIs”, en Cuadernos de Ufología nº 13, 2ª época. [96]: Gaston, Patrice (1975): Desapariciones misteriosas, Plaza & Janés, Barcelona. Trad. de Gaston (1973): Disparitions Mysterieuses, Robert Laffont, París. [108]: Guijarro, Josep (1994): Infiltrados: seres de otras dimensiones entre nosotros, Sangrilá, Barcelona. [109]: Guyatt, David (1997): “Police State of Mind”, en Fortean Times nº 95. [97]: Gillmor, D. S. (ed.) (1969): Scientific Study of Unidentified Flying Objects, Bantam Books, New York. [110]: Hall, Richard H. (ed.) (1964): The UFO Evidence, NICAP, Washington D.C. [98]: Giraud, J. (ed.) (1979): Les soucoupes volantes, le grand refus, Grupo Gabriel. [111]: Harbinsson, W.A. (1995): Projekt UFO: The Case for Man-made Flying [99]: González, José Gregorio (1995): Los 241 Apéndice bibliogràfico Saucers, Boxtree, Hardback. XXVII, 3. [112]: Hendry, Allan (1979): The UFO Handbook, Doubleday & Co., New York. [123]: Jung, Carl Gustav (1961): Sobre cosas que se ven en el cielo, Diana, México. Trad. de Jung (1958): Flying Saucers: A Modern Myth of Things Seen in the Skies. [113]: Henning, Richard (1971): Grandes enigmas del Universo, Plaza & Janés, Barcelona. Trad. de Henning (1950): Wo lag das Paradies?, Verlag des Druckhauses Tempelhof, Berlin. [124]: Keel, John A. (1969): “The Principle of Transmogrification”, en Flying Saucer Review, julio-agosto. [114]: Hildgard, Ernest R. (1977): Divided Consciousness: Multiple Controls in Human Thought and Action, Wiley, New York. [125]: Keel, John A. (1970-71): “Mystery Aeroplanes of the 1930s”, en Flying Saucer Review XVI, 3; XVI, 4; XVII, 4 y XVII, 5. [115]: Hobana, Ion y Weverbergh, Julien (1976): Les OVNI en URSS et dans les pays de l’Est, Robert Laffont Editeur, París. [126]: Keel, John A. (1973): UFOs: Operation Trojan Horse, Abacus, London. [127]: Keyhoe, Donald E. (1974): Los Desconocidos del espacio, Pomaire, Barcelona. Trad. de Keyhoe (1973): Aliens from space, Doubleday & co., New York. [116]: Hopkins, Budd (1981): Missing Time, Richard Marek Publishers, New York. [117]: Hopkins, Budd (1988): Intrusos: las increíbles visitas a Copley Woods, EDAF, Madrid. Trad. de Hopkins (1987): Intruders, Rosentone / Wender, New York. [128]: Khuon, Ernst von (ed.) (1976): Los dioses vinieron de las estrellas, ATE, Barcelona. [118]: Hufford, David J. (1982): The Terror that Comes in the Night, University of Pennsylvania Press, Philadelphia. [129]: Klass, Philip J. (1968): UFOsIdentified, Random House, NewYork. [130]: Klass, Philip J. (1989): “Majestic 12”, en International UFO Reporter. [119]: Huyghe, Patrick (1996): The Field Guide to Extraterrestrials, Hodder & Stoughton, London. [131]: Klass, Philip J. (1989): UFO Abductions: A dangerous game, Prometheus Books, Buffalo. [120]: Hynek, Josef Allen (1972): The UFO Experience, Henry Regnery Co., Chicago. [121]: Jacobs, David M. (1993): Vida Secreta, Ediciones B, Barcelona. Trad. de Jacobs (1992): Secret Life. Firsthand Accounts of UFO Abductions, Simon & Schuster, New York. [132]: Kottmeyer, Martin (1990): “La ovnilogía como un sistema desarrollado de paranoia”, en [236]. [133]: Kottmeyer, Martin (1990): “Entirely Unpredisposed”, en Magonia nº 35. [122]: Jamaludin, Ahmad (1981): “Classification of levels of humanoid intelligence”, en Flying Saucer Review [134]: Kottmeyer, Martin (1994): “Alienating Fancies”, en Magonia nº 50. 242 Apéndice bibliogràfico [135]: Kottmeyer, Martin (1994): “El error del platillo”, en Perspectivas Ufológicas nº 2. [145]: Lawson, Alvin H. (1979): “Aliens roots: six UFO entity types and some possible earthly ancestors”, en MUFON Symposium Proceedings. [136]: Krassa, Peter (1982): Hombres de negro, Martínez Roca, Barcelona. Trad. de Krassa (1980): Phantome des Schreckens, Caesar Verlag, Salzburgo. [146]: Lawson, Alvin H. (1981): “Hypnosis of imaginary UFO ‘Abductees’” en Journal of UFO Studies, vol. 1-1. [137]: Kuhn, Thomas S. (1994): La estructura de las revoluciones científicas, Fondo de Cultura Económica, Madrid. Trad. de Kuhn (1970): The Structure of Scientific Revolutions, 2ª ed., University of Chicago Press. [147]: Lawson, Alvin H. (1990): “Una teoría comprobable sobre los informes sobre raptos efectuados por OVNIs: la hipótesis de los recuerdos perinatales (HRP)”, en [236]. [148]: Leaning, F. E. (1925): “An introductory study of hypnagogic phenomena”, en Proceedings of the Society for Psychical Research, vol. 94. [138]: Kusche, Lawrence David (1977): El misterio del triángulo de las Bermudas solucionado, Sagitario ediciones, Barcelona. Trad. de Kusche (1975): The Bermuda Triangle Mystery-Solved, Prometheus Books, Buffalo. [149]: Ledwoch, Janusz (1996): Luftwafe 1946, Widjawnictwo militaria, Warszawa [139]: Kusche, Lawrence David (1980): The Disappearance of Flight 19, Harper & Row, New York. [150]: Lewis, James (1995): The Gods Have Landed, State University of New York Press, New York. [140]: Kyle, David A. (1976): A Pictorial History of Science Fiction, Hamlyn, London. [151]: Loftus, Elizabeth (1979): Eyewitness Testimony, Harvard University Press, Cambridge (Massachusetts). [141]: Kyle, David A. (1977): The Illustrated Book of Science Fiction Ideas & Dreams, Hamlyn, London. [152]: Loftus, Elizabeth “Remembering Dangerously”, Skeptical Inquirer 19-2. [142]: Laffite, Felipe y Osuna, Manuel (1970): “Más datos sobre el caso de Morón de la Frontera”, en Stendek nº 3. (1995): en The [153]: Mack, John E. (1994): Abduction: Human Encounters with Aliens, Simon & Schuster, London. [143]: Lagrange, Pierre (1990): “Pendleton 24-26 de junio 1947: tres días en la vida de Kenneth Arnold y de los platillos volantes. Algunas consideraciones sobre la forma de escribir la historia de los platillos volantes”, en Cuadernos de Ufología nº 8, 2ª época. [154]: Magee, J. M. (1978): “Maureen Puddy’s Third Encounter”, en Flying Saucer Review XXIV, 3. [155]: McCampbell, James M. (1973): Ufology, Jaymac Company, Belmont. [144]: Lagrange, Pierre (1996): La rumeur de Roswell, Edition La Découverte, Paris. [156]: McClure, Kevin (1983): The evidence 243 Apéndice bibliogràfico for visions of the Virgin Mary, The Aquarian Press, Wellingborough. Occupants & Critters, Communications, New York. [157]: McKay, Christopher P. (1990): “La vida en Marte”, en Mundo Científico nº 108. [169]: Nicholls, Peter (ed.) (1979): The Encyclopedia of Science Fiction, Granada Publishing Ltd., London. [158]: McKay, David S. et al. (1996): “Search for Past Life on Mars: Possible Relic Biogenic Activity in Martian Meteorite ALH 84001”, en Science 273 (5277): 924-930. Global [170]: Oberg, James E. (1982): UFOs & Outer Space Mysteries, Donning Co. Publishers, Norfolk. [171]: Pedrajo, Manuel (1954): Los platillos volantes y la evidencia, ed. del autor, Santander. [159]: Méheust, Bertrand (1978): Science Fiction et soucoupes volantes, Mercure de France, París. [172]: Pereira, Jader U. (1971-72): “Los Extra-terrestres”, en Stendek nº 5 a 10. Trad. de Pereira (1970-71): “Les Extra-terrestres”, en Phénomènes Spatiaux, nº 24, 25, 27, 28 y 29. [160]: Méheust, Bertrand (1985): Soucoupes volantes et folklore, Mercure de France, París. [161]: Meurger, Michel (1996): “Surgeons from Outside”, en Fortean Studies, vol. 3. [173]: Perera, Ramos (1990): Las creencias de los españoles: La tierra de María Santísima, Mondadori, Madrid. [162]: Michel, Aimé (1963): Los misteriosos platillos volantes, Pomaire, Barcelona. Trad. de Michel (1958): Mystérieux Objets Célestes, Arthaud, Paris. [174]: Persinger, Michael A. y Lafrenière, G. (1977): Space-Time Transients and Unusual Events, Nelson-Hall, Chicago. [163]: Michel, Aimé (1979): “The ‘Cat-flap’ Effect”, en Flying Saucer Review XXV, 5. [175]: Persinger, Michael A. (1989): “Variables geofísicas y comportamiento: IX. Esperadas consecuencias clínicas de la proximidad a luminosidades tipo OVNI”, en Cuadernos de Ufología nº 5, 2ª época. [164]: Miranda, Justo (s/f ): Strange Phenomena in the German Sky, ed. del autor. [165]: Monnerie, Michel (1977): Et si les OVNI n’existaient pas?, Les Humanoïdes Associes, Paris. [176]: Persinger, Michael A. (1990): “La experiencia del “visitante” y la personalidad: el factor del lóbulo temporal”, en [236]: [166]: Montejo Aguilera, José Juan y Berché Cruz, Carles (1994): “Terremoto en Ummo: los hilos del montaje”, en Cuadernos de Ufología nº 16-17, 2ª época. [177]: Petit Gancedo, Antonio (1995): “24 de junio de 1947: los 9 discos de Arnold”, en Cuadernos de Ufología nº 19-20, 2ª época. [167]: Morey Ripoll, Matías (1996): “Caos y casuística ufológica”, en Cuadernos de Ufología nº 19-20, 2ª época. [178]: Peyró, Miguel (1979): ¿OVNIS? Sí, pero..., Ed. 7 1/2, Barcelona. [168]: Musgrave, John B. (1979): UFO [179]: Pflock, Karl T. (1995): “Roswell, un 244 Apéndice bibliogràfico cuento aleccionador: hechos y fantasías, lecciones y secuelas”, en Cuadernos de Ufología nº 19-20, 2ª época. [191]: Ribera Jordà, Antonio (1966): El gran enigma de los platillos volantes, Pomaire, Barcelona. [180]: Phillips, Ted (1975): Physical traces associated with UFO sightings, CUFOS. [192]: Ribera Jordà, Antonio y Farriols, Rafael (1969): Un caso perfecto, Pomaire, Barcelona. [181]: Pilkington, Mark (1997): “What’s on your mind?”, en Magonia, 58. [193]: Ribera Jordà, Antonio (1976): Los doce triángulos de la muerte, ATE, Barcelona. [182]: Pinillos, Jesús (1995): “La revolución Stealth”, en Avion Revue nº 162. [194]: Ribera Jordà, Antonio (1979): El misterio de Ummo, Plaza & Janés, Barcelona. [183]: Pinotti, Roberto (1974): Ufo: La congiura del silenzio, Armenia Editore, Milano. [195]: Ribera Jordà, Antonio (1981): Secuestrados por extraterrestres, Planeta, Barcelona. [184]: Pinvidic, Thierry (ed.)(1993): OVNI: vers une anthropologie d’un mythe contemporain, Ed. Heimdal, Bayeux. [196]: Ribera Jordà, Antonio (1983): Las máquinas del Cosmos, Planeta, Barcelona. [185]: Plana Crivillén, Joan (1994): “El OVNI más grande jamás visto”, en Papers d’OVNIS nº 1. [197]: Ribera Jordà, Antonio (1984): En el túnel del tiempo, Planeta, Barcelona. [198]: Ribera Jordà, Antonio (1985): Ummo: la increíble verdad, Plaza & Janés, Barcelona. [186]: Plana Crivillén, Joan (1995): “¿Estamos preparados?”, en Papers d’OVNIS nº 13. [199]: Rimmer, John (1984): The Evidence for Alien Abductions, The Aquarian Press, Wellingborough. [187]: Plana Crivillén, Joan (1995): “Organismos oficiales de estudio OVNI en el mundo”, en Papers d’OVNIS nº 21-22. [188]: Powels, Louis y Bergier, Jacques (1962): El retorno de los brujos, Plaza & Janés, Barcelona. Trad. de Powels y Bergier (1960): Le matin des magiciens, Gallimard, París. [200]: Ring, Kenneth (1995): El Proyecto Omega: experiencias cercanas a la muerte, encuentros con OVNIs y la mente planetaria, Más Allá Libros, Madrid. Trad. de Ring (1992): The Omega Project, Morrow, New York. [189]: Regis Jr., Edwards (1985): Extraterrestrials: Science and alien intelligence, Cambridge University Press, Cambridge. [201]: Ritrovato, Joseph W. (1995): “Analyzing UFO Waves”, en MUFON UFO Journal nº 323. [202]: Robiou Lamarche, Sebastián (1973): “Epigrafía OVNI”, en Stendek nº 14. [190]: Rey Brea, Óscar (1972): “Algo sobre las fotografías del supuesto OVNI de San José de Valderas”, en Stendek nº 9. [203]: Rogerson, Peter (1993-94): “Notes 245 Apéndice bibliogràfico Towards a Revisionist History Abductions,” en Magonia, nº 46 a 50. of intelligence (CETI), Massachussetts Institute of Technology, Boston. [204]: Rojcewicz, Peter (1987): “The ‘Men in Black’ Experience and Tradition: Analogies with the Traditional Devil Hypothesis”, en Journal of American Folklore, nº 100. [214]: Sagan, Carl (1995): Un punto azul pálido, Planeta , Barcelona. Trad. de Sagan (1994): Pale blue dot, Random House, New York. [215]: Sandell, Roger (1996): “Editorial: De Roswell a Oklahoma”, en CdU. Suplemento Internacional nº 2. [205]: Ruesga Montiel, José (1988): “Próspera Muñoz: un caso divulgado y poco conocido”, en Cuadernos de Ufología nº 4, 2ª época. [216]: Schaefer, Vincent J. y Day, John A. (1983): Guía de Campo de la Atmósfera, Ediciones Omega, Barcelona. Trad. de Schaefer y Day (1983): A Field Guide to the Atmosphere, Houghton Mifflin Co., Boston. [206]: Ruiz Noguez, Luis (1990): “Centellas o rayos en bola”, en Cuadernos de Ufología nº 7, 2ª época. [207]: Ruiz Noguez, Luis (1993): “Anillos de Hadas”, en Perspectivas Ufológicas 1-1 [217]: Schatzman, Evry (1994): Los niños de Urania, Salvat editores, Barcelona. Trad. de Schatzman (1986): Les enfants d’Uranie, Éditions du Seuil, Paris. [208]: Ruppelt, Edward (1956): Report on Unidentified Flying Objects, Ace Books, New York. [218]: Schnabel, Jim (1993): Round in circles, Hamish Hamilton, London. [209]: Rutkowski, Chris (1989): “Terremotos y fenómenos luminosos”, en Cuadernos de Ufología nº 5, 2ª época. [219]: Schnabel, Jim (1994): Dark White, Hamish Hamilton, London. [210]: Rutledge, Harley D.(1981): Project Identification: The first scientific field study of UFO phenomena, Prentice-Hall, New Jersey. [220]: Schneider, Adolf (1977): Visitantes del Universo, Plaza & Janés, Barcelona. Trad. de Schneider (1973): Besucher aus dem all, Hermann Bauer Verlag, Friburg. [211]: Sagan, Carl y Page, Thornton (eds.) (1972): UFOs. A Scientific Debate, W.W. Norton, New York. [221]: Schwarz, Berthold Eric (1977-78): “Talks with Betty Hill”, en Flying Saucer Review XXIII, 2; XXIII, 3 y XXIII, 4. [212]: Sagan, Carl y Shklovskii, I. S. (1981): Vida inteligente en el Universo, Reverté, Barcelona. Trad. de Sagan y Shklovskii (1966): Intelligent Life in the Universe, Holden-Day Inc., San Francisco. [222]: Scornaux, Jacques y Piens, Christiane (1976): A la recherche des OVNI, Marabout, Bruxelles. [223]: Scornaux, Jacques (1979): “Un ensayo de clasificación de apariciones y desapariciones ‘in situ’”, en Stendek nº 37. [213]: Sagan, Carl (1993): Comunicación con inteligencias extraterrestres, RBA editores, Barcelona. Trad. de Sagan (1973): Communication with extraterrestrial [224]: Sheaffer, Robert (1994): Veredicto 246 Apéndice bibliogràfico OVNI, Tikal, Gerona. Trad. de Sheaffer (1981): The UFO Veredict, Prometheus Books, New York. [236]: Stillings, Dennis et al. (1990): Lo imaginario en el contacto OVNI, Héptada, Madrid. Trad. de Stillings (1989): Ciberbiological studies of the imaginal component in the UFO contact experience, USA. [225]: Sider, Jean (1990): Ces OVNIs qui font peur, Editions Axis Mundi, Paris. [226]: Sierra, Javier (1990): “Encuentros cercanos con lo absurdo (comentarios y anotaciones al libro ‘The Matrix’)”, en Cuadernos de Ufología, nº 9-10, 2ª época. [237]: Stilo, Giuseppe (1994): “I dischi di Hitler”, en Il Giornale dei Misterii, nº 268. [238]: Stilo, Giuseppe (1994): “I dischi di Hitler”, en Il Giornale dei Misterii, nº 269. [227]: Sierra, Javier (1991): “Noticias basura sobre los extraterrestres”, en Más Allá, nº monográfico OVNIS, septiembre. [239]: Stilo, Giuseppe (1993-94): “UFO e Nazismo”, en Il Giornale dei Misterii nº 264-271 [228]: Sierra, Javier (1995): Roswell: secreto de estado, EDAF, Madrid. [240]: Story, Ronald D. (ed.) (1980): The Encyclopedia of UFOs, Doubleday and co., New York. [229]: Smith, Willy (1990): “La saga de Gulf Breeze”, en Cuadernos de Ufología nº 9-10, 2ª época. [241]: Strand, Erling (1988): “Proyecto Hessdalen”, en Cuadernos de Ufología nº 2, 2ª época. [230]: Smith, Willy (1992): “Extrañeza”, en Cuadernos de Ufología nº 13, 2ª época. [242]: Strieber, Whitley (1988): Comunión, Plaza & Janés, Barcelona. Trad. de Strieber (1987): Communion, Century Hutchinson, New York. [231]: Smith, Willy (1992): “Del Libro Azul al Proyecto UNICAT,” monografía 1 de Cuadernos de Ufología, Santander. [232]: Spaulding, William J. y Adrian, Fred (1981): “Análisis por ordenador de una fotografía OVNI en Mallorca”, en Stendek nº 43. [243]: Stringfield, Leonard H. (1979): “Retrievals of the Third Kind”, en Flying Saucer Review XXV, 4; XXV, 5 y XXV, 6. [233]: Spencer, John et al. (1987): “Computers in ufology”, en [82]. [244]: Stringfield, Leonard H. (1980): The UFO Crash / Retrieval Syndrome. Status Report II, MUFON, Seguin. [234]: Sprague de Camp, L. y Ley, Willy (1967): De la Atlántida al Dorado, Caralt, Barcelona. [245]: Stringfield, Leonard H. (1982): UFO Crash / Retrievals: Amassing the Evidence. Status Report III, ed. del autor, Ohio. [235]: Steiger, Brad (1977): Proyecto Libro Azul, EDAF, Madrid. Trad. de Steiger (1976): Project Blue Book, Ballantine Books, New York. [246]: Sullivan, Walter (1975): No estamos solos, Noguer, Barcelona. Trad. de Sullivan (1964): We Are Not Alone, McGraw-Hill, New York. 247 Apéndice bibliogràfico [247]: Toselli, Paolo (1994): La famosa invasione delle vipere volanti, RCS Libri & Grandi Opere SpA., Milano. sin disparar, Ed. 29, Barcelona. Trad. de Vesco (1968): Intercettateli senza sparare, Mursia, Milano. [248]: Urrero Peña, Guzmán (1994): El cine de ciencia-ficción, Royal Books, Barcelona. [258]: Watson, Nigel (1992): “Down to Earth,” en Magonia nº 43. [249]: Vallée, Jacques F. (1964): “A descriptive study of the entities associated with Type-I sightings”, en Flying Saucer Review X, 1 y X, 3. [259]: Watson, Nigel (1993): Seeing and Believing. UFOs and aliens in film and TV, Valis Books, London. [260]: Wieder, Irwin (1993): “The Willamette Pass Oregon UFO Photo Revisited: An Explanation”, en Journal of Scientific Exploration, vol. 7-2. [250]: Vallée, Jacques y Vallée, Janine (1967): Fenómenos Insólitos del espacio, Pomaire, Barcelona. Trad. de Vallée y Vallée (1966): Les Phénomènes insolites de l’espace, La Table Ronde, Paris. [261]: Wilbur, W.H. (1960): “El misterio de los globos japoneses”, en Selecciones del Reader’s Digest. [251]: Vallée, Jacques (1967): “Algunas constantes en los aterrizajes de OVNI”, en [1]. [262]: Wilkins, Harold T. (1954): Flying Saucers on the Attack, Citadel Press, New York. [252]: Vallée, Jacques (1972): Pasaporte a Magonia, Plaza & Janés, Barcelona. Trad. de Vallée (1969): Passport to Magonia, Henry Regnery and co., Chicago. [263]: Zerpa, Fabio (1979): Los Hombres de Negro y los OVNIS, Plaza & Janés, Barcelona. [253]: Vallée, Jacques (1981): El colegio invisible, Diana, México. Trad. de Vallée (1975): The Invisible College, E.P. Dutton and co., Inc., New York. [264]: Ziguel, Félix (1968): Observation des OVNI en URSS, vol. I, ed. del autor. [254]: Vallée, Jacques (1990): Confrontations, Ballantine Books, New York. [265]: Ziman, John (1981): La credibilidad de la ciencia, Alianza editorial, Madrid. Trad. de Ziman (1978): Reliable Knowledge. An exploration of the grounds for belief in science, Cambridge University Press, Cambridge. [255]: Vallée, Jacques (1994): “Anatomy of a Hoax: The Philadelphia Experiment Fifty Years Later”, en Journal of Scientific Exploration, vol. 8-1. [266]: Zinsstag, Lou y Good, Timothy (1983): George Adamski, the untold story, CETI Publications, Gran Bretaña. [267]: Zurcher, Eric (1979): Les Apparitions d’Humanoïdes, Editions Alain Lefeuvre, Marseille. [256]: Valls Girol, Enrique y Ruesga Montiel, José (1993): “La fotografía infrarroja y el fenómeno OVNI”, en Cuadernos de Ufología nº 14, 2ª época. [257]: Vesco, Renato (1968): Interceptadlos 248
https://openalex.org/W2132726017	https://iimmun.ru/iimm/article/download/120/119	Russian	null	ENTEROBACTERIACAE, PRODUCING ESBLS AND METALLO-β-LACTAMASE NDM-1, ISOLATED IN HOSPITALS OF BALTIC REGION COUNTRIES	Infekciâ i immunitet	2,014	cc-by	5,064	Оригинальные статьи Оригинальные статьи Инфекция и иммунитет 2013, Т. 3, № 1, с. 29–36 ШТАММЫ ЭНТЕРОБАКТЕРИЙ, ПРОДУЦИРУЮЩИЕ БЕТА-ЛАКТАМАЗЫ РАСШИРЕННОГО СПЕКТРА И МЕТАЛЛО- β-ЛАКТАМАЗУ NDM-1, ВЫДЕЛЕННЫЕ В СТАЦИОНАРАХ В СТРАНАХ БАЛТИЙСКОГО РЕГИОНА С.А. Егорова1, Л.А. Кафтырева1, Л.В. Липская2, И.Б. Коноваленко3, М.Ф. Пясетская4, Т.С. Курчикова5, Н.Б. Ведерникова6, О.Т. Морозова7, М.В. Смирнова8, Л.Н. Попенко9, М.И. Любушкина9, Ю.А. Савочкина10, М.А. Макарова1, Л.В. Сужаева1, Ю.В. Останкова1, М.Н. Иванова11, А.М. Павелкович11, П. Наабер12, Э. Сепп12, С. Кыльялг12, И. Mицюлявичeнe13, А. Балоде14 1 ФБУН НИИ эпидемиологии и микробиологии имени Пастера, Санкт-Петербург 2 Городская клиническая больница № 40, Санкт-Петербург 3 Городская клиническая больница № 31, Санкт-Петербург 4 Детская городская клиническая больница № 5, Санкт-Петербург 5 Детская городская больница № 17, Санкт-Петербург 6 Городская больница Святого Великомученика Георгия, Санкт-Петербург 7 Детская городская больница № 1, Санкт-Петербург 8 Городская Мариинская больница, Санкт-Петербург 9 НИИ скорой помощи имени И.И. Джанелидзе, Санкт-Петербург 10 ФБУН Центральный НИИ эпидемиологии, Москва 11 Восточная Центральная больница, Таллинн, Эстония 12 Университет Тарту, Тарту, Эстония 13 Вильнюсскaя городская клиническая больница, Вильнюс, Литва 14 Университетская клиническая больница им. Пaула Страдыня, Рига, Латвия 1 ФБУН НИИ эпидемиологии и микробиологии имени Пастера, Санкт-Петербург 2 Городская клиническая больница № 40, Санкт-Петербург 3 Городская клиническая больница № 31, Санкт-Петербург 4 Детская городская клиническая больница № 5, Санкт-Петербург 5 Детская городская больница № 17, Санкт-Петербург 6 Городская больница Святого Великомученика Георгия, Санкт-Петербург 7 Детская городская больница № 1, Санкт-Петербург 8 Городская Мариинская больница, Санкт-Петербург 9 НИИ скорой помощи имени И.И. Джанелидзе, Санкт-Петербург 10 ФБУН Центральный НИИ эпидемиологии, Москва 11 Восточная Центральная больница, Таллинн, Эстония 12 Университет Тарту, Тарту, Эстония 13 Вильнюсскaя городская клиническая больница, Вильнюс, Литва 14 Университетская клиническая больница им. Пaула Страдыня, Рига, Латвия 14 Университетская клиническая больница им. Пaула Страдыня, Рига, Латвия Резюме. Изучали распространенность штаммов, устойчивых к цефалоспоринам расширенного спектра и карбапенемам, среди K. pneumoniae и E. coli, выделенных из клинического материала пациентов семи стационаров Санкт-Петербурга в период с января по май 2012 г. Доля штаммов, устойчивых к цефало- споринам, колебалась в различных стационарах: E. coli — от 7,8 до 50%, K. pneumoniae — от 25,4 до 88,4%. Устойчивость была обусловлена продукцией бета-лактамаз расширенного спектра семейства СТХ-М, которые относились главным образом к группе СТХ-М-1, а также СТХ-М-2 и СТХ-М-9. В трех стациона- рах выявили 22 полирезистентных штамма K. pneumoniae, устойчивых к карбапенемам. Метод MАLDI- TOF MS показал, что резистентность к карбапенемам у всех штаммов была обусловлена продукцией карбапенемаз, которые согласно результатам тестов ROSCO Diagnostica относились к классу металло- β-лактамаз. У всех изученных штаммов выявлен ген металло-β-лактамазы New Delhi metallo-β-lactamase (blaNDM-1). Результаты проведенных исследований показали, что в странах Балтийского региона (вклю- чая Россию, Санкт-Петербург) ведущим механизмом резистентности к цефалоспоринам штаммов E. ENTEROBACTERIACAE, PRODUCING ESBLS AND METALLO-β-LACTAMASE NDM-1, ISOLATED IN HOSPITALS OF BALTIC REGION COUNTRIES Egorova S.A., Kaftyreva L.A., Lipskaya L.V., Konovalenko I.B., Pyasetskaya M.F., Kurchikova T.S., Vedernikova N.B., Morozova O.T., Smirnova M.V., Popenko L.N., Lubushkina M.I., Savochkina J.A., Makarova M.A., Suzhaeva L.V., Ostankova J.V., Ivanova M.N., Pavelkovich A.M., Naaber P., Sepp E., K ljalg S., Miciuleviciene J., Balode A. Egorova S.A., Kaftyreva L.A., Lipskaya L.V., Konovalenko I.B., Pyasetskaya M.F., Kurchikova T.S., Vedernikova N.B., Morozova O.T., Smirnova M.V., Popenko L.N., Lubushkina M.I., Savochkina J.A., Makarova M.A., Suzhaeva L.V., Ostankova J.V., Ivanova M.N., Pavelkovich A.M., Naaber P., Sepp E., K ljalg S., Miciuleviciene J., Balode A. Abstract. We studied the prevalence of K. pneumoniae and E. coli resistance to extended spectrum cephalosporins and carbapenems, isolated from patients of eight hospitals in St-Petersburg from January to May, 2012. Prevalence of cephalosporin resistant isolates varied in different hospitals: E. coli – from 7,8 to 50%, K. pneumoniae – from 25,4 to 88,4%. Isolates produced extended spectrum beta-lactamases СТХ-М, mainly СТХ-М-1, also СТХ-М-2 and СТХ-М-9. Twenty two carbapenem-resistant K. pneumoniae strains (also resistant to other antimicrobials) were isolated in three hospitals. MALDI-TOF MS showed that carbapenem resistance was caused by carbapenemase. Carbapenemases of all isolates belonged to metallo-β-lactamases according to results of the ROSCO Diagnostica tests. The gene coding production of New Delhi metallo-β-lactamase (blaNDM-1) were detected in all strains. Our data confirmed that the main cephalosporin resistance mechanism of E. coli и K. pneumoniae in Baltic region (including Russia, St-Petersburg) is CTX-M-1 production. For the first time in Russia K. pneumoniae strains producing metallo-β-lactamases NDM-1 were isolated in several hospitals of St-Petersburg. (Infekc. immun., 2013, vol. 3, N 1, p. 29–36) Key words: K. pneumoniae, E. coli, resistance, СТХ-М, NDM-1. Key words: K. pneumoniae, E. coli, resistance, СТХ-М, NDM-1. ных округов и показывают, что штаммы энте- робактерий, продуцирующих бета-лактамазы расширенного спектра (БЛРС), карбапенем- резистентных P. aeruginosa и A. baumanii на про- тяжении многих лет циркулируют в российских стационарах, особенно в отделениях реанима- ции и интенсивной терапии. Такие микроорга- низмы способны вызывать у пациентов тяже- лые состояния, часто являющиеся причиной продления срока госпитализации, повышения уровня летальности, а также увеличения эконо- мических затрат на лечение. Другой стороной проблемы антибиотикорезистентности являет- ся глобальное распространение определенных полирезистентных клонов микроорганизмов, примером которого в настоящее время является ситуация с распространением резистентности к карбапенемам у K. pneumoniae и E. coli, продуци- рующих металло-бета-лактамазу NDM-1 (New Delhi metallo-β-lactamase). ENTEROBACTERIACAE, PRODUCING ESBLS AND METALLO-β-LACTAMASE NDM-1, ISOLATED IN HOSPITALS OF BALTIC REGION COUNTRIES Развитие авиасооб- щений приводит к тому, что сегодня врач может столкнуться с пациентом, который несколько дней назад находился в стационаре в другой части света. «Международными» пациентами могут оказаться люди, эвакуированные из мест постоянного проживания в результате военных действий или природных катастроф, а также туристы, которые оказываются госпитализи- рованными во время путешествий в результа- те несчастных случаев или в целях получения дешевой медицинской помощи (стоматология, офтальмология, пластическая и кардиохи- рургия). Госпитализация в стационары неко- торых стран может быть сопряжена с риском ШТАММЫ ЭНТЕРОБАКТЕРИЙ, ПРОДУЦИРУЮЩИЕ БЕТА-ЛАКТАМАЗЫ РАСШИРЕННОГО СПЕКТРА И МЕТАЛЛО- β-ЛАКТАМАЗУ NDM-1, ВЫДЕЛЕННЫЕ В СТАЦИОНАРАХ В СТРАНАХ БАЛТИЙСКОГО РЕГИОНА coli поступила в редакцию 15.02.2013 отправлена на доработку 17.02.2013 принята к печати 06.03.2013 Адрес для переписки: Егорова Светлана Александровна, к.м.н., научный сотрудник лаборатории кишечных инфекций ФБУН НИИЭМ имени Пастера 197101, Санкт-Петербург, ул. Мира, 14, ФБУН НИИЭМ имени Пастера. Тел.: (812) 232-48-83 (служебн.); +7 911 959-95-19 (моб.). E-mail: egorova72@mail.ru © Егорова С.А. и соавт., 2013 Адрес для переписки: Егорова Светлана Александровна, к.м.н., научный сотрудник лаборатории кишечных инфекций ФБУН НИИЭМ имени Пастера 29 С.А. Егорова и др. Инфекция и иммунитет и K. pneumoniae, выделенных в стационарах, является продукция СТХ-М-1. Впервые в России выделены штаммы K. pneumoniae, продуцирующие металло-β-лактамазу NDM-1, причем одновременно в несколь- ких стационарах Санкт-Петербурга. и K. pneumoniae, выделенных в стационарах, является продукция СТХ-М-1. Впервые в России выделены штаммы K. pneumoniae, продуцирующие металло-β-лактамазу NDM-1, причем одновременно в несколь- ких стационарах Санкт-Петербурга. Ключевые слова: K. pneumoniae, E. coli, резистентность, СТХ-М, NDM-1. Ключевые слова: K. pneumoniae, E. coli, резистентность, СТХ-М, NDM-1. Key words: K. pneumoniae, E. coli, resistance, СТХ-М, NDM-1. Введение Для проведения реакции циклического секвенирования использовали наборы реаген- тов BigDye Terminator v1.1 (Applied Biosystems, США) и праймеры NDM-seq-F (CgC-ATT-AgC- CgC-TgC-ATT-gA) и NDM-seq-R (ggg-Cgg-AAT- ggC-TCA-TCA). Разделение меченых фрагментов ДНК осуществляли с помощью генетического анализатора ABI Prism 3500 (Applied Biosystems, США). Анализ нуклеотидных последовательно- стей осуществляли, используя программы, пред- ставленные в свободном доступе в Интернете, и проводили сравнение с последовательностями генов blaNDM-1,2…7, опубликованными в базе данных GenBank (http://www.ncbi.nlm.nih.gov). Результаты инфицирования полирезистентными клонами микроорганизмов, которые перемещаются вме- сте с пациентами из стационаров одной стра- ны в стационары другой страны, где они ранее не циркулировали. Очевидно, что в отношении таких микроорганизмов мониторинг должен осуществляться на международном уровне. диффузионным методом (ДДМ) согласно МУК 4.12.1890-04, рекомендациям Европейского Коми- тета по определению чувствительности к анти- биотикам (EUCAST) (http://www.eucast.org/cli ni cal_ breakpoints) с использованием среды Мюл лера– Хинтон и дисков производства Oxoid (Велико- британия). МИК имипенема определяли ме- то дом Е-тестов (BioMerieux, Франция). Интер- пре тацию результатов тестирования проводили согласно критериям EUCAST, 2011 г. У нечув- ствительных штаммов механизм резистентно- сти — гидролиз эртапенема — выявляли методом времяпролетной масс-спектрометрии (MАLDI- TOF MS) на приборе MALDI Biotyper Microflex LT (Bruker Daltonics) согласно протоколу [18]. Результат гидролиза был визуализирован и про- анализирован с помощью спектрограмм. Диф- ференциацию различных видов бета-лактамаз проводили, выявляя синергизм между ЦРС, кар- бапенемами и ингибиторами, специфичными для различных видов бета-лактамаз (для БЛРС — клавулановая кислота, для AmpC — клокса- циллин, для металло-бета-лактамаз (МБЛ) — дипиколиновая кислота, для КРС — боронико- вая кислота) с использованием наборов «ESBL + AmpC Screen ID Kit» и «KPC+MBL Confirm ID Kit» (Rosco Diagnostica, Дания). Детекцию ге- нов бета-лактамаз проводили методом ПЦР в режиме реального времени с использованием праймеров и флуоресцентно-меченых олигону- клеотидных зондов, описанных ранее, семейств СТХ-М (групп СТХ-М-1, СТХ-М-2, СТХ-М-9, СТХ-М-8/25), ТЕМ, SHV, KPC, IMP, VIM, OXA- 48, NDM [6, 9, 11]. Для проведения ПЦР в режи- ме реального времени использовали амплифи- катор с системой оптической детекции CFX 96 (BioRad, США) или RotorGene Q (QIAGEN, Гер- мания). Для проведения реакции циклического секвенирования использовали наборы реаген- тов BigDye Terminator v1.1 (Applied Biosystems, США) и праймеры NDM-seq-F (CgC-ATT-AgC- CgC-TgC-ATT-gA) и NDM-seq-R (ggg-Cgg-AAT- ggC-TCA-TCA). Разделение меченых фрагментов ДНК осуществляли с помощью генетического анализатора ABI Prism 3500 (Applied Biosystems, США). Анализ нуклеотидных последовательно- стей осуществляли, используя программы, пред- ставленные в свободном доступе в Интернете, и проводили сравнение с последовательностями генов blaNDM-1,2…7, опубликованными в базе данных GenBank (http://www.ncbi.nlm.nih.gov). Существуют различные международные организации, занимающиеся вопросами анти- биотикорезистентности. Введение Специалисты Санкт- Петербурга (бактериологи, эпидемиологи, ин- фекционисты, хирурги) являются членами BARN (Baltic Antimicrobial Resistance Net — Сеть по надзору за антибиотикорезистентно- стью в странах Балтийского региона), глав- ной целью которой является противодействие распространению резистентности в странах Балтийского региона. BARN организует меж- дународные научно-практические проекты, касающиеся углубленного изучения антибио- тикорезистентности. В 2012 г. стартовал международный проект, основной целью которого являлось улучшение детекции и надзора за резистентностью, вы- званной БЛРС, у штаммов Enterobacteriaceae в странах Балтийского региона (Эстония, Лат- вия, Литва и Россия, Санкт-Петербург). В за- дачи проекта входило внедрение единого фено- типического скрининга и алгоритма детекции бета-лактамаз, включая БЛРС и карбапенема- зы, а также развитие научного сотрудничества между Россией и странами Евросоюза. В про- екте участвовала 21 лаборатория из Латвии, Литвы, Эстонии и Санкт-Петербурга (лабора- тории семи многопрофильных стационаров). Научное руководство проектом осуществляли научно-исследовательские учреждения: Санкт- Петербургский НИИЭМ имени Пастера, Уни- верситет Тарту и Шведский Институт по Кон- тролю Заболеваний. Материалы и методы В период с 1 января по 31 мая 2012 г. в 21 ста- ционаре проводили скрининг штаммов K. pneu- moniae и E. coli, нечувствительных (устойчивых и с промежуточной чувствительностью) к цефа- лоспоринам расширенного спектра (ЦРС) (цефтазидиму, цефотаксиму, цефтриаксону) и карбапенемам (меропенему, имипенему), вы- деленных в этот период из различного клиниче- ского материала (кровь, моча, мокрота, раневое отделяемое). Повторные изоляты, выделенные от одного пациента, в исследование не вклю- чали. Штаммы выделяли и идентифицировали классическими бактериологическими метода- ми с использованием стандартных селектив- ных и дифференциально-диагностических сред для энтеробактерий. Чувствительность к АМП определяли согласно нормативным докумен- там, принятым в странах-участниках, диско- Введение В современных условиях проблема лекарст- венной устойчивости микроорганизмов приоб- рела глобальный характер. Полирезис тент ные штаммы являются причиной возникновения тя желых форм внутрибольничных гнойно-сеп- тических инфекций, а также разнообразных ин- фекционных заболеваний. В борьбе с антибио- тикорезистентностью основным направлением является организация системы мониторинга циркуляции резистентных микроорганизмов и генов, детерминирующих устойчивость к ан- тимикробным препаратам (АМП). Единая стан- дартизированная методология оп ре деления чувст вительности микроорганизмов к АМП и единые критерии интерпретации, основан- ные на современных знаниях о механиз мах ре- зистентности и о фармакодинамике/фармако- кинетике АМП, позволят улучшить качество исследований и проводить эффективный мони- торинг резистентности не только на уровне от- дельного стационара, но и региона или страны. В настоящее время требования к организации микробиологического мониторинга в учреж- дениях здравоохранения изложены в СанПиН 2.1.3.2630-10 «Санитарно-эпи демиологические требования к организациям, осуществляющим медицинскую деятель ность». В Российской Федерации проводятся науч- ные исследования, результаты которых отража- ют ситуацию по распространению резистент- ности к АМП у различных микроорганизмов на территориях практически всех федераль- 30 Энтеробактерии, продуцирующие БЛРС и NDM-1 2013, Т. 3, № 1 диффузионным методом (ДДМ) согласно МУК 4.12.1890-04, рекомендациям Европейского Коми- тета по определению чувствительности к анти- биотикам (EUCAST) (http://www.eucast.org/cli ni cal_ breakpoints) с использованием среды Мюл лера– Хинтон и дисков производства Oxoid (Велико- британия). МИК имипенема определяли ме- то дом Е-тестов (BioMerieux, Франция). Интер- пре тацию результатов тестирования проводили согласно критериям EUCAST, 2011 г. У нечув- ствительных штаммов механизм резистентно- сти — гидролиз эртапенема — выявляли методом времяпролетной масс-спектрометрии (MАLDI- TOF MS) на приборе MALDI Biotyper Microflex LT (Bruker Daltonics) согласно протоколу [18]. Результат гидролиза был визуализирован и про- анализирован с помощью спектрограмм. Диф- ференциацию различных видов бета-лактамаз проводили, выявляя синергизм между ЦРС, кар- бапенемами и ингибиторами, специфичными для различных видов бета-лактамаз (для БЛРС — клавулановая кислота, для AmpC — клокса- циллин, для металло-бета-лактамаз (МБЛ) — дипиколиновая кислота, для КРС — боронико- вая кислота) с использованием наборов «ESBL + AmpC Screen ID Kit» и «KPC+MBL Confirm ID Kit» (Rosco Diagnostica, Дания). Детекцию ге- нов бета-лактамаз проводили методом ПЦР в режиме реального времени с использованием праймеров и флуоресцентно-меченых олигону- клеотидных зондов, описанных ранее, семейств СТХ-М (групп СТХ-М-1, СТХ-М-2, СТХ-М-9, СТХ-М-8/25), ТЕМ, SHV, KPC, IMP, VIM, OXA- 48, NDM [6, 9, 11]. Для проведения ПЦР в режи- ме реального времени использовали амплифи- катор с системой оптической детекции CFX 96 (BioRad, США) или RotorGene Q (QIAGEN, Гер- мания). Механизмы устойчивости к карбапенемам Штаммы, устойчивые к карбапенемам, во всех странах были выявлены только среди K. pneumo- niae (137 штаммов). Для 22 штаммов, выделенных в Санкт-Петербурге в трех стационарах, метод MULDI-TOF MS показал, что при инкубации эртапенема с микробной суспензией, приготов- ленной из нечувствительных штаммов, препарат подвергался гидролизу. Этот факт свидетель- ствовал о том, что резистентность к карбапене- мам у этих штаммов была обусловлена продук- цией бета-лактамаз, разрушающих карбапенемы (карбапенемаз). У остальных 115 штаммов устой- чивость к карбапенемам была обусловлена дру- гими механизмами резистентности. Результа- ты фенотипических подтверждающих тестов (выявленный синергизм между меропенемом и дипиколиновой кислотой) у всех 22 штаммов свидетельствовали о том, что продуцируемые карбапенемазы относились к классу металло- бета-лактамаз. Результаты За 5 месяцев 2012 г. в четырех странах в рам- ках проекта было выделено 13 130 штаммов E. coli и K. pneumoniae. В семи стационарах Санкт-Петербурга из различного клиничес- кого материала было выделено 1135 штаммов E. coli (устойчивы к ЦРС — 23,1%) и 536 штам- мов K. pneumoniae (устойчивы к ЦРС — 66,7%). 31 С.А. Егорова и др. Инфекция и иммунитет 22 штамма K. pneumoniae дополнительно харак- теризовались устойчивостью к карбапенемам (4,1%). Механизмы устойчивости к БЛРС В стационарах всех стран были выделены штаммы, устойчивые к ЦРС. Подтверждающие тесты (синергизм с клавулановой кислотой) по- казали, что устойчивость была обусловлена про- дукцией БЛРС. В Санкт-Петербурге доля устой- чивых штаммов составляла: среди E. coli — 23% (от 7,8 до 50% в зависимости от профиля стацио- нара), среди K. pneumoniae – 67% (от 25,4 до 88,4%) (рис. 1). Во всех странах устойчивость была обу- словлена продукцией «классических» БЛРС се- мейства СТХ-М, которые относились к группам СТХ-М-1, СТХ-М-2, СТХ-М-9 и СТХ-М-8/25. У штаммов K. pneumoniae преобладала продукция СТХ-М-1: во всех странах доля таких штаммов превышала 80%. У штаммов E. coli в трех стра- нах, включая Россию, преобладала продукция СТХ-М-1 (более 80%), в Литве — СТХ-М-1 (около 50%) и СТХ-М-9 (около 30%). В Санкт-Петербурге 85% штаммов E. coli и 97% штаммов K. pneumoniae продуцировали БЛРС СТХ-М-1. Кроме того, 13% штаммов E. coli продуцировали СТХ-М груп- пы 9, и 2% штаммов K. pneumoniae — СТХ-М гру- пы 2. Во всех странах выявлена незначительная доля штаммов, у которых устойчивость была обусловлена продукцией БЛРС, не относящихся к СТХ-М, SHV, TEM, которые не удалось иденти- фицировать на данном этапе работы (рис. 2 и 3). Географическое распространение штаммов, про- дуцирующих цефотаксимазы различных групп, показано на рис. 4. Дальнейшие молекулярно-генетические ис- следования выявили у всех 22 штаммов гены blaNDM. Гены других карбапенемаз (ОХА-48, KPC, VIM, GIM и IMP) обнаружены не были. Полученные сиквенсы полностью соответ- ствовали гену blaNDM-1. Нуклеотидная по- следовательность одного из штаммов депони- рована в GenBank (GenBank accession number BankIt160 1193 Seq1 KC534 856). У всех 22 штаммов K. pneumoniae, продуци- рующих NDM-1, зоны задержки роста вокруг дисков с имипенемом и меропенемом, а также МИК имипенема соответствовали категориям «устойчивый» или «промежуточный» согласно Рисунок 1. Доля штаммов (%) E. coli и K. pneumoniae, устойчивых к ЦРС, в стационарах Санкт-Петербурга, январь–май 2012 г. Рисунок 1. Доля штаммов (%) E. coli и K. pneumoniae, устойчивых к ЦРС, в стационарах Санкт-Петербурга, январь–май 2012 г. 32 Энтеробактерии, продуцирующие БЛРС и NDM-1 2013, Т. 3, № 1 критериям EUCAST (S ≥ 22 мм или ≤ 2,0 мг/л; R < 16 мм или > 8,0 мг/л). критериям EUCAST (S ≥ 22 мм или ≤ 2,0 мг/л; R < 16 мм или > 8,0 мг/л). критериям EUCAST (S ≥ 22 мм или ≤ 2,0 мг/л; R < 16 мм или > 8,0 мг/л). Наши результаты также свидетельствуют, что в странах Балтийского региона, включая Рос- сию (Санкт-Петербург), в стационарах широко распространены штаммы K. Устойчивость к препаратам других групп Все штаммы K. pneumoniae и E. coli, про- дуцирующие бета-лактамазы, включая МБЛ, характеризовались множественной устойчи- востью к АМП других групп: фторхиноло- нам (ципрофлоксацину, офлоксацину), ами- ногликозидам (гентамицину, тобрамицину, амикацину), тетрациклину, хлорамфениколу и ко-тримоксазолу. Более 90% штаммов были чувствительны к нитрофуранам, 100% — к по- лимиксину. Рисунок 2. Доля штаммов (%) K. pneumoniae, продуцирующих БЛРС семейства СТХ-М различных групп, в странах Балтийского региона Рисунок 3. Доля штаммов (%) E. coli, продуцирующих БЛРС семейства СТХ-М различных групп, в странах Балтийского региона Рисунок 2. Доля штаммов (%) K. pneumoniae, продуцирующих БЛРС семейства СТХ-М различных групп, в странах Балтийского региона Механизмы устойчивости к БЛРС pneumoniae и E. coli, устойчивые к ЦРС за счет продукции БЛРС СТХ-М-1 класса. Наиболее частой локализацией карбапенем- резистентных штаммов являлись нижние ды- хательные (10 штаммов) и мочевыводящие пути (7 штаммов). Три штамма были выделены из ра- невого отделяемого и два штамма – из крови. Карбапенемы часто являются единствен- ными АМП, которые сохраняют активность против полирезистентных грамотрицательных микроорганизмов, в частности продуцирую- щих БЛРС. Поэтому распространение штам- мов, устойчивых к этой группе препаратов, представляет серьезную угрозу для здравоохра- нения. Наше исследование показало, что в че- тырех странах Балтийского региона выявлены Обсуждение ЦРС и карбапенемы отличаются высокой активностью в отношении широкого спектра микроорганизмов. В условиях нарастания мно- жественной устойчивости возбудителей, в том числе к бета-лактамным препаратам за счет продукции бета-лактамаз, карбапенемы оста- ются препаратами резерва для терапии тяжелых инфекций, особенно у пациентов в отделениях реанимаций и интенсивной терапии. Наиболее эпидемиологически значимым механизмом ре- зистентности к бета-лактамам у энтеробактерий является продукция различных бета-лактамаз (в том числе БЛРС и МБЛ). В странах Евро- союза доля инвазивных штаммов (выделенных из крови и ликвора) E. coli и K. pneumoniae, про- дуцирующих БЛРС, колеблется. Наименьшие показатели отмечены в Швеции — 3 и 2,3% со- ответственно, наиболее высокие — на Кипре (36% E. coli) и в Болгарии (81% K. pneumoniae) [5]. В начале 90-х гг. XX века впервые были описа- ны цефотаксимазы семейства СТХ-М, кото- рые в настоящее время распространены на всех континентах. Способностью продуцировать СТХ-М характеризуются энтеробактерии, вы- зывающие внутрибольничные и внебольнич- ные инфекции, а также заболевания, передаю- щиеся с пищевыми продуктами. Так, вспышка, возникшая в 16 странах Европы летом 2011 г., показала, что многие пищевые продукты могут быть активными факторами передачи пищевых инфекций, вызванных полирезистентными штаммами E. coli, способными продуцировать не только экзотоксины, вызывающие ослож- нения, угрожающие жизни, но и БЛРС семей- ства СТХ-М [1]. Исследования, периодически проводимые в РФ, показывают, что в стацио- нарах различных городов распространенность штаммов K. pneumoniae и E. coli, устойчивых к ЦРС, в последние годы превышает 80% [2, 3]. Рисунок 2. Доля штаммов (%) K. pneumoniae, продуцирующих БЛРС семейства СТХ-М различных групп, в странах Балтийского региона региона Рисунок 3. Доля штаммов (%) E. coli, продуцирующих БЛРС семейства СТХ-М различных групп, в странах Балтийского региона Рисунок 3. Доля штаммов (%) E. coli, продуцирующих БЛРС семейства СТХ-М различных групп, в странах Балтийского региона Рисунок 3. Доля штаммов (%) E. coli, продуцирующих БЛРС семейства СТХ-М различных групп, в странах Балтийского региона 33 С.А. Егорова и др. Инфекция и иммунитет штаммы K. pneumoniae, резистентные к карбапе- немам с различными механизмами резистент- ности. Штаммы, продуцирующие МБЛ, были выявлены только в России, в трех стационарах Санкт-Петербурга. дуцирующим МБЛ группы VIM, а продукция NDM-1 выявлена только у штамма Acinetobacter nosocomialis [4, 17]. Продукция этого фермента штаммами Enterobacteriaceae ранее не описана. В настоящее время выявлены 3 варианта NDM, возникших в результате точечных му- таций в гене NDM-1. NDM-2 была выявлена у штамма A. baumanii, выделенного от пациента, перевезенного из египетского госпиталя в ста- ционар в Германии. В последствии клональное распространение A. baumanii, продуцирующего NDM-2, было описано в Израиле. Обсуждение были пересмотрены в сторону ужесточения и строго ориентированы на достижение клинической эффективности антимикробной терапии. Тем не менее, идентификация конкретного специ- фического фермента может быть полезна для проведения инфекционного контроля и эпиде- миологических исследований. Штаммы энтеробактерий, устойчивые к кар- бапенемам за счет продукции NDM, выявляют обычными рутинными лабораторными метода- ми, включая ДДМ или методы серийных разве- дений, с дальнейшим проведением различных подтверждающих тестов (модифицированный Hodge-тест, тесты синергизма с ЭДТА и др.). Широко распространенные автоматизирован- ные системы (например, Vitek) также показыва- ют хорошую чувствительность при выявлении устойчивости, обусловленной МБЛ, хотя и ха- рактеризуются низкой специфичностью при дифференциации различных видов карбепене- маз. Подтверждение продукции металло-бета- лактамазы класса NDM требует проведения ПЦР или ДНК-секвенирования, что возможно только в специализированных лабораториях. Профилактика распространения штаммов- продуцентов карбапенемаз основана на раннем выявлении носителей таких штаммов в стацио- наре. Скринингу подлежат пациенты, которые были госпитализированы в одной стране, а затем переехали в другую страну, и пациенты из груп- пы риска (госпитализированные в ОРИТ, пере- несшие операцию по трансплантации органов и др.). В отношении таких пациентов должны применяться мероприятия, ограничивающие контакты с другими пациентами и медицинским персоналом до получения результатов лабора- торного обследования. Для быстрого выявления таких штаммов разработаны и используются специальные среды: хромогенные и содержащие различные концентрации карбапенемов. Тем не менее, в настоящее время отсутствует универ- сальная среда, позволяющая с высокой чувстви- тельностью и специфичностью выявлять штам- мы, продуцирующие все виды карбапенемаз. Оснащение оборудованием для молекулярно- генетических исследований расширяет анали- тические возможности лабораторий по детекции механизмов резистентности. Проведенные нами исследования показа- ли, что ДДМ и Е-тесты обладают достаточной чувствительностью для выявления штаммов, продуцирующих NDM. Зоны задержки роста вокруг дисков с имипенемом и меропенемом у всех 22 штаммов соответствовали категориям «устойчивый» или «промежуточный», при этом использование для скрининга двух препаратов из группы карбапенемов повышало вероят- ность выявления таких штаммов. Наши данные подтвердили, что большинство штаммов, про- дуцирующих NDM-1, резистентны практиче- ски ко всем АМП, используемым для лечения тяжелых инфекций. Полимиксин сохраняет активность in vitro, однако в последние годы этот препарат не используется широко в кли- нической практике, его клиническая эффек- тивность при лечении инфекций, вызванных энтеробактерия ми, недостаточно доказана. В разработке и осуществлении мер по сдержи- ванию резистентности к АМП штаммов микро- организмов, вызывающих инфекционный про- цесс у пациентов, находящихся на стационарном лечении, должны принимать участие специали- сты разного профиля: от главного врача стацио- нара до санитарки. Обсуждение NDM-4 был выявлен у штамма E. coli от пациента, госпита- лизированного в Индии, а также от пациента, поступившего в стационар Франции из Каме- руна. Последний вариант, NDM-5, обнаружен у штамма E. coli, выделенного от пациента, ко- торый также в анамнезе имел случай госпита- лизации в Индии [10]. р ур У энтеробактерий описана продукция раз- личных классов МБЛ (VIM, IMP и др.). В 2008 г. в Великобритании были впервые выделены штаммы K. pneumoniae и E. coli, продуцирующие новый класс МБЛ, так называемые New Delhi metallo-β-lactamase (NDM) [12]. По данным Ев- ропейского Центра по Контролю Заболеваемо- сти (ECDC) к марту 2011 г. в 13 странах Европы зарегистрировано 106 случаев выделения NDM- продуцирующих энтеробактерий (K. pneumoniae, E. coli, Enterobacter spp.) [19]. В США к марту 2012 г. отмечено 13 случаев выделения NDM- 1-продуцирующих энтеробактерий [7]. Также случаи выделения таких штаммов были отме- чены в Австралии, Канаде, Японии, Кении [8, 13, 15, 16]. Эпидемиологические данные сви- детельствуют о том, что большинство случаев инфекции, зарегистрированных в Европе, свя- заны с посещением стран Индийского субкон- тинента (Индия, Пакистан) или Балканского полуострова (Косово, Сербия, Босния, Герце- говина). В различных городах Индии NDM- продуцирующие штаммы составляют до 38,5% всех карбапенем-резистентных клинических изолятов Enterobacteriaceae, а также обнаружены в сточных и поверхностных водах [20]. В Рос- сии был зарегистрирован лишь один случай инфекции, вызванный штаммом E. coli, про- По сравнению с другими карбапенемазами, NDM обладает некоторыми особенностями, которые обуславливают ее особую значимость для здравоохранения. Гены blaNDM-1 выяв- лены у клонально неродственных штаммов, обнаружены в объектах окружающей среды (водопроводная вода и вода поверхностных водоемов) на Индийском субконтиненте. Наи- более часто гены blaNDM-1 приобретают штам- мы K. pneumoniae, типичного нозокомиального возбудителя, а также штаммы E. coli, основного возбудителя внебольничных инфекций МВП. Источником выделения таких штаммов может быть значительная часть населения планеты, Рисунок 4. Группы БЛРС класса СТХ-М у штаммов Enterobacteriacae, выделенных в стационарах стран Балтийского региона Рисунок 4. Группы БЛРС класса СТХ-М у штаммов Enterobacteriacae, выделенных в стационарах стран Балтийского региона 34 Энтеробактерии, продуцирующие БЛРС и NDM-1 2013, Т. 3, № 1 поскольку в странах Индийского субконтинен- та проживает более 1,4 млрд человек. В некото- рых регионах Пакистана до 20% населения могут являться носителями NDM-1-продуцирующих штаммов [14]. поскольку в странах Индийского субконтинен- та проживает более 1,4 млрд человек. В некото- рых регионах Пакистана до 20% населения могут являться носителями NDM-1-продуцирующих штаммов [14]. поскольку пограничные концентрации для це- фалоспоринов и карбапенемов в 2012 г. Обсуждение Меры по борьбе с резистент- ностью к АМП состоят из двух самостоятельных направлений: устранение факторов, способству- ющих селекции резистентных микроорганизмов и распространению резистентных микроорга- низмов во внешней среде и восприимчивой попу- ляции. Успешное решение этих задач в условиях стационаров требует знаний эпидемиологиче- ских, микробиологических, фармакодинамиче- ских и молекулярно-генетических особенностей формирования резистентности у конкретного штамма микроорганизма. Полученные нами данные свидетельствуют о том, что в настоящее время для стационаров Санкт-Петербурга становится актуальной про- блема циркуляции возбудителей, обладаю- щих множественной устойчивостью к АМП, включая препараты резерва – карбапенемы. Причем такие штаммы встречаются не только среди Pseudomonas spp. и Acinetobacter spp., что было описано ранее, но также впервые выявле- ны штаммы K. pneumoniae, характеризующиеся продукцией металло-бета-лактамазы NDM-1, первые находки которой описаны в мире отно- сительно недавно. Список литературы Кафтырева Л.А., Егорова С.А., Макарова М.А., 1. Забровская А.В., Матвеева З.Н., Сужаева Л.В., Артамонова Ю.А. Характеристика биологичес- ких свойств E. coli O104:H4 — возбудителя круп- ной пищевой вспышки, возникшей в Германии в мае 2011 г. // Клиническая лабораторная диа- гностика. — 2012. — № 1. — С. 44–46. Kumarasamy K.K., Toleman M.A., Walsh T.R., Baga- 12. ria J., Butt F., Balakrishnan R., Chaudhary U., Dou- mith M., Giske C.G., Irfan S., Krishnan P., Kumar A.V., Maharjan S., Mushtaq S., Noorie T., Paterson D.L., Pear- son A., Perry C., Pike R., Rao B., Ray U., Sarma J.B., Sharma M., Sheridan E., Thirunarayan M.A., Turton J., Upadhyay S., Warner M., Welfare W., Livermore D.M., Woodford N. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study // Lancet Infect. Dis. — 2010. — Vol. 10, N 9. — P. 597–602. Решедько Г.К., Шебников А.Г., Морозов М.В., Ре- 2. шедько Л.А. Escherichia coli как возбудитель но- зокомиальных инфекций в ОРИТ // Клиничес- кая микробиология и антимикробная химиоте- рапия. — 2011. — Т. 13, № 4. — С. 314–321. Рябкова Е.Л., Иванчик Н.В., Сухорукова М.В., 3. Шебников А.Г., Решедько Г.К. Резистентность нозокомиальных штаммов Escherichia coli в ста- ционарах России // Клиническая микробиоло- гия и антимикробная химиотерапия. — 2009. — Т. 11, № 2. — С. 161–169. Mulvey M.R., Grant J.M., Plewes K., Roscoe D., 13. Boyd D.A. New Delhi metallo-β-lactamase in Kleb- siella pneumoniae and Escherichia coli, Canada // Emerg. Infect. Dis. — 2011. — Vol. 17, N 1. — P. 103–106. Эйдельштейн М.В., Склеенова Е.Ю., Шевчен- 4. ко О.В. Распространенность и молекулярная эпи- демиология грамотрицательных бактерий, про- дуцирующих металло-бета-лактамазы, в России, Беларуси и Казахстане // Клиническая микро- биология и антимикробная химиотерапия. — 2012. — Т. 14, № 2. — С. 132–152. Nordmann P., Naas T., Poirel L. Global spread of car- 14. bapenemase-producing Enterobacteriacae // Emerg. Infect. Dis. — 2011. — Vol. 17, N 10. — P. 1791–1798. Poirel L., Lagrutta E., Taylor P., Pham J., Nordmann P. 15. Emergence of metallo-β-lactamase NDM-1-producing multidrug-resistant Escherichia coli in Australia // Anti- microb. Agents Chemother. — 2010. — Vol. 54, N 11. — P. 4914–4916. Antimicrobial resistance surveillance in Europe 2011. 5. Surveillance report http://www.ecdc.europa.eu/en/ publications/Publications/antimicrobial-resistance- surveillance-europe-2011.pdf Poirel L., Revathi G., Bernabeu S., Nordmann P. De- 16. tection of NDM-1-producing Klebsiella pneumoniae in Kenya // Antimicrob. Agents Chemother. — 2011. — Vol. 55, N 2. Благодарность Авторы статьи выражают благодарность BARN (Сети по надзору за антибиотикорезис- тентностью в странах Балтийского региона) за оказание финансовой и научной поддержки в ходе реализации проекта «Изучение эпидемио- логии БЛРС-продуцирующих энтеробактерий в странах Балтийского региона». В настоящее время в международных реко- мендациях (EUCAST, CLSI) детекция специ- фического механизма резистентности (включая продукцию конкретной β-лактамазы — БЛРС, карбапенемазы KPC или МБЛ) не требуется для определения тактики лечения пациента, 35 С.А. Егорова и др. Инфекция и иммунитет Egorova S., Kaftyreva L., Grimont P.A.D., Weill F.-X. 11. Prevalence and characterization of extended-spect- rum cephalosporin-resistant nontyphoidal Salmonella isolates in adults in Saint Petersburg, Russia (2002– 2005) // Microb. Drug. Resist. — 2007. — Vol. 13, N 2. — P. 102–107. Список литературы — P. 934–936. Birkett C.I., Ludlam H.А., Woodford N., Brown D.F.J., 6. Brown N.M., Roberts M.T.M., Milner N., Cur- ran M.D. Real-time TaqMan PCR for rapid detection and typing of genes encoding CTX-M extendedspec- trum b-lactamases // J. Medical. Microbiol. — 2007. — Vol. 56. — P. 52–55. Shevchenko O.V., Mudrak D.Y., Skleenova E.Y., Kozy- 17. reva V.K., Ilina E.N., Ikryannikova L.N., Ale xand- rova I.A., Sidorenko S.V., Edelstein M.V. First detec- tion of VIM-4metallo-beta-lactamase-producing Es- cherichia coli in Russia // Clin. Microbiol. Infect. — 2012. — Vol. 18. — P. 214–217. Carbapenem-resistant Enterobacteriaceae containing 7. New Delhi metallo-beta-lactamase in two patients — Rhode Island, March 2012. CDC Morbidity and Mor- tality Weekly Report. — 2012. — Vol. 61, N 24. — P. 446– 448. — Режим доступа: http://www.cdc.gov/mmwr/pre- view/mmwrhtml/mm6124a3.htm?s_cid=mm6124a3_w. Sparbier K., Schubert S., Weller U., Boogen C., Kos- 18. trzewa M. Matrix-assisted laser desorption ioniza- tion — time of flight mass spectrometry-based func- tional assay for rapid detection of resistance against β-lactam antibiotics // J. Clin. Microbiol. — 2012. — Vol. 50, N 3. — P. 927–937. Chihara S., Okuzumi K., Yamamoto Y., Oikawa S., 8. Hishinuma A. First case of New Delhi metallo-β- lactamase 1-producing Escherichia coli infection in Japan // Clin. Infect. Dis. — 2011. — Vol. 52, N 1. — P. 153–154. Updated ECDC risk assessment on the spread of New 19. Delhi metallo-β-lactamase and its variants within Europe. ECDC Technical report. (http://www.ecdc. europa.eu/en/publications/Publications/1111_TER_ Risk-assessment-NDM.pdf). Danny C.T. Ong, Tse-Hsien Koh, Nur Syahidah, 9. Prabha Krishnan, Thean Yen Tan. Rapid detection of the blaNDM-1 gene by real-time PCR // J. An- timicrob. Chemother. — 2011. — Vol. 66, N 7. — P. 1647–1649. Walsh T.R., Weeks J., Livermore D.M., Toleman M.A. 20. Dissemination of NDM-1 positive bacteria in the New Delhi environment and its implications for hu- man health: an environmental point prevalence study // Lancet Infect. Dis. — 2011. — Vol. 11, N 5. — P. 355–362. Dortet L., Poirel L., Anguel N., Nordmann P. New 10. Delhi metallo β-lactamase 4-producing Escherichia coli in Cameroon // Emerg. Infect. Dis. — 2012. — Vol. 18, N 9. — P. 1540–1542. 36
https://openalex.org/W3047031083	https://www.europeanproceedings.com/files/data/article/10030/10875/article_10030_10875_pdf_100.pdf	English	null	Metaphorical Representation Of “Social Networks” Concept In The Russian Online Discourse	The European Proceedings of Social & Behavioural Sciences	2,020	cc-by	3,934	(a) Kemerovo State University, 6 Krasnaya str., Kemerovo, Russian Federation, Olnik25@mail.ru (b) Kemerovo State University, 6 Krasnaya str., Kemerovo, Russian Federation, r961nav@rambler.ru (a) Kemerovo State University, 6 Krasnaya str., Kemerovo, Russian Federation, Olnik25@mail.ru (b) Kemerovo State University, 6 Krasnaya str., Kemerovo, Russian Federation, r961nav@rambler.ru www.europeanproceedings.com DOI: 10.15405/epsbs.2020.08.84 Olga Kondratyeva (a), Anna Novoklinova (b) Corresponding author (a) Kemerovo State University, 6 Krasnaya str., Kemerovo, Russian Federation, Olnik25@mail.ru (b) Kemerovo State University, 6 Krasnaya str., Kemerovo, Russian Federation, r961nav@rambler.ru ywords: Metaphorical representation, conceptual metaphor, concept, online discourse, social networks. 2. Problem Statement An important and yet insufficiently studied aspect of social networks is the metaphorical interpretation in different types of discourse (science, media, network, everyday-life, etc.). The need for research is determined by the fact that, social networks terminology is largely metaphorical (cf. social network, page, gallery, etc.). Besides, analyzing and assessing social networks phenomenon, IT specialists, common users, linguists, journalists actively use metaphors to explicate the most significant features of the relatively new phenomenon in communication. An important and yet insufficiently studied aspect of social networks is the metaphorical interpretation in different types of discourse (science, media, network, everyday-life, etc.). The need for research is determined by the fact that, social networks terminology is largely metaphorical (cf. social network, page, gallery, etc.). Besides, analyzing and assessing social networks phenomenon, IT specialists, common users, linguists, journalists actively use metaphors to explicate the most significant features of the relatively new phenomenon in communication. 1. Introduction Rapid development of technology at the end of the XXth – beginning of XXIth century gave rise to a fundamentally new space of communication – social networks – and to a new type of communication – online discourse, which is significantly different from other types of discourse. Discourse-making signs are (1) common interests of participants and their connection to a single electronic resource, (2) polymodality of messages (visual, audio and text components), (3) the possibility to receive an instant response to the content, (4) the reduced distance of communication, in which the boundary between private and public communication formats is dissolved (Karasik, 2018, p. 164). Linguists immediately took the interest in the new discourse: the research object has been the language of social networks, genres, virtual linguistic persona, typology of network characters, online strategies and tactics, a virtual conceptual system formation (Akhrenova, 2016; Dobrinskaya, 2016; Frischmann, 2018; Karasik, 2018; Language and speech on the Internet, 2018; Pavlov, 2017; Prokofiev, 2017, etc.). Abstract The article features metaphorical representation of “social networks” concept in the Russian online discourse dated 2014-2019. The research material was posts from Odnoklassniki, Twitter, Vkontakte, Facebook Photostrana, Facebook, LiveJournal, LiveInternet and Diary networks. The metaphorical modeling method revealed the metaphors by which users of social networks interpret and evaluate the new communicative space. We identified and ranked the dominant models with content analysis method. We found, that metaphors used by participants of virtual communication allow explicating significant features of social networks as well as characterizing their functions. In particular, social networks are perceived as a space in which communication is carried out (ocean), where they spend a lot of time (home), as a storage of diverse (not always reliable) information (ocean, garbage hole). The complex structure of social networks (web, network, jungle) has also been noted. The Russian online discourse dominates metaphors with pronounced negative pragmatic potential, most of which actualizes addiction on social networks, the difficulty of returning to reality (web, swamp, jungle, disease). Metaphors representing the need for restrictions in the use of social networks or the complete rejection (metaphors of uprooting from the whirlpool, escape from home, getting rid of garbage, healing from disease, detoxifications, etc.), reflecting users’ awareness of the problem and their willingness to find a solution. Thus, the analysis of metaphorical representations of “social networks” concept allows analyzing cognitive mechanisms involved in understanding the new communicative phenomenon, as well as explicating the social problems associated with the new format of communication. The article features metaphorical representation of “social networks” concept in the Russian online discourse dated 2014-2019. The research material was posts from Odnoklassniki, Twitter, Vkontakte, Facebook Photostrana, Facebook, LiveJournal, LiveInternet and Diary networks. The metaphorical modeling method revealed the metaphors by which users of social networks interpret and evaluate the new communicative space. We identified and ranked the dominant models with content analysis method. 2357-1330 © 2020 Published by European Publisher. 2357-1330 © 2020 Published by European Publisher. Keywords: Metaphorical representation, conceptual metaphor, concept, online discourse, social networks. T This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial 4.0 Unported License, permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. https://doi.org/10.15405/epsbs.2020.08.84 Corresponding Author: Olga Kondratyeva Selection and peer-review under responsibility of the Organizing Committee of the conference eISSN: 2357-1330 5. Research Methods We used the method of metaphorical modeling to analyze the language, consisting in the defining of sources of metaphoric expansion used in online discourse for representation of “social networks” concept, the identification of metaphorical transfer and the explication of assessments of a cognizable phenomenon contained in the metaphors. We also used content analysis method, which allows determining the frequency of metaphoric models. In statistical processing of the material the percentage distribution of metaphoric models with their subsequent ranking was applied. At the final stage, we interpreted the information, providing linguistic and cultural comment. 6. Findings The study showed that most metaphors representing “social networks” concept are determined by English origin “the original terminology, which is based on the Russian-speaking usage gets a new understanding, acquires specific cultural texts and connotations” (Kakorina, 2013, p. 145). Metaphors of web, water reservoir, swamp, wilds, garbage hole, home and disease are used most actively for the representation of the concept (target sphere) in online discourse. 3. Research Questions The study focused on two issues: first, defining the role of a metaphor in conceptualization, interpretation and assessment of reality, the article demonstrates the effect of metaphorical cognitive mechanisms in the exploration of a new conceptual space – social networks. Secondly, the online discourse as research material will reveal the features of metaphorical interpretation and assessment of a new communicative phenomenon immediately by its users, and not by observers in case of studying of “social networks” concept in other types of discourse (Galichkina, 2016; Kondratyeva, 2019; Makeeva & Borodulina, 2017, 2018; Tomaszewski, 2011; Voiskunsky, 2001, etc.). In addition, the study will highlight the most disputable moments of modern virtual communication. 710 https://doi.org/10.15405/epsbs.2020.08.84 Corresponding Author: Olga Kondratyeva Selection and peer-review under responsibility of the Organizing Committee of the conference eISSN: 2357-1330 https://doi.org/10.15405/epsbs.2020.08.84 Corresponding Author: Olga Kondratyeva Selection and peer-review under responsibility of the Organizing Committee of the conference eISSN: 2357-1330 4. Purpose of the Study The purpose is to analyze the metaphors representing “social networks” concept in the Russian online discourse. The research material was the posts taken from VKontakte, Facebook, Odnoklassniki, Twitter, Fotostrana, LiveJournal, LiveInternet and Diary social networks. Language material was collected through social media monitoring services Socialseek and Blogs.yandex. The research file included 1687 metaphors dated 2014-2019 The purpose is to analyze the metaphors representing “social networks” concept in the Russian online discourse. The research material was the posts taken from VKontakte, Facebook, Odnoklassniki, Twitter, Fotostrana, LiveJournal, LiveInternet and Diary social networks. Language material was collected through social media monitoring services Socialseek and Blogs.yandex. The research file included 1687 metaphors dated 2014-2019. 6.1. Web metaphor (23%) The metaphor is connected with the metonymic transfer from the generic social networks concept – the Internet, metaphorical by its nature in the language source, to its part – social networks: WWW is an abbreviation of “World Wide Web”, “World Wide Web”.... Many believe that the World Wide Web, WWW, is the Internet, although this is certainly not the case (eponim2008.livejournal.com). Translating the word net into Russian, the rich semantic potential of the word is revealed. For such metaphors “semes of the direct meaning of the word, its components “device for catching fish or birds, etc. “, “thread knotting”, ‘threads crossing”, “knotting” (Kakorina, 2013, p. 146), on the basis of which the figurative meaning 'to be bound into a single system' arises: The world has long been quite firmly linked in the social Internet networks. Invisible threads intertwined in a bizarre macrame of Facebook, Vkontakte and twitter, sometimes captivate weak minds and inexperienced souls into the virtual world so deep that real life is already seems to them bland and boring ( 711 https://doi.org/10.15405/epsbs.2020.08.84 Corresponding Author: Olga Kondratyeva Selection and peer-review under responsibility of the Organizing Committee of the conference eISSN: 2357-1330 The representatives of the metaphors are network, web and related verbs to entangle/confuse, etc.: On the web of social networks. Social networks have entangled my generation, probably, since I was an eleven-grader, if not earlier (pay.diary.ru›~Cjoe/? from=20); We were all mired in the web of social networks, or rather got confused (patri0t.livejournal.com). The metaphor has negative connotations in Russian linguoculture: the word-play to get into the network often indicates addiction to virtual reality. 6.2. Water space metaphor (14%) Marine theme “from the very beginning of the formation of the Internet metaphoric language dominated both English and French” (Makeeva & Borodulina, 2017, p. 9). As part of the metaphorical transfer “ocean – Internet” in the English discourse there were metaphors to surf the Internet, to navigate, navigation system, which were calqued in Romance, German and Slavic languages (see more details Jamet, 2010; Juanals, 2004; Kakorina, 2013; Makeeva & Borodulina, 2017). There is similar metaphorical understanding of social networks. Mainly, the metaphor compares search for information and online communication with sliding on the water surface or with full immersion in water space. Representatives of the metaphorical model are verbs to surf, to drift, characterizing the search for information in the network, as well as following one site / page to another without a search strategy or definite objective: That's where you need to surf. Oddly enough, but every day I find more and more resources in the boundless ocean of social networks (natalya-kiriche.livejournal.com); What do people do in social networks, spending here a few hours daily? Drift in search of new information (paradoxov.livejournal.com). Spending time in social networks is metaphorically interpreted as immersion in water, which is facilitated by the use of verbs to dive, to plunge, dip: And how do volunteers live? We decided to dive into social networks and find it out (ok.ru›newslab/topic/69331416320853); I can't wait to come home and quickly plunge into social networks! (nickkein.livejournal.com); I personally dive into social networks every night like a diver to the depth! (vk.com›wall253001020). The return to the real world in metaphorical meaning the verb to come out / up is used: Having removed his profile, he come out to the real world and saw that this world is not so boring (molli-dolli.livejournal.com). 6.5. Forest metaphor (wilds, jungle) (11%) Metaphors of wilds highlight the idea of difficult orientation in the virtual world and the tricky exit from the world of social communications. It includes lexemes, denoting impassable forest areas – woods, wilds, jungle: We will guide you through the wilds of empty communities over the abyss of content right where abandoned publics with millions of people have frozen in silence (vk.com›wall-26760927? own=1); Going through the jungle of social networks, I came across the full version of the poem, (aged- hare.livejournal.com). The jungle metaphor additionally actualizes conflict behavior of the users: Guide to Survival in the “Jungle” of Social Networks (by SecWorld); Digital Jungle: how to avoid danger in social networks (vbudushee.ru›upload/iblock). 6.4. Garbage hole metaphor (9%) Actualizes negative characteristics of the information and the way it is presented in social networks: unreliable, disorderly, undifferentiated: Social networks are garbage. Reliable information may be found, but it takes pains to check for its accuracy (gusev-a-v.livejournal.com). Representatives of the model are garbage, cesspool: I’ve deleted my profile in VKontakte (I haven't been there for a long time), Odnoklassniki (even longer), MirTesen, PhotoStrana, My Circle, Facebook, and several other social dumps (liveinternet.ru/users/ylogru); I’ve found a new cesspool. The abyss of social networks suppressing our minds (@Eledantes).Such lexemes as dirt, stick, etc.: Another dirt from the social network (ok.ru›rznnovosti); People wished they hadn’t got stuck in social networks (30rus.livejournal.com) 6.3. Swamp metaphor (17%) It is an implementation of water metaphor with a negative connotation, since it is used to indicate negative information found in social networks: Social networks (especially FB) are increasingly reminiscent of the swamp: the same bad smelling and sticky matter. News feeds are full of gossip, squabbles, arguments (ikrima.livejournal.com); In the abyss of social networks, users drown in fecal whirlpools, Opinion leaders horse around on the shit- waves, scoffing and spitting ( manyunya1116.livejournal.com). In addition, verbs to suck, to tighten, to get bogged, explicating immersion in social networks for a long time, the difficulty of getting out of virtual space: How not to get bogged down in the “swamp” of social networks and move forward? (vk.com›wall-100123914? own=1); The abyss of social networks can suck at any time of the day (OK. Nadezhda Fatyanova). Swamp metaphors represent Internet addiction mostly. 712 https://doi.org/10.15405/epsbs.2020.08.84 Corresponding Author: Olga Kondratyeva Selection and peer-review under responsibility of the Organizing Committee of the conference eISSN: 2357-1330 https://doi.org/10.15405/epsbs.2020.08.84 Corresponding Author: Olga Kondratyeva Selection and peer-review under responsibility of the Organizing Committee of the conference eISSN: 2357-1330 https://doi.org/10.15405/epsbs.2020.08.84 Corresponding Author: Olga Kondratyeva Selection and peer-review under responsibility of the Organizing Committee of the conference eISSN: 2357-1330 6.6. Home metaphor (12%) The metaphor is of English origin: Internet resources, their separate parts, interface elements are interpreted with house/home metaphor, e.g., Home Page, Gallery, News Room. The similar metaphorization principle is observed in social networks. Home metaphor is realized through the nomination of site visitors: residents, inhabitants, guests or regulars (depending on the degree of involvement or the amount of time spent in the network): The unfortunate inhabitants of Facebook. A person, who once joined the popular social network, finds it difficult to quit using it (matveychev-oleg.livejournal.com); Permanent residents of the network join in groups (alexhitrov.livejournal.com); Last night “VKontakte” regulars found that all the downloaded audio files disappeared from their profiles (vk.com›wall-125503122? q); My former “barrack-mate” turned out to be a regular of social networks... (minorowsky. livejournal.com›); Social networks сan make life more comfortable. Provided that every guest of social networks thinks about the probable consequences... (mihalych_org). Some users’ nicknames are also indicative in this respect, e.g., Live_in the network and Livininet: Living in the network. 36 years old, Tynda (Amur region), Russia; Live online. 42 years old, Moscow, Russia (ok.ru›search/profiles/Livein-network). Home metaphors are also created by verbal vocabulary, denoting living in the house or visiting the house – to live, call on, live, walk/enter, move, be registered in: OK, social networks won. All people went to live in social networks (tema.livejournal.com); I wonder, what social networks you live in (minakovas.livejournal.com); Social networks: Who goes there and why? (rubin65.livejournal.com); Those who got registered in social networks, cannot be called polyglots ( 713 https://doi.org/10.15405/epsbs.2020.08.84 Corresponding Author: Olga Kondratyeva Selection and peer-review under responsibility of the Organizing Committee of the conference eISSN: 2357-1330 Spending a lot of time in social networks is also represented by means of the verb to stay/to sit. It is indicative that users exploit the home metaphor: “Staying in Facebook, LiveJournal, the computer, Vkontakte. It feels like an invisible room, limited space around us. That didn’t use to be the case – one cannot sit/stay in a book/video, although it seems to be, in fact, the same temporal move to another, virtual, world (ru-etymology.livejournal.com). Account deleting or refusal to communicate virtually is described by leaving metaphors: Escape from social networks. Action Plan (shakkar.livejournal.com); Are you ready to leave social networks (za- y-ac.livejournal.com); Leaving social networks is the best solution to combat this addiction. 6.7. Disease metaphor (16%) Social media users are actively discussing the dangers of excessive pastime in the virtual world, using medical metaphors. Such lexemes as disease, treatment, medicine, etc. are of frequent occurrence: IS SOCIAL NETWORK A DISEASE? (liveinternet.ru/users/buvay_sar); Do you consider social media addiction a disease? And if so, what should be the treatment? (k-poli.livejournal.com). Most users are concerned about the steady dependence on social networks. They compare their condition – when they cannot visit their favorite resources – with withdrawal syndrome: How has your life changed after deleting your profile? (in particular “Vkontakte”). What urged you to do it? How long did the withdrawal syndrome last? (And did you have it? (justforus.diary.ru›p209549234.htm); To forbid myself to sit in a social network (and at the same time test my addiction) was difficult. So hard that there even were withdrawal symptoms... (jennifer-hot.livejournal.com). Many users also reflect on finding treatment for the virtual dependence, using metaphors of hygiene and detoxification: Digital hygiene: how to use social networks, not letting them use you (karhu53.livejournal.com); Social detox. I’ve deleted Facebook profile and messengers from the phone, life immediately sparkled (shri-boomer.livejournal.com). Morbid metaphor is a bright indicator of the presence of social problems and has the greatest force in terms of emotional impact. 6.6. Home metaphor (12%) Not everyone can break their habits and log out of (=leave) the profile never to return there again (ok.ru›shakkar/topic/65978042919741). Home metaphors create the image of a space replacing a real home in which users spend much time (often to the detriment of the real communication). 7. Conclusion The study of metaphors representing “social networks” concept in the online discourse revealed several trends: 1. Metaphors used by participants of virtual communication allow explicating significant features of social networks as a complex phenomenon as well as characterizing their functions. In particular, social networks are perceived by users as a space in which communication is carried out (ocean) and where they spend a lot of time (home), as a storage of diverse information (ocean, garbage hole). The complex structure of social networks (web, network, jungle) has been also noted. 714 https://doi.org/10.15405/epsbs.2020.08.84 Corresponding Author: Olga Kondratyeva Selection and peer-review under responsibility of the Organizing Committee of the conference eISSN: 2357-1330 2. Online discourse metaphors are less diverse than, e.g., in media and science discourses (Kondratyeva, 2019; Voiskunsky, 2001), e.g., such metaphors as of a mirror, labyrinth, or a frontal were unpopular. It is probably due to a rather low cultural level of average users. 3. Metaphors with strongly-marked negative pragmatic potential (tightening web, sticky swamp, large garbage hole, impassable wilds, severe disease) are dominating. Moreover, metaphors used in online discourse increase the tension degree compared to the same metaphors of media discourse (The swamp is filled with faeces, A rubbish dump is transformed into a garbage hole, etc.). 3. Metaphors with strongly-marked negative pragmatic potential (tightening web, sticky swamp, large garbage hole, impassable wilds, severe disease) are dominating. Moreover, metaphors used in online discourse increase the tension degree compared to the same metaphors of media discourse (The swamp is filled with faeces, A rubbish dump is transformed into a garbage hole, etc.). Most metaphors represent the addiction on social networks, the difficulty of returning from the virtual world to reality (web, swamp, jungle, disease). Compared to the media discourse, there is increased number of metaphors that represent the need for restrictions in their use or complete rejection (Coming out of a whirlpool, escape from home, getting rid of garbage, recovering from a disease, detoxification, etc.). This clearly demonstrates not only the problem itself, but also the users’ concern about finding a solution to it. Acknowledgments The research was supported by the Russian Foundation for Basic Research, research project No. 19-012-00202 “Everyday political communication in social networks: a comprehensive linguistic analysis”. References Akhrenova, N. A. (2016). Internet-lingvistika: novaya paradigm v opisanii yazyka interneta [Internet linguistics: a new paradigm in the description of the Internet language]. Bulletin of the Moscow Regional State University. Series: Linguistics, 3, 8–14. [In Russ.]. Dobrinskaya, D. E. (2016). Sotsiologicheskoe osmyslenie interneta; teoreticheskie podhody k osmysleniyu seti] [Sociological understanding of the Internet: theoretical approaches to the study of the network. Bulletin of Moscow University. Issue 18. Political Science and Sociology, 22(3), 21–38. https://doi.org/10.24290/1029-3736-2016-22-3-27-38 [In Russ.]. Dobrinskaya, D. E. (2016). Sotsiologicheskoe osmyslenie interneta; teoreticheskie podhody k osmysleniyu seti] [Sociological understanding of the Internet: theoretical approaches to the study of the network. Bulletin of Moscow University. Issue 18. Political Science and Sociology, 22(3), 21–38. https://doi.org/10.24290/1029-3736-2016-22-3-27-38 [In Russ.]. Frischmann, В. (2018). The Misleading Power of Internet Metaphors. Scientific American. https://blogs. scientificamerican.com/observations/the-misleading-power-of-internet-metaphors/ Frischmann, В. (2018). The Misleading Power of Internet Metaphors. Scientific American. https://blogs. scientificamerican.com/observations/the-misleading-power-of-internet-metaphors/ Galichkina, Е. (2016). Internet Conceptual Metaphors. Journal of Siberian Federal University. Humanities & Social Sciences, 9, 470–480. [In Russ.]. Galichkina, Е. (2016). Internet Conceptual Metaphors. Journal of Siberian Federal University. Humanities & Social Sciences, 9, 470–480. [In Russ.]. Jamet, D. L. (2010). What do Internet metaphors reveal about the perception of the Internet. Metaphorik, 18(2), 17–32. Juanals, B. (2004). L'arbre, le labyrinthe et l'océan. Les métaphores du savoir, des Lumières au numérique. Communication et langages, 139(1), 101–110. Kakorina, E. V. (2013). Metafory interneta v rechi komp’yuternyh polzovateley [Metaphors of the Internet in the speech of computer users]. Man and language in the communicative space: collection of scientific articles. Krasnoyarsk, 145–150. (In Russ.) Karasik, V. I. (2018). Setevoy diskurs: zhanrovye harakteristiki. Yazyk. Tekst. Diskurs [Network discourse: genre characteristics]. In G. Manayenko (Ed.), Language. Text. Discourse. Stavropol, (152–167). [In Russ.]. Kondratyeva, O. N. (2019). Metafory v mediadiskurse kak sposob interpretatsii predmetnoi oblasti ‘sotsialnye seti’ [Metaphors in the Mediadiscourse as the Way of Interpreting the new Subject Domain (оn an Example of a Subject Domain «Social Network»]. Issues of cognitive linguistics, 2, 85–94. https://doi.org/10.20916/1812-3228-2019-2-85-94 [In Russ.] 715 https://doi.org/10.15405/epsbs.2020.08.84 Corresponding Author: Olga Kondratyeva Selection and peer-review under responsibility of the Organizing Committee of the conference eISSN: 2357-1330 Makeeva, M. N., & Borodulina, N. Yu. (2017). Kognitivnyi aspect ispolzovaniya metafory prostranstva v internete [Cognitive aspect of the use of space metaphor on the Internet]. Filologicke vedomosti, 3, 8–15. Retrieved from http: // sociosphera.com/files/conference /2017/Filologicke_vedomosti_3- 17/8-15_m_n_makeeva_n_yu_borodulina.pdf [In Russ.]. Makeeva, M. N., & Borodulina, N. Yu. (2018). https://doi.org/10.15405/epsbs.2020.08.84 Corresponding Author: Olga Kondratyeva Selection and peer-review under responsibility of the Organizing Committee of the conference eISSN: 2357-1330 References Metafora ‘Internet-vodnoe prostranstvo’ v pechatnyh SMI (na osnove content-analiza resursov natsionalnogo korpusa russkogo yazyka) [Metaphor “Internet- water space” in print media] (based on content analysis of resources of the national corps of the Russian language)]. Issues of cognitive linguistics, 2, 13–20. https://doi.org/10.20916/1812-3228- 2018-2-13-20 [In Russ.]. Pavlov, A. M. (2017). Ponyatie setevogo diskursa v sovremennoi longvistike [The concept of network discourse in modern linguistics]. Science and Education: a new time. Scientific and methodical journal, 1(2), 9–13. [In Russ.]. Prokofiev, A. V. (2017). Razvitie diskursa Internet-prostranstva I osobennosti ego yazykovoi organizatsii [Development of the discourse of the Internet space at the present stage and features of its linguistic organization]. Bulletin of the Moscow Regional State University. Series: Linguistics, 5, 85–96. https://doi.org/10.18384/2310-712X-2017-5-85-96 [In Russ.]. Prokofiev, A. V. (2017). Razvitie diskursa Internet-prostranstva I osobennosti ego yazykovoi organizatsii [Development of the discourse of the Internet space at the present stage and features of its linguistic organization]. Bulletin of the Moscow Regional State University. Series: Linguistics, 5, 85–96. https://doi.org/10.18384/2310-712X-2017-5-85-96 [In Russ.]. omaszewski, Z. (2011). Conceptual Metaphors of the World Wide Web Retrieved from http://www2. hawaii.edu/~ztomasze/ling440/webmetaphors.html Tomaszewski, Z. (2011). Conceptual Metaphors of the World Wide Web Retrieved from http://www hawaii.edu/~ztomasze/ling440/webmetaphors.html oiskunsky, A. E. (2001). Metafory Interneta [Metaphors of the Internet]. Issues of Philosophy, 11, 64- 79. [In Russ.]. Yazyk i rech' v internete: lichnost', obshchestvo, kommunikatsiya, kul'tura [Language and speech on the Internet: personality, society, communication, culture] (2018). A.V. Dolzhikova, V.V. Barabash (Ed.). Articles of the II International Scientific and Practical Conference. Moscow. Yazyk i rech' v internete: lichnost', obshchestvo, kommunikatsiya, kul'tura [Language and speech on the Internet: personality, society, communication, culture] (2018). A.V. Dolzhikova, V.V. Barabash (Ed.). Articles of the II International Scientific and Practical Conference. Moscow. 716
https://openalex.org/W2170536105	https://europepmc.org/articles/pmc3397854?pdf=render	English	null	ADP-ribosylation Factor 1 Protein Regulates Trypsinogen Activation via Organellar Trafficking of Procathepsin B Protein and Autophagic Maturation in Acute Pancreatitis	Journal of biological chemistry/The Journal of biological chemistry	2,012	cc-by	10,608	ADP-ribosylation Factor 1 Protein Regulates Trypsinogen Activation via Organellar Trafficking of Procathepsin B Protein and Autophagic Maturation in Acute Pancreatitis□ S p g for publication,November 30, 2011, and in revised form, April 24, 2012 Published, JBC Papers in Press,May 8, 2012, DOI 10.1074/ Lidiya Orlichenko‡, Donna B. Stolz§, Pawan Noel‡, Jaideep Behari‡, Shiguang Liu‡, and Vijay P. Singh‡1 From the Departments of ‡Medicine and §Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15213 Background: Autophagy and cathepsin B-mediated trypsin generation may be deleterious in acute pancreatitis. The role of ARF1 in the process is unknown. ARF1 in the process is unknown. Results: BFA-mediated ARF1 inhibition prevents caerulein-induced processing of procathepsin B and perturbs autophagic maturation. Conclusion: ARF1 dependent trafficking of procathepsin B and autophagic maturation result in trypsinogen activation Results: BFA-mediated ARF1 inhibition prevents caerulein-induced processing of procathepsin B and perturbs autophagic maturation. Conclusion: ARF1-dependent trafficking of procathepsin B and autophagic maturation result in trypsinogen activation. Significance: ARF1 plays a significant role in acute pancreatitis. Several studies have suggested that autophagy might play a deleterious role in acute pancreatitis via intra-acinar activation of digestive enzymes. The prototype for this phenomenon is cathepsin B-mediated trypsin generation. To determine the organellar basis of this process, we investigated the subcellular distribution of the cathepsin B precursor, procathepsin B. We found that procathepsin B is enriched in Golgi-containing microsomes, suggesting a role for the ADP-ribosylation (ARF)- dependent trafficking of cathepsin B. Indeed, caerulein treat- ment increased processing of procathepsin B, whereas a known ARF inhibitor brefeldin A (BFA) prevented this. Similar treat- ment did not affect processing of procathepsin L. BFA-mediated ARF1 inhibition resulted in reduced cathepsin B activity and consequently reduced trypsinogen activation. However, forma- tion of light chain 3 (LC3-II) was not affected, suggesting that BFA did not prevent autophagy induction. Instead, sucrose den- sity gradient centrifugation and electron microscopy showed that BFA arrested caerulein-induced autophagosomal matura- tion. Therefore, ARF1-dependent trafficking of procathepsin B and the maturation of autophagosomes results in cathepsin B-mediated trypsinogen activation induced by caerulein. during pancreatitis (6, 7). In addition, the formation of large heterogeneous vacuoles containing lysosomal and digestive enzymes in the post-Golgi compartment (8, 9) has previously been noted in pancreatitis. Acinar cells contain both procathepsin B (10), which has an N terminus inhibitory propeptide (11), and the glycosylated active forms of cathepsin B (12). Procathepsin B is known to inhibit cathepsin B activity via the propeptide (11). This work was supported by a startup package from the University of Pitts- burgh Department of Medicine (to V. P. S.). □ S This article contains supplemental Figs. 1–5. 1 To whom correspondence should be addressed: Dept. of Medicine, Division of Gastroenterology, Hepatology and Nutrition, University of Pittsburgh, 566, Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261. Tel.: 412-648-9573; Fax: 412-383-8913; E-mail: singhv2@upmc.edu. 2 The abbreviations used are: ARF, ADP-ribosylation factor; BFA, brefeldin A; CXCL, CXC ligand; LC3, microtubule-associated protein light chain 3; GTPS, guanosine 5-3-O-(thio)triphosphate; Z, benzyloxycarbonyl; MCA, 4-methyl-coumarin-7-amide; CER, caerulein; WB, Western blot. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 29, pp. 24284–24293, July 13, 2012 © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 29, pp. 24284–24293, July 13, 2012 © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 29, pp. 24284–24293, July 13, 2012 © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. EXPERIMENTAL PROCEDURES When indicated, acini were treated with 50 M BFA at 37 °C for 2 h, whereas controls were kept in the same conditions without BFA. Results of in vitro studies are reported as averages from at least three independent experiments. g p Sucrose Gradient Centrifugation—Fractions were prepared from mouse pancreata homogenized in 0.3 M sucrose and spun in a ST 40R centrifuge (Thermo Fisher Scientific Inc.) as described previously (30). The zymogen fraction was separated from the postnuclear supernatant after centrifugation at 3,000 g for 10 min. Mitochondrial and microsomal fractions were precipitated from zymogen supernatant at 7,800 g and 40,000 rpm, respectively. The cytosolic fraction represented the supernatant of the microsomal fraction. For Golgi fractions analysis, purified microsomes were loaded onto 2.5 M sucrose gradient cushion according to previously described protocol in Ref. 30. The individual submicrosomal sucrose gradient frac- tions were collected from the top of the tube and boiled in 1 Laemmli buffer before Western blot analysis. p Western Blotting, RT-PCR, and Real-time PCR—Proteins were extracted from pancreas or acini after homogenization with a Potter-Elvehjem homogenizer in homogenization buffer containing 50 mM Tris at pH 7.2, 150 mM NaCl, 0.5 mM EDTA, 1 mM EGTA, 2 mM dithiothreitol, 1 mM Na3VO4, 25 mM NaF, 1% Nonidet P-40, and Complete protease inhibitor mixture (Roche Diagnostics). Lysates were boiled in 1 Laemmli sam- ple buffer before Western blot analysis according to standard procedures as described previously (23) using the following antibodies: GM130, p115, and syntaxin 6 (BD Transduction Laboratories), ARF1 and ARF6 (Thermo Fisher Scientific Inc.), LC3 (Cell Signaling Technology, Boston, MA), cathepsin B (Sigma-Aldrich), cathepsin L and cathepsin B antibody raised against its propeptide domain (Abcam, Cambridge, MA), chy- motrypsin (Millipore, Billerica, MA), and actin (Santa Cruz Biotechnology Inc., Santa Cruz, CA). Polyclonal antibody against pancreatic lipases was a kind gift from Dr. Mark Lowe (University of Pittsburgh). RNA from cells or tissue was isolated using TRIzol reagent (Invitrogen), and real-time PCR was con- ducted as described previously (24) using proprietary primers for CXCL1, CXCL2, TNF-, and 18S RNA from Applied Bio- systems (Invitrogen). Immunofluorescence Microscopy—Immunofluorescence mi- croscopy was done on pancreatic tissue cryosections after embedding in Tissue-Tek OCT (optimal cutting temperature compound) (Sakura Finetek USA, Inc., Torrance, CA) or on acini plated on plain glass coverslips. These were fixed with 2% paraformaldehyde and processed as described previously (23, 31). ADP-ribosylation Factor 1 Protein Regulates Trypsinogen Activation via Organellar Trafficking of Procathepsin B Protein and Autophagic Maturation in Acute Pancreatitis□ S Removal of this procathepsin B propeptide and subsequent activation to cathepsin B is secured in acidic compartments (13) only after procathepsin B glycosylation and sorting out in the trans-Golgi network. Although the co-localization of cathepsin B with tryp- sinogen in the autophagosome-like vacuoles was hypothesized to play an important role in pancreatitis (10, 14, 15) and studies have shown that autophagy is essential to the generation of intracellular trypsin (16), the regulation of procathepsin B traf- ficking in this phenomenon and how the organelles involved in trypsinogen activation contribute to autophagosome formation or maturation are unknown. Golgi membranes seem to be involved in autophagosome maturation (17) and, by extension, proteins involved in recruit- ing adaptor proteins and lipid-modifying enzymes to the Golgi, such as ARFs2 may be involved in this. BFA is a noncompetitive specific inhibitor of ARF GTPases (18, 19) and prevents GTP exchange mediated by the guanine nucleotide exchange factors of ARFs. The function of ARF1 in recruiting adaptor proteins and lipid-modifying enzymes on the Golgi is well established (20), whereas ARF6 is typically active on the cell surface (21), regulating phenomena such as endocytosis. Because previous studies showed that BFA disrupts Golgi transport and disman- tles the Golgi stacks in pancreatic acinar cells (22), we chose to mechanistically study the organellar basis of procathepsin B processing in trypsinogen activation along with post-Golgi trafficking in the progression of autophagy during acute pancreatitis. Chiari (1) proposed more than a century ago that pancreatitis is an autodigestive disease. Several models of acute pancreatitis have shown intra-acinar activation of the zymogen trypsinogen to trypsin as playing an important role in disease pathogenesis (2–4). This process is thought to be important in pancreatitis because trypsin is a “master activator” of the digestive enzyme cascade. The role of lysosomal proteases in the pathogenesis of pancreatitis is supported by studies showing a marked increase in activity of the lysosomal enzyme cathepsin B (which is known to activate trypsinogen to trypsin (5)) in the zymogen fraction 24284 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 29•JULY 13, 2012 EXPERIMENTAL PROCEDURES and measured overall cathepsin B or cathepsin L activities irre- spective of the isoforms. Amylase release into the medium was measured as described previously (4, 26). Effects of procathep- sin B propeptide (amino acids 25–60) (Bachem, Torrance, CA) on cathepsin B activity were studied by preincubating various concentrations of the peptide with acinar lysates for 15 min as described previously (27). Animals and Animal Procedures—8-week-old CD-1 mice or 80–100-gm Sprague-Dawley rats were purchased from Charles River Laboratories (Wilmington, MA) and housed with a 12-h light/dark cycle, at temperatures from 21 to 25 °C, fed standard laboratory chow, and allowed to drink ad libitum. Animals were acclimatized for at least 2 days before use. Caerulein and the cathepsin B substrate (Z-Arg-Arg-MCA) were pur- chased from Bachem (King of Prussia, PA). Specific antibodies and reagent sources are described below. All other reagents and chemicals were purchased from Sigma. Animal experiments were approved by the Institutional Animal Care and Use Com- mittee of the University of Pittsburgh (Pittsburgh, PA). p y Models of Pancreatitis—Pancreatitis was induced in rats and mice using caerulein. Mice were given hourly injections (10) of caerulein (50 mcg/kg) intraperitoneally and sacrificed 1 h after the last injection as described previously (4, 28). Pancrea- titis was induced in rats with a single dose of 20 mcg/kg of caerulein given intraperitoneally as described previously (23). Rats were sacrificed 6 h after induction of pancreatitis. Blood and pancreas were harvested as described previously (4, 28). BFA (25 mg/kg) or its vehicle (0.1 ml, 50% dimethyl sulfoxide (DMSO)) was administered by intraperitoneal injection 1 h before the induction of pancreatitis as described previously (29). Animal studies included 6–8 rodents per experimental group. Preparation and Use of Pancreatic Acini—Acini were har- vested as described previously (23) and stored at 4 °C until fur- ther use. Viability before use was more than 95%, as indicated by trypan blue exclusion. When indicated, acini were treated with 50 M BFA at 37 °C for 2 h, whereas controls were kept in the same conditions without BFA. Results of in vitro studies are reported as averages from at least three independent experiments. Preparation and Use of Pancreatic Acini—Acini were har- vested as described previously (23) and stored at 4 °C until fur- ther use. Viability before use was more than 95%, as indicated by trypan blue exclusion. EXPERIMENTAL PROCEDURES A and B, WBs of ARF1 (A) and ARF6 (B) pulldowns from pancreatic acini after treatment with 100 nM CER at the indicated times show basal activity (Bas) and an increase in ARF1 but not ARF6 activation. BFA (50 M) blocks CER-induced ARF1 activation (BFA, 1B). C, no activation of ARF6 was noted. The positive control GTPS, but not GDP, induces both ARF1 and ARF6 activation. Dual Regulation of Trypsinogen Activation by ARF1 Dual Regulation of Trypsinogen Activation by ARF1 Dual Regulation of Trypsinogen Activation by ARF1 FIGURE 1. BFA dismantles the Golgi by inhibiting ARF1 activation. A1–A4, immunostaining of pancreatic acini with antibodies against the Golgi marker, P115 (shown in green) and actin (shown in red) under basal conditions (A1) or treated with 100 nM caerulein (CER) alone for 15 min (A2); 50 M BFA for 2 h (A3); or BFA followed by CER treatment (A4). These changes demonstrate that BFA dismantles the Golgi without causing major changes in actin distribution. Arrows indicate basal relocation of the F-actin. Note that BFA did not prevent caerulein-induced blebbing (A2 and A4). A and B, WBs of ARF1 (A) and ARF6 (B) pulldowns from pancreatic acini after treatment with 100 nM CER at the indicated times show basal activity (Bas) and an increase in ARF1 but not ARF6 activation. BFA (50 M) blocks CER-induced ARF1 activation (BFA, 1B). C, no activation of ARF6 was noted. The positive control GTPS, but not GDP, induces both ARF1 and ARF6 activation. dependent phenomena, such as endocytosis (21, 34), which is also involved in trypsinogen activation (35, 36). Because cathepsin B can activate trypsinogen to trypsin (5), we exam- ined the effect of BFA on trafficking of cathepsin B and its pre- cursor procathepsin B. empty, those containing both dense and non-dense content, and those containing only dense granules. Statistical Analysis—All values are presented as means S.E. There were at least three independent experiments done for each all data presented from in vitro studies. Differences between two groups were analyzed by unpaired Student’s t test. A two-tailed p value of less than 0.05 was considered to indicate statistical significance. For electron microscopy analysis, differ- ences between two groups were compared using a Fisher’s exact test. Previous studies have shown an increase in cathepsin B activ- ity in the zymogen fraction to be associated with trypsinogen activation (4, 6, 10, 37, 38). EXPERIMENTAL PROCEDURES After blocking with 5% normal goat serum, tissue cryosec- tions were incubated with primary antibodies (GM130 1:200, p115 1:200, or ARF1 1:50) for 1 h at room temperature, washed, and incubated with secondary antibodies (Alexa Fluor 488- or Alexa Fluor 594-conjugated, diluted 1:500) rhodamine-conju- gated phalloidin (100 nM) (Invitrogen) with or without DRAQ5 (1:5000) for 30 min. After washing and mounting in ProLong Gold (Invitrogen), confocal imaging (1 m thick) was done using a Zeiss LSM510 confocal microscope (Thornwood, NJ), and processed using Adobe Photoshop 6.0 (Adobe Systems, Mountain View, CA). GST Pulldown Assays—Binding assays with the glutathione resin-bound GST-GGA3-PBD fusion protein were performed either on protein lysates from murine or rat pancreas or on isolated acini using active ARF1 (or ARF6) pulldown and detec- tion kit from Thermo Fisher Scientific Inc. Binding assays with GTPS or GDP served as an internal control according to the manufacturer’s protocol. Activity Assays—Cathepsin B activity was assayed spectro- fluorometrically at 38 °C in 0.1 M sodium phosphate buffer (pH 6.0) containing 5 mM dithiothreitol and EDTA using the sub- strate Z-Arg-Arg-MCA (25). Trypsin was measured in acinar cell homogenates as described previously (4, 7). Cathepsin L activity was measured according to Wartmann et al. (40). Both cathepsin B and cathepsin L assays were normalized to DNA Electron Microscopy—Pancreatic tissue was cut into 2 m pieces immediately after harvesting, fixed in Karnovsky’s fixa- tive, and processed as described previously (32). Sections (70 nm) were imaged on a Jeol JEM-1011 transmission electron microscope. For quantification of autophagosome contents, all areas that could be imaged from each experimental group at 4,000 were taken, and autophagosomes were classified into JULY 13, 2012•VOLUME 287•NUMBER 29 JULY 13, 2012•VOLUME 287•NUMBER 29 24285 JOURNAL OF BIOLOGICAL CHEMISTRY FIGURE 1. BFA dismantles the Golgi by inhibiting ARF1 activation. A1–A4, immunostaining of pancreatic acini with antibodies against the Golgi marker, P115 (shown in green) and actin (shown in red) under basal conditions (A1) or treated with 100 nM caerulein (CER) alone for 15 min (A2); 50 M BFA for 2 h (A3); or BFA followed by CER treatment (A4). These changes demonstrate that BFA dismantles the Golgi without causing major changes in actin distribution. Arrows indicate basal relocation of the F-actin. Note that BFA did not prevent caerulein-induced blebbing (A2 and A4). EXPERIMENTAL PROCEDURES We found that 100 nM caerulein significantly increased cathepsin B activity in the zymogen frac- tion (p 0.05 versus basal; Fig. 2C), which was prevented by BFA (Fig. 2C). On Western blot analysis of acinar homogenates, however, supraphysiologic caerulein caused a reduction in the amount of active cathepsin B, i.e. its 25- and 29-kDa bands, in both mice (Fig. 2, D and E1) and rat acini (supplemental Fig. 1A) (39), although there was no change in overall cathepsin B activ- ity (Fig. 2E2). On the contrary, calculation of cathepsin B activ- ity per amount of protein showed a significant increase with 100 nM caerulein, and this increase was significantly blunted by BFA (280 37.4% versus 150 29% control, p 0.035) (Fig. 2E3). We also observed that caerulein caused complete processing of the 34-kDa procathepsin B that contains an inhibitory propep- tide (Fig. 2D) and predominantly localizes to the Golgi-contain- ing fraction under control conditions (Fig. 3B1). This caerulein- mediated procathepsin B processing to mature cathepsin B was prevented by BFA (Fig. 2D). Because this BFA-mediated reten- tion of procathepsin B was associated with a decrease in cathep- sin B activity (Fig. 2, C and E3), we tested to see whether the propeptide, which may be formed during the maturation of cathepsin B, may have an inhibitory role. The dose-dependent inhibition of cathepsin B activity by the propeptide fragment confirmed its inhibitory role and that of procathepsin B in our system (Fig. 2F). RESULTS ARF1 Inhibition Prevents Procathepsin B Processing and Trafficking from the Golgi, Reducing Caerulein-induced Tryp- sinogen Activation—The Golgi stacks under control or basal conditions were compact, with a crescent-like arrangement and no visible vesiculation (Fig. 1A1). Caerulein (100 nM) caused the Golgi to extend more apically in an antegrade fash- ion (i.e. toward the apical actin) (Fig. 1A2). Treatment of con- trol acini with BFA resulted in dismantling and vesiculation of the Golgi (Fig. 1A3) in resting acini, as described previously by Farquhar and co-workers (33). BFA reduced caerulein-induced antegrade Golgi extension (Fig. 1, A2 and A4). Although we observed constitutive ARF1 activity under basal conditions (Fig. 1B), which is consistent with the changes noted in Fig. 1A3 and by Farquhar and co-workers (33), a further increase in ARF1 activity was noted in acinar cells within 2 min of adding caerulein (Fig. 1B), although this effect was blocked by BFA. Caerulein did not activate ARF6 (Fig. 1C). BFA also prevented caerulein-induced trypsin generation in mice (Fig. 2A; 2.3 0.1 versus 1.0 0.1-fold control, p 0.01) and rat acini (Fig. 2B; 18.6 4.0 versus 10.3 1.3-fold control, p 0.04). Thus, through its inhibition of ARF1, BFA dismantled the Golgi stacks and prevented the caerulein-induced antegrade Golgi extension, resulting in reduced trypsinogen activation. These data suggest that BFA prevents trypsinogen activation via an ARF1-dependent, Golgi-based phenomena and not an ARF6- Recently, pharmacological inhibition of cathepsin L has been shown to increase the amount of active trypsin in rodent mod- els of acute pancreatitis (10) and prevent trypsinogen activation in humans (40). This suggested that an imbalance between VOLUME 287•NUMBER 29•JULY 13, 2012 24286 24286 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 29•JULY 13, 2012 Dual Regulation of Trypsinogen Activation by ARF1 Dual Regulation of Trypsinogen Activation by ARF1 FIGURE 2. BFA prevents trypsinogen activation and retains procathepsin B during treatment of pancreatic acini with 100 nM CER in vitro. A and B, trypsin activity assay in acinar cells after 30 min under CER stimulation demonstrates that BFA prevents CER-induced trypsinogen activation in both mouse (A) and rat acini (B). Data are presented as -fold change when compared with untreated acini (CON). RESULTS Histograms (E3) represent the quantification of the cathepsin B activity per the average protein content of the active, 25-kDa, and 29-kDa cathepsin B isoform under the same treatment condition as described in D from at least three independent experiments. The histograms show a dramatic increase in the cathepsin B activity/protein upon stimulation with CER, which is significantly inhibited by 50 M BFA. F, quantification of the inhibitory effect of procathepsin B peptide on cathepsin B activity in acinar cell lysates. indicates a significant (p 0.05) decrease in activity at the corresponding peptide concentration compared to the activity in its absence. cathepsin L and cathepsin B could play a role in intra-acinar trypsinogen activation in pancreatitis. We, therefore, com- pared the processing and maturation of procathepsin L with procathepsin B in acinar cells treated with 100 nM caerulein with or without BFA. Our experiments consistently demon- strated that although the amount of 34-kDa form of procathep- sin B was greatly reduced by supraphysiologic caerulein (Figs. 2D and Fig. 6B), equal amounts of the unprocessed 42-kDa form of procathepsin L were present in all acinar cell lysates regardless of the treatment (see Fig. 6C). There were no changes in the protein level of the mature 25-kDa form of cathepsin L (see Fig. 6C). Likewise, calculation of cathepsin L activity per amount of DNA showed no changes with 100 nM caerulein (see Fig. 6A) with or without BFA. Therefore, it is likely that BFA affects caerulein-induced trypsinogen activation via altering the processing of procathepsin B and not procathepsin L. caerulein released cathepsin B inhibition by removing its pro- peptide from the N terminus of procathepsin B. Interestingly, the early caerulein-induced trypsinogen activation was not accompanied by an increase in LC3-II (supplemental Fig. 1B), which is a marker of autophagic activity. BFA Inhibits ARF1 in Vivo and Reduces Trypsinogen Activa- tion and Severity of Caerulein-induced Pancreatitis without Preventing Induction of Autophagy—We next sought to deter- mine whether caerulein also activated ARF1, which regulates transport from the endoplasmic reticulum to the Golgi and between Golgi cisternae (41) during pancreatitis. Caerulein- induced pancreatitis at 6 h resulted in activation of ARF1 that was prevented by BFA (Fig. 3A). In control animals, when assessed by immunocytochemistry, ARF1 had a diffuse cyto- plasmic appearance (supplemental Fig. 2, B and C, ovals) that shifted to supranuclear areas under pancreatitis conditions (supplemental Fig. 2E, dashed ovals). RESULTS C, cathepsin B activity assayed under conditions described in A and B shows that BFA reduces cathepsin B activation quantified as the ratio of the cathepsin B activity in the zymogen (1,300 g) versus the lysosomal (12,000 g) fraction. BAS, basal activity. D, WB of cathepsin B isoforms present in pancreatic acini shows that the 34-kDa procathepsin B (CathB) isoform is presentintotalcelllysateunderbasalconditionandinacinipretreatedwithBFAfollowedbystimulationwithCERbutnotincellstreatedwithCERalone.E1–E3, densitometry (E1) of the protein bands from WB shown in D demonstrates the reduction in the amount of active 25- and 29-kDa cathepsin B (CB) isoforms present in acini under CER-treated conditions, whereas the activity of cathepsin B in total cell lysates remains unchanged (E2). Histograms (E3) represent the quantification of the cathepsin B activity per the average protein content of the active, 25-kDa, and 29-kDa cathepsin B isoform under the same treatment condition as described in D from at least three independent experiments. The histograms show a dramatic increase in the cathepsin B activity/protein upon stimulation with CER, which is significantly inhibited by 50 M BFA. F, quantification of the inhibitory effect of procathepsin B peptide on cathepsin B activity in acinar cell lysates. * indicates a significant (p 0.05) decrease in activity at the corresponding peptide concentration compared to the activity in its absence. Dual Regulation of Trypsinogen Activation by ARF1 FIGURE 2. BFA prevents trypsinogen activation and retains procathepsin B during treatment of pancreatic acini with 100 nM CER in vitro. A and B, trypsin activity assay in acinar cells after 30 min under CER stimulation demonstrates that BFA prevents CER-induced trypsinogen activation in both mouse (A) and rat acini (B). Data are presented as -fold change when compared with untreated acini (CON). C, cathepsin B activity assayed under conditions described in A and B shows that BFA reduces cathepsin B activation quantified as the ratio of the cathepsin B activity in the zymogen (1,300 g) versus the lysosomal (12,000 g) fraction. BAS, basal activity. D, WB of cathepsin B isoforms present in pancreatic acini shows that the 34-kDa procathepsin B (CathB) isoform is presentintotalcelllysateunderbasalconditionandinacinipretreatedwithBFAfollowedbystimulationwithCERbutnotincellstreatedwithCERalone.E1–E3, densitometry (E1) of the protein bands from WB shown in D demonstrates the reduction in the amount of active 25- and 29-kDa cathepsin B (CB) isoforms present in acini under CER-treated conditions, whereas the activity of cathepsin B in total cell lysates remains unchanged (E2). RESULTS To assess the distribution of zymogens (Zymo) and microsomes in subcellular fractions, the antibodies against chymotrypsin (Chymo), a protein marker of zymogen granules, and GM130, a Golgi marker, were used. Cathepsin B antibody was used to assess its localization in subcellular fractions under the different treatment conditions. PNS, postnuclear supernatant; Mito, mitochondrial fraction. C and D, trypsin activity assay done on pancreatic tissue from mouse (C) and rat (D) treated under conditions described in A and B1–B3. Histograms represent trypsin activity per DNA content in mouse and rat pancreatic homogenates. E, WB analysis of mouse pancreata showsaninductionofautophagyinmurinepancreasduring10-hCER-inducedpancreatitisthatisnotpreventedbyBFAtreatment.F,histogramsrepresentthe means of at least three densitometries of WB shown in E. pancreatitis; BFA also reduced the severity of rat caerulein pan- creatitis (data not shown). Interestingly, although BFA pre- vented the translocation of the zymogen marker chymotrypsin to the microsomal fraction (Fig. 3B2, discussed below), it did not prevent the induction of autophagy, as evidenced by the formation of LC3-II (Fig. 3, E and F). only electron-dense granules in both rat and mouse models (Fig. 5D and supplemental Fig. 3). High power electron microscopy analysis showed these empty vacuoles to have double membranes (supplemental Fig. 4), consistent with their autophagic origin. We independently verified these findings in subfractionation of microsomes. This revealed that although the zymogen marker chymotrypsin is absent, the endolysosomal marker syn- taxin 6 (42) enriches in fractions lighter than the Golgi marker GM130 under control conditions (Fig. 5F1). During pancreati- tis, syntaxin 6 co-localizes with GM130 and chymotrypsin (consistent with relocation of chymotrypsin noted in Fig. 3B2) in LC3-II-enriched fractions during caerulein-induced pancre- atitis (Fig. 5F2). This suggests that co-localization of digestive enzymes and lysosomal hydrolases (6, 9, 38, 43) occurs in mature autophagosomes during pancreatitis. As predicted by the morphological effects we saw with electron microscopy, BFA prevented the entry of the Golgi marker GM130 and chy- motrypsin into the LC3-II-rich compartments but did not ARF1 Inhibition Prevents Autophagic Maturation during Pancreatitis—Recent studies have shown that autophagy is needed for trypsinogen activation during pancreatitis (10, 16). Our observations above on the blunting effect of BFA on tryp- sinogen activation (Fig. 3, C and D) and severity of pancreatitis (Fig. 4, A–D) but not induction of autophagy (Fig. 3, E and F) prompted us to ask whether inhibiting ARF1 affected matura- tion of the autophagosomes. RESULTS We then studied the sub- cellular localization of procathepsin B, which normally concen- trates in the Golgi-enriched microsomal fraction (Fig. 3B1). Caerulein-induced ARF1 activation was associated with loss of procathepsin B from the microsomal fraction. This was pre- vented by BFA (Fig. 3B3), which also reduced trypsinogen acti- vation in both rat and mouse caerulein-induced pancreatitis (Fig. 3, C and D). BFA also reduced the parameters of local severity (i.e. necrosis (Fig. 4, A and B), edema (Fig. 4C), and serum amylase (Fig. 4D)) without affecting mRNA up-regula- tion of the inflammatory mediators TNF-, CXCL1, and CXCL2 in pancreatic tissue (Fig. 4, E1–E3) in mouse caerulein To better characterize the rapid maturation of procathepsin B caused by supraphysiologic caerulein, we assessed the cell homogenates from acini treated with 100 nM caerulein with or without BFA by Western blotting using an antibody raised against cathepsin B propeptide region. Our experimental data have shown that the loss of the 34-kDa form of procathepsin B was accompanied by an increase in the amount of the 6-kDa propeptide in the lysates stimulated with 100 nM caerulein when compared with untreated controls or lysates pretreated with BFA (see Fig. 6B). As cathepsin B activity per protein amount was significantly increased with supraphysiologic caerulein (Fig. 2E3), it is likely that the stimulation with 100 nM JULY 13, 2012•VOLUME 287•NUMBER 29 24287 JULY 13, 2012•VOLUME 287•NUMBER 29 JOURNAL OF BIOLOGICAL CHEMISTRY 24287 Dual Regulation of Trypsinogen Activation by ARF1 FIGURE 3. BFA blocks ARF1 activation, retains procathepsin B in the microsomal fraction, and prevents trypsinogen activation during caerulein- induced pancreatitis in vivo. A, WB of ARF1 pulldowns from mice with 6-h CER-induced acute pancreatitis using GST-GGA3-PBD fusion protein show an increase in ARF1 activation. The activation is completely blocked by BFA. CON, control. B1–B3, WBs of subcellular fractions from pancreas of control mice (B1), mice with 6-h CER-induced pancreatitis (B2, Ptts.), and mice pretreated with BFA during 6-h CER pancreatitis (B3, Ptts. BFA) show procathepsin B (ProcathB) being retained in a microsomal fraction (Micro) in control and BFA-treated mice but not in mice with 6-h pancreatitis. To assess the distribution of zymogens (Zymo) and microsomes in subcellular fractions, the antibodies against chymotrypsin (Chymo), a protein marker of zymogen granules, and GM130, a Golgi marker, were used. Cathepsin B antibody was used to assess its localization in subcellular fractions under the different treatment conditions. RESULTS PNS, postnuclear supernatant; Mito, mitochondrial fraction. C and D, trypsin activity assay done on pancreatic tissue from mouse (C) and rat (D) treated under conditions described in A and B1–B3. Histograms represent trypsin activity per DNA content in mouse and rat pancreatic homogenates. E, WB analysis of mouse pancreata showsaninductionofautophagyinmurinepancreasduring10-hCER-inducedpancreatitisthatisnotpreventedbyBFAtreatment.F,histogramsrepresentthe means of at least three densitometries of WB shown in E. Dual Regulation of Trypsinogen Activation by ARF1 FIGURE 3. BFA blocks ARF1 activation, retains procathepsin B in the microsomal fraction, and prevents trypsinogen activation during caerulein- induced pancreatitis in vivo. A, WB of ARF1 pulldowns from mice with 6-h CER-induced acute pancreatitis using GST-GGA3-PBD fusion protein show an increase in ARF1 activation. The activation is completely blocked by BFA. CON, control. B1–B3, WBs of subcellular fractions from pancreas of control mice (B1), mice with 6-h CER-induced pancreatitis (B2, Ptts.), and mice pretreated with BFA during 6-h CER pancreatitis (B3, Ptts. BFA) show procathepsin B (ProcathB) being retained in a microsomal fraction (Micro) in control and BFA-treated mice but not in mice with 6-h pancreatitis. To assess the distribution of zymogens (Zymo) and microsomes in subcellular fractions, the antibodies against chymotrypsin (Chymo), a protein marker of zymogen granules, and GM130, a Golgi marker, were used. Cathepsin B antibody was used to assess its localization in subcellular fractions under the different treatment conditions. PNS, postnuclear supernatant; Mito, mitochondrial fraction. C and D, trypsin activity assay done on pancreatic tissue from mouse (C) and rat (D) treated under conditions described in A and B1–B3. Histograms represent trypsin activity per DNA content in mouse and rat pancreatic homogenates. E, WB analysis of mouse pancreata showsaninductionofautophagyinmurinepancreasduring10-hCER-inducedpancreatitisthatisnotpreventedbyBFAtreatment.F,histogramsrepresentthe means of at least three densitometries of WB shown in E. FIGURE 3. BFA blocks ARF1 activation, retains procathepsin B in the microsomal fraction, and prevents trypsinogen activation during caerulein- induced pancreatitis in vivo. A, WB of ARF1 pulldowns from mice with 6-h CER-induced acute pancreatitis using GST-GGA3-PBD fusion protein show an increase in ARF1 activation. The activation is completely blocked by BFA. CON, control. B1–B3, WBs of subcellular fractions from pancreas of control mice (B1), mice with 6-h CER-induced pancreatitis (B2, Ptts.), and mice pretreated with BFA during 6-h CER pancreatitis (B3, Ptts. BFA) show procathepsin B (ProcathB) being retained in a microsomal fraction (Micro) in control and BFA-treated mice but not in mice with 6-h pancreatitis. RESULTS Electron microscopy showed that autophagosomal maturation culminated in the formation of compound vacuoles containing both electron-dense and non- dense components during caerulein-induced pancreatitis (Fig. 5, C and E). BFA significantly altered this process, with the predominant population of vacuoles being empty or containing VOLUME 287•NUMBER 29•JULY 13, 2012 24288 JOURNAL OF BIOLOGICAL CHEMISTRY 24288 FIGURE 4. BFA reduces severity of CER-induced pancreatitis without affecting inflammatory cytokine up-regulation. A1–A4, BFA reduces pancreatic necrosis. Pancreatic tissue from control mice (A1), mice with 10-h CER-induced pancreatitis (A2), and mice pretreated either with vehicle (A3) or with BFA (A4) was dissected for histology and quantified for the amount of necrotic cell death per total area of pancreatic tissue. Representative images are shown. B, histograms from quantified histology data described in A1–A4 show the decrease (p 0.01) in the amount of necrotic area in mice treated with BFA relative to vehicle control (CON). VEH, vehicle. C and D, BFA reduces pancreatic edema (C) and serum amylase (D) at the end of 10 h of pancreatitis. E1–E3, BFA does not decrease the severity of pancreatitis by interfering with the transcriptional regulation of TNF- (E1), CXCL1 (E2), or CXCL2 (E3) in the pancreas. Histograms represent the -fold increase in the amount of TNF-, CXCL1, and CXCL2 transcripts relative to control as assessed by real-time PCR. Dual Regulation of Trypsinogen Activation by ARF1 Dual Regulation of Trypsinogen Activation by ARF1 IGURE 4. BFA reduces severity of CER-induced pancreatitis without affecting inflammatory cytokine up-regulation. A1–A4, BFA reduces pancreatic necrosis. Pancreatic tissue from control mice (A1), mice with 10-h CER-induced pancreatitis (A2), and mice pretreated either with vehicle (A3) or with BFA (A4) was dissected for histology and quantified for the amount of necrotic cell death per total area of pancreatic tissue. Representative images are shown. B hi f ifi d hi l d d ib d i A1 A4 h h d ( 0 01) i h f i i i d i h BFA l i ces severity of CER-induced pancreatitis without affecting inflammatory cytokine up-regulation. A1–A4, BFA re FIGURE 4. BFA reduces severity of CER-induced pancreatitis without affecting inflammatory cytokine up-reg FIGURE 4. BFA reduces severity of CER-induced pancreatitis without affecting inflammatory cytokine up-regulation. A1–A4, BFA reduces pancreatic necrosis. RESULTS Pancreatic tissue from control mice (A1), mice with 10-h CER-induced pancreatitis (A2), and mice pretreated either with vehicle (A3) or with BFA (A4) was dissected for histology and quantified for the amount of necrotic cell death per total area of pancreatic tissue. Representative images are shown. B, histograms from quantified histology data described in A1–A4 show the decrease (p 0.01) in the amount of necrotic area in mice treated with BFA relative to vehicle control (CON). VEH, vehicle. C and D, BFA reduces pancreatic edema (C) and serum amylase (D) at the end of 10 h of pancreatitis. E1–E3, BFA does not decrease the severity of pancreatitis by interfering with the transcriptional regulation of TNF- (E1), CXCL1 (E2), or CXCL2 (E3) in the pancreas. Histograms represent the -fold increase in the amount of TNF-, CXCL1, and CXCL2 transcripts relative to control as assessed by real-time PCR. affect syntaxin 6 (Fig. 5, E and F3). Our studies also demonstrate that supraphysiologic caerulein affects neither cathepsin L activity nor the protein amount or its precursor procathepsin L, whereas it depletes procathepsin B (Fig. 6). activity by an unprocessed protein prosequence shown for the subtilisin family of proteases (45). During the later phase of pathogenic trypsinogen activation, at 6 h after caerulein, ARF1 is activated, promoting the forma- tion of double-membrane compound vacuoles containing both zymogen granules and microsomal components. In this phase, BFA prevented autophagic maturation at the step when the double-membrane autophagic vacuoles were empty or con- tained only zymogen granules. Fig. 7 presents a schema illus- trating these distinct phenomena. DISCUSSION F2, 6-h CER treatment induces co-localization of chymotrypsin, syntaxin 6, and LC3-II with the Golgi marker GM130 in heavy fractions (fractions 7–9) demonstrated by isopycnic centrifugation of pancreata from animals with pancreatitis. The WB shows a biochemical equivalent to the EM images in C. F3, BFA pretreatment results in changes in the autophagosomal density leading to a shift of LC3-II to the lighter sucrose density fractions with the loss of chymotrypsin co-localization with the Golgi marker GM130. This is consistent with the increase in empty autophagosomes noted by EM (D). FIGURE 5. BFA prevents autophagosomal maturation. A–D, representative electron microscopy images (10,000) of tissue sections from control pancreas (A), pancreas of BFA-treated rat (B), rat with 6-h CER pancreatitis (C), and one with 6-h pancreatitis pretreated with BFA (D). C, pancreatitis results in autopha- gosomes containing both dense (zymogen dense-like) and non-dense granule components. D, BFA results in several of them being empty. E, the table shows quantification of the autophagosomal contents measured on electron microscopy images. There is a significant increase in the number of empty autophago- somes (p 0.03) and autophagosomes containing only dense granules (gran., p 0.001) in the CER BFA group when compared with the CER group. F1–F3, WBs of submicrosomal fractions from control mice (F1), mice with 6-h CER-induced pancreatitis (F2), and pretreated with BFA before CER pancreatitis (F3). F1, WB of pancreatic lysates after their isopycnic centrifugation in 20% (fraction 4) to 50% (fractions 9 and 10) sucrose gradient demonstrate the distribution of zymogen marker chymotrypsin (Chymo), the endolysosomal marker syntaxin 6, the autophagosomal marker LC3-II, and the Golgi marker GM130 under control conditions. F2, 6-h CER treatment induces co-localization of chymotrypsin, syntaxin 6, and LC3-II with the Golgi marker GM130 in heavy fractions (fractions 7–9) demonstrated by isopycnic centrifugation of pancreata from animals with pancreatitis. The WB shows a biochemical equivalent to the EM images in C. F3, BFA pretreatment results in changes in the autophagosomal density leading to a shift of LC3-II to the lighter sucrose density fractions with the loss of chymotrypsin co-localization with the Golgi marker GM130. This is consistent with the increase in empty autophagosomes noted by EM (D). DISCUSSION Our studies show that trypsinogen activation during acute pancreatitis occurs by two different mechanisms. During the initial 30 min following caerulein administration, procathepsin B, which contains the inhibitory propeptide, undergoes in- creased processing independent of autophagy. The procathep- sin B maturation releases the inhibition of cathepsin B activity, leading to trypsinogen activation. In this phase, we observed that ARF1 inhibition by BFA dismantled the Golgi and pre- vented its antegrade extension (as would be induced by caeru- lein), leading to the retention of procathepsin B and its inhibi- tion of trypsinogen activation. The fact that the 34-kDa procathepsin B can inhibit cathepsin B activity has been previ- ously demonstrated in a study, where an addition of mature cathepsin B in catalytic amounts to a non-autoactivatable var- iant of proenzyme did not result in the significant processing of procathepsin B (44). The concept is also supported by an early finding of intermolecular in trans complementation of protein Although caerulein-induced maturation of procathepsin B at 30 min may contribute to the increase in cathepsin B activity in the zymogen fraction (Fig. 1C) and subsequent trypsinogen activation, how this occurs, i.e. whether by accelerated process- ing or alternative trafficking of procathepsin B, remains to be determined. The accumulation of the 6-kDa propeptide, along with procathepsin B processing, and the increase in cathepsin B activity per amount of protein in response to supraphysiologic caerulein favors accelerated processing; however, the subcellu- lar organelles to which the propeptide is trafficked remain unknown. Similarly, the reasons for reduced cathepsin B amounts in acinar lysates despite caerulein-induced matura- tion of procathepsin B remain to be explored. Although this JULY 13, 2012•VOLUME 287•NUMBER 29 24289 JOURNAL OF BIOLOGICAL CHEMISTRY 24289 JULY 13, 2012•VOLUME 287•NUMBER 29 Dual Regulation of Trypsinogen Activation by ARF1 Dual Regulation of Trypsinogen Activation by ARF1 FIGURE 5. BFA prevents autophagosomal maturation. A–D, representative electron microscopy images (10,000) of tissue sections from control pancreas (A), pancreas of BFA-treated rat (B), rat with 6-h CER pancreatitis (C), and one with 6-h pancreatitis pretreated with BFA (D). C, pancreatitis results in autopha- gosomes containing both dense (zymogen dense-like) and non-dense granule components. D, BFA results in several of them being empty. E, the table shows quantification of the autophagosomal contents measured on electron microscopy images. DISCUSSION There is a significant increase in the number of empty autophago- somes (p 0.03) and autophagosomes containing only dense granules (gran., p 0.001) in the CER BFA group when compared with the CER group. F1–F3, WBs of submicrosomal fractions from control mice (F1), mice with 6-h CER-induced pancreatitis (F2), and pretreated with BFA before CER pancreatitis (F3). F1, WB of pancreatic lysates after their isopycnic centrifugation in 20% (fraction 4) to 50% (fractions 9 and 10) sucrose gradient demonstrate the distribution of zymogen marker chymotrypsin (Chymo), the endolysosomal marker syntaxin 6, the autophagosomal marker LC3-II, and the Golgi marker GM130 under control conditions. F2, 6-h CER treatment induces co-localization of chymotrypsin, syntaxin 6, and LC3-II with the Golgi marker GM130 in heavy fractions (fractions 7–9) demonstrated by isopycnic centrifugation of pancreata from animals with pancreatitis. The WB shows a biochemical equivalent to the EM images in C. F3, BFA pretreatment results in changes in the autophagosomal density leading to a shift of LC3-II to the lighter sucrose density fractions with the loss of chymotrypsin co-localization with the Golgi marker GM130. This is consistent with the increase in empty autophagosomes noted by EM (D). FIGURE 5. BFA prevents autophagosomal maturation. A–D, representative electron microscopy images (10,000) of tissue sections from control pancreas (A), pancreas of BFA-treated rat (B), rat with 6-h CER pancreatitis (C), and one with 6-h pancreatitis pretreated with BFA (D). C, pancreatitis results in autopha- gosomes containing both dense (zymogen dense-like) and non-dense granule components. D, BFA results in several of them being empty. E, the table shows quantification of the autophagosomal contents measured on electron microscopy images. There is a significant increase in the number of empty autophago- somes (p 0.03) and autophagosomes containing only dense granules (gran., p 0.001) in the CER BFA group when compared with the CER group. F1–F3, WBs of submicrosomal fractions from control mice (F1), mice with 6-h CER-induced pancreatitis (F2), and pretreated with BFA before CER pancreatitis (F3). F1, WB of pancreatic lysates after their isopycnic centrifugation in 20% (fraction 4) to 50% (fractions 9 and 10) sucrose gradient demonstrate the distribution of zymogen marker chymotrypsin (Chymo), the endolysosomal marker syntaxin 6, the autophagosomal marker LC3-II, and the Golgi marker GM130 under control conditions. VOLUME 287•NUMBER 29•JULY 13, 2012 DISCUSSION Recent studies have shown that intra-acinar trypsinogen activation may not be due to a cathepsin B-mediated process alone and that the balance between cathepsin B and cathepsin L, as activating and inactivating enzymes, may determine the final level of trypsinogen activation (10, 40). In our experimen- tal settings, we observed neither overall changes in the cathep- sin L activity nor changes in amounts of the 42-kDa procathep- sin L or 25-kDa mature cathepsin L with supraphysiologic caerulein. Thus, cathepsin L did not replicate the increase in cathepsin B activity associated with the maturation of proca- thepsin B under the same conditions. Our data do not refute the role of cathepsin L in trypsinogen inactivation but clearly dem- onstrate that its processing is not affected by BFA. Therefore, the effect of BFA on trypsinogen activation results from its effect on the activity of cathepsin B as discussed above. may be due to cathepsin B degradation, secretion into the extra- cellular environment (46) remains a possibility. The dismantling of Golgi by BFA may be associated with trapping of procathepsin B in the rough endoplasmic reticu- lum, thus preventing procathepsin B maturation and subse- quent activation; this argues against a preformed mature com- partment (e.g. lysosomes) contributing to cathepsin B-induced trypsinogen activation. However, BFA did not fully prevent the caerulein-induced increase in cathepsin B activity (Fig. 2E3), so there may be other mechanisms, such as endocytosis (36), that contribute to early trypsinogen activation. Although ARF6 is known to regulate endocytosis (21), we did not detect any caerulein-induced activation of ARF6 in pancreatic acini at 2 and 30 min (Fig. 1C). Additionally, because BFA induced mor- phological changes in control acini (Fig. 1A3), there may be ongoing ARF-dependent Golgi trafficking in resting cells, as has been shown previously (33). The novel finding that BFA inhibition of ARF1 prevents autophagic maturation and trypsinogen activation noted at 6 h 24290 VOLUME 287•NUMBER 29•JULY 13, 2012 24290 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 29•JULY 13, 2012 FIGURE 6. Supraphysiologic caerulein affects neither cathepsin L activity nor protein amount or its precursor procathepsin L, whereas depleting procathepsin B. A, cathepsin L activity under the mentioned conditions shows no significant changes. Bas, basal activity. B, WB of lysates from murine pancreatic acini treated for 30 min with 0.1 M caerulein (0.1 M CER) shows the depletion of 34-kDa procathepsin B, which is prevented by 50 M BFA. Dual Regulation of Trypsinogen Activation by ARF1 Supraphysiologic caerulein affects neither cathepsin L activity nor protein amount or its precursor procathepsin L, whereas depleting procathepsin B. A, cathepsin L activity under the mentioned conditions shows no significant changes. Bas, basal activity. B, WB of lysates from murine pancreatic acini treated for 30 min with 0.1 M caerulein (0.1 M CER) shows the depletion of 34-kDa procathepsin B, which is prevented by 50 M BFA. Concomitant with the depletion of procathepsin B (proCath B) by 0.1 M caerulein, its propeptide appears in the acinar cell homogenates in the form of a 6-kDa fragment. Lipase is shown as a loading control. Cath B, cathepsin B. C, WB of lysates from murine pancreatic acini treated for 30 min with 0.1 M caerulein shows that this does not induce depletion of 42-kDa procathepsin L or change the amounts of mature (25 kDa) cathepsin L (Cath L). Supraphysiologic caerulein induces numerous deleterious phenomena other than autophagy in acinar cells, including actin reorganization, blebbing (23, 31), mitochondrial depolar- ization (28, 55, 56), sustained elevations in cytosolic calcium (55), and generation of inflammatory mediators (24, 57). In our studies, BFA did not prevent up-regulation of inflammatory mediator mRNA (i.e. TNF-) or of the neutrophil attractant chemokines CXCL1 or CXCL2 and did not prevent F-actin localization or blebbing or change the pattern of caerulein-in- duced amylase secretion (supplemental Fig. 5). These findings indicate that BFA acted selectively on the processing of proca- thepsin B and the organellar trafficking events described above. Although BFA could indeed have affected similar phenomena in other cells, including inflammatory cells involved in pancre- atitis, the acinar cell-specific role of ARF1 in regulating autophagic maturation and the severity of pancreatitis would remain to be determined when such animals are available because genetic deletion of ARF1 (Knockout Mouse Project Repository) or ARF6 (58) is embryonically lethal, and cultured acinar cells that may be adenovirally transfected with ARFs or their mutants do not activate trypsinogen (24), whereas main- taining polarity. FIGURE 7. Two distinct pathways regulate trypsinogen activation. During the first 30 min after supraphysiologic caerulein administration, ARF1 inhibi- tion by BFA results in the retention of the inhibitory procathepsin B (shown in red circles) and consequent reduction of cathepsin B (shown in green circles) activity. 6 h later, after autophagy induction, autophagy maturation is arrested, and consequent trypsin generation is prevented by ARF1 inhibition. PAS, Pre-autophagosomal structure. FIGURE 7. DISCUSSION Concomitant with the depletion of procathepsin B (proCath B) by 0.1 M caerulein, its propeptide appears in the acinar cell homogenates in the form of a 6-kDa fragment. Lipase is shown as a loading control. Cath B, cathepsin B. C, WB of lysates from murine pancreatic acini treated for 30 min with 0.1 M caerulein shows that this does not induce depletion of 42-kDa procathepsin L or change the amounts of mature (25 kDa) cathepsin L (Cath L). Dual Regulation of Trypsinogen Activation by ARF1 late trypsin generation during pancreatitis. Although recent studies suggest a role for autophagy (16) or its derangement (10) in the generation of intracellular trypsin, the induction of autophagy (e.g. by fasting (47) or cessation of camostat feeding (48)) results in pancreatic atrophy (47) but not in pancreatitis or the generation of trypsin (10). Interestingly, Nevalainen and Janigan (47) did not find zymogen granules in autophagosomes generated during fasting. Similarly, although LAMP-2-defi- cient mice, which have impaired lysosomal-autophagosomal fusion, experience massive autophagy and atrophy of the pan- creas (49), pancreatitis has not been documented during the lifespan of these mice. At 6 h of caerulein-induced acute pan- creatitis, however, we see the formation of mature autophago- somes and the generation of trypsin, concurrent with pancrea- titis (4, 50). Our data therefore suggest that autophagic maturation is required for late trypsin generation during pan- creatitis and that induction of autophagy alone is not sufficient for this. Although the pathologic role of trypsin generated during pancreatitis has been argued (51), the reduction in morphologic changes of necrosis by BFA (Fig. 4, A and B) supports the role of active intracellular trypsin generated as a consequence of autophagy contributing to pancreatic necrosis in this study. Recent studies showing necrosis in mice with acinar cells car- rying an autoactivating trypsinogen (52) support this phenom- enon. This is additionally supported by the finding that SPINK3 (the murine homolog of human SPINK1) knock-out mice have massive autophagy (53), generate more trypsin (54), and have elevated serum amylase. FIGURE 6. Supraphysiologic caerulein affects neither cathepsin L activity nor protein amount or its precursor procathepsin L, whereas depleting procathepsin B. A, cathepsin L activity under the mentioned conditions shows no significant changes. Bas, basal activity. B, WB of lysates from murine pancreatic acini treated for 30 min with 0.1 M caerulein (0.1 M CER) shows the depletion of 34-kDa procathepsin B, which is prevented by 50 M BFA. Concomitant with the depletion of procathepsin B (proCath B) by 0.1 M caerulein, its propeptide appears in the acinar cell homogenates in the form of a 6-kDa fragment. Lipase is shown as a loading control. Cath B, cathepsin B. C, WB of lysates from murine pancreatic acini treated for 30 min with 0.1 M caerulein shows that this does not induce depletion of 42-kDa procathepsin L or change the amounts of mature (25 kDa) cathepsin L (Cath L). FIGURE 6. REFERENCES L., and Saluja, A. K. (2002) Thermal stress-induced HSP70 mediates protection against intrapancreatic trypsinogen activation and acute pan- creatitis in rats. Gastroenterology 122, 156–165 7. Saluja, A. K., Donovan, E. A., Yamanaka, K., Yamaguchi, Y., Hofbauer, B., and Steer, M. L. (1997) Cerulein-induced in vitro activation of trypsinogen in rat pancreatic acini is mediated by cathepsin B. Gastroenterology 113, 304–310 27. Chagas, J. R., Ferrer-Di Martino, M., Gauthier, F., and Lalmanach, G. (1996) Inhibition of cathepsin B by its propeptide: use of overlapping peptides to identify a critical segment. FEBS Lett. 392, 233–236 8. Hiraoka, T., Watanabe, E., Mochinaga, M., Tashiro, S., Miyauchi, Y., Na- kamura, I., and Yokoyama, I. (1984) Intraoperative irradiation combined with radical resection for cancer of the head of the pancreas. World J. Surg. 8, 766–771 28. Singh, V. P., Bren, G. D., Algeciras-Schimnich, A., Schnepple, D., Navina, S., Rizza, S. A., Dawra, R. K., Saluja, A. K., Chari, S. T., Vege, S. S., and Badley, A. D. (2009) Nelfinavir/ritonavir reduces acinar injury but not inflammation during mouse caerulein pancreatitis. Am. J. Physiol. Gastro- intest. Liver Physiol 296, G1040–G1046 9. Saito, I., Hashimoto, S., Saluja, A., Steer, M. L., and Meldolesi, J. (1987) Intracellular transport of pancreatic zymogens during caerulein supra- maximal stimulation. Am. J. Physiol. 253, G517–G526 29. Sausville, E. A., Duncan, K. L., Senderowicz, A., Plowman, J., Randazzo, P. A., Kahn, R., Malspeis, L., and Grever, M. R. (1996) Antiproliferative effect in vitro and antitumor activity in vivo of brefeldin A. Cancer J. Sci. Am. 2, 52–58 10. Mareninova, O. A., Hermann, K., French, S. W., O’Konski, M. S., Pandol, S. J., Webster, P., Erickson, A. H., Katunuma, N., Gorelick, F. S., Gukovsky, I., and Gukovskaya, A. S. (2009) Impaired autophagic flux mediates acinar cell vacuole formation and trypsinogen activation in rodent models of acute pancreatitis. J. Clin. Invest. 119, 3340–3355 30. Saraste, J., Palade, G. E., and Farquhar, M. G. (1986) Temperature-sensi- tive steps in the transport of secretory proteins through the Golgi complex in exocrine pancreatic cells. Proc. Natl. Acad. Sci. U.S.A. 83, 6425–6429 11. Fox, T., de Miguel, E., Mort, J. S., and Storer, A. C. (1992) Potent slow- binding inhibition of cathepsin B by its propeptide. Biochemistry 31, 12571–12576 31. Torgerson, R. R., and McNiven, M. A. (1998) The actin-myosin cytoskel- eton mediates reversible agonist-induced membrane blebbing. J. Cell Sci. 111, 2911–2922 12. Caglic, D., Pungercar, J. REFERENCES 21. Donaldson, J. G. (2003) Multiple roles for Arf6: sorting, structuring, and signaling at the plasma membrane. J. Biol. Chem. 278, 41573–41576 1. Chiari, H. (1906) About the relationship between the pancreas and fat necrosis. Zbl. Pathol. 17, 798–799 2. Waldthaler, A., Schütte, K., and Malfertheiner, P. (2010) Causes and mechanisms in acute pancreatitis. Dig. Dis. 28, 364–372 22. Hendricks, L. C., McClanahan, S. L., McCaffery, M., Palade, G. E., and Farquhar, M. G. (1992) Golgi proteins persist in the tubulovesicular rem- nants found in brefeldin A-treated pancreatic acinar cells. Eur. J. Cell Biol. 58, 202–213 3. Halangk, W., Lerch, M. M., Brandt-Nedelev, B., Roth, W., Ruthenbuerger, 3. Halangk, W., Lerch, M. M., Brandt-Nedelev, B., Roth, W., Ruthenbuerger, M., Reinheckel, T., Domschke, W., Lippert, H., Peters, C., and Deussing, J. (2000) Role of cathepsin B in intracellular trypsinogen activation and the onset of acute pancreatitis. J. Clin. Invest. 106, 773–781 23. Singh, V. P., and McNiven, M. A. (2008) Src-mediated cortactin phospho- rylation regulates actin localization and injurious blebbing in acinar cells. Mol. Biol. Cell 19, 2339–2347 4. Singh, V. P., Saluja, A. K., Bhagat, L., van Acker, G. J., Song, A. M., Soltoff, S. P., Cantley, L. C., and Steer, M. L. (2001) Phosphatidylinositol 3-kinase- dependent activation of trypsinogen modulates the severity of acute pan- creatitis. J. Clin. Invest. 108, 1387–1395 24. Orlichenko, L. S., Behari, J., Yeh, T. H., Liu, S., Stolz, D. B., Saluja, A. K., and Singh, V. P. (2010) Transcriptional regulation of CXC-ELR chemokines KC and MIP-2 in mouse pancreatic acini. Am. J. Physiol. Gastrointest. Liver Physiol 299, G867–G876 5. Figarella, C., Miszczuk-Jamska, B., and Barrett, A. J. (1988) Possible lyso- somal activation of pancreatic zymogens. Activation of both human trypsinogens by cathepsin B and spontaneous acid. Activation of human trypsinogen 1. Biol. Chem. Hoppe Seyler 369, (suppl.) 293–298 25. Mach, L., Mort, J. S., and Glössl, J. (1994) Maturation of human proca- thepsin B: proenzyme activation and proteolytic processing of the precur- sor to the mature proteinase, in vitro, are primarily unimolecular pro- cesses. J. Biol. Chem. 269, 13030–13035 6. Van Acker, G. J., Weiss, E., Steer, M. L., and Perides, G. (2007) Cause-effect relationships between zymogen activation and other early events in secre- tagogue-induced acute pancreatitis. Am. J. Physiol. Gastrointest. Liver Physiol. 292, G1738–G1746 26. Bhagat, L., Singh, V. P., Song, A. M., van Acker, G. J., Agrawal, S., Steer, M. REFERENCES R., Pejler, G., Turk, V., and Turk, B. (2007) Gly- cosaminoglycans facilitate procathepsin B activation through disruption of propeptide-mature enzyme interactions. J. Biol. Chem. 282, 33076–33085 32. Stolz, D. B., Ross, M. A., Salem, H. M., Mars, W. M., Michalopoulos, G. K., and Enomoto, K. (1999) Cationic colloidal silica membrane perturbation as a means of examining changes at the sinusoidal surface during liver regeneration. Am. J. Pathol. 155, 1487–1498 13. Mort, J. S., and Buttle, D. J. (1997) Cathepsin B. Int. J. Biochem. Cell Biol. 29, 715–720 33. Hendricks, L. C., McClanahan, S. L., Palade, G. E., and Farquhar, M. G. (1992) Brefeldin A affects early events but does not affect late events along the exocytic pathway in pancreatic acinar cells. Proc. Natl. Acad. Sci. U.S.A. 89, 7242–7246 14. Hofbauer, B., Saluja, A. K., Lerch, M. M., Bhagat, L., Bhatia, M., Lee, H. S., Frossard, J. L., Adler, G., and Steer, M. L. (1998) Intra-acinar cell activation of trypsinogen during caerulein-induced pancreatitis in rats. Am. J. Physiol. 275, G352–G362 34. Naslavsky, N., Weigert, R., and Donaldson, J. G. (2003) Convergence of non-clathrin- and clathrin-derived endosomes involves Arf6 inactivation and changes in phosphoinositides. Mol. Biol. Cell 14, 417–431 15. Steer, M. L., Meldolesi, J., and Figarella, C. (1984) Pancreatitis: the role of lysosomes. Dig. Dis. Sci. 29, 934–938 35. Raraty, M., Ward, J., Erdemli, G., Vaillant, C., Neoptolemos, J. P., Sutton, R., and Petersen, O. H. (2000) Calcium-dependent enzyme activation and vacuole formation in the apical granular region of pancreatic acinar cells. Proc. Natl. Acad. Sci. U.S.A. 97, 13126–13131 16. Hashimoto, D., Ohmuraya, M., Hirota, M., Yamamoto, A., Suyama, K., Ida, S., Okumura, Y., Takahashi, E., Kido, H., Araki, K., Baba, H., Miz- ushima, N., and Yamamura, K. (2008) Involvement of autophagy in tryp- sinogen activation within the pancreatic acinar cells. J. Cell Biol. 181, 1065–1072 36. Sherwood, M. W., Prior, I. A., Voronina, S. G., Barrow, S. L., Woodsmith, J. D., Gerasimenko, O. V., Petersen, O. H., and Tepikin, A. V. (2007) Ac- tivation of trypsinogen in large endocytic vacuoles of pancreatic acinar cells. Proc. Natl. Acad. Sci. U.S.A. 104, 5674–5679 17. Geng, J., and Klionsky, D. J. (2010) The Golgi as a potential membrane source for autophagy. Autophagy 6, 950–951 37. Willemer, S., Bialek, R., and Adler, G. (1990) Localization of lysosomal and digestive enzymes in cytoplasmic vacuoles in caerulein-pancreatitis. His- tochemistry 94, 161–170 18. Donaldson, J. Dual Regulation of Trypsinogen Activation by ARF1 Two distinct pathways regulate trypsinogen activation. During the first 30 min after supraphysiologic caerulein administration, ARF1 inhibi- tion by BFA results in the retention of the inhibitory procathepsin B (shown in red circles) and consequent reduction of cathepsin B (shown in green circles) activity. 6 h later, after autophagy induction, autophagy maturation is arrested, and consequent trypsin generation is prevented by ARF1 inhibition. PAS, Pre-autophagosomal structure. In summary, we demonstrate that ARF1 regulates trypsino- gen activation by two different mechanisms in two different phases: by procathepsin B processing during an early phase and by the transfer of microsomal contents into maturing autopha- gosomes during the later phase. The relevance of these mecha- nisms of trypsinogen activation to other diverse etiologies of pancreatitis, some of which are triggered by drugs that induce of pancreatitis reconciles the historically relevant phenomenon of co-localization during pancreatitis (6, 9, 38, 43) to a complex multistep process and demonstrates that this maturation resulting in the formation of macroautophagic vacuoles con- taining dense and microsomal components is required for this JULY 13, 2012•VOLUME 287•NUMBER 29 JULY 13, 2012•VOLUME 287•NUMBER 29 24291 JOURNAL OF BIOLOGICAL CHEMISTRY JULY 13, 2012•VOLUME 287•NUMBER 29 Dual Regulation of Trypsinogen Activation by ARF1 C. L. (1999) Brefeldin A acts to stabilize an abortive ARF-GDP-Sec7 do- main protein complex: involvement of specific residues of the Sec7 do- main. Mol. Cell 3, 275–285 C. L. (1999) Brefeldin A acts to stabilize an abortive ARF-GDP-Sec7 do- main protein complex: involvement of specific residues of the Sec7 do- main. Mol. Cell 3, 275–285 autophagy (e.g. L-asparaginase) (59), whereas others are caused by poorly understood mechanisms (e.g. alcohol), would be interesting to explore in the future. 20. Lippincott-Schwartz, J., Cole, N. B., and Donaldson, J. G. (1998) Building a secretory apparatus: role of ARF1/COPI in Golgi biogenesis and mainte- nance. Histochem. Cell Biol. 109, 449–462 VOLUME 287•NUMBER 29•JULY 13, 2012 Dual Regulation of Trypsinogen Activation by ARF1 M. (1989) Pancreatic duct obstruction in rabbits causes digestive zymogen and lysosomal enzyme colocalization. J. Clin. Invest. 84, 1260–1266 M. (1989) Pancreatic duct obstruction in rabbits causes digestive zymogen and lysosomal enzyme colocalization. J. Clin. Invest. 84, 1260–1266 (2000) Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2-deficient mice. Nature 406, 902–906 (2000) Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2-deficient mice. Nature 406, 902–906 50. Van Acker, G. J., Saluja, A. K., Bhagat, L., Singh, V. P., Song, A. M., and Steer, M. L. (2002) Cathepsin B inhibition prevents trypsinogen activation and reduces pancreatitis severity. Am. J. Physiol. Gastrointest. Liver Physiol 283, G794–800 39. Jane, D. T., Morvay, L., Dasilva, L., Cavallo-Medved, D., Sloane, B. F., and Dufresne, M. J. (2006) Cathepsin B localizes to plasma membrane caveolae of differentiating myoblasts and is secreted in an active form at physiolog- ical pH. Biol. Chem. 387, 223–234 51. Klöppel, G., Dreyer, T., Willemer, S., Kern, H. F., and Adler, G. (1986) Human acute pancreatitis: its pathogenesis in the light of immunocyto- chemical and ultrastructural findings in acinar cells. Virchows Arch. A Pathol. Anat. Histopathol. 409, 791–803 40. Wartmann, T., Mayerle, J., Kähne, T., Sahin-Tóth, M., Ruthenbürger, M., Matthias, R., Kruse, A., Reinheckel, T., Peters, C., Weiss, F. U., Sendler, M., Lippert, H., Schulz, H. U., Aghdassi, A., Dummer, A., Teller, S., Halangk, W., and Lerch, M. M. (2010) Cathepsin L inactivates human trypsinogen, whereas cathepsin L deletion reduces the severity of pancreatitis in mice. Gastroenterology 138, 726–737 52. Gaiser, S., Daniluk, J., Liu, Y., Tsou, L., Chu, J., Lee, W., Longnecker, D. S., Logsdon, C. D., and Ji, B. (2011) Intracellular activation of trypsinogen in transgenic mice induces acute but not chronic pancreatitis. Gut 60, 1379–1388 41. D’Souza-Schorey, C., and Chavrier, P. (2006) ARF proteins: roles in mem- brane traffic and beyond. Nat. Rev. Mol. Cell Biol. 7, 347–358 42. Wendler, F., and Tooze, S. (2001) Syntaxin 6: the promiscuous behavior of a SNARE protein. Traffic 2, 606–611 53. Ohmuraya, M., Hirota, M., Araki, M., Mizushima, N., Matsui, M., Mizu- moto, T., Haruna, K., Kume, S., Takeya, M., Ogawa, M., Araki, K., and Yamamura, K. (2005) Autophagic cell death of pancreatic acinar cells in serine protease inhibitor Kazal type 3-deficient mice. Gastroenterology 129, 696–705 43. Watanabe, O., Baccino, F. M., Steer, M. L., and Meldolesi, J. Dual Regulation of Trypsinogen Activation by ARF1 (1984) Supra- maximal caerulein stimulation and ultrastructure of rat pancreatic acinar cell: early morphological changes during development of experimental pancreatitis. Am. J. Physiol. 246, G457–G467 54. Ohmuraya, M., Hirota, M., Araki, K., Baba, H., and Yamamura, K. (2006) Enhanced trypsin activity in pancreatic acinar cells deficient for serine protease inhibitor Kazal type 3. Pancreas 33, 104–106 44. Mach, L., Schwihla, H., Stüwe, K., Rowan, A. D., Mort, J. S., and Glössl, J. (1993) Activation of procathepsin B in human hepatoma cells: the conver- sion into the mature enzyme relies on the action of cathepsin B itself. Biochem. J. 293, 437–442 55. Sutton, R., Criddle, D., Raraty, M. G., Tepikin, A., Neoptolemos, J. P., and Petersen, O. H. (2003) Signal transduction, calcium, and acute pancreati- tis. Pancreatology 3, 497–505 45. Zhu, X. L., Ohta, Y., Jordan, F., and Inouye, M. (1989) Pro-sequence of subtilisin can guide the refolding of denatured subtilisin in an intermolec- ular process. Nature 339, 483–484 56. Gukovskaya, A. S., Gukovsky, I., Jung, Y., Mouria, M., and Pandol, S. J. (2002) Cholecystokinin induces caspase activation and mitochondrial dysfunction in pancreatic acinar cells: roles in cell injury processes of pancreatitis. J. Biol. Chem. 277, 22595–22604 46. Hirano, T., Saluja, A., Ramarao, P., Lerch, M. M., Saluja, M., and Steer, M. L. (1991) Apical secretion of lysosomal enzymes in rabbit pancreas occurs via a secretagogue regulated pathway and is increased after pan- creatic duct obstruction. J. Clin. Invest. 87, 865–869 57. Grady, T., Liang, P., Ernst, S. A., and Logsdon, C. D. (1997) Chemokine gene expression in rat pancreatic acinar cells is an early event associated with acute pancreatitis. Gastroenterology 113, 1966–1975 47. Nevalainen, T. J., and Janigan, D. T. (1974) Degeneration of mouse pan- creatic acinar cells during fasting. Virchows Arch. B Cell Pathol. 15, 107–118 58. Suzuki, T., Kanai, Y., Hara, T., Sasaki, J., Sasaki, T., Kohara, M., Maehama, T., Taya, C., Shitara, H., Yonekawa, H., Frohman, M. A., Yokozeki, T., and Kanaho, Y. (2006) Crucial role of the small GTPase ARF6 in hepatic cord formation during liver development. Mol. Cell. Biol. 26, 6149–6156 48. Crozier, S. J., Sans, M. D., Lang, C. H., D’Alecy, L. G., Ernst, S. A., and Williams, J. A. (2008) CCK-induced pancreatic growth is not limited by mitogenic capacity in mice. Am. J. Physiol. Gastrointest. Liver Physiol 294, G1148–G1157 59. Bunpo, P., Dudley, A., Cundiff, J. K., Cavener, D. R., Wek, R. REFERENCES G., Finazzi, D., and Klausner, R. D. (1992) Brefeldin A inhib- its Golgi membrane-catalyzed exchange of guanine nucleotide onto ARF protein. Nature 360, 350–352 19. Peyroche, A., Antonny, B., Robineau, S., Acker, J., Cherfils, J., and Jackson, 38. Saluja, A., Saluja, M., Villa, A., Leli, U., Rutledge, P., Meldolesi, J., and Steer, 24292 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 29•JULY 13, 2012 VOLUME 287•NUMBER 29•JULY 13, 2012 VOLUME 287•NUMBER 29•JULY 13, 2012 Dual Regulation of Trypsinogen Activation by ARF1 C., and Anthony, T. G. (2009) GCN2 protein kinase is required to activate amino acid deprivation responses in mice treated with the anticancer agent L-as- paraginase. J. Biol. Chem. 284, 32742–32749 49. Tanaka, Y., Guhde, G., Suter, A., Eskelinen, E. L., Hartmann, D., Lüll- mann-Rauch, R., Janssen, P. M., Blanz, J., von Figura, K., and Saftig, P. JOURNAL OF BIOLOGICAL CHEMISTRY 24293 JOURNAL OF BIOLOGICAL CHEMISTRY 24293
https://openalex.org/W2040028481	https://europepmc.org/articles/pmc4224878?pdf=render	English	null	Analysis of hepatitis non‐treatment causes in a cohort of HCV and HCV/HIV infected patients	Journal of the International AIDS Society	2,014	cc-by	605	Analysis of hepatitis non-treatment causes in a cohort of HCV and HCV/HIV infected patients Pereira, Karen; Cla´udia Miranda, Ana; Baptista, Teresa; Peres, Susana; Antunes, Isabel; Ventura, Fernando; Borges, Fernando and Mansinho, Kamal Infectious Diseases, CHLO, E.P.E, Hospital de Egas Moniz, Lisboa, Portugal. Introduction: The decision to start hepatitis C virus (HCV) treatment and its timing remains controversial. As new treatment regimens are approved, it is essential to identify patients eligible for each regimen in a timed and tailored approach. This study aims to identify the reasons to defer treatment of chronic hepatitis C infection in both HCV and HCV/HIV infected patients. Material and Methods: Retrospective observational study of a cohort of HCV chronically infected patients with or without HIV infection, followed in an infectious disease clinic in Lisbon. Demographic, epidemiological, clinical, immunologic and virologic data were collected. Statistical analysis was performed with Microsoft Office†- Excel 2012. Kolmogorov-Smirnov, t-test, Chi-square and correlation analysis were performed for a significant p valueB0.05. Results: The study included 669 patients, 225 patients infected with HCV (group A) and 444 patients co-infected with HCV/HIV (group B). The comparative analysis of those groups (A vs. B) showed: mean age was 49.4 years versus 46.9 (pB0.01), mean time since HCV diagnosis was 9.5 versus 14.6 years (p0.558) both groups shared a male predominance and HCV acquisition due to intravenous drug use. Regarding genotype characterization, the predominant was 1a in both groups (pB0.01). Evaluation of IL28B polymorphism revealed CC 15.5% (A) versus 9.45% (B) (pB0.01). Group B mean TCD4 count was 585 cells/mL (mean percentage 27.1%). There was spontaneous viral clearance in 10.7% (A) versus 4.1% (B) (pB0.01). There were treated 52.0% (A) versus 32.2% (B) patients (pB0.01). For the untreated ones (107 group A vs 270 group B), no reason was identified for treatment deferral in 32.5% (A) versus 48.0% (B) patients. The most frequent reasons for deferring treatment were: withdrawal to follow-up (33.7%), active staging of disease (7.2%), alcohol abuse (6.0%) and advanced age (6.0%) in group A versus low TCD4 cell count (17.1%), loss to follow-up (7.5%), poor adherence (7.5%) and alcohol abuse (3.2%) in group B. Conclusions: One of the highlighted cause for treatment deferral in both mono and co-infected patients was withdrawal to follow-up. In co-infected patients, low TCD4 cell count and poor adherence, also gain prominence, suggesting that strategies to improve retention in care may be needed. Copyright: – 2014 Pereira K et al; licensee International AIDS Society. This is an Open Access article distributed under the terms of the Creative Commons Attribution 3.0 Unported (CC BY 3.0) License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Poster Sessions Abstract P113 Poster Sessions Analysis of hepatitis non-treatment causes in a cohort of HCV and HCV/HIV infected patients Additionally, emergence of direct-acting antiviral agents is expected to reduce these determinants in starting treatment, namely reduce the impact of low TCD4 count in co-infected patients. Published 2 November 2014 Abstracts of the HIV Drug Therapy Glasgow Congress 2014 Pereira K et al. Journal of the International AIDS Society 2014, 17(Suppl 3):19645 h // / d h / / l / / \| h //d d / Pereira K et al. Journal of the International AIDS Society 2014, 17(Suppl 3):19645 http://www.jiasociety.org/index.php/jias/article/view/19645 \| http://dx.doi.org/10. Published 2 November 2014 Copyright: – 2014 Pereira K et al; licensee International AIDS Society. This is an Open Access article distributed under the terms of the Creative Commons Attribution 3.0 Unported (CC BY 3.0) License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1
https://openalex.org/W4243061117	https://www.nature.com/articles/s41467-020-14427-5.pdf	English	null	Author Correction: Blood–brain barrier permeable nano immunoconjugates induce local immune responses for glioma therapy	Nature communications	2,020	cc-by	277	© The Author(s) 2020 Author Correction: Blood–brain barrier permeable nano immunoconjugates induce local immune responses for glioma therapy Author Correction: Blood–brain barrier permeable nano immunoconjugates induce local immune responses for glioma therapy Anna Galstyan, Janet L. Markman , Ekaterina S. Shatalova, Antonella Chiechi, Alan J. Korman, Rameshwar Patil , Dmytro Klymyshyn, Warren G. Tourtellotte, Liron L. Israel, Oliver Braubach , Vladimir A. Ljubimov, Leila A. Mashouf, Arshia Ramesh, Zachary B. Grodzinski, Manuel L. Penichet, Keith L. Black, Eggehard Holler , Tao Sun, Hui Ding, Alexander V. Ljubimov & Julia Y. Ljubimova rrection to: Nature Communications https://doi.org/10.1038/s41467-019-11719-3, published online 28 This Article contained an error in Fig. 1. In ﬁgure 1g, the PD-1 antigen, green oval, was incorrectly shown binding to a-msTfR instead of a-PD1. This error has now been corrected in the HTML and PDF versions of the article. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. NATURE COMMUNICATIONS \| (2020) 11:701 \| https://doi.org/10.1038/s41467-020-14427-5 \| www.nature.com/naturecommunicatio
https://openalex.org/W4236422806	https://kula.uvic.ca/index.php/kula/article/download/109/211	English	null	Indigitization	KULA	2,018	cc-by	1,073	PROJECT REPORT Rachel Bickel and Sarah Dupont University of British Columbia, CA Corresponding author: Rachel Bickel (r.bickel@alumni.ubc.ca) Corresponding author: Rachel Bickel (r.bickel@alumni.ubc.ca) Indigitization is a British Columbia-based collaborative initiative between Indigenous communities and organizations, the Irving K. Barber Learning Centre (IKBLC), the Museum of Anthropology (MOA), the UBC iSchool at the University of British Columbia (UBC), and the University of Northern British Columbia (UNBC) Archives and Special Collections, to facilitate capacity building in Indigenous information management. This project is committed to clarifying processes and identifying issues in the conservation, digitization, and management of Indigenous community knowledge. It does so by providing information resources through the Indigitization toolkit and by enabling community-led audio cassette digitization projects through grant funding and training. Indigitization seeks to grow and work with a network of practitioners to develop effective practices for the management of digital heritage that support the goals of individual communities. Keywords: culture; language; First Nations; Aboriginal; Indigenous; audio cassette; community archives; British Columbia Keywords: culture; language; First Nations; Aboriginal; Indigenous; audio cassette; community archives; British Columbia Bickel, Rachel and Sarah Dupont. 2018. Indigitization. KULA: knowledge creation, dissemination, and preservation studies 2(1): 11. DOI: https://doi. org/10.5334/kula.56 Indigitization g Many people who have worked with community heritage institutions know that navigating best practices established by the information professions can be a challenge, particularly when they are developed with larger institutional settings in mind. This navigation is especially challenging if best practices cannot speak to the unique nature of culturally situated materials, such as Indigenous contexts. How can information professionals address the unnecessary barriers created by their own fields to better support the people and organizations best equipped to manage and make decisions about their collections and community knowledge? These are the core questions which gave rise to the Indigitization program (http://www. indigitization.ca/) and continue to drive the work that we do. Tlingit artist, Alison O. Marks. Tlingit artist, Alison O. Marks. Tlingit artist, Alison O. Marks. Tlingit artist, Alison O. Marks. Art. 11, page 2 of 2 Bickel and Dupont: Indigitization Indigitization is a British Columbia-based collaboration between the Irving K. Barber Learning Centre (IKBLC) and the Museum of Anthropology (MOA) at the University of British Columbia (UBC), the UBC iSchool, and the University of Northern British Columbia (UNBC) Archives and Special Collections. It grew out of an understanding that vitally important cultural heritage information on legacy media formats is increasingly at risk. For First Nations, the need to make these materials accessible is intensified by language revitalization efforts, as well as extensive rights and titles endeavors. The role digitization plays in support of these activities cannot be overstated, and yet finding the resources necessary to undertake such projects is a challenge. One of the resources needed to start this work was a step-by-step manual written for a non-technical audience. The Indigitization Toolkit was developed as an online manual intended to help First Nations organizations plan and conduct scalable digitization projects. In addition, Indigitization put together a buying guide with a set of hardware recommendations specifically for audio-cassette digitization. A barrier First Nations face in accessing grant funding for this work is the common requirement by agencies that they make the digitized materials open access (OA). In making the case that IKBLC should establish a new grant program for First Nations cultural heritage workers, our team successfully advocated that First Nations retaining control of the digitized materials is fundamental to the values of the program. To date, the Indigitization program has co-funded 44 projects and helped First Nations in BC build their internal capacity and digitize almost 11,000 tapes. As Indigitization grows, it needs to stay relevant. Thanks to feedback from previous participants about where the program should focus next, the work is starting to take a slightly different shape. Beyond developing additional training opportunities throughout the province for other formats, particularly reel-to-reel and VHS (and continually updating our Toolkit), there is a growing interest in the ethical questions surrounding Indigenous information stewardship. While there is a clear value in Indigitization’s participation in scholarly activities (such as publishing and presenting at conferences), these activities take time away from other efforts that might more directly benefit First Nations, including further developing curriculum materials for additional formats, offering remote training opportunities and workshops, or funding development for the Indigitization program. Tlingit artist, Alison O. Marks. This is a constant negotiation for all involved with the program, but also a very generative one. It keeps pushing us to stay true to the questions which guide this work, to keep looking at the big picture, and to identify new ways to support the critically important work being done by First Nations and Aboriginal organizations. Competing Interests p g The authors have no competing interests to declare. How to cite this article: Bickel, Rachel and Sarah Dupont. 2018. Indigitization. KULA: knowledge creation, dissemination, and preservation studies 2(1): 11. DOI: https://doi.org/10.5334/kula.56 Submitted: 06 July 2018 Accepted: 31 July 2018 Published: 29 November 2018 Copyright: © 2018 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See http://creativecommons.org/ licenses/by/4.0/. KULA: knowledge creation, dissemination, and preservation studies is a peer-reviewed open access journal published by Ubiquity Press OPEN ACCESS How to cite this article: Bickel, Rachel and Sarah Dupont. 2018. Indigitization. KULA: knowledge creation, dissemination, and preservation studies 2(1): 11. DOI: https://doi.org/10.5334/kula.56 How to cite this article: Bickel, Rachel and Sarah Dupont. 2018. Indigitization. KULA: knowledge creation, dissemination, and preservation studies 2(1): 11. DOI: https://doi.org/10.5334/kula.56 Published: 29 November 2018 Submitted: 06 July 2018 Accepted: 31 July 2018 Copyright: © 2018 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See http://creativecommons.org/ licenses/by/4.0/. Copyright: © 2018 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See http://creativecommons.org/ licenses/by/4.0/. KULA: knowledge creation, dissemination, and preservation studies is a peer-reviewed open access journal published by Ubiquity Press OPEN ACCESS KULA: knowledge creation, dissemination, and preservation studies is a peer-reviewed open access journal published by Ubiquity Press OPEN ACCESS
https://openalex.org/W2962100168	https://amb-express.springeropen.com/counter/pdf/10.1186/s13568-019-0834-1	English	null	Preparation and characterization of a single-domain antibody specific for the porcine epidemic diarrhea virus spike protein	AMB express	2,019	cc-by	7,506	© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Abstract Porcine epidemic diarrhea (PED) is a diarrheal disease of swine caused by porcine epidemic diarrhea virus (PEDV). It is characterized by acute watery diarrhea, dehydration and vomiting in swine of all ages and is especially fatal for neonatal and postweaning piglets. The spike protein of PEDV plays an important role in mediating virus attachment and fusion to target cells, and recent studies also reported that the neutralizing epitopes of the spike protein were mainly located in the S1 subunit, which makes it a candidate for vaccine development and clinical diagnosis. In this study, we successfully constructed an immune phage display single-domain antibody library with a library size of 3.4 × 106. A single-domain antibody, named S7, specific for the spike protein of PEDV was identified from the phage display single-domain antibody library. S7 could be expressed in a soluble form in E. coli, bound to the spike protein of PEDV in ELISA and stained the PEDV virus in Vero cells, but it showed no neutralization activity on PEDV. These results indicated the potent application of the S7 antibody as an imaging probe or as a candidate for the development of a diagnostic assay. Keywords: Porcine epidemic diarrhea virus (PEDV), Spike protein, Bactrian camel, Single-domain antibody, Nanobody, Phage display antibody library, Immunofluorescence, Neutralization activity Preparation and characterization of a single‑domain antibody specific for the porcine epidemic diarrhea virus spike protein Fuxiang Bao1,2, Lixin Wang1, Xinxin Zhao1, Ting Lu1, A. Mi Na1, Xuefei Wang1, Jinshan Cao1,2* Bao et al. AMB Expr (2019) 9:104 https://doi.org/10.1186/s13568-019-0834-1 Bao et al. AMB Expr (2019) 9:104 https://doi.org/10.1186/s13568-019-0834-1 Open Access Introduction (Lee 2015; Song et al. 2015b). In the late autumn of 2010, a large-scale outbreak of diarrhea characterized by severe watery diarrhea, dehydration with high morbidity and mortality emerged in a swine farm in southern China, and it has affected an estimated millions of piglets and greatly threatened the global swine industry. The accu- mulated evidence suggests that the causative agent of the disease was PEDV with a possible variation (Li et al. 2012; Song et al. 2015a; Tian et al. 2013). (Lee 2015; Song et al. 2015b). In the late autumn of 2010, a large-scale outbreak of diarrhea characterized by severe watery diarrhea, dehydration with high morbidity and mortality emerged in a swine farm in southern China, and it has affected an estimated millions of piglets and greatly threatened the global swine industry. The accu- mulated evidence suggests that the causative agent of the disease was PEDV with a possible variation (Li et al. 2012; Song et al. 2015a; Tian et al. 2013). Porcine epidemic diarrhea (PED) is a diarrheal disease of swine caused by the porcine epidemic diarrhea virus (PEDV). It is characterized by acute watery diarrhea, dehydration and vomiting in swine of all ages and is espe- cially fatal for neonatal and postweaning piglets. The dis- ease was first described in Europe in the early seventies, and the PEDV virus was first isolated in Belgium and the United Kingdom in 1978. Since the nineties, several out- breaks have been reported in Asian countries, such as South Korea, China and Japan, and they have been char- acterized by relatively high mortality rates ranging from 50 to 95%, causing severe damage to the swine industry PEDV is a member of the family Coronaviridae and has a single-stranded, positive-sense RNA genome. PEDV mainly infects piglets and can infect several cell lines in vitro, such as Vero cells. The complete genome of PEDV comprises approximately 280,000 nucleotides (nt) with 5′ and 3′ ends containing untranslated regions (UTRs). The remaining genomic sequences contain seven open reading frames (ORFs) and encode four proteins: spike (S), envelope (E), membrane (M) and nucleoprotein Correspondence: jinshancao@imau.edu.cn; yanandu@126.com 1 College of Veterinary Medicine, Inner Mongolia Agricultural University, No. 306, Zhaowuda Road, Saihan District, Huhhot 010018, China Full list of author information is available at the end of the article Bao et al. AMB Expr (2019) 9:104 Page 2 of 10 Table 1 PCR primers Primers Sequences S1 5′-CGGAATTCGCACCTGCCGTCGTTGTT-3′ S2 5′-CTAGCTCGAGACCGTACTTGGTGATGACAAT-3′ P1 5′-GTCCTGGCTGCTCTTCTACAAGG-3′ P2 5′-GGTACGTGCTGTTGAACTGTTCC-3′ P3 5′-CCAGCCGGCCATGGCTGAKTBCAGCTGGTGGAGTCTGG-3′ P4 5′-GGACTAGTGCGGCCGCTGAGGAGACRGTGACCWGGGT-3′ P5 5′-ATCTTAATTACTGGCCCAGCCGGCCATGGCTGAKGTB CAGCTGCAGGCGTCTGGRGGAGG-3′ P6 5′-ATTGCGTCAGCTATTAGTGCGGCCGCTGAGGAGACRGTG ACCWGGGTCC-3′ R1 5′-CCATGATTACGCCAAGCTTTGGAGCC-3′ R2 5′-CCATGATTACGCCAAGCTTTGGAGCC-3′ (N) (Rasmussen et al. 2018; Yang et al. 2014). The spike protein of PEDV is a type one membrane glycopro- tein consisting of the N terminal S1 and C terminal S2 subunits and plays an important role in mediating virus attachment and fusion to target cells. The spike pro- tein contains a multidomain architecture and has been reported to bind to carbohydrate (sialic acid) and ami- nopeptidase N molecules in porcine cells (Li et al. 2016; Meng et al. 2014; Sun et al. 2018). The neutralizing epitopes of the spike protein were identified in recent studies, and that suggested that the epitopes were mainly located in the S1 subunit (Chang et al. 2019; Li et al. 2017). The recombinant expressed S1 protein can also induce protective immunity in piglets (Makadiya et al. 2016; Oh et al. 2014). These factors make the spike pro- tein of PEDV a putative candidate for developing an effi- cient protective vaccine. Table 1 PCR primers was synthesized with the GoScript Reverse Transcription System (Promega, USA). The truncated S gene sequence was amplified by PCR, cloned into the pET-28a vector by EcoRI and XhoI (TaKaRa, Japan) digestion and ligation, and transformed into E. coli Transetta-DE3 (Transgene, Beijing, China). Expression of the recombinant proteins was induced by 1 mM IPTG and analyzed by SDS-PAGE electroporation, after which the proteins were purified by Ni–NTA Agarose (Qiagen, Germany) under denatur- ing conditions. The purified S protein was transformed onto a PVDF membrane (Bio-Rad, USA) for further western blot detection by incubation with Rabbit Anti- 6× His Polyclonal Antibody (Sangon Biotech, Shanghai, China) at a 1:2000 dilution in PBS and then with 1:20,000 PBS-diluted Goat Anti-Rabbit IgG Antibody (GenScript, Nanjing, China). The protein band was visualized with ImageQuant LAS 4000 (GE Healthcare, USA) by adding the Pierce™ ECL Western Blotting Substrate (Thermo Scientific, USA) onto the membrane. Although there has been some success in developing a vaccine for PEDV, it is noteworthy that no effective vac- cine is available in the market to protect newborn piglets. Bactrian camel immunization A healthy 5-year-old female Alashan Bactrian camel was chosen for PEDV immunization. The cell culture medium containing 105 pfu/ml PEDV strain CV777 inactivated by formaldehyde was mixed with equal volume of Freund’s Complete Adjuvant (FCA) (Sigma-Aldrich, USA) and subcutaneously injected into the camel cervical area for the first immunization, and Freund’s Incomplete Adju- vant (FIA) (Sigma-Aldrich, USA) was used for the sec- ond and third immunizations. The time interval between immunizations was 10 days, and the camel blood was taken from the jugular vein 10 days after each injection to prepare the serum for evaluation of the PEDV heavy chain antibody (IgG2) titer. The camel was farmed in the isolated Gobi area of the Alxa Left Banner in Inner Mon- golia and was provided free access to water and food. The experimental procedures were performed in accordance An attenuated vaccine has been designed for use in sows to protect neonatal piglets in some Asian countries; how- ever, the efficiency of the vaccine still needs to be verified (Paudel et al. 2014; Song et al. 2007). Passive immuniza- tion by oral administration of egg yolk antibodies (IgY) obtained from immunized chickens and the colostrum obtained from immunized cows has been shown to pre- vent and treat PEDV infection in newborn piglets (Lee et al. 2015). Early diagnosis and corresponding preven- tion and treatment measures are key points for managing PEDV infection, which requires effective antibodies for both diagnostic assays and treatment. In this study, we immunized the Alashan Bactrian camel with PEDV and established a phage display anti- body library. The spike protein of PEDV was cloned, expressed and purified and was used as the antigen to enrich and select a virus-specific single-domain antibody (sdAb). The PEDV-specific single-domain antibody was expressed and purified, and the specificity of the anti- body was determined. Materials and methodsi Expression and purification of the PEDV spike protein d h bl h d k Construction and screening of Bactrian camel phage display antibody libraryi One hundred fifty milliliters of blood was taken from the jugular vein of the immunized camel, and periph- eral blood monocytes (PBMCs) were obtained with a PBMC isolation kit (TBD science, Tianjin Haoyang Bio- logical Manufacture CO., LTD) according to the manu- facturer’s instructions. Total RNA was extracted from the PBMCs with TRIzol reagent (Invitrogen, USA), and first strand cDNA was synthesized with the GoScript™ Reverse Transcription System (Promega, USA) by using Oligo (dT) 12–18 (Invitrogen, USA). The VHH frag- ment was amplified with a nested PCR method by using the primers listed in Table 1. In the first round of PCR, a fragment containing the leader sequence to the CH2 region of the IgG (900 bp for VH and 600 bp for VHH) was amplified with primers P1 and P2, and the 600 bp fragment was purified by 1% agarose gel electroporation and used as the template for the second round of PCR. The second round of PCR with primers P3 and P4, which anneal to the VHH FR1 and FR4 regions, respectively, amplified the VHH. SfiI and NotI restrictions sites were introduced in the VHH fragment by the third round of PCR with primers P5 and P6. The PCR-amplified VHH fragments were digested by SfiI and NotI (New England Biolabs, UK) and ligated into the pCANTAB 5E plasmid (GE Healthcare, USA), cleaved by the same restriction enzymes, with T4 ligase (New England Biolabs, UK). The ligation products were electro-transformed into compe- tent E. coli TG1 for antibody library construction. The antibody library size was estimated by calculating the colony forming units (CFU) of 10 serial dilutions of the library on a 2 × YT-AG plate, and the electro-transfor- mation efficiency was evaluated by the PCR method with the pCANTAB 5E phagemid vector sequencing primers R1 and R2 (Table 1).h Individual clones with target specificity were identi- fied with phage ELISA. Briefly, 10 μg/ml of S protein was coated on the Stripwel microplate overnight at 4 °C. After blocking with 2% skimmed milk solution, 100 μl of recombinant phages prepared from 48 randomly picked clones were added to each well, and M13K07 helper phage was used as a negative control and PBS was used as a blank control. Then, 100 μl of HRP/anti-M13 mono- clonal antibody (GE Healthcare, USA) was added to each well, followed by TMB substrate (Promega, USA) for vis- ualization. Expression and purification of the PEDV spike protein i Based on the published spike protein gene sequences of the PEDV CV777 strain and its antigenic properties, we designed a pair of primers (S1 and S2) to anneal to truncated PEDV spike protein 444–770 amino acid and added the EcoRI and XhoI restriction sites at the end of the sequences (Table 1). PEDV virus strain CV777 (obtained from Wuhan Institute of Virology, China Acad- emy of Sciences) genomic RNA was extracted from the PEDV-infected Vero cell culture medium with TRIzol reagent (Invitrogen, USA), and the first strand cDNA Bao et al. AMB Expr (2019) 9:104 Bao et al. AMB Expr (2019) 9:104 Page 3 of 10 with the institutional and national guidelines and regu- lations and were approved by the Animal Care and Use Committee of Inner Mongolia Agriculture University. coated with S protein at 20 μg/ml and incubated at RT for 1 h. The bound phages were eluted with 100 μl of 100 mM triethylamine and neutralized with 50 μl of 1 M Tris–HCl. Half of the eluted phages were added to 10 ml E. coli TG1 (OD600 nm = 0.5), and then 109 cfu/ml of M13K07 was added. The culture medium was changed to 100 ml 2 × YT medium containing 100 μg/ml ampicillin and 70 μg/ml kanamycin. The phages were harvested and purified with PEG/NaCl for a new round of enrichment. The E. coli TG1 cells infected with the eluted phages from the third round of enrichment were grown on 2 × YT-AG plates. Construction and screening of Bactrian camel phage display antibody libraryi The plate was read at 450 nm in a microplate reader, and absorbance of experimental group/negative control ≥ 2.1 was considered positive. Expression and purification of the sdAbh i The plasmids of the positive clones from phage ELISA were isolated, and the VHH gene was digested with NcoI and NotI and ligated to a modified pET-25b vector that contained the 38-amino acid sequence of streptavidin binding protein (SBP) between the NotI and XhoI restric- tion sites (Additional file 1: Fig. S1) The resulting vector was transformed into competent E. coli Transetta-DE3 (Transgene, Beijing, China). Expression of the recom- binant sdAb was induced by 1 mM IPTG, and then the proteins were purified by the Ni–NTA Agarose (Qia- gen, Germany) under native conditions. The expression and purification of the sdAb was analyzed by SDS-PAGE electroporation and western blot with HRP-streptavidin (Solarbio Life Sciences, Beijing, China). The antibody library was diluted in 2 × YT medium containing 100 μg/ml ampicillin and 2% glucose to an OD600 nm = 0.3 and incubated at 37 °C for 1 h with 250 r/min agitation. Recombinant phages were rescued by superinfection of bacteria with M13K07 helper phage (GE Healthcare), and the phages were purified by add- ing 1/5 volume of PEG/NaCl on ice for 1 h and cen- trifuged at 4 °C for 20 min at 10,000g. Then, the phage pellets were resuspended in PBS and filtered through a 0.22 μm membrane for further screening. Afterwards, 100 μl 1 × 1011 pfu/ml of recombinant phages was mixed with an equal volume of 2% skimmed milk solution and added to a 96-well Stripwel microplate (Corning, USA) Immunocytochemistry y y Vero cells (obtained from Wuhan Institute of Virology, China Academy of Sciences) were seeded on a petri dish, infected with 1 ml of PEDV mixed with 1 ml of pancrea- tin (10 μg/ml) and incubated at 37 °C for 1 h. The super- natant was removed, and the cells were incubated with Dulbecco’s modified Eagle medium (DMEM) contain- ing 1% fetal bovine serum (Gibco, USA) at 37 °C with 5% CO2 for 48 h. The culture medium was removed from cells when the cytopathic effect (CPE) reached 50–70%. The cells were washed with PBS once, fixed with 4% paraformaldehyde (Solarbio, Beijing, China) for 10 min, washed with PBST three times and blocked with 3% BSA at 37 °C for 1 h. The fixed cells were incubated with 150 μg purified recombinant sdAb diluted in PBS at 4 °C over- night, and an equal volume of PBS was added to another dish of infected cell to use as a negative control. After three washes, the cells were incubated with 1:300 diluted FITC-streptavidin (Solarbio, Beijing, China) at 37 °C for 30 min and washed with PBST five times. The cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) solution for 5 min at room temperature and washed five times with PBST, and fluorescent signals were detected by confocal microscopy (ZEISS, LSM-800). Binding activity and specificity of the sdAbfi i Different concentrations of the purified recombinant sdAb were added to a 10 μg/ml S protein-coated 96-well Stripwell microplate (Corning, USA), and the protein extracted from the bacterial cells transformed with the empty pET-25b vector containing both an SBP-tag and a 6× His tag was used as a negative control. PBS was used as a blank control. The binding activity of each well was detected with HRP-streptavidin (Solarbio Life Sciences, Beijing, China) at a 1:10,000 concentration and visualized Bao et al. AMB Expr (2019) 9:104 Bao et al. AMB Expr (2019) 9:104 Page 4 of 10 with the TMB solution, and the plate was read at 450 nm in a microplate reader. was obtained from the amplification as shown in Fig. 1a, and the sequencing result showed that the gene fragment corresponded to the 1330–2310 bp of the PEDV spike protein gene (444–770 amino acids, data not shown). The recombinant spike protein with a 36.5 kDa molecu- lar weight was expressed in the periplasm of E. coli in soluble form by ligating the gene fragment to the pET- 28a vector and transforming it in E. coli. After purifica- tion with a Ni–NTA Agarose, we successfully obtained a high-purity recombinant spike protein as shown by SDS- PAGE analysis in Fig. 1b, and by western blotting analysis using an anti-6× His tag antibody (Fig. 1c). Construction and screening of a single‑domain antibody phage display libraryh The diagram of the construction and screening of a sin- gle-domain antibody phage display library is indicated in Fig. 2. The PCR-amplified VHH gene was ligated into the pCANTAB 5E plasmid and transformed into E. coli TG1, and 5.49 × 106 transformants were obtained. We randomly picked 24 clones and amplified them with the R1 and R2 pCANTAB 5E sequencing primers, and the results showed that 15 clones were obtained from the ~ 400 bp amplicons (Additional file 1: Fig S2). We calculated the positive rate of the transformation to be 62.5%; thus, the real size of the antibody library was 3.4 × 106. After rescue by M13K07 helper phage, the phage dis- play antibody library was screened against PEDV spike protein by three rounds of panning. The enrichment fac- tor of the output to input phage was increased with the panning procedure and with a 30-fold increase in phage recovery after the third round of panning compared with the first round (Table 2). We randomly picked 96 clones from the third elution to evaluate the binding activity to the spike protein by phage ELISA, and 20 these clones showed high OD450 nm values (Fig. 3). Expression and purification of a recombinant sdAb l d h l h h h h b d Expression and purification of a recombinant sdAb We selected the S7 clones with the highest binding activ- ity to the spike protein from phage ELISA for further expression and characterization. SDS-PAGE results showed that the recombinant S7 antibody was expressed in a soluble form in the supernatant of the cell lysate with an expected molecular weight of 20 kDa, and con- centrated recombinant S7 antibody was eluted from the Ni–NTA Agarose as shown in Fig. 4a. The purified recombinant S7 antibody was verified with HRP-strepta- vidin, which binds to the 38-amino acid SBP fusion part- ner of the sdAb in western blot analysis, and a 20-kDa specific band was detected (Fig. 4b). Statistical analysis GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA, USA) was used for all univariate statistical analyses. The date of neutralization activity was presented as the mean value ± SEMs and analyzed by Student’s t-test. P values < 0.05 were considered to be significant. Neutralizing activityhi The purified recombinant sdAb was used in a microti- ter neutralization assay in Vero cells. Recombinant sdAb at 100, 50, 25 and 12.5 μg/ml final concentrations were incubated with 100 times the tissue culture infectious dose 50 (TCID50) of PEDV strain CV777 at 37 °C for 1 h and added to the Vero cells in 96-well plates for 72 h. Cells incubated with PBS were used as a negative control. The cytopathic effect and the neutralization activity of the recombinant sdAb antibody were calculated by com- paring the changes in the TCID50 value. All experiments were repeated three times. Immunocytochemistry PEDV in Vero cells was detected with the recombinant S7 antibody and visualized with FITC-streptavidin, and the fluorescent signal and images were obtained through confocal microscopy. The results showed that the PEDV virions in Vero cells could be detected by the recombi- nant S7 antibody outside the cell nucleus, while the PEDV-free Vero cells were not stained by the recombi- nant S7 antibody (Fig. 6). Discussion h PEDV is the causative agent of porcine epidemic diar- rhea, which is an acute and highly contagious viral dis- ease of swine. Pigs of all ages and breeds are susceptible to PEDV, and infections of suckling piglets 1–5 days old were the most serious, with the highest infection rate and mortality of 100%. The main symptoms of the dis- ease are vomiting, severe diarrhea and dehydration. The PEDV genome is a single-stranded RNA belonging to the coronavirus family. The total length of the PEDV genome is approximately 28 Kb, containing at least 7 open reading frames (Chen et al. 2019; Nefedeva et al. 2019). The spike protein of PEDV is composed of 1383 amino acids, including signal peptide (1–18 aa), neutrali- zation epitopes (499–638 aa, 748–755 aa, 764–771 aa and 1368–1374 aa), transmembrane region (1334–1356 aa), and a short cytoplasmic region in the middle. The spike protein plays a key regulatory role in the binding of the virus to the receptor. It is involved in the binding to cell receptors and in membrane fusion, which can indirectly Binding activity and specificity of the recombinant sdAbhi neutralize PEDV. PEDV incubated with 100, 50, 25 and 12.5 μg/ml final concentrations of recombinant S7 anti- body showed TCID50/0.1 ml ranging from 105 to 105.25, which is not significantly different from the control group incubated with PBS that showed TCID50/0.1 ml of 105.25 (Fig. 7). Binding activity and specificity of the recombinant sdAb The binding activity and specificity of the recombinant S7 antibody were analyzed by ELISA. The results showed that OD450 nm values increased with increasing recom- binant S7 antibody concentrations, and the S7 antibody showed a very strong binding activity to the spike protein even with a concentration of 1 μg/ml. The OD450 nm value of the recombinant S7 antibody to an irrelevant 6× His- tagged protein was similar to that of PBS used as blank control, which indicated that the recombinant S7 anti- body bound specifically to the spike protein (Fig. 5). xpression and purification of PEDV spike proteinh The truncated spike gene of PEDV was PCR amplified with a pair of specific primers. A single specific gene frag- ment with a molecular weight of approximately 1000 bp Bao et al. AMB Expr (2019) 9:104 Page 5 of 10 Fig. 1 Cloning, expression and purification of the PEDV spike protein. a PCR amplification of the truncated spike gene of PEDV corresponding to the 1330–2310 bp of the spike protein gene (444–770 amino acids). Lanes 1–2, PCR amplification products of the PEDV spike gene. b SDS-PAGE analysis of the expression and purification of the PEDV spike protein. Lanes 1–2, the supernatant of E. coli cell lysate that were transformed with the PEDV spike protein gene after induction. Lanes 3–4, PEDV spike protein eluted from Ni–NTA Agarose. c Western blot analysis of the PEDV spike protein with an anti-6× His antibody. Lanes 1–2, the purified PEDV spike protein. Lane M, molecular weight marker Fig. 1 Cloning, expression and purification of the PEDV spike protein. a PCR amplification of the truncated spike gene of PEDV corresponding to the 1330–2310 bp of the spike protein gene (444–770 amino acids). Lanes 1–2, PCR amplification products of the PEDV spike gene. b SDS-PAGE analysis of the expression and purification of the PEDV spike protein. Lanes 1–2, the supernatant of E. coli cell lysate that were transformed with the PEDV spike protein gene after induction. Lanes 3–4, PEDV spike protein eluted from Ni–NTA Agarose. c Western blot analysis of the PEDV spike protein with an anti-6× His antibody. Lanes 1–2, the purified PEDV spike protein. Lane M, molecular weight marker Neutralizing activityhf The PEDV spike protein-specific phages were enriched by three rounds of biopanning Table 2 Enrichment of sdAb-displaying phage by panning with the spike protein of PEDV a Enriching factor = Output (clones)/input (clones) Round Input (clones) Output (clones) Enriching factora 1 5 × 109 1 × 106 2 × 10−4 2 5 × 109 6 × 107 1.2 × 10−2 3 5 × 109 3 × 107 6 × 10−3 Table 2 Enrichment of sdAb-displaying phage by panning with the spike protein of PEDV Table 2 Enrichment of sdAb-displaying phage by panning with the spike protein of PEDV Greenberg et al. 1995; Hamers-Casterman et al. 1993). Due to their single-domain nature and excellent proper- ties, sdAbs have become ideal for developing sensitive diagnostic assays, immuno-imaging probes, and immu- notherapeutics, especially for infectious diseases and tumors (Beghein and Gettemans 2017; Iezzi et al. 2018; Wilken and McPherson 2018). Considering the broad application of sdAbs on the diagnosis and treatment of viral diseases, in this study we identified and character- ized a sdAb specific for the spike protein of PEDV. Greenberg et al. 1995; Hamers-Casterman et al. 1993). Due to their single-domain nature and excellent proper- ties, sdAbs have become ideal for developing sensitive diagnostic assays, immuno-imaging probes, and immu- notherapeutics, especially for infectious diseases and tumors (Beghein and Gettemans 2017; Iezzi et al. 2018; Wilken and McPherson 2018). Considering the broad application of sdAbs on the diagnosis and treatment of viral diseases, in this study we identified and character- ized a sdAb specific for the spike protein of PEDV. a Enriching factor = Output (clones)/input (clones) regulate virus invasion and stimulate the host to produce neutralizing antibodies, making it the main target for the development of novel genetically engineered vaccines and antibodies that are used for diagnostic and disease prevention and treatment (Kim et al. 2018). i We constructed a camel immune phage display library of single-domain antibodies with a library size of 3.4 × 106 by immunizing a Bactrian camel with PEDV, ligating the VHH gene to the pCANTAB 5E plasmid and transforming it in E. coli TG1. The truncated S gene of PEDV corresponding to the 444–770 amino acids of the PEDV spike protein, which covers most neutralizing epitopes of the spike protein, was cloned, expressed and purified to use as an antigen to screen spike protein-spe- cific sdAbs. Neutralizing activityhf The effect of recombinant S7 antibody on the infectious titer of PEDV was analyzed by TCID50 assay. The results demonstrated that the recombinant S7 antibody failed to Bao et al. AMB Expr (2019) 9:104 Page 6 of 10 Fig. 2 Schematic diagram of the construction and screening of a single-domain phage display library. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood samples of a Bactrian camel immunized with PEDV, and total RNA was extracted from the PBMCs and reverse transcribed to cDNA. The VHH gene fragments with restriction enzyme sequences were amplified with nested PCR. In the first round of PCR, the gene fragment (~ 600 bp) containing the VHH, hinge region and CH2 domain was amplified from the cDNA, and then the VHH gene was amplified by a pair of primers specific for VHH gene FR1 to FR4 using the product of the first PCR (600 bp product). SfiI and NotI restriction sites were added on the third round of PCR. The VHH gene was ligated into the pCANTAB 5E plasmid and transformed into E. coli TG1, and after rescue by the M13K07 helper phage, the VHH gene was displayed on the phage surface. The PEDV spike protein-specific phages were enriched by three rounds of biopanning Fig. 2 Schematic diagram of the construction and screening of a single-domain phage display library. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood samples of a Bactrian camel immunized with PEDV, and total RNA was extracted from the PBMCs and reverse transcribed to cDNA. The VHH gene fragments with restriction enzyme sequences were amplified with nested PCR. In the first round of PCR, the gene fragment (~ 600 bp) containing the VHH, hinge region and CH2 domain was amplified from the cDNA, and then the VHH gene was amplified by a pair of primers specific for VHH gene FR1 to FR4 using the product of the first PCR (600 bp product). SfiI and NotI restriction sites were added on the third round of PCR. The VHH gene was ligated into the pCANTAB 5E plasmid and transformed into E. coli TG1, and after rescue by the M13K07 helper phage, the VHH gene was displayed on the phage surface. Neutralizing activityhf After three rounds of panning and selection, a spike protein-specific sdAb named S7 was selected and characterized. Single-domain antibodies are a unique kind of antibody that naturally lacks a light chain and the CH1 region of IgG. They are found in camelids and nurse sharks, and they possess the unique properties of small molecular size (15 kDa, 1/10 of conventional antibody), low immu- nogenicity, strong tissue penetrating ability, high bind- ing affinity and good stability (Arbabi-Ghahroudi 2017; Bao et al. AMB Expr (2019) 9:104 Page 7 of 10 Fig. 3 The PEDV spike protein-specific recombinant phages were identified by phage ELISA. The recombinant phages of 96 clones that were randomly picked from the third round of panning were added to microplates coated with the PEDV spike protein, and the bound phages were detected with HRP/anti-M13 monoclonal antibody. M13K07 helper phage was used as a negative control Fig. 3 The PEDV spike protein-specific recombinant phages were identified by phage ELISA. The recombinant phages of 96 clones that were randomly picked from the third round of panning were added to microplates coated with the PEDV spike protein, and the bound phages were detected with HRP/anti-M13 monoclonal antibody. M13K07 helper phage was used as a negative control Fig. 3 The PEDV spike protein-specific recombinant phages were identified by phage ELISA. The recombinant phages of 96 clones that were randomly picked from the third round of panning were added to microplates coated with the PEDV spike protein, and the bound phages were detected with HRP/anti-M13 monoclonal antibody. M13K07 helper phage was used as a negative control results showed that HRP-streptavidin could bind to the purified recombinant S7 antibody fused to the SBP-tag. This simplified detection strategy may favor the appli- cation of sdAbs as detection agents in immunoassays or immuno-imaging and is similar to the use of quan- tum dots to label sdAbs for tracer materials (Modi et al. 2018; Wang et al. 2014). Fig. 4 Expression and purification of the S7 antibody. The gene fragment of the S7 antibody was ligated into a modified pET-25b vector and fusion expression with SBP Tag. The recombinant S7 antibody was purified with Ni–NTA agarose and verified with HRP-streptavidin in western blot analysis. Lane M, molecular weight marker. Lane 1, the supernatant of E. coli cell lysate that were transformed with the S7 antibody gene after induction. Neutralizing activityhf Lane 2, the flow-through from Ni–NTA Agarose after incubation with the S7 antibody. Lane 3, elution 1. Lane 4, elution 2. Lane WB, western blot analysis of the purified S7 antibody The specificity of the S7 antibody to the spike protein and PEDV were further assessed by ELISA, immuno- cytochemistry and neutralization experiments. ELISA results demonstrated that the S7 antibody could spe- cifically bind to the spike protein with strong binding activity, even at a 1 μg/ml (50 pmol) concentration, demonstrating that the S7 antibody could strongly bind to the spike protein, which is in accordance with pub- lished data on sdAbs. Due to their excellent properties of smaller size, permeability and stability, sdAbs have been widely used as probes for immuno-imaging and diagnostic assays. In the present study, PEDV-infected Vero cells were stained with the S7 antibody followed by FITC-streptavidin in a direct immunofluorescence assay. The Vero cells infected with PEDV were nicely stained by the S7 antibody, and the control Vero cells without PEDV infection had no signal. These results suggest the potent application of the S7 antibody as a nanoprobe for the detection of PEDV in living cells. Unfortunately, we did not find any neutralization effects of the S7 antibody on PEDV infection in the current study, which suggests that the S7 antibody is not a suit- able passive immunization agent or therapeutic anti- body. The results were in accordance with the recent findings that antibodies raised by the spike protein of Fig. 4 Expression and purification of the S7 antibody. The gene fragment of the S7 antibody was ligated into a modified pET-25b vector and fusion expression with SBP Tag. The recombinant S7 antibody was purified with Ni–NTA agarose and verified with HRP-streptavidin in western blot analysis. Lane M, molecular weight marker. Lane 1, the supernatant of E. coli cell lysate that were transformed with the S7 antibody gene after induction. Lane 2, the flow-through from Ni–NTA Agarose after incubation with the S7 antibody. Lane 3, elution 1. Lane 4, elution 2. Lane WB, western blot analysis of the purified S7 antibody Fig. 4 Expression and purification of the S7 antibody. The gene fragment of the S7 antibody was ligated into a modified pET-25b vector and fusion expression with SBP Tag. The recombinant S7 antibody was purified with Ni–NTA agarose and verified with HRP-streptavidin in western blot analysis. Lane M, molecular weight marker. Neutralizing activityhf Lane 1, the supernatant of E. coli cell lysate that were transformed with the S7 antibody gene after induction. Lane 2, the flow-through from Ni–NTA Agarose after incubation with the S7 antibody. Lane 3, elution 1. Lane 4, elution 2. Lane WB, western blot analysis of the purified S7 antibody For convenience of detection, we fused the S7 anti- body gene to an SBP-tag in the pET-25b vector. The SBP-tag is a 38-amino acid peptide and can strongly bind to streptavidin with an equilibrium dissociation constant of 2.5 nM (Keefe et al. 2001; Yang and Ver- aksa 2017). The S7 antibody was expressed in a soluble form in E. coli with a high yield, and the western blot Bao et al. AMB Expr (2019) 9:104 Page 8 of 10 Fig. 5 The binding activity and specificity of the S7 antibody were analyzed by indirect ELISA. 5, 1 and 0.2 μg/ml final concentrations of S7 antibody were added to PEDV spike protein-coated microplates, and the binding of S7 antibody was detected with HRP-streptavidin. Protein extracts from E. coli transformed with the modified pET-25b vector were used as a negative control, and PBS solution was used as a blank control PEDV with different binding epitopes showed distinct neutralizing effects on different strains of PEDV (Li et al. 2017). In conclusion, a Bactrian camel immune phage display antibody library was constructed, and a PEDV spike pro- tein-specific sdAb S7 was isolated from the library. The soluble S7 antibody fused with an SBP-tag specifically bound to the spike protein with high binding activity in an ELISA and could nicely stain the PEDV-infected Vero cells in an immunofluorescence assay but had no PEDV neutralizing activity. In brief, the S7 antibody can be a useful nanoprobe for potent application in PEDV diag- nostic assays or for tracing PEDV in living cells to study virus-host interactions. Fig. 5 The binding activity and specificity of the S7 antibody were analyzed by indirect ELISA. 5, 1 and 0.2 μg/ml final concentrations of S7 antibody were added to PEDV spike protein-coated microplates, and the binding of S7 antibody was detected with HRP-streptavidin. Protein extracts from E. coli transformed with the modified pET-25b vector were used as a negative control, and PBS solution was used as a blank control Additional file Additional file 1. Additional figures. Additional file 1. Additional figures. Funding Thi d This study was funded by the National Natural Science Foundation of China (No. 81660297), the Research Program of Science and Technology at Universi- ties of Inner Mongolia Autonomous Region (No. NJZY069), The Innovation Fund for Young Talent at College of Veterinary Medicine, Inner Mongolia Agricultural University (No. 2015QNJJ01). Li CH, Li WT, de Esesarte EL, Guo HB, van den Elzen P, Aarts E, van den Born E, Rottier PJM, Bosch BJ (2017) Cell attachment domains of the porcine epidemic diarrhea virus spike protein are key targets of neutralizing antibodies. J Virol 91:e00273–e00317 Makadiya N, Brownlie R, van den Hurk J, Berube N, Allan B, Gerdts V, Zakhartchouk A (2016) S1 domain of the porcine epidemic diarrhea virus spike protein as a vaccine antigen. Virology Journal 13:57 Consent for publication Oh J, Lee KW, Choi HW, Lee C (2014) Immunogenicity and protective efficacy of recombinant S1 domain of the porcine epidemic diarrhea virus spike protein. Arch Virol 159:2977–2987 Not applicable. Availability of data and materials Please contact the authors for all requests. Meng FD, Suo SQGW, Zarlenga DS, Cong YY, Ma XW, Zhao Q, Ren XF (2014) A phage-displayed peptide recognizing porcine aminopeptidase N is a potent small molecule inhibitor of PEDV entry. Virology 456:20–27 References A b b Gh h Arbabi-Ghahroudi M (2017) Camelid single-domain antibodies: historical perspective and future outlook. Front Immunol 8:1589 Beghein E, Gettemans J (2017) Nanobody technology: a versatile toolkit for microscopic imaging, protein-protein interaction analysis, and protein function exploration. Front Immunol 8:771 Chang CY, Cheng IC, Chang YC, Tsai PS, Lai SY, Huang YL, Jeng CR, Pang VF, Chang HW (2019) Identification of neutralizing monoclonal antibodies targeting novel conformational epitopes of the porcine epidemic diar- rhoea virus spike protein. Sci Rep 9(1):2529 Chen P, Wang K, Hou Y, Li H, Li X, Yu L, Jiang Y, Gao F, Tong W, Yu H, Yang Z, Tong G, Zhou Y (2019) Genetic evolution analysis and pathogenicity assessment of porcine epidemic diarrhea virus strains circulating in part of China during 2011–2017. Infect Genet Evol 69:153–165 Greenberg AS, Avila D, Hughes M, Hughes A, McKinney EC, Flajnik MF (1995) A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks. Nature 374:168–173 Fig. 7 Neutralization activity assessment of the S7 antibody. The neutralization effect of the S7 antibody on PEDV was analyzed in Vero cells. Different concentrations of S7 antibody were incubated with PEDV, and the TCID50 was determined by the Reed Muench method. PBS was used as a negative control i Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R (1993) Naturally occurring antibodies devoid of light chains. Nature 363:446–448 g Iezzi ME, Policastro L, Werbajh S, Podhajcer O, Canziani GA (2018) Single- domain antibodies and the promise of modular targeting in cancer imag- ing and treatment. Front Immunol 9:273 g Keefe AD, Wilson DS, Seelig B, Szostak JW (2001) One-step purification of recombinant proteins using a nanomolar-affinity streptavidin-binding peptide, the SBP-Tag. Protein Expr Purif 23:440–446 Kim SH, Cho BH, Lee KY, Jang YS (2018) N-terminal domain of the spike protein of porcine epidemic diarrhea virus as a new candidate molecule for a mucosal vaccine. Immune Netw 18:e21 p g The authors declare that they have no competing interests. p g The authors declare that they have no competing interests. Authors’ contributions Lee C (2015) Porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus. Virol J 12:193 BFX, DYN and CJS conceived and designed the experiments. BFX, WLX, ZXX, LT, AMN, WXF, and DYN carried out the experiments. BFX, WLX, DYN and CJS analyzed the data. BFX, WLX, ZXX, LT, AMN, and WXF contributed reagents/ materials/analysis tools. BFX, DYN and CJS wrote the paper. BFX, DYN and CJS contributed to the study, interpretation of the studies, Analysis of the data and review of the manuscript. BFX, DYN and CJS supervised the project. All authors read and approved the final manuscript. Lee DH, Jeon YS, Park CK, Kim S, Lee DS, Lee C (2015) Immunoprophylactic effect of chicken egg yolk antibody (IgY) against a recombinant S1 domain of the porcine epidemic diarrhea virus spike protein in piglets. Adv Virol 160:2197–2207 Li W, Li H, Liu Y, Pan Y, Deng F, Song Y, Tang X, He Q (2012) New variants of por- cine epidemic diarrhea virus, China, 2011. Emerg Infect Dis 18:1350–1353 Li W, van Kuppeveld FJM, He Q, Rottier PJM, Bosch BJ (2016) Cellular entry of the porcine epidemic diarrhea virus. Virus Res 226:117–127 Acknowledgements The authors thank the staff at the Animal Disease Control and Prevention Center of Alxa Left Banner for their kind help with Bactrian camel immuniza- tion and blood sampling. Fig. 6 Immunocytochemistry analysis of the binding activity of S7 antibody to PEDV in living cells. The Vero cells infected with PEDV were incubated with S7 antibody and subsequently detected with FITC-streptavidin (a), and the uninfected Vero cells were used as a negative control (b). Cell nuclei were stained with DAPI. The merged images showed the localization of PEDV in Vero cells (a), but no fluorescent signal was detected in the PEDV-free Vero cells (b) Fig. 6 Immunocytochemistry analysis of the binding activity of S7 antibody to PEDV in living cells. The Vero cells infected with PEDV were incubated with S7 antibody and subsequently detected with FITC-streptavidin (a), and the uninfected Vero cells were used as a negative control (b). Cell nuclei were stained with DAPI. The merged images showed the localization of PEDV in Vero cells (a), but no fluorescent signal was detected in the PEDV-free Vero cells (b) Bao et al. AMB Expr (2019) 9:104 Page 9 of 10 Page 9 of 10 Page 9 of 10 Fig. 7 Neutralization activity assessment of the S7 antibody. The neutralization effect of the S7 antibody on PEDV was analyzed in Vero cells. Different concentrations of S7 antibody were incubated with PEDV, and the TCID50 was determined by the Reed Muench method. PBS was used as a negative control Received: 23 June 2019 Accepted: 5 July 2019 Ethics approval and consent to participate Blood samples from a 5-year-old female Bactrian camel that was farmed in the Gobi Desert of the Alxa Left Banner in Inner Mongolia were collected accord- ing to the Animal Ethics Procedures and Guidelines of the People’s Republic of China. The current study was approved by the Animal Care and Use Commit- tee of Inner Mongolia Agriculture University. Modi S, Higgs NF, Sheehan D, Griffin LD, Kittler JT (2018) Quantum dot conjugated nanobodies for multiplex imaging of protein dynamics at synapses. Nanoscale 10:10241–10249 Nefedeva M, Titov I, Malogolovkin A (2019) Molecular characteristics of a novel recombinant of porcine epidemic diarrhea virus. Arch Virol 164:1199–1204 Competing interests Paudel S, Park JE, Jang H, Hyun BH, Yang DG, Shin HJ (2014) Evaluation of antibody response of killed and live vaccines against porcine epidemic diarrhea virus in a field study. Vet Q 34:194–200 Competing interests The authors declare that they have no competing interests. Author details 1 i Rasmussen TB, Boniotti MB, Papetti A, Grasland B, Frossard JP, Dastjerdi A, Hulst M, Hanke D, Pohlmann A, Blome S, van der Poel WHM, Steinbach F, Blanchard Y, Lavazza A, Botner A, Belsham GJ (2018) Full-length genome sequences of porcine epidemic diarrhoea virus strain CV777; Use of NGS to analyse genomic and sub-genomic RNAs. PLoS ONE 13:e0193682 1 College of Veterinary Medicine, Inner Mongolia Agricultural University, No. 306, Zhaowuda Road, Saihan District, Huhhot 010018, China. 2 Key Laboratory of Clinical Diagnosis and Treatment Techniques for Animal Disease, Ministry of Agriculture (LDTA), Huhhot, China. Page 10 of 10 Bao et al. AMB Expr (2019) 9:104 Song DS, Oh JS, Kang BK, Yang JS, Moon HJ, Yoo HS, Jang YS, Park BK (2007) Oral efficacy of vero cell attenuated porcine epidemic diarrhea virus DR13 strain. Res Vet Sci 82:134–140 to nanobodies as immunofluorescence probes for nanometric micros- copy. Bioconjug Chem 25:2205–2211 y Wilken L, McPherson A (2018) Application of camelid heavy-chain variable domains (VHHs) in prevention and treatment of bacterial and viral infec- tions. Int Rev Immunol 37:69–76 Song D, Huang D, Peng Q, Huang T, Chen Y, Zhang T, Nie X, He H, Wang P, Liu Q, Tang Y (2015a) Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea viruses associated with outbreaks of severe diarrhea in piglets in Jiangxi, China 2013. PLoS ONE 10:e0120310 Yang L, Veraksa A (2017) Single-step affinity purification of ERK signaling complexes using the streptavidin-binding peptide (SBP) Tag. Methods Mol Biol 1487:113–126 p g g Song D, Moon H, Kang B (2015b) Porcine epidemic diarrhea: a review of cur- rent epidemiology and available vaccines. Clin Exp Vaccine Res 4:166–176 Yang DQ, Ge FF, Ju HB, Wang J, Liu J, Ning K, Liu PH, Zhou JP, Sun QY (2014) Whole-genome analysis of porcine epidemic diarrhea virus (PEDV) from eastern China. Arch Virol 159:2777–2785 Sun YG, Li R, Jiang LG, Qiao SL, Zhi YB, Chen XX, Xie S, Wu JW, Li XW, Deng RG, Zhang GP (2018) Characterization of the interaction between recom- binant porcine aminopeptidase N and spike glycoprotein of porcine epidemic diarrhea virus. Int J Biol Macromol 117:704–712 Publisher’s Note S N Tian Y, Yu Z, Cheng K, Liu Y, Huang J, Xin Y, Li Y, Fan S, Wang T, Huang G, Feng N, Yang Z, Yang S, Gao Y, Xia X (2013) Molecular characterization and phylo- genetic analysis of new variants of the porcine epidemic diarrhea virus in Gansu, China in 2012. Viruses 5:1991–2004 Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Wang Y, Cai E, Rosenkranz T, Ge P, Teng KW, Lim SJ, Smith AM, Chung HJ, Sachs F, Green WN, Gottlieb P, Selvin PR (2014) Small quantum dots conjugated
https://openalex.org/W3035178703	https://www.journals.vu.lt/ekonomika/article/download/17361/16527	Lithuanian	null	Congratulations to Professor Leonas Simanauskas	Ekonomika	2,004	cc-by	981	The Editorial Board dedicates this issue to Professor Leonas Simanauskas on the occasion of his 70th birthday and extends wannest congratulations and wishes. His talent, dedication and academic achieve- ments are an outstanding example of aca- demic excellence and knowledge. His openness, approachability and sincerity in sharing his academic values and dedi- cation toward preserving these ideals are admired and respected by colleagues, doctoral students and graduate and un- dergraduate students alike. thesis, in which he analyzed the planning of autotransport systems through the use of mathematical and computing methods. His later research works were summa- rized in his habilitation thesis entitled "The Planning Methods of Production Management Information Systems", which he defended in 1989. Professor Simanauskas has published over 100 articles in Lithuanian and for- eign prestigious journals. He has pub- lished eight books, among them two monographs, four textbooks and two methodological and pedagogical books. Professor Simanauskas' career is as follows. In 1953-1958 he was a student of Vilnius University, after which he worked as chief engineer at the Vilnius computer machine factory, and later as head of the engineering department and chief specialist of the Scientific Research Coordinating Committee of the Scien- tific Council at the factory. His scien- tific potential was already apparent in 1962, when he published his first co- authored book on electronic computing techniques. Science was and has re- mained one of his greatest values, which led him to pursue it further. From 1963 to 1968 he continued his research and studies as a doctoral candidate at the Moscow Institute of Economic Statis- tics. In 1968 he defended his doctoral For over two decades Professor Simanauskas has shared his time, energy and experience with undergraduate and graduate students at Vilnius University and many doctoral students, thus contrib- uting to the development of a new gen- eration of young scholars. He is well- known and respected throughout Lithua- nia and teaches and supervises doctoral students at other universities, such as Vilnius Gediminas Technical University and Siauliai University. We are proud of his academic and teaching career dedicated to Vilnius Univeristy, where he has been Professor for more than ten years and served as head of the departments of Economic Cybernetics and Economic Informatics. His academic 163 and service achievements have been ac- knowledged through pennanent positions on the boards of both the Faculty of Ec0- nomics and Vllnius University. The Vilnius University appreciates and thanks Profes- sor Simanauskas for his dedication and fruitful service as President of the Univer- sity Economic Board and Financial Com- mittee. His academic colleages at the Faculty of Economics and throught Lithuania are grateful for his persistence in reviving the journal "Ekonomika" and for his many-year service as Editor-in-Chief of the journal. With great respect for Professor Simanauskas' achievements we wish our colleague all the best and many more creative years in his academic endeavors. The Editorial Board The Editorial Board 164 cSo/'erbiamas profesoriau, cSo/'erbiamas profesoriau, kand.) disertacija, kurioje nagrinėti au- totransporto darbo planavimo naudojant matematinius metodus ir skaičiavimo techniką metodai. Vėlesni mokslo tiria- mojo darbo rezultatai apibendrinti ha- bilituoto daktaro disertacijoje .. Gamy- bos valdymo išskirstytų informacinių sistemų projektavimo metodai", apgin- toje 1989 metais. cSo/'erbiamas profesoriau, nuoširdžiai sveikiname Jus gražaus ir gar- bingo jubiliejaus proga. Jūsų talentas, darbštumas ir mokslinis kūrybiškumas yra gražus mokslinės etikos ir pasišventi- mo mokslo kūrybai bei žinių skleidimui pavyzdys. Mokslo kolegos, doktorantai ir studentai vertina ir gerbia Jus už atviru- mą ir nuoširdumą dalinantis mokslo ver- tybėmis ir principingumą saugant šias vertybes nuo primityvaus populizmo ir di- letantizmo. Mokslinio darbo rezultatai paskelb- ti per 100 mokslinių publikacijų Lietu- vos, užsienio tarptautiniuose bei leidi- niuose su cituojamu indeksu. Išleistos 8 knygos, iš kurių 2 monografijos, 4 va- dovėliai, 2 mokslinio metodinio pobū džio knygos. Savo kelyje i mokslo aukštumas ir pri- pažinimą nuosekliai perėjote studijų, ga- mybos ir mokslo pakopas: 1953-1958 m. studijos Vilniaus universitete, po jų dar- bas Vilniaus skaičiavimo mašinų gamyk- loje, einant vyr. inžinieriaus, vadovau- jančiojo inžinieriaus, biuro viršininko ir tos pačios imo nės Mokslo tarybos Moks- linio tyrimo darbų koordinavimo komi- teto vyr. specialisto pareigas. Jau 1962 m. išryškėjo Jūsų mokslinis polinkis ir kū rybiškumas, kai su bendraautoriais pa- rengėte ir išleidote pirmąją knygą, kurioje skaitytojus supažindinote su to meto elek- tronine skaičiavimo technika. Mokslas Jums buvo ir liko vertybe, kuri lėmė to- lesni Jūsų gyvenimo kelią ir siekius. 1%3- 1968 metais studijos ir mokslo tiriamasis darbas Maskvos ekonomikos statistikos instituto aspirantūroje. 1968 m. sėkmingai apginta daktaro (tuo metu ekon. m. Jau keletą dešimtmečių negailėdami jėgų savo žinias ir patyrimą Jūs pertei- kiate Vilniaus universiteto bakalauro ir magistro programų studentams, vadovau- dami doktorantams svariai prisidedate prie jaunosios mokslininkų kartos ugdy- mo. Esate žinomas, vertinamas ir kvie- čiamas dėstyti, vadovauti doktorantams taip pat Vilniaus Gedimino technikos bei Šaulių universitetuose. Mums malonu, kad Jūsų mokslinė pedagoginė veikla ir kompetencija bren- do, plėtojosi ir atsiskleidė Vilniaus uni- versitete, kur išaugote iki profesoriaus ir daugiau kaip 10 metų vadovavote iš pradžių Ekonominės kibernetikos, vė liau Ekonominės informacijos kated- 165 roms. Mokslinio pedagoginio darbo nuopelnai, asmeninis autoritetas pripa- žinti išrenkant Jus, galima sakyti, nuola- tiniu Ekonomikos fakulteto tarybos ir ke- lių kadencijų Vilniaus universiteto tarybos nariu. Vilniaus universiteto bendruomenė vertina ir dėkoja už Jūsų nuoširdų ir vai- singą darbą einant universiteto Ūkio tary- bos pirmininko, o vėliau Finansų komisi- jos pirmininko pareigas. roms. cSo/'erbiamas profesoriau, Mokslinio pedagoginio darbo nuopelnai, asmeninis autoritetas pripa- žinti išrenkant Jus, galima sakyti, nuola- tiniu Ekonomikos fakulteto tarybos ir ke- lių kadencijų Vilniaus universiteto tarybos nariu. Vilniaus universiteto bendruomenė vertina ir dėkoja už Jūsų nuoširdų ir vai- singą darbą einant universiteto Ūkio tary- bos pirmininko, o vėliau Finansų komisi- jos pirmininko pareigas. vu Ekonomikos fakulteto bei Lietu- vos ekonomikos mokslo kolegos dėkingi už Jūsų pasiaukojamą darbą atgaivinant mokslo darbų "Ekonomika" leidybą ir sėkmingą ilgameti darbą šio leidinio vyr. redaktoriumi. žavėdamiesi Jūsų mokslinės kūry bos brandumu ir gausumu linkime Jums, mielas kolega, kuo geriausios sveikatos bei ilgų ir kūrybingų metų. Redaktorių kolegija 166
https://openalex.org/W3011433641	https://pureadmin.qub.ac.uk/ws/files/221324624/Integrated.pdf	English	null	Current-Source Single-Phase Module Integrated Inverters for PV Grid-Connected Applications	IEEE access	2,020	cc-by	11,803	Published in: IEEE Access Document Version: Publisher's PDF, also known as Version of record Document Version: Publisher's PDF, also known as Version of record Queen's University Belfast - Research Portal: Link to publication record in Queen's University Belfast Research Portal Queen s University Belfast Research Portal: Link to publication record in Queen's University Belfast Research Portal Publisher rights © 2020 The Authors. This is an open access article published under a Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. Publisher rights © 2020 The Authors. This is an open access article published under a Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. 0 0 e u o s This is an open access article published under a Creative Commons Attribution License (https://creativecommons.org/ which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited Current-source single-phase module integrated inverters for PV grid- connected applications co ec ed app ca o s Darwish, A., Alotaibi, S., & Elgenedy, M. A. (2020). Current-source single-phase module integrated inverters for PV grid-connected applications. IEEE Access, 8, 53082-53096. https://doi.org/10.1109/ACCESS.2020.2981552 Take down policy Th R h P Take down policy The Research Portal is Queen's institutional repository that provides access to Queen's research output. Every effort has been made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the Research Portal that you believe breaches copyright or violates any law, please contact openaccess@qub.ac.uk. Open Access This research has been made openly available by Queen's academics and its Open Research team. We would love to hear how access to this research benefits you. – Share your feedback with us: http://go.qub.ac.uk/oa-feedback General rights g Copyright for the publications made accessible via the Queen's University Belfast Research Portal is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Take down policy The Research Portal is Queen's institutional repository that provides access to Queen's research output. Every effort has been made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the Research Portal that you believe breaches copyright or violates any law, please contact openaccess@qub.ac.uk. Current-source single-phase module integrated inverters for PV grid- connected applications Darwish, A., Alotaibi, S., & Elgenedy, M. A. (2020). Current-source single-phase module integrated inverters for PV grid-connected applications. IEEE Access, 8, 53082-53096. https://doi.org/10.1109/ACCESS.2020.2981552 Current-source single-phase module integrated inverters for PV grid- connected applications Darwish, A., Alotaibi, S., & Elgenedy, M. A. (2020). Current-source single-phase module integrated inverters for PV grid-connected applications. IEEE Access, 8, 53082-53096. https://doi.org/10.1109/ACCESS.2020.2981552 IEEE POWER & ENERGY SOCIETY SECTION eceived February 16, 2020, accepted March 6, 2020, date of pu Received February 16, 2020, accepted March 6, 2020, date of publication March 17, 2020, date of current version March 26, 2020. Digital Object Identifier 10.1109/ACCESS.2020.2981552 Current-Source Single-Phase Module Integrated Inverters for PV Grid-Connected Applications AHMED DARWISH 1, SAUD ALOTAIBI 1, AND MOHAMED A. ELGENEDY 2,3, (Senior Member, IEEE) 1Department of Engineering, Lancaster University, Lancaster LA1 4WY, U.K. 2Electrical Engineering Department, University of Strathclyde, Glasgow G1 1XQ, U.K. 3Department of Electrical Power Engineering, Alexandria University, Alexandria 21526, Egypt Corresponding author: Ahmed Darwish (a.badawy@lancaster.ac.uk) ABSTRACT This paper presents a modular grid-connected single-phase system based on series-connected current-source module integrated converters (MICs). The modular conﬁguration improves the reliability, redundancy and scalability of photovoltaic (PV) distributed generators. In this system, each PV panel is connected to a dc/ac inverter to permit individual Maximum Power Point Tracking (MPPT) operation for each panel. Thus, the harvested power from the PV system will increase signiﬁcantly. There are four different inverter topologies suitable to be used as MICs with different performances in terms of ﬁltering elements size, power losses, efﬁciency, output voltage range, and high frequency transformers’ size. For the MPPT control, the oscillating even order harmonic components should be eliminated from the inverter’s input side otherwise the maximum power cannot be extracted. The proposed modulation scheme in this paper will ease the control of inverter’s input and output sides. Therefore, the 2nd order harmonic in the input current can be eliminated without adding new active semiconductor switches. A repetitive controller coupled with proportional-resonant controllers are employed to achieve accurate tracking for grid side as well as input side currents. Comparisons and performance evaluations for the proposed MICs are presented and validated with 1 kVA prototype controlled by TMS320F29335 DSP. INDEX TERMS Photovoltaic generation, series-connected, power decoupling, Cuk converter, Sepic con- verter. FIGURE 1. Grid-connected PV system: (a) I-V and P-V curves and (b) Modular configuration. IEEE POWER & ENERGY SOCIETY SECTION Open Access Thi h Open Access This research has been made openly available by Queen's academics and its Open Research team. We would love t this research benefits you. – Share your feedback with us: http://go.qub.ac.uk/oa-feedback Download date:24. Oct. 2024 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0 I. INTRODUCTION Because it is simple to assem- ble, it can be used by users without professional knowledge and skills as ‘‘plug-and-play’’ systems [5]. On the downside, the micro-inverter is required to provide high boosting ratio to match the PV panels’ voltages to the grid’s ac voltage level and hence the semiconductor losses are increased and the power density may be reduced [5]. Due to the increased voltage and current stresses, the micro-inverter’s reliability will be affected. Also, without central high voltage dc-bus the micro-inverter is required to decouple the instantaneous pulsating 2nd order harmonic energy components generated in single-phase systems otherwise the MPPT will not be achieved [6]. As a compromise between centralised, string and ac module structures, the series-connected modular struc- ture can improve the performance of the grid-tied PV system signiﬁcantly [7]. As shown in Fig. 1b, each module in this system is connected to a single PV panel while the output sides of the symmetrical modules are connected in series to match the output voltage at the point of common cou- pling (PCC) to the grid voltage. Thus, the voltage and cur- rent stresses in the module inverters are shared between the inverters’ devices and hence the reliability increases, and the power losses decreases. Moreover, the modular structure of the series-connected system provides a degree of redundancy and scalability. FIGURE 2. Isolated converters (a) C5 (Cuk), (b) F5, (c) G5 (Sepic), (d) P5. To eliminate the leakage currents ﬂowing between the PV neutral point and the ac network ground which create hazards, and affect the operation and lifetime, the selected modules are isolated with small-size HF transformer cores [10]–[12]. The transformer cores’ sizes can be reduced by increasing the switching frequency of the semiconductor devices in the inverter. Moreover, the isolating HF transformers provide voltage boosting and reduce Electro-Magnetic Interference (EMI). Examining the different structures of power electronic converters, four buck-boost converters can be found with the features stated earlier [12], [13]. These converters are demon- strated in Fig. 2 and will be labelled in this paper as C5 (Cuk), F5, G5 (SEPIC), and P5. The single-phase descendants from these converters are generated and shown in Fig. 3. g g These four inverter modules can be employed in the mod- ular PV system in Fig. 1b provided that the input power is decoupled from output. This paper proposes modiﬁed modu- lation schemes for the single-phase inverters in Fig. I. INTRODUCTION The remarkable development in the installed capacity of distributed generators (DGs) increases the international aspi- ration for improving the performance of power inverters employed in renewable energy systems (RESs) such as photo- voltaic (PV) systems [1]. The employed inverters are required to have small size, light weight and low power losses. The inverter’s power losses are generated from several sources including conduction, switching losses in the semiconductor devices, and equivalent series resistance (ESR) in the induc- tors and capacitors. Another implicit source of PV system’s power losses is when the PV panel is not able to produce its maximum power because of drawing time-variant currents by the employed inverter. In this case, the maximum power point tracking (MPPT) controller fails to operate the system at the peak of the P–V curve, see Fig. 1a. As reviewed in [2]–[4], the PV systems can be classiﬁed into three main FIGURE 1. Grid-connected PV system: (a) I-V and P-V curves and (b) Modular configuration. categories: string inverter, centralised inverter, and ac module. Traditionally, the centralised and the string inverter systems are preferred for their power density, reduced cost and power losses. Thus, they dominated the commercial market. In the centralised inverter systems, PV modules are connected in series to boost the dc voltage bus. Then, a centralised inverter generates the necessary ac voltages and currents to be fed to The associate editor coordinating the review of this manuscript and approving it for publication was Pierluigi Siano . 53082 53082 VOLUME 8, 2020 VOLUME 8, 2020 A. Darwish et al.: Current-Source Single-Phase Module Integrated Inverters for PV Grid-Connected Applications FIGURE 2. Isolated converters (a) C5 (Cuk), (b) F5, (c) G5 (Sepic), (d) P5. the residential ac loads or to be injected into the local grid. Thus, the PV panels share the same dc current and the MPPT operation becomes complicated. Although the centralised inverter systems reduce the power losses inside the power converters, the harvested energy is reduced because of the MPPT may not be achieved in all circumstances. In addition, the central inverter transfers the total power and therefore the system’s scalability is restricted [3]. In ac module sys- tem, the PV panels are connected to micro-inverters which boosts the low dc voltage from a single panel to higher ac voltage at the grid frequency [4]. I. INTRODUCTION Because each PV panel is connected to a micro-inverter, the output current of each panel can be independently controlled and therefore a better MPPT performance can be achieved. Because it is simple to assem- ble, it can be used by users without professional knowledge and skills as ‘‘plug-and-play’’ systems [5]. On the downside, the micro-inverter is required to provide high boosting ratio to match the PV panels’ voltages to the grid’s ac voltage level and hence the semiconductor losses are increased and the power density may be reduced [5]. Due to the increased voltage and current stresses, the micro-inverter’s reliability will be affected. Also, without central high voltage dc-bus the micro-inverter is required to decouple the instantaneous pulsating 2nd order harmonic energy components generated in single-phase systems otherwise the MPPT will not be achieved [6]. As a compromise between centralised, string and ac module structures, the series-connected modular struc- ture can improve the performance of the grid-tied PV system signiﬁcantly [7]. As shown in Fig. 1b, each module in this system is connected to a single PV panel while the output sides of the symmetrical modules are connected in series to match the output voltage at the point of common cou- pling (PCC) to the grid voltage. Thus, the voltage and cur- rent stresses in the module inverters are shared between the inverters’ devices and hence the reliability increases, and the power losses decreases. Moreover, the modular structure of the series-connected system provides a degree of redundancy and scalability. the residential ac loads or to be injected into the local grid. Thus, the PV panels share the same dc current and the MPPT operation becomes complicated. Although the centralised inverter systems reduce the power losses inside the power converters, the harvested energy is reduced because of the MPPT may not be achieved in all circumstances. In addition, the central inverter transfers the total power and therefore the system’s scalability is restricted [3]. In ac module sys- tem, the PV panels are connected to micro-inverters which boosts the low dc voltage from a single panel to higher ac voltage at the grid frequency [4]. Because each PV panel is connected to a micro-inverter, the output current of each panel can be independently controlled and therefore a better MPPT performance can be achieved. VOLUME 8, 2020 I. INTRODUCTION 3 where the output bridge switches are operated independently with respect to the input side to provide an additional degree of freedom. In this way, both input and output sides’ currents can be controlled together and the even harmonics in the input currents will be eliminated and hence the MPPT controller will be able to maximise the output power of the system. Because the proposed converters and their descendants have two or more right-half-plane (RHP) zeros, the classical con- trollers will not be able to give the required gain at 50 Hz (i.e to reduce the sensitivity function S(s) gain) and provide the required stability margins in the same time. Therefore, this paper presents a control scheme based on Feed-Forward Repetitive Control (RC) with a Proportional-resonant (PR) controller to ensure that the reference signals can be tracked and the stability conditions will be satisﬁed. The proposed controller can provide good disturbance rejections at the mul- tiples of the controller periodic signals. The PR controllers are responsible for tracking the reference signals with zero steady-state errors. To draw constant currents from the PV panels and extract the maximum power, two current components in the input side current should be minimised. The ﬁrst is the high fre- quency (HF) current ripple which can be eliminated by an input ﬁltering such as capacitors or inductors [8]. In most of cases, a bulky electrolytic capacitor is employed as an input ﬁlter and therefore the power density is reduced. Moreover, the lifetime of this capacitor is halved for 10 ◦C increase in the temperature and hence, the inverter’s reliability is limited [8]. With current-sourced inverters, the HF current ripple is eliminated with an input inductor and, if necessary, small plastic capacitors [9]. The second current component to be eliminated is the 2nd order harmonic current which is generated from the operation of single-phase inverters. This component should be decoupled to enable the MPPT controller to settle at the peak of the P–V curve and harvest the maximum available power in the solar source. The rest of this paper is organised as follows: Section. II presents the operation’s concept of the inverters. Section III presents the modiﬁed modulation techniques of the inverters. 53083 53083 VOLUME 8, 2020 VOLUME 8, 2020 A. Darwish et al.: Current-Source Single-Phase Module Integrated Inverters for PV Grid-Connected Applications FIGURE 3. I. INTRODUCTION Single-phase inverter modules: (a) Cuk, (b) Sepic, (c) F5 and (d) P5. a complementary manner with the input side switch. Conse- quently, only the input or the output sides’ currents can be controlled. In these inverters, the output current is controlled to inject the required power into the local grid while the input side current is left uncontrolled. The published single-phase inverters can be categorized into three main types. The ﬁrst is the differential inverter as presented in [14] and [17], see Fig. 4a. In this conﬁguration, two two-switch two-diodes Cuk converters are connected differentially across the ac load. Each converter, by controlling its input side switch, pro- duces one half-cycle of the output voltage and current. The unfolding-type Cuk inverter has been proposed in [15], see Fig. 4b. In this type, the rectiﬁed voltage is generated across C3 with controlling the input switch S1 and then this volt- age is directed to the output load using the bridge switches S2 →S5. This means that the controlling device is S1 while the switches (S2 and S5) are ON for the positive half-cycle of the generated 50/60 Hz voltage while (S3 and S4) are ON for the negative half-cycle. In the bridgeless Cuk inverter proposed in [16], see Fig. 4c, the bridge has been moved before the output stage to improve the efﬁciency. With the same modulation concept, the input switch S1 is responsible for shaping the output voltage while S2 →S5 are only direc- tors for the positive and negative parts of the ac waveforms. Because only one switch is responsible for generating the output voltage/current in the abovementioned inverters, it is only possible to control either the output or the input sides. Because of the oscillating power nature in ac single-phase operation, the input current will be composed of a dc plus a 2nd order harmonic components if only the output side is controlled. It is can be deduced from curves in Fig. 1a and the input currents of the inverters in Fig. 4 that the operational points with the time-variant input currents will be oscillating between the points 2 and 3 of the I-V curve. This causes the power to be oscillating between points 4 and 3 which prevents achieving the PV maximum power and increases the temperature of the PV module, and decrease its lifetime [10]. I. INTRODUCTION The next section presents the modiﬁed mod- ulation scheme for the proposed inverters in order to ﬁx this problem and eliminate the even harmonics from the input side currents. FIGURE 3. Single-phase inverter modules: (a) Cuk, (b) Sepic, (c) F5 and (d) P5. III. MODULATION SCHEMES The generic operation of the modular system is explained in and Section IV. Section V presents the RC control scheme used to operate the system. Section VI presents the experi- mental results of the MIC PV system using the four inverters. The parameter’s selection process as well as comparisons between the different modules are presented in section VII. Without loss of generality, the analysis and principle of operation will be explained for the Ćuk-based module, in Fig. 3a, and the operation of the other candidates can then be deduced similarly. The main difference between these inverters is in the element that stores the oscillating energy. A discussion regarding to this issue will be considered later in this paper. To operate the Cuk module in continuous current mode (CCM), the input and output inductors (Lin and Lout) will be assumed large enough. The module has one switch at the input side Sin and four switches at the output bridge S1 →S4. II. INVERTER MODULES Some single-phase descendants of the Cuk and Sepic con- verters have been published in [14]–[17]. In all these single-phase inverters, the output switches are operating in 53084 VOLUME 8, 2020 53084 VOLUME 8, 2020 A. Darwish et al.: Current-Source Single-Phase Module Integrated Inverters for PV Grid-Connected Applications FIGURE 5. Operating modes of the Cuk inverter. (a) M1, (b) M2 and (c) M3. FIGURE 4. Cuk-based inverters: (a) Differential configuration in [14] and [17] (b) Unfolding-type in [15] and (c) Bridgeless type in [16]. FIGURE 5. Operating modes of the Cuk inverter. (a) M1, (b) M2 and (c) M3. ﬂows through freewheeling diodes D2 and D3 (or D1 and D4 if it is in the negative half cycle). Cp and Cs are charging leading io to decrease. This mode, where the input current is increasing while the output current is decreasing, does not exist in the Cuk converter and its inverter descendants described presented in [14], [15]. Because it adds an additional degree of freedom, this mode enables the decoupling of the input and output cur- rents behaviors and therefore it helps in controlling both input and output currents. The theoretical waveforms of the proposed inverter are shown in Fig. 6. FIGURE 4. Cuk-based inverters: (a) Differential configuration in [14] and [17] (b) Unfolding-type in [15] and (c) Bridgeless type in [16]. A. MODES OF OPERATION There are three operating modes for this inverter from the perspective of the middle capacitors Cp and Cs, charging mode M1, discharging mode M2, and charging mode M3. If ig is positive, the operation can be described as: B. AVERAGE MODEL OF THE INVERTER (6) It is shown that Pac is composed of a 2nd order ac components, hence the desired capacitor voltage vCt can be assumed as: vCt(t) = VCdc + VCac sin(2ωt + φ) (7) (7) Solving (6) and (7) yields: φ = tan−1 ωLoIg cos 2γ −Vg sin 2γ Vg cos 2γ + ωLgIg sin 2γ (8) Vcac = VgIg cos 2γ + ωLoI2 g cos 2γ 4ωCtVcdc cos φ (9) (8) (9) (9) Consequently, the oscillating component of the inverter’s power can be eliminated from the input side by controlling the capacitors voltage vCt as in (7), (8) and (9). The required duty cycle ratios for that can be calculated from the state-space representation in (2). The ﬁrst row if this representation can be written separately as: FIGURE 6. Theoretical waveforms of the Cuk inverter. (a) Positive half cycle and (b) negative half cycle. +   1 Lin 0 0 0 0 0 0 0 0 −1 Lg   vin vg (2) (2) diin(t) dt = D(t) −1 HLin vCt(t) + 1 Lin Vin (10) (10) It is desired to keep the input current constant with time and therefore (10) can be re-written as: Using the average modelling method in [9], [12], the Cuk-based module is averaged along the switching cycle ts and the average model as in (2). where the state vector x(t) = [iin(t) vCt(t) io(t) vo(t) ig(t)] and y(t) = ig(t) is the grid current. The state vCt(t) is HvCp(t) + vCs(t), H is the turns ratio (Ns/Np), Ct is CpCs/(Cp + H2Cs), d1(t) is the duty cycle ratio of M2, d2(t) is the duty cycle ratio of M3 while D(t) is the duty cycle ratio of switch Sin. B. AVERAGE MODEL OF THE INVERTER p p • During M1 (0 ≤t < toff ), Sin is turned OFF while one of the switches S1 or S4 is switched ON. The input current iin decreases and ﬂows through Cp which stores energy, see Fig. 5a and Fig.6. Meanwhile, the output current io is ﬂowing through D2, S4, (or D3 and S1). When the grid current ig reverses direction in the negative half-cycle, the output current passes through S2 and D4 (or D1 and S3) while the input side remains the same. Assuming that d1 and d2 are the duty cycle ratios of modes M2 and M3 respectively, they can be expressed as: d1 = t1 ts , d2 = t2 ts and D = d1 + d2 (1) ˙x(t) =   0 D −1 HLin 0 0 0 1 −D HCeq 0 d2 −d1 HCeq 0 0 0 d1 −d2 Lo 0 −1 Lo 0 0 0 1 Co 0 −1 Co 0 0 0 1 Lg 0   x(t) 53085 (1) • During M2 ( toff ≤t < toff +t1), Sin is turned ON leading iin to increase. As in Fig. 5b, io ﬂows through S1 and S4. As Cp and Cs discharge during this mode, their energy transfers to Lo and the output current increases. During the negative cycle of ig, the output current ﬂows through S2 and S3 while the input side remains the same. • During M3 (toff + t1 ≤t < ts), Sin is turned ON and the input current is still increasing. The output current • During M3 (toff + t1 ≤t < ts), Sin is turned ON and the input current is still increasing. The output current VOLUME 8, 2020 A. Darwish et al.: Current-Source Single-Phase Module Integrated Inverters for PV Grid-Connected Applications A. Darwish et al.: Current-Source Single-Phase Module Integrated Inverters for PV Grid-Connected Applications CtvCt(t)dvCt(t) dt = −Pac(t)= VgIg cos(2ωt −γ ) 2 (6) FIGURE 6. Theoretical waveforms of the Cuk inverter. (a) Positive half cycle and (b) negative half cycle. B. AVERAGE MODEL OF THE INVERTER 0 = D(t) −1 HLin vCt(t) + 1 Lin Vin D(t) = vCt(t) −Hvin vCt(t) (11) (11) In the same way, the third row of the state-space model is written as: The grid voltage and current can be expressed as: vg = Vg sin(ωt) (3a) ig = Ig sin(ωt −γ ) (3b) dio(t) dt = 1 −D(t) Lo vCt(t) −d2(t) −d1(t) Lo vo(t) (12) (12) (3b) Then, the instantaneous power injected to the grid (Pgrid) can be calculated from: Arranging (12), the duty cycle ratios can be found from: Arranging (12), the duty cycle ratios can be found from: d1(t) = Lodio dt 2vCt(t) + vo(t) −d1(t) 2vCt(t) + D(t) 2 (13a) d2(t) = D(t) −d1(t) (13b) Pgrid(t) = vg(t).ig(t) (4a) Pgrid(t) = Pdc + Pac(t) (4b) Pdc = VgIg cos γ 2 (4c) Pac = VgIg 2 sin(2ωt −γ −π 2 ) (4d) (13a) (13b) (4c) The duty cycle ratios can be calculated from (13) assuming that io ≈ig and vo ≈vg. These duty cycle ratios are compared with carrier signals at the switching frequency fs = 1/ts and used to operate the inverter’s switches. Thus, the 2nd order component can be eliminated from the input side. With the duty-cycle ratios d1 and d2, the inverter will be able to gen- erate the required output sinusoidal current with keeping the input current constant with time which is necessary for MPPT operation. The calculations errors due to the parameters’ mismatches, operational variations or external disturbances will be corrected by the closed-loop controller which will be presented later in this paper. (4d) From (4d), the oscillating part of the inverter’s power can be obtained from the known grid voltage and current (Vg and Ig). As stated earlier, the additional mode of operation will add a new control parameter d2 which will allow for controlling the capacitors voltages vCt. This voltage can be controlled to gen- erate the oscillating power component instead of supplying it from the input side as: Pac(t)+CtvCt(t)dvCt(t) dt = 0 (5) (5) 53086 VOLUME 8, 2020 VOLUME 8, 2020 A. Darwish et al.: Current-Source Single-Phase Module Integrated Inverters for PV Grid-Connected Applications If the cables losses are neglected, the total output power in normal conditions is calculated from: If the cables losses are neglected, the total output power in normal conditions is calculated from: IV. MODULAR SYSTEM The MIC based system is shown in Fig. A. NORMAL CONDITIONS The grid current ig ﬂows through each modules’ ac sides and determines the active and reactive power ﬂow. Fig. 7 shows the phasor diagram and illustrate the system’s currents and voltages with respect to the grid voltage vg. FIGURE 7. Phasor diagram of the modular converter in Fig. 1b. FIGURE 8. I–V and P–V characteristics of shaded PV arrays. FIGURE 7. Phasor diagram of the modular converter in Fig. 1b. The inverters’ voltages can be expressed as: voj = Voj sin(ωt + θj) (14 vPCC = Vo1 sin(ωt + θ1) + Vo2 sin(ωt + θ2) + . . . Von sin(ωt + θn) = n X i=1 voj = vg + igZg (15 voj = Voj sin(ωt + θj) (14) vPCC = Vo1 sin(ωt + θ1) + Vo2 sin(ωt + θ2) + . . . Von sin(ωt + θn) = n X i=1 voj = vg + igZg (15) FIGURE 8. I–V and P–V cha To explain that, ass other (n-k) modules ar can be expressed as: ′ (14) FIGURE 8. I–V and P–V characteristics of shaded PV arrays. To explain that, assume that k modules are shaded while other (n-k) modules are unshaded. The new grid current ig‘(t) can be expressed as: = n X i=1 voj = vg + igZg (15) (15) i′ g(t) = kVo_sh sin(ωt+θsh)+(n−k)Vo sin(ωt+θ)−Vg sin(ωt) Zg = I′ g sin(ωt −γ ) (21) i′ g(t) = kVo_sh sin(ωt+θsh)+(n−k)Vo sin(ωt+θ)−Vg sin(ωt) Zg i′ g(t) where θj is the module voltage phase angle, γ is the grid power factor angle, and Zg is the grid impedance. In normal operation, the modules’ output voltages have the same mag- nitude and phase angle as: g = I′ g sin(ωt −γ ) (21) (21) = I′ g sin(ωt −γ ) Vo1 = Vo2 = · · · = Von (16) θ1 = θ2 = · · · = θn (17) (16) (17) where Vo_sh and θsh are the amplitude and the phase shift angle of the shaded modules’ output voltages respectively while Ig‘ is the amplitude of the new grid current. B. PARTIAL SHADING CONDITIONS Fig. 8 shows the I–V and P–V characteristics during shading conditions where the shaded PV module is expected to reduce its maximum power point. Although the characteristics of the PV modules vary with their type and connection, the P–V curve will always have reduced peak value in shading conditions. Thus, the optimum points of the shaded modules move to (Vin_sh, Iin_sh, and Pmod_sh) so the grid current should be changed to another value in order to extract the maximum available power from the shaded and unshaded PV modules. B. AVERAGE MODEL OF THE INVERTER 1b where the out- put voltages of the series n modules (vo1 to von) are added together to generate the required total voltage, vPCC, at the point of common coupling (PCC). The four candidates shown in Fig. 3 can be used as inverter module with different advantages and disadvantages. The input PV modules can be connected separately to each inverter module (as shown in Fig. 1b) in case of low modules’ currents while several modules can be connected to one PV array if the PV arrays’ currents are high enough. If the input current of the inverter modules (output of the PV modules) is left uncontrolled, the operating point will be oscillating between points 3 and 4 in the power curve shown in Fig. 1a. So, the extracted power will be limited to almost have the available power from the PV module. Ptotal = nVoIg cos(θ −γ ) 2 ≈VgIg cos(γ ) 2 (19) (19) The power of each module is calculated from: P mod _u = VinIin = VoIg cos(θ −γ ) 2 ≈VgIg cos(θ −γ ) 2n (20) (20) If the desired operating point is known from the MPPT calculations at the optimum points (Vin, Iin, and Pmod_u), the reference grid current ig can be obtained from (18), (19) and (20). If the desired operating point is known from the MPPT calculations at the optimum points (Vin, Iin, and Pmod_u), the reference grid current ig can be obtained from (18), (19) and (20). A. NORMAL CONDITIONS The power of the shaded modules is calculated from: Starting from the normal conditions, the active power is evenly shared between the modules, the grid current is expressed as: P mod _sh = Vin_shIin_sh ≈ Vo_shI′ g cos(θsh −γ ) 2 (22) (22) ig(t) = n [Vo sin(ωt + θ)] −Vg sin(ωt) Zg = Im sin(ωt −γ ) The total power harvested from the system becomes: The total power harvested from the system becomes: (23) P′ total = kVin_shIin_sh + (n −k)VinIin (18) VOLUME 8, 2020 53087 53087 A. Darwish et al.: Current-Source Single-Phase Module Integrated Inverters for PV Grid-Connected Applications FIGURE 9. Block diagram of the proposed controller. FIGURE 9. Block diagram of the proposed controller. The power injected into the grid can be expressed as: The power injected into the grid can be expressed as: is used as the duty cycle ratio input to the inverter module. To obtain the transfer function of the Cuk inverter Ginv(z), the state space representation of the inverter is linearized as in [18] to obtain the small-signal model as in (27) where the superscript ‘∼’ refers to the incremental variation of a state while the subscript ‘e’ stands for the equilibrium point of a state. P′ total = (n −k)VoI′ g cos(θ −γ ) + kVo_shI′ g cos(θsh −γ ) 2 ≈ VgI′ g cos(γ ) 2 (24) (24) To extract the maximum available power from the shaded and unshaded PV modules, the new reference value is set to I‘∗ g which can be obtained from solving (22), (23) and (24). To extract the maximum available power from the shaded and unshaded PV modules, the new reference value is set to I‘∗ g which can be obtained from solving (22), (23) and (24). ˙˜x(t) =   0 De −1 HLin 0 0 0 1 −De HCeq 0 d2e −d1e HCeq 0 0 0 d1e −d2e Lo 0 −1 Lo 0 0 0 1 Co 0 −1 Co 0 0 0 1 Lg 0   ˜x(t) +   Vcte HLin Vcte HLin −(Iine + Ige) HCeq (Ige −Iine) HCeq Vcte Lo −Vcte Lo 0 0 0 0   ˜d1 ˜d2 ˜y = 0 0 0 0 1 ˜x(t) (27) V. CLOSED-LOOP CONTROL The two RHP zeros in the Cuk inverter’s transfer function complicate the control design. The RC-based control schemes learn the behaviour of the system with many repetition sam- ples and use this information to reduce the system error and improve the tracking error in the next trial [18]–[20]. So, it can improve tracking accuracy with several repeti- tions [19]. The controller in Fig. 9 is composed of three control loops with different tasks and this will be explained in the following subsections.  g  +   Vcte HLin Vcte HLin −(Iine + Ige) HCeq (Ige −Iine) HCeq Vcte Lo −Vcte Lo 0 0 0 0   ˜d1 ˜d2 ˜y = 0 0 0 0 1 ˜x(t) (27) D. CONTROL SYSTEM DESIGN The overall control system comprises of four gains (k, kp, kr and kr‘) and a LPF Q(z). The gains and the LPF should be selected so that the roots of the system are always inside the unity circle. This can be ensured from the following steps: Ginv(z)= ˜ig(z) ˜d1(z) = m5z5+m4z4+m3z3+m2z2+m1z+m0 n5z5+n4z4+n3z3+n2z2+n1z + n0 (29) 0 (29) a) The LPF can be expressed as: Without the RC controller, the closed-loop transfer T(z) to control the output current ig can be expressed as: Q(z) = α0 + α1z−1 + α2z−2 + ... + αnz−n (34) (34) T(z) = ˜ig(z) I∗g (z) = GPR(z)Ginv(z) 1 + GPR(z)Ginv(z) (30) where n is the ﬁlter’s order (30) The roots of the LPF are all inside the unity circle if the ﬁlter’s gains are selected as: B. FEED-FORWARD RC CONTROLLER α0 + α1 + α2 + ... + αn = 1 (35) (35) The feed-forward RC controller is shown in Fig. 9. The internal model principle (IMP) states that a closed-loop controller is able to eliminate the steady state error of a system if the sampling frequency of the controller is a multiple of the periodic signal which is desired to be con- trolled [19]. The required integral action can be modelled as unit delays. The ratio (N) of the sampling frequency (fs) and the controlled signal’s frequency (f ) deﬁnes the number of memory locations required. The Feedforward RC controller includes the internal model as delays followed by a low-pass ﬁlter (LPF) Q(z). This LPF increases the robustness of the system by reducing the high-frequency ripples in the pro- cessed signals. From Fig. 8, the transfer function of the RC controller is: b) To stabilise the closed-loop transfer function T(z), the roots of (1-kT(z)) = 0 should be kept inside the unit circle. This can be achieved by the careful choice of the gains k, kp and kr. From the small-signal model in (27), the poles of the Cuk inverter transfer function Ginv(z) will move to the RHP with increasing the duty cycle ratios or the input voltage. Also, the values of the two zeros will become smaller. This means that the stability margins of the inverter will are inversely pro- portional with the invert’s power. Thus, the controller gains should be designed at the maximum expected power for the inverter. b) To stabilise the closed-loop transfer function T(z), the roots of (1-kT(z)) = 0 should be kept inside the unit circle. This can be achieved by the careful choice of the gains k, kp and kr. From the small-signal model in (27), the poles of the Cuk inverter transfer function Ginv(z) will move to the RHP with increasing the duty cycle ratios or the input voltage. Also, the values of the two zeros will become smaller. This means that the stability margins of the inverter will are inversely pro- portional with the invert’s power. Thus, the controller gains should be designed at the maximum expected power for the inverter. A. OUTPUT PR CONTROLLER A PR controller tuned at the grid angular frequency ω is able to provide high gain at this frequency. The PR controller’s transfer function in the continuous s-domain is: GPR(s) = kp + krs s2 + ω2 (25) (27) (25) where kp and kr are the proportional and resonant gains of the controller respectively. The transfer function in (25) can be discretised using Tustin method as: Based on the small signal model in (27), the s-domain transfer function between the control input d1 to the output ig can be deduced as: GPR(z)=kp+ 2krts(1−z−2) 4+(ωts)2+2[(ωts)2−4]z−1+[(ωts)2+4]z−2 (26) Ginv(s)= ˜ig(s) ˜d1(s) = a3s3 + a2s2 + a1s + a0 b5s5 + b4s4 + b3s3 + b2s2 + b1s + b0 (28) z (26) (28) As shown in the proposed controller in Fig. 9, the reference grid current I∗ g (z) is compared with the actual measured value of the grid current to give the errors signal E(z) which is fed to the PR controller. Then, the output of the PR controller where the parameters ai for i = 1,2,3 and bj for j = 1, . . . , 5 are omitted for brevity. To design the controller in discrete-time domain, the transfer function is obtained using VOLUME 8, 2020 53088 VOLUME 8, 2020 A. Darwish et al.: Current-Source Single-Phase Module Integrated Inverters for PV Grid-Connected Applications Tustin method and arranged as: Tustin method and arranged as: D. C Tustin method and arranged as: Tustin method and arranged as: B. FEED-FORWARD RC CONTROLLER c) The roots of 1 + GR(z)Ginv(z) = 0 are located inside the unit circle by adjusting the resonant controller’s gain kr‘ The eliminating time required until iin(t) is constant (and the the 2nd order harmonic is removed) is not critical. Thus, kr‘ can be chosen freely only to keep GR(z)Ginv(z) is stable without the necessity of the controller to be fast. GFFRC(z) = UF(z) E(z) = z−NQ(z) 1 −z−N (31) (31) where N = fs/f . The poles of GFFRC(z) are located at 2πjf where j = 0, 1, 2 . . . J (J = N/2). As shown from (31), the RC controller provides very high gain if the LPF gain is 1. Thus, the closed-loop system with the RC controller can eliminate the steady-state error and reject any arising disturbances. The error transfer function of the overall system can be found from: FIGURE 10. Experimental rig. E(z) I∗g (z) = k(1 + GPR(z)Ginv(z))−1(1 −z−N) 1 −z−NQ(z)(1 −kT(z) (32) (32) C. INPUT CURRENT CONTROLLER The input current controller is responsible for correcting the calculation errors from equation (13) due to parameters variations or external disturbances. Same as the PR transfer function in (25) and (26), the resonant controller is tuned at double the grid frequency are expressed as: GR(s) = k‘ rs s2 + 4ω2 (33a) GR(z) = 0.5k‘rts(1 −z−2) 1 + (ωts)2 + [(ωts)2 −2]z−1 + [(ωts)2 + 1]z−2 (33b) (33a) FIGURE 10. Experimental rig. FIGURE 10. Experimental rig. VI. EXPERIMENTAL RESULTS Fig. 10 shows the experimental prototype for the modular energy conversion system. The modules have changeable Fig. 10 shows the experimental prototype for the modular energy conversion system. The modules have changeable where kr‘ is the resonant gain of the controller. VOLUME 8, 2020 A. Darwish et al.: Current-Source Single-Phase Module Integrated Inverters for PV Grid-Connected Applications FIGURE 11. Experimental results of Cuk-based modular system: (a) input currents, (b) Capacitors voltages, (c) module’s output voltages, and (d) grid voltage and current. TABLE 1. System’s parameters. TABLE 1. System’s parameters. terminals to ease testing different inverter modules. The sys- tem is controlled by TMS32028335 DSP and has the same parameters in Table 1 to inject 1 kW into the grid. The nominal power of each module in this system is 250 W and the maximum allowed power is limited to 500 W and the maximum input voltage is 250V. FIGURE 11. Experimental results of Cuk-based modular system: (a) input currents, (b) Capacitors voltages, (c) module’s output voltages, and (d) grid voltage and current. FIGURE 12. Experimental results of Sepic-based system: (a) input currents, (b) Ls currents, (c) output voltages, and (d) grid voltage/current. At these conditions, the worst-case transfer function for the Cuk inverter can be obtained from (29) as: Ginv(z)= ˜ig(z) ˜d1(z) = −0.01z3−0.02z2+0.05z+0.04 z5+0.6z4+0.45z3−0.5z2+0.25z −0.15 (36) (36) The PR controller gains kp and kr are chosen as 1.5 and 1.8 respectively so the gain and phase margins of the closed loop transfer function T(z) in (28) are 30 dB and 80 ◦respec- tively. Practically, three samples are sufﬁcient for the LPF to perform the required ﬁltering and hence it is chosen as Q(z) = 0.15z−2 + 0.15z−1 + 0.7. For the FFRC controller, the roots of 1-kT(z) will be all inside the unit circle if the gain k = 1.5. The input’s side resonant controller gain can be chosen independently as a small value where kr‘ = 0.2. The same steps have been taken to ﬁne-tune the con- trol loops’ gains for other three inverters Sepic, F5 and P5. Fig. 11 shows the operation of the system when operated by four Cuk-based modules in the normal condition. Fig 11a shows the input current of the four modules in the steady state where the 2nd order harmonic is eliminated in all modules. Fig. 11b shows the voltages across Cp and Cs. Fig. VI. EXPERIMENTAL RESULTS It should be noted that if more than one PV module are completely shaded, the unshaded modules will be required to generate higher output voltages to compensate for the differ- ence and match the voltage to the grid. If the output voltages FIGURE 15. FFT of the inverters’ output currents: (a) Cuk, (b) Sepic, (c) F5, and P5. FIGURE 16. Partial shading conditions: (a) input voltages/currents, (b) module’s output voltages, (c) capacitors voltages, and (d) grid voltage and current. and performances. All modules have the same semiconductor stresses but they have different current and voltage ripples and therefore the passive elements’ values will vary in order to achieve the same operating conditions. A. PARAMETERS SELECTION The detailed parameter selection process for switched mode power supplies has already been discussed in many publica- tions [8]–[10] and [13]. This subsection will present a practi- cal methods to select the passive elements and the transformer FIGURE 15. FFT of the inverters’ output currents: (a) Cuk, (b) Sepic, (c) F5, and P5. FIGURE 15. FFT of the inverters’ output currents: (a) Cuk, (b) Sepic, (c) F5, and P5. FIGURE 13. Experimental results of F5-based system: (a) input currents, (b) Ls currents, (c) output voltages, and (d) grid voltage/current. FIGURE 16. Partial shading conditions: (a) input voltages/currents, (b) module’s output voltages, (c) capacitors voltages, and (d) grid voltage and current. FIGURE 14. Experimental results of P5-based system: (a) input currents, (b) Lp / Ls currents, (c) output voltages, and (d) grid voltage/current. FIGURE 16. Partial shading conditions: (a) input voltages/currents, (b) module’s output voltages, (c) capacitors voltages, and (d) grid voltage and current. FIGURE 14. Experimental results of P5-based system: (a) input currents, (b) Lp / Ls currents, (c) output voltages, and (d) grid voltage/current. and performances. All modules have the same semiconductor stresses but they have different current and voltage ripples and therefore the passive elements’ values will vary in order to achieve the same operating conditions. conditions where the current controllers of all modules oper- ate the system at the peaks of the power curves to extract the maximum available power during shading conditions. It should be noted that if more than one PV module are completely shaded, the unshaded modules will be required to generate higher output voltages to compensate for the differ- ence and match the voltage to the grid. A. PARAMETERS SELECTION The detailed parameter selection process for switched mode power supplies has already been discussed in many publica- tions [8]–[10] and [13]. This subsection will present a practi- cal methods to select the passive elements and the transformer cores. VI. EXPERIMENTAL RESULTS If the output voltages of the unshaded modules exceed the maximum allowed stress on the switches, the MPPT will not be achievable and the system output power will be restricted. 1) PASSIVE ELEMENTS For Cuk-based module as an example, the relation between input and output inductors and currents can be found from: VI. EXPERIMENTAL RESULTS 11c shows the output voltages of the four modules. Fig. 11d shows the output current with the grid voltage. FIGURE 12. Experimental results of Sepic-based system: (a) input currents, (b) Ls currents, (c) output voltages, and (d) grid voltage/current. and vin for the 1st (unshaded) and the 4th (shaded) mod- ules. Fig 16b shows the output voltages vo1 and vo4 of the 1st and 4th modules where the controller changes the unshaded and shaded modules to keep their sum close to the grid voltage. The capacitors’ voltages of the 1st and 4th modules are shown in Fig. 16c. Finally, the grid voltage with current is shown in Fig. 16d. In this case, the new reference current to the FFRC and PR controllers is calcu- lated from (21) to extract 50W of the shaded modules while keeping the unshaded modules at 250W. As shown from Fig. 16b, the voltages vo of unshaded modules increase while they decrease for the shaded modules as calculated from (21). The resultant power can be calculated as ≈600 W which is the maximum assumed power to be available from the input sources. Fig. 17 shows the I-V and power curves of the different shaded and unshaded modules at the steady-state The same results for the system when operated by Sepic, F5 and P5-based modules are shown in Fig. 12, 13, and 14 respectively. In these normal conditions, the grid voltage is shared equally by the four modules. Fig. 15 shows the Fast Fourier Transform (FFT) for the four inverters’ output currents. Experimental case studies are carried out to mimic the performance of the system during partial shading condi- tions. At the shading time tsh, the power of the 3rd and 4th Cuk-based modules drops to 20% while the 1st and 2nd modules’ powers remain unchanged. Fig. 16a shows iin 53090 53090 VOLUME 8, 2020 VOLUME 8, 2020 A. Darwish et al.: Current-Source Single-Phase Module Integrated Inverters for PV Grid-Connected Applications FIGURE 13. Experimental results of F5-based system: (a) input currents, (b) Ls currents, (c) output voltages, and (d) grid voltage/current. FIGURE 14. Experimental results of P5-based system: (a) input currents, (b) Lp / Ls currents, (c) output voltages, and (d) grid voltage/current. conditions where the current controllers of all modules oper- ate the system at the peaks of the power curves to extract the maximum available power during shading conditions. VII. PARAMETERS SELECTION AND COMPARISON . PARAMETERS SELECTION AND COMPARISON This section presents the steps for parameters selection pro- cess for different inverters and comparisons between the different candidates’ modules in terms of their sizes, losses Lin = Vin1t 1Iin = DVin fs1Iin (37) (37) 53091 VOLUME 8, 2020 A. Darwish et al.: Current-Source Single-Phase Module Integrated Inverters for PV Grid-Connected Applications TABLE 2. Formulas for parameters selection. FIGURE 17. IV curves of the modules: (a) unshaded modules (1 and 2), (b) shaded modules (3 and 4). TABLE 2. Formulas for parameters selection. TABLE 2. Formulas for parameters selection. FIGURE 17. IV curves of the modules: (a) unshaded modules (1 and 2), (b) shaded modules (3 and 4). If the maximum acceptable limit factor for the input and output currents’ ripples are xLin and xLo respectively, so Lin and Lo can be expressed as: xLin = 1Iin(peak) Iin and xLo = 1io(peak) ig (38a) Lin = DmaxVin fsIinxLin (38b) Lo = (1 −Dmax)Vo fsigxLo (38c) (38a) (38b) Similarly, the Cuk module’s capacitors are chosen as: Cp > PdcH(1 + H) fsxCp(Vg + nHVin)(Vcdc + Vcm) (42a) Cs > Pdc(1 + H) fsxCp(Vg + nHVin)(Vcdc + Vcm) (42b) (38c) (42a) Substituting in equations (4) and (37), the modules’ inductors should be chosen as: (42b) Finally, the output current THD is further reduced by the aid of the output capacitor Co. The output capacitor is selected as: Lin > nVgV 2 in (Vg + nHVin)fsPdcxLin (39a) Lo > VinV 2 g H cos(γ ) 2fsPdcxLin(Vg + nHVin) (39b) (39a) Co = Ig 2πVgfsxo where xo = 1ig(peak) 1io(peak) (43) (43) (39b) In the same way, the passive elements of the different modules can be deduced and listed in Table 2. II. In practice, the values of these passive elements are chosen to keep the voltage and current limits below 10% while the THD of the output current should be kept below 5% to meet grid standards. In the same way, the passive elements of the different modules can be deduced and listed in Table 2. II. In practice, the values of these passive elements are chosen to keep the voltage and current limits below 10% while the THD of the output current should be kept below 5% to meet grid standards. VII. PARAMETERS SELECTION AND COMPARISON Similarly, the primary and secondary capacitors are expressed as: as: as: Cp = Iin1t 1Vcp = Iin(1 −D) fsxcpVcp (40a) Cs = Iin H1t 1Vcp = Iin(1 −D) HfsxcsVcs (40b) (40a) B. PERFORMANCE EVALUATION In order to evaluate the performances of the different mod- ules, the proposed system has been tested in the normal operation with the four single-phase inverter modules. TABLE 3. Transformer’s parameters. FIGURE 18. Passive elements breakdown: (a) Cuk, (b) Sepic, (c) F5 and (d) P5. experiments, the slight differences in the THD of the output currents in Fig. 15 occur due to the low order 3rd, 5th, and 7th because they have different transfer functions and therefore different poles locations. These low order harmonics will increase if the passive elements in the inverters are increased in case of reducing the switching frequency. In Fig. 15, it has been shown that the THD of the output currents is always less than 5% as pre-calculated from Table 4. However, if the low order harmonics increased and lead the THD to exceed 5%, harmonic eliminators can be used in the control scheme to remove these components as shown in [1], but this will add to the complexity of the control system. For energy storage elements such as Cp, Cs in Cuk-module or Ls and Lp in other modules, the values are chosen to ensure that the element will withstand the peak value and to avoid saturation. Fig. 18 shows a comparison between the sizes of different 2) TRANSFORMERS’ DESIGN 2) TRANSFORMERS’ DESIGN (40b) It is required to minimise the volume, size and weight of the transformer core in the different inverter topologies. Nanocrystalline ribbon materials can reduce the transformer cores’ size signiﬁcantly due to its high magnetic perme- ability which exceeds 30,000 H/m [21]. The ﬁrst step is to calculate the rms voltage across the transformer terminals. where xcp and xcs are the maximum acceptable voltage ripple factor for the capacitors’ voltages as: xcp = 1vcp(peak) vcp and xcs = 1vcs(peak) vcs (41) (41) 53092 VOLUME 8, 2020 VOLUME 8, 2020 A. Darwish et al.: Current-Source Single-Phase Module Integrated Inverters for PV Grid-Connected Applications TABLE 4. Passive elements’ values of the operated system. FIGURE 18. Passive elements breakdown: (a) Cuk, (b) Sepic, (c) F5 and (d) P5. experiments, the slight differences in the THD of the output currents in Fig. 15 occur due to the low order 3rd, 5th, and 7th because they have different transfer functions and therefore different poles locations. These low order harmonics will TABLE 4. Passive elements’ values of the operated system. TABLE 4. Passive elements’ values of the operated system. The rms voltages at the rated power are listed in Table 3. Then, the speciﬁed magnetizing inductance is calculated from [22]: L∗ m = Vrms 2πfsIrms kLm (44) (44) It is required to satisfy this magnetizing inductance while keeping the ﬂux density below its maximum value. The constant kLm depends on the type of the converter and is subjected for ﬁne tuning. Then, the magnetising inductance Lm is calculated from [22] as: Lm = N 2AcµoµrSF lc (45) (45) where N is the number of turns, µo and µr are the permeabil- ities of the air and the core’s materials, lc is the mean length of the magnetic path, Ac is the cross section area of the cores and SF is the number of cores needed to achieve the required area product Ap. The values of Ac and lc are obtained from the datasheet of the core and Ap is calculated from [23]: where N is the number of turns, µo and µr are the permeabil- ities of the air and the core’s materials, lc is the mean length of the magnetic path, Ac is the cross section area of the cores and SF is the number of cores needed to achieve the required area product Ap. 2) TRANSFORMERS’ DESIGN The values of Ac and lc are obtained from the datasheet of the core and Ap is calculated from [23]: FIGURE 18. Passive elements breakdown: (a) Cuk, (b) Sepic, (c) F5 and (d) P5. Ap = 4 √ 2 × VA Boptfs (46a) Bopt = 1.3 × 104 VA × f 5/12 s (46b) (46a) (46b) Finally, it is required to conﬁrm that the transformer ﬂux density will be below the saturation limits Bsat of the core as: Bopt ≤Bsat (47) (47) The resultant values of this process are listed in Table 3 and have been used to design the transformer cores. The resultant values of this process are listed in Table 3 and have been used to design the transformer cores. TABLE 3. Transformer’s parameters. 1) PASSIVE ELEMENTS AND EFFICIENCIES The passive elements of each module have been selected to keep the high-frequency current and voltage ripples = 5% of the nominal current and voltage values at the rated power. These passive elements of the different elements are calcu- lated from Table 2 and listed in Table 4. Therefore, the THD of the output currents should be close and less than 5% for all inverters. Although the high-frequency ripples in the output currents are the same for all inverters as shown in the 53093 VOLUME 8, 2020 A. Darwish et al.: Current-Source Single-Phase Module Integrated Inverters for PV Grid-Connected Applications FIGURE 19. Simulated efficiencies of different inverters configurations. FIGURE 20. Comparative analysis of different inverter modules. FIGURE 19. Simulated efficiencies of different inverters configurations. FIGURE 20. Comparative analysis of different inverter modules. FIGURE 20. Comparative analysis of different inverter modules. FIGURE 19. Simulated efficiencies of different inverters configurations. FIGURE 21. Simulated efficiencies of Cuk inverters configurations. modules. A comparison between the efﬁciencies of the dif- ferent module inverters (from Fig. 3) is conducted using MATLAB/SIMULINK and plotted in Fig. 19. Cuk-based module requires bigger capacitor to store the oscillating energy while all other components are relatively small. As shown in the efﬁciency comparison in Fig. 19, this module has the best efﬁciency performance as it experiences the least equivalent Series Resistance (ESR) and transformer core losses. For Sepic-based module, the ESR losses increases as the energy is stored in Ls which has relatively higher para- sitic resistance when compared with the capacitors leads and therefore the total efﬁciency decreased. As the transformer core transfers the energy instantaneously between the primary and secondary sides, small cores can be used for isolation in case of Cuk, Sepic, and P5. F5-based modules have the worst efﬁciency when compared with the other candidates, this comes from the fact that bigger transformer magnetic core should be used as it stores the energy temporarily and then releases this energy after period of time. P5-based modules employ the smallest capacitors as the average voltages across Cp and Cs are always zero. FIGURE 21. Simulated efficiencies of Cuk inverters configurations. respect to the input electrical power from the dc source. However, when the other Cuk conﬁgurations are connected to PV panels, the 2nd order harmonic component in the input current, inevitably, will lead to power oscillation between points 3 and 4 in Fig. 1a. 1) PASSIVE ELEMENTS AND EFFICIENCIES This will reduce the extracted PV power below its maximum available point and hence will reduce the effective efﬁciency. TABLE 5. Controller gains’ values. TABLE 5. Controller gains’ values. p The ESR losses are lower than F5 and Sepic-based modules losses as the oscillating energy is stored in the two induc- tors (Lp & Ls). It should be mentioned that although Sepic, F5, P5-based modules have lower efﬁciency and bigger sizes than the Cuk-based module, they have the advantage of using smaller capacitors which may be an important feature for improving the total system’s reliability especially when fewer modules are employed with high boosting ratio Vo/Vin. With fewer modules and higher boosting ratios, higher voltage stresses are generated across the capacitors which may reduce the system’s reliability in these conditions. Fig. 20a shows the practical efﬁciencies of the inverter modules at Vin = 50V. 2) DYNAMIC PERFORMANCES COMPARISON This subsection discusses the dynamic responses of the dif- ferent inverter topologies during grid instability conditions. Table 5 shows the controller gains’ values in order to achieve 30 dB and 80◦stability margins for all inverters. The Cuk inverter Transfer Function is fourth-order and the control sys- tem can reach bandwidth of ≈1.8 kHz. This can be increased if the optional capacitor Co is reduced or removed. Sepic inverter requires higher values of Co and Ls when compared with the Cuk inverter and therefore the bandwidth is lower. F5 has the highest bandwidth as it needs lower total capac- itances and inductances. P5 has the highest order among all A comparison between the efﬁciencies of the different Cuk inverters conﬁguration (from Fig. 4) is plotted in Fig. 21. The comparison is conducted using the same passive elements and devices in Table 1. The efﬁciency of the proposed Cuk inverter is close to the differential-Cuk when Vin is low. The proposed Cuk inverter is superior to other Cuk conﬁgurations when the output power increase because the reduced losses in its input inductor. It should be noted that these efﬁciencies are calculated considering the inverters’ power losses with 53094 VOLUME 8, 2020 VOLUME 8, 2020 A. Darwish et al.: Current-Source Single-Phase Module Integrated Inverters for PV Grid-Connected Applications FIGURE 22. Dynamic response of the four inverters: (a) Cuk, (b) Sepic, (c) F5 and (d) P5. of the highest order and therefore the control design is more difﬁcult. In this context, the F5 inverter is preferred when the switching frequency is low (<5kHz) and large passive elements are employed to reduce the current and voltage ripples. • Despite of the abovementioned drawbacks, P5 inverter’s capacitors have zero average voltages and therefore small capacitors can be used. This increases the reliabil- ity of the inverter and reduces the size of the transformer. Therefore, the P5 inverter is favoured if the switching frequency can be increased (>50kHz). • Despite of the abovementioned drawbacks, P5 inverter’s capacitors have zero average voltages and therefore small capacitors can be used. This increases the reliabil- ity of the inverter and reduces the size of the transformer. • In general, the efﬁciencies of the Sepic, F5, and P5 inverters are lower than the Cuk inverter as they store the oscillating energy inside the shunt inductors Ls. For this reason, the ESR of Ls affects the power losses signiﬁcantly. VIII. CONCLUSION The paper presents a modular energy conversion structure for grid-connected PV system based on four single-phase buck-boost isolated current source inverters. The four mod- ules have been investigated in terms of power losses, size, performance and voltage/current ripples. The proposed sys- tem is capable of controlling the output active and reactive power injected to the grid while operating the PV panels at the maximum power points. The MPPT operation is achieved by storing the oscillating power component in storage elements inside the inverters. The modular system offers modularity, scalability and improve the reliability by reducing the voltage and current stresses across each module’s passive elements as well as providing redundant modules in case of module’s failure. Moreover, the modular structure can operate during partial shading conditions if the individual modules’ currents and voltages are measured. P5 shows the worst performance as it takes more than one cycle to recover the current after the fault. This comes from the fact that P5 stems from a ﬁfth-order converter and therefore it is difﬁcult to increase the controller bandwidth with a single loop controller. A solution to this issue has been proposed in [24] where an additional loop can increase the controller bandwidth and hence its speed. Cuk and Sepic inverters’ speed to recover the grid currents are between F5 and P5. 2) DYNAMIC PERFORMANCES COMPARISON • The Sepic inverter shows a good trade-off between the size, efﬁciency, complexity of the control design, and reliability if moderate module number, voltage level, and switching frequency have been used. FIGURE 22. Dynamic response of the four inverters: (a) Cuk, (b) Sepic, (c) F5 and (d) P5. inverters and therefore the control design process is restricted. Fig. 22 shows the response of the four inverters when the grid voltage dropped to 20% of its nominal value to mimic a phase-to-ground fault. Because F5 inverter has the high- est bandwidth in its control system, the current controller recovered the current in less than half-cycle to enable the grid protection devices to operate without destroying the inverter’s switches. 3) OVERALL EVALUATION Following to the previous analyses and results, important ﬁndings and recommendations can be made as: Following to the previous analyses and results, important ﬁndings and recommendations can be made as: Following to the previous analyses and results, important ﬁndings and recommendations can be made as: REFERENCES degrees in electrical engineering from the Fac- ulty of Engineering, Alexandria University, Egypt, in 2008 and 2012, respectively, and the Ph.D. degree in electrical engineering from the Electric and Electronic Engineering Department, Univer- sity of Strathclyde, Glasgow, U.K., in 2016. AHMED DARWISH received the B.Sc. and M.Sc. degrees in electrical engineering from the Fac- ulty of Engineering, Alexandria University, Egypt, in 2008 and 2012, respectively, and the Ph.D. degree in electrical engineering from the Electric and Electronic Engineering Department, Univer- sity of Strathclyde, Glasgow, U.K., in 2016. AHMED DARWISH received the B.Sc. and M.Sc. degrees in electrical engineering from the Fac- ulty of Engineering, Alexandria University, Egypt, in 2008 and 2012, respectively, and the Ph.D. degree in electrical engineering from the Electric and Electronic Engineering Department, Univer- sity of Strathclyde, Glasgow, U.K., in 2016. p [8] A. Darwish, A. M. Massoud, D. Holliday, S. Ahmed, and B. W. Williams, ‘‘Single-stage three-phase differential-mode buck-boost inverters with continuous input current for PV applications,’’ IEEE Trans.Power Elec- tron., vol. 31, no. 12, pp. 8218–8236, Dec. 2016. From 2009 to 2012, he was a Research Assis- tant at Texas A&M University at Qatar. In 2015, he was a Postdoctoral Research Associate with the PEDEC Group, University of Strathclyde, where he worked for two years. He joined Lancaster University as a Lecturer in electrical engineering, in 2017. His research interests include dc–dc converters, multilevel convert- ers, electric machines, digital control of power electronic systems, energy conversion, renewable energy, and power quality. From 2009 to 2012, he was a Research Assis- tant at Texas A&M University at Qatar. In 2015, he was a Postdoctoral Research Associate with the PEDEC Group, University of Strathclyde, where he worked for two years. He joined Lancaster University as a Lecturer in electrical engineering, in 2017. His research interests include dc–dc converters, multilevel convert- ers, electric machines, digital control of power electronic systems, energy conversion, renewable energy, and power quality. [9] A. D. Badawy, ‘‘Current source DC-DC and DC-AC converters with continuous energy ﬂow,’’ Ph.D. dissertation, Dept. Electron. Elect. Eng., Univ. Strathclyde, Glasgow, Scotland, 2015. [10] B. W. Williams, ‘‘Generation and analysis of canonical switching cell DC- to-DC converters,’’ IEEE Trans. Ind. Electron., vol. 61, no. 1, pp. 329–346, Jan. 2013. [11] M. R. Islam, Y. Guo, and J. Zhu, ‘‘A high-frequency link multilevel cascaded medium-voltage converter for direct grid integration of renew- able energy systems,’’ IEEE Trans. Power Electron., vol. REFERENCES 29, no. 8, pp. 4167–4182, Aug. 2014. [12] A. Darwish, A. Elserougi, A. S. Abdel-Khalik, S. Ahmed, A. Massoud, D. Holliday, and B. W. Williams, ‘‘A single-stage three-phase DC/AC inverter based on Ćuk converter for PV application,’’ in Proc. 7th IEEE GCC Conf. Exhib. (GCC), Nov. 2013, pp. 384–389. [13] A. Darwish and M. A. Elgenedy, ‘‘Current-source modular medium- voltage grid-connected system with high-frequency isolation for pho- tovoltaic applications,’’ IEEE Trans. Energy Convers., vol. 34, no. 1, pp. 255–266, Mar. 2019. SAUD ALOTAIBI received the B.Sc. degree in electrical engineering from Taif University, Taif, Saudi Arabia, in 2012, and the M.Sc. degree in electrical engineering from the University of Nottingham, Nottingham, U.K., in 2016. He is currently pursuing the Ph.D. degree with the Department of Engineering, Lancaster University, Lancaster, U.K. [14] S. Mehrnami and S. K. Mazumder, ‘‘Discontinuous modulation scheme for a differential-mode Ćuk inverter,’’ IEEE Trans. Power Electron., vol. 30, no. 3, pp. 1242–1254, Mar. 2015. [15] B. Han, J. S. Lee, and M. Kim, ‘‘Repetitive controller with phase-lead compensation for Ćuk CCM inverter,’’ IEEE Trans. Ind. Electron., vol. 65, no. 3, pp. 2356–2367, Mar. 2018. [16] B. Han, J.-S. Lai, and M. Kim, ‘‘Bridgeless Ćuk-derived single power conversion inverter with reactive-power capability,’’IEEE Trans. Power Electron., vol. 35, no. 3, pp. 2629–2645, Mar. 2020. Since 2013, he has been with the College of Engineering, Shaqra University, where he holds a lecturer position. His areas of interests include power electronics, dc–dc converters, multilevel converters, energy conversion, and power quality. Since 2013, he has been with the College of Engineering, Shaqra University, where he holds a lecturer position. His areas of interests include power electronics, dc–dc converters, multilevel converters, energy conversion, and power quality. [17] A. Darwish, A. Massoud, D. Holliday, S. Ahmed, and B. Williams, ‘‘Generation, performance evaluation and control design of single-phase differential-mode buck–boost current-source inverters,’’ IET Renew. Power Gener., vol. 10, no. 7, pp. 916–927, Jul. 2016. [18] N. Rabia, ‘‘Advanced repetitive control of grid converters for power qual- ity improvement,’’ Ph.D. dissertation, College Eng., Univ. Canterbury, Christchurch, New Zealand, 2015. [19] Z.-X. Zou, K. Zhou, Z. Wang, and M. Cheng, ‘‘Frequency-adaptive fractional-order repetitive control of shunt active power ﬁlters,’’ IEEE Trans. Ind. Electron., vol. 62, no. 3, pp. 1659–1668, Mar. 2015. [20] B. A. Francis and W. M. Wonham, ‘‘The internal model principle for linear multivariable regulators,’’ Appl. Math. Optim., vol. 2, pp. 170–194, Jun. 1975. REFERENCES [1] M. Mirjafari, S. Harb, and R. S. Balog, ‘‘Multiobjective optimization and topology selection for a module-integrated inverter,’’ IEEE Trans. Power Electron., vol. 30, no. 8, pp. 4219–4231, Aug. 2015. • The efﬁciency of the Cuk inverter in Fig. 3a is the highest and therefore it is favoured to maximise the harvested power from the system. However, as the oscillating energy is stored in the capacitors Cp and Cs, the reliabil- ity may be reduced when the operating voltage increases, or the number of modules is reduced and the Cuk inverter will not be suitable in this case as the voltage stresses across the capacitors will be high. [2] S. B. Kjaer, J. K. Pedersen, and F. Blaabjerg, ‘‘A review of single-phase grid-connected inverters for photovoltaic modules,’’ IEEE Trans. Ind. Appl., vol. 41, no. 5, pp. 1292–1306, Sep. 2005. [3] S. B. Kjaer, J. K. Pedersen, and F. Blaabjerg, ‘‘Power inverter topologies for photovoltaic modules-a review,’’ in Proc. Conf. Rec. IEEE Ind. Appl. Conf. 37th IAS Annu. Meeting, Oct. 2002, pp. 782–788. [4] Q. Li and P. Wolfs, ‘‘A review of the single phase photovoltaic mod- ule integrated converter topologies with three different DC link conﬁg- urations,’’ IEEE Trans. Power Electron., vol. 23, no. 3, pp. 1320–1333, May 2008. • The F5 inverter has the highest losses, lowest efﬁciency, and the biggest transformer size. [5] D. Leuenberger and J. Biela, ‘‘PV-module-integrated AC inverters (AC Modules) with subpanel MPP tracking,’’ IEEE Trans. Power Electron., vol. 32, no. 8, pp. 6105–6118, Aug. 2017. • From the control point of view, the transfer function of F5 inverter enables for achieving high bandwidth and hence the control design is less complex. This affects the dynamic response during faults and instabilities. [6] T. Shimizu, K. Wada, and N. Nakamura, ‘‘Flyback-type single-phase utility interactive inverter with power pulsation decoupling on the DC input for an AC photovoltaic module system,’’ IEEE Trans. Power Electron., vol. 21, no. 5, pp. 1264–1272, Sep. 2006. On the other hand, the P5 inverter’s transfer function is 53095 VOLUME 8, 2020 A. Darwish et al.: Current-Source Single-Phase Module Integrated Inverters for PV Grid-Connected Applications [7] L. Zhang, K. Sun, Y. W. Li, X. Lu, and J. Zhao, ‘‘A distributed power control of series-connected module-integrated inverters for PV grid-tied applications,’’ IEEE Trans. Power Electron., vol. 33, no. 9, pp. 7698–7707, Sep. 2018. AHMED DARWISH received the B.Sc. and M.Sc. REFERENCES MOHAMED A. ELGENEDY (Senior Member, IEEE) received the B.Sc. (Hons.) and M.Sc. degrees in electrical engineering from Alexandria University, Egypt, in 2007 and 2010, respectively, and the Ph.D. degree in electrical and electronic engineering from the University of Strathclyde, Glasgow, U.K., in 2018. From 2011 to 2013, he was with Spiretronic LLC, Houston, TX, USA, as a Research Engineer. From 2013 to 2014, he was a Research Associate at Texas A&M University at Qatar, Doha, Qatar. He is currently a Postdoctoral Research Associate with the PEDEC Group, Strathclyde University, Glasgow, U.K. He is also an Assistant Lecturer with the Electrical Engineering Department, Faculty of Engineering, Alexandria University. His research interests include high power electronics, pulse power generator, electric machine drives, energy conversion, electric vehicles, and renewable energy. [21] R. Burdt, K. McDonald, R. Curry, B. Huhman, P. Melcher, R. Ness, and C. Huang, ‘‘Evaluation of nanocrystalline materials, amorphous alloys and ferrites for repetitive-magnetic pulse compression applications,’’ in Proc. IEEE Pulsed Power Conf., Jun. 2005, pp. 843–847. [22] Y. Du, S. Baek, S. Bhattacharya, and A. Q. Huang, ‘‘High-voltage high- frequency transformer design for a 7.2kV to 120 V/240 V 20kVA solid state transformer,’’ in Proc. 36th Annu. Conf. IEEE Ind. Electron. Soc. (IECON), Nov. 2010, pp. 493–498. [23] O. Aldosari, L. A. Garcia Rodriguez, J. C. Balda, and S. K. Mazumder, ‘‘Design trade-offs for Medium- and high-frequency transformers for isolated power converters in distribution system applications,’’ in Proc. 9th IEEE Int. Symp. Power Electron. for Distrib. Gener. Syst. (PEDG), Jun. 2018, pp. 1–7. [24] A. Darwish, A. S. Abdel-Khalik, A. Elserougi, S. Ahmed, and A. Massoud, ‘‘Fault current contribution scenarios for grid-connected voltage source inverter-based distributed generation with an LCL ﬁlter,’’ Electr. Power Syst. Res., vol. 104, pp. 93–103, Nov. 2013. 53096 VOLUME 8, 2020
https://openalex.org/W2124633704	https://europepmc.org/articles/pmc3605679?pdf=render	English	null	Nuclear Envelope Composition Determines the Ability of Neutrophil-type Cells to Passage through Micron-scale Constrictions	Journal of biological chemistry/The Journal of biological chemistry	2,013	cc-by	9,771	* This work was supported by a Cross-Disciplinary Fellowship of the Interna- tional Human Frontiers Science Program (HFSP) (to A. C. R.) and National Institutes of Health Grants R01 NS059348 and R01 HL082792 and Depart- ment of Defense Breast Cancer Idea Award BC102152 (to J. L.). This work was also supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) (to W. L. U. and I. A. E.), an American Heart Asso- ciation fellowship (AHA Award 09POST2320042) (to D. E. J.), and HFSP Grant RGP0004/2005-C102, National Science Foundation (NSF) Contracts DMR-1006546 and DBI-649865, and Harvard Materials Research Science and Engineering Center (MRSEC) Contract DMR-0820484. Amy C. Rowat‡§¶1, Diana E. Jaalouk§2, Monika Zwerger§3, W. Lloyd Ung¶, Irwin A. Eydelnant¶, Don E. Olins, Ada L. Olins, Harald Herrmann, David A. Weitz¶, and Jan Lammerding§‡‡ Amy C. Rowat‡§¶1, Diana E. Jaalouk§2, Monika Zwerger§3, W. Lloyd Ung¶, Irwin A. Eydelnant¶, Don E. Olins, Ada L. Olins, Harald Herrmann, David A. Weitz¶, and Jan Lammerding§‡‡ From the ‡Department of Integrative Biology and Physiology, UCLA, Los Angeles, California 90095, the §Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, Massachusetts 02139, the ¶Department of Physics and School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts 02138, the Department of Pharmaceutical Sciences, College of Pharmacy, University of New England, Portland, Maine 04103, the Division of Molecular Genetics, German Cancer Research Center, D-69120 Heidelberg, Germany, and the ‡‡Weill Institute for Cell and Molecular Biology, Department of Biomedical Engineering, Cornell University, Ithaca, New York 14853 Background: The unusual nuclear shape of neutrophils has been speculated to facilitate their passage through confined spaces. Results: Levels of nuclear protein lamin A modulate cell passage through micron-scale pores. Conclusion: The unique protein composition of neutrophil nuclei facilitates their deformation; lobulated nuclear shape is not essential. Si ifi Alt d l l iti t d i ll ld i t ll th h h i l i l ckground: The unusual nuclear shape of neutrophils has been speculated to facilitate their passage through sults: Levels of nuclear protein lamin A modulate cell passage through micron-scale pores. Results: Levels of nuclear protein lamin A modulate cell passage through micron-scale pores. Conclusion: The unique protein composition of neutrophil nuclei facilitates their deformation; lobulated nuclear shape is not essential. Conclusion: The unique protein composition of neutrophil nuclei facilitates their deformation; lobula essential. Significance: Altered nuclear envelope composition, as reported in cancer cells, could impact cell passage through physiological gaps. Neutrophils are characterized by their distinct nuclear shape, which is thought to facilitate the transit of these cells through pore spaces less than one-fifth of their diameter. We used human promyelocytic leukemia (HL-60) cells as a model system to investigate the effect of nuclear shape in whole cell deform- ability. We probed neutrophil-differentiated HL-60 cells lack- ing expression of lamin B receptor, which fail to develop lobu- lated nuclei during granulopoiesis and present an in vitro model for Pelger-Huët anomaly; despite the circular morphology of their nuclei, the cells passed through micron-scale constrictions on similar timescales as scrambled controls. 1 To whom correspondence should be addressed: Dept. of Integrative Biol- ogyandPhysiology,TerasakiLifeSciencesBldg.1125,610CharlesE.Young Dr. S., Los Angeles, CA 90095. Tel.: 310-825-4026; Fax: 310-206-9184; E-mail: rowat@ucla.edu. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 12, pp. 8610–8618, March 22, 2013 © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 12, pp. 8610–8618, March 22, 2013 © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 12, pp. 8610–8618, March 22, 2013 © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. g g □ S This article contains supplemental Methods and Figs. S1–S5. 2 Present address: Dept. of Biology, American University of Beirut, Beirut 1107 2020, Lebanon. 3 Present address: Dept. of Biochemistry, University of Zurich, 8057 Zurich, Switzerland. 4 The abbreviations used are: HL-60, human promyelocytic leukemia; LamA OE, lamin A-overexpressing; LBR, lamin B receptor; LBR KD, LBR knockdown; ATRA, all-trans-retinoic acid; Bis-Tris, 2-(bis(2-hydroxyethyl)amino)-2- (hydroxymethyl)propane-1,3-diol. Physical Properties of the Neutrophil Nucleus Analysis of Protein Expression—Cell lysates were prepared during the process of granulopoiesis, as recapitulated in vitro using human promyelocytic leukemia (HL-60)4 cells, major alterations occur in the expression levels of two key nuclear envelope proteins; the integral nuclear membrane protein, lamin B receptor (LBR), is strongly up-regulated, whereas there is a concurrent decrease in levels of lamin A, a key structural protein that forms a network underlying the inner nuclear membrane and imparts the nucleus with mechanical stability (11–13). Thus, although the unique shape of the neutrophil nucleus could facilitate the passage of these cells through nar- row constrictions, we hypothesized that reduced levels of lamin A could enhance nuclear deformability and thereby facilitate the passage of cells through micron-scale constrictions. during the process of granulopoiesis, as recapitulated in vitro using human promyelocytic leukemia (HL-60)4 cells, major alterations occur in the expression levels of two key nuclear envelope proteins; the integral nuclear membrane protein, lamin B receptor (LBR), is strongly up-regulated, whereas there is a concurrent decrease in levels of lamin A, a key structural protein that forms a network underlying the inner nuclear membrane and imparts the nucleus with mechanical stability (11–13). Thus, although the unique shape of the neutrophil nucleus could facilitate the passage of these cells through nar- row constrictions, we hypothesized that reduced levels of lamin A could enhance nuclear deformability and thereby facilitate the passage of cells through micron-scale constrictions. To dissect the role of nuclear shape and nuclear envelope composition in the passage of cells through constrictions that mimic physiological gaps, we used all-trans-retinoic acid (ATRA)-stimulated HL-60 cells to recapitulate granulopoiesis; this in vitro system is widely used for structural and functional assays of white blood cells (14–16). We probed the ability of cells to transit through micron-scale constrictions and investi- gated the effects of both altered nuclear shape and altered lamin A expression levels. Our results show that levels of lamin A have a predominant effect on the ability of cells to passage through narrow constrictions, whereas the altered shape of the neutro- phil nucleus is not essential for rapid passage through micron- scale pores. Amy C. Rowat‡§¶1, Diana E. Jaalouk§2, Monika Zwerger§3, W. Lloyd Ung¶, Irwin A. Eydelnant¶, Don E. Olins, Ada L. Olins, Harald Herrmann, David A. Weitz¶, and Jan Lammerding§‡‡ We then investi- gated the unique nuclear envelope composition of neutrophil- differentiated HL-60 cells, which may also impact their deform- ability; although lamin A is typically down-regulated during granulopoiesis, we genetically modified HL-60 cells to generate a subpopulation of cells with well defined levels of ectopic lamin A. The lamin A-overexpressing neutrophil-type cells showed similar functional characteristics as the mock controls, but they had an impaired ability to pass through micron-scale constric- tions. Our results suggest that levels of lamin A have a marked effect on the ability of neutrophils to passage through micron- scale constrictions, whereas the unusual multilobed shape of the neutrophil nucleus is less essential. The passage of cells through narrow spaces is critical in phys- iological and disease processes from immune response to metastasis. For example, neutrophils are required to rapidly traverse constrictions that are much smaller than their own diameter of 7–8 m: during perfusion through capillaries with diameters as small as 2 m or during migration through tran- sendothelial and interstitial spaces ranging from 0.1 to 10 m (1). The ability of neutrophils to transit through narrow con- strictions is essential; increased cell stiffness results in retention of neutrophils in arteries and capillaries (2), as well as accumu- lation in postcapillary venules leading to inflammation in the vascular bed (3). Although the mechanical properties of neutrophils can be regulated by cytoskeletal filaments such as actin (4–6) and microtubules (7), the hallmark multilobed nuclear morphology has long been thought to facilitate the deformation of neutro- phils through narrow spaces (8, 9); a round-shaped nucleus could sterically hinder the deformation of a cell through a nar- row pore, whereas the multilobed neutrophil nucleus could aid cell passage as individual lobes could be sequentially “threaded” through constrictions. Indeed, cells with lobulated nuclear shape show less retention in 8-m porous membranes as com- pared with their progenitors with round nuclei (10). However, it remains unclear to what extent this hyperlobulated nuclear shape is required for neutrophils to deform through narrow gaps; tightly regulated modifications in nuclear envelope pro- tein composition also occur during granulopoiesis. Specifically, 2 Present address: Dept. of Biology, American University of Beirut, Beirut 1107 2020, Lebanon. 3 3 Present address: Dept. of Biochemistry, University of Zurich, 8057 Zurich, Switzerland. VOLUME 288•NUMBER 12•MARCH 22, 2013 8610 JOURNAL OF BIOLOGICAL CHEMISTRY 8610 Physical Properties of the Neutrophil Nucleus Retroviral Transduction—We generated the stably modified lamin A-overexpressing (LamA OE) cells from the parent HL-60/S4 cell line by retroviral transduction (21–23) with the bicistronic vector (pRetroX-IRES-ZsGreen1, Clontech) for lamin A and the fluorophore reporter Zoanthus green fluores- cent protein (ZsGreen1) with the 5 Moloney murine leukemia virus LTR as the promoter. Cloning of the wild-type prelamin A into the bicistronic retroviral vector was performed as follows: the insert was generated by cutting pSVK3-prelamin A (24) (kind gift from Howard J. Worman) with SmaI and SalI; this was ligated to the vector obtained from cutting pEGFP-C1 (Clon- tech) with Ecl136II and SalI resulting in a shuttle vector, which was subsequently digested with XmaI, blunted with Klenow, and then cut with BglII. The insert from the latter digestion was then ligated to the vector generated from cutting the pRetroX- IRES-ZsGreen1 with BamHI and blunted with Klenow fol- lowed by BglII digestion. Transfection of the resultant pRetro-prelamin A-IRES-ZsGreen 1 expression vector into the 293GPG retroviral packaging cell line (kind gift from Rich- ard C. Mulligan) was performed using Lipofectamine Plus rea- gent (Invitrogen) based on the manufacturer’s specifications and previous protocols with minor modifications (21–23). A ZsGreen1 retrovector without lamin A insert was used to gen- erate the mock control cells. Viral supernatant was collected daily for 6 consecutive days, filtered through 0.45-m pores, and stored at 20 °C. Later, the viral supernatants collected per batch were thawed and pooled, and viral titer was determined by viral infection of mouse embryo fibroblasts. Two rounds of viral transduction of HL-60/S4 cells were then performed using unconcentrated viral supernatant supplemented with 6–8 g/ml Polybrene (Sigma-Aldrich) at a multiplicity of infection of 25–50. Gene transfer efficiency was assayed 5 days after ret- roviral infection by flow cytometry probing ZsGreen1 levels; because ZsGreen1 and lamin A are derived from the same bicis- tronic mRNA transcript, we sorted individual cells based on ZsGreen1 levels into a subpopulation of cells with well defined, elevated expression levels by fluorescence-activated cell sorting (Aria II, BD Biosciences or MoFlo, Beckman Coulter) into cal- cium-free PBS buffer. The resulting subpopulation does not derive from a single clone, but is rather generated by the highest expressing cells that may contain multiple insertions; the 5 LTR promoter is relatively weak, and we observed an 20–30- fold increase in lamin A levels in comparison with the mock controls. Physical Properties of the Neutrophil Nucleus Circularity for a nuclear cross-section is 4A/P2, where A is the cross-sectional area and P is the perimeter. FIGURE 1. Ectopic expression of lamin A increases passage time through FIGURE 1. Ectopic expression of lamin A increases passage time through microfluidic constriction channels. A, schematic overview of the microflu- idic device and close-up of the 5-m constrictions. Pressurizing the reservoir drives the cell suspension through the inlet, and cells passage through the channels with 5-m constrictions, as shown in the inset. Scale bar, 20 m. B, time-sequence images of day 4/ATRA-treated cells passaging through 5-m constrictions. Scrmbl Ctrl, scrambled control; Mock Ctrl, mock control. C, LBR KD cells have similar passage times as the scrambled controls despite the round shape of their nucleus, which has been speculated to sterically hinder the passage of cells through narrow pores. D and E, LamA OE cells take longer to passage the constrictions than mock control cells. In all box plots, the white bar denotes the population median, boxes are the 25th and 75th percentiles, and lines show the 10th and 90th percentiles. n.s., p 0.05 for LBR KD versus scrambled control; *, p 0.001 for LamA OE versus mock control. n 300 cells for each cell type. Error bars represent S.E. over three independent experiments. Transwell Migration Assay—We used membranes with 3- and 8-m pore sizes (Millipore) and FBS as chemoattractant (27, 28). Day 4/ATRA-treated cells were resuspended to 5 106 cells/ml in RPMI without FBS. We placed media with and with- out FBS in the bottom well and cells in the top well and then incubated the plate at 37 °C, 5% CO2 for 2 h. We then removed the membrane insert, labeled cells in the bottom well with Hoechst, and imaged each well by microscopy using a 10/0.25 Ph1 objective (A-Plan, Zeiss), charge-coupled device camera (AxioCam MRm, Zeiss), and DAPI filter set. We determined the number of cells per well using image analysis (ImageJ). tion is required for a cell to passage through a pore (supplemen- tal Fig. S1). We forced the neutrophil-type cells (day 4/ATRA- treated HL-60 cells) to transit through these micron-scale pores using pressure to drive a flow of cell suspension through the channels; we monitored the passage of cells as a function of time. EXPERIMENTAL PROCEDURES Cell Culture—HL-60/S4 cells were maintained in RPMI 1640 medium with L-glutamine (Invitrogen), 10% fetal bovine serum (FBS), and 1% penicillin:streptomycin (Gemini Bio-Products, West Sacramento, CA). We generated scrambled control cells to compare with HL-60/S4 cells with stable shRNA-mediated knockdown of LBR (LBR KD cells) (17). To induce differentia- tion into neutrophil-type cells, we added ATRA at a final con- centration of 5 M to 1 105 cells/ml; ethanol was used as vehicle control. We probed nuclear shape and nuclear envelope composition at days 0, 3, and 5 after ATRA treatment; we per- formed functional assays of neutrophil-type cells at 4 days after ATRA treatment, when cells display characteristics of neutro- phils (11, 18). Microfluidic Deformation—Soft lithography was used to fab- ricate microfluidic channels in polydimethylsiloxane (Sylgard 184 silicone elastomer, Dow Corning) (19). Devices were bonded to #1.5-thickness coverglasses. We drove the flow of cells by applying 28 kilopascals (4 p.s.i.) of pressure to a tube of 2.5 106 cells/ml with 0.1% F127 (Pluoronic F-127, Invitrogen) to minimize surface adhesion (20). Images were acquired at 300 frames/s with a high speed camera (Miro ex4, Vision Research, Wayne, NJ) mounted on an inverted light microscope (Zeiss Observer) with 10/0.25 Ph1 objective (A-Plan, Zeiss). The resulting image sequences were analyzed using a custom-writ- ten program (MATLAB) to extract the time for cell passage through the first constriction. Analysis of Protein Expression—Cell lysates were prepared from 5 106 cells using urea lysis buffer with final concentra- tions of 9 M urea, 10 mM Tris-HCl (pH 8), 10 M EDTA, 500 M phenylmethylsulfonyl fluoride, 20 l of -mercaptoethanol, and 1 l/ml protease inhibitor mixture (Sigma). All steps were performed at 4 °C. Proteins were separated on a 4-12% Bis-Tris gel with 1 MOPS running buffer and then transferred onto activated transfer membranes, blocked, and labeled using horseradish peroxidase-conjugated antibodies (Bio-Rad). We used protein standard (Invitrogen SeeBlue Plus2) for size calibra- tion and used -tubulin as a loading control because its levels remainconstantthroughoutdifferentiation(11).Primaryantibod- ies used for probing are described in the supplemental Methods. MARCH 22, 2013•VOLUME 288•NUMBER 12 8611 JOURNAL OF BIOLOGICAL CHEMISTRY MARCH 22, 2013•VOLUME 288•NUMBER 12 Physical Properties of the Neutrophil Nucleus FIGURE 1. Ectopic expression of lamin A increases passage time through microfluidic constriction channels. A, schematic overview of the microflu- idic device and close-up of the 5-m constrictions. Pressurizing the reservoir drives the cell suspension through the inlet, and cells passage through the channels with 5-m constrictions, as shown in the inset. Scale bar, 20 m. B, time-sequence images of day 4/ATRA-treated cells passaging through 5-m constrictions. Scrmbl Ctrl, scrambled control; Mock Ctrl, mock control. C, LBR KD cells have similar passage times as the scrambled controls despite the round shape of their nucleus, which has been speculated to sterically hinder the passage of cells through narrow pores. D and E, LamA OE cells take longer to passage the constrictions than mock control cells. In all box plots, the white bar denotes the population median, boxes are the 25th and 75th percentiles, and lines show the 10th and 90th percentiles. n.s., p 0.05 for LBR KD versus scrambled control; , p 0.001 for LamA OE versus mock control. n 300 cells for each cell type. Error bars represent S.E. over three independent experiments. Expression levels were quantified by optical density analysis using ImageJ (National Institutes of Health). Cell Surface Marker Analysis—To assay expression levels of CD11b, we used Fc receptor polyclonal human IgG (Sigma) as a blocking agent and labeled 106 cells with Alexa Fluor 700 mouse anti-human-CD11b (BD Pharmingen). We analyzed fluores- cence levels by flow cytometry (LSR II, BD Biosciences). Respiratory Burst Assay—We determined superoxide radi- cal production of day 4/ATRA-treated cells using luminol- enhanced chemiluminescence (Diogenes reagent, National Diagnostics, Atlanta, GA) following stimulation by phorbol 12-myristate 13-acetate (25, 26), as per the manufacturer’s instructions. Cells were activated by the addition of phorbol 12-myristate 13-acetate (stock solution 1 mg/ml dimethyl sulf- oxide (DMSO), Sigma) to a final concentration of 11 M; we recorded luminescence values after 30 min using a plate reader (SpectraMax M5). Nuclear Shape Analysis—We incubated cells with Hoechst 33342 (1 g/ml, Invitrogen) for 30 min at 37 °C. We then placed the cells on a glass slide pretreated with poly-L-lysine (0.01% w/v in water) by centrifuging a 20-l drop of cell suspension at 1,000 rpm for 5 s. Images were acquired using a 20/0.5 Ph2 objective (EC Plan Neofluar, Zeiss), DAPI filter set, and charge- coupled device camera (AxioCam MRm, Zeiss). Analysis of nuclear geometry was performed using ImageJ. Physical Properties of the Neutrophil Nucleus When a cell arrives at a constriction, it is subjected to physical forces resulting from external stresses due to the pres- sure drop across the cell trapped in the constriction; these stresses cause the cell to deform and passage through the pore. Given the dimensions of a single pore, a pressure of 28 kilopas- cals corresponds to approximately micronewton-scale forces. The rate at which the cell deforms largely depends on the applied stress (driving pressure) as well as the global mechani- cal properties of the cell and nucleus (5, 29–32) As individual cells deformed through the 5-m constrictions of the microflu- idic device, we imaged their passage using a high speed camera (Fig. 1, B and D). By automated image analysis, we determined the time required for the cell to passage through the first 5-m constriction, which we define as its passage time. Given these millisecond timescales of cell passage at a driving pressure of 28 Two-dimensional Migration Assay—Glass-bottomed dishes (World Precision Instruments, Sarasota, FL) were coated with human fibronectin (10 g/ml in Hanks’ balanced salt solution without calcium and magnesium, Gemini Bio-products). Cells were seeded onto the dishes, and images were acquired at 1-min intervals over 3 h (5% CO2, 37 °C) using a Zeiss microscope outfitted with an automated stage (Applied Scientific Instru- ments, Eugene, OR), 10/0.3 Ph objective (EC-Plan Neofluar, Zeiss), and charge-coupled device camera (AxioCam MRm, Zeiss); x-y positions of cells were extracted from the resultant movies (ImageJ), and trajectory analysis was performed using MATLAB. RESULTS B and C, quantitative analysis of lamin A and LBR protein levels normalized first to -tubulin and then to day 0 for each protein in each cell line. Error bars represent S.E. of 3–5 independent experiments; where not visible, they are smaller than the symbols. Based on immunoblot analysis, base-line levels of lamin A are estimated to be 20–30 greater in theLamAOEcellsascomparedwiththemockcontrolcells(supplementalFig. S2); for this reason, two separate axes are plotted for each cell line. D, expres- sion levels of the cell surface antigen, CD11b, a hallmark of neutrophils, increaseduringdifferentiationforallcelllines.Left,representativehistograms of data from a single flow cytometry experiment showing the distribution of CD11b expression levels at day 4 after ATRA stimulation. Right, graphs show- ingmedianvaluesofCD11bafterATRAtreatmentwiththevaluesforeachcell line normalized to day 0 for each independent experiment. Error bars repre- sent S.E. over three independent experiments. Scrmbl Ctrl, scrambled control. E, respiratory burst assay that probes superoxide production using a lumines- cence assay 30 min after stimulation by phorbol myristate acetate, indicating that all cells show normal functional characteristics of neutrophils. Lumines- cence values are relative to the mock and scrambled control for the left and right panels, respectively. Data represent the average of three independent experiments; error bars represent the S.E. n.s., p 0.05 for LBR KD versus scrambled control; , p 0.05 for LamA OE versus mock control. To test the effect of increased lamin A levels on cell passage through narrow constrictions, we generated a LamA OE HL-60 cell line by retroviral transduction. The resulting subpopulation of high expressing cells exhibits lamin A levels that are about 20–30-fold higher than the mock-modified cells (supplemental Fig. S2A). Although lamin A expression levels in the LamA OE cells are greater than those in unmodified HL-60 cells, they are comparable with physiological levels in other somatic cells such as mouse embryo fibroblast cells (supplemental Fig. S2B). To confirm that the ectopic lamin A is properly localized to the nuclear envelope, we conducted immunofluorescence and con- focal imaging (supplemental Fig. S3). ATRA treatment, and performed immunoblotting (Fig. 2A). As expected, unmodified and mock-modified cells displayed a strong up-regulation of LBR during granulopoiesis with a con- current decrease in lamin A levels (Fig. 2, B and C), confirming previous observations (11). In contrast, LamA OE cells have increased levels of lamin A that further increased during gran- ulopoiesis (Fig. RESULTS E, respiratory burst assay that probes superoxide production using a lumines- cence assay 30 min after stimulation by phorbol myristate acetate, indicating that all cells show normal functional characteristics of neutrophils. Lumines- cence values are relative to the mock and scrambled control for the left and right panels, respectively. Data represent the average of three independent experiments; error bars represent the S.E. n.s., p 0.05 for LBR KD versus scrambled control; , p 0.05 for LamA OE versus mock control. To assess the effect of hypolobulated or round-shaped nuclei on the passage of neutrophil-type cells through micron-scale constrictions, we used LBR KD cells as an in vitro system. In contrast to the control cells that exhibit strong up-regulation of LBR during differentiation and develop lobulated nuclei, LBR KD cells show only trace levels of LBR expression and maintain round nuclei (17). Nevertheless, despite their round nuclei, LBR KD cells exhibited similar passage times as compared with the scrambled control cells (Fig. 1B). These observations suggest that the multilobed shapes of nuclei in mature neutrophils pro- vide no significant advantage in the time required for cells to deform through 5-m constrictions. Generating Neutrophil-type Cells with Increased Lamin A Expression—Because the above experiments indicate that lob- ulated nuclear shape is not essential for neutrophil-type cell passage through narrow constrictions, we hypothesized that the unique molecular composition of the nuclear envelope in neutrophils could determine the ability of cells to deform. One possible origin may be the low levels of the key structural pro- tein of the nucleus, lamin A; this protein is normally down- regulated by over 90% in ATRA-stimulated HL-60 cells after 4–5 days of stimulation (Fig. 2B) (11, 14). Given the essential role of lamin A in nuclear mechanical stability (12, 13, 30), we postulated that preventing lamin A down-regulation could reduce nuclear deformability and impair cell passage through pores. Because the LBR KD neutrophil-type cells that have round nuclei have similar reduced lamin A expression levels as unmodified and mock-modified cells, this may also explain their unaltered passage times (17). FIGURE 2. Genetically modified HL-60 cells show typical characteristics of neutrophils after ATRA stimulation. A, representative immunoblots for lamins A/C, B1, B2, and LBR with -tubulin as loading control. Cell lysates are collected from LamA OE and mock control (Mock Ctrl) cells at days 0, 3, and 5 after ATRA stimulation. RESULTS Lobulated Nuclear Shape Is Not Essential for Cell Transit— To probe the ability of neutrophil-type cells with round or lob- ulated nuclei to deform through narrow gaps, we designed a microfluidic device with precisely defined constrictions of 5-m width (Fig. 1A); this width is less than the typical 7–10-m diameter of HL-60 nuclei, such that nuclear deforma- VOLUME 288•NUMBER 12•MARCH 22, 2013 8612 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288•NUMBER 12•MARCH 22, 2013 FIGURE 2. Genetically modified HL-60 cells show typical characteristics of neutrophils after ATRA stimulation A representative immunoblots for Physical Properties of the Neutrophil Nucleus Physical Properties of the Neutrophil Nucleus y p p kilopascals, this microfluidic assay primarily probes the passive mechanical behavior of the cell, as actin remodeling and pro- tein expression changes occur on timescales of several minutes and more (33). Although actin can contribute to the cortical stiffness of neutrophils (4–6), we confirmed that the actin makes little contribution to these measurements by treating a subset of neutrophil-type cells with cytochalasin D to disrupt actin polymerization; this treatment had no effect on passage times (data not shown), indicating that the deformability of the nucleus has a pivotal role in the passage of cells through micron-scale pores. FIGURE 2. Genetically modified HL-60 cells show typical characteristics of neutrophils after ATRA stimulation. A, representative immunoblots for lamins A/C, B1, B2, and LBR with -tubulin as loading control. Cell lysates are collected from LamA OE and mock control (Mock Ctrl) cells at days 0, 3, and 5 after ATRA stimulation. B and C, quantitative analysis of lamin A and LBR protein levels normalized first to -tubulin and then to day 0 for each protein in each cell line. Error bars represent S.E. of 3–5 independent experiments; where not visible, they are smaller than the symbols. Based on immunoblot analysis, base-line levels of lamin A are estimated to be 20–30 greater in theLamAOEcellsascomparedwiththemockcontrolcells(supplementalFig. S2); for this reason, two separate axes are plotted for each cell line. D, expres- sion levels of the cell surface antigen, CD11b, a hallmark of neutrophils, increaseduringdifferentiationforallcelllines.Left,representativehistograms of data from a single flow cytometry experiment showing the distribution of CD11b expression levels at day 4 after ATRA stimulation. Right, graphs show- ingmedianvaluesofCD11bafterATRAtreatmentwiththevaluesforeachcell line normalized to day 0 for each independent experiment. Error bars repre- sent S.E. over three independent experiments. Scrmbl Ctrl, scrambled control. Physical Properties of the Neutrophil Nucleus C, box plots show the circularity of nuclei at days 0, 3, and 5 after ATRA treatment. The white bar denotes the population median, boxes are the 25th and 75th percentiles, and lines show the 10th and 90th percentiles. To evaluate statistical significance, we compared the medians of at least 3 independent experiments for each cell type. Day 0, unmodified to scrambled control. n.s., p 0.05; , p 0.05. Nuclei from over 300 individual cells were analyzed for each cell type. HL-60 cells after 3–5 days following ATRA treatment (11, 18). To investigate the effect of altered nuclear envelope composition on this shape transition, we imaged Hoechst-stained nuclei by flu- orescence microscopy over the differentiation time course. To quantify changes in nuclear shape, we analyzed the circularity of nuclei, defined as 4A/P2, where A is the cross-sectional area and P is the perimeter of an individual nuclear cross-section. For a perfect circle, the circularity value equals one; lower val- ues reflect deviations from a circular shape. In the undifferen- tiated state, all cell lines have nuclei with predominantly circu- lar shape and similarly high circularity values, representative of round nuclei (Fig. 3, A–C). Another hallmark of neutrophil cells is their respiratory burst response upon exposure to phagocytotic stimuli, such as yeast or bacteria. To probe this functional characteristic of the modified neutrophil-type cells, we stimulated cells with phor- bol myristate acetate and measured subsequent superoxide production (Fig. 2E). LBR KD cells showed similar response as compared with the scrambled controls. LamA OE cells exhib- ited a small yet statistically significant 15% reduction in super- oxide production as compared with the mock controls. Overall, these experiments suggest that the functional and biochemical characteristics of the genetically modified neutrophil-type cells are generally maintained despite their altered nuclear envelope composition. After 3 days of ATRA treatment, the unmodified, mock, and scrambled control cells exhibited nuclei with large invagina- tions; circularity values correspondingly showed a lower median and greater variability, reflecting these irregular nuclear shapes. By contrast, LBR KD cells retained their round shape, as reflected by the higher circularity values, even after 5 days of ATRA treatment (Fig. 3A) (17). The nuclei of LamA OE cells showed some morphological changes but failed to develop the characteristic lobulations seen in the unmodified and mock control cells (Fig. 3A, supplemental Fig. Physical Properties of the Neutrophil Nucleus FIGURE 3. Nuclear shape transition during granulopoiesis requires lamin A down-regulation and LBR up-regulation. A, fluorescent images of Hoechst-stained nuclei acquired at day 0 and day 5 after ATRA treatment. All images were acquired at the same magnification. Scale bar, 5 m. B, to quan- titatively describe nuclear shape, the circularity of the nucleus is defined as 4A/P2. Histograms show the distribution for each cell type at days 0 and 5 after ATRA treatment. C, box plots show the circularity of nuclei at days 0, 3, and 5 after ATRA treatment. The white bar denotes the population median, boxes are the 25th and 75th percentiles, and lines show the 10th and 90th percentiles. To evaluate statistical significance, we compared the medians of at least 3 independent experiments for each cell type. Day 0, unmodified to scrambled control. n.s., p 0.05; , p 0.05. Nuclei from over 300 individual cells were analyzed for each cell type. elevated basal levels of LBR as compared with the mock con- trols, with a similar 4-fold increase in LBR levels during gran- ulocytic differentiation. We also probed levels of other struc- tural proteins that could contribute to cell deformability (4–6); during differentiation in both the mock-modified and LamA OE cells, actin levels showed minor variations, and other struc- tural nuclear proteins, including lamin B1 and B2, showed a decrease in expression levels (Fig. 2) (34). Although we cannot exclude the possible contribution of lamin B1 and B2 down- regulation to altered cellular mechanical properties, we antici- pate that the observed changes in lamin B1/B2 levels would have little effect on nuclear mechanical properties in compari- son with the lamin A up-regulation; lamins A/C have a predom- inant role in nuclear shape stability and stiffness (12, 13, 30), whereas lamin B1 does not have any significant effect on nuclear mechanical stability (13). y Genetically Modified Cells Display Characteristics of Neutrophils—To test whether the genetically modified HL-60 cells still undergo normal granulopoiesis, we assayed essential functional, biochemical, and proteomic characteristics that define neutrophils. One metric to assess the differentiation of HL-60 cells into neutrophil-type cells is to measure cell density following ATRA stimulation; decreased proliferation rates are an indicator of successful differentiation as cells exit the cell cycle to commit to their differentiation into neutrophils (35). RESULTS 2, B and C), possibly due to an ATRA-sensitive element in the ectopic promoter region. LamA OE cells showed Protein Composition of LamA OE Cells—To characterize how protein levels of the LamA OE cells change during granu- lopoiesis, we monitored expression levels of major structural proteins over the differentiation time course; we induced the HL-60 cells to differentiate into neutrophil-type cells by ATRA treatment, collected cell lysates at days 0, 3, and 5 following MARCH 22, 2013•VOLUME 288•NUMBER 12 8613 MARCH 22, 2013•VOLUME 288•NUMBER 12 JOURNAL OF BIOLOGICAL CHEMISTRY Physical Properties of the Neutrophil Nucleus Both LBR KD and LamA OE cells showed a similar progressive decrease in proliferation rates over the days following ATRA treatment as compared with the scrambled and mock controls (supplemental Fig. S4). As a more direct assay of differentiation into neutrophil-type cells, we measured expression levels of the cell surface marker, CD11b, a subunit of a heterodimeric adhe- sion glycoprotein, which is widely used as a marker for neutro- phils (36). After 4 days of ATRA treatment, CD11b levels were increased for all cell types as compared with the undifferenti- ated HL-60 cells and vehicle-treated controls (supplemental Fig. S5). These results confirmed that the HL-60 cells are dif- ferentiating into neutrophil-type cells. Importantly, we observed that all cell lines show significant increase in CD11b levels following ATRA stimulation, with levels varying slightly between cell lines (Fig. 2D); this demonstrates that the changes in nuclear envelope composition do not markedly affect differ- entiation efficiency. FIGURE 3. Nuclear shape transition during granulopoiesis requires lamin A down-regulation and LBR up-regulation. A, fluorescent images of Hoechst-stained nuclei acquired at day 0 and day 5 after ATRA treatment. All images were acquired at the same magnification. Scale bar, 5 m. B, to quan- titatively describe nuclear shape, the circularity of the nucleus is defined as 4A/P2. Histograms show the distribution for each cell type at days 0 and 5 after ATRA treatment. C, box plots show the circularity of nuclei at days 0, 3, and 5 after ATRA treatment. The white bar denotes the population median, boxes are the 25th and 75th percentiles, and lines show the 10th and 90th percentiles. To evaluate statistical significance, we compared the medians of at least 3 independent experiments for each cell type. Day 0, unmodified to scrambled control. n.s., p 0.05; , p 0.05. Nuclei from over 300 individual cells were analyzed for each cell type. FIGURE 3. Nuclear shape transition during granulopoiesis requires lamin A down-regulation and LBR up-regulation. A, fluorescent images of Hoechst-stained nuclei acquired at day 0 and day 5 after ATRA treatment. All images were acquired at the same magnification. Scale bar, 5 m. B, to quan- titatively describe nuclear shape, the circularity of the nucleus is defined as 4A/P2. Histograms show the distribution for each cell type at days 0 and 5 after ATRA treatment. Physical Properties of the Neutrophil Nucleus S3); the lack of severe lobulation that is typical for normal neutrophil cells illustrates that down-regulation of lamin A expression during neutrophil differentiation could also be required for the lobulated nuclear shape of mature neutrophils. Lamin A Expression Alters Nuclear Lobulations during Granulopoiesis—A key hallmark of granulopoiesis is the transi- tion from round to multilobed nuclear shape, which is observed in VOLUME 288•NUMBER 12•MARCH 22, 2013 8614 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288•NUMBER 12•MARCH 22, 2013 8614 FIGURE 4. Impaired migration of LamA OE cells through narrow constric- tions. To probe the active migration of cells through micron-scale pores, we use a Transwell migration assay. A and D, representative images from a single experiment showing Hoechst-stained nuclei of cells that have passed through 3- or 8-m pores. Scale bar, 100 m. Ctrl, control; Scrmbl Ctrl, scram- bled control; Mock Ctrl, mock control. B and E, migration efficiency is defined as the number of cells that passaged through the porous membrane relative to the corresponding scrambled or mock control. Bars represent averages from at least three independent experiments; error bars represent S.E. **, p 0.001 for LamA OE versus mock control. C and F, two-dimensional migration experiments were performed by tracking the positions of individual cells at 1-min intervals over 3 h. Traces of three representative cells for each cell type show the total distance traveled and the directionality of movement over the three-hour time-lapse experiment. Axes are 150 m with 50-m increments. Migration speed over the entire trajectory is computed from the individual traces of over 50 cells for each cell type. Mean values for each LBR KD and LamA OE cells are normalized to their respective controls. n.s., p 0.05; , p 0.05; *, p 0.001. Absolute velocities of cells are: LBR KD, 4.4–5.1 m/min; scrambled control, 3.5–4.2 m/min; LamA OE, 2.2–4.7 m/min; mock con- trol, 2.8–4.8 m/min. Physical Properties of the Neutrophil Nucleus Physical Properties of the Neutrophil Nucleus Increased Lamin A Expression Delays Cell Passage through Pores—Lamin A is a crucial modulator of nuclear deformability (13, 30, 37). To probe the effects of increased lamin A levels on the ability of cells to deform through physiological gaps, we measured the passage time of LamA OE neutrophil-type cells and mock controls when forced through the 5-m constric- tions of our microfluidic device (Fig. 1, A and D). Physical Properties of the Neutrophil Nucleus The LamA OE neutrophil-type cells exhibited a 3-fold increase in median pas- sage time as compared with the mock controls (Fig. 1E); these results indicate that increased density of lamin protein at the nuclear envelope may impair the ability of LamA OE neutro- phil-type cells to passage through the 5-m constrictions. Taken together, our results show that lamin A levels have an important effect on the ability of cells to passage through 5-m constrictions; physiological down-regulation of lamin A following ATRA-induced differentiation of HL-60 cells results in faster passage through the micron-scale constric- tions, whereas ectopically increased expression of lamin A results in slower passage of LamA OE-neutrophils through the 5-m constrictions. Active Migration through Pores Is Impaired in LamA OE Cells—The results of our microfluidic experiments illustrate that altered expression of lamin A can substantially alter the passive deformability of cells. However, a critical function of neutrophils is their ability to actively migrate through narrow constrictions. To test migration efficiency, we used a transwell migration assay to probe the ability of cells to migrate through 3- and 8-m pores; we monitored the number of cells that migrate through the pores after 2 h and determined the migra- tion efficiency relative to the respective control cells. As seen in the passive deformation results obtained by microfluidic assays, the LBR KD cells exhibited similar migration efficiency as the scrambled control cells (Fig. 4, A and B), further substantiating that neutrophil-type cells with round nuclei can exhibit equiv- alent passage efficiency through micron-scale pores. By con- trast, the LamA OE cells showed a marked reduction in migra- tion through 3-m pores (Fig. 4, D and E). The impaired migration was less severe in the experiments with 8-m pores (Fig. 4, D and E); because deformation through 8-m pores requires smaller deformations of nuclei, these results are con- sistent with our observations that nuclear deformation rate- limits the passage of cells through micron-scale constrictions. FIGURE 4. Impaired migration of LamA OE cells through narrow constric- tions. To probe the active migration of cells through micron-scale pores, we use a Transwell migration assay. A and D, representative images from a single experiment showing Hoechst-stained nuclei of cells that have passed through 3- or 8-m pores. Scale bar, 100 m. Ctrl, control; Scrmbl Ctrl, scram- bled control; Mock Ctrl, mock control. Physical Properties of the Neutrophil Nucleus B and E, migration efficiency is defined as the number of cells that passaged through the porous membrane relative to the corresponding scrambled or mock control. Bars represent averages from at least three independent experiments; error bars represent S.E. , p 0.001 for LamA OE versus mock control. C and F, two-dimensional migration experiments were performed by tracking the positions of individual cells at 1-min intervals over 3 h. Traces of three representative cells for each cell type show the total distance traveled and the directionality of movement over the three-hour time-lapse experiment. Axes are 150 m with 50-m increments. Migration speed over the entire trajectory is computed from the individual traces of over 50 cells for each cell type. Mean values for each LBR KD and LamA OE cells are normalized to their respective controls. n.s., p 0.05; , p 0.05; ***, p 0.001. Absolute velocities of cells are: LBR KD, 4.4–5.1 m/min; scrambled control, 3.5–4.2 m/min; LamA OE, 2.2–4.7 m/min; mock con- trol, 2.8–4.8 m/min. DISCUSSION It has long been speculated that the lobulated shape of the neutrophil nucleus is ”a special adaptation for passing through vessel walls“ (8). However, here we show that nuclear shape alone does not always determine the timescale for neutrophil deformation through micron-scale pores; neutrophil-type cells with round nuclei resulting from LBR knockdown (17) show unaltered passage efficiency through pores down to 3 m, as probed using both passive deformation through 5-m micro- fluidic constrictions, as well as active migration through 3- and 8-m porous membranes. To address the possibility that a general migration defect underlies the impaired transwell migration efficiency of the LamA OE cells, we performed two-dimensional migration assays; cells exhibited velocities from 2 to 5 m/min, consistent with previous observations of neutrophil migration (38). The LBR KD cells showed a slightly increased velocity as compared with the scrambled control cells (Fig. 4C). Importantly, LamA OE cells exhibited similar migration velocities as the mock con- trol cells (Fig. 4F), indicating that the observed differences in the transwell assay cannot be attributed to general defects in their migration. Taken together, our experiments indicate that the density of lamin A at the nuclear envelope is crucial in facilitating the passage of cells through micron-scale constrictions. These LBR KD cells also provide an in vitro model for Pelger- Huët anomaly; the nuclei from neutrophils of these individuals are round or bilobulated (36, 37) due to a complete or partial lack of functional LBR. The extent to which the altered nuclear shape of Pelger-Huët anomaly neutrophils affects their ability to passage through micron-scale constrictions has been incon- clusive (26, 39–41). Some previous studies of these neutrophils discovered altered migration (26, 39, 40); however, these pri- mary neutrophils also exhibited bilobular nuclei, and differ- MARCH 22, 2013•VOLUME 288•NUMBER 12 8615 MARCH 22, 2013•VOLUME 288•NUMBER 12 JOURNAL OF BIOLOGICAL CHEMISTRY Physical Properties of the Neutrophil Nucleus Physical Properties of the Neutrophil Nucleus (14–16). HL-60 cells also exhibit similar mechanical properties; recent measurements of cell compliance using an optical stretcher confirmed that in vitro differentiation of HL-60 cells into neutrophil-type cells recapitulates the 3–6-fold increase in cell deformability observed in primary neutrophils and their CD34 precursor cells (31). DISCUSSION A direct comparison of the abso- lute passage times through micron-sized constriction between primary neutrophils and HL-60-derived neutrophil-type cells is complicated by the fact that HL-60 cells are typically larger than primary neutrophils (12-m versus 7–8-m median diame- ter, respectively) and exhibit substantially larger transit times through microfluidic constriction channels (29). Consequently, we have focused our study on HL-60 cells and vary protein levels within the same cell type; this has enabled us to clearly illustrate the importance of nuclear envelope composition, par- ticularly the levels of lamins A/C, on the ability of cells to pass through narrow constrictions during perfusion and migration. FIGURE 5. Lamin A levels, rather than nuclear shape, are a primary deter- minant of the efficiency of cell passage through narrow constrictions. A schematic illustration summarizing the effects of nuclear shape and lamin A expression levels on the ability of cells to deform through narrow constric- tions is shown. The ratio of LamA to LBR expression levels is estimated from immunoblots. Undifferentiated (unmodified or mock-modified) HL-60 cells, as well as the LBR KD neutrophil-type cells, exhibit efficient passage, despite their round nuclear shape. By contrast, lamin A overexpression results in impaired passage, both through the constricted 5-m channels of a micro- fluidic device, as well as the 3- and 8-m pores of the transwell migration assay. Undifferentiated LamA OE cells with more circular nuclei and lower levels of LBR require even longer time to passage through narrow constrictions. It is intriguing to speculate that changes in levels of lamin A expression may have implications for cellular deformability in a variety of physiological processes and diseases (45). For exam- ple, certain types of cancer cells have reduced levels of lamin A expression as compared with their nonmalignant progenitors (46, 47). Akin to neutrophils, large deformations of cancer cells and their nuclei are required during deformation through micron-scale constrictions (48) in extravasation and metasta- sis. Ultimately, a deeper knowledge of the molecular basis of cellular and nuclear deformability will provide unique insights into the mechanical aspects of cell biology and possibly new therapeutic approaches. ences in migratory ability could result from other phenotypic differences. Here we used LBR KD cells as an in vitro system to specifically investigate the effect of the hypolobulated nucleus on cell passage through micron-scale constrictions. DISCUSSION Despite the round shape of LBR KD nuclei, which could sterically hinder the passage of nuclei through constrictions, these cells exhib- ited similar passage efficiencies through micron-scale constric- tions as compared with the scrambled control cells with multi- lobed nuclei (Fig. 1). Although LBR KD neutrophil-type cells have an atypical round nuclear shape, they have similarly low levels of lamin A as the unmodified controls (17). Here, we show that lamin A expression levels, rather than the shape of the cell nucleus, can be a major determinant of the timescale of cell passage through micron-scale gaps (Fig. 5). By contrast, nonmechanical func- tions of these cells are not substantially affected by changes in nuclear envelope composition. Although other types of white blood cells with ovoid-shaped nuclei, such as macrophages, also undergo transendothelial migration, their deformations occur on a slower timescale as compared with neutrophils (31). Indeed, monocyte/macrophage-differentiated HL-60 cells also show increased levels of lamin A/C expression relative to neu- trophil-type cells (34). Acknowledgments—We acknowledge the Center for Nanoscale Sys- tems, Harvard University, for access to the profilometer and Bino Varghese for advice on data analysis protocols. Cell sorting was per- formed by Brian Tilton, Bauer Center at Harvard University, and Jeff Calimlim in the UCLA Jonsson Comprehensive Cancer Center (JCCC) and Center for AIDS Research Flow Cytometry Core Facility. The UCLA JCCC and Center for AIDS Research Flow Cytometry Core Facility are supported by National Institutes of Health Awards CA-16042 and AI-28697 and by the JCCC, the UCLA AIDS Institute, the David Geffen School of Medicine at UCLA, and the UCLA Chan- cellor’s Office. If irregular nuclear shape is not essential for the deformabil- ity of neutrophil cells, then why do their nuclei exhibit this distinct shape? One possibility is that the multilobed nucleus could simply result from the marked changes in nuclear enve- lope protein composition. Indeed, lamin A levels impact the mechanical stability of the nuclear envelope, whereas ectopic overexpression of LBR can increase nuclear membrane surface area (42, 43) (Fig. 5). Alternatively, the unusual multilobed nuclear shape may facilitate other neutrophil functions, such as phagocytosis, the formation of neutrophil extracellular traps, or migration through even smaller 1-m constrictions of the endothelium, either between or through cells (44). Physical Properties of the Neutrophil Nucleus 6. Ting-Beall, H. P., Lee, A. S., and Hochmuth, R. M. (1995) Effect of cytocha- lasin D on the mechanical properties and morphology of passive human neutrophils. Ann. Biomed. Eng. 23, 666–671 and Berliner, N. (2008) Mouse neutrophils lacking lamin B-receptor ex- pression exhibit aberrant development and lack critical functional re- sponses. Exp. Hematol. 36, 965–976 and Berliner, N. (2008) Mouse neutrophils lacking lamin B-receptor ex- pression exhibit aberrant development and lack critical functional re- sponses. Exp. Hematol. 36, 965–976 7. Lautenschläger, F., Paschke, S., Schinkinger, S., Bruel, A., Beil, M., and Guck, J. (2009) The regulatory role of cell mechanics for migration of differentiating myeloid cells. Proc. Natl. Acad. Sci. U.S.A. 106, 15696–15701 27. Auguste, K. I., Jin, S., Uchida, K., Yan, D., Manley, G. T., Papadopoulos, M. C., and Verkman, A. S. (2007) Greatly impaired migration of implanted aquaporin-4-deficient astroglial cells in mouse brain toward a site of in- jury. FASEB J. 21, 108–116 8. Hirsch, J. G. (1959) Immunity to infectious diseases: review of some con- cepts of Metchnikoff. Bacteriol. Rev. 23, 48–60 28. Muinonen-Martin, A. J., Veltman, D. M., Kalna, G., and Insall, R. H. (2010) An improved chamber for direct visualisation of chemotaxis. PLoS One 5, e15309 9. Erzurum, S. C., Kus, M. L., Bohse, C., Elson, E. L., and Worthen, G. S. (1991) Mechanical properties of HL60 cells: role of stimulation and differ- entiation in retention in capillary-sized pores. Am. J. Respir. Cell Mol. Biol. 5, 230–241 29. Rosenbluth, M. J., Lam, W. A., and Fletcher, D. A. (2008) Analyzing cell mechanics in hematologic diseases with microfluidic biophysical flow cy- tometry. Lab. Chip 8, 1062–1070 10. Downey, G. P., Doherty, D. E., Schwab, B., 3rd, Elson, E. L., Henson, P. M., and Worthen, G. S. (1990) Retention of leukocytes in capillaries: role of cell size and deformability. J. Appl. Physiol. 69, 1767–1778 30. Pajerowski, J. D., Dahl, K. N., Zhong, F. L., Sammak, P. J., and Discher, D. E. (2007) Physical plasticity of the nucleus in stem cell differentiation. Proc. Natl. Acad. Sci. U.S.A. 104, 15619–15624 11. Olins, A. L., Herrmann, H., Lichter, P., and Olins, D. E. (2000) Retinoic acid differentiation of HL-60 cells promotes cytoskeletal polarization. Exp. Cell Res. 254, 130–142 31. Ekpenyong, A. E., Whyte, G., Chalut, K., Pagliara, S., Lautenschläger, F., Fiddler, C., Paschke, S., Keyser, U. F., Chilvers, E. R., and Guck, J. Physical Properties of the Neutrophil Nucleus Nucleus 1, 506–512 18. Meyer, P. A., and Kleinschnitz, C. (1990) Retinoic acid induced differen- tiation and commitment in HL-60 cells. Environ. Health Perspect. 88, 179–182 37. Rowat, A. C., Lammerding, J., Herrmann, H., and Aebi, U. (2008) Towards an integrated understanding of the structure and mechanics of the cell nucleus. BioEssays 30, 226–236 19. Duffy, D. C., McDonald, J. C., Schueller, O. J. A., and Whitesides, G. M. (1998) Rapid prototyping of microfluidic systems in poly(dimethylsilox- ane). Anal. Chem. 70, 4974–4984 38. Gonzalez, A. L., El-Bjeirami, W., West, J. L., McIntire, L. V., and Smith, C. W. (2007) Transendothelial migration enhances integrin-dependent human neutrophil chemokinesis. J. Leukocyte Biol. 81, 686–695 39. Park, B. H., Dolen, J., and Snyder, B. (1977) Defective chemotactic migra- tion of polymorphonuclear leukocytes in Pelger-Huet anomaly. Proc. Soc. Exp. Biol. Med. 155, 51–54 20. Gómez-Sjöberg, R., Leyrat, A. A., Pirone, D. M., Chen, C. S., and Quake, S. R. (2007) Versatile, fully automated, microfluidic cell culture system. Anal Chem. 79, 8557–8563 40. Repo, H., Vuopio, P., Leirisalo, M., Jansson, S. E., and Kosunen, T. U. (1979) Impaired neutrophil chemotaxis in Pelger-Huët anomaly. Clin. Exp. Immunol. 36, 326–333 21. Ory, D. S., Neugeboren, B. A., and Mulligan, R. C. (1996) A stable human- derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes. Proc. Natl. Acad. Sci. U.S.A. 93, 11400–11406 41. Matsumoto, T., Harada, Y., Yamaguchi, K., Matsuzaki, H., Sanada, I., Yo- shimura, T., Honda, M., and Tanaka, R. (1984) Cytogenetic and functional studies of leukocytes with Pelger-Huët anomaly. Acta Haematol. 72, 264–273 22. Jaalouk, D. E., Lejeune, L., Couture, C., and Galipeau, J. (2006) A self- inactivating retrovector incorporating the IL-2 promoter for activation- induced transgene expression in genetically engineered T-cells. Virol. J. 3, 97 42. Ellenberg, J., Siggia, E. D., Moreira, J. E., Smith, C. L., Presley, J. F., Worman, H. J., and Lippincott-Schwartz, J. (1997) Nuclear membrane dynamics and reassembly in living cells: targeting of an inner nuclear membrane protein in interphase and mitosis. J. Cell Biol. 138, 1193–1206 23. Spitzer, D., Wu, X., Ma, X., Xu, L., Ponder, K. P., and Atkinson, J. P. (2006) Cutting edge: treatment of complement regulatory protein deficiency by retroviral in vivo gene therapy. J. Immunol. 177, 4953–4956 24. Boguslavsky, R. L., Stewart, C. L., and Worman, H. J. (2006) Nuclear lamin A inhibits adipocyte differentiation: implications for Dunnigan-type fa- milial partial lipodystrophy. Physical Properties of the Neutrophil Nucleus (2012) Viscoelastic properties of differentiating blood cells are fate- and function- dependent. PLoS One 7, e45237 12. Lammerding, J., Schulze, P. C., Takahashi, T., Kozlov, S., Sullivan, T., Kamm, R. D., Stewart, C. L., and Lee, R. T. (2004) Lamin A/C deficiency causes defective nuclear mechanics and mechanotransduction. J. Clin. Invest. 113, 370–378 32. Bathe, M., Shirai, A., Doerschuk, C. M., and Kamm, R. D. (2002) Neutro- phil transit times through pulmonary capillaries: the effects of capillary geometry and fMLP-stimulation. Biophys. J. 83, 1917–1933 13. Lammerding, J., Fong, L. G., Ji, J. Y., Reue, K., Stewart, C. L., Young, S. G., and Lee, R. T. (2006) Lamins A and C but not lamin B1 regulate nuclear mechanics. J. Biol. Chem. 281, 25768–25780 33. Cano, M. L., Lauffenburger, D. A., and Zigmond, S. H. (1991) Kinetic analysis of F-actin depolymerization in polymorphonuclear leukocyte ly- sates indicates that chemoattractant stimulation increases actin filament number without altering the filament length distribution. J. Cell Biol. 115, 677–687 14. Olins, A. L., Buendia, B., Herrmann, H., Lichter, P., and Olins, D. E. (1998) Retinoic acid induction of nuclear envelope-limited chromatin sheets in HL-60. Exp. Cell Res. 245, 91–104 34. Olins, A. L., Herrmann, H., Lichter, P., Kratzmeier, M., Doenecke, D., and Olins, D. E. (2001) Nuclear envelope and chromatin compositional differ- ences comparing undifferentiated and retinoic acid- and phorbol ester- treated HL-60 cells. Exp. Cell Res. 268, 115–127 15. Collins, S. J. (1987) The HL-60 promyelocytic leukemia cell line: prolifer- ation, differentiation, and cellular oncogene expression. Blood 70, 1233–1244 35. Fleck, R. A., Athwal, H., Bygraves, J. A., Hockley, D. J., Feavers, I. M., and Stacey, G. N. (2003) Optimization of nb-4 and hl-60 differentiation for use in opsonophagocytosis assays. In Vitro Cell Dev. Biol. Anim. 39, 235–242 16. Gallagher, R., Collins, S., Trujillo, J., McCredie, K., Ahearn, M., Tsai, S., Metzgar, R., Aulakh, G., Ting, R., Ruscetti, F., and Gallo, R. (1979) Char- acterization of the continuous, differentiating myeloid cell line (HL-60) from a patient with acute promyelocytic leukemia. Blood 54, 713–733 36. Gaines, P., and Berliner, N. (2005) Differentiation and characterization of myeloid cells. in Current Protocols in Immunology, Unit 22F 5, John Wiley & Sons, Inc., New York 17. Olins, A. L., Ernst, A., Zwerger, M., Herrmann, H., and Olins, D. E. (2010) An in vitro model for Pelger-Huët anomaly: stable knockdown of lamin B receptor in HL-60 cells. REFERENCES 1. Doerschuk, C. M., Beyers, N., Coxson, H. O., Wiggs, B., and Hogg, J. C. (1993) Comparison of neutrophil and capillary diameters and their rela- tion to neutrophil sequestration in the lung. J. Appl. Physiol. 74, 3040–3045 2. Worthen, G. S., Schwab, B., 3rd, Elson, E. L., and Downey, G. P. (1989) Mechanics of stimulated neutrophils: cell stiffening induces retention in capillaries. Science 245, 183–186 3. Downey, G. P., Worthen, G. S., Henson, P. M., and Hyde, D. M. (1993) Neutrophil sequestration and migration in localized pulmonary inflam- mation. Capillary localization and migration across the interalveolar sep- tum. Am. Rev. Respir. Dis. 147, 168–176 4. Tsai, M. A., Waugh, R. E., and Keng, P. C. (1998) Passive mechanical behavior of human neutrophils: effects of colchicine and paclitaxel. Bio- phys. J. 74, 3282–3291 Here we have used HL-60 cells, which are a well established in vitro model system to study white blood cell lineages; for example, the resulting neutrophil-type cells show similar struc- tural and functional characteristics as primary neutrophils 5. Tsai, M. A., Frank, R. S., and Waugh, R. E. (1994) Passive mechanical behavior of human neutrophils: effect of cytochalasin B. Biophys. J. 66, 2166–2172 8616 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288•NUMBER 12•MARCH 22, 2013 VOLUME 288•NUMBER 12•MARCH 22, 2013 26. Gaines, P., Tien, C. W., Olins, A. L., Olins, D. E., Shultz, L. D., Carney, L., Physical Properties of the Neutrophil Nucleus Hum. Mol. Genet. 15, 653–663 43. Ma, Y., Cai, S., Lv, Q., Jiang, Q., Zhang, Q., Sodmergen, Zhai, Z., and Zhang, C. (2007) Lamin B receptor plays a role in stimulating nuclear envelope production and targeting membrane vesicles to chromatin dur- ing nuclear envelope assembly through direct interaction with importin . J. Cell Sci. 120, 520–530 25. Teufelhofer, O., Weiss, R. M., Parzefall, W., Schulte-Hermann, R., Mick- sche, M., Berger, W., and Elbling, L. (2003) Promyelocytic HL60 cells express NADPH oxidase and are excellent targets in a rapid spectropho- tometric microplate assay for extracellular superoxide. Toxicol. Sci. 76, 376–383 44. Feng, D., Nagy, J. A., Pyne, K., Dvorak, H. F., and Dvorak, A. M. (1998) Neutrophils emigrate from venules by a transendothelial cell pathway in response to FMLP. J. Exp. Med. 187, 903–915 26. Gaines, P., Tien, C. W., Olins, A. L., Olins, D. E., Shultz, L. D., Carney, L., MARCH 22, 2013•VOLUME 288•NUMBER 12 8617 MARCH 22, 2013•VOLUME 288•NUMBER 12 JOURNAL OF BIOLOGICAL CHEMISTRY JOURNAL OF BIOLOGICAL CHEMISTRY 8 47. Zink, D., Fischer, A. H., and Nickerson, J. A. (2004) Nuclear structure in cancer cells. Nat. Rev. Cancer 4, 677–687 48. Fu, Y., Chin, L. K., Bourouina, T., Liu, A. Q., and VanDongen, A. M. (2012) Nuclear deformation during breast cancer cell transmigration. Lab. Chip 12, 3774–3778 45. Campbell, M. S., Lovell, M. A., and Gorbsky, G. J. (1995) Stability of nu- clear segments in human neutrophils and evidence against a role for mi- crofilaments or microtubules in their genesis during differentiation of HL60 myelocytes. J. Leukoc. Biol. 58, 659–666 y y 46. de Las Heras, J. I., Batrakou, D. G., and Schirmer, E. C. (2012) Cancer biology and the nuclear envelope: A convoluted relationship. Semin. Can- VOLUME 288•NUMBER 12•MARCH 22, 2013 cer Biol., in press 47. Zink, D., Fischer, A. H., and Nickerson, J. A. (2004) Nuclear structure in cancer cells. Nat. Rev. Cancer 4, 677–687 48. Fu, Y., Chin, L. K., Bourouina, T., Liu, A. Q., and VanDongen, A. M. (2012) Nuclear deformation during breast cancer cell transmigration. Lab. Chip 12, 3774–3778 45. Campbell, M. S., Lovell, M. A., and Gorbsky, G. J. (1995) Stability of nu- clear segments in human neutrophils and evidence against a role for mi- crofilaments or microtubules in their genesis during differentiation of HL60 myelocytes. J. Leukoc. Biol. 58, 659–666 46. de Las Heras, J. I., Batrakou, D. G., and Schirmer, E. C. (2012) Cancer biology and the nuclear envelope: A convoluted relationship. Semin. Can- Physical Properties of the Neutrophil Nucleus cer Biol., in press 45. Campbell, M. S., Lovell, M. A., and Gorbsky, G. J. (1995) Stability of nu- clear segments in human neutrophils and evidence against a role for mi- crofilaments or microtubules in their genesis during differentiation of HL60 myelocytes. J. Leukoc. Biol. 58, 659–666 47. Zink, D., Fischer, A. H., and Nickerson, J. A. (2004) Nuclear structure in cancer cells. Nat. Rev. Cancer 4, 677–687 48. Fu, Y., Chin, L. K., Bourouina, T., Liu, A. Q., and VanDongen, A. M. (2012) Nuclear deformation during breast cancer cell transmigration. Lab. Chip 12, 3774–3778 y y 46. de Las Heras, J. I., Batrakou, D. G., and Schirmer, E. C. (2012) Cancer biology and the nuclear envelope: A convoluted relationship. Semin. Can- VOLUME 288•NUMBER 12•MARCH 22, 2013 8618 JOURNAL OF BIOLOGICAL CHEMISTRY 8618
https://openalex.org/W1605684955	http://www.scielo.br/pdf/rsp/v25n4/07.pdf	Spanish; Castilian	null	Infant undernourishment, the development coefficient and its relation to the environment: a pilot study	null	1,991	cc-by	5,171	Introducción hogares. Actualmente, se encuentra al momento del ingreso de los niños internados en los Centros de Recuperación Nutricional de la Corporación para la Nutrición Infantil (CONIN) un CD normal, aproximadamente en un 30%. Numerosos estudios han demostrado que la desnutrición infantil produce un retardo en el creci- miento y éste puede ser más o menos severo de- pendiendo del grado de nutrición3,4,6,10,14,16,17,25. Esta situación nos motivó para investigar que factores de tipo sociocultural podrían estar incidien- do en el desarrollo psicomotor normal de un lac- tante desnutrido. En este momento, prácticamente no se encuentran lactantes con desnutrición prima- ria severa en nuestro país, y CONIN está recuperan- do a los lactantes con desnutrición moderada (70- 80% de nutrición). También se ha dicho que la desnutrición afec- taría el desarrollo psicomotor del lactante, y por lo tanto, dejaría secuelas en el desarrollo psicológico posterior7,18,21,26. Sin embargo, esto último no es tan evidente cuando se evalúa el Coeficiente de Desarrollo (CD) de los lactantes desnutridos. En un estudio realizado sobre el lenguaje ges- tual de la madre durante el acto de darle de comer a su hijo lactante con desnutrición severa (60-70% de nutrición según Tablas del NCHS)15, se en- contró que había un 35% de lactantes con CD nor- mal2,24. Todos estos niños estaban viviendo en sus Desnutrición infantil, coeficiente de desarrollo y su relación con el medio ambiente: un estudio piloto Infant undernourishment, the development coefficient and its relation to the environment: a pilot study M. de Ia Luz Alvarez, Ximena Concha, Margarita Elordi, Cecilia Lamilla, Carolina Ramos, Paulina Perez ALVAREZ, M. L. et al.. Desnutrición infantil, coeficiente de desarrollo y su relación con el medio ambiente: un estudio piloto. Rev. Saúde públ., S. Paulo, 25: 282-8, 1991. Fueron estudiados los factores socioculturales que podrían estar influyendo en el desarrollo psicomotor normal del lactante desnutrido. La muestra consistió en 32 díadas madrelactante: 16 tenían CD normal (Grupo A) y 16 CD bajo lo normal (Grupo B) según el Test de Bailey aplicado a los lactantes recién ingresados a un Centro de Recuperación Nutricional. Los resultados muestran que había algunas diferencias en los antecedentes de los lactantes: Los lactantes del Grupo A eran producto de un embarazo deseado (p<.05), y fueron menos separados del lado de su madre (p<.007) que aquellos del Grupo B. No se encontraron diferencias en los antecedentes socioculturales y demográficos de las madres. El medio externo de los lactantes fue diferente en los aspectos observados en el barrio: hubo menos aspectos negativos en el Grupo A que en los del B (p<.001). El medio interno de los lactantes, también fue diferente en lo relacionado con la madre. Madres del Grupo A percibían más afección por parte de su pareja (p<.008), eran más sensitivas (sentimientos de alegría y de sufrimientos) (p<.003) y estimulaban más a su hijo (p<.004) que aquellas del Grupo B. Estos resultados muestran que el medio externo e interno en el cuál se desenvolvían los lactantes con CD normal y CD bajo lo normal eran diferentes. Esto podría dar cuenta de las diferencias en el desarrollo psicomotor entre lactantes desnutridos pertenecientes a un bajo estrato socioeconómico. Descriptores: Desarrollo infantil. Factors socioeconómicos. Trastornos de la nutrición infantil. * Instituto de Nutrición de la Universidad de Chile. ** Escuela de Trabajo Social del Instituto Professional de Santiago. Separatas/Reprints: M. de la Luz Alvarez - Casilla 138-11, Santiago , Chile. * Instituto de Nutrición de la Universidad de Chile. ** Escuela de Trabajo Social del Instituto Professional de Santiago. Separatas/Reprints: M. de la Luz Alvarez - Casilla 138-11, Santiago , Chile. Procedimiento Lo primero que se hizo fue la observación de la relación madre-lactante sin saber a que grupo pertenecía el niño a fin de evitar algún sesgo en la re-colección de la información. Luego se visitó a la madre en su domicilio para realizar las entre- vistas y la observación de la casa y del barrio. Posteriormente se conoció el resultado del Test de Bailey para saber en que grupo se colocaría al lactante. El Test de Bailey fue aplicado por espe- cialistas de CONIN en los Centros de Recupera- ción Nutricio-nal dentro de la semana de ingresso del lactante. Esto es parte de la rutina de atención que se da al lactante y no hubo interferencia con la observación. 2) Observaciones estructuradas: a) Medición del medio ambiente interno y externo que rodea al lactante: a') en el hogar: presencia de colores pre- dominantes en el jardín si había, por ej. plantas o flores; en los muros, (externos de la casa e inter- nos), o sea el color que tenían, lo mismo en los muebles; si había orden, ej. que no hubiera cosas tiradas que no correspondieran a lo que un niño puede hacer uso tales como juguetes, y la presen- cia de animales domésticos, a") en el barrio: pre- sencia a 6 cuadras a la redonda de aspectos negati- vos (basurales, canales abiertos, venta de alcohol clandestina, y otros) y positivos (centros de abas- tecimientos, canchas de deportes, agrupaciones co- munitarias, templos, movilización). Todo esto se marcaba con una X significando la presencia de lo que se buscaba. b) Relación madre-lactante: fre- cuencia y calidad de la estimulación que la madre daba a su hijo internado en el Centro de Recupera- ción Nutricional. Se observó durante 10 min. lo que la madre hacía del punto de vista verbal (ha- blarle, cantarle), gestual (caricias, besos) y lúdico (juegos ya fuera con juguetes o con sus propias manos) al momento de visitar por segunda vez al niño. Se hizo así, para dar tiempo a la madre a que El análisis de datos se hizo a través de índices: - NSE está compuesto de 65 puntos máximo y un mínimo de 13. Se considera NSE bajo cuando se obtiene un puntaje de 40 y más puntos. En esta población que es considerada deprivada, los pun- tajes fluctuaron entre 45 y 60 puntos correspon- diendo a un nivel medio bajo y muy bajo. Material y Método La muestra está formada por 32 diadas madre- lactante: 16 con CD normal (Grupo A) y 16 con CD bajo lo normal (Grupo B) según el Test de Bailey aplicado al ingreso del lactante al Centro de Recuperación Nutricional. El cien por ciento del grupo A fue calificado por dicho test como normal y el 31,2% como dudoso y 68,8% como anormal en el grupo B. Todos los lactantes provenían del área urbana de Santiago (Chile). Ambos grupos pertenecían a un nivel socioeconómico (NSE) bajo, las madres no diferían en nivel de escolari- dad, ni en edad ([] 19,62 grupo A y 20,12 grupo B), ni en años de convivencia con su pareja ( [] 3,06 grupo A y 4.37 grupo B). Los grupos fueron pareados por edad del lactante. El [] de edad del grupo A fue de 11.688 meses y la del grupo B fue de 12.688. La DS en el primer grupo es de 5.029 y en el segundo de 4.785 meses, pero no hay dife- rencia entre ambos grupos (p< 0,5579). Las técnicas aplicadas fueron: se habituara a la nueva situación. se habituara a la nueva situación. área urbana de Santiago (Chile). Ambos grupos pertenecían a un nivel socioeconómico (NSE) bajo, las madres no diferían en nivel de escolari- dad, ni en edad ([] 19,62 grupo A y 20,12 grupo B), ni en años de convivencia con su pareja ( [] 3,06 grupo A y 4.37 grupo B). Los grupos fueron pareados por edad del lactante. El [] de edad del grupo A fue de 11.688 meses y la del grupo B fue de 12.688. La DS en el primer grupo es de 5.029 y en el segundo de 4.785 meses, pero no hay dife- rencia entre ambos grupos (p< 0,5579). Las técnicas aplicadas fueron: Para realizar esta observación se actuó en pare- ja y sin que la madre observada se diera cuenta que ella era objeto de estudio, pues se actuaba den- tro de la sala como una persona más del Centro de Recuperación Nutricional. Las conductas observa- das se registraban en una hoja diseñada para este fin con las variables antes mencionadas en cada in- dicador específico. Una vez terminada la observa- ción se comparaba lo registrado en cada hoja y sólo se aceptó aquellas observaciones en que había un 90% de coincidencia en la conducta observada. Material y Método Las observadoras fueron entrenadas previamente en la misma situación de los Centros de Recupera- ción de CONIN. 1) Entrevistas: a) Encuesta Socioeconómica para medir el NSE con un instrumento que discri- mina en poblaciones relativamente homogéneas1. Esta escala incluye 13 variables cuyo puntaje es de 1 a 6 puntos. Las variables que incluye dicho ins- trumento son: número de personas que viven en el hogar, presencia del padre del lactante en el hogar, escolaridad y actividad del jefe de hogar, proprie- dad de la casa en que viven, calidad de la casa, sis- tema de abastecimiento de agua, disposición de ex- cretas, seguridad social, promiscuidad, pieza independiente para cocinar, recolección de basuras y algunos bienes en el hogar los cuáles dan un solo puntaje. b) Encuesta Sociocultural para medir al- gunos antecedentes del embarazo, parto y separa- ciones de la madre antes de que el lactante ingrese a CONIN; aspectos afectivos en su relación de pa- reja y expresión de sentimientos (alegrías y penas). Procedimiento j y y j - Expresión de penas (encuesta sociocultural) está compuesto por 6 indicadores cuyo puntaje fluctuó entre un máximo de 6 puntos y un mínimo de 0. La escala es de: 0-2 = alta expresión de penas; 2-4 = moderada expresión de penas; 4-6 = baja ex- presión de penas. Esto se obtuvo a través de una pregunta que decía: podría nombrar 3 penas o co- sas que la hayan hecho sufrir el año pasado? Se consignaba el hecho que fuera capaz de mencionar una o más penas y el tipo de ella (material, afectiva o ambas). Si la madre era capaz de mencionar 3 pe- nas y las tres de tipo afectivo obtenía 0 puntos, lo cuál indicaba que era capaz de expresar externa- mente sus penas. En la medida que no era capaz de decirlo, su puntaje aumentaba y demostraba menor capacidad de expresión. p p - Expresión de alegrías está constituido por 6 indicadores cuyo puntaje va de un máximo de 6 dre en el tiempo de la observación. El máximo de conductas observadas fueron 105 y el mínimo 14. Se consideró baja estimulación aquella que obtuvo entre 1 y 50 puntos y alta, aquella que presentó en- tre 51 y 105 puntos. puntos a un mínimo de 0. La escala es de: 0-2 = baja expresión de alegrías; 2-4 = moderada expre- sión de alegrías; 4-6 = alta expresión de alegrías. Esto se obtuvo preguntando a la madre que dijiera 3 alegrías o acontecimientos que la hicieron feliz el año anterior. También se registró el tipo de la alegría como en el caso anterior. y p Se aplicaron las pruebas de Fisher por ser una muestra pequeña, el t de Student y la Regresión Logística Múltiple. g - índice sentirse rechazada: este índice com- prende una serie de preguntas sobre la vida pasada desde el nacimiento hasta que formó su propia fa- milia. Se preguntó si se había sentido rechazada, por quien (padre, madre, abuelos, tíos hermanos) y cómo se lo demostraron (formas: materiales, afecti- vas o ambas). Los puntajes flucturon entre 5 y 0. A mayor puntaje menor es el rechazo percibido por la madre en su infancia y adolescencia. Resultados El primero se encuentra significativamente menos en el grupo A y el segundo, está significativamente más presente en dicho grupo en relación con el grupo B (p <.01). Los aspectos negativos del bar- rio encontrados fueron la presencia significativa- mente menor de basurales y de canales abiertos en el grupo A en relación al grupo B (p < 0,0009 y p < 0,02 respectivamente). El índice de aspectos negativos del barrio fue significativamente inferior en el grupo A en relación al grupo B (p < 0,003). Los aspectos positivos del barrio tales como la presencia de centros de abastecimientos (p <0,001), canchas de deportes (p < 0,01), agrupa- ciones (p < 0,01) y templos, iglesias (p < 0,02) fueron significativamente mayores en el grupo A en relación al grupo B. A nivel del índice de as- pectos positivos las familias del grupo A presenta- ron significativamente mayores aspectos positivos que aquellas del grupo B (p < 0,001). q q g p (p , ) La Tabla 3 señala la situación de la madre y su relación con el lactante. Un tercio de las ma- dres del grupo A percibía afecto por parte de su pareja contra ninguna en el grupo B (p < 0,008). La mayoría de las madres del grupo A fueron ca- paces de manifestar las alegrías vividas el año an- terior contra menos de la mitad de aquellas del grupo B( 81,3 % vs 43,7%). Las alegrías manifes- tadas fueron más afectivas en el grupo A que aquellas del grupo B (68,7% vs 37,5%). Las pe- nas también fueron más expresadas por las ma- dres del grupo A que aquellas del grupo B (75% vs 37,5%) y fueron de tipo afectivas (p < 0,03). Finalmente las madres del grupo A estimulan significativamente más a su hijo lactante que aquellas del grupo B (p < 0,004). q g p (p , ) La Tabla 4 presenta la Regresión Logística Múltiple realizada a fin de explicar, en parte, el CD del lactante desnutrido. El índice de expresión de penas está correlacionado negativamente con el CD: a mayor índice de expresión de penas (o incapacidad de expresión) sentidas por la madre, menor es el CD del lactante (p < 0,01). La per- cepción de la madre de haber sido castigada en su familia se correlaciona positivamente con el CD (p < 0,02). Resultados Primeramente se presentará algunos antecedentes del lactante. Más de la mitad de los lactantes del gru- po A provienen significativamente más de un emba- razo deseado contra menos de la mitad de los del gru- po B (p < 0,05). Más de la mitad de las madres de los lactantes del grupo A recibieron alimentación suple- mentaria durante el embarazo contra menos del ter- cio de aquellas del grupo B (p <0,05). Las semanas de gestación no difieren entre ambos grupos, lo mismo el tipo de parto que fue mayoritariamente normal (87,6% vs 81,2%). Tampoco difieren los grupo en el peso de nacimiento que fue sobre 2.500 grs. en am- bos grupos. Se encontró que los lactantes del grupo A tuvieron significativamente menos separaciones del lado de su madre que los lactantes del grupo B (p < .007), antes de ingresar al Centro de Recuperación Nutricional de CONIN. Estas separaciones de 15 días tienden a ser menos en el grupo A que en el B (p < 0,09). Finalmente, el promedio peso/ edad del lac- tante al ingreso a CONIN fue más alto en el grupo A que en el B (p < 0,05), presentando ambos una desnu- y - Aspectos negativos del barrio: está formado por 7 indicadores como los mencionados anterior- mente. El puntaje oscila entre 0 y 7 puntos. Cero significa la ausencia de lo observado. La escala va de: 0-2.3 = barrio no negativo; 2.3-4.6 = barrio más o menos negativo; 4.6-7= barrio negativo. g g - Aspectos positivos del barrio: está formado por 8 indicadores según los ítems señalados ante- riormente. El puntaje va de 0 a 8 puntos indicando 0 punto la ausencia de lo observado. La escala va de: 0-2.6 = barrio no positivo; 2.6-5.2 = barrio más o menos positivo; 5.2-8 = barrio positivo. p ; p - Estimulación materna: está formado por todas las conductas verbales (hablar, cantar, repetir soni- dos al niño), gestuales (besar, sonreir, acariciar) y lúdicas (pasar objetos para que juegue el niño, pa- searlo en la pieza, jugar con él) que realizó la ma- trición moderada. Sin embargo, no hay diferencia en el promedio peso/talla entre ambos grupos (Tabla 1). p p g p ( ) La Tabla 2 presenta el medio ambiente externo del lactante. En relación a los colores, la única diferencia encontrada es en el azul y amarillo. Discusión y Conclusiones La aceptación del retardo en el desarrollo psi- co-motor del lactante desnutrido ha impedido lle- var a cabo otro tipo de investigaciones que de- muestren que esta afirmación no es tan cierta. En estudios realizados hace años atrás se encontró que los lactantes con desnutrición severa presenta- ban un CD normal, pero esto quedó a un nivel restringido de difusión, ya que iba contra todo lo conocido y aceptado hasta ese momento13. Con el tiempo, la CONIN a través de sus Centros de Re- cuperación Nutricional también encontró que al- rededor del 30% de los lactantes desnutridos pre- sentaban un CD normal y no sabía a qué atribuirlo. Por esto, se estudió fundamentalmente el medio en el cual se desenvolvía el lactante. El fin fue detec- tar que factores podrían asociarse y correlacio- narse con el desarrollo psico-motor del lactante, ya que este desarrollo se basa principalmente en los estímulos que recibe19,20. q Primeramente se encontró que los antecedentes de los lactantes difieren en diversos aspectos. Los lactantes con CD normal presentan aspectos positi- vos tan importantes como: haber sido un hijo de- seado (p < 0,05), haber sufrido muchísimo menos separaciones de la madre antes de ingresar a un Centro de Recuperación Nutricional de CONIN (p < 0,007) y que estas separaciones tienden a ser • más cortas en tiempo (p < 0,09). Muchas de estas separaciones se debieron a hospitalizaciones del lactante, lo cual indica la mayor morbilidad y fue- ron anteriores a la internación en el Centro de Re- cuperación Nutricional de CONIN. Otras se debie- ron a que la madre dejaba al lactante en casa de la abuela para que se lo cuidara. Pese a estas separa- ciones, los lactantes del grupo A se vieron menos afectados por ella como es habitual en muchos ca- sos de hospitalizaciones prolongadas8, porque habría otros factores que lo favorecieron. Luego, los aspectos menos negativos y más posi- tivos del barrio (p < 0,003 y p < 0,001 respectiva- mente) se encontraron en el grupo A. Resultados El índice de aspectos negativos del barrio se correlaciona negativamente con el CD, mientras más negativo es el barrio (o sea, mayor puntaje obtenido) más bajo es el CD del lactante (p < 0,03). Finalmente, el índice de rechazo perci- bido por la madre en su familia de origen presen- ta una tendência a correlacionarse positivamente con el CD (p < 0,07). Es decir, a mayor puntaje nificaría que las diferencias en el CD posible- mente no podrían ser atribuidas en este caso, a es- tos factores. Sin embargo, la relación peso/edad, al momento de ingresar al Centro de Recuperación Nutricional de CONIN, es significativa, aunque dentro del rango de desnutrición moderada. Los lactantes del grupo A presentaron un peso superior al de aquellos del grupo B (p < 0,05), pero no se encontró diferencia en la relación peso/talla. El he- cho de no encontrar diferencia significativa entre ambos grupos en relación al NSE, señalaría que éste "per se" no condicionaría el medio ambiente en el cual se desenvuelve el lactante y que existiría otros factores que estarían influyendo. El NSE me- dido por los 13 indicadores que se usaron en este caso está mostrando las características inmediatas del jefe de hogar y de la vivienda, lo cuál no inva- lida las características externas del medio, en este caso, del barrio en que está ubicada la familia. El medio externo que rodea al lactante es significati- vamente diferente entre los dos grupos. Por ejem- plo, a nivel de colores se encontró que el azul está más presente en el grupo A (p < 0,01) y el amaril- lo menos (p < 0,01). Es indudable que los colores ejercen una estimulación visual y en este caso, el color más estimulante se encontró en los lactantes con CD normal. del índice de Rechazo significa que la madre fue menos rechazada y a su vez tiende el niño a tener mayor CD. Discusión y Conclusiones The infants' external environment was different in aspects observed within the neighborhood: there were fewer negative aspects in Group A than in Group B (p < .003) and more positive in Group A than in B (p < .001). The infant's internal environment as related to the mother was also different. Mothers of Group A perceived more affection from their partner (p < .008), were more sensitive (to feelings of joy and suffering) (p < .003) and stimulated their infant (p < .004) more than those of Group B. These results show that the internal and external environments in which the infant with normal and below normal DQ evolves were different between the two groups. This could explain the differences in psychomotor development among undernourished infants belonging to the lower socioeconomic strata. Esto se reafirma con la correlación encontrada respecto a la expresión de penas (p < 0,01). p p p (p ) Pero también es importante destacar la relación observada entre la madre y el lactante. Esta indicó que existía una alta estimulación por parte de la ma- dre hacia el lactante en aquellas madres del grupo A (p < 0,004). Se ha demostrado que el lactante neces- sita ser estimulado para lograr su desarrollo psico- motor5,9,11,12,22,23, por lo tanto, estas madres al actu- ar en forma más estimulante, junto con presentar un ambiente externo más positivo, tener más capacidad de expresar sus penas y alegrías y tener una tenden- cia a hab cia y adolescencia, podrían haber contribuido en el CD normal que presentó el lactante del grupo A. ALVAREZ, M. L. et al. [Desnutrição infantil, coefi- ciente do desenvolvimento e sua relação com o meio ambiente: um estudo piloto.] Rev. Saúde publ., S. Paulo, 25: 282-8, 1991. Estudam-se os fatores sócio-culturais que poderiam estar influenciando no desenvolvimento psicomotor normal do lactente desnutrido. A amostra consistiu de 32 pares mãe-criança: 16 com coeficiente de desenvolvimento (CD) normal (grupo A) e 16 com CD abaixo do normal (grupo B) de acordo com o teste de Bailey aplicado a lactentes que ingressavam a um centro de recuperação nutricional. Havia diferenças nos antecedentes das crianças: os lactentes do grupo A eram fruto de gravidez desejada (p < 0,05), e foram menos se- parados de suas mães (p < 0,007) que os do grupo B. Não se encontravam diferenças nos antecedentes demo- gráficos e sócio-culturais das mães. Discusión y Conclusiones Esto podría indicar que las condiciones del barrio ayudarían a la madre a sacar al niño fuera del hogar por diferentes razones: llevarlo cuando ella hace las compras, pues tiene centros de abastecimientos cercanos; lle- varlo a la cancha de foot-ball los fines de semana, ya que esta es una entretención muy difundida en los barrios y es gratuita; llevarlo a la iglesia o tem- plo, pues es habitual asistir a los servicios si se prac- tica una religión. Esto se corrobora con la correla- ción negativa encontrada: a mayores aspectos negativos del barrio, menor CD presenta el lactante (p < 0,03). Estos resultados están acordes con otros estudios los cuáles valoran la calidad del barrio 13. Las características de tipo sociodemográficas de la madre, tampoco difieren. Los grupos presen- taron similar baja escolaridad, edad y años de con- vivencia con su pareja. Esto señalaría que las dife- rencias de CD del lactante se deberían a otras características propias de la madre tales como: mayor equilibrio afectivo en las madres del grupo A que en aquellas del grupo B. Las primeras sien- ten mayor afección por parte de su pareja (p < 0,0008) lo cual podría significar que al sentirse queridas, ellas también transmitan ese cariño a su hijo; son capaces de expresar penas y alegrías (p < 0,003) de tipo afectivo lo cual indicaría una mayor sensibilidad que las otras madres. También se encontró que durante el embarazo de los lactantes con CD normal, la madre recibió más suplementación alimentaria que las otras (p < 0.05). Esto podría indicar que estuvo más contro- lada por el equipo de salud que las otras madres y que podría haber recibido más información acerca de los cuidados que debería dar a su hijo, tanto du- rante el embarazo, como del recién nacido. Esto es parte de la rutina de trabajo de las matronas y de las enfermeras de los Consultorios Periféricos. El hecho que no se haya encontrado ninguna diferencia respecto a las semanas de gestación, ni al tipo de parto, ni en el peso de nacimiento, sig- sido menos rechazadas durante su infan- infants of Group A were the product of a wished-for pregnancy (p < .05), and were separated less from their mother's side (p < .007) than was the case in Group B. No differences were found in the socio-cultural and demographic background of the mothers. Discusión y Conclusiones Em relação ao meio ambiente externo observado no bairro de residência, ha- via menos aspectos negativos no grupo A que no B (p < 0,001). O meio interno também foi diferente em relação às mães: As do grupo A recebiam mais carinho por parte do companheiro (p < 0,008), eram mais sensíveis (senti- mentos de alegria e sofrimento) ( p < 0,003) e estimula- vam mais os seus filhos (p < 0,006) que as do grupo B. Os resultados mostraram que os meios internos e externos no qual se desenvolvem crianças com CD abaixo do normal eram diferentes. Isto poderia explicar as dife- renças no desenvolvimento psicomotor em desnutridos pertencentes a baixos níveis sócio-econômicos. Keywords: Child development. Socioeconomic factors. Infant nutrition disorders. Keywords: Child development. Socioeconomic factors. Infant nutrition disorders. References 1. ALVAREZ, M.L.; WURGAFT, R; SALAZAR, M.E. Me- diciones de nivel socioeconomico bajo urbano en fami- lias con lactante desnutrido. Arch. latinoamer. Nutr., 32: 650-62, 1982. 2. ALVAREZ, M.L.; WURGAFT, F.; WILDER, H. Non ver- bal language in mothers with malnourished infants: a pilot study. Soc. Sci. - Med., 16: 1365-9,1982. 3. ASTBURY, J.; ORGILL, A.A.; BAJUK, B.; YU, V.Y.H. Sequelae of growth failure in appropiate for gestational age, very low birth-weigh infants. Develop. Med. Child Neurol. 28: 472-9, 1986. 4. BRAZELTON, B.T.; TRONICK, E; LECHTIG, A.; LA- SKY, R.E.; KLEIN, R. E. The behavior of nutritionally deprived Guatemalan infants. Develop. Med. Child Neurol., 19: 364-72, 1977. 5. BROFENBRENNER, U. Ecology of the family as a con- text for human development research perspectives. De- velop. Psycol., 22: 723-42, 1986. 6. CATER, J.I. Correlates of low birth weight. Child Care Hlth Develop., 6: 267-77, 1980. 7. CELEDON, J.M. & ANDRACA, I. de. Psychomotor devel- opment during treatment of severely marasmic infants. Early hum. Develop., 3: 276-285,1979. Descritores: Desenvolvimento infantil. Fatores sócio- econômicos. Distúrbios da nutrição infantil, compli- cações. 8. CELEDON, J.M.; CSASZAR, D.; MIDDLETON, J.: AN- DRACA, L. de. The effect of treatment of marasmic in- fants according to age of admission. J. ment. Defic. Res., 24: 27-35,1980. ALVAREZ, M. L. et al [Infant undernourishment, the development coefficient and its relationship to the environment: a pilot study.] Rev. Saúde públ., S. Paulo, 25: 282-8, 1991. The socio-cultural factors that might be influencing the normal psychomotor development of the undernourished infant are studied. The sample consisted of 32 mother-infant dyads: 16 having normal DQ (Group A) and 16 having a below normal DQ (Group B) according to Bailey's Test applied to infants who entered a Nutritional Recuperation Center. Results showed that there were some differences in the infants' backgrounds: 9. COHN, J.F. & TRONICK, E.Z. Mother-infant face-to-face interaction: the sequence of dyadic status at 3,6 and 9 months. Develop. Psycol, 23: 68-77, 1987. 10. CRAVIOTO, J. & DELICARDIE, E. La malnutrición pre- coz en la primera infancia & algunas de suas posteri- ores repercusiones en los individuos y en la comunidad. Aliment. y Nutr. 2: 2-12,1976. 11. ESTRADA, P.; ARSENIO, W.F.; HESS, R.D.; HOLLO- WAY, S.D. Affective quality of the mother-child rela- tionship: longitudinal consequences for children's school-relevant cognitive functioning. Develop. Psy- chol., 23: 210-5, 1987. York, John Wiley, 1979. p. 519-48. 12. FEIRING, C; FOX, N.A.; JASKIN, J.; LEWIS, M. References The re- lation between social support, infant risk status and mother-infant interaction. Develop. Psychol., 23: 400-5, 1987. y 20. PARKE, R.D. Children's home environments: social and cognitive effects. In: Altman, I. & Wohlwill, J.F., eds. Children and environment: human behavior and envi- ronment. New York, Plenum, 1978. v. 3. p. 33-81. 13.GARBARINO, J. & SHERMAN, D. High-risk neigbord- hoods and high risk families: the human ecology of child maltreatment. Child Develop., 51: 188-98,1980. ronment. New York, Plenum, 1978. v. 3. p. 33-81. 21.POLLIT, E. & LEWIS, N. Nutrition and educational achievement. Part II: Correlations between nutritional and behavioral test indicators within populations where malnutrition is not a major public health problem. Food Nutr. Bull. 2 (4): 33-7, 1980. 14. GRAVES, P.L. Nutrition and infant behavior: a replication study in the Katmandu, Valley Nepal. Amer. J. clin. Nutr. 31: 541-51,1978. ( ) , 22. RUDDY, M.G. & BORNSTEIN, M.H. Cognitive correlates of infant attention and maternal stimulation over the first year of life. Child Develop., 53: 183-8,1982. 15.HAMILL, P.V.V.; DRIZD, T.A.; JOHNSON, C.L.; REED, R.B.; ROCHE, A.F.; MOORE, W.M. Physical growth: National Center for Health Statistics percentiles. Amer. J. clin. Nutr., 32: 607-29,1979. y p 23. SANDEEP, P. & PUSHPA, M. Deprivation and cognitive development in children. Child Psychiatry Quart. 14 (2): 47-54, 1981. , , 16. JOHNSTON, F.E.; NEWMAN, B.; CRAVIOTO, J.; DELI- CARDIE, E.; SCHOLL, T. A factor analysis of corre- lates of nutritional status in Mexican children, birth to 3 years. In: Greene L.S. & Johnston, F.E., eds. Social and biological predictors of nutritional status physical growth and neurological development. New York, Aca- demic Press, 1980. p. 291-307. 24. WURGAFT, F. Factores socioculturales y su relación con la desnutrición del lactante: estudio en madres de nivel so- cioe en Antropologia-Universidad de Chile]. 25. ZESKIND, P. S. & RAMEY, C.T. Fetal malnutrition: an ex- perimental study of its consequences on infant develop- ment in two caregiving environments. Child Develop. 49: 1155-62,1978. , p 17. LASKY. R.E.; KLEIN, R.E.; YARBROUGH, C.; ENGLE, P.L.; LECHTIG, A.; MARTORELL, R. The relationship between physical growth and infant behavioral develop- ment in rural Guatemala. Child. Develop., 52: 219-26, 1981. 26. ZESKIND, P.S. & RAMEY, G.T. Preventing intellectual and interactional sequelae of fetal malnutrition: a longi- tudinal, transactional and synergistic approach to devel- opment. Child Develop., 52: 213-8, 1981. 18. LASKY, R.E.; KLEIN, R.; YARBROUGH, C; SELLARS, M.J.; KAGAN, J. Recebido para publicação em 12/11/1990. Reapresentado em 29/05/1991. Aprovado para publicação em 3/06/1991. References Social interactions of Guatemalan in- fants. J. Cross cult. PsychoL, 14 (1): 17-28, 1983. Recebido para publicação em 12/11/1990. Reapresentado em 29/05/1991. Aprovado para publicação em 3/06/1991. y ( ) , 19. OSOFSKY, J.D. & CONNORS, K. Mother-infant interac- tion: an integrative view of a complex system. In: Osof- sky, J., ed. The handbook of infant development. New
https://openalex.org/W4248652866	https://ejournal.uncen.ac.id/index.php/KEUDA/article/download/1251/1055	Indonesian	null	Studi Komparatif Pendanaan Pembangunan Daerah dengan Penerimaan Umum Daerah, Obligasi Daerah, dan Pembayaran Ketersediaan Layanan	Keuda: Jurnal Kajian Ekonomi dan Keuangan Daerah	2,020	cc-by-sa	7,306	199 199 \| Jurnal Kajian Ekonomi & Keuangan Daerah, Volume 5, Nomor 2, September 2020 : 199-224 Keywords: regional bonds, availability payment, regional funding. Keywords: regional bonds, availability payment, regional funding. Supriyadi Supriyadi supriyadi@mail.ugm.ac.id Abstract The development of regional infrastructures is necessary to improve the service for the welfare of the people. In fact, not all local governments have a budget to fund the infrastructure development. This study compared the efficient funding source for regional infrastructure development between the funding from regional general acceptance with regional bonds and availability payment. This study refers to the case of the development of Wates Regional General Hospital (RSUD Wates) of Kulon Progo Regency. This study uses an interpretive paradigm with a descriptive qualitative research approach. The results show that funding with regional bonds is more efficient than funding with availability payment and funding from general regional acceptance. The regional bond scheme provides a funding efficiency of IDR501.71 billion compared to using regional general acceptance, while alternative funding for availability payment provides an efficiency of IDR430.69 billion compared to using sources from regional general acceptance. The difference shows that regional bonds are more efficient at IDR 71.02 billion compared to alternative funding for availability payment. STUDI KOMPARATIF PENDANAAN PEMBANGUNAN DAERAH DENGAN PENERIMAAN UMUM DAERAH, OBLIGASI DAERAH, DAN PEMBAYARAN KETERSEDIAAN LAYANAN Roby Aditiya roby.aditiya@uin-alauddin.ac.id Supriyadi supriyadi@mail.ugm.ac.id Roby Aditiya roby.aditiya@uin-alauddin.ac.id PENDAHULUAN Era otonomi daerah dan desentralisasi fiskal menuntut kemandirian daerah untuk dapat melakukan tugas dan tanggungjawab dalam memberikan pelayanan yang terbaik untuk meningkatkan kesejahteraan bagi masyarakat (Halim, 2014). Oleh karena itu, pemerintah daerah dituntut untuk dapat melakukan pembangunan diberbagai sektor. Salah satunya ialah pembangunan infrastruktur yang memadai. Pembangunan infrastruktur diyakini mampu memberikan multiplier effect dalam menggerakkan sektor rill, menyerap tenaga kerja, meningkatkan investasi, meningkatkan konsumsi masyarakat dan pemerintah, memicu kegiatan produksi serta meningkatkan perekonomian (Esfahani & Ramı´rez, 2003; Daroedono, 2004; Sahoo & Dash, 2009; Palei, 2015). \| Jurnal Kajian Ekonomi & Keuangan Daerah, Volume 5, Nomor 2, September 2020 : 199-224 200 Hanya saja, pembangunan infrastruktur daerah membutuhkan dana yang besar yang berimplikasi terhadap ketidakmampuan ruang fiskal pemerintah daerah untuk memenuhi kebutuhan pembangunan infrastruktur tersebut. Oleh karena itu, pemerintah daerah masih menggantungkan pendanaannya pada penerimaan umum daerah berupa dana perimbangan yang dikucurkan oleh pemerintah pusat baik dalam bentuk dana bagi hasil, dana alokasi umum, maupun dana alokasi khusus (Hamid, 2003; Kusumadewi & Rahman, 2007; Dewi & Kaluge, 2011; Inayati & Setiawan, 2017). Adanya ketimpangan antara kebutuhan pembangunan dan kemampuan pendanaan tersebut, mengharuskan pemerintah daerah mencari alternatif pendanaan lainnya. Di Indonesia, beberapa kebijakan telah dikeluarkan oleh pemerintah pusat untuk memberikan ruang kepada pemerinah daerah dalam mencari alternatif pendanaan pembangunan infrastruktur daerah. Alternatif pendanaan tersebut dapat dilakukan dengan skema pinjaman daerah maupun dengan skema kerjasama pemerintah daerah dan badan usaha (KPDBU) atau yang biasa disebut public private partnership (PPP) (Elmi, 2005; Djunedi, 2008; Kurniawan, Pudjianto, & Wicaksono, 2009; Fatimah, 2011; Ambarwati, 2016). Ketentuan mengenai pinjaman daerah diatur dalam Peraturan Pemerintah (PP) Nomor 30 Tahun 2011 tentang Pinjaman Daerah.. Salah satu skema pinjaman daerah dalam PP tersebut yang dewasa ini menarik perhatian pemerintah daerah di Indonesia ialah obligasi daerah. Beberapa pemerintah provinsi di Indonesia bahkan telah melakukan kajian persiapan penerbitan obligasi daerah (Dewi & Kaluge, 2011). Obligasi daerah diyakini dapat mempermudah dalam memperoleh dana yang besar untuk pembangunan infrastruktur, juga dapat menjadi solusi dan memberikan manfaat bagi masyarakat, investor dan pelaku pasar modal (Elmi, 2005). Dengan obligasi daerah, pemerintah daerah diharapkan dapat melakukan akselesari pembangunan tanpa bergantung semata-mata dari transfer pemerintah pusat (Surachman & Setiawan, 2016; Dewi & Kaluge, 2011). Selain obligasi daerah, terdapat alternatif pendanaan pembangunan daerah berupa pembayaran ketersediaan layanan. PENDAHULUAN Alternatif ini merupakan salah satu bentuk dari skema \| Jurnal Kajian Ekonomi & Keuangan Daerah, Volume 5, Nomor 2, September 2020 : 199-224 201 Kerjasama Pemerintah Daerah dengan Badan Usaha (KPDBU) (Sharma & Cui, 2012). Konsep KPDBU banyak diterapkan oleh pemerintah daerah dalam rangka mengajak investor swasta, domestik maupun asing untuk bekerjasama dalam penyediaan infrastruktur (Sharma & Cui, 2012; Djunedi, 2008; Masitoh, 2014; Grout, 2005). Mekanisme pendanaan dengan skema pembayaran ketersediaan layanan telah diatur dalam Peraturan Menteri Dalam Negeri (Permendagri) No. 96 Tahun 2016 tentang Pembayaran Ketersediaan Layanan dalam Rangka Kerjasama Pemerintah Daerah dengan Badan Usaha dalam Penyediaan Infrastruktur di Daerah. Kedua alternatif pendanaan tersebut merupakan alternatif pendanaan pembangunan infrastruktur daerah yang lebih mutakhir dibandingkan menggunakan penerimaan umum daerah dalam anggaran pendapatan dan belanja daerah (APBD). Oleh karena itu, menjadi menarik untuk dikaji lebih mendalam terkait tingkat efisiensinya dibandingkan menggunakan penerimaan umum daerah dalam anggaran pendapatan dan belanja daerah (APBD). Studi komparatif alternatif pendanaan pembangunan infrastruktur daerah ini mengacu pengembangan Rumah Sakit Umum Daerah (RSUD) Wates Kabupaten Kulon Progo. Pemilihan kabupaten tersebut sebagai objek penelitian dengan pertimbangan bahwa Kabupaten Kulon Progo merupakan salah satu kabupaten di Indonesia yang sebagian besar belanjanya masih berfokus pada belanja operasi dibanding dengan belanja modal. Rata-rata belanja modal Pemerintah Kabupaten Kulon Progo dalam laporan realisasi anggaran (LRA) dari tahun 2013 hingga tahun 2016 hanya sebesar 16,58% dari total belanja daerah. Kondisi ini mengindikasikan bahwa Pemerintah Kabupaten Kulon Progo memiliki kesulitan untuk mengalokasikan anggaran yang memadai untuk pembangunan infrastruktur daerah yang notabene membutuhkan dana yang besar. Kesulitan pendanaan tersebut kontradiktif dengan status Kabupaten Kulon Progo yang akan menjadi salah satu pintu masuk di kawasan Daerah Istimewah Yogyakarta (DIY). Hal ini disebabkan oleh adanya Mega Proyek Pembangunan Bandara DIY di Kulon Progo. Adanya bandara tersebut tentu menuntut respon dari pemerintah setempat untuk \| Jurnal Kajian Ekonomi & Keuangan Daerah, Volume 5, Nomor 2, September 2020 : 199-224 202 menyediakan fasilitas-fasilitas umum yang dapat memberikan efek pengganda dari adanya bandara tersebut guna mendorong peningkatan aktifitas ekonomi daerah. Salah satu fasilitas pendukung yang sedang dibangun oleh Pemerintah Kabupaten Kulon Progo ialah perluasan bangunan dan fasilitas Rumah Sakit Umum Daerah (RSUD) Wates Kabupaten Kulon Progo. Perluasan dan peningkatan fasilitas tersebut sebagai upaya untuk mentransformasi RSUD Wates menjadi RSUD Pendidikan Kabupaten Kulon Progo yang berkelas internasional. Total anggaran yang dibutuhkan sebagaimana yang tercantum dalam Studi Kelayakan Perluasan Bangunan dan Fasilitas RSUD Wates Kabupaten Kulon Progo ialah sebesar Rp485,69 milyar. PENDAHULUAN Dana sebesar itu belum mampu disediakan oleh Pemerintah Kabupaten Kulon Progo dalam satu atau dua periode anggaran jika menggunakan penerimaan umum daerah. Atas dasar adanya ketimpangan antara kebutuhan dana dengan kemampuan keuangan daerah maka perlu dilakukan studi komparatif alternatif pendanaan pembangunan infrastruktur daerah yang lebih mutakhir. Penelitian ini diharapkan mampu menunjukkan tingkat efisiensi pendanaan pembangunan infrastruktur daerah dengan obligasi daerah dan pembayaran ketersediaan layanan dibandingkan dengan pendanaan dari penerimaan umum daerah. Selain itu hasil penelitian diharapkan menjadi pertimbangan Pemerintah Kabupaten Kulon Progo dan pemerintah lainnya untuk menggunakan alternatif pendanaan obligasi daerah atau pembayaran ketersediaan layanan dalam proyek pembangunan infrastruktur daerah. Obligasi Daerah Salah satu jenis pinjaman daerah yang selama ini diharapakan dapat dimanfaatkan oleh pemerintah daerah untuk menghimpun dana dari masyarakat ialah pinjaman daerah dengan skema obligasi daerah (Ambarwati, 2016; Dewi & Kaluge, 2011; Elmi, 2005; Purnomo, 2015). Obligasi daerah didefinisikan sebagai surat berharga yang menunjukkan Jurnal Kajian Ekonomi & Keuangan Daerah, Volume 5, Nomor 2, September 2020 : 199-224 203 bahwa penerbit obligasi (pemerintah daerah) menjamin sejumlah dana kepada masyarakat (pembeli obligasi) dan memiliki kewajiban untuk membayar secara berkala (pokok, bunga, dan denda) pada waktu yang telah ditentukan (Darmadji & Fakhruddin, 2006; Sitorus, 2015; Purnomo, 2015). Obligasi daerah merupakan salah satu skema pembiayaan yang cukup populer di negara maju dan berkembang (Hamid, 2003; Elmi, 2005; Purnomo, 2015). Hal ini karena pembiayaan dengan obligasi daerah selain mempermudah dalam memperoleh dana pembangunan infrastruktur, juga dapat menjadi solusi dan memberikan manfaat bagi masyarakat, investor dan pelaku pasar modal ( (Hamid, 2003; Elmi, 2005). Beberapa tahap harus dilakukan dan dilalui pemerintah daerah sebelum menerbitkan obligasi daerah di pasar modal. Tahap-tahap tersebut meliputi persiapan di daerah, persetujuan Menteri Keuangan, tahap pra-registrasi dan registrasi, hingga tahap penawaran umum (DJPK, 2007). 1. Persiapan di Daerah 1. Persiapan di Daerah Pemerintah daerah dalam upaya melakukan penerbitan obligasi daerah perlu membentuk sebuah tim persiapan penerbitan obligasi daerah. Tim persiapan bertanggung jawab untuk menyiapkan rencana kegiatan investasi yang akan dibiayai. Dalam ketentuan perubahan pasal 8 ayat (2) dalam PMK 180/PMK.07 Tahun 2015 setidaknya telah disebutkan enam tahap yang harus dilakukan pemerintah daerah dalam melakukan persiapan penerbitan obligasi daerah. a. Pemerintah daerah menentukan kegiatan yang akan didanai dengan obligasi daerah. b. Membuat Kerangka Acuan Kegiatan. a. Pemerintah daerah menentukan kegiatan yang akan didanai dengan obligasi daera c. Langkah selanjutnya adalah membuat perhitungan batas kumulatif pinjaman. Batas kumulatif pinjaman daerah yaitu jumlah sisa pinjaman daerah ditambah jumlah pinjaman yang akan ditarik tidak melebihi 75% dari jumlah penerimaan umum APBD tahun sebelumnya. d. Selanjutnya menghitung rasio kemampuan keuangan daerah yang dikenal dengan istilah debt service coverage ratio (DSCR). DSCR menjadi penting untuk dijadikan dasar dalam d. Selanjutnya menghitung rasio kemampuan keuangan daerah yang dikenal dengan istilah debt service coverage ratio (DSCR). DSCR menjadi penting untuk dijadikan dasar dalam \| Jurnal Kajian Ekonomi & Keuangan Daerah, Volume 5, Nomor 2, September 2020 : 199-224 204 menentukan apakah pinjaman yang diambil nantinya akan menyulitkan kondisi keuangan entitas atau tidak (Noor, 2009). Rumus yang dapat digunakan dalam menghitung DSCR tercantum dalam penjelasan PP No. 30 Tahun 2011 sebagai berikut. e. Selanjutnya pemerintah daerah mengajukan permohonan persetujuan prinsip kepada Dewan Perwakilan Rakyat Daerah (DPRD). Setidaknya ada tiga perihal yang harus tertuang dalam surat persetujuan prinsip DPRD yaitu: (1) nilai bersih maksimal obligasi daerah yang akan diterbitkan pada saat penetapan APBD, (2) kesediaan pembayaran pokok dan bunga sebagai akibat penerbitan obligasi daerah, dan (3) kesediaan pembayaran segala biaya yang timbul dari penerbitan obligasi daerah. f. Terakhir dalam tahap persiapan ialah membentuk struktur organisasi, perangkat kerja, dan sumber daya manusia unit pengelola obligasi daerah. f. Terakhir dalam tahap persiapan ialah membentuk struktur organisasi, perangkat kerja, dan sumber daya manusia unit pengelola obligasi daerah. 2. Pesetujuan Menteri Keuangan 2. Pesetujuan Menteri Keuangan Langkah selanjutnya ialah pemerintah daerah mengajukan usul penerbitan obligasi daerah untuk mendapatkan persetujuan kepada Menteri Keuangan dalam hal ini Direktur Jenderal Perimbangan Keuangan (DJPK, 2007). Untuk memberikan persetujuan, setidaknya Kementerian Keuangan melakukan penilaian meliputi penilaian administrasi dan penilaian keuangan. Surat usulan penerbitan obligasi daerah harus dilengkapi dengan dokumen- dokumen sebagai berikut. a. Kerangka Acuan Kegiatan. b. Laporan keuangan pemerintah daerah selama 3 (tiga) tahun terakhir. b. Laporan keuangan pemerintah daerah selama 3 (tiga) tahun terakhir. c. Peraturan daerah mengenai APBD tahun berkenaan. d. Perhitungan jumlah kumulatif pinjaman pemerintah daerah dan defisit APBD. e. Perhitungan rasio kemampuan keuangan daerah untuk mengembalikan pinjaman atau debt service coverage ratio (DSCR). f. Surat persetujuan prinsip dari Dewan Perwakilan Rakyat Daerah. \| Jurnal Kajian Ekonomi & Keuangan Daerah, Volume 5, Nomor 2, September 2020 : 199-224 205 g. Struktur organisasi, perangkat kerja, dan sumber daya manusia unit pengelola obligasi daerah. g. Struktur organisasi, perangkat kerja, dan sumber daya manusia unit pengelola obligasi daerah. 4. Penawaran Umum Tahap selanjutnya adalah penawaran umum dan pencatatan. Jangka waktu yang dibutuhkan selama proses penawaran umum, terhitung sejak pernyataan pendaftaran dinyatakan efektif oleh OJK hingga obligasi daerah dibeli oleh investor dan dicatatkan di bursa efek adalah kurang lebih 10 hari kerja (DJPK, 2007). 3. Pra-registrasi dan Registrasi Pada tahap pra-regisrasi dan registrasi sebenarnya sudah mulai memasuki skema penerbitan obligasi di pasar modal. Tahap pra-registrasi meliputi langkah-langkah yang harus ditempuh sebelum mengajukan pernyataan pendaftaran ke Otoroitas Jasa Keuangan (OJK) yang diantaranya menunjuk profesi-profesi penunjang, melakukan uji tuntas (due diligence), pemeringkatan efek, persiapan pendaftaran, pembuatan janji-janji terkait dan penetapan struktur obligasi daerah (DJPK, 2007). Jika persyaratan telah lengkap dan memenuhi kriteria maka OJK akan mengeluarkan pernyataan efektif dalam rangka penawaran umum obligasi daerah. Pembayaran Ketersediaan Layanan (Availability payment) Kerjasama pemerintah daerah dengan badan usaha (KPDBU) atau public private partnership (PPP) merupakan alternatif lain yang dapat dimanfaatkan oleh pemerintah daerah untuk mendapatkan bantuan dana dalam pembangunan infrastruktur. (Djunedi, 2008; Kurniawan, Pudjianto, & Wicaksono, 2009; Masitoh, 2014). Salah satu skema pendanaan dalam KPDBU ialah skema Pembayaran Ketersediaan Layanan (Availability Payment) (Sharma & Cui, 2012). Skema pembayaran ketersediaan layanan menjadi salah satu skema pendanaan yang ditawarkan oleh pemerintah pusat melalui Peraturan Pemerintah (PP) No. 38 Tahun 2015 tentang Kerjasama Pemerintah dengan Badan Usaha (KPBU) dalam Penyediaan Infrastruktur. Dalam PP tersebut menyatakan bahwa \| Jurnal Kajian Ekonomi & Keuangan Daerah, Volume 5, Nomor 2, September 2020 : 199-224 206 pembayaran ketersediaan layanan adalah pembayaran secara berkala oleh Menteri/Kepala Lembaga/Kepala Daerah kepada Badan Usaha Pelaksana atas tersedianya layanan infrastruktur yang sesuai dengan kualitas dan/atau kriteria sebagaimana ditentukan dalam perjanjian KPBU. Skema ini memiliki tujuan untuk (1) memastikan ketersediaan layanan yang berkualitas kepada masyarakat secara berkesinambungan dan (2) mengoptimalkan nilai guna dari APBD (time value of money) dalam penyediaan layanan. Tahap pelaksanaan KPBU dengan skema pembayaran ketersediaan layanan dimulai dengan perencanaan pembayaran ketersediaan layanan, penyiapan pembayaran ketersediaan layanan, dan transaksi pembayaran ketersediaan layanan. 1. Tahap Perencanaan Pembayaran ketersediaan layanan 1. Tahap Perencanaan Pembayaran ketersediaan layanan Tahap perencanaan dimaksudkan untuk memperoleh informasi mengenai kebutuhan penyediaan infrastruktur yang dapat dikerjasamakan dengan badan usaha dan untuk mendukung koordinasi perencanaan dan pengembangan rencana pembayaran ketersediaan layanan serta melakukan keterbukaan informasi kepada masyarkat mengenai rencana pembayaran ketersediaan layanan. Tahap perencanaan pembayaran ketersediaan layanan dijelaskan dalam pasal 7 hingga 13 Permendagri No. 96 Tahun 2016 yang terdiri dari beberapa kegiatan yaitu sebagai berikut. a. Pemerintah daerah melakukan penyusunan rencana anggaran dana pembayaran ketersediaan layanan sesuai dengan peraturan perundang-undangan dengan memperhatikan setiap tahap pelaksanaan KPBU dengan skema pembayaran ketersediaan layanan. b. Langkah kedua ialah dentifikasi dan penetapan pembayaran ketersediaan layanan. Dalam rangka melakukan identifikasi, maka pemerintah daerah menyusun studi pendahuluan dan melakukan konsultasi publik. b. Langkah kedua ialah dentifikasi dan penetapan pembayaran ketersediaan layanan. Dalam rangka melakukan identifikasi, maka pemerintah daerah menyusun studi pendahuluan dan melakukan konsultasi publik. c. Selanjutnya pengambilan keputusan apakah akan melanjutkan atau menghentikan pembangunan skema pembayaran ketersediaan layanan. c. Selanjutnya pengambilan keputusan apakah akan melanjutkan atau menghentikan pembangunan skema pembayaran ketersediaan layanan. 2. 3. Tahap Transaksi Setelah seluruh syarat-syarat terpenuhi dari tahap perencanaan hingga penyiapan, maka selanjutnya adalah tahap transaksi. Dalam Permendagri No. 96 Tahun 2016, ketentuan mengenai tahap transaksi dibahas dari pasal 23 hingga pasal 34. Setidaknya terdapat 3 kegiatan dalam tahap transaksi ini, yaitu: a. pengadaan Badan Usaha Pelaksana yang mencakup persiapan dan pelaksanaan pengadaan Badan Usaha Pelaksana. b. Penandatangan pernjanjian KPBU dengan skema pembayaran ketersediaan layanan. b. Penandatangan pernjanjian KPBU dengan skema pembayaran ketersediaan layanan. c. Pemenuhan pendanaan (financial close). Pembayaran Ketersediaan Layanan (Availability payment) Tahap Penyiapan 07 \| Jurnal Kajian Ekonomi & Keuangan Daerah, Volume 5, Nomor 2, September 2020 : 199-224 207 Ketika memutuskan untuk melanjutkan pembangunan infrastruktur dengan skema pembayaran ketersediaan layanan, maka langkah selanjutnya adalah penyiapan KPBU dengan skema pembayaran ketersediaan layanan. Langkah-langkah tersebut telah tertuang dalam Permendagri No. 96 Tahun 2016 pada pasal 14 hingga pasal 22 sebagai berikut. a. Langkah pertama adalah pra studi kelayakan investasi. Kajian ini terdiri dari berbagai aspek antara lain: (1) kajian hukum dan kelembangaan, (2) kajian teknis, (3) kajian ekonomi dan sosial, (4) kajian lingkungan dan sosial, (5) kajian bentuk kerjasama dalam penyediaan infrastruktur, (6) kajian risiko, (7) kajian kebutuhan dukungan Pemerintah dan/atau Jaminan Pemerintah, serta (8) kajian mengenai hal-hal yang perlu ditindaklanjuti. b. Selanjutnya melakukan konsultasi publik dan penjajakan minat pasar (market sounding). b. Selanjutnya melakukan konsultasi publik dan penjajakan minat pasar (market sounding). c. Untuk melancarkan proses pembayaran ketersediaan layanan, maka pemerintah daerah dapat mengajukan usulan terhadap dukungan Pemerintah dalam pembayaran ketersediaan layanan. Kebutuhan Dana Pengembangan RSUD Wates Anggaran yang dibutuhkan untuk pengembangan RSUD Wates Kabupaten Kulon Progo tersebut sebesar Rp272,18 milyar untuk fisik bangunan dan Rp213,51 milyar untuk pengadaan alat-alat kesehatan. Dengan demikian total anggaran yang dibutuhkan untuk pengembangan RSUD tersebut sebesar Rp485,69 milyar. METODE PENELITIAN Penelitian ini menggunakan paradigma interpretif dengan pendekatan penelitian kualitatif deskriptif. Data penelitian yang dikumpulkan dalam penelitian ini adalah data \| Jurnal Kajian Ekonomi & Keuangan Daerah, Volume 5, Nomor 2, September 2020 : 199-224 208 sekunder berupa Rencana Pengembangan RSUD Wates Kabupaten Kulon Progo, Laporan Keuangan Kabupaten Kulon Progo, serta dokumen pendukung lainnya yang kemudian dianalisis dan diinterpretasikan sesuai kebutuhan penelitian. Analisis data dilakukan dengan dua tahap utama. Analisis pertama berfokus pada kajian mengenai simulasi investasi pada proyek pengembangan infrastruktur RSUD Wates Kabupaten Kulon Progo dengan skema obligasi daerah dan pembayaran ketersediaan layanan. Beberapa hal yang harus dianalisis antara lain (1) Batas Maksimum Pinjaman Daerah, (2) Debt Service Coverage Ratio (DSCR), dan Menghitung Total Avilability Payment. Perhitungan simulasi obligasi daerah mengacu pada Peraturan Menteri Keuangan Nomor 180/PMK.07 Tahun 2015 tentang Perubahan atas Peraturan Menteri Keuangan Nomor 111/PMK.07 Tahun 2012 tentang Tata Cara Penerbitan dan Pertanggungjawaban Obligasi Daerah, serta mengacu pada Panduan Penerbitan Obligasi Daerah yang dikeluarkan oleh Direktorat Jenderal Perimbangan Keuangan, Departemen Keuangan Republik Indonesia. Perhitungan simulasi Pembayaran Ketersediaan Layanan mengacu pada Peraturan Menteri Perencanaan Pembangunan Nasional atau Kepala Badan Perencanaan Pembangunan Nasional Nomot 4 Tahun 2015 tentang Tata Cara Pelaksanaan Kerjasama Pemerintah Dengan Badan Usaha Dalam Penyediaan Infrastruktur, serta mengacu pada perhitungan pembayaran ketersediaan layanan dalam Laporan Akhir Kajian Awal Pra Studi Kelayakan KPBU RSUD Krian Kabupaten Sidoarjo. Analisis kedua yaitu analisis komparatif alternatif pendanaan antara obligasi daerah dan pembayaran ketersediaan layanan dengan pendanaan dari penerimaan umum daerah. Pendekatan yang digunakan dalam analisis komparatif alternatif pendanaan ialah time value of money dengan melihat present value masing-masing alternatif pendanaan tersebut. Angka-angka proyeksi dalam penelitian ini dihitung menggunakan metode trend linear kuadrat terkecil (least square). Jurnal Kajian Ekonomi & Keuangan Daerah, Volume 5, Nomor 2, September 2020 : 199-224 209 Simulasi Obligasi Daerah Simulasi yang disusun berfokus kepada aspek kemampuan keuangan Kabupaten Kulon Progo untuk menerbitkan obligasi daerah. Dalam Studi Kelayakan Pengembangan RSUD Wates, telah dilakukan analisis ekonomi dan keuangan dengan hasil net present value (NPV) sebesar Rp80,98 milyar, internal rate of return (IRR) sebesar 9,16%, payback period (PBC) selama 20 Tahun 8 Bulan, dan profitability index (PI) sebesar 1,150. Berikut ini langkah-langkah yang dilakukan untuk menyususn simulasi penerbitan obligasi daerah. 1. Menghitung Batas Maksimum Pinjaman Daerah Sebelum menentukan batas maksimum pinjaman daerah, maka perlu dilakukan perhitungan penerimaan umum daerah pada tahun sebelum mengajukan pendanaan dengan obligasi daerah. Penerimaan umum daerah dihitung dengan rumus sebagai berikut. PU = PD – (DAK + DD + DP + L) Dimana: PU = Penerimaan Umum PD = Total Penerimaan Daerah DAK = Dana Alokasi Khusus DD = Dana Darurat DP = Pembayaran Pokok Pinjaman L = Pendapatan Pendanaan Tertentu Tahun dasar obligasi daerah dalam penelitian ini diasumsikan mulai tahun 2017. Oleh karena itu, penerimaan umum APBD akan menggunakan data-data pada tahun anggaran 2016. Tabel 1 Penerimaan Umum Daerah Tahun 2016 (dalam milyar rupiah) Tabel 1 Penerimaan Umum Daerah Tahun 2016 (dalam milyar rupiah) 210 Jurnal Kajian Ekonomi & Keuangan Daerah, Volume 5, Nomor 2, September 2020 : 199-224 Keterangan Rp Total Penerimaan Daerah 2016 1.358,07 Pengurangan: 1 Dana Alokasi Khusus 212,67 2 Dana Darurat - 3 Pembayaran Pokok Pinjaman Sebelumnya - 4 Pendapatan Pendanaan Tertentu: Bagian Pendapatan sebagai Transfer Bagi Hasil 9,81 Bagian Pendapatan yang Ditransfer ke Desa 135,04 Pendapatan telah Ditentukan Peruntukannya 27,65 Pendapatan Dana Penyesuaian 99,74 Penerimaan Umum Tahun 2016 873,16 Sumber: LRA APBD Kulon Progo Tahun 2016 (data diolah) Total penerimaan umum APBD Kabupaten Kulon Progo Tahun Anggaran 2016 ialah sebesar Rp873,16 milyar. Dengan demikian, maka batas maksimum pinjaman Kabupaten Kulon Progo berdasarkan penerimaan umum tersebut sebagai berikut. BMPD = (75% x PU) - Pinjaman Sebelumnya BMPD = (75% x Rp873,16 M) - Rp5 M BMPD = Rp654,87 M - Rp5 M BMPD = Rp649,87 M Total penerimaan umum APBD Kabupaten Kulon Progo Tahun Anggaran 2016 ialah sebesar Rp873,16 milyar. Dengan demikian, maka batas maksimum pinjaman Kabupaten Kulon Progo berdasarkan penerimaan umum tersebut sebagai berikut. BMPD = (75% x PU) - Pinjaman Sebelumnya BMPD = (75% x Rp873,16 M) - Rp5 M BMPD = Rp654,87 M - Rp5 M BMPD = Rp649,87 M Pemerintah Kabupaten Kulon Progo sebelumnya telah memiliki pinjaman sebesar Rp5 milyar dari Bank Pembangunan Daerah (BPD) DIY. Dengan demikian batas maksimum pinjaman yang boleh ditarik oleh Pemerintah Kabupaten Kulon Progo yaitu sebesar Rp649,87 milyar. Berdasarkan perhitungan batas maksimum pinjaman daerah, maka Pemerintah Kabupaten Kulon Progo memenuhi syarat untuk melakukan pinjaman daerah dengan skema obligasi daerah sebesar Rp485,69 milyar. 2. Menghitung Debt Coverage Service Ratio 2. Menghitung Debt Coverage Service Ratio Debt Coverage Service Ratio (DSCR) menunjukkan kemampuan keuangan daerah untuk melakukan pengembalian pinjaman selama periode pinjaman. Nilai DSCR pemerintah daerah untuk dianggap layak melakukan pinjaman ialah di atas 2,5. \| Jurnal Kajian Ekonomi & Keuangan Daerah, Volume 5, Nomor 2, September 2020 : 199-224 211 Perhitungan DSCR ini membutuhkan beberapa asumsi dan data-data pendukung. Berikut ini asumsi-asumsi yang digunakan untuk menghitung DSCR dalam konteks penerbitan obligasi daerah oleh Pemerintah Kabupaten Kulon Progo. a. Obligasi daerah Pemerintah Kabupaten Kulon Progo diterbitkan pada tahun 2017 dengan bunga 7,17%. Bunga 7,17% dianggap cukup bersaing apabila dibandingkan dengan beberapa alternatif lainnya dalam investasi keuangan pada tahun 2017. Misalnya rata- rata imbal hasil dari Obligasi Negara Ritel (ORI) pada tahun 2017 diprediksi berkisar antara 7% sampai 7,50% (www.infovesta.com). Sementara itu, suku bunga deposito pada tahun 2017 diperkirakan berada pada kisaran 7,02% (m.kontan.co.id). Tabel 1 Penerimaan Umum Daerah Tahun 2016 (dalam milyar rupiah) Jika berkaca juga dari pengalaman Pemerintah Kulon Progo yang sebelumnya telah melakukan pinjaman pada BPD DIY dengan suku bunga 11,5%, maka suku bunga 7,17% dianggap berada pada batas aman. b. Obligasi daerah Pemerintah Kabupaten Kulon Progo diasumsikan dengan tenor 10 tahun. Dengan demikian, jika obligasi daerah dimulai pada tahun 2017, maka akan jatuh tempo pada tahun 2026. Pemilihan jangka waktu 10 tahun agar Pemerintah Kabupaten Kulon Progo memiliki waktu yang cukup untuk mengumpulkan dana untuk pembayaran pokok pinjaman serta mengurangi sensitifitas dengan inflasi. c. Bunga obligasi daerah dibayarkan setiap tahun dengan nilai tetap, sementara untuk pokok obligasi daerah akan dibayarkan pada saat jatuh tempo yaitu di tahun 2026. Agar pembayaran pokok obligasi tidak memberatkan APBD pada saat jatuh tempo, maka pemerintah akan membentuk dana cadangan yang disimpan khusus untuk pembayaran pokok obligasi di masa mendatang. Pembentukan dana cadangan akan dimulai pada saat aset yang dibangun mulai beroperasi dengan baik. Untuk menentukan berapa dana yang harus dicadangkan setiap tahunnya, maka akan dilakukan perhitungan nilai anuitas majemuk. d. Masa konstruksi dengan sumber pendanaan dari obligasi daerah diyakini akan lebih cepat satu tahun dibandingkan dengan menggunakan dana dari penerimaan umum \| Jurnal Kajian Ekonomi & Keuangan Daerah, Volume 5, Nomor 2, September 2020 : 199-224 212 daerah. Dengan demikian, masa konstruksi menjadi hanya 2 tahun dari 4 tahun pada rencana pembangunan dalam dokumen Pemerintah Kabupaten Kulon Progo. Pertimbangan tersebut karena dana untuk kebutuhan pembangunan infrastruktur akan tersedia lebih cepat ketika pendanaannya bersumber dari obligasi daerah dibandingkan ketika bersumber dari penerimaan umum di APBD. e. Belanja wajib yang digunakan dalam perhitungan DSCR ialah belanja pegawai ditambah dengan transfer ke desa. f. Nilai dasar dalam perhitungan proyeksi DSCR pada tahun 2013 hingga 2016 menggunakana data dalam Laporan Realisasi APBD Kabupaten Kulon Progo tahun anggaran 2013 hingga 2016. Sementara nilai dalam tahun 2017 menggunakan nilai dalam APBD Kabupaten Kulon Progo tahun anggaran 2017 dan nilai untuk tahun 2018 hingga 2026 menggunakan angka proyeksi yang diperoleh dari perhitungan metode trend linear kuadrat terkecil (least square). Hasil perhitungan proyeksi DSCR selama perode obligasi yaitu tahun 2017 hingga 2026 dapat dilihat pada Tabel 2. Syarat pemerintah daerah agar dapat melakukan pinjaman daerah dengan skema obligasi daerah ialah harus memiliki nilai DSCR minimal 2,5. Berdasarkan hasil perhitungan DSCR, maka Pemerintah Kabupaten Kulon Progo memiliki kemampuan dimasa depan untuk membayar pinjaman obligasi daerah yang ditarik dari publik sebesar Rp485,69 milyar. Tahun DSCR 2026 6,91 Simulasi Pembayaran ketersediaan layanan Tahun DSCR 2026 6,91 Simulasi Pembayaran ketersediaan layanan Tabel 1 Penerimaan Umum Daerah Tahun 2016 (dalam milyar rupiah) Tabel 2 Hasil Perhitungan DSCR Tahun DSCR 2017 6,68 2018 5,51 2019 5,63 2020 5,75 2021 6,08 2022 6,31 2023 6,46 2024 6,61 2025 6,76 Tabel 2 Hasil Perhitungan DSCR 213 Jurnal Kajian Ekonomi & Keuangan Daerah, Volume 5, Nomor 2, September 2020 : 199-224 Simulasi Pembayaran ketersediaan layanan Simulasi yang akan disusun berfokus pada aspek keuangan atau besaran pembayaran ketersediaan layanan setiap tahunnya yang akan dibayarkan oleh Pemerintah Kabupaten Kulon Progo selama masa konsesi KPBU. Sebelum menyusun simulasi pendanaan dengan skema pembayaran ketersediaan layanan untuk pengembangan RSUD Wates Kabupaten Kulon Progo, maka terlebih dahulu ditentukan asumsi-asumsi yang akan digunakan dalam simulasi tersebut. 1. KPDBU dengan skema pembayaran ketersediaan layanan akan mulai dilakukan pada tahun 2017 dengan jangka waktu pembayaran ketersediaan layanan yaitu 10 tahun. Pada tahun 2017 akan dimulai kegiatan kontruksi. Jangka waktu kontruksi dengan skema pembayaran ketersediaan layanan diyakini selesai dalam 2 tahun. Jangka waktu 2 tahun ditentukan dengan pertimbangan bahwa badan usaha yang memenangkan lelang merupakan badan usaha yang memiliki kemampuan finansial untuk menutupi kebutuhan pembangunan infrastruktur tersebut. Selain itu, dengan skema pembayaran ketersediaan layanan maka badan usaha akan termotivasi untuk segera menyelesaikan pembangunan infrastruktur agar pembayaran atas ketersediaan layanan dapat segera dilakukan. Dengan demikian, layanan akan tersedia pada tahun 2019 sehingga pembayaran ketersediaan layanan juga akan dimulai pada tahun 2019 selama 10 tahun dan akan berakhir pada tahun 2028. 2. Capital structure untuk pembangunan diperbolehkan 30% dari modal sendiri dan 70% dari pinjaman. 3. Pinjaman yang ditarik badan usaha untuk membantu modal sendiri diasumsikan selama 7 tahun dengan bunga pinjaman sebesar 10,35% per tahun. Bunga pinjaman tersebut diambil dari rata-rata bunga pinjaman korporasi pada akhir tahun 2016 di Bank Rakyat Indonesia, Bank Negara Indonesia, Bank Mandiri, Bank Tabungan Negara, dan Bank \| Jurnal Kajian Ekonomi & Keuangan Daerah, Volume 5, Nomor 2, September 2020 : 199-224 214 Central Asia (www.bi.go.id). Pinjaman akan dimulai pada tahun 2018 dengan pertimbangan bahwa dana untuk konstruksi pada tahun 2017 masih akan ditutupi dari modal sendiri badan usaha sebesar 30% dari total kebutuhan pendanaan. Oleh karena itu, pinjaman sebaiknya dimulai pada tahun 2018 untuk menghindari risiko bunga. 4. Capital expenditure (capex) yang digunakan ialah jumlah dari kebutuhan pendanaan untuk fisik gedung dan alat-alat kesehatan dengan total sebesar Rp485,69 ditambah dengan bunga yang timbul selama masa konstruksi atau interest during construction (IDC). 5. Operating expenditure (opex) dimulai sebesar 10% dari capex sebelum IDC dan akan terus naik sebesar 5% setiap tahunnya. 6. Margin yang diberikan kepada badan usaha pelaksana sebesar 35% dari total capex dan opex tiap tahun. Pertimbangan tersebut karena pada dasarnya badan usaha pelaksana ialah organisasi sektor privat yang profit oriented. Oleh karena itu, margin sebesar 35% dianggap akan mampu menarik minat investor atau badan usaha untuk berinvestasi. 6. Simulasi Pembayaran ketersediaan layanan Margin yang diberikan kepada badan usaha pelaksana sebesar 35% dari total capex dan opex tiap tahun. Pertimbangan tersebut karena pada dasarnya badan usaha pelaksana ialah organisasi sektor privat yang profit oriented. Oleh karena itu, margin sebesar 35% dianggap akan mampu menarik minat investor atau badan usaha untuk berinvestasi. 7. Tarif Pajak Penghasilan Badan ditentukan sebesar 25% yang merupakan tarif Pajak Penghasilan Badan sesuai dengan peraturan perpajakan di Indonesia. 8. Asumsi-asumsi ekonomi dan keuangan mengacu pada Studi Kelayakan Perluasan bangunan dan fasilitas RSUD Wates Kabupaten Kulon Progo yang telah disusun oleh Pemerintah Kabupaten Kulon Progo. 9. Biaya lain-lain diasumsikan Rp0 (nol rupiah). Berdasarkan asumsi-asumsi di atas, maka dapat dilakukan simulasi pembayaran ketersediaan layanan yang akan dibayarkan oleh Pemerintah Kabupaten Kulon Progo setiap tahun selama masa konsesi KPBU dengan skema pembayaran ketersediaan layanan. Pertama yang dilakukan yaitu dengan menghitung total capex ditambah IDC. Tabel 3 Total Capex ditambah IDC (dalam milyar rupiah) CAPEX Rp Modal Sendiri (30% dari Total Investasi) 145,71 Tabel 3 Total Capex ditambah IDC (dalam milyar rupiah) CAPEX Rp Modal Sendiri (30% dari Total Investasi) 145,71 215 Jurnal Kajian Ekonomi & Keuangan Daerah, Volume 5, Nomor 2, September 2020 : 199-224 Modal Pinjaman (70% dari total Investasi) 339,98 Capex sebelum IDC (1) 485,69 IDC: - Tahun 1 (2018) 35,19 Capex + IDC 520,88 Tabel 3 menunjukkan bahwa bunga yang timbul dalam masa konstruksi aset yang akan dikapitalisasi ke dalam capex yaitu sebesar Rp35,19 milyar. Dengan demikian, total capex setelah ditambah dengan IDC ialah sebesar Rp520,88 milyar. Setelah capex ditambah IDC ditentukan, maka selanjutnya dapat dilakukan perhitungan pembayaran ketersediaan layanan. Tabel 4 Perhitungan Pembayaran ketersediaan layanan (dalam milyar rupiah) Tahun Capex Opex Margin AP 2019 52,09 48,57 35,23 135,89 2020 52,09 51,00 51,00 139,17 2021 52,09 53,55 53,55 142,61 2022 52,09 56,22 56,22 146,22 2023 52,09 59,04 59,04 150,02 2024 52,09 61,99 62,98 154,00 2025 52,09 65,09 65,09 158,19 2026 52,09 68,34 68,34 162,58 2027 52,09 71,76 71,76 167,19 2028 52,09 75,35 75,35 172,04 Jumlah 520,88 610,90 395,12 1.527,90 Berdasarkan tabel 4, total pembayaran ketersediaan layanan selama 10 tahun yang harus dibayar oleh Pemerintah Kabupaten Kulon Progo selaku Penanggung Jawab Proyek Kerjasama adalah sebesar Rp1.527,90 milyar. Analisis Komparatif Alternatif Pendanaan Tabel 5 Hasil Analisis Komparatif Alternatif Pendanaan (dalam milyar rupiah) Penerimaan Umum APBD PV Investasi Fisik Gedung 243,02 PV Investasi Alat Kesehatan 184,51 PV Pengorbanan atas Layanan yang Tidak Diberikan 105,58 PV Operating Expenditure 320,28 PV Pengorbanan atas Alokasi Anggaran yang Ditunda 427,52 Total PV Penerimaan Umum APBD (A) 1.280,91 Tabel 5 Hasil Analisis Komparatif Alternatif Pendanaan (dalam milyar rupiah) Penerimaan Umum APBD PV Investasi Fisik Gedung 243,02 PV Investasi Alat Kesehatan 184,51 PV Pengorbanan atas Layanan yang Tidak Diberikan 105,58 PV Operating Expenditure 320,28 PV Pengorbanan atas Alokasi Anggaran yang Ditunda 427,52 Total PV Penerimaan Umum APBD (A) 1.280,91 \| Jurnal Kajian Ekonomi & Keuangan Daerah, Volume 5, Nomor 2, September 2020 : 199-224 216 Obligasi Daerah PV Dana Cadangan Untuk Pembayaran Pokok Obligasi Daerah dikurangi Bunga 167,83 PV Bunga Obligasi 249,68 PV Operating Expenditure 361,69 Total PV Obligasi Daerah (B) 779,20 Pembayaran ketersediaan layanan PV Pembayaran ketersediaan layanan 918,13 Present Value PPh Badan yang akan diterima (67,91) Total PV Pembayaran ketersediaan layanan (C) 850,22 Tingkat Diskonto 8,32% Selisih (A) dan (B) 501,71 Selisih (A) dan (C) 430,69 Selisih (C) dan (B) 71.02 Langkah-langkah yang dilakukan dalam analisis ini ialah dengan mengidentifikasi potensi-potensi pengorbanan ekonomi pada masing-masing alternatif pendanaan, kemudian dibandingkan dengan potensi pengorbanan ekonomi jika pembangunan tersebut dilakukan secara tradisional atau menggunakan dana penerimaan umum daerah. Analsis alternatif pendanaan ini dilakukan dengan pendekatan time value of money. Penggunaan time value of money dalam perbandingan alternatif pendanaan karena pada umumnya nilai uang sekarang lebih berharga dari pada nilai uang di masa mendatang. Seluruh potensi pengorbanan ekonomi harus dipastikan telah diperhitungkan dalam setiap alternatif pendanaan untuk memastikan bahwa perbandingan alternatif pendanaan tersebut apple to apple. Dalam analisis time value of money tersebut, tingkat diskonto yang digunakan ialah 8,32%. Tingkat diskonto tersebut mengacu pada cost of capital yang dihitung dengan menjumlahkan antara tingkat suku bunga obligasi sebesar 7,17%, social opportunity cost sebesar 1,00% dan Social Rate of Time Preference sebesar 0,15%. Social opportunity cost dan Social Rate of Time Preference merupakan pertimbangan yang harus ada dalam organisasi sektor publik karena hal yang menjadi pertimbangan utamanya ialah kualitas layanan yang dapat diterima oleh masyarakat. \| Jurnal Kajian Ekonomi & Keuangan Daerah, Volume 5, Nomor 2, September 2020 : 199-224 217 Present Value Penerimaan Umum APBD Berdasarkan Tabel 5 tersebut, ketika pembangunan infrastruktur menggunakan dana dari penerimaan umum APBD, maka terdapat total present value potensi pengorbanan ekonomi sebesar Rp1.280,91 milyar. Nilai tersebut merupakan hasil penjumlahan dari present value investasi fisik gedung, present value investasi alat kesehatan, present value pengorbanan ekonomi atas layanan yang tidak diberikan, present value operating expenditure dan present value pengorbanan ekonomi atas alokasi anggaran yang ditunda. Present value investasi fisik gedung sebesar Rp243,02 milyar merupakan nilai sekarang dari seluruh alokasi dana yang akan dikeluarkan untuk membangun fisik gedung RSUD Wates. Dalam Studi Kelayakan Perluasan Gedung dan Fasilitas RSUD Wates Kabupaten Kulon Progo, penyediaan dana untuk kebutuhan fisik gedung dilakukan secara bertahap selama 3 tahun yaitu tahun 2017 hingga tahun 2019. Oleh karena itu, dana yang dikeluarkan pada tahun kedua dan ketiga di-present value-kan. Present value investasi alat kesehatan sebesar Rp184,50 milyar merupakan nilai sekarang dari seluruh alokasi dana yang akan dikeluarkan untuk pengadaan alat-alat kesehatan RSUD Wates. Dalam Studi Kelayakan Perluasan Gedung dan Fasilitas RSUD Wates Kabupaten Kulon Progo, penyediaan dana untuk pengadaan alat-alat kesehatan dilakukan secara bertahap selama 2 tahun yaitu tahun 2018 dan tahun 2019. Oleh karena itu, dana yang dikeluarkan pada tahun tersebut di-present value-kan. Present value pengorbanan ekonomi atas layanan yang tidak diberikan sebesar Rp105,58 milyar merupakan nilai sekarang dari potensi atas layanan jasa yang ditaksir akan diperoleh jika perluasan bangunan dan fasilitas RSUD Wates dapat beroperasi pada tahun 2019. Layanan kesehatan yang tidak diberikan dihitung sebagai salah satu pengorbanan ekonomi yang harus ditanggung, karena jika menggunakan skema obligasi daerah atau pembayaran ketersediaan layanan, maka pada tahun 2019 perluasan bangunan dan fasilitas RSUD Wates mulai beroperasi dengan baik dan ditaksir dapat memberikan jasa layanan sebesar Rp123,84 milyar. Jasa layanan tersebut diperoleh dari proyeksi \| Jurnal Kajian Ekonomi & Keuangan Daerah, Volume 5, Nomor 2, September 2020 : 199-224 218 pendapatan RSUD Wates tahun 2019 dalam Studi Kelayakan Perluasan Bangunan dan Fasilitas RSUD Wates Kabupaten Kulon Progo. Present value opex sebesar Rp320,28 milyar merupakan nilai sekarang dari biaya operasi yang ditaksir akan keluar pada saat hasil perluasan bangunan dan fasilitas RSUD Wates mulai beroperasi. Nilai dari opex ditentukan sama dengan nilai opex dari ketiga alternatif pendanaan. Infrastruktur tersebut mulai beroperasi pada tahun keempat, sehingga opex yang diperhitungkan ialah opex yang timbul sejak tahun keempat. Present Value Penerimaan Umum APBD Present value pengorbanan ekonomi atas alokasi anggaran yang ditunda sebesar Rp427,52 milyar merupakan nilai sekarang dari potensi alokasi anggaran yang ditunda untuk kebutuhan lainnya karena harus dialokasi pada proyek perluasan bangunan dan fasilitas RSUD Wates Kabupaten Kulon Progo. Pengorbanan ekonomi tersebut seharusnya dapat dialokasikan pada kebutuhan dan pembangunan lainnya jika saja Pemerintah Kabupaten Kulon Progo menggunakan alternatif pendanaan obligasi daerah atau pembayaran ketersediaan layanan dalam mendanai proyek perluasan bangunan dan fasilitas RSUD Wates Kabupaten Kulon Progo. Nilai pengorbanan ekonomi tersebut ialah sesuai dengan nilai kebutuhan investasi untuk fisik gedung dan alat kesehatan proyek perluasan bangunan dan fasilitas RSUD Wates Kabupaten Kulon Progo. Present Value Obligasi Daerah Pada Tabel 5 menunjukkan bahwa skema pendanaan obligasi daerah terdapat total present value sebesar Rp779,20 milyar. Nilai tersebut merupakan hasil penjumlahan dari present value dana cadangan untuk pembayaran pokok obligasi daerah dikurangi bunga, present value bunga obligasi, dan present value opex. Dengan pendanaan menggunakan obligasi daerah, maka konstruksi diperkirakan hanya 2 tahun, karena penyediaan dana dengan obligasi daerah diyakini akan lebih cepat satu tahun dibandingkan dengan menggunakan dana dari penerimaan umum daerah. \| Jurnal Kajian Ekonomi & Keuangan Daerah, Volume 5, Nomor 2, September 2020 : 199-224 219 Present value dana cadangan untuk pembayaran pokok obligasi daerah dikurangi bunga sebesar Rp167,83 milyar merupakan nilai sekarang dari dana yang dicadangkan mulai tahun ketiga hingga tahun kesepuluh, dikurangi dengan present value atas bunga yang timbul dari dana cadangan tersebut. Dana cadangan tersebut disimpan dalam bentuk deposito dengan tujuan untuk mengantisipasi pembayaran pokok obligasi daerah pada saat jatuh tempo di tahun kesepuluh atau pada tahun 2026. Pembentukan dana cadangan dimulai pada saat infrastruktur tersebut mulai beroperasi yaitu pada tahun 2019. Oleh karena itu, dana candangan akan dibentuk selama 8 tahun. Dana yang dicadangkan setiap tahun sebesar Rp47,31 milyar. Angka tersebut diperoleh dari perhitungan nilai anuitas majemuk dengan anuitas yaitu pokok obligasi daerah sebesar Rp485,69 milyar dan tingkat suku bunga sesuai suku bunga deposito yaitu 7,02%. Present value bunga obligasi sebesar Rp249,68 milyar merupakan akumulasi nilai sekarang dari bunga obligasi. Sebagaimana telah dijelaskan sebelumnya bahwa bunga obligasi sebesar 7,17% dari pokok obligasi dan akan dibayarkan setiap tahunnya. Present value opex sebesar Rp361,69 milyar merupakan nilai sekarang dari biaya operasi yang ditaksir akan keluar pada saat hasil perluasan bangunan dan fasilitas RSUD Wates mulai beroperasi. Nilai dari opex ditentukan sama dengan nilai opex dari ketiga alternatif pendanaan. Sebagaimana telah dijelaskan sebelumnya bahwa dengan skema obligasi daerah, maka masa konstruksi hanya 2 tahun, maka pada tahun 2019 infrastruktur tersebut muai beroperasi. Oleh karena itu, opex akan mulai diperhitungkan pada tahun ketiga atau tahun infrastruktur mulai beroperasi. Present Value Pembayaran ketersediaan layanan Pada Tabel 5 juga dapat dilihat bahwa present value potensi pengorbanan ekonomi yang akan ditanggung ketika menggunakan skema pendanaan pembayaran ketersediaan layanan yaitu sebesar Rp850,22 milyar. Angka tersebut merupakan total present value \| Jurnal Kajian Ekonomi & Keuangan Daerah, Volume 5, Nomor 2, September 2020 : 199-224 220 pembayaran ketersediaan layanan dikurangi dengan present value PPh Badan yang akan diterima. Present value pembayaran ketersediaan layanan sebesar Rp918,13 milyar merupakan akumulasi nilai sekarang dari pembayaran ketersediaan layanan yang akan dibayarkan oleh Pemerintah Kabupaten Kulon Progo selaku PJPK setiap tahunnya. Sementara itu, present value PPh Badan yang diterima sebesar Rp67,91 milyar merupakan pajak yang akan dibayarkan oleh badan usaha pelaksana atas aliran kas bersih yang diterima setelah dikurangi dengan pembiayaan. Present value PPh Badan yang diterima akan mengurangi total biaya yang ditanggung Pemerintah Kulon Progo karena PPh Badan ini merupakan salah satu aliran kas masuk ke Pemerintah Kabupaten Kulon Progo sebagai satu kesatuan entitas dengan seluruh tingkat pemerintahan di Indonesia. Setelah mengetahui total pengorbanan ekonomi yang ditanggung oleh Pemerintah Kabupaten Kulon Progo pada masing-masing alternatif pendanaan tersebut, maka selanjutnya dapat ditentukan alternatif pendanaan mana yang membutuhkan pengorbanan ekonomi lebih rendah untuk mengetahui alternatif pendanaan yang paling efisien. Selisih pengorbanan ekonomi yang dikeluarkan oleh Pemerintah Kabupaten Kulon Progo pada masing-masing alternatif tersebut dapat dilihat pada Grafik 1. Berdasarkan Grafik 1, dapat dilihat bahwa skema pendanaan obligasi daerah dan pembayaran ketersediaan layanan membutuhkan pengorbanan ekonomi lebih rendah dibanding ketika menggunakan pendanaan dari penerimaan umum daerah. Selain itu, skema pendanaan obligasi daerah juga membutuhkan pengorbanan ekonomi lebih rendah dibanding ketika menggunakan skema pembayaran ketersediaan layanan. 221 Jurnal Kajian Ekonomi & Keuangan Daerah, Volume 5, Nomor 2, September 2020 : 199-224 1,280.91 779.20 850.22 0 501.71 430.69 PENERIMAAN UMUM APBD OBLIGASI DAERAH PEMBAYARAN KETERSEDIAAN LAYANAN Grafik Perbandingan Alternatif Pendanaan (dalam milyar rupiah) Pengorbanan Ekonomi Selisih Pengorbanan Ekonomi Grafik 1 Ringkasan Perbandingan Alternatif Pendanaan Grafik Perbandingan Alternatif Pendanaan (dalam milyar rupiah) Grafik 1 Ringkasan Perbandingan Alternatif Pendanaan Selisih pengorbanan ekonomi dari pendanaan penerimaan umum daerah dengan obligasi daerah yaitu sebesar Rp501,71 milyar. Selisih pengorbanan ekonomi antara pendanaan dari penerimaan umum daerah dengan pembayaran ketersediaan layanan yaitu sebesar Rp430,69 milyar. Sedangkan selisih antara alternatif pendanaan obligasi daerah dengan pembayaran ketersediaan layanan yaitu sebesar Rp71,02 milyar. Present Value Pembayaran ketersediaan layanan Dengan demikian, apabila Pemerintah Kabupaten Kulon Progo menggunakan skema pendanaan obligasi daerah dalam perluasan bangunan dan fasilitas RSUD Wates Kabupaten Kulon Progo, maka dapat melakukan efisiensi dana sebesar Rp501,71 milyar dibanding ketika menggunakan sumber pendanaan dari penerimaan umum daerah dan dapat melakukan efisiensi dana sebesar Rp71,02 milyar dibanding ketika menggunakan alternatif pendanaan pembayaran ketersediaan layanan. SIMPULAN Penelitian ini bertujuan untuk mengetahui alternatif pendanaan yang paling efisien antara obligasi daerah dan pembayaran ketersediaan layanan dibandingkan dengan pendanaan dari penerimaan umum daerah. Penelitian ini mengacu pada proyek \| Jurnal Kajian Ekonomi & Keuangan Daerah, Volume 5, Nomor 2, September 2020 : 199-224 222 pengembangan RSUD Wates Kabupaten Kulon Progo. Berdasarkan pembahasan yang telah dijelaskan, maka hasil penelitian ini dapat disimpulkan sebagai berikut. 1. Alternatif pendanaan yang paling efisien adalah alternatif pendanaan obligasi daerah jika dibandingkan dengan pembayaran ketersediaan layanan dan pendanaan dari penerimaan umum daerah. 2. Apabila Pemerintah Kabupaten Kulon Progo memilih alternatif pendanaan skema obligasi daerah dengan tenor 10 tahun dan bunga 7,17%, maka dapat melakukan efisiensi dana sebesar Rp501,71 milyar dibanding ketika menggunakan sumber pendanaan dari penerimaan umum daerah. 3. Apabila Pemerintah Kabupaten Kulon Progo memilih alternatif pendanaan skema pembayaran ketersediaan layanan dengan masa konsesi 10 tahun dan margin 35% dari total capex dan opex tiap tahun, maka dapat melakukan efisiensi dana sebesar Rp430,69 milyar dibanding ketika menggunakan sumber pendanaan dari penerimaan umum daerah. 3. Apabila Pemerintah Kabupaten Kulon Progo memilih alternatif pendanaan skema pembayaran ketersediaan layanan dengan masa konsesi 10 tahun dan margin 35% dari total capex dan opex tiap tahun, maka dapat melakukan efisiensi dana sebesar Rp430,69 milyar dibanding ketika menggunakan sumber pendanaan dari penerimaan umum daerah. DAFTAR PUSTAKA DAFTAR PUSTAKA Ambarwati, D. I. (2016). Penerbitan Obligasi Daerah Dalam Menunjang Ketahanan Ekonomi Daerah: Studi pada Pemerintah Provinsi Sulawesi Selatan. Yogyakarta: Universitas Gadjah Mada. Angggaran Pendapatan Dan Belanja Daerah Kabupaten Kulon Progo Tahun Anggaran 2017. Bank Indonesia. (2016). http://www.bi.go.id/id/perbankan/suku-bunga-dasar/Default.aspx Darmadji, T., & Fakhruddin. (2006). Pasar Modal di Indonesia Pendekatan Tanya Jawab. Jakarta: Salemba Empat. Daroedono. (2004). Pengembangan Lembaga Keuangan dan Investasi Infrastruktur. Info Kajian Bappenas. Dewi, O., & Kaluge, D. (2011, Oktober). Analisis Peluang Penerbitan Obligasi Daerah Sebagai Alternatif Pembiayaan Daerah. Jurnal of Indonesia Applied Economics, Vol 5(2), 157-171. DJPK. (2007). Direktorat Jenderal Perimbangan Keuangan, Departemen Keuangan Republik Indonesia. Panduan Penerbitan Obligasi Daerah. Jakarta. Djunedi, P. (2008). Implementasi Public Private Partnership dan Dampaknya ke APBN. Direktorat Jenderal Anggaran. \| Jurnal Kajian Ekonomi & Keuangan Daerah, Volume 5, Nomor 2, September 2020 : 199-224 223 Elmi, B. (2005). Analisa Obligasi Daerah Untuk Membiayai Pembangunan Daerah (Municipal Bond): Kasus Pemerintah Daerah Provinsi Jawa Barat. Kajian Ekonomi dan Keuangan. Esfahani, H. S., & Ramı´rez, M. T. (2003). Institutions, infrastructure, and economic growth. Journal of Development Economics , 443-477. Fatimah, D. S. (2011). Pinjaman Daerah dan Public Private Partnership Sebagai Alternatif Pembiayaan Pembangunan Bandar Udara Perintis Di Kabupaten Labuhanbatu. Yogyakarta: Universitas Gadjah Mada. Grout, P. (2005). Value For Money Measurements in Public Private Partnerships. European Investment Bank (EIB), 33-56. Halim, A. (2014). Manajemen Keuangan Sektor Publik: Problematika Penerimaan dan Pengeluaran Pemerintah (Anggaran Pendapatan dan Belanja Negara/Daerah). Jakarta: Salemba Empat. Inayati, N. I., & Setiawan, D. (2017). Fenomena Flypaper Effect pada Belanja Daerah Kabupaten/Kota di Indonesia. Jurnal Ekonomi dan Keuangan, 220-239. Infovesta. 2017. Pilihan Investasi Di Tahun 2017. 25 Februari 2017. Diakses dari https://www.infovesta.com/index/article/articleread;jsessionid=E1357E039E6015F33 6991AC7C65040A9.NGXA/ff3babe7-b0a1-4243-9320-8dc14e3 45a46 pada tanggal 15 Agustus 2017. Kontan. (2016). Bunga Deposito Berpeluang Turun Hingga Akhir Tahun. Diakses dari http://m.kontan.co.id/news/bunga-deposito-berpeluang-turun-hingga-akhir-tahun pada tanggal 20 Agustus 2017. Kurniawan, E. S., Pudjianto, B., & Wicaksono, Y. I. (2009). Analisis Potensi Penerapan Kerjasama Pemerintah Swasta (KPS) Dalam Pengembangan Infrastruktur Transportasi di Perkotaan. Jurnal Teknik, Vol. 30(No. 3), 147-155. Kusumadewi, D. A., & Rahman, A. (2007). Flypaper Effect pada Dana Alokasi Umum (DAU) dan Pendapatan Asli Daerah (PAD) Terhadap Belanja Daerah pada Kabupaten/Kota di Indonesia. Jurnal Akuntansi dan Auditing Indonesia (JAAI), 67-80. aporan Keuangan Pemerintah Daerah Kabupaten Kulon Progo Tahun Anggaran 2013. Laporan Keuangan Pemerintah Daerah Kabupaten Kulon Progo Tahun Anggaran 2014. Laporan Keuangan Pemerintah Daerah Kabupaten Kulon Progo Tahun Anggaran 2015. aporan Keuangan Pemerintah Daerah Kabupaten Kulon Progo Tahun Anggaran 2016. Palei, T. (2015). Assessing The Impact of Infrastructure on Economic Growth and Global Competitiveness. Procedia Economics and Finance 23, hal. 168-175. DAFTAR PUSTAKA Laporan Akhir Kajian Awal Pra Studi Kelayakan KPBU RSUD Krian Kabupaten Sidoarjo Tahun 2016. Masitoh, H. (2014, Januari). Public Private Partnership (PPP) Pengelolaan Aset Daerah: Studi Deskriptif tentang Kemitraan antara Perusahaan Daerah Pasar Surya (PDPS) Surabaya dengan PT Arwinto Intan Wijaya (AIW) dalam Pembangunan dan Pengembangan Darmo Trade Centre (DTC) Surabaya. Kebijakan dan Manajemen Publik, Vol 2(1). Noor, H. F. (2009). Investasi: Pengelolaan Keuangan Bisnis dan Pengembangan Ekonomi Masyarakat. Jakarta: Indeks. Palei, T. (2015). Assessing The Impact of Infrastructure on Economic Growth and Global Competitiveness. Procedia Economics and Finance 23, hal. 168-175. 224 Jurnal Kajian Ekonomi & Keuangan Daerah, Volume 5, Nomor 2, September 2020 : 199-224 Peraturan Menteri Keuangan Nomor 111/PMK.07 Tahun 2012 tentang Tata Cara Penerbitan dan Pertanggungjawaban Obligasi Daerah. Peraturan Menteri Keuangan Nomor 111/PMK.07 Tahun 2012 tentang Tata Cara Penerbitan dan Pertanggungjawaban Obligasi Daerah. Peraturan Pemerintah Nomor 30 Tahun 2011 tentang Pinjaman Daerah. Peraturan Menteri Keuangan Nomor 180/PMK.07 Tahun 2015 tentang Perubahan atas Peraturan Menteri Keuangan Nomor 111/PMK.07 Tahun 2012 tentang Tata Cara Penerbitan dan Pertanggungjawaban Obligasi Daerah. Peraturan Menteri Perencanaan Pembangunan Nasional atau Kepala Badan Perencanaan Pembangunan Nasional Nomot 4 Tahun 2015 tentang Tata Cara Pelaksanaan Kerjasama Pemerintah Dengan Badan Usaha Dalam Penyediaan Infrastruktur. Peraturan Pemerintah Nomor 38 Tahun 2015 tentang Kerjasama Pemerintah dengan Badan Usaha dalam Penyediaan Infrastruktur. Peraturan Menteri Dalam Negeri Nomor 96 Tahun 2016 tentang Pembayaran Ketersediaan Layanan dalam Rangka Kerjasama Pemerintah Daerah dengan Badan Usaha dalam Penyediaan Infrastruktur di Daerah. PT Sarana Multi Infrastruktur. 2017. Katalis Untuk Infrastruktur Indonesia Yang Lebih Baik. Diakses dari https://www.ptsmi.co.id/id/tentang-kami/tentang-pt-smi/ pada tanggal 28 Mei 2017. Purnomo, B. S. (2015). Obligasi Daerah: Alternatif Investasi Bagi Masyarakat dan Sumber Pendanaan Bagi Pemerintah Daerah. Bandung: Alfabeta. Sahoo, P., & Dash, R. K. (2009). Infrastructure Development and Economic Growth in India. Journal of the Asia Paciﬁc Economy, 14, 351-365. Sharma, D., & Cui, Q. (2012). Design of Concession and Annual Payments for Availability Payment Public Private Partnership (PPP) Projects. Construction Research Congress 2012. Sitorus, T. (2015). Pasar Obligasi Indonesia: Teori dan Praktik (Edisi 1). Jakarta: Raja Grafindo Persada. Surachman, E. N., & Setiawan, H. (2016). Municipal Bonds as The Financing Strategy for Urban Infrastructure: Case Study of Jakarta MRT. Jurnal Keuangan dan Perbankan, 369-381. Tukuboya, M. W., Nurjaman, H. N., & Suryani, F. (2018). Public Private Partnership pada Konservasi dan Pembangunan Infrastruktur Pengaman Pantai; Studi Kasus Bali. Ikraith Teknologi, 32-40. Undang-Undang Nomor 23 Tahun 2014 tentang Pemerintah Daerah.
https://openalex.org/W2811166885	https://europepmc.org/articles/pmc6073265?pdf=render	English	null	Recent Insights on Alzheimer’s Disease Originating from Yeast Models	International journal of molecular sciences	2,018	cc-by	19,114	Recent Insights on Alzheimer’s Disease Originatin from Yeast Models David Seynnaeve 1 ID , Mara Del Vecchio 1, Gernot Fruhmann 1, Joke Verelst 1, Melody Cools 1, Jimmy Beckers 1, Daniel P. Mulvihill 2 ID , Joris Winderickx 1 ID and Vanessa Franssens 1,* David Seynnaeve 1 ID , Mara Del Vecchio 1, Gernot Fruhmann 1, Joke Verelst 1, Melody Cools 1 immy Beckers 1, Daniel P. Mulvihill 2 ID , Joris Winderickx 1 ID and Vanessa Franssens 1,* 1 Functional Biology, KU Leuven, Kasteelpark Arenberg 31, 3000 Leuven, Belgium; david.seynnaeve@kuleuven.be (D.S.); mara.delvecchio@kuleuven.be (M.D.V.); gernot.fruhmann@kuleuven.be (G.F.); joke.verelst@kuleuven.be (J.V.); melody.cools@kuleuven.be (M.C.); jimmy.beckers@student.kuleuven.be (J.B.); joris.winderickx@kuleuven.be (J.W.) 2 School of Biosciences, University of Kent, Canterbury CT2 7NJ, Kent, UK; D.P.Mulvihill@kent.ac.uk * Correspondence: vanessa.franssens@kuleuven.be; Tel.: +32-16320381; Fax: +32-16321967 Received: 14 June 2018; Accepted: 30 June 2018; Published: 3 July 2018 Abstract: In this review article, yeast model-based research advances regarding the role of Amyloid-β (Aβ), Tau and frameshift Ubiquitin UBB+1 in Alzheimer’s disease (AD) are discussed. Despite having limitations with regard to intercellular and cognitive AD aspects, these models have clearly shown their added value as complementary models for the study of the molecular aspects of these proteins, including their interplay with AD-related cellular processes such as mitochondrial dysfunction and altered proteostasis. Moreover, these yeast models have also shown their importance in translational research, e.g., in compound screenings and for AD diagnostics development. In addition to well-established Saccharomyces cerevisiae models, new upcoming Schizosaccharomyces pombe, Candida glabrata and Kluyveromyces lactis yeast models for Aβ and Tau are brieﬂy described. Finally, traditional and more innovative research methodologies, e.g., for studying protein oligomerization/aggregation, are highlighted. ywords: Alzheimer’s disease; yeast; Tau; amyloid β; ubiquitin; aggregation; oligomerization; prion Int. J. Mol. Sci. 2018, 19, 1947; doi:10.3390/ijms19071947 International Journal of Molecular Sciences International Journal of Molecular Sciences 1.2. Yeast as a Model Organism to Study AD: Advantages Studies to gain more insights on AD primarily make use of human cell lines and transgenic mouse models. However, yeast cell models are playing an increasingly important role in unravelling the fundamental disease aspects of AD. In fact, the yeast S. cerevisiae is a very widely studied single-celled model organism. With more than 6000 genes distributed on 16 chromosomes, its genome was the ﬁrst eukaryotic genome to be completely sequenced in 1996 [11]. Since then, it has been estimated that nearly 31% of yeast genes have human orthologues [12]. Beyond the laboratory yeast strains, many different natural, brewery and clinical isolates exist and all have a core genome of about 5000 shared genes [12]. Yeast reproduction is through mitosis of either a haploid or a diploid cell. Haploids are of 2 different mating type (a or α) and a haploid cell can only mate with a cell of the opposite mating type. Mating leads to the formation of a diploid cell that can either continue to exist and bud as a diploid or, under conditions of stress, produce spores by meiosis. Spores can then later give rise to haploid cells [12]. Haploidy implies that gene-knockout strains can easily be obtained. In 2001, a collection of isogenic yeast strains, each deleted for one of the 6000 putative open reading frames (ORFs), was created [12]. This allowed for the easy phenotypic analysis of mutants, paving the way to determining gene function. In addition, yeast cells share many conserved biological processes such as cell cycle progression, protein turnover, vesicular trafﬁcking and signal transduction with cells of higher eukaryotes [13], including human neurons. Its short generation time (1.5 h on rich medium), means that it can be very easily cultured. Thanks to its susceptibility to simple genetic and environmental manipulations, this model organism has become a valuable tool to shed more light on the complex and fundamental intracellular mechanisms underlying neurodegenerative diseases. p y g g So-called “humanized yeast model systems” have been constructed and used as a tool to investigate the molecular mechanisms involved in several neurological disorders [14,15]. The main advantage of using yeast is its reduced complexity compared to the mammalian models. On the contrary, Tau and Aβ have no functional yeast orthologues. Heterologous expression of Tau and Aβ can be highly informative and provides useful new insights into the pathobiology of these proteins in vivo. 1.2. Yeast as a Model Organism to Study AD: Advantages At the same time, yeast is an excellent screening tool for compounds that may be useful in treatment and/or prevention of AD. 1.1. Alzheimer’s Disease (AD) Alzheimer’s disease (AD) is the most common neurodegenerative disease worldwide. It accounts for approximately 60–70% of all dementia cases and affects about 6% of the population aged over 65 (late-onset AD), whereas 2–10% of patients suffer from early-onset AD [1,2]. Currently, around 50 million individuals live with this devastating chronic disease and it has been estimated that the number will increase up to approximately 106 million people by 2050 due to an increasing aging population [2,3]. At the cellular level, AD is characterized by an irreversible and progressive loss of neuronal structure and function within certain regions of the brain including the hippocampus and neocortical brain, leading to cognitive dysfunction and dementia [4]. Widespread experimental evidence also suggests that AD is characterized by synaptic dysfunction early on in the disease process, disrupting communication within neural circuits important for memory formation and other cognitive functions such as intellectuality and comprehensive capacity [5–7]. Therefore, damage to these brain structures results in memory loss, language difﬁculties and learning deﬁcits that are typically observed within early stages of clinical manifestation of AD. In addition, upon disease progression, a decline in other cognitive domains occurs which will result in the complete inability to function independently in basic daily activities [7]. Besides AD having Int. J. Mol. Sci. 2018, 19, 1947; doi:10.3390/ijms19071947 www.mdpi.com/journal/ijms www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2018, 19, 1947 2 of 26 a profound impact on the life quality of patients, this chronic disease also imposes a huge economic burden on healthcare systems globally with an associated cost which is estimated will exceed $1 trillion by 2050 [8]. The neuronal damage is related to the accumulation of misfolded proteins into extracellular and intracellular aggregates, consisting of Aβ peptides or protein Tau, respectively [9,10]. It is not yet clear whether the presence of these two hallmarks is the cause of AD or mainly the result of a cascade of cellular events including oxidative stress, mitochondrial dysfunction and apoptosis. Either way, the exact mechanism by which these proteins damage neurons is still unknown. 2.1. Protein Tau: Structure, Functions and Modiﬁcations 2.1. Protein Tau: Structure, Functions and Modiﬁcations Protein Tau, encoded by the 16 exon long microtubule (MT)-associated protein Tau (MAPT) gene located on chromosome 17q21.31, is present in neuronal and glial axons, but has also been detected outside of cells [17–22]. Tau is natively unfolded and has the tendency to adapt a paperclip-like shape, in which the N- and C-terminal domains and repeat regions are all closely located to each other (see below) [23]. It is a MT associated protein, susceptive to dynamic (de-) phosphorylation. These modiﬁcations inﬂuence its main cellular function which consists of regulating MT dynamic instability i.e., the process of polymerization (rescue) and depolymerization (catastrophe) [24–27]. Besides being involved in the regulation of MT dynamics, Tau has functions in regulating axonal transport/elongation/maturation, synaptic plasticity and maintaining DNA and RNA integrity [28–37]. It is clear that Tau is involved in numerous processes and that loss of Tau function can initiate neurotoxicity through disruption of various processes in which it is involved. For a more complete overview of physiological and pathological Tau functions, we refer to [38]. A ﬁrst mechanism by which Tau function is regulated is alternative splicing. Due to alternative splicing of exon 2, 3 and 10 of the MAPT gene, Tau can be present in 6 different isoforms differing in the number of N-terminal inserts (1 or 2) and conserved 18 amino acid long repeats (3 to 4) in the MT binding region (C-terminal region or assembly domain) [39,40]. Tau isoforms with 4 repeat regions show a stronger interaction with MT and are more efﬁcient in MT assembly [41–43]. The N-terminal projection region of the protein is located adjacent to a proline rich region and has a role in MT spacing and stabilization [44,45]. In addition, it was proposed that this domain, which projects away from MT, interacts with cell organelles such as the plasma membrane, mitochondria and actin filaments [46–51]. This binding could be facilitated via an interaction between the PXXP motifs in the proline-rich region and the SH3 domains of the src-family non-receptor tyrosine kinases (e.g., kinase FYN) [52–54]. Note that this plasma membrane interaction might play an important role in vesicle-mediated secretion and therefore impact the cell-to-cell spreading of protein Tau. It was proposed that the Tau-FYN interaction may regulate the post-synaptic targeting of FYN, and thereby mediate Aβ-induced excitotoxicity [23]. 1.3. Yeast as a Model Organism to Study AD: Limitations Despite being a powerful and simpliﬁed model system, yeast also has its natural limitations. As a unicellular organism, the most important limitation for neurodegenerative disease research is the analysis of disease aspects that focus on multicellularity and cell–cell interactions. These interactions include synaptic transmissions, axonal transport, glial-neuronal interactions, immune and inﬂammatory responses and many neuronal specializations that are likely to play an important role in neurodegeneration, but cannot be recapitulated in yeast [16]. Moreover, it is also impossible to study the cognitive aspects of AD in yeast cells. This review discusses the ﬁndings of more recent studies on neurodegenerative disorders conducted using different yeast species. Int. J. Mol. Sci. 2018, 19, 1947 3 of 26 2.1. Protein Tau: Structure, Functions and Modiﬁcations Additional proposed pathways for cell-to-cell transfer are tunneling nanotubes and trans-synaptic spreading [55–58]. This spreading process is still ill-deﬁned and it still needs to be proven if spreading of (a) toxic Tau species is sufﬁcient or necessary for the induction of a tauopathy. Guo and colleague published a comprehensive article on this emerging ﬁeld within Tau biology [59]. Finally, the proline-rich domain of Tau has a role in facilitating the binding of the MT binding region to the MT [60,61]. Tau function is also regulated by several post-translational modiﬁcations including phosphorylation, glycosylation, truncation, nitration, isomerisation, acetylation, glycation, ubiquitination, deamidation, methylation, sumoylation and oxidation [38]. Tau phosphorylation has been studied extensively. The protein contains 80 putative serine/threonine and 5 potential tyrosine phosphorylation sites, of which the majority is phosphorylated in vitro, on the 2N/4R (2 N-terminal inserts and 4 amino acid repeat regions) isoform. Tau is phosphorylated by numerous kinases, grouped in 4 different classes [62,63]. More recently, GSK3α, GSK3β, MAPK13 and AMP-activated protein kinase were found to play an actual role in in vivo Tau phosphorylation using different cell lines [64,65]. Tau (de-) phosphorylation is an important factor inﬂuencing Tau’s afﬁnity for MT, thereby regulating its role in MT (de-) polymerization. On the other hand, aberrant phosphorylation (so-called hyperphosphorylation) on several epitopes (e.g., Thr181, Thr231, Ser202, Ser205, Ser214, Ser396, Ser404, Ser409, and Ser422), which severely affects Tau’s MT binding capacity and stabilizing properties [66–68] can lead to an increased propensity of Tau to subsequently oligomerize and aggregate into paired helical ﬁlaments (PHF) and neuroﬁbrillary tangles (NFT) [66]. These NFT are characteristic for a group of neurodegenerative diseases called tauopathies including AD. Int. J. Mol. Sci. 2018, 19, 1947 4 of 26 The aggregation is due to a redistribution of mainly MT bound to unbound Tau, which facilitates Tau-Tau interactions made possible by 2 hexapeptide motives in repeat regions 2 and 3, which can adapt β-sheet structures [69]. While the repeat domain makes up the PHF core, the N- and C-terminal Tau region form a “coat” around this core [23]. Hyperphosphorylation can also induce pathology through other mechanisms. It can ﬁrst of all lead to Tau missorting from axons to the somatodendritic compartment, which might cause synaptic dysfunction. Another consequence is affected substrate recognition, which leads to an altered proteasomal degradation [70]. 2.1. Protein Tau: Structure, Functions and Modiﬁcations Phosphorylation is, however, only one potential covalent modiﬁcation Tau can undergo and it should be noted that this modiﬁcation alone is not sufﬁcient to cause aggregation. Phosphorylation at some sites (e.g., Ser214 and Ser262) in the repeat domain can even protect against aggregation [69]. Thus, it is suggested that phosphorylation might facilitate this process, and therefore serves as an indirect aggregation inducer, and that other factors are involved as well. Indeed, other modiﬁcations, and especially truncation, can be equally important for disease development. Tau truncated at Glu391 and Asp421, for example, has been identiﬁed as an event following phosphorylation and facilitating Tau ﬁlament formation. Tau truncation can even induce neurodegeneration independently of Tau aggregation through the formation of speciﬁc Tau fragments [69]. Tau truncation disrupts the paperclip-like structure, thereby promoting Tau aggregation. Tau ubiquitination, on the other hand, is considered a protective strategy of the cell to get rid of toxic Tau intermediates and accumulations of hyperphosphorylated Tau are mainly found in cells with a defective or malfunctioning ubiquitin/proteasome system. The latter can be caused by oxidative stress due to mitochondrial malfunctioning, illustrating the complex cellular pathways involved in the induction of Tau-mediated toxicity. For a more complete overview of Tau post-translational modiﬁcations and their consequences on Tau pathology, we refer to [38,71]. The Tau aggregation process itself seems to be a requirement for Tau-induced toxicity and although recent papers are pointing towards the soluble mono- or oligomeric hyperphosphorylated Tau species as being the toxic Tau forms, their relative contribution remains largely unclear. The insoluble aggregated structures are thought to act as protective structures by sequestering the toxic species [72–76]. Numerous Tau mutations, either causing AD or other tauopathies such as frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), have been documented over the years and can either be missense, silent or causing a deletion. Depending on the mutation’s nature and its gene location, the mutation can directly disturb Tau’s MT binding capacity, thereby increasing Tau’s tendency for aggregation, or indirectly by affecting the 4R:3R ratio by inﬂuencing Tau splicing [77–85]. Most of these mutations are nicely documented on the “Alzforum” website. 2.2. From Complementary Disease Models to AD Diagnostics Historically, the AD ﬁeld has been dominated by research supporting Aβ having the main role in pathogenesis. Only after the discovery of several MAPT mutations in FTDP-17 did Tau research receive a signiﬁcant and rightful boost. Indeed, both in vitro and in vivo studies show evidence that Tau is required for Aβ-mediated neurotoxicity [86]. Therefore, only a limited amount of research articles on the pathological aspects of protein Tau using the yeast S. cerevisiae as a model organism have been published to date [87–89]. Figure 1 gives a visual overview of human Tau processes and modiﬁcations in S. cerevisiae. These studies have already been extensively reviewed, so a brief summary will be given. Upon overexpression in S. cerevisiae, Tau becomes hyperphosphorylated and acquires several pathological phospho-epitopes (AD2 (p-Ser396/p-Ser404), AT8 (p-Ser202/p-Ser205), AT270 (p-Thr181), AT180 (p-Thr231/p-Ser235) AT100 (p-Thr212/p-Ser214) and PG5 (p-Ser409)). Moreover, it was possible to detect the disease-relevant conformational epitope recognized by the MC1 antibody. 5 of 26 5 of 26 Int. J. Mol. Sci. 2018, 19, 1947 I t J M l S i 2018 19 Figure 1. Humanized yeast model expressing human protein Tau: overview of Tau processes and modifications in S. cerevisiae. Double arrows indicate a bidirectional/reversible reaction and dashed lines specify the promoter and expressed human Tau gene on the plasmid. ‘TPI’; Triosephosphate isomerase promoter, ‘P’; phosphate group. Figure 1. Humanized yeast model expressing human protein Tau: overview of Tau processes and modiﬁcations in S. cerevisiae. Double arrows indicate a bidirectional/reversible reaction and dashed lines specify the promoter and expressed human Tau gene on the plasmid. ‘TPI’; Triosephosphate isomerase promoter, ‘P’; phosphate group. Figure 1. Humanized yeast model expressing human protein Tau: overview of Tau processes and modifications in S. cerevisiae. Double arrows indicate a bidirectional/reversible reaction and dashed lines specify the promoter and expressed human Tau gene on the plasmid. ‘TPI’; Triosephosphate isomerase promoter ‘P’; phosphate group Figure 1. Humanized yeast model expressing human protein Tau: overview of Tau processes and modiﬁcations in S. cerevisiae. Double arrows indicate a bidirectional/reversible reaction and dashed lines specify the promoter and expressed human Tau gene on the plasmid. ‘TPI’; Triosephosphate isomerase promoter, ‘P’; phosphate group. Pho85 and Mds1 protein kinases, yeast orthologues of human Tau kinases Cdk5 and GSK3β, respectively, were shown to play a key role in modulating Tau phosphorylation. It was suggested that Pho85 may have a direct or indirect inhibitory effect on the activity of Mds1. 2.2. From Complementary Disease Models to AD Diagnostics Upon deletion of Pho85, phosphorylation of Tau was enriched on the AD2 and PG5 epitopes. Accordingly, the MC1- reactive Tau fraction was also higher. Tau aggregation in this pho85∆ strain was assessed by measuring the sarkosyl-insoluble Tau (SinT) fraction and it was proposed that Tau epitopes PG5 and AT100 might play a crucial role in the accumulation of SinT aggregates, since these epitopes were especially enriched in the insoluble fraction [87]. The importance of phosphorylation of the PG5 epitope for Tau aggregation was also confirmed in a follow-up study in which aggregation of several Tau mutants was assessed. In addition, PG5 epitope phosphorylation is detrimental for Tau’s MAPT function, illustrated by lack of Tau binding to taxol-stabilized MT from porcine Tubulin in vitro Pho85 and Mds1 protein kinases, yeast orthologues of human Tau kinases Cdk5 and GSK3β, respectively, were shown to play a key role in modulating Tau phosphorylation. It was suggested that Pho85 may have a direct or indirect inhibitory effect on the activity of Mds1. Upon deletion of Pho85, phosphorylation of Tau was enriched on the AD2 and PG5 epitopes. Accordingly, the MC1-reactive Tau fraction was also higher. Tau aggregation in this pho85∆strain was assessed by measuring the sarkosyl-insoluble Tau (SinT) fraction and it was proposed that Tau epitopes PG5 and AT100 might play a crucial role in the accumulation of SinT aggregates, since these epitopes were especially enriched in the insoluble fraction [87]. The importance of phosphorylation of the PG5 epitope for Tau aggregation was also conﬁrmed in a follow-up study in which aggregation of several Tau mutants was assessed. In addition, PG5 epitope phosphorylation is detrimental for Tau’s MAPT function, illustrated by lack of Tau binding to taxol-stabilized MT from porcine Tubulin in vitro [88,89]. , y g p [88,89]. Despite all of this, Tau 2N/4R and 2N/3R expression does not induce an impaired growth phenotype in S. cerevisiae [87,88,90]. The latter is not necessarily expected since little attention was paid to the extent of formation of early stage, presumably toxic, soluble (oligomeric) Tau species in these studies. The possibility exists that these oligomeric Tau species are rapidly sequestered in inert Despite all of this, Tau 2N/4R and 2N/3R expression does not induce an impaired growth phenotype in S. cerevisiae [87,88,90]. 2.2. From Complementary Disease Models to AD Diagnostics The latter is not necessarily expected since little attention was paid to the extent of formation of early stage, presumably toxic, soluble (oligomeric) Tau species in these studies. The possibility exists that these oligomeric Tau species are rapidly sequestered in inert aggregates as a cell protection mechanism. p y g p p y q aggregates as a cell protection mechanism. As described above, several other post-translational modifications are expected to contribute to tauopathy development, besides phosphorylation. Therefore, it might be highly interesting to verify if these modifications are also recapitulated in yeast, and if so, to what extend they could offer an As described above, several other post-translational modiﬁcations are expected to contribute to tauopathy development, besides phosphorylation. Therefore, it might be highly interesting to verify if these modiﬁcations are also recapitulated in yeast, and if so, to what extend they could offer an explanation for the (lack of) aggregation/toxicity in yeast cells. p y , , y explanation for the (lack of) aggregation/toxicity in yeast cells. On top of that, it was found that oxidative stress and mitochondrial dysfunction, independently On top of that, it was found that oxidative stress and mitochondrial dysfunction, independently of Tau phosphorylation, also strongly induce Tau aggregation in yeast cells [88]. It is also worth Int. J. Mol. Sci. 2018, 19, 1947 6 of 26 mentioning that inducing oxidative stress resulted in Tau dephosphorylation, in accordance with other results obtained from human, rat and mice neuronal cells [91,92]. One potential mechanism is oxidative stress-induced Pin1 activation. Pin1, a peptidyl prolyl cis/trans isomerase, can then subsequently activate Phosphatase 2a. De Vos and colleagues reported, in accordance with this ﬁnding, that a dysfunctional Ess1, Pin1’s yeast orthologue, increases Tau hyperphosphorylation [93]. On the other hand, other studies point out that oxidative stress does not alter Tau phosphorylation or even induces Tau hyperphosphorylation. These studies were performed using a Dropsophila melanogaster model and human neuronal cells [94], respectively. The interplay between oxidative stress and Tau phosphorylation, therefore, needs more attention in future studies to further elucidate their relationship. Tau was also puriﬁed, using anion-exchange chromatography, from the previously mentioned pho85∆S. cerevisiae strain, maintaining its hyperphosphorylated MC1-reactive state, and could subsequently seed aggregation of wt 2N/4R Tau protein puriﬁed from a wt strain in vitro [87]. The possibility of purifying these stable, pathologically-relevant, Tau structures from S. 2.2. From Complementary Disease Models to AD Diagnostics cerevisiae cells paved the way for using yeast-puriﬁed Tau as an antigen source for mice immunization [95]. This strategy offers a signiﬁcant beneﬁt over E. coli based Tau puriﬁcations and antibody generation [76,96, 97], since Tau is not post-translationally modiﬁed in bacterial cells. Although oligomerization can be induced by use of, for example, arachidonic acid or heparin, there is no evidence that these artiﬁcially formed oligomers/aggregates are the actual toxic species and, therefore, that the produced monoclonal antibodies recognize pathologically relevant Tau species. ADx215, an antibody developed by immunizing mice with 2N/4R Tau puriﬁed from a pho85∆ S. cerevisiae strain, is capable of detecting both mono- and oligomeric Tau protein [95]. This antibody was recently successfully implemented in a digital enzyme-linked immunosorbent assay (ELISA) platform and able to detect attomolar concentrations of Tau protein, thereby unlocking the potential of Tau as a serum-based AD biomarker [98]. So, over the last decade, yeast has developed from a reliable model organism, merely used to gain more understanding of pathological Tau features such as aggregation and phosphorylation, to a highly suitable platform model for disease-relevant antigen production. In the NFT of transgenic mice models, α-synuclein can co-localize with Tau and it has been shown that α-synuclein can seed Tau aggregation in vitro and in vivo and even enhance Tau’s toxicity in mice models [99–103]. It is, therefore, clear that the interplay between both proteins is important. Yeast models have been developed that enable the study of both proteins and the resulting effects on toxicity and aggregation [104,105]. Episomal expression of wt and A53T α-synuclein and wt and P301L Tau resulted in increased phosphorylation on the AD2 epitope and Tau aggregation, but no growth-inhibiting effect was detected [105]. The latter was in contrast to a previously reported study [104], where synergistic toxicity was observed upon stable genomic integration of plasmids expressing wt α-synuclein and wt Tau. This is in accordance with increased α-synuclein inclusion formation and Tau phosphorylation/aggregation [105]. 2.3. Future Perspectives Baker’s yeast has been of interest to humans since the existence of brewing and bread-making. Since these two activities have been subject to continuous improvement, research on S. cerevisiae’s physiology was mainly application-driven. In contrast, focus on the ﬁssion yeast S. pombe was mainly interested-driven and initial studies were performed to gain more insights in its mating type system and sexual and cell division cycle [106]. Nevertheless, S. pombe might offer great potential as a complementary model to study Tau biology since several features such as the cell division machinery, cell polarity and cytoskeleton organization are more closely related to higher eukaryotes compared to S. cerevisiae [106–108]. This organism could, therefore, be advantageous to study Tau characteristics such as in vivo MT binding, which has not been observed so far in budding yeast models most likely due to critical gene sequence differences. So far, binding to porcine MT has only been shown for yeast Int. J. Mol. Sci. 2018, 19, 1947 7 of 26 extracted Tau [88]. Indeed, ﬂuorescence microscopy studies indicate potential binding of protein Tau to MT in vivo in S. pombe cells. Moreover, preliminary data points out that several Tau epitopes also become phosphorylated in S. pombe (data not shown). The precise role(s) of each of the ﬁssion yeast Tau kinase orthologues remains unresolved. Heinisch and colleague also proposed several arguments why the milk yeast K. lactis could serve as a useful model to study Tau biology, more speciﬁcally the effects of energy signaling and oxidative stress on Tau aggregation [109]. K. lactis has several advantages over the traditional baker’s yeast model. For example, a respiratory metabolism more resembling that of mammalian cells. Moreover, K. lactis did not undergo a whole genome duplication throughout evolution which limits the number of redundant gene functions. This ensures a more easily trackable phenotype upon single gene deletions [110]. Experimental methodologies used in the aforementioned reports, e.g., SinT assay or ﬂuorescence microscopy using a green ﬂuorescent protein (GFP)-tagged Tau protein, are well suited for the study of Tau aggregation, but lack applicability for analysis of Tau oligomerization. Since the current consensus is that oligomeric, rather than aggregated, Tau is the toxic Tau species, neat technologies to study oligomerization could enhance yeast’s value as a model for the study of neurodegenerative diseases such as AD. An example is the use of a split-GFP sensor system. Several split-GFP technologies are described in [111–115]. 3.2. From Heterologously Expressed APP to Secretory Pathway-Targeted Aβ Peptides 3.2. From Heterologously Expressed APP to Secretory Pathway-Targeted Aβ Peptides Since Aβ peptides are generated by Secretase cleavage of APP, modelling of Aβ pathology in yeast cells can be done via a number of approaches. The APP, or the Aβ peptides can be heterologously expressed in yeast. Although there are no orthologues of the human Secretases in yeast present, both α- and β-secretase activity has been reported [154,155], with the yeast proteases Yap3 and Mkc7 suggested to exhibit α-secretase activity [156,157]. γ-secretase activity was successfully reconstituted in S. cerevisiae upon combined expression of APP-based substrates and human γ- secretase, resulting in the production of Aβ40, Aβ42, and Aβ43 [158,159] (Figure 2). Since Aβ peptides are generated by Secretase cleavage of APP, modelling of Aβ pathology in yeast cells can be done via a number of approaches. The APP, or the Aβ peptides can be heterologously expressed in yeast. Although there are no orthologues of the human Secretases in yeast present, both α- and β-secretase activity has been reported [154,155], with the yeast proteases Yap3 and Mkc7 suggested to exhibit α-secretase activity [156,157]. γ-secretase activity was successfully reconstituted in S. cerevisiae upon combined expression of APP-based substrates and human γ- secretase, resulting in the production of Aβ40, Aβ42, and Aβ43 [158,159] (Figure 2). Figure 2. Humanized yeast model expressing human proteins, APP and γ-secretase: overview of Secretase-mediated APP processing and Aβ peptide production. The scissors icon indicates cleavage of the respective proteins. Figure 2. Humanized yeast model expressing human proteins, APP and γ-secretase: overview of Secretase-mediated APP processing and Aβ peptide production. The scissors icon indicates cleavage of the respective proteins. Figure 2. Humanized yeast model expressing human proteins, APP and γ-secretase: overview of Secretase-mediated APP processing and Aβ peptide production. The scissors icon indicates cleavage of the respective proteins. Figure 2. Humanized yeast model expressing human proteins, APP and γ-secretase: overview of Secretase-mediated APP processing and Aβ peptide production. The scissors icon indicates cleavage of the respective proteins. As mentioned above, the γ-secretase complex consists of 4 different components and the influence of different combinations of Presenilin and Aph1 proteins on function and substrate specificity of the γ-secretase was tested in a yeast system [160,161]. Although differences were observed, the results were not well in line with parallel studies performed in mammalian cells, which, they reasoned, could be explained by the lack of additional proteins (e.g., GSAP and CD147) that affect γ-secretase function and that are not present in yeast. 3.1. Protein Aβ: Structure, Function and Aggregation Glycoprotein amyloid precursor protein (APP) plays an important role in numerous biological activities, ranging from neuronal development and homeostasis to signaling and intracellular transport [116–119]. After synthesis in the endoplasmatic reticulum (ER), the protein is subsequently transported from the Golgi apparatus to the plasma membrane where it is cleaved by α- and γ-secretase or β- and γ-secretase following the non-amyloidogenic or amyloidogenic pathway, respectively [120,121]. This cleavage yields several Aβ species with amino acid sequences varying from 40 to 51 amino acids with Aβ40 and Aβ42 being the ﬁnal fragments [122,123]. While β-secretase activity is primarily mediated by BACE1, γ-secretase activity actually requires the presence of 4 proteins; Presenilin 1 or 2, Nicastrin, Presenilin enhancer 2 and Anterior pharynx defective 1 (Aph1) [124]. These peptides can then be released in the extracellular space where they can bind to a variety of receptors or they remain associated with the plasma membrane and lipid raft structures [119]. The amyloidogenic pathway is central in the so-called “amyloid cascade hypothesis”, which states that the formed Aβ structures sequentially oligomerize and aggregate thereby causing neurotoxicity and dementia [125]. Aβ peptides can aggregate in different structural forms i.e., soluble oligomers, protoﬁbrils, but also insoluble amyloid ﬁbrils and all of them feature β-sheet structures [126,127]. While the oligomers may spread throughout the brain, the ﬁbrils can further assemble into plaques, which are commonly found in the neocortex of the AD patient brains [128]. However, there is no direct correlation between amyloid plaques and the loss of synapses and neurons in AD patient brains [129–131]. In fact, cognitive deﬁcits appear before plaque deposition or the deposition of insoluble amyloid ﬁbrils. Similarly to protein Tau, it is suggested that the Aβ oligomers trigger synapse dysfunction and memory impairment [132,133]. Extracellular receptor-bound Aβ oligomers were proposed to induce neurotoxic effects by causing mitochondrial dysfunction and oxidative stress in neuronal cells, which can cause a massive calcium inﬂux [134]. This can then impair the ability of cells to conduct normal physiological functions [135]. It should be mentioned, however, that it is highly possible that different Aβ forms may contribute to neurodegeneration at different disease stages [135]. A proposed link between Aβ and Tau pathology is the Aβ aggregation-mediated kinase activation, which results in Tau hyperphosphorylation Int. J. Mol. Sci. 2018, 19, 1947 8 of 26 (see above) that in turn results in NFT formation. 3.1. Protein Aβ: Structure, Function and Aggregation This is, however, only one possible hypothesis linking these two proteins and their interplay is presumed to be more complex [135]. Other secondary, toxicity inducing, effects of Aβ aggregation are the involvement of the innate immune system and inﬂammatory responses [136–138]. Int. J. Mol. Sci. 2018, 19, x 8 of 26 To maintain Aβ protein homeostasis both in the brain and in plasma, production of Aβ is To maintain Aβ protein homeostasis both in the brain and in plasma, production of Aβ is counterbalanced by mechanisms such as proteolytic degradation [139–141], active transport via the blood brain barrier [142–147] and deposition of Aβ in insoluble aggregates [148,149]. This does not only involve neurons, but also other cells of the neurovascular unit, such as astrocytes [150–152]. Disruption of any of these processes might result in neuropathology. Cathepsin B, for example, was identiﬁed as a major Aβ-degrading enzyme and its expression level is altered in the brain of AD patients [153]. β p p , p β counterbalanced by mechanisms such as proteolytic degradation [139–141], active transport via the blood brain barrier [142–147] and deposition of Aβ in insoluble aggregates [148,149]. This does not only involve neurons, but also other cells of the neurovascular unit, such as astrocytes [150–152]. Disruption of any of these processes might result in neuropathology. Cathepsin B, for example, was identified as a major Aβ-degrading enzyme and its expression level is altered in the brain of AD patients [153]. 3.2. From Heterologously Expressed APP to Secretory Pathway-Targeted Aβ Peptides 3.2. From Heterologously Expressed APP to Secretory Pathway-Targeted Aβ Peptides This setup offers a neat in vivo system to screen for modulators of oligomerization. A second, independent study yielded a similar result in that the Aβ42-Sup35 fusion was able to form aggregates, although less stable compared to Sup35 aggregates, and restore the [PSI+] phenotype of Sup35 lacking the prion forming domain [165]. To investigate Aβ42’s location and interactions, it was fused to GFP. Apart from inducing a growth defect, Aβ42 also induced a heat shock response [166]. The latter is in correspondence with data obtained from AD patients that indicate that heat shock protein expression is upregulated in AD as a protective measure [167]. A study on oligomerization/aggregation modiﬁers using this Aβ42-GFP construct suggested that folinic acid might assist in preventing Aβ42 misfolding and aggregation [168]. Recently, yeast was also used as a model to screen for rationally designed compounds [169]. More speciﬁcally, Thioﬂavin assays, circular dichroism measurements and transmission electron microscopy were used to assess the efﬁciency of peptidomimetic inhibitors to inhibit Aβ42 aggregation by targeting non-covalent interactions (Table 1). This way, two compounds were able to rescue yeast from Aβ42-induced toxicity. Yeast also served as an excellent tool to shed more light on the mechanism of action of the anti-histamine latrepirdine (Dimebon™) [170], which showed promising aggregate clearing activity in vivo (Table 1). The compound was suggested to upregulate the sequestering of aggregated GFP-Aβ42 into autophagic-like vesicles which get targeted for degradation. Autophagy plays a crucial role in the removal of aggregated or misfolded proteins, such as Aβ, in neurodegenerative diseases [171–173] and impaired clearance of autophagic vesicles is also observed in the brains of AD mice models and patients [171–173]. Highly similar results were obtained in other cell and animal models: Steele and colleagues reported that the treatment of cultured mammalian cells with latrepirdine led to enhanced mTOR- and Atg5-dependent autophagy. Moreover, latrepirdine treatment of TgCRND8 transgenic mice was associated with improved learning behavior and with a reduction in accumulation of Aβ42 [174]. Finally, a yeast-based screen identiﬁed clioquinol and dihydropyrimidine-thiones as compounds being able to ameliorate Aβ toxicity in a synergistic, metal-dependent, way via different mechanisms such as increasing Aβ turnover, restoring vesicle trafﬁcking and oxidative stress protection [175,176] (Table 1). Again, also in transgenic mice models, treatment with clioquinol (analogue) compounds inhibited Aβ accumulation [177] and resulted in a dramatic improvement in learning and memory, accompanied by marked inhibition of AD-like neuropathology [178]. 3.2. From Heterologously Expressed APP to Secretory Pathway-Targeted Aβ Peptides 3.2. From Heterologously Expressed APP to Secretory Pathway-Targeted Aβ Peptides Moreover, the same group found that Nicastrin can be dispensable for protease activity of double-mutated γ-secretase, e.g., F411Y/S438P [162]. This first group of yeast models, in which pre-Aβ components (i.e., APP-like substrates and Secretases) are expressed, offers the possibility to screen for components and drugs that interfere with As mentioned above, the γ-secretase complex consists of 4 different components and the inﬂuence of different combinations of Presenilin and Aph1 proteins on function and substrate speciﬁcity of the γ-secretase was tested in a yeast system [160,161]. Although differences were observed, the results were not well in line with parallel studies performed in mammalian cells, which, they reasoned, could be explained by the lack of additional proteins (e.g., GSAP and CD147) that affect γ-secretase function and that are not present in yeast. Moreover, the same group found that Nicastrin can be dispensable for protease activity of double-mutated γ-secretase, e.g., F411Y/S438P [162]. This ﬁrst group of yeast models, in which pre-Aβ components (i.e., APP-like substrates and Secretases) are Int. J. Mol. Sci. 2018, 19, 1947 9 of 26 expressed, offers the possibility to screen for components and drugs that interfere with Aβ peptide generation and, therefore, have therapeutic potential. The focus, however, has shifted more towards the expression of the actual Aβ peptides in yeast cells. This has one major beneﬁt since it limits the amount of heterologously expressed proteins in yeast and, therefore, also any potential side reactions which result in non-Aβ pathology associated phenotypes. In this context, the prion-forming capability of S. cerevisiae has also been exploited to gain more insight in Aβ’s oligomerization and aggregation capability. Sup35, for example, is a translation termination factor and has the natural propensity to form propagating infectious amyloid aggregates which results in a prion phenotype “[PSI+]” [163]. Bagriantsev and colleague fused the MRF (middle- and release factor domain) of the protein to Aβ42 and screened for the ability of an ade1-14 strain to grown on medium lacking adenine. They showed that fusion of Aβ42 to this MRF domain resulted in a similar phenotype as did the N (N-terminal domain) MRF protein, which makes up the entire amino acid sequence of the Sup35 protein. The N-terminal domain is required and sufﬁcient for induction of the prion properties. Aβ42 induced oligomerization, resulting in the inability of Sup35 to terminate translation, enabled growth of the ade1-14 strain on medium lacking adenine by restoring adenine prototrophy [164]. 3.2. From Heterologously Expressed APP to Secretory Pathway-Targeted Aβ Peptides 3.2. From Heterologously Expressed APP to Secretory Pathway-Targeted Aβ Peptides Finally, a study assessed the clinical effect of clioquinol analogue PBT2 using human patient cohorts. Compared to the placebo group, Aβ CSF concentration was reduced upon treating AD patients with PBT2. In addition, some cognitive test results indicated an improvement in AD patients treated with the clioquinol analogue [179]. These research and clinical studies highlight the fact that yeast-based compound Int. J. Mol. Sci. 2018, 19, 1947 Int. J. Mol. Sci. 2018, 19, 1947 10 of 26 screenings are extremely valuable to identify promising molecules that ameliorate Aβ pathology. Secondly, in several cases, the proposed mechanism of action of a compound, based on insights obtained from yeast research, was conﬁrmed by other, often more comprehensive, pathological AD models [174]. Int. J. Mol. Sci. 2018, 19, x 10 of 26 Aβ peptides are generated at the plasma membrane and can subsequently be secreted and re-uptaken in the cell and eventually be found in the cytosol, mitochondria, secretory pathway and autophagosomes [180]. To recapitulate Aβ’s multi-compartment trafﬁcking, Treusch and colleagues fused a Kar2 sequence to the N-terminus of Aβ42, targeting the peptide to the ER [181] (Figure 3). After cleavage of this sequence, Aβ42 is released in the secretory pathway. The presence of a cell wall prevents diffusing of the peptides in the medium, thereby allowing interaction with the plasma membrane and endocytosis. Cell growth was decreased after expression of Aβ42 using a multicopy plasmid and galactose-inducible promoter and this in contrast to Aβ40. Screening of an overexpression library consisting of >5000 ORFs yielded several suppressors and enhancers of Aβ42 toxicity. Int. J. Mol. Sci. 2018, 19, x 10 of 26 autophagosomes [180]. To recapitulate Aβ’s multi-compartment trafficking, Treusch and colleagues fused a Kar2 sequence to the N-terminus of Aβ42, targeting the peptide to the ER [181] (Figure 3). After cleavage of this sequence, Aβ42 is released in the secretory pathway. The presence of a cell wall prevents diffusing of the peptides in the medium, thereby allowing interaction with the plasma membrane and endocytosis. Cell growth was decreased after expression of Aβ42 using a multicopy plasmid and galactose-inducible promoter and this in contrast to Aβ40. Screening of an overexpression library consisting of >5000 ORFs yielded several suppressors and enhancers of Aβ42 toxicity. Figure 3. Humanized yeast model expressing GFP-fused Aβ peptides tagged with an endoplasmatic reticulum (ER) or Golgi complex targeting sequence. 3.2. From Heterologously Expressed APP to Secretory Pathway-Targeted Aβ Peptides 3.2. From Heterologously Expressed APP to Secretory Pathway-Targeted Aβ Peptides Treusch and colleagues expressed Aβ42 N- terminally tagged with the Kar2 sequence, while D’Angelo and colleagues expressed Aβ42/ARC N- terminally tagged with the α prepro-leader sequence (with and without a C-terminal GFP tag). The scissors icon indicates cleavage of the respective proteins. Figure 3. Humanized yeast model expressing GFP-fused Aβ peptides tagged with an endoplasmatic reticulum (ER) or Golgi complex targeting sequence. Treusch and colleagues expressed Aβ42 N-terminally tagged with the Kar2 sequence, while D’Angelo and colleagues expressed Aβ42/ARC N-terminally tagged with the α prepro-leader sequence (with and without a C-terminal GFP tag). The scissors icon indicates cleavage of the respective proteins. Figure 3. Humanized yeast model expressing GFP-fused Aβ peptides tagged with an endoplasmatic reticulum (ER) or Golgi complex targeting sequence. Treusch and colleagues expressed Aβ42 N- terminally tagged with the Kar2 sequence, while D’Angelo and colleagues expressed Aβ42/ARC N- terminally tagged with the α prepro-leader sequence (with and without a C-terminal GFP tag). The scissors icon indicates cleavage of the respective proteins. Figure 3. Humanized yeast model expressing GFP-fused Aβ peptides tagged with an endoplasmatic reticulum (ER) or Golgi complex targeting sequence. Treusch and colleagues expressed Aβ42 N-terminally tagged with the Kar2 sequence, while D’Angelo and colleagues expressed Aβ42/ARC N-terminally tagged with the α prepro-leader sequence (with and without a C-terminal GFP tag). The scissors icon indicates cleavage of the respective proteins. Figure 3. Humanized yeast model expressing GFP-fused Aβ peptides tagged with an endoplasmatic reticulum (ER) or Golgi complex targeting sequence. Treusch and colleagues expressed Aβ42 N- terminally tagged with the Kar2 sequence, while D’Angelo and colleagues expressed Aβ42/ARC N- terminally tagged with the α prepro-leader sequence (with and without a C-terminal GFP tag). The scissors icon indicates cleavage of the respective proteins. Figure 3. Humanized yeast model expressing GFP-fused Aβ peptides tagged with an endoplasmatic reticulum (ER) or Golgi complex targeting sequence. Treusch and colleagues expressed Aβ42 N-terminally tagged with the Kar2 sequence, while D’Angelo and colleagues expressed Aβ42/ARC N-terminally tagged with the α prepro-leader sequence (with and without a C-terminal GFP tag). The scissors icon indicates cleavage of the respective proteins. PICALM (phosphatidylinositol-binding Clathrin assembly protein), of which Yap1801 and Yap1802 are the yeast homologues [182], was one of the toxicity suppressor hits and is one the most highly validated AD risk factors. The exact role of PICALM in AD is unknown, but it is thought to play a role in APP trafficking [183]. 3.2. From Heterologously Expressed APP to Secretory Pathway-Targeted Aβ Peptides 3.2. From Heterologously Expressed APP to Secretory Pathway-Targeted Aβ Peptides In another article [184], the same yeast model was used to study the effects of native Aβ and in addition to previously shown lower growth rate, a reduced respiratory rate and elevated levels of reactive oxygen species (ROS) were exhibited. These are hallmarks of mitochondrial and ubiquitin-proteasome system dysfunction, which also occur in neurons and peripheral tissues of AD patients [185], and nicely illustrate the applicability of such a yeast model to study the role of Aβ in cell stress and damage. In fact, these results were in accordance with ﬁndings obtained from a yeast model after prolonged exposure to cytosolic Aβ42. Several signs of mitochondrial dysfunction were observed, including increased ROS production, decreased mitochondrial membrane potential and reduced oxygen consumption [184]. A major question that remains is how Aβ peptides actually are taken up in the mitochondria. y p In a follow-up study, using a more systems biology approach, the interplay between ER stress and the unfolded protein response (UPR) were studied upon constitutive expression of Aβ40 and Aβ42 [186]. In comparison to Aβ40 which only induced mild stress, Aβ42 expression resulted in prolonged high stress and an UPR failing to cope with the unfolded protein load resulting in cellular dysfunction, a shorter chronological lifespan and deregulation of lipid metabolism. These results are highly relevant for other diseases as well, especially cancer and diabetes due to the emerging role of the UPR in these diseases. Using a similar strategy by fusing the mating type factor α prepro-leader sequence to Aβ, another group also showed that targeting Aβ in the secretory pathway is essential for toxicity in yeast [182] (Figure 3). The researchers tested both native and C-terminally GFP-tagged Aβ42 and AβARC and detected aggregate formation and a more profound toxic effect in case of the prepro-Aβ-linker-GFP constructs, especially AβARC for which they measured a decrease in respiratory rate. They also suggested that Hsp104 could play a role in mediating this toxicity by favoring the conversion of large aggregates into smaller oligomeric species. Western blot data showed a decreased protein level in the case of native Aβ peptides, which indicated that the GFP moiety might have a stabilizing effect. These results were not in line with the results published by Treusch and colleagues, since in their research native Aβ42 expression resulted in signiﬁcant toxicity when ending up in the secretory pathway. Another difference was the presumed role of PICALM. 3.2. From Heterologously Expressed APP to Secretory Pathway-Targeted Aβ Peptides 3.2. From Heterologously Expressed APP to Secretory Pathway-Targeted Aβ Peptides Since Aβ perturbs endocytotic trafficking, it was suggested that PICALM has a role in restoring this process. These findings were backed up by data obtained from PICALM (phosphatidylinositol-binding Clathrin assembly protein), of which Yap1801 and Yap1802 are the yeast homologues [182], was one of the toxicity suppressor hits and is one the most highly validated AD risk factors. The exact role of PICALM in AD is unknown, but it is thought to play a role in APP trafﬁcking [183]. Since Aβ perturbs endocytotic trafﬁcking, it was suggested that 11 of 26 Int. J. Mol. Sci. 2018, 19, 1947 PICALM has a role in restoring this process. These ﬁndings were backed up by data obtained from rat cortical neurons, in which Aβ-induced cell death was partly prevented upon PICALM expression [181]. PICALM has a role in restoring this process. These ﬁndings were backed up by data obtained from rat cortical neurons, in which Aβ-induced cell death was partly prevented upon PICALM expression [181]. A more recent study also reported on the beneﬁcial role of PICALM, since it was able to reduce Aβ42 oligomerization [183] (Table 1). In another article [184], the same yeast model was used to study the effects of native Aβ and in addition to previously shown lower growth rate, a reduced respiratory rate and elevated levels of reactive oxygen species (ROS) were exhibited. These are hallmarks of mitochondrial and ubiquitin-proteasome system dysfunction, which also occur in neurons and peripheral tissues of AD patients [185], and nicely illustrate the applicability of such a yeast model to study the role of Aβ in cell stress and damage. In fact, these results were in accordance with ﬁndings obtained from a yeast model after prolonged exposure to cytosolic Aβ42. Several signs of mitochondrial dysfunction were observed, including increased ROS production, decreased mitochondrial membrane potential and reduced oxygen consumption [184]. A major question that remains is how Aβ peptides actually are taken up in the mitochondria. PICALM has a role in restoring this process. These ﬁndings were backed up by data obtained from rat cortical neurons, in which Aβ-induced cell death was partly prevented upon PICALM expression [181]. A more recent study also reported on the beneﬁcial role of PICALM, since it was able to reduce Aβ42 oligomerization [183] (Table 1). 3.2. From Heterologously Expressed APP to Secretory Pathway-Targeted Aβ Peptides 3.2. From Heterologously Expressed APP to Secretory Pathway-Targeted Aβ Peptides Protein Toxicity Modiﬁers Description Other Models References Tau Pin1 (yeast homologue Ess1) Depletion of Pin1 isomerase activity results in reduced growth of Tau expressing yeast cells. mouse model [93,196,197] Aβ peptidomimetic inhibitors Inhibition of Aβ42 aggregation by peptidomimetics. - [169] Aβ latrepirdine (Dimebon™) Latrepirdine induces autophagy and decreases the intracellular GFP-Aβ42 levels in yeast. Hela cells, mouse model [170,174] Aβ clioquinol Small molecule screen identiﬁed several 8-hydroxyquinolines, including clioquinol, that ameliorate Aβ toxicity. mouse model, nematode model [175–179] Aβ dihydropyrimidine-thiones Phenotypic small molecule yeast screen identiﬁed dihydropyrimidine-thiones that rescue Aβ-induced toxicity in a metal dependent manner. nematode model [176] Aβ PICALM (yeast homologues Yap1801, Yap1802) Screening of overexpression library yielded suppressors and enhancers of Aβ42 toxicity, including the PICALM suppressor. rat cortical neurons [181–183] 4. Humanized Yeast Models to Study Frameshift Ubiquitin Mutant UBB+1 Biology Table 1. Overview of Tau and Aβ toxicity modiﬁers identiﬁed using yeast-based screens. Protein Toxicity Modiﬁers Description Other Models References Tau Pin1 (yeast homologue Ess1) Depletion of Pin1 isomerase activity results in reduced growth of Tau expressing yeast cells. mouse model [93,196,197] Aβ peptidomimetic inhibitors Inhibition of Aβ42 aggregation by peptidomimetics. - [169] Aβ latrepirdine (Dimebon™) Latrepirdine induces autophagy and decreases the intracellular GFP-Aβ42 levels in yeast. Hela cells, mouse model [170,174] Aβ clioquinol Small molecule screen identiﬁed several 8-hydroxyquinolines, including clioquinol, that ameliorate Aβ toxicity. mouse model, nematode model [175–179] Aβ dihydropyrimidine-thiones Phenotypic small molecule yeast screen identiﬁed dihydropyrimidine-thiones that rescue Aβ-induced toxicity in a metal dependent manner. nematode model [176] Aβ PICALM (yeast homologues Yap1801, Yap1802) Screening of overexpression library yielded suppressors and enhancers of Aβ42 toxicity, including the PICALM suppressor. rat cortical neurons [181–183] 4. Humanized Yeast Models to Study Frameshift Ubiquitin Mutant UBB+1 Biology Table 1. Overview of Tau and Aβ toxicity modiﬁers identiﬁed using yeast-based screens. 4. Humanized Yeast Models to Study Frameshift Ubiquitin Mutant UBB+1 Biology Yeast models expressing the frameshift Ubiquitin mutant UBB+1 have also been developed. UBB+1 accumulation is found in neurons of all AD patients, but absent in those of Parkinson’s disease patients, and co-localizes with the MC1 marker, i.e., NFT [198]. How UBB+1 is related to aberrant and phosphorylated Tau protein, both spatially and temporally, still needs to be elucidated [198]. The authors suggested that these mutant proteins may be responsible for the lack of multi-ubiquitination of the hyperphosphorylated Tau fraction found in the NFT [199]. 3.2. From Heterologously Expressed APP to Secretory Pathway-Targeted Aβ Peptides 3.2. From Heterologously Expressed APP to Secretory Pathway-Targeted Aβ Peptides These UBB+1 molecules are unable to bind to lysine residues in target molecules, since they lack the COOH-terminal glycine residue in the ﬁrst repeat region, which is essential for subsequent multi-ubiquitination and activation of the proteasomal machinery [200]. Upon expression of UBB+1 in yeast, the protein becomes a substrate of the UPR and accumulated UBB+1 impairs the UPR both in yeast and mammalian cells [201–204]. This results in an accumulation of polyubiquitinated substrates which do not get degraded, partially accomplished by the inhibition of deubiquitinating enzymes [205]. Despite this impairment, no toxicity is observed. By contrast, upon prolonged expression of high levels of UBB+1, cell death and mitochondrial dysfunction were observed in neuronal cells and yeast models [203,206,207]. Interestingly to keep in mind here is the fact that UBB+1 can be a toxic protein by itself, but it could also act as a potent modiﬁer of toxicity of other neurotoxic proteins, such as Tau and Aβ. Therefore, yeast models combining expression of these proteins in combination with UBB+1 could unravel molecular mechanisms important in AD, such as UPS dysfunction and mitochondrial activity [208]. 3.2. From Heterologously Expressed APP to Secretory Pathway-Targeted Aβ Peptides 3.2. From Heterologously Expressed APP to Secretory Pathway-Targeted Aβ Peptides In the paper published by D’Angelo and colleagues, deletion of Yap1801 and Yap1802 resulted in a decrease in Aβ-induced toxicity. Upon expression of PICALM, this toxic effect was partly restored. Therefore, more research is necessary to shed light on the actual role of PICALM in Aβ-induced toxicity. Since it is clear that AD, mitochondrial dysfunction and altered proteostasis are linked to one another, two studies also more closely investigated the interplay between the Pitrilysin Metallopeptidase 1 (PITRM1), an oligopeptide-digesting mitochondrial matrix enzyme, and Aβ. In addition to its role in cleaving the mitochondrial targeting sequence (MTS) of proteins imported across the inner mitochondrial membrane, it also disposes mitochondrial Aβ [187–190]. However, in a ﬁrst study, it was shown that the accumulation of Aβ peptides inhibits the activity of Cym1, which is the yeast PITRM1 orthologue, leading to impaired MTS processing and accumulation of precursor proteins [191]. In a second study, the effect of a missense mutation in this enzyme was documented using yeast by modelling the R183Q mutation in the Cym1 protein [192]. This resulted in a reduced Aβ42 degradation compared to wt Cym1, suggesting a pathogenic role of this mutated protein, displaying similar behavior as in human beings. Cenini and colleagues reported that Aβ peptides, especially Aβ42, inhibited mitochondrial protein import by affecting an early process step when newly synthesized mitochondrial polypeptides are exposed to the cytosolic environment, rather than affecting mitochondrial membrane potential, TOM and TIM (Translocase of the outer and inner membrane, respectively) or respiratory chain metabolic protein complex composition [193]. These ﬁndings are in contrast to the study described earlier in this Int. J. Mol. Sci. 2018, 19, 1947 12 of 26 paragraph [191], in which Aβ peptides indirectly interfered with the processing of imported precursor proteins to the mature and active forms, which is a late step of the mitochondrial import reaction. Finally, a C. glabrata model was used to assess toxicity of extracellular chemically-synthesized Aβ [194] by determining the viable colony count, using a water-based assay. It was shown that Aβ did bind the plasma membrane of C. glabrata, but the exact mechanism by which Aβ kills C. glabrata remains to be determined. Interestingly, upon oligomerization Aβ loses its toxic effect, while Aβ has a protective function against sodium hydroxide toxicity [195]. Table 1. Overview of Tau and Aβ toxicity modiﬁers identiﬁed using yeast-based screens. 5. Studying Prion Characteristics of Aβ and Tau in Yeast Prions are self-propagating infection protein species. They were ﬁrst discovered as causative agents in mammalian diseases like Creutzfeldt-Jakob or Scrapie [209,210]. There, a normal protein (PrPC) conformationally changes into a malicious and infectious PrPSc prion protein [211]. Besides these disease-causing prions, a plethora of prions with mostly unknown function has been discovered also in yeast [212,213]. Except for Podospora anserina’s [Het-s] prion, most functions of all these prions are unclear and all are toxic, or at least growth-inhibitory, but still are supposed to be beneﬁcial for the Int. J. Mol. Sci. 2018, 19, 1947 13 of 26 survival of cells under stress conditions [214–216]. To ensure optimal survival but limit malicious effects, prion formation has to be tightly controlled and carefully balanced. Several factors promote or inhibit prion formation like Hsp104, Hsp70, Sse1, Cur1, Btn2 [217–221]. Interestingly, some of these factors, like Hsp104, promote and inhibit prion formation depending on co-factors and expression levels [222,223]. [ ] Many human diseases are described or at least suspected to be prion diseases such as type 2 diabetes mellitus, AD or Huntington’s disease [209,224,225]. Most prions are amyloid and, as its name indicates, Aβ for example stacks β-sheets to form toxic amyloid oligomers which were shown to be transmittable and infectious in mice [226–228]. If Aβ is a prion or not is still to be discussed but more and more hints are pointing towards it [227,229]. It is almost accepted that the second key-player in AD, Tau, might be a prion as well. Several studies point towards the MT binding domain of Tau being responsible for aggregation and prionization [230,231]. Several of the yeast models to study AD discussed above [181,182,191,232] are not only used to study the impact of Aβ or Tau on biochemical pathways and on organelles, but also the prion characteristics of these proteins are the focus of research. Evidence for Aβ and Tau being prions were found in mice or other higher eukaryotic model organisms but not in yeast. Tau is hyperphosphorylated and forms aggregates but it is hardly toxic in yeast models [95]. Also, transmission of neither aggregated Tau nor Aβ from affected yeast to healthy strains has been shown so far. But still, there are excellent and robust yeast in vivo techniques to study prion domains and push this ﬁeld towards greater success. 5. Studying Prion Characteristics of Aβ and Tau in Yeast Brachmann and colleagues extended a model developed by Schlumperger and colleagues which makes use of the Ure2 prion system in yeast [233,234]. By replacing promoters of reporter genes by the Ure2 suppressed DAL5 promotor (pDAL5) it is possible to track Ure2-prion strength. If Ure2 is in a non-prion state it binds Gln3, the transcription factor activating pDAL5. When Ure2 forms its prion, [URE3], it releases Gln3 and, thus, induces the reporter gene expression through pDAL5. By replacing Ure2’s own prion domain by any protein domain, one can easily test if it is a prion domain. By making use of different reporter genes it is not only possible to check for a domain to be a prion domain in a black-white manner, like with the URA reporter, but it is also possible to measure the strength of a prion domain by using ADE2 as a reporter. The “redness” of the reporter strain indicates the strength of prion formation and thus the release of Gln3 from unprionized Ure2. Another technique based on a similar principle is the recently developed yTRAP [235]. Here, suspected prions are fused to a synthetic transcription factor, the synTA. When the protein is soluble and thus not prionized, it allows the synTA to bind the promotor and induce expression of a ﬂuorescent protein, in this case mNeonGreen or mKate2. When aggregated, the transcription factor cannot reach its promotor and the expression of the reporter gene is suppressed. An overview of several traditional and more innovative yeast techniques that play(ed) a crucial role in unravelling Tau and Aβ functions such as protein–protein interaction and prion formation can be found in Table 2. Table 2. Summary of yeast-based techniques applicable in studies on proteins involved in neurodegenerative diseases. Technique Used for Description Split-GFP system [111–115,236] Protein–protein interaction GFP ﬂuorescence is reconstituted when its two subunits are in close proximity. Synthetic genetic array [237] Synthetic lethality Approach for the systematic construction of double mutants for large-scale mapping of synthetic genetic interactions. Yeast two-hybrid [238] Protein–protein interaction Protein interaction leads to reporter gene expression. Prion-forming assay [233] Prion forming The prion domain of the yeast Ure2 prion is replaced by a potential prion domain of any protein. Reporter gene expression is induced if this domain can complement for the Ure2 prion domain. Yeast transcriptional reporting aggregating proteins (yTRAP) [235] Prion forming High-throughput quantitative prion forming assay. Uses ﬂuorescence as quantiﬁable reporter. 5. Studying Prion Characteristics of Aβ and Tau in Yeast Table 2. Summary of yeast-based techniques applicable in studies on proteins involved in neurodegenerative diseases. Int. J. Mol. Sci. 2018, 19, 1947 14 of 26 14 of 26 References 1. Prince, M.; Guerchet, M.; Prina, M. The Epidemiology and Impact of Dementia: Current State and Future Trends. Available online: http://www.who.int/mental_health/neurology/en/ (accessed on 26 March 2015). 1. Prince, M.; Guerchet, M.; Prina, M. The Epidemiology and Impact of Dementia: Current State and Future Trends. Available online: http://www.who.int/mental_health/neurology/en/ (accessed on 26 March 2015). 2. Prince, M.; Bryce, R.; Albanese, E.; Wimo, A.; Ribeiro, W.; Ferri, C.P. The global prevalence of dementia: A systematic review and metaanalysis. Alzheimer’s Dement. 2013, 9, 63–75. [CrossRef] [PubMed] 2. Prince, M.; Bryce, R.; Albanese, E.; Wimo, A.; Ribeiro, W.; Ferri, C.P. The global prevalence of dementia: A systematic review and metaanalysis. Alzheimer’s Dement. 2013, 9, 63–75. [CrossRef] [PubMed] 4. Norfray, J.F.; Provenzale, J.M. Alzheimer’s Disease: Neuropathologic Findings and Recent Advances in Imaging. Am. J. Roentgenol. 2004, 182, 3–13. [CrossRef] [PubMed] 4. Norfray, J.F.; Provenzale, J.M. Alzheimer’s Disease: Neuropathologic Findings and Recent Advances in Imaging. Am. J. Roentgenol. 2004, 182, 3–13. [CrossRef] [PubMed] 5. Marsh, J.; Alifragis, P. Synaptic dysfunction in Alzheimer’s disease: The effects of amyloid beta on synaptic vesicle dynamics as a novel target for therapeutic intervention. Neural Regen. Res. 2018, 13, 616–623. [CrossRef] [PubMed] 5. Marsh, J.; Alifragis, P. Synaptic dysfunction in Alzheimer’s disease: The effects of amyloid beta on synaptic vesicle dynamics as a novel target for therapeutic intervention. Neural Regen. Res. 2018, 13, 616–623. [CrossRef] [PubMed] 6. Selkoe, D.J. Alzheimer’s Disease Is a Synaptic Failure. Science 2002, 298, 789–791. [CrossRef] [PubM 6. Selkoe, D.J. Alzheimer s Disease Is a Synaptic Failure. Science 2002, 298, 789 791. [CrossRef] [PubMed] 7. Backman, L.; Jones, S.; Berger, A.-K.; Laukka, E.J.; Small, B.J. Multiple cognitive deﬁcits during the transition to Alzheimer’s disease. J. Intern. Med. 2004, 256, 195–204. [CrossRef] [PubMed] 8. Lang, A.E. Clinical trials of disease-modifying therapies for neurodegenerative diseases: The challenges and the future. Nat. Med. 2010, 16, 1223–1226. [CrossRef] [PubMed] 9. Irie, K.; Murakami, K.; Masuda, Y.; Morimoto, A.; Ohigashi, H.; Ohashi, R.; Takegoshi, K.; Nagao, M.; Shimizu, T.; Shirasawa, T. Structure of β-amyloid ﬁbrils and its relevance to their neurotoxicity: Implications for the pathogenesis of Alzheimer’s disease. J. Biosci. Bioeng. 2005, 99, 437–447. [CrossRef] [PubMed] 10. Ittner, L.M.; Götz, J. Amyloid-β and tau—A toxic pas de deux in Alzheimer’s disease. Nat. Rev. Neurosci. 2011, 12, 67–72. [CrossRef] [PubMed] 11. Goffeau, A.; Barrell, B.G.; Bussey, H.; Davis, R.W.; Dujon, B.; Feldmann, H.; Galibert, F.; Hoheisel, J.D.; Jacq, C.; Johnston, M.; et al. 6. Conclusions It is clear from the above discussed articles that yeast has proven its value in modern AD research. Although S. cerevisiae models are most prevalent, it is inspiring to see that alternative models such as S. pombe or C. glabrata are gaining popularity. The pathobiology of proteins such as Tau and Aβ is robustly recapitulated in yeast. Research using these models has shed more light on the oligomerization/aggregation and prion properties of these proteins, including their role in mitochondrial dysfunction and altered proteostasis, which are two important pathological AD-related cellular processes. This, in combination with the intrinsic beneﬁts of using yeast such as speed and lower costs of research, puts these humanized yeast models in a unique position as a complementary model organism. Therefore, yeast may play a crucial role in overcoming the major future challenges in AD research, including identifying the relationship between all these different pathological AD-related processes. Author Contributions: D.S. wrote the majority of this manuscript. Other listed authors provided ﬁgures/tables, wrote speciﬁc sections and/or reviewed the content. Funding: David Seynnaeve is supported by an Aspirant fellowship (1101317N) of Research Foundation-Flanders (FWO) and Vanessa Franssens by a post-doctoral fellowship (1287517N) of FWO. Joke Verelst is supported by an FWO SBO Fellowship (1S80418N). Mara Del Vecchio, Gernot Fruhmann and Melody Cools are ﬁnanced through FWO SBO (S006617N) and KU Leuven funding granted to Joris Winderickx. Work in Daniel P. Mulvihill’s lab is supported by the University of Kent and by funding from the Biotechnology and Biological Sciences Research Council Acknowledgments: Human head and scissors icon made by Freepik from www.ﬂaticon.com. Conﬂicts of Interest: The authors declare no conﬂicts of interest. References Light and electron microscope localization of the microtubule-associated tau protein in rat brain. J. Neurosci. 1988, 8, 1846–1851. [CrossRef] [PubMed] 18. Papasozomenos, S.C.; Binder, L.I. Phosphorylation determines two distinct species of tau in the central nervous system. Cell Motil. Cytoskelet. 1987, 8, 210–226. [CrossRef] [PubMed] microtubule associated tau protein in rat brain. J. Neurosci. 1988, 8, 1846 1851. [CrossRef] [PubMed] 18. Papasozomenos, S.C.; Binder, L.I. Phosphorylation determines two distinct species of tau in the central nervous system. Cell Motil. Cytoskelet. 1987, 8, 210–226. [CrossRef] [PubMed] 19. Binder, L.I.; Frankfurter, A.; Rebhun, L.I. The distribution of tau in the mammalian central nervous system. J. Cell Biol. 1985, 101, 1371–1378. [CrossRef] [PubMed] 20. Wilhelmsen, K.C.; Lynch, T.; Pavlou, E.; Higgins, M.; Nygaard, T.G. Localization of disinhibition-dementia-parkinsonism-amyotrophy complex to 17q21-22. Am. J. Hum. Genet. 1994, 55, 1159–1165. [PubMed] 1. Pooler, A.M.; Phillips, E.C.; Lau, D.H.W.; Noble, W.; Hanger, D.P. Physiological release of endogenous ta stimulated by neuronal activity. EMBO Rep. 2013, 14, 389–394. [CrossRef] [PubMed] 22. Yamada, K. Extracellular Tau and Its Potential Role in the Propagation of Tau Pathology. Front. Neurosci. 2017, 11. [CrossRef] [PubMed] 23. Ittner, L.M.; Ke, Y.D.; Delerue, F.; Bi, M.; Gladbach, A.; van Eersel, J.; Wölﬁng, H.; Chieng, B.C.; Christie, M.J.; Napier, I.A.; et al. Dendritic Function of Tau Mediates Amyloid-β Toxicity in Alzheimer’s Disease Mouse Models. Cell 2010, 142, 387–397. [CrossRef] [PubMed] 24. Drechsel, D.N.; Hyman, A.A.; Cobb, M.H.; Kirschner, M.W. Modulation of the dynamic instability of tubulin assembly by the microtubule-associated protein tau. Mol. Biol. Cell 1992, 3, 1141–1154. [CrossRef] [PubMed] 24. Drechsel, D.N.; Hyman, A.A.; Cobb, M.H.; Kirschner, M.W. Modulation of the dynamic instability of tubulin assembly by the microtubule-associated protein tau. Mol. Biol. Cell 1992, 3, 1141–1154. [CrossRef] [PubMed] 25. Crowther, R.A. Straight and paired helical ﬁlaments in Alzheimer disease have a common structural unit. l d [ f] [ b d] assembly by the microtubule associated protein tau. Mol. Biol. Cell 1992, 3, 1141 1154. [CrossRef] [PubMed] 25. Crowther, R.A. Straight and paired helical ﬁlaments in Alzheimer disease have a common structural unit. Proc. Natl. Acad. Sci. USA 1991, 88, 2288–2292. [CrossRef] [PubMed] 26. Wischik, C.M.; Crowther, R.A.; Stewart, M.; Roth, M. Subunit structure of paired helical ﬁlaments in 25. Crowther, R.A. Straight and paired helical ﬁlaments in Alzheimer disease have a common structural unit. Proc. Natl. Acad. Sci. USA 1991, 88, 2288–2292. [CrossRef] [PubMed] 26. References Life with 6000 genes. Science 1996, 274, 563–567. [CrossRef] 12. Botstein, D.; Chervitz, S.A.; Cherry, J.M. Yeast as a model organism. Science 1997, 277, 1259–1260. [CrossRef] [PubMed] 13. Petranovic, D.; Tyo, K.; Vemuri, G.N.; Nielsen, J. Prospects of yeast systems biology for human health: Integrating lipid, protein and energy metabolism. FEMS Yeast Res. 2010, 10, 1046–1059. [CrossRef] [PubMed] Int. J. Mol. Sci. 2018, 19, 1947 15 of 26 15 of 26 14. Winderickx, J.; Delay, C.; De Vos, A.; Klinger, H.; Pellens, K.; Vanhelmont, T.; Van Leuven, F.; Zabrocki, P. Protein folding diseases and neurodegeneration: Lessons learned from yeast. Biochim. Biophys. Acta Mol. Cell Res. 2008, 1783, 1381–1395. [CrossRef] [PubMed] 14. Winderickx, J.; Delay, C.; De Vos, A.; Klinger, H.; Pellens, K.; Vanhelmont, T.; Van Leuven, F.; Zabrocki, P. Protein folding diseases and neurodegeneration: Lessons learned from yeast. Biochim. Biophys. Acta Mol. Cell Res. 2008, 1783, 1381–1395. [CrossRef] [PubMed] 5. Tenreiro, S.; Munder, M.C.; Alberti, S.; Outeiro, T.F. Harnessing the power of yeast to unravel the molec basis of neurodegeneration. J. Neurochem. 2013, 127, 438–452. [CrossRef] [PubMed] 16. Mohammadi, S.; Saberidokht, B.; Subramaniam, S.; Grama, A. Scope and limitations of yeast as a model organism for studying human tissue-speciﬁc pathways. BMC Syst. Biol. 2015, 9, 96. [CrossRef] [PubMed] 16. Mohammadi, S.; Saberidokht, B.; Subramaniam, S.; Grama, A. Scope and limitations of yeast as a model organism for studying human tissue-speciﬁc pathways. BMC Syst. Biol. 2015, 9, 96. [CrossRef] [PubMed] 17. Migheli, A.; Butler, M.; Brown, K.; Shelanski, M. Light and electron microscope localization of the microtubule-associated tau protein in rat brain. J. Neurosci. 1988, 8, 1846–1851. [CrossRef] [PubMed] 18. Papasozomenos, S.C.; Binder, L.I. Phosphorylation determines two distinct species of tau in the central nervous system Cell Motil Cytoskelet 1987 8 210 226 [CrossRef] [PubMed] g y g p p y y , , [ ] [ ] 17. Migheli, A.; Butler, M.; Brown, K.; Shelanski, M. Light and electron microscope localization of the microtubule-associated tau protein in rat brain. J. Neurosci. 1988, 8, 1846–1851. [CrossRef] [PubMed] 18. Papasozomenos, S.C.; Binder, L.I. Phosphorylation determines two distinct species of tau in the central 17. Migheli, A.; Butler, M.; Brown, K.; Shelanski, M. Light and electron microscope localization of the microtubule-associated tau protein in rat brain. J. Neurosci. 1988, 8, 1846–1851. [CrossRef] [PubMed] 17. Migheli, A.; Butler, M.; Brown, K.; Shelanski, M. References Wischik, C.M.; Crowther, R.A.; Stewart, M.; Roth, M. Subunit structure of paired helical ﬁlam Alzheimer’s disease. J. Cell Biol. 1985, 100, 1905–1912. [CrossRef] [PubMed] 27. Ichihara, K.; Kitazawa, H.; Iguchi, Y.; Hotani, H.; Itoh, T.J. Visualization of the stop of microtubule depolymerization that occurs at the high-density region of microtubule-associated protein 2 (MAP2). J. Mol. Biol. 2001, 312, 107–118. [CrossRef] [PubMed] 28. Violet, M.; Delattre, L.; Tardivel, M.; Sultan, A.; Chauderlier, A.; Caillierez, R.; Talahari, S.; Nesslany, F.; Lefebvre, B.; Bonnefoy, E.; et al. A major role for Tau in neuronal DNA and RNA protection in vivo under physiological and hyperthermic conditions. Front. Cell. Neurosci. 2014, 8. [CrossRef] [PubMed] 29. Frandemiche, M.L.; De Seranno, S.; Rush, T.; Borel, E.; Elie, A.; Arnal, I.; Lante, F.; Buisson, A. Activity-Dependent Tau Protein Translocation to Excitatory Synapse Is Disrupted by Exposure to Amyloid-Beta Oligomers. J. Neurosci. 2014, 34, 6084–6097. [CrossRef] [PubMed] 30. Stamer, K.; Vogel, R.; Thies, E.; Mandelkow, E.; Mandelkow, E.-M. Tau blocks trafﬁc of organelles, neuroﬁlaments, and APP vesicles in neurons and enhances oxidative stress. J. Cell Biol. 2002, 156, 1051–1063. [CrossRef] [PubMed] 31. Dixit, R.; Ross, J.L.; Goldman, Y.E.; Holzbaur, E.L.F. Differential Regulation of Dynein and Kinesin Motor Proteins by Tau. Science 2008, 319, 1086–1089. [CrossRef] [PubMed] 32. Konzack, S.; Thies, E.; Marx, A.; Mandelkow, E.-M.; Mandelkow, E. Swimming against the Tide: Mobility of the Microtubule-Associated Protein Tau in Neurons. J. Neurosci. 2007, 27, 9916–9927. [CrossRef] [PubMed] 32. Konzack, S.; Thies, E.; Marx, A.; Mandelkow, E.-M.; Mandelkow, E. Swimming against the Tide: Mobility of the Microtubule-Associated Protein Tau in Neurons. J. Neurosci. 2007, 27, 9916–9927. [CrossRef] [PubMed] 33. Vershinin, M.; Carter, B.C.; Razafsky, D.S.; King, S.J.; Gross, S.P. Multiple-motor based transport and its 33. Vershinin, M.; Carter, B.C.; Razafsky, D.S.; King, S.J.; Gross, S.P. Multiple-motor based transport and its regulation by Tau. Proc. Natl. Acad. Sci. USA 2007, 104, 87–92. [CrossRef] [PubMed] 34. Utton, M.A.; Noble, W.J.; Hill, J.E.; Anderton, B.H.; Hanger, D.P. Molecular motors implicated in the axonal transport of tau and alpha-synuclein. J. Cell Sci. 2005, 118, 4645–4654. [CrossRef] [PubMed] 35. Kanaan, N.M.; Morﬁni, G.A.; LaPointe, N.E.; Pigino, G.F.; Patterson, K.R.; Song, Y.; Andreadis, A.; Fu, Y.; Brady, S.T.; Binder, L.I. Pathogenic Forms of Tau Inhibit Kinesin-Dependent Axonal Transport through a Mechanism Involving Activation of Axonal Phosphotransferases. J. Neurosci. 2011, 31, 9858–9868. [CrossRef] [PubMed] 16 of 26 Int. J. Mol. Sci. 2018, 19, 1947 16 of 26 36. References Magnani, E.; Fan, J.; Gasparini, L.; Golding, M.; Williams, M.; Schiavo, G.; Goedert, M.; Amos, L.A.; Spillantini, M.G. Interaction of tau protein with the dynactin complex. EMBO J. 2007, 26, 4546–4554. [CrossRef] [PubMed] 7. Caceres, A.; Kosik, K.S. Inhibition of neurite polarity by tau antisense oligonucleotides in primary cereb neurons. Nature 1990, 343, 461–463. [CrossRef] [PubMed] 38. Wang, Y.; Mandelkow, E. Tau in physiology and pathology. Nat. Rev. Neurosci. 2016, 17, 22–35. [CrossRef] [PubMed] 39. Goedert, M.; Spillantini, M.G.; Potier, M.C.; Ulrich, J.; Crowther, R.A. Cloning and sequencing of the cDNA encoding an isoform of microtubule-associated protein tau containing four tandem repeats: Differential expression of tau protein mRNAs in human brain. EMBO J. 1989, 8, 393–399. [PubMed] 40. Himmler, A.; Drechsel, D.; Kirschner, M.W.; Martin, D.W. Tau consists of a set of proteins with repeated C-terminal microtubule-binding domains and variable N-terminal domains. Mol. Cell. Biol. 1989, 9, 1381–1388. [CrossRef] [PubMed] 41. Rosenberg, K.J.; Ross, J.L.; Feinstein, H.E.; Feinstein, S.C.; Israelachvili, J. Complementary dimerization of microtubule-associated tau protein: Implications for microtubule bundling and tau-mediated pathogenesis. Proc. Natl. Acad. Sci. USA 2008, 105, 7445–7450. [CrossRef] [PubMed] 42. Bunker, J.M.; Wilson, L.; Jordan, M.A.; Feinstein, S.C. Modulation of microtubule dynamics by tau in living cells: Implications for development and neurodegeneration. Mol. Biol. Cell 2004, 15, 2720–2728. [CrossRef] [PubMed] 43. Lu, M.; Kosik, K.S. Competition for microtubule-binding with dual expression of tau missense and splice isoforms. Mol. Biol. Cell 2001, 12, 171–184. [CrossRef] [PubMed] 44. Derisbourg, M.; Leghay, C.; Chiappetta, G.; Fernandez-Gomez, F.-J.; Laurent, C.; Demeyer, D.; Carrier, S.; Buée-Scherrer, V.; Blum, D.; Vinh, J.; et al. Role of the Tau N-terminal region in microtubule stabilization revealed by new endogenous truncated forms. Sci. Rep. 2015, 5, 9659. [CrossRef] [PubMed] 45. Chen, J.; Kanai, Y.; Cowan, N.J.; Hirokawa, N. Projection domains of MAP2 and tau determine spacings between microtubules in dendrites and axons. Nature 1992, 360, 674–677. [CrossRef] [PubMed] 46. Sattilaro, R.F.; Dentler, W.L.; LeCluyse, E.L. Microtubule-associated proteins (MAPs) and the organization of actin ﬁlaments in vitro. J. Cell Biol. 1981, 90, 467–473. [CrossRef] [PubMed] 47. Correas, I.; Padilla, R.; Avila, J. The tubulin-binding sequence of brain microtubule-associated proteins, tau and MAP-2, is also involved in actin binding. Biochem. J. 1990, 269, 61–64. [CrossRef] [PubMed] 48. Rendon, A.; Jung, D.; Jancsik, V. Interaction of microtubules and microtubule-associated proteins (MAPs) with rat brain mitochondria. Biochem. J. 1990, 269, 555–556. [CrossRef] [PubMed] 49. References Cell 1997, 8, 353–365. [CrossRef] [PubMed] 61. Sillen, A.; Barbier, P.; Landrieu, I.; Lefebvre, S.; Wieruszeski, J.-M.; Leroy, A.; Peyrot, V.; Lippens, G. NMR Investigation of the Interaction between the Neuronal Protein Tau and the Microtubules. Biochemistry 2007. [CrossRef] [PubMed] 2. Hanger, D.P.; Seereeram, A.; Noble, W. Mediators of tau phosphorylation in the pathogenesis of Alzheim disease. Expert Rev. Ther. 2009, 9, 1647–1666. [CrossRef] [PubMed] 63. Sergeant, N.; Bretteville, A.; Hamdane, M.; Caillet-Boudin, M.-L.; Grognet, P.; Bombois, S.; Blum, D.; Delacourte, A.; Pasquier, F.; Vanmechelen, E.; et al. Biochemistry of Tau in Alzheimer’s disease and related neurological disorders. Expert Rev. Proteom. 2008, 5, 207–224. [CrossRef] [PubMed] 64. Domise, M.; Didier, S.; Marinangeli, C.; Zhao, H.; Chandakkar, P.; Buée, L.; Viollet, B.; Davies, P.; Marambaud, P.; Vingtdeux, V. AMP-activated protein kinase modulates tau phosphorylation and tau pathology in vivo. Sci. Rep. 2016, 6, 26758. [CrossRef] [PubMed] 65. Cavallini, A.; Brewerton, S.; Bell, A.; Sargent, S.; Glover, S.; Hardy, C.; Moore, R.; Calley, J.; Ramachandran, D.; Poidinger, M.; et al. An unbiased approach to identifying tau kinases that phosphorylate tau at sites associated with alzheimer disease. J. Biol. Chem. 2013, 288, 23331–23347. [CrossRef] [PubMed] 66. Trinczek, B.; Biernat, J.; Baumann, K.; Mandelkow, E.M.; Mandelkow, E. Domains of tau protein, differential phosphorylation, and dynamic instability of microtubules. Mol. Biol. Cell 1995, 6, 1887–1902. [CrossRef] [PubMed] 67. Hanger, D.P.; Anderton, B.H.; Noble, W. Tau phosphorylation: The therapeutic challenge for neurodegenerative disease. Trends Mol. Med. 2009, 15, 112–119. [CrossRef] [PubMed] 68. Lu, P.-J.; Wulf, G.; Zhou, X.Z.; Davies, P.; Lu, K.P. The prolyl isomerase Pin1 restores the function of Alzheimer-associated phosphorylated tau protein. Nature 1999, 399, 784–788. [CrossRef] [PubMed] 69. von Bergen, M.; Friedhoff, P.; Biernat, J.; Heberle, J.; Mandelkow, E.M.; Mandelkow, E. Assembly of tau protein into Alzheimer paired helical ﬁlaments depends on a local sequence motif ((306)VQIVYK(311)) forming beta structure. Proc. Natl. Acad. Sci. USA 2000, 97, 5129–5134. [CrossRef] [PubMed] 70. Dickey, C.A.; Kamal, A.; Lundgren, K.; Klosak, N.; Bailey, R.M.; Dunmore, J.; Ash, P.; Shoraka, S.; Zlatkovic, J.; Eckman, C.B.; et al. The high-afﬁnity HSP90-CHIP complex recognizes and selectively degrades phosphorylated tau client proteins. J. Clin. Investig. 2007, 117, 648–658. [CrossRef] [PubMed] 71. Šimi´c, G.; Babi´c Leko, M.; Wray, S.; Harrington, C.; Delalle, I.; Jovanov-Miloševi´c, N.; Bažadona, D.; Buée, L.; de Silva, R.; Giovanni, G.D.; et al. Tau protein hyperphosphorylation and aggregation in alzheimer’s disease and other tauopathies, and possible neuroprotective strategies. References Brandt, R.; Léger, J.; Lee, G. Interaction of tau with the neural plasma membrane mediated by tau’s amino-terminal projection domain. J. Cell Biol. 1995, 131, 1327–1340. [CrossRef] [PubMed] 50. Zmuda, J.F.; Rivas, R.J. Actin disruption alters the localization of tau in the growth cones of cerebellar granule neurons. J. Cell Sci. 2000, 113, 2797–2809. [PubMed] 51. Manczak, M.; Reddy, P.H. Abnormal interaction between the mitochondrial ﬁssion protein Drp1 and hyperphosphorylated tau in Alzheimer’s disease neurons: Implications for mitochondrial dysfunction and neuronal damage. Hum. Mol. Genet. 2012, 21, 2538–2547. [CrossRef] [PubMed] 52. Bhaskar, K.; Hobbs, G.A.; Yen, S.-H.; Lee, G. Tyrosine phosphorylation of tau accompanies disease progression in transgenic mouse models of tauopathy. Neuropathol. Appl. Neurobiol. 2010, 36, 462–477. [CrossRef] [PubMed] 53. Lee, G.; Newman, S.T.; Gard, D.L.; Band, H.; Panchamoorthy, G. Tau interacts with src-family non-receptor tyrosine kinases. J. Cell Sci. 1998, 111, 3167–3177. [PubMed] 54. Lee, G.; Thangavel, R.; Sharma, V.M.; Litersky, J.M.; Bhaskar, K.; Fang, S.M.; Do, L.H.; Andreadis, A.; Van Hoesen, G.; Ksiezak-Reding, H. Phosphorylation of Tau by Fyn: Implications for Alzheimer’s Disease. J. Neurosci. 2004, 24, 2304–2312. [CrossRef] [PubMed] 55. Dujardin, S.; Bégard, S.; Caillierez, R.; Lachaud, C.; Delattre, L.; Carrier, S.; Loyens, A.; Galas, M.-C.; Bousset, L.; Melki, R.; et al. Ectosomes: A New Mechanism for Non-Exosomal Secretion of Tau Protein. PLoS ONE 2014, 9, e100760. [CrossRef] [PubMed] 56. Calafate, S.; Buist, A.; Miskiewicz, K.; Vijayan, V.; Daneels, G.; de Strooper, B.; de Wit, J.; Verstreken, P.; Moechars, D. Synaptic Contacts Enhance Cell-to-Cell Tau Pathology Propagation. Cell Rep. 2015, 11, 1176–1183. [CrossRef] [PubMed] Int. J. Mol. Sci. 2018, 19, 1947 17 of 26 17 of 26 57. Tardivel, M.; Bégard, S.; Bousset, L.; Dujardin, S.; Coens, A.; Melki, R.; Buée, L.; Colin, M. Tunneling nanotube (TNT)-mediated neuron-to neuron transfer of pathological Tau protein assemblies. Acta Neuropathol. Commun. 2016, 4, 117. [CrossRef] [PubMed] 58. Wang, Y.; Balaji, V.; Kaniyappan, S.; Krüger, L.; Irsen, S.; Tepper, K.; Chandupatla, R.; Maetzler, W.; Schneider, A.; Mandelkow, E.; Mandelkow, E.-M. The release and trans-synaptic transmission of Tau via exosomes. Mol. Neurodegener. 2017, 12, 5. [CrossRef] [PubMed] 59. Guo, J.L.; Lee, V.M.Y. Cell-to-cell transmission of pathogenic proteins in neurodegenerative diseases. Nat. Med. 2014, 20, 130–138. [CrossRef] [PubMed] 60. Goode, B.L.; Denis, P.E.; Panda, D.; Radeke, M.J.; Miller, H.P.; Wilson, L.; Feinstein, S.C. Functional interactions between the proline-rich and repeat regions of tau enhance microtubule binding and assembly. Mol. Biol. 75. Maeda, S.; Sahara, N.; Saito, Y.; Murayama, M.; Yoshiike, Y.; Kim, H.; Miyasaka, T.; Murayama, S.; Ikai, A.; Takashima, A. Granular Tau Oligomers as Intermediates of Tau Filaments. Biochemistry 2007, 46, 3856–3861. [CrossRef] [PubMed] References Biomolecules 2016, 6, 2–28. [CrossRef] [PubMed] 72. Berger, Z.; Roder, H.; Hanna, A.; Carlson, A.; Rangachari, V.; Yue, M.; Wszolek, Z.; Ashe, K.; Knight, J.; Dickson, D.; et al. Accumulation of Pathological Tau Species and Memory Loss in a Conditional Model of Tauopathy. J. Neurosci. 2007, 27, 3650–3662. [CrossRef] [PubMed] 73. Bretteville, A.; Planel, E. Tau aggregates: Toxic, inert, or protective species? J. Alzheimer’s Dis. 2008, 14, 431–436. [CrossRef] 74. Cowan, C.M.; Mudher, A. Are tau aggregates toxic or protective in tauopathies? Front. Neurol. 2013, 4, 1–13. [CrossRef] [PubMed] 75. Maeda, S.; Sahara, N.; Saito, Y.; Murayama, M.; Yoshiike, Y.; Kim, H.; Miyasaka, T.; Murayama, S.; Ikai, A.; Takashima, A. Granular Tau Oligomers as Intermediates of Tau Filaments. Biochemistry 2007, 46, 3856–3861. [CrossRef] [PubMed] Int. J. Mol. Sci. 2018, 19, 1947 18 of 26 18 of 26 76. Lasagna-Reeves, C.A.; Castillo-Carranza, D.L.; Sengupta, U.; Sarmiento, J.; Troncoso, J.; Jackson, G.R.; Kayed, R. Identiﬁcation of oligomers at early stages of tau aggregation in Alzheimer’s disease. FASEB J. 2012, 26, 1946–1959. [CrossRef] [PubMed] 77. Hutton, M.; Lendon, C.L.; Rizzu, P.; Baker, M.; Froelich, S.; Houlden, H.; Pickering-Brown, S.; Chakraverty, S.; Isaacs, A.; Grover, A.; et al. Association of missense and 5′-splice-site mutations in tau with the inherited dementia FTDP-17. Nature 1998, 393, 702–705. [CrossRef] [PubMed] 78. Poorkaj, P.; Bird, T.D.; Wijsman, E.; Nemens, E.; Garruto, R.M.; Anderson, L.; Andreadis, A.; Wiederholt, W.C.; Raskind, M.; Schellenberg, G.D. Tau is a candidate gene for chromosome 17 frontotemporal dementia. Ann. Neurol. 1998, 43, 815–825. [CrossRef] [PubMed] 79. Spillantini, M.G.; Murrell, J.R.; Goedert, M.; Farlow, M.R.; Klug, A.; Ghetti, B. Mutation in the tau gene in familial multiple system tauopathy with presenile dementia. Proc. Natl. Acad. Sci. USA 1998, 95, 7737–7741. [CrossRef] [PubMed] 80. Spillantini, M.G.; Bird, T.D.; Ghetti, B. Frontotemporal dementia and Parkinsonism linked to chromosome 17: A new group of tauopathies. Brain Pathol. 1998, 8, 387–402. [CrossRef] [PubMed] 81. Rademakers, R.; Cruts, M.; van Broeckhoven, C. The role of tau (MAPT) in frontotemporal dementia and related tauopathies. Hum. Mutat. 2004, 24, 277–295. [CrossRef] [PubMed] 82. Dermaut, B.; Kumar-Singh, S.; Rademakers, R.; Theuns, J.; Cruts, M.; Van Broeckhoven, C. Tau is central in the genetic Alzheimer–frontotemporal dementia spectrum. Trends Genet. 2005, 21, 664–672. [CrossRef] [PubMed] 83. D’Souza, I.; Poorkaj, P.; Hong, M.; Nochlin, D.; Lee, V.M.; Bird, T.D.; Schellenberg, G.D. References Missense and silent tau gene mutations cause frontotemporal dementia with parkinsonism-chromosome 17 type, by affecting multiple alternative RNA splicing regulatory elements. Proc. Natl. Acad. Sci. USA 1999, 96, 5598–5603. [CrossRef] [PubMed] 84. Barghorn, S.; Zheng-Fischhöfer, Q.; Ackmann, M.; Biernat, J.; von Bergen, M.; Mandelkow, E.M.; Mandelkow, E. Structure, microtubule interactions, and paired helical ﬁlament aggregation by tau mutants of frontotemporal dementias. Biochemistry 2000, 39, 11714–11721. [CrossRef] [PubMed] 85. Hong, M.; Zhukareva, V.; Vogelsberg-Ragaglia, V.; Wszolek, Z.; Reed, L.; Miller, B.I.; Geschwind, D.H.; Bird, T.D.; McKeel, D.; Goate, A.; et al. Mutation-speciﬁc functional impairments in distinct tau isoforms of hereditary FTDP-17. Science 1998, 282, 1914–1917. [CrossRef] [PubMed] 86. Roberson, E.D.; Scearce-Levie, K.; Palop, J.J.; Yan, F.; Cheng, I.H.; Wu, T.; Gerstein, H.; Yu, G.-Q.; Mucke, L. Reducing Endogenous Tau Ameliorates Amyloid β-Induced Deﬁcits in an Alzheimer’s Disease Mouse Model. Science 2007, 316, 750–754. [CrossRef] [PubMed] 87. Vandebroek, T.; Vanhelmont, T.; Terwel, D.; Borghgraef, P.; Lemaire, K.; Snauwaert, J.; Wera, S.; Van Leuven, F.; Winderickx, J. Identiﬁcation and isolation of a hyperphosphorylated, conformationally changed intermediate of human protein tau expressed in yeast. Biochemistry 2005, 44, 11466–11475. [CrossRef] [PubMed] 88. Vanhelmont, T.; Vandebroek, T.; De Vos, A.; Terwel, D.; Lemaire, K.; Anandhakumar, J.; Franssens, V.; Swinnen, E.; Van Leuven, F.; Winderickx, J. Serine-409 phosphorylation and oxidative damage deﬁne aggregation of human protein tau in yeast. FEMS Yeast Res. 2010, 10, 992–1005. [CrossRef] [PubMed] 89. Vandebroek, T.; Terwel, D.; Vanhelmont, T.; Gysemans, M.; Van Haesendonck, C.; Engelborghs, Y.; Winderickx, J.; Van Leuven, F. Microtubule binding and clustering of human Tau-4R and Tau-P301L proteins isolated from yeast deﬁcient in orthologues of glycogen synthase kinase-3beta or cdk5. J. Biol. Chem. 2006, 281, 25388–25397. [CrossRef] [PubMed] 90. De Vos, A.; Anandhakumar, J.; Van den Brande, J.; Verduyckt, M.; Franssens, V.; Winderickx, J.; Swinnen, E. Yeast as a model system to study tau biology. Int. J. Alzheimers Dis. 2011, 2011, 428970. [CrossRef] [PubMed] 91. Zambrano, C.A.; Egaña, J.T.; Núñez, M.T.; Maccioni, R.B.; González-Billault, C. Oxidative stress promotes tau dephosphorylation in neuronal cells: The roles of cdk5 and PP1. Free Radic. Biol. Med. 2004, 36, 1393–1402. [CrossRef] [PubMed] 92. Kang, S.W.; Kim, S.J.; Kim, M.S. Oxidative stress with tau hyperphosphorylation in memory impaired 1,2-diacetylbenzene-treated mice. Toxicol. Lett. 2017, 279, 53–59. [CrossRef] [PubMed] 93. De Vos, A.; Bynens, T.; Rosseels, J.; Coun, C.; Ring, J.; Madeo, F.; Galas, M.-C.; Winderickx, J.; Franssens, V. References The peptidyl prolyl cis/trans isomerase Pin1/Ess1 inhibits phosphorylation and toxicity of tau in a yeast model for Alzheimer’s disease. AIMS Mol. Sci. 2015, 2, 144–160. [CrossRef] Int. J. Mol. Sci. 2018, 19, 1947 19 of 26 19 of 26 94. Su, B.; Wang, X.; Lee, H.; Tabaton, M.; Perry, G.; Smith, M.A.; Zhu, X. Chronic oxidative stress causes increased tau phosphorylation in M17 neuroblastoma cells. Neurosci. Lett. 2010, 468, 267–271. [CrossRef] [PubMed] 95. Rosseels, J.; Van Den Brande, J.; Violet, M.; Jacobs, D.; Grognet, P.; Lopez, J.; Huvent, I.; Caldara, M.; Swinnen, E.; Papegaey, A.; et al. Tau monoclonal antibody generation based on humanized yeast models: Impact on tau oligomerization and diagnostics. J. Biol. Chem. 2015, 290, 4059–4074. [CrossRef] [PubMed] Impact on tau oligomerization and diagnostics. J. Biol. Chem. 2015, 290, 4059–4074. [CrossRef] [PubMed] 96. Castillo-Carranza, D.L.; Gerson, J.E.; Sengupta, U.; Guerrero-Muñoz, M.J.; Lasagna-Reeves, C.A.; Kayed, R. Speciﬁc Targeting of Tau Oligomers in Htau Mice Prevents Cognitive Impairment and Tau Toxicity Following Injection with Brain-Derived Tau Oligomeric Seeds. J. Alzheimer Dis. 2014, 40, S97–S111. [CrossRef] [PubMed] 97. Patterson, K.R.; Remmers, C.; Fu, Y.; Brooker, S.; Kanaan, N.M.; Vana, L.; Ward, S.; Reyes, J.F.; Philibert, K.; Glucksman, M.J.; et al. Characterization of preﬁbrillar Tau oligomers in vitro and in Alzheimer disease. J Biol Chem 2011 286 23063 23076 [C R f] [P bM d] 96. Castillo-Carranza, D.L.; Gerson, J.E.; Sengupta, U.; Guerrero-Muñoz, M.J.; Lasagna-Reeves, C.A.; Kayed, R. Speciﬁc Targeting of Tau Oligomers in Htau Mice Prevents Cognitive Impairment and Tau Toxicity Following Injection with Brain-Derived Tau Oligomeric Seeds. J. Alzheimer Dis. 2014, 40, S97–S111. [CrossRef] [PubMed] 97. Patterson, K.R.; Remmers, C.; Fu, Y.; Brooker, S.; Kanaan, N.M.; Vana, L.; Ward, S.; Reyes, J.F.; Philibert, K.; Glucksman, M.J.; et al. Characterization of preﬁbrillar Tau oligomers in vitro and in Alzheimer disease. J. Biol. Chem. 2011, 286, 23063–23076. [CrossRef] [PubMed] 98. Pérez-Ruiz, E.; Decrop, D.; Ven, K.; Tripodi, L.; Leirs, K.; Rosseels, J.; van de Wouwer, M.; Geukens, N.; De Vos, A.; Vanmechelen, E.; et al. Digital ELISA for the quantiﬁcation of attomolar concentrations of Alzheimer’s disease biomarker protein Tau in biological samples. Anal. Chim. Acta 2018, 1015, 74–81. [CrossRef] [PubMed] 99. Giasson, B.I.; Forman, M.S.; Higuchi, M.; Golbe, L.I.; Graves, C.L.; Kotzbauer, P.T.; Trojanowski, J.Q.; Lee, V.M.Y. Initiation and synergistic ﬁbrillization of tau and alpha-synuctein. Science 2003, 300, 636–640. [CrossRef] [PubMed] 100. References [CrossRef] [PubMed] Int. J. Mol. Sci. 2018, 19, 1947 20 of 26 20 of 26 113. Cabantous, S.; Terwilliger, T.C.; Waldo, G.S. Protein tagging and detection with engineered self-assembling fragments of green ﬂuorescent protein. Nat. Biotechnol. 2005, 23, 102–107. [CrossRef] [PubMed] 114. Cabantous, S.; Waldo, G.S. In vivo and in vitro protein solubility assays using split GFP. Nat. Methods 2006, 3, 845–854. [CrossRef] [PubMed] 115. Foglieni, C.; Papin, S.; Salvadè, A.; Afroz, T.; Pinton, S.; Pedrioli, G.; Ulrich, G.; Polymenidou, M.; Paganetti, P. Split GFP technologies to structurally characterize and quantify functional biomolecular interactions of FTD-related proteins. Sci. Rep. 2017, 7, 14013. [CrossRef] [PubMed] 116. Priller, C.; Bauer, T.; Mitteregger, G.; Krebs, B.; Kretzschmar, H.A.; Herms, J. Synapse Formation and Function Is Modulated by the Amyloid Precursor Protein. J. Neurosci. 2006, 26, 7212–7221. [CrossRef] [PubMed] 117. Turner, P.R.; O’Connor, K.; Tate, W.P.; Abraham, W.C. Roles of amyloid precursor protein and its fragments in regulating neural activity, plasticity and memory. Prog. Neurobiol. 2003, 70, 1–32. [CrossRef] 118. Duce, J.A.; Tsatsanis, A.; Cater, M.A.; James, S.A.; Robb, E.; Wikhe, K.; Leong, S.L.; Perez, K.; Johanssen, T.; Greenough, M.A.; et al. Iron-Export Ferroxidase Activity of β-Amyloid Precursor Protein Is Inhibited by Zinc in Alzheimer’s Disease. Cell 2010, 142, 857–867. [CrossRef] [PubMed] 119. Chen, G.; Xu, T.; Yan, Y.; Zhou, Y.; Jiang, Y.; Melcher, K.; Xu, H.E. Amyloid beta: Structure, biology and structure-based therapeutic development. Acta Pharmacol. Sin. 2017, 38, 1205–1235. [CrossRef] [PubMed] 119. Chen, G.; Xu, T.; Yan, Y.; Zhou, Y.; Jiang, Y.; Melcher, K.; Xu, H.E. Amyloid beta: Structure, biology and structure-based therapeutic development. Acta Pharmacol. Sin. 2017, 38, 1205–1235. [CrossRef] [PubMed] 120. Haass, C.; Koo, E.H.; Mellon, A.; Hung, A.Y.; Selkoe, D.J. Targeting of cell-surface β-amyloid precursor protein to lysosomes: Alternative processing into amyloid-bearing fragments. Nature 1992, 357, 500–503. [C R f] [P bM d] 120. Haass, C.; Koo, E.H.; Mellon, A.; Hung, A.Y.; Selkoe, D.J. Targeting of cell-surface β-amyloid precursor protein to lysosomes: Alternative processing into amyloid-bearing fragments. Nature 1992, 357, 500–503. [CrossRef] [PubMed] 121. Joshi, G.; Wang, Y. Golgi defects enhance APP amyloidogenic processing in Alzheimer’s disease. BioEssays 2015, 37, 240–247. [CrossRef] [PubMed] 122. Olsson, F.; Schmidt, S.; Althoff, V.; Munter, L.M.; Jin, S.; Rosqvist, S.; Lendahl, U.; Multhaup, G.; Lundkvist, J. Characterization of Intermediate Steps in Amyloid Beta (Aβ) Production under Near-native Conditions. J. Biol. Chem. 2014, 289, 1540–1550. [CrossRef] [PubMed] 123. References Takami, M.; Nagashima, Y.; Sano, Y.; Ishihara, S.; Morishima-Kawashima, M.; Funamoto, S.; Ihara, Y. γ-Secretase: Successive Tripeptide and Tetrapeptide Release from the Transmembrane Domain of β-Carboxyl Terminal Fragment. J. Neurosci. 2009, 29, 13042–13052. [CrossRef] [PubMed] 124. Wolfe, M.S. Inhibition and modulation of γ-secretase for Alzheimer’s disease. Neurotherapeutics 391–398. [CrossRef] [PubMed] 125. Hensley, K.; Carney, J.M.; Mattson, M.P.; Aksenova, M.; Harris, M.; Wu, J.F.; Floyd, R.A.; Butterﬁeld, D.A. A model for beta-amyloid aggregation and neurotoxicity based on free radical generation by the peptide: Relevance to Alzheimer disease. Proc. Natl. Acad. Sci. USA 1994, 91, 3270–3274. [CrossRef] [PubMed] 126. .Benzinger, T.L.; Gregory, D.M.; Burkoth, T.S.; Miller-Auer, H.; Lynn, D.G.; Botto, R.E.; Meredith, S.C. Propagating structure of Alzheimer’s beta-amyloid(10-35) is parallel beta-sheet with residues in exact register. Proc. Natl. Acad. Sci. USA 1998, 95, 13407–13412. [CrossRef] [PubMed] 127. Yu, L.; Edalji, R.; Harlan, J.E.; Holzman, T.F.; Lopez, A.P.; Labkovsky, B.; Hillen, H.; Barghorn, S.; Ebert, U.; Richardson, P.L.; et al. Structural Characterization of a Soluble Amyloid β-Peptide Oligomer. Biochemistry 2009, 48, 1870–1877. [CrossRef] [PubMed] 128. Haass, C.; Schlossmacher, M.G.; Hung, A.Y.; Vigo-Pelfrey, C.; Mellon, A.; Ostaszewski, B.L.; Lieberburg, I.; Koo, E.H.; Schenk, D.; Teplow, D.B.; et al. Amyloid β-peptide is produced by cultured cells during normal metabolism. Nature 1992, 359, 322–325. [CrossRef] [PubMed] 129. Terry, R.D.; Masliah, E.; Salmon, D.P.; Butters, N.; DeTeresa, R.; Hill, R.; Hansen, L.A.; Katzman, R. Physical basis of cognitive alterations in alzheimer’s disease: Synapse loss is the major correlate of cognitive impairment. Ann. Neurol. 1991, 30, 572–580. [CrossRef] [PubMed] p 130. Masliah, E.; Terry, R.D.; Alford, M.; DeTeresa, R.; Hansen, L.A. Cortical and subcortical patterns of synaptophysinlike immunoreactivity in Alzheimer’s disease. Am. J. Pathol. 1991, 138, 235–246. [PubMed] 130. Masliah, E.; Terry, R.D.; Alford, M.; DeTeresa, R.; Hansen, L.A. Cortical and subcortical patterns of synaptophysinlike immunoreactivity in Alzheimer’s disease. Am. J. Pathol. 1991, 138, 235–246. [PubMed] 131. Masliah, E.; Achim, C.L.; Ge, N.; DeTeresa, R.; Terry, R.D.; Wiley, C.A. Spectrum of human immunodeﬁciency virus-associated neocortical damage. Ann. Neurol. 1992, 32, 321–329. [CrossRef] [PubMed] 131. Masliah, E.; Achim, C.L.; Ge, N.; DeTeresa, R.; Terry, R.D.; Wiley, C.A. Spectrum of human immunodeﬁciency virus-associated neocortical damage. Ann. Neurol. 1992, 32, 321–329. [CrossRef] [PubMed] 132. Mucke, L.; Masliah, E.; Yu, G.Q.; Mallory, M.; Rockenstein, E.M.; Tatsuno, G.; Hu, K.; Kholodenko, D.; Johnson-Wood, K.; McConlogue, L. References Jensen, P.H.; Hager, H.; Nielsen, M.S.; Hojrup, P.; Gliemann, J.; Jakes, R. alpha-synuclein binds to Tau and stimulates the protein kinase A-catalyzed tau phosphorylation of serine residues 262 and 356. J. Biol. Chem. 1999, 274, 25481–25489. [CrossRef] [PubMed] 101. Duka, T.; Rusnak, M.; Drolet, R.E.; Duka, V.; Wersinger, C.; Goudreau, J.L.; Sidhu, A. Alpha-Synuclein Induces Hyperphosphorylation of Au in the Mptp Model of Parkinsonism. FASEB J. 2006, 20, 2302–2312. [CrossRef] [PubMed] 102. Waxman, E.A.; Giasson, B.I. Induction of Intracellular Tau Aggregation Is Promoted by α-Synuclein Seeds and Provides Novel Insights into the Hyperphosphorylation of Tau. J. Neurosci. 2011, 31, 7604–7618. [CrossRef] [PubMed] 103. Frasier, M.; Walzer, M.; McCarthy, L.; Magnuson, D.; Lee, J.M.; Haas, C.; Kahle, P.; Wolozin, B. Tau phosphorylation increases in symptomatic mice overexpressing A30P α-synuclein. Exp. Neurol. 2005, 192, 274–287. [CrossRef] [PubMed] 104. Zabrocki, P.; Pellens, K.; Vanhelmont, T.; Vandebroek, T.; Grifﬁoen, G.; Wera, S.; Van Leuven, F.; Winderickx, J. Characterization of α-synuclein aggregation and synergistic toxicity with protein tau in yeast. FEBS J. 2005, 272, 1386–1400. [CrossRef] [PubMed] 105. Ciaccioli, G.; Martins, A.; Rodrigues, C.; Vieira, H.; Calado, P. A powerful yeast model to investigate the synergistic interaction of α-synuclein and tau in neurodegeneration. PLoS ONE 2013, 8, e55848. [CrossRef] [PubMed] 106. Hoffman, C.S.; Wood, V.; Fantes, P.A. An Ancient Yeast for Young Geneticists: A Primer on the Schizosaccharomyces pombe Model System. Genetics 2015, 201, 403–423. [CrossRef] [PubMed] 107. Balasubramanian, M.K.; Bi, E.; Glotzer, M. Comparative analysis of cytokinesis in budding yeast, ﬁssion yeast and animal cells. Curr. Biol. 2004, 14, R806–R818. [CrossRef] [PubMed] 108. Humphrey, T.; Pearce, A. Cell cycle molecules and mechanisms of the budding and ﬁssion yeasts. Methods Mol. Biol. 2005, 296, 3–29. [PubMed] 109. Heinisch, J.J.; Brandt, R. Signaling pathways and posttranslational modiﬁcations of tau in Alzheimer’s disease: The humanization of yeast cells. Microb. Cell 2016, 3, 135–146. [CrossRef] [PubMed] 110. Rodicio, R.; Heinisch, J.J. Yeast on the milky way: Genetics, physiology and biotechnology of Kluyveromyces lactis. Yeast 2013, 30, 165–177. [CrossRef] [PubMed] 111. Barnard, E.; McFerran, N.V.; Trudgett, A.; Nelson, J.; Timson, D.J. Development and implementation of split-GFP-based bimolecular ﬂuorescence complementation (BiFC) assays in yeast. Biochem. Soc. Trans. 2008, 36, 479–482. [CrossRef] [PubMed] 112. Cabantous, S.; Nguyen, H.B.; Pedelacq, J.D.; Koraïchi, F.; Chaudhary, A.; Ganguly, K.; Lockard, M.A.; Favre, G.; Terwilliger, T.C.; Waldo, G.S. A new protein-protein interaction sensor based on tripartite split-GFP association. Sci. Rep. 2013, 3, 02854. References High-level neuronal expression of abeta 1-42 in wild-type human amyloid protein precursor transgenic mice: Synaptotoxicity without plaque formation. J. Neurosci. 2000, 20, 4050–4058. [CrossRef] [PubMed] Int. J. Mol. Sci. 2018, 19, 1947 21 of 26 21 of 26 133. Hsia, A.Y.; Masliah, E.; McConlogue, L.; Yu, G.Q.; Tatsuno, G.; Hu, K.; Kholodenko, D.; Malenka, R.C.; Nicoll, R.A.; Mucke, L. Plaque-independent disruption of neural circuits in Alzheimer’s disease mouse models. Proc. Natl. Acad. Sci. USA 1999, 96, 3228–3233. [CrossRef] [PubMed] 34. Canevari, L.; Abramov, A.Y.; Duchen, M.R. Toxicity of amyloid beta peptide: Tales of calcium, mitochon and oxidative stress. Neurochem. Res. 2004, 29, 637–650. [CrossRef] [PubMed] 135. Mattson, M.P. Pathways towards and away from Alzheimer’s disease. Nature 2004, 430, 631–639. [CrossRef] [PubMed] 136. Weggen, S.; Eriksen, J.L.; Das, P.; Sagi, S.A.; Wang, R.; Pietrzik, C.U.; Findlay, K.A.; Smith, T.E.; Murphy, M.P.; Bulter, T.; et al. A subset of NSAIDs lower amyloidogenic Aβ42 independently of cyclooxygenase activity. Nature 2001, 414, 212–216. [CrossRef] [PubMed] 137. Neniskyte, U.; Neher, J.J.; Brown, G.C. Neuronal Death Induced by Nanomolar Amyloid β Is Mediated by Primary Phagocytosis of Neurons by Microglia. J. Biol. Chem. 2011, 286, 39904–39913. [CrossRef] [PubMed] 137. Neniskyte, U.; Neher, J.J.; Brown, G.C. Neuronal Death Induced by Nanomolar Amyloid β Is Mediated by Primary Phagocytosis of Neurons by Microglia. J. Biol. Chem. 2011, 286, 39904–39913. [CrossRef] [PubMed] 138. Heneka, M.T.; Golenbock, D.T.; Latz, E. Innate immunity in Alzheimer’s disease. Nat. Immunol. 2015, 16, 229 236 [C R f] [P bM d] 138. Heneka, M.T.; Golenbock, D.T.; Latz, E. Innate immunity in Alzheimer’s disease. Nat. Immunol. 2015, 16, 229–236. [CrossRef] [PubMed] 139. Iwata, N.; Tsubuki, S.; Takaki, Y.; Watanabe, K.; Sekiguchi, M.; Hosoki, E.; Kawashima-Morishima, M.; Lee, H.-J.; Hama, E.; Sekine-Aizawa, Y.; et al. Identiﬁcation of the major Aβ1–42-degrading catabolic pathway in brain parenchyma: Suppression leads to biochemical and pathological deposition. Nat. Med. 2000, 6, 143–150. [CrossRef] [PubMed] 140. Iwata, N.; Tsubuki, S.; Takaki, Y.; Shirotani, K.; Lu, B.; Gerard, N.P.; Gerard, C.; Hama, E.; Lee, H.J.; Saido, T.C. Metabolic Regulation of Brain Abeta by Neprilysin. Science 2001, 292, 1550–1552. [CrossRef] [PubMed] 141. Selkoe, D.J. Clearing the brain’s amyloid cobwebs. Neuron 2001, 32, 177–180. [CrossRef] 142. DeMattos, R.B.; Bales, K.R.; Cummins, D.J.; Dodart, J.-C.; Paul, S.M.; Holtzman, D.M. Peripheral anti-Aβ antibody alters CNS and plasma Aβ clearance and decreases brain Aβ burden in a mouse model of Alzheimer’s disease. Proc. Natl. Acad. Sci. References Proteolytic processing and secretion of human beta-amyloid precursor protein in yeast. Evidence for a yeast secretase activity. J. Biol. Chem. 1994, 269, 27799–27802. [PubMed] 155. Hines, V.; Zhang, W.; Ramakrishna, N.; Styles, J.; Mehta, P.; Kim, K.S.; Innis, M.; Miller, D.L. The expression and processing of human beta-amyloid peptide precursors in Saccharomyces cerevisiae: Evidence for a novel endopeptidase in the yeast secretory system. Cell. Mol. Biol. Res. 1994, 40, 273–284. [PubMed] 156. Zhang, W.; Espinoza, D.; Hines, V.; Innis, M.; Mehta, P.; Miller, D.L. Characterization of β-amyloid peptide precursor processing by the yeast Yap3 and Mkc7 proteases. Biochim. Biophys. Acta Mol. Cell Res. 1997, 1359, 110–122. [CrossRef] 157. Komano, H.; Seeger, M.; Gandy, S.; Wang, G.T.; Krafft, G.A.; Fuller, R.S. Involvement of cell surface glycosyl-phosphatidylinositol-linked aspartyl proteases in alpha-secretase-type cleavage and ectodomain solubilization of human Alzheimer beta-amyloid precursor protein in yeast. J. Biol. Chem. 1998, 273, 31648–31651. [CrossRef] [PubMed] 158. Edbauer, D.; Winkler, E.; Regula, J.T.; Pesold, B.; Steiner, H.; Haass, C. Reconstitution of γ-secretase activity. Nat. Cell Biol. 2003, 5, 486–488. [CrossRef] [PubMed] 159. Yagishita, S.; Futai, E.; Ishiura, S. In vitro reconstitution of gamma-secretase activity using yeast microsomes. Biochem. Biophys. Res. Commun. 2008, 377, 141–145. [CrossRef] [PubMed] 160. Yonemura, Y.; Futai, E.; Yagishita, S.; Suo, S.; Tomita, T.; Iwatsubo, T.; Ishiura, S. Comparison of presenilin 1 and presenilin 2 γ-secretase activities using a yeast reconstitution system. J. Biol. Chem. 2011, 286, 44569–44575. [CrossRef] [PubMed] 161. Yonemura, Y.; Futai, E.; Yagishita, S.; Kaether, C.; Ishiura, S. Speciﬁc combinations of presenilins and Aph1s affect the substrate speciﬁcity and activity of γ-secretase. Biochem. Biophys. Res. Commun. 2016, 478, 1751–1757. [CrossRef] [PubMed] 162. Futai, E.; Yagishita, S.; Ishiura, S. Nicastrin is dispensable for gamma-secretase protease activity in the presence of speciﬁc presenilin mutations. J. Biol. Chem. 2009, 284, 13013–13022. [CrossRef] [PubMed] 163. Wickner, R.B.; Edskes, H.K.; Kryndushkin, D.; McGlinchey, R.; Bateman, D.; Kelly, A. Prion diseases of yeast: Amyloid structure and biology. Semin. Cell Dev. Biol. 2011, 22, 469–475. [CrossRef] [PubMed] 64. Bagriantsev, S.; Liebman, S. Modulation of Abeta42 low-n oligomerization using a novel yeast repo system. BMC Biol. 2006, 4, 32. [CrossRef] [PubMed] 165. von der Haar, T.; Jossé, L.; Wright, P.; Zenthon, J.; Tuite, M.F. Development of a novel yeast cell-based system for studying the aggregation of Alzheimer’s disease-associated Abeta peptides in vivo. Neurodegener. Dis. 2007, 4, 136–147. [CrossRef] [PubMed] 166. References USA 2001, 98, 8850–8855. [CrossRef] [PubMed] 143. Zlokovic, B.V.; Yamada, S.; Holtzman, D.; Ghiso, J.; Frangione, B. Clearance of amyloid beta-peptide from brain: Transport or metabolism? Nat. Med. 2000, 6, 718. [CrossRef] [PubMed] 144. Martel, C.L.; Mackic, J.B.; Matsubara, E.; Governale, S.; Miguel, C.; Miao, W.; McComb, J.G.; Frangione, B.; Ghiso, J.; Zlokovic, B. V Isoform-speciﬁc effects of apolipoproteins E2, E3, and E4 on cerebral capillary sequestration and blood-brain barrier transport of circulating Alzheimer’s amyloid beta. J. Neurochem. 1997, 69, 1995–2004. [CrossRef] [PubMed] 145. Ghersi-Egea, J.F.; Gorevic, P.D.; Ghiso, J.; Frangione, B.; Patlak, C.S.; Fenstermacher, J.D. Fate of cerebrospinal ﬂuid-borne amyloid beta-peptide: Rapid clearance into blood and appreciable accumulation by cerebral arteries. J. Neurochem. 1996, 67, 880–883. [CrossRef] [PubMed] 146. Maness, L.M.; Banks, W.A.; Podlisny, M.B.; Selkoe, D.J.; Kastin, A.J. Passage of human amyloid beta-protein 1–40 across the murine blood-brain barrier. Life Sci. 1994, 55, 1643–1650. [CrossRef] 147. Zlokovic, B.V.; Martel, C.L.; Mackic, J.B.; Matsubara, E.; Wisniewski, T.; Mccomb, J.G.; Frangione, B.; Ghiso, J. Brain Uptake of Circulating Apolipoproteins J and E Complexed to Alzheimer′s Amyloid β. Biochem. Biophys. Res. Commun. 1994, 205, 1431–1437. [CrossRef] [PubMed] 148. Hardy, J.; Selkoe, D.J. The Amyloid Hypothesis of Alzheimer’s Disease: Progress and Problems on the Road to Therapeutics. Science 2002, 297, 353–356. [CrossRef] [PubMed] 49. Tanzi, R.E.; Moir, R.D.; Wagner, S.L. Clearance of Alzheimer’s Aβ Peptide. Neuron 2004, 43, 605– [CrossRef] [PubMed] 150. Wyss-Coray, T.; Loike, J.D.; Brionne, T.C.; Lu, E.; Anankov, R.; Yan, F.; Silverstein, S.C.; Husemann, J. Adult mouse astrocytes degrade amyloid-β in vitro and in situ. Nat. Med. 2003, 9, 453–457. [CrossRef] [PubMed] 151. Koistinaho, M.; Lin, S.; Wu, X.; Esterman, M.; Koger, D.; Hanson, J.; Higgs, R.; Liu, F.; Malkani, S.; Bales, K.R.; et al. Apolipoprotein E promotes astrocyte colocalization and degradation of deposited amyloid-β peptides. Nat. Med. 2004, 10, 719–726. [CrossRef] [PubMed] 152. Zlokovic, B.V.; Deane, R.; Sallstrom, J.; Chow, N.; Miano, J.M. Neurovascular pathways and Alzheimer amyloid beta-peptide. Brain Pathol. 2005, 15, 78–83. [CrossRef] [PubMed] 153. Cataldo, A.M.; Hamilton, D.J.; Barnett, J.L.; Paskevich, P.A.; Nixon, R.A. Properties of the endosomal-lysosomal system in the human central nervous system: Disturbances mark most neurons in populations at risk to degenerate in Alzheimer’s disease. J. Neurosci. 1996, 16, 186–199. [CrossRef] [PubMed] 22 of 26 Int. J. Mol. Sci. 2018, 19, 1947 154. Zhang, H.; Komano, H.; Fuller, R.S.; Gandy, S.E.; Frail, D.E. References Caine, J.; Sankovich, S.; Antony, H.; Waddington, L.; Macreadie, P.; Varghese, J.; Macreadie, I. Alzheimer’s Abeta fused to green ﬂuorescent protein induces growth stress and a heat shock response. FEMS Yeast Res. 2007, 7, 1230–1236. [CrossRef] [PubMed] 167. Hamos, J.E.; Oblas, B.; Pulaski-Salo, D.; Welch, W.J.; Bole, D.G.; Drachman, D.A. Expression of heat shock proteins in Alzheimer’s disease. Neurology 1991, 41, 345–350. [CrossRef] [PubMed] 168. Macreadie, I.; Lotﬁ-Miri, M.; Mohotti, S.; Shapira, D.; Bennett, L.; Varghese, J. Validation of folate in a convenient yeast assay suited for identiﬁcation of inhibitors of Alzheimer’s amyloid-beta aggregation. J. Alzheimer’s Dis. 2008, 15, 391–396. [CrossRef] 169. Rajasekhar, K.; Suresh, S.N.; Manjithaya, R.; Govindaraju, T. Rationally Designed Peptidomimetic Mod of Aβ Toxicity in Alzheimer’s Disease. Sci. Rep. 2015, 5, 8139. [CrossRef] [PubMed] 170. Bharadwaj, P.R.; Verdile, G.; Barr, R.K.; Gupta, V.; Steele, J.W.; Lachenmayer, M.L.; Yue, Z.; Ehrlich, M.E.; Petsko, G.; Ju, S.; et al. Latrepirdine (dimebon) enhances autophagy and reduces intracellular GFP-Aβ42 levels in yeast. J. Alzheimer’s Dis. 2012, 32, 949–967. [CrossRef] [PubMed] 171. .Nixon, R.A.; Wegiel, J.; Kumar, A.; Yu, W.H.; Peterhoff, C.; Cataldo, A.; Cuervo, A.M. Extensive involvement of autophagy in Alzheimer disease: An immuno-electron microscopy study. J. Neuropathol. Exp. Neurol. 2005, 64, 113–122. [CrossRef] [PubMed] 172. Yu, W.H.; Cuervo, A.M.; Kumar, A.; Peterhoff, C.M.; Schmidt, S.D.; Lee, J.-H.; Mohan, P.S.; Mercken, M.; Farmery, M.R.; Tjernberg, L.O.; et al. Macroautophagy—A novel β-amyloid peptide-generating pathway activated in Alzheimer’s disease. J. Cell Biol. 2005, 171, 87–98. [CrossRef] [PubMed] Int. J. Mol. Sci. 2018, 19, 1947 23 of 26 173. Lee, J.-H.; Yu, W.H.; Kumar, A.; Lee, S.; Mohan, P.S.; Peterhoff, C.M.; Wolfe, D.M.; Martinez-Vicente, M.; Massey, A.C.; Sovak, G.; et al. Lysosomal Proteolysis and Autophagy Require Presenilin 1 and Are Disrupted by Alzheimer-Related PS1 Mutations. Cell 2010, 141, 1146–1158. [CrossRef] [PubMed] 174. Steele, J.W.; Lachenmayer, M.L.; Ju, S.; Stock, A.; Liken, J.; Kim, S.H.; Delgado, L.M.; Alfaro, I.E.; Bernales, S.; Verdile, G.; et al. Latrepirdine improves cognition and arrests progression of neuropathology in an Alzheimer’s mouse model. Mol. Psychiatry 2013, 18, 889–897. [CrossRef] [PubMed] 175. Matlack, K.E.S.; Tardiff, D.F.; Narayan, P.; Hamamichi, S.; Caldwell, K.A.; Caldwell, G.A.; Lindquist, S. Clioquinol promotes the degradation of metal-dependent amyloid-β (Aβ) oligomers to restore endocytosis and ameliorate Aβ toxicity. Proc. Natl. Acad. Sci. USA 2014, 111, 4013–4018. [CrossRef] [PubMed] 176. References Tardiff, D.F.; Brown, L.E.; Yan, X.; Trilles, R.; Jui, N.T.; Barrasa, M.I.; Caldwell, K.A.; Caldwell, G.A.; Schaus, S.E.; Lindquist, S. Dihydropyrimidine-Thiones and Clioquinol Synergize To Target β-Amyloid Cellular Pathologies through a Metal-Dependent Mechanism. ACS Chem. Neurosci. 2017, 8, 2039–2055. [CrossRef] [PubMed] 177. Cherny, R.A.; Atwood, C.S.; Xilinas, M.E.; Gray, D.N.; Jones, W.D.; McLean, C.A.; Barnham, K.J.; Volitakis, I.; Fraser, F.W.; Kim, Y.-S.; et al. Treatment with a Copper-Zinc Chelator Markedly and Rapidly Inhibits β-Amyloid Accumulation in Alzheimer’s Disease Transgenic Mice. Neuron 2001, 30, 665–676. [CrossRef] 178. Adlard, P.A.; Cherny, R.A.; Finkelstein, D.I.; Gautier, E.; Robb, E.; Cortes, M.; Volitakis, I.; Liu, X.; Smith, J.P.; Perez, K.; et al. Rapid Restoration of Cognition in Alzheimer’s Transgenic Mice with 8-Hydroxy Quinoline Analogs Is Associated with Decreased Interstitial Aβ. Neuron 2008, 59, 43–55. [CrossRef] [PubMed] 179. Lannfelt, L.; Blennow, K.; Zetterberg, H.; Batsman, S.; Ames, D.; Harrison, J.; Masters, C.L.; Targum, S.; Bush, A.I.; Murdoch, R.; et al. Safety, efﬁcacy, and biomarker ﬁndings of PBT2 in targeting Aβ as a modifying therapy for Alzheimer’s disease: A phase IIa, double-blind, randomised, placebo-controlled trial. Lancet Neurol. 2008, 7, 779–786. [CrossRef] 180. LaFerla, F.M.; Green, K.N.; Oddo, S. Intracellular amyloid-β in Alzheimer’s disease. Nat. Rev. Neurosci. 2007, 8, 499–509. [CrossRef] [PubMed] 181. .Treusch, S.; Hamamichi, S.; Goodman, J.L.; Matlack, K.E.S.; Chung, C.Y.; Baru, V.; Shulman, J.M.; Parrado, A.; Bevis, B.J.; Valastyan, J.S.; et al. Functional links between Aβ toxicity, endocytic trafﬁcking, and Alzheimer’s disease risk factors in yeast. Science 2011, 334, 1241–1245. [CrossRef] [PubMed] y 182. D’Angelo, F.; Vignaud, H.; Di Martino, J.; Salin, B.; Devin, A.; Cullin, C.; Marchal, C. A yeast model for amyloid-β aggregation exempliﬁes the role of membrane trafﬁcking and PICALM in cytotoxicity. Dis. Model. Mech. 2013, 6, 206–216. [CrossRef] [PubMed] 183. Park, S.-K.; Ratia, K.; Ba, M.; Valencik, M.; Liebman, S.W. Inhibition of Aβ42 oligomerization in yeast by a PICALM ortholog and certain FDA approved drugs. Microb. Cell 2016, 3, 53–64. [CrossRef] [PubMed] 183. Park, S.-K.; Ratia, K.; Ba, M.; Valencik, M.; Liebman, S.W. Inhibition of Aβ42 oligomerization in yeast by a PICALM ortholog and certain FDA approved drugs. Microb. Cell 2016, 3, 53–64. [CrossRef] [PubMed] 184. Chen, X.; Petranovic, D. Amyloid-β peptide-induced cytotoxicity and mitochondrial dysfunction in yeast. FEMS Yeast Res. 2015, 15, fov061. [CrossRef] [PubMed] g pp g [ ] [ ] 184. Chen, X.; Petranovic, D. Amyloid-β peptide-induced cytotoxicity and mitochondrial dysfunction in yeast. FEMS Yeast Res. References 2015, 15, fov061. [CrossRef] [PubMed] 185. Fukui, H.; Moraes, C.T. The mitochondrial impairment, oxidative stress and neurodegeneration connection: Reality or just an attractive hypothesis? Trends Neurosci. 2008, 31, 251–256. [CrossRef] [PubMed] 186. Chen, X.; Bisschops, M.M.M.; Agarwal, N.R.; Ji, B.; Shanmugavel, K.P.; Petranovic, D. Interplay of Energetics and ER Stress Exacerbates Alzheimer’s Amyloid-β (Aβ) Toxicity in Yeast. Front. Mol. Neurosci. 2017, 10, 232. [CrossRef] [PubMed] 187. Stahl, A.; Moberg, P.; Ytterberg, J.; Panﬁlov, O.; Brockenhuus Von Lowenhielm, H.; Nilsson, F.; Glaser, E. Isolation and identiﬁcation of a novel mitochondrial metalloprotease (PreP) that degrades targeting presequences in plants. J. Biol. Chem. 2002, 277, 41931–41939. [CrossRef] [PubMed] 188. Alikhani, N.; Berglund, A.-K.; Engmann, T.; Spånning, E.; Vögtle, F.-N.; Pavlov, P.; Meisinger, C.; Langer, T.; Glaser, E. Targeting Capacity and Conservation of PreP Homologues Localization in Mitochondria of Different Species. J. Mol. Biol. 2011, 410, 400–410. [CrossRef] [PubMed] 189. Alikhani, N.; Guo, L.; Yan, S.; Du, H.; Pinho, C.M.; Chen, J.X.; Glaser, E.; Yan, S.S. Decreased proteolytic activity of the mitochondrial amyloid-β degrading enzyme, PreP peptidasome, in Alzheimer’s disease brain mitochondria. J. Alzheimer’s Dis. 2011, 27, 75–87. [CrossRef] [PubMed] 190. Teixeira, P.F.; Glaser, E. Processing peptidases in mitochondria and chloroplasts. Biochim. Biophys. Acta-Mol. Cell Res. 2013, 1833, 360–370. [CrossRef] [PubMed] Int. J. Mol. Sci. 2018, 19, 1947 24 of 26 24 of 26 191. Mossmann, D.; Vögtle, F.-N.; Taskin, A.A.; Teixeira, P.F.; Ring, J.; Burkhart, J.M.; Burger, N.; Pinho, C.M.; Tadic, J.; Loreth, D.; et al. Amyloid-β Peptide Induces Mitochondrial Dysfunction by Inhibition of Preprotein Maturation. Cell Metab. 2014, 20, 662–669. [CrossRef] [PubMed] 192. Brunetti, D.; Torsvik, J.; Dallabona, C.; Teixeira, P.; Sztromwasser, P.; Fernandez-Vizarra, E.; Cerutti, R.; Reyes, A.; Preziuso, C.; D’Amati, G.; et al. Defective PITRM1 mitochondrial peptidase is associated with A amyloidotic neurodegeneration. EMBO Mol. Med. 2016, 8, 176–190. [CrossRef] [PubMed] 193. Cenini, G.; Rub, C.; Bruderek, M.; Voos, W. Amyloid β-peptides interfere with mitochondrial preprotein import competence by a coaggregation process. Mol. Biol. Cell 2016, 27, 3257–3272. [CrossRef] [PubMed] 194. Bharadwaj, P.; Waddington, L.; Varghese, J.; Macreadie, I.G. A new method to measure cellular toxicity of non-ﬁbrillar and ﬁbrillar Alzheimer’s Abeta using yeast. J. Alzheimer’s Dis. 2008, 13, 147–150. [CrossRef] 195. Dubey, A.K.; Bharadwaj, P.R.; Varghese, J.N.; Macreadie, I.G. Alzheimer’s Amyloid-β Rescues Yeast from Hydroxide Toxicity. J. Alzheimer’s Dis. 2009, 18, 31–33. [CrossRef] [PubMed] 196. Liou, Y.-C.; Sun, A.; Ryo, A.; Zhou, X. References Z.; Yu, Z.-X.; Huang, H.-K.; Uchida, T.; Bronson, R.; Bing, G.; Li, X.; et al. Role of the prolyl isomerase Pin1 in protecting against age-dependent neurodegeneration. Nature 2003, 424, 556–561. [CrossRef] [PubMed] 197. Kondo, A.; Albayram, O.; Zhou, X.Z.; Lu, K.P. Pin1 Knockout Mice: A Model for the Study of Tau Pathology in Alzheimer’s Disease. Methods Mol Biol. 2017, 1523, 415–425. [PubMed] 198. van Leeuwen, F.W.; de Kleijn, D.P.; van den Hurk, H.H.; Neubauer, A.; Sonnemans, M.A.; Sluijs, J.A.; Köycü, S.; Ramdjielal, R.D.; Salehi, A.; Martens, G.J.; et al. Frameshift mutants of beta amyloid precursor protein and ubiquitin-B in Alzheimer’s and Down patients. Science 1998, 279, 242–247. [CrossRef] [PubMed] j j Köycü, S.; Ramdjielal, R.D.; Salehi, A.; Martens, G.J.; et al. Frameshift mutants of beta amyloid precursor protein and ubiquitin-B in Alzheimer’s and Down patients. Science 1998, 279, 242–247. [CrossRef] [PubMed] 199. Morishima-Kawashima, M.; Hasegawa, M.; Takio, K.; Suzuki, M.; Titani, K.; Ihara, Y. Ubiquitin is conjugated with amino-terminally processed tau in paired helical ﬁlaments. Neuron 1993, 10, 1151–1160. [CrossRef] 199. Morishima-Kawashima, M.; Hasegawa, M.; Takio, K.; Suzuki, M.; Titani, K.; Ihara, Y. Ubiquitin is conjugated with amino-terminally processed tau in paired helical ﬁlaments. Neuron 1993, 10, 1151–1160. [CrossRef] with amino-terminally processed tau in paired helical ﬁlaments. Neuron 1993, 10, 1151–1160. [CrossRef] 200. Hilt, W.; Wolf, D.H. Proteasomes: Destruction as a programme. Trends Biochem. Sci. 1996, 21, 96–102. [CrossRef] 200. Hilt, W.; Wolf, D.H. Proteasomes: Destruction as a programme. Trends Biochem. Sci. 1996, 21, 96–102. [CrossRef] 201. van Tijn, P.; de Vrij, F.M.S.; Schuurman, K.G.; Dantuma, N.P.; Fischer, D.F.; van Leeuwen, F.W.; Hol, E.M. Dose-dependent inhibition of proteasome activity by a mutant ubiquitin associated with neurodegenerative disease. J. Cell Sci. 2007, 120, 1615–1623. [CrossRef] [PubMed] 202. Lindsten, K.; de Vrij, F.M.S.; Verhoef, L.G.G.C.; Fischer, D.F.; van Leeuwen, F.W.; Hol, E.M.; Masucci, M.G.; Dantuma, N.P. Mutant ubiquitin found in neurodegenerative disorders is a ubiquitin fusion degradation substrate that blocks proteasomal degradation. J. Cell Biol. 2002, 157, 417–427. [CrossRef] [PubMed] 203. Braun, R.J.; Sommer, C.; Leibiger, C.; Gentier, R.J.G.; Dumit, V.I.; Paduch, K.; Eisenberg, T.; Habernig, L.; Trausinger, G.; Magnes, C.; et al. Accumulation of Basic Amino Acids at Mitochondria Dictates the Cytotoxicity of Aberrant Ubiquitin. Cell Rep. 2015, 10, 1557–1571. [CrossRef] [PubMed] g g g Trausinger, G.; Magnes, C.; et al. Accumulation of Basic Amino Acids at Mitochondria Dictates the Cytotoxicity of Aberrant Ubiquitin. Cell Rep. 2015, 10, 1557–1571. References Biochemistry 2018, 57, 1285–1292. [CrossRef] [PubMed] 217. Wu, Y.-X.; Greene, L.E.; Masison, D.C.; Eisenberg, E. Curing of yeast [PSI+] prion by guanidine inactivation of Hsp104 does not require cell division. Proc. Natl. Acad. Sci. USA 2005, 102, 12789–12794. [CrossRef] [PubMed] 218. Wegrzyn, R.D.; Bapat, K.; Newnam, G.P.; Zink, A.D.; Chernoff, Y.O. Mechanism of prion loss after Hsp104 inactivation in yeast. Mol. Cell. Biol. 2001, 21, 4656–4669. [CrossRef] [PubMed] 219. Moriyama, H.; Edskes, H.K.; Wickner, R.B. [URE3] prion propagation in Saccharomyces cerevisiae: Requirement for chaperone Hsp104 and curing by overexpressed chaperone Ydj1p. Mol. Cell. Biol. 2000, 20, 8916–8922. [CrossRef] [PubMed] 220. Kryndushkin, D.S.; Shewmaker, F.; Wickner, R.B. Curing of the [URE3] prion by Btn2p, a Batten disease-related protein. EMBO J. 2008, 27, 2725–2735. [CrossRef] [PubMed] 221. Wickner, R.B.; Bezsonov, E.; Bateman, D.A. Normal levels of the antiprion proteins Btn2 and Cur1 cure most newly formed [URE3] prion variants. Proc. Natl. Acad. Sci. USA 2014, 111, E2711–E2720. [CrossRef] [PubMed] 222. O’Driscoll, J.; Clare, D.; Saibil, H. Prion aggregate structure in yeast cells is determined by the Hsp104-Hsp110 disaggregase machinery. J. Cell Biol. 2015, 211, 145–158. [CrossRef] [PubMed] 223. Romanova, N.V.; Chernoff, Y.O. Hsp104 and prion propagation. Protein Pept. Lett. 2009, 16, 598–605. 222. O’Driscoll, J.; Clare, D.; Saibil, H. Prion aggregate structure in yeast cells is determined by the Hsp104-Hsp110 disaggregase machinery. J. Cell Biol. 2015, 211, 145–158. [CrossRef] [PubMed] 23. Romanova, N.V.; Chernoff, Y.O. Hsp104 and prion propagation. Protein Pept. Lett. 2009, 16, 598– [CrossRef] [PubMed] 224. Mukherjee, A.; Soto, C. Prion-Like Protein Aggregates and Type 2 Diabetes. Cold Spring Harb. Perspect. Med. 2017, 7, a024315. [CrossRef] [PubMed] 225. Moore, R.C.; Xiang, F.; Monaghan, J.; Han, D.; Zhang, Z.; Edström, L.; Anvret, M.; Prusiner, S.B. Huntington Disease Phenocopy Is a Familial Prion Disease. Am. J. Hum. Genet. 2001, 69, 1385–1388. [CrossRef] [PubMed] 225. Moore, R.C.; Xiang, F.; Monaghan, J.; Han, D.; Zhang, Z.; Edström, L.; Anvret, M.; Prusiner, S.B. Huntington Disease Phenocopy Is a Familial Prion Disease. Am. J. Hum. Genet. 2001, 69, 1385–1388. [CrossRef] [PubMed] 226. Abbott, A. The red-hot debate about transmissible Alzheimer’s. Nature 2016, 531, 294–297. [CrossRef] [PubMed] 226. Abbott, A. The red-hot debate about transmissible Alzheimer’s. Nature 2016, 531, 294–297. [CrossRef] [PubMed] 227. Olsson, T.T.; Klementieva, O.; Gouras, G.K. Prion-like seeding and nucleation of intracellular amyloid-β. Neurobiol. Dis. 2018, 113, 1–10. [CrossRef] [PubMed] 228. Lu, J.-X.; Qiang, W.; Yau, W.-M.; Schwieters, C.D.; Meredith, S.C.; Tycko, R. References [CrossRef] [PubMed] 204. Tank, E.M.H.; True, H.L. Disease-Associated Mutant Ubiquitin Causes Proteasomal Impairment and Enhances the Toxicity of Protein Aggregates. PLoS Genet. 2009, 5, e1000382. [CrossRef] [PubMed] 205. Krutauz, D.; Reis, N.; Nakasone, M.A.; Siman, P.; Zhang, D.; Kirkpatrick, D.S.; Gygi, S.P.; Brik, A.; Fushman, D.; Glickman, M.H. Extended ubiquitin species are protein-based DUB inhibitors. Nat. Chem. Biol. 2014, 10, 664–670. [CrossRef] [PubMed] 206. De Vrij, F.M.S.; Sluijs, J.A.; Gregori, L.; Fischer, D.F.; Hermens, W.T.J.M.C.; Goldgaber, D.; Verhaagen, J.; Van Leeuwen, F.W.; Hol, E.M. Mutant ubiquitin expressed in Alzheimer’s disease causes neuronal death. FASEB J. 2001, 15, 2680–2688. [CrossRef] [PubMed] 207. Tan, Z.; Sun, X.; Hou, F.-S.; Oh, H.-W.; Hilgenberg, L.G.W.; Hol, E.M.; van Leeuwen, F.W.; Smith, M.A.; O’Dowd, D.K.; Schreiber, S.S. Mutant ubiquitin found in Alzheimer’s disease causes neuritic beading of mitochondria in association with neuronal degeneration. Cell Death Differ. 2007, 14, 1721–1732. [CrossRef] [PubMed] 208. Braun, R.J. Ubiquitin-dependent proteolysis in yeast cells expressing neurotoxic proteins. Front. Mol. Neurosci. 2015, 8, 8. [CrossRef] [PubMed] Colby, D.W.; Prusiner, S.B. Prions. Cold Spring Harb. Perspect. Biol. 2011, 3, a006833. [CrossRef] [PubMed] 209. Colby, D.W.; Prusiner, S.B. Prions. Cold Spring Harb. Perspect. Biol. 2011, 3, a006833. [CrossRef] [PubMed] 210. Prusiner, S.B. Creutzfeldt-Jakob disease and scrapie prions. Alzheimer Dis. Assoc. Disord. 1989, 3, 52–78. [CrossRef] [PubMed] 210. Prusiner, S.B. Creutzfeldt-Jakob disease and scrapie prions. Alzheimer Dis. Assoc. Disord. 1989, 3, 52–78. [CrossRef] [PubMed] 211. Safar, J.G. Molecular pathogenesis of sporadic prion diseases in man. Prion 2012, 6, 108–115. [CrossRef] [PubMed] 25 of 26 Int. J. Mol. Sci. 2018, 19, 1947 212. Wickner, R.B.; Shewmaker, F.P.; Bateman, D.A.; Edskes, H.K.; Gorkovskiy, A.; Dayani, Y.; Bezsonov, E.E. Yeast Prions: Structure, Biology, and Prion-Handling Systems. Microbiol. Mol. Biol. Rev. 2015, 79, 1–17. [CrossRef] [PubMed] 213. Wickner, R.B.; Edskes, H.K.; Son, M.; Bezsonov, E.E.; DeWilde, M.; Ducatez, M. Yeast Prions Comp Functional Prions and Amyloids. J. Mol. Biol. 2018. [CrossRef] [PubMed] 214. Riek, R.; Saupe, S.J. The HET-S/s Prion Motif in the Control of Programmed Cell Death. Cold Spring Harb. Perspect. Biol. 2016, 8, a023515. [CrossRef] [PubMed] 215. Coustou, V.; Deleu, C.; Saupe, S.; Begueret, J. The protein product of the het-s heterokaryon incompatibility gene of the fungus Podospora anserina behaves as a prion analog. Proc. Natl. Acad. Sci. USA 1997, 94, 9773–9778. [CrossRef] [PubMed] 216. Wickner, R.B.; Bezsonov, E.E.; Son, M.; Ducatez, M.; DeWilde, M.; Edskes, H.K. Anti-Prion Systems in Yeast and Inositol Polyphosphates. References Molecular Structure of β-Amyloid Fibrils in Alzheimer’s Disease Brain Tissue. Cell 2013, 154, 1257–1268. [CrossRef] [PubMed] 229. Coalier, K.A.; Paranjape, G.S.; Karki, S.; Nichols, M.R. Stability of early-stage amyloid-β(1–42) aggregation species. Biochim. Biophys. Acta Proteins Proteom. 2013, 1834, 65–70. [CrossRef] [PubMed] 230. Holmes, B.B.; Diamond, M.I. Prion-like properties of Tau protein: The importance of extracellular Tau as a therapeutic target. J. Biol. Chem. 2014, 289, 19855–19861. [CrossRef] [PubMed] 231. .Kaufman, S.K.; Sanders, D.W.; Thomas, T.L.; Ruchinskas, A.J.; Vaquer-Alicea, J.; Sharma, A.M.; Miller, T.M.; Diamond, M.I. Tau Prion Strains Dictate Patterns of Cell Pathology, Progression Rate, and Regional Vulnerability In Vivo. Neuron 2016, 92, 796–812. [CrossRef] [PubMed] 232. Fruhmann, G.; Seynnaeve, D.; Zheng, J.; Ven, K.; Molenberghs, S.; Wilms, T.; Liu, B.; Winderickx, J.; Franssens, V. Yeast buddies helping to unravel the complexity of neurodegenerative disorders. Mech. Ageing Dev. 2017, 161, 288–305. [CrossRef] [PubMed] 233. Brachmann, A.; Toombs, J.A.; Ross, E.D. Reporter assay systems for [URE3] detection and analysis. Methods 2006, 39, 35–42. [CrossRef] [PubMed] 234. Schlumpberger, M.; Prusiner, S.B.; Herskowitz, I. Induction of distinct [URE3] yeast prion strains. Mol. Cell. Biol. 2001, 21, 7035–7046. [CrossRef] [PubMed] Int. J. Mol. Sci. 2018, 19, 1947 26 of 26 26 of 26 235. Newby, G.A.; Kiriakov, S.; Hallacli, E.; Kayatekin, C.; Tsvetkov, P.; Mancuso, C.P.; Bonner, J.M.; Hesse, W.R.; Chakrabortee, S.; Manogaran, A.L.; et al. A Genetic Tool to Track Protein Aggregates and Control Prion Inheritance. Cell 2017, 171, 966–979.e18. [CrossRef] [PubMed] 236. Chun, W.; Waldo, G.S.; Johnson, G.V.W. Split GFP complementation assay: a novel approach to quantitatively measure aggregation of tau in situ: Effects of GSK3β activation and caspase 3 cleavage. J. Neurochem. 2007, 103, 2529–2539. [CrossRef] [PubMed] 237. Zhao, L.; Yang, Q.; Zheng, J.; Zhu, X.; Hao, X.; Song, J.; Lebacq, T.; Franssens, V.; Winderickx, J.; Nystrom, T.; et al. A genome-wide imaging-based screening to identify genes involved in synphilin-1 inclusion formation in Saccharomyces cerevisiae. Sci. Rep. 2016, 6, 30134. [CrossRef] [PubMed] y g g g g y g y p inclusion formation in Saccharomyces cerevisiae. Sci. Rep. 2016, 6, 30134. [CrossRef] [PubMed] 238. Yu, Y.; Li, Y.; Zhang, Y. Screening of APP interaction proteins by DUALmembrane yeast two-hybrid system. Int. J. Clin. Exp. Pathol. 2015, 8, 2802–2808. [PubMed] 238. Yu, Y.; Li, Y.; Zhang, Y. Screening of APP interaction proteins by DUALmembrane yeast two-hybrid system. Int. J. Clin. Exp. Pathol. 2015, 8, 2802–2808. [PubMed] © 2018 by the authors. References Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W4235772251	https://figshare.com/articles/thesis/Theoretical_and_Experimental_Investigation_of_Thermodiffusion_Soret_Effect_in_a_Porous_Medium/14653563/1/files/28135335.pdf	English	null	Theoretical and Experimental Investigation of Thermodiffusion (Soret Effect) in a Porous Medium	null	2,023	cc-by	54,028	1-1-2010 Theoretical And Experimental Investigation Of Thermodiffusion (Soret Effect) In A Porous Medium Tawfiq J. Jaber Ryerson University Follow this and additional works at: http://digitalcommons.ryerson.ca/dissertations Part of the Mechanical Engineering Commons Part of the Mechanical Engineering Commons Ryerson University Digital Commons @ Ryerson Ryerson University Digital Commons @ Ryerson Recommended Citation Jaber, Tawfiq J., "Theoretical And Experimental Investigation Of Thermodiffusion (Soret Effect) In A Porous Medium" (2010). Theses and dissertations. Paper 1449. This Dissertation is brought to you for free and open access by Digital Commons @ Ryerson. It has been accepted for inclusion in Theses and dissertations by an authorized administrator of Digital Commons @ Ryerson. For more information, please contact bcameron@ryerson.ca. Author’s Declaration I hereby declare that I am the sole author of this dissertation. I hereby declare that I am the sole author of this dissertation. I authorize Ryerson University to lend this dissertation to other institutions or individuals for the purpose of scholarly research. Tawfiq Jaber Tawfiq Jaber I further authorize Ryerson University to reproduce this dissertation by photocopying or by other means, in total or in part, at the request of other institutions or individuals for the purpose of scholarly research. Tawfiq Jaber ii ii Abstract Theoretical and Experimental Investigation of Thermodiffusion (Soret effect) in a Porous Medium Doctor of Philosophy TAWFIQ J. JABER Mechanical Engineering Ryerson University 2010 Theoretical and Experimental Investigation of Thermodiffusion (Soret effect) in a Porous Medium Doctor of Philosophy TAWFIQ J. JABER Mechanical Engineering Ryerson University 2010 Doctor of Philosophy TAWFIQ J. JABER Mechanical Engineering Ryerson University 2010 Thermodiffusion (the Soret effect) is important for the study of compositional variation in hydrocarbon reservoirs. The development of research history, theoretical modeling and applications to multicomponent hydrocarbon mixtures is included in this work. The Firoozabadi model appears to be an appropriate model for thermodiffusion estimation for hydrocarbon mixtures, and it is derived based on the equation of entropy generation rate and four postulates in non-equilibrium thermodynamics. Two equations of state, the Peng-Robinson Equation of State (PR-EoS) and the volume translated Peng Robinson Equation of State (vt-PR-EoS), have been used to estimate the thermodynamic properties of mixtures. In this work, different cases are presented: first, a new thermodiffusion cell designed to perform high pressure measurements in a porous medium has been validated at atmospheric pressure. Two systems were investigated, (1) 1,2,3,4-tetrahydronaphtalene (THN) and n-dodecane (nC12), and (2) isobutylbenzene (IBB) and n-dodecane at 50% of mass fraction. Experimental results revealed an excellent agreement with benchmark values and a good agreement with theoretical data. Second, the thermal expansion and concentration expansion coefficients and the viscosity of mixtures are necessary properties for the determination of the thermodiffusion coefficient. The densities of binaries of nC12, IBB and THN for pressures from 0.1 to 20 MPa and a temperature centred on 25°C, were measured. By a derivative method, the thermal expansion and In this work, different cases are presented: first, a new thermodiffusion cell designed to perform high pressure measurements in a porous medium has been validated at atmospheric pressure. Two systems were investigated, (1) 1,2,3,4-tetrahydronaphtalene (THN) and n-dodecane (nC12), and (2) isobutylbenzene (IBB) and n-dodecane at 50% of mass fraction. Experimental results revealed an excellent agreement with benchmark values and a good agreement with theoretical data. Second, the thermal expansion and concentration expansion coefficients and the viscosity of mixtures are necessary properties for the determination of the thermodiffusion coefficient. The densities of binaries of nC12, IBB and THN for pressures from 0.1 to 20 MPa and a temperature centred on 25°C, were measured. By a derivative method, the thermal expansion and iii concentration expansion coefficients were determined. Viscosities were directly measured using a high pressure high temperature viscometer. Theoretical and Experimental Investigation of Thermodiffusion (Soret effect) in a Porous Medium Doctor of Philosophy TAWFIQ J. JABER Mechanical Engineering Ryerson University 2010 Finally, the thermosolutal convections of two ternary mixtures, methane (C1), n-butane (nC4) and n-dodecane (nC12) at a pressure of 35.0 MPa and nC12, THN, and IBB at atmospheric pressure, in a porous medium, were investigated over a wide range of permeability. The effect of permeability in the homogeneous and heterogeneous porous media on fluid transport was studied with consideration of thermodiffusion and molecular diffusion. In the analysis of the homogeneous porous medium, it was found that, for permeability below 300 mD, the thermodiffusion for both mixtures was dominant; and above this level, buoyancy convection became the dominant mechanism. Also, the viscosity was found to influence the evaluation of the molecular and thermodiffusion coefficients. In the case of the heterogeneous porous medium, the impact of the permeability ratio on the composition of the mixture components, velocity in the porous medium and on the separation ratio was investigated. It was found that the heterogeneity of porous medium has a significant influence on the composition of the mixture components. iv iv Acknowledgements I would like to thank Professor Ziad Saghir, my supervisor, for his guidance during my PhD program; I am grateful to him for all precious advices. I would also like to thank Dr. Henri Bataller, who helped me during my experimental part at Laboratoire des Fluides Complexes, Pau University, France. Also, I would like to thank my Supervisory Committee Members: Dr. Jacob Friedman and Dr. Marcello Papini for their valuable suggestions. Special thanks go to Prof. Greg Kawall, the Director of graduate studies. I acknowledge the financial support of the Canadian Space Agency (CSA), the Natural Sciences and Engineering Research Council of Canada (NSERC) and Ryerson University. I would like to express my deepest gratitude to my wife Rihab Jaralla. I thank her for her love, and patience. I would like to dedicate this thesis to my family members, my wife and my children, Mohamad, Ali and Hussain. v v Table of Contents Table of Contents Author’s Declaration .................................................................................................. ii Abstract ..........................................................................................................................iii Acknowledgements...................................................................................................... v Table of Contents........................................................................................................ vi List of Tables ................................................................................................................ ix List of Figures............................................................................................................... xi Nomenclature ............................................................................................................. xiv Chapter 1 ........................................................................................................................ 1 1.1 Introduction............................................................................................................... 1 1.2 Research Objectives.................................................................................................. 2 1.3 Thesis Organization .................................................................................................. 3 1.4 Literature Review...................................................................................................... 4 1.4.1 Theoretical models for thermal diffusion factor ................................................ 4 1.4.2 Experimental methods for thermal diffusion factor......................................... 11 1.5 Basic Concept and Equations for Diffusion............................................................ 14 1.5.1 Fundamental properties of porous media......................................................... 14 1.5.2 Diffusion flux................................................................................................... 16 Chapter 2 ...................................................................................................................... 20 Theoretical Derivation of Firoozabadi Model for Multicomponent Mixtures and Governing Equations in a Porous Medium................................................................... 20 2.1 Introduction............................................................................................................. 20 2.2 Equation of State..................................................................................................... 25 2.2.1 Peng-Robinson Equation of State (PR-EoS).................................................... 26 2.2.2 The volume translated Peng-Robinson Equation of State (vtPR-EoS)............ 28 2.3 Governing equations in porous medium................................................................. 28 2.3.1 The mass continuity equation .......................................................................... 29 2.3.2 Darcy’s law...................................................................................................... 30 2.3.3 The thermal energy conservation equation...................................................... 31 Chapter 3 ...................................................................................................................... 33 vi Measurement of Soret Coefficient in Porous Medium: Comparison with Numerical Model................................................................................................................................ 33 3.1 Introduction............................................................................................................. 33 3.2 Experimental Setup................................................................................................. 34 3.2.1 Experimental description of the apparatus....................................................... 34 3.2.2 Theoretical relations......................................................................................... 41 3.2.3 Analysis approach............................................................................................ 42 3.3 Results and Discussion ........................................................................................... 43 3.3.1 Strategy for the validation of the cell............................................................... 43 3.3.2 Set-up in vertical position................................................................................ 45 3.3.3 Experimental cell set-up in the horizontal position ......................................... 52 3.4 Numerical Model .................................................................................................... 54 3.4.1 Model description ............................................................................................ 54 3.4.2 Mathematical model......................................................................................... 56 3.4.4 Comparison of experimental data with numerical model................................ 57 3.5 Summary................................................................................................................. 62 Chapter 4 ...................................................................................................................... 63 Experimental and Theoretical Estimation of the Thermal Expansion Coefficient, Concentration Expansion Coefficient and Viscosity for Binary Mixtures under Pressure up to 20 MPa................................................................................................................. 63 4.1 Introduction............................................................................................................. 63 4.2 Experimental Setup................................................................................................. 64 4.3 Theoretical Approach.............................................................................................. 71 4.4 Results and Discussion ........................................................................................... 73 4.5 Summary................................................................................................................. 76 Chapter 5 ...................................................................................................................... 77 Soret Effect for a Ternary Mixture in a Porous Cavity: Modeling with Variable Diffusion Coefficients and Viscosity ................................................................................................ 77 5.1 Introduction............................................................................................................. 77 5. 3 Model Description ................................................................................................. 79 5.4 Governing Equations and Boundary Conditions .................................................... 80 5.4 Molecular Diffusion and Thermodiffusion Model ................................................. 82 5.5 Numerical Solution Procedure................................................................................ 87 5.6 Results and Discussion ........................................................................................... 90 5.6.1 Comparison between experimental data and numerical calculation................ 90 5.6.2 Permeability effect on composition distribution.............................................. 91 vii 5.6.3 Viscosity effect on diffusion coefficients ...................................................... 100 5.8 Summary............................................................................................................... 103 Chapter 6 .................................................................................................................... List of Tables Table 3.1: Benchmark values for thermodiffusion coefficient DT, molecular diffusion coefficient D and Soret coefficient T S and contrast factors   T P c n , /   at 1 atm and T = 25 °C. .44 Table 3.2: Measured difference of temperature T, adjustment parameters A, B and C, molecular diffusion coefficient in free liquid D, Soret coefficient ST and (IBB-nC12 50 wt%, Tmean D and Soret coefficient T S and contrast factors   T P c n , /   at 1 atm and T = 25 °C. .44 Table 3.2: Measured difference of temperature T, adjustment parameters A, B and C, molecular diffusion coefficient in free liquid D, Soret coefficient ST and (IBB-nC12 50 wt%, Tmean = 25 °C, o 5 C T   , 1 atm). ........................................................................................48 Table 3.3: Adjustment parameters A, B and C, molecular diffusion coefficient in free liquid D, Soret coefficient ST (IBB-nC12 50 wt%, o o 25 C, 10 C mean T T    , 1 atm)..............51 Table 3.4: Adjustment parameters A, B and C, molecular diffusion coefficient in free liquid D, Soret coefficient ST (THN-nC12 50 wt%., Tmean = 25 °C, o 5 C T   , 1 atm).............52 Table 3.5: Adjustment parameters A, B and C, molecular diffusion coefficient in free liquid D, Soret coefficient ST (IBB-nC12, 50 wt%., Tmean = 25 °C, o 5 C T   , 1 atm, horizontal position). ........................................................................................................................54 Table 3.6: Physical properties of the porous material and fluid mixture, Tmean=25 oC and 1 atm.55 Table 3.7: Model in vertical position, the coefficient values for porous medium, Tmean= 25 oC and 1 atm...............................................................................................................................61 Table 3.8: Theoretical and experimental molecular diffusion and Soret coefficients for three binary mixtures, Tmean= 25 oC and 1 atm (for 1.4 tortuosity 1.4). ..................................61 Table 4.1: Pure component parameters for PR-EoS and vt-PR-EoS.............................................65 Table 4.2: Theoretical and experimental densities for binary mixtures (50 wt% and 25 °C)........74 Table 4.3: Theoretical and experimental thermal expansion coefficient for binary mixtures (50 wt% and 25 °C)..............................................................................................................74 Table 4.4: Theoretical and experimental concentration expansion coefficient for binary mixtures (50 wt% and 25 °C)........................................................................................................75 Table 4.5: Theoretical and experimental viscosities for binary mixtures (50 wt% and 25 °C).....76 Table 5.1: Physical properties of the mixture and porous material, Tave =303 K. .........................80 Table 5.2: Mesh size sensitivity. Table of Contents 105 Three-Dimensional Study of Permeability Effect on Convection in Heterogeneous Porous Medium Filled with a Ternary Hydrocarbon Mixture .................................................... 105 6.1 Introduction........................................................................................................... 105 6.2 Model Description ................................................................................................ 108 6.3 Governing Equations ............................................................................................ 111 6.3.1 Conservation of mass..................................................................................... 111 6.3.2 Conservation of momentum........................................................................... 112 6.3.3 Conservation of energy.................................................................................. 113 6.4 Boundary Conditions ............................................................................................ 114 6.5 Numerical Solution Procedure.............................................................................. 115 6.6 Results and Discussion ......................................................................................... 117 6.6.1 Variation of concentration with permeability................................................ 117 6.6.2 Comparison of flow patterns.......................................................................... 124 6.6.3 Separation ratio.............................................................................................. 127 6.7 Summary............................................................................................................... 129 Chapter 7 .................................................................................................................... 130 Conclusions and Recommendations ............................................................................... 130 7.1 Conclusions........................................................................................................... 130 7.2 Contributions......................................................................................................... 133 7.3 Recommendations................................................................................................. 134 Appendix A................................................................................................................. 145 Custom-Made Software Package.................................................................................... 145 Appendix B................................................................................................................. 148 The measurement value of the Density of the three binary Mixtures......................... 148 Appendix C................................................................................................................. 161 Physical and Thermodynamic Properties of Pure Hydrocarbon Components............ 161 viii List of Tables ...................................................................................................89 Table 5.3: The thermodiffusion coefficients (experimental and theoretical) of nC12-THN-IBB ternary mixture (33.3-33.3-33.4 wt%) at 25 oC and 1 atm.............................................90 Table 6.1: Physical properties of the fluid mixture and porous material at T0=303.15 K and p0=1 atm................................................................................................................................110 Table 6.2: Physical properties of the mixture components at T0=303.15 K................................110 Table 3.2: Measured difference of temperature T, adjustment parameters A, B and C, molecular diffusion coefficient in free liquid D, Soret coefficient ST and (IBB-nC12 50 wt%, Tmean ix Table B.1: Experimental densities of the binary IBB-nC12 (50 wt% mass fraction) as function of the temperature and the pressure..................................................................................149 Table B.2: Experimental densities of the binary THN-nC12 (50 wt% mass fraction) as function of the temperature and the pressure. ............................................................................151 Table B.3: Experimental densities of the binary THN-IBB (50 wt% mass fraction) as function of the temperature and the pressure..................................................................................153 Table B.4: Experimental densities of the binary IBB-nC12 (T0=25 oC) as function of the mass fraction of the densest component and the pressure.....................................................155 Table B.5: Experimental densities of the binary THN-nC12 (T0=25 oC) as function of the mass fraction of the densest component and the pressure.....................................................157 Table B.6: Experimental densities of the binary THN-IBB (T0=25 oC) as function of the mass fraction of the densest component and the pressure.....................................................159 Table C.1: Pure hydrocarbon components parameters which used in this work Tmean=25 oC, (Daubert and Danner 1989)..........................................................................................161 x x List of Figures Figure 3.1: Experimental setup. M1, M2 and M3 are mirrors; BS1 and BS2 are beam-splitters; V1, V2, V3 and V4 are valves used for the filling of the cell. ...............................................36 Figure 3.2: Cell design and geometrical configuration. ................................................................37 Figure 3.3: Photo of the porous medium used...............................................................................38 Figure 3.4: Interferogram recorded with a) the cell in horizontal position and b) the cell in vertical position..............................................................................................................38 Figure 3.5: Gray level and adjustment of the first 150 pixels of the line 500 of an interferogram (THN/nC12 mixture 50 wt%; Tmean = 25 °C; o 3 C   T ; time = 512 s).......................42 Figure 3.6: Temperature versus time in dead-volumes for the system IBB-nC12 (50 wt%, Tmean= 25 °C, 5 T   oC , 1atm, vertical position). ..................................................................45 Figure 3.7: Phase difference versus time (nC12-IBB, 50 wt%., Tmean = 25 °C, 5 T   oC, 1 atm, vertical position ). ..........................................................................................................47 Figure 3.8: Second kinetic of the phase difference evolution for different durations of experiments (IBB-nC12, 50 wt%, 5 T   oC, 1 atm, vertical position ).......................47 Figure 3.9: Temperature versus time in dead-volumes for the system IBB-nC12 (50 wt%, Tmean= 25 °C, o 10 C T   , 1atm, vertical position)..................................................................49 Figure 3.10: Phase difference versus time (nC12-IBB, 50 wt%, Tmean = 25 °C, T   5 oC, 1 atm, vertical position). ...........................................................................................................50 Figure 3.11: Second kinetic of the phase difference evolution for different durations of experiments (IBB-nC12, 50 wt%, o 10 C T   , 1 atm, vertical position ). ...............…50 Figure 3.12: Second kinetic of the phase difference evolution for different durations of experiments (THN-nC12, 50 wt%, T  5 oC, 1 atm, vertical position )......................51 Figure 3.13: Interferogram (IBB-nC12, 50 wt%, Tmean = 25 °C, o 5 C T   , 1 atm, horizontal position) images time recorded for the cell: a) 0 s, b) 1 min and c) 10 min.................53 Figure 3.14: Second kinetic of the phase difference evolution for different durations of experiments (IBB-nC12, 50 wt%, o 5 C T   , 1 atm, horizontal position ).................53 Figure 3.15: Schematic diagram of the porous medium and the boundary conditions. List of Figures ................55 Figure 3.16: Temperature distribution along the vertical position................................................57 Figure 3.17: THN mass fraction distributions in the centre of the cavity along the vertical position THN nC12 mixture 59 xi Figure 3.18: nC12 mass fraction distributions in the centre of the cavity along the vertical position, THN-nC12 mixture .........................................................................................59 Figure 3.19: Density distribution for three binary mixtures in the centre cavity along the vertical direction. ........................................................................................................................60 Figure 4.1: Density of IBB-nC12 mixture as a function of the temperature and for different pressures (0.1-20 MPa). .................................................................................................68 Figure 4.2: Density of IBB-nC12 mixture at 25°C as function of the mass fraction of the densest component and for different pressures (0.1-20 MPa). ...................................................69 Figure 4.3: Density of THN-nC12 mixture at 25°C as a function of the temperature and for different pressures (0.1-20 MPa)....................................................................................69 Figure 4.4: Density of the THN-nC12 mixture at 25°C as function of the mass fraction of the densest component and for different pressures (0.1-20 MPa)........................................70 Figure 4.5: Density of the THN-IBB mixture at 25°C as a function of the temperature and for different pressures (0.1-20 MPa)....................................................................................70 Figure 4.6: Density of THN-IBB mixture at 25°C as function of the mass fraction of the densest component and for different pressures (0.1-20 MPa). ...................................................71 Figure 5.1: Schematic diagram of the horizontal porous cavity and boundary conditions ...........79 Figure 5.2: Nusselt number with mesh size...................................................................................89 Figure 5.3: Density of nC12-IBB-THN mixture vs. temperature..................................................91 Figure 5.4: Methane (C1) mole fraction distributions along the centre of the cavity. ..................92 Figure 5.5: n-butane (nC4) mole fraction distributions along the centre of the cavity .................93 Figure 5.6: C1/nC4 compositional distributions in the centre x-z plane of the C1-nC4-nC12 ternary mixture...............................................................................................................97 Figure 5.7: nC12/THN compositional distribution in the centre x-z plane of the nC12-THN-IBB ternary mixture...............................................................................................................98 Figure 5.8: Permeability vs. separation ratio of C1-nC4-nC12.....................................................99 Figure 5.9: Permeability vs. separation ratio of the nC12-THN-IBB ternary mixture..................99 Figure 5.10: Viscosity variation with temperature of n-dodecane and n-butane.........................101 Figure 5.11: Thermodiffusion coefficient distributions for methane and n-butane along the horizontal direction (κ =102 mD).................................................................................102 Figure 5.12: Variation of the molecular diffusion coefficient of methane along the horizontal cavity (κ =102 mD). .....................................................................................................103 Figure 6.1: Schematic diagram of the rectangular porous medium and boundary conditions. List of Figures ...108 Figure 6.2: Heterogeneous porous medium configurations, x-z plane........................................109 xii Figure 6.3: n-dodecane (nC12) mole fraction along the horizontal direction ( / 10 /10 f s  κ κ ), Conf.1...........................................................................................................................119 Figure 6.4: n-dodecane (nC12) mole fraction along the horizontal direction ( / 10 /10 f s  κ κ ), Conf.2...........................................................................................................................119 Figure 6.5: THN mole fraction along the horizontal direction ( / 10 /10 f s  κ κ ), Conf.1.........120 Figure 6.6: THN mole fraction along the horizontal direction ( / 10 /10 f s  κ κ ), Conf.2.........120 Figure 6.7: n-dodecane (nC12) mole fraction along the horizontal direction ( / 10000 /10 f s  κ κ ), Conf.1. ....................................................................................122 Figure 6.8: n-dodecane (nC12) mole fraction along the horizontal direction ( / 10000 /10 f s  κ κ ), Conf.2. ....................................................................................122 Figure 6.9: THN mole fraction along the horizontal direction ( / 10000 /10 f s  κ κ ), Conf.1...123 Figure 6.10: THN mole fraction along the horizontal direction ( / 10000 /10 f s  κ κ ), Conf.2.123 Figure 6.11: The magnitude of velocity a long the horizontal direction ( / 10 /10 f s  κ κ ), Conf.1. ......................................................................................................................................125 Figure 6.12: The magnitude of velocity a long the horizontal direction ( / 10 /10 f s  κ κ ), Conf.2. ......................................................................................................................................125 Figure 6.13: The magnitude of velocity a long the horizontal direction ( / 10000 /10 f s  κ κ ), Conf.1...........................................................................................................................126 Figure 6.14: The magnitude of velocity a long the horizontal direction ( / 10000/10 f s  κ κ ), Conf.2...........................................................................................................................126 Figure 6.15: Separation ratio as a function of the permeability ratio, Conf.1.............................128 Figure 6.16: Separation ratio as a function of the permeability ratio, Conf.2.............................129 Figure A.1: Front-screen of the LabView-Refr_In software.......................................................147 Figure B.1: Experimental setup of DMA HPM, Israfilov et al. (2009). .....................................148 xiii Nomenclature p C The heat capacity at constant pressure, [J. Nomenclature kg-1 K-1] D The molecular or Fick’s diffusion coefficient in a free fluid, [m2.s-1] * D The molecular diffusion coefficient in a porous medium, [m2.s-1] T D The thermodiffusion coefficient in a free fluid, [m2.s-1 K-1] * T D The thermodiffusion coefficient in a porous medium, [m2.s-1 K-1] ij D The Maxwell-Stephan diffusion coefficient, [m2.s-1] if The fugacity of component i, [N.m-2] g The gravitational acceleration [m/s2] i H The partial molar enthalpy of component i, [J/mol] I The light intensity iJ The molar diffusive flux of component i, [ 2 mole/m /s ] ij The mass diffusion flux of component i, [ 2 kg/m /s] mi J  The molar diffusion flux vector [mole.m-2.s-1] k The thermal conductivity [W.m-1.K-1] ik The wave number of the beam with refractive index ni ijk The interaction parameter between the ith and jth components Lij The Onsager coefficient or the phenomenological coefficient M The molecular weight of the mixture, [kg/kmol] i M The molecular weight of component i, [kg/kmol] mD The milli-darcy (1 mD= 9.87x10-16 m2) is a unit of permeability Nu The Nusselt number, [-] n The number of components in the mixture [-] P The pressure, [Pa] Pc The critical pressure, [Pa] i Q The heat of transport [J/mol] * i Q The net heat of transport [J/mol] xiv q The separation ratio [-] R The universal gas constant, 8.314 [J/mol/K] T S The Soret coefficient, [K-1], vis The shift parameter of component i in vtPR-EoS T The temperature [K] cT The critical temperature, [K] rT The reduced temperature, [-] t Time [s] i U The partial molar internal energy of component i, [J/mole] u The velocity component in x-direction [m.s-1] v The velocity component in y-direction [m.s-1] V  The fluid velocity vector (u, v, w) [m.s-1] w The velocity component in z-direction [m.s-1] x The spatial coordination in x-direction [m] ix The molar fraction of component i, [-] y The spatial coordination in y-direction [m] Z The compressibility factor [-] z The spatial coordination in z-direction [m] q The separation ratio [-] R The universal gas constant, 8.314 [J/mol/K] T S The Soret coefficient, [K-1], vis The shift parameter of component i in vtPR-EoS T The temperature [K] cT The critical temperature, [K] rT The reduced temperature, [-] t Time [s] i U The partial molar internal energy of component i, [J/mole] u The velocity component in x-direction [m.s-1] v The velocity component in y-direction [m.s-1] V  The fluid velocity vector (u, v, w) [m.s-1] w The velocity component in z-direction [m.s-1] x The spatial coordination in x-direction [m] ix The molar fraction of component i, [-] y The spatial coordination in y-direction [m] Z The compressibility factor [-] z The spatial coordination in z-direction [m] Greek Symbols T  The thermal diffusion factor, (-) c  The coefficient of concentration expansion [-] T  The Coefficient of thermal expansion [1/K] vap i U  The cohesive energy (also called the vaporization energy), [J/mol] visc i U  The viscous energy, [J/mol]  The porosity [-]  The phase difference between the two laser beams at the point of th Greek Symbols Greek Symbols T  The thermal diffusion factor, (-) c  The coefficient of concentration expansion [-] T  The Coefficient of thermal expansion [1/K] vap i U  The cohesive energy (also called the vaporization energy), [J/mol] visc i U  The viscous energy, [J/mol]  The porosity [-]  The phase difference between the two laser beams at the point of the interference field, [rad] κ The permeability [m2] xv xv k The wave number of the beams in air k  The chemical potential of component k, [J/mol]  The dynamic viscosity of the fluid mixture [kg.m-1.s-1]  The fluid mixture density [kg/m3] m  The molar density of fluid mixture [kmole/m3]  The rate of entropy production, [J/mol/s]  The mass fraction [-] w The pulsation of the two laser beams ω The acentric factor [-]  The mixture viscosity parameter r The relaxation time, [s] i The ratio of cohesive energy and viscous energy i  The volume fraction of molecules moving into a hole left by a molecule of type i Subscripts e Effective fl Fluid mixture m Molar quantities, s Porous media or matrix xvi 1.1 Introduction In a fluid mixture, diffusion is the general term used to describe the motion of one species with respect to another. The thermodiffusion phenomenon, also known as the Soret effect ( T S ), is a coupling between a temperature gradient and its resulting mass flux in a multicomponent system. The Soret effect was discovered by German physicist Carl Ludwig (Ludwig, 1856) and later more deeply analysed by Swiss physicist Charles Soret (Soret, 1879 and 1880). This phenomenon appears in a wide range of physical and chemical processes in nature, and researchers have contributed to its study over the years, including studies of oil reservoir analysis, tar sand extraction, fluid separation, distillation, material processing and biotechnological applications. Comprehensive diffusion theories and accurate diffusion experiments are of great importance to the optimization of diffusion-dominant industrial processes. It is interesting to note that the thermodiffusion and molecular diffusion (also called Fickian diffusion, Fick 1855) are coupled processes; molecular diffusion tends to decrease a composition gradient which was created due to the thermodiffusion. This work concentrates on the study of hydrocarbon mixtures (binary and ternary) in porous media. A new thermodiffusion cell designed to measure the molecular diffusion coefficient and the Soret coefficient of hydrocarbon binary systems in a porous medium, which is based on optical digital interferometry, is presented. Also, the most recently developed non-equilibrium thermodynamics model by Firoozabadi has been applied. The three dimensional study of the thermodiffusion process in porous media is the most realistic study, which will lead to the most complete understanding of the natural laws applicable to the thermodiffusion process. The thermodiffusion in multicomponent systems, like ternary mixtures, is much more complicated than in binary mixtures for both experimental and theoretical work. There have been extensive experimental and theoretical studies of the molecular diffusion 1 1 coefficient in binary mixtures in the literature. Experimental data on the binary thermodiffusion coefficient in porous media are much scarcer than for molecular diffusion. On the multicomponent thermodiffusion in liquid, very few models have been published. The Shukla-Firoozabadi (2000) multicomponent model (also known as the Firoozabadi model), based on their binary mixture model, is adopted to carry out a comparison with some latest results by Platten et al. (2004) and Leahy-Dios et al. (2005) by using two equations of state: Peng-Robinson and volume translated Peng-Robinson, respectively. 1.2 Research Objectives The main objectives in this research are as follows: 1) To study and derive the Firoozabadi model for multicomponent mixtures, and obtain full understanding of this model and its application. 1) To study and derive the Firoozabadi model for multicomponent mixtures, and obtain full understanding of this model and its application. 2) To measure molecular diffusion and thermodiffusion coefficients of binary mixtures of n-dodecane, isobutylbenzene and 1,2,3,4-tetrahydronaphthalene using an optical digital interferometer, to compare the results with benchmark values and theoretical values as well, and to prove the reliability of the digital interferometry technique. The theoretical values are determined by a computer code that can be used to simulate a thermodiffusion process as a function of temperature, pressure, and composition in a fluid mixture to investigate the Soret effect, and a FORTRAN program (in-house code) applying Firoozabadi’s model was developed. 3) To numerically simulate the thermodiffusion for a hydrocarbon ternary mixture in a porous medium by applying different parameters such as the viscosity effect and a different range of permeabilites. 4) To simulate the thermodiffusion in a heterogeneous porous medium by applying different fracture orientations and study the effect of multi-permeability and multi-porosity on the mixture behaviour. 2 2 1.4.1 Theoretical models for thermal diffusion factor In the thermodynamics of an irreversible process, there are two energy quantities: the heat of transport, i Q , which is the amount of energy transported across a given reference plane per mole of the ith component. The heat of transport is interpreted as the energy difference between the energy required when a molecule of component i moves out of its position in a temperature field and the energy given up when its left hole is filled by one of its neighbour molecules. * i Q is the net heat of transport, (de Groot 1945), which is defined by: * i i i Q Q H   (1.1) where i H is the partial molar enthalpy of the ith component in the solution. In terms of the heat of transport, the flux equation for the ith component in a multicomponent constant pressure system (Dougherty and Drickamer 1955) is given by: where i H is the partial molar enthalpy of the ith component in the solution. In terms of the heat of transport, the flux equation for the ith component in a multicomponent constant pressure system (Dougherty and Drickamer 1955) is given by:   1 1 1 n n k i ik k k i k i i T J L Q H x T x                    (1.2) (1.2) where iJ is the flux of the component i, k is the chemical potential of component k, T is the absolute temperature, ix is the mole fraction of component i, and ik L are phenomenological coefficients. For a binary mixture, the composition gradient resulting from a temperature gradient in the steady state ( iJ ) for the first component is given by: where iJ is the flux of the component i, k is the chemical potential of component k, T is the absolute temperature, ix is the mole fraction of component i, and ik L are phenomenological coefficients. 1.3 Thesis Organization This thesis consists of seven chapters and is organized as follows: This thesis consists of seven chapters and is organized as follows:  Chapter 1 presents an introduction to the thermodiffusion, literature review and the basic concept and diffusion equations.  In Chapter 2, the theoretical derivation of the Firoozabadi model, that has been used to estimate the Soret coefficient for a multicomponent mixture, is presented; the governing equations for porous media are explained; these equations are mass continuity, Darcy’s law and thermal energy conservation.  In Chapter 3, the experimental technique used to measure the molecular diffusion coefficient and Soret coefficient for binary hydrocarbon fluid mixtures in a porous medium is presented and experimental data are discussed and compared with the benchmark values and numerical results.  In Chapter 4, the comparison between experimental and theoretical estimation of the thermal expansion coefficient, concentration expansion coefficient, density and viscosity for three binary hydrocarbon mixtures under pressure up to 20 MPa are described and discussed.  In Chapter 5, the theoretical model of thermosolutal convections of two hydrocarbon ternary mixtures in a homogenous porous medium under different permeabilities is carried out and discussed.  In Chapter 6, a three-dimensional study of the permeability effect on convection in a heterogeneous porous medium filled with a ternary hydrocarbon mixture is described and discussed.  Finally, Chapter 7 presents the conclusions and recommendations. 3 1.4.1 Theoretical models for thermal diffusion factor For a binary mixture, the composition gradient resulting from a temperature gradient in the steady state ( iJ ) for the first component is given by:     * 1 1 1 1 1 1 1 1 1 1 1 / / x Q H T Q T x x x T x x T             (1.3) (1.3) where Eq. 1.1 has been used. In the same manner, the second component is: where Eq. 1.1 has been used. 1.4.1 Theoretical models for thermal diffusion factor In the same manner, the second component is: 4   * 1 2 2 2 2 1 / x Q T x x x T       (1.4) (1.4) using Eqs 1.3 and 1.4, with the Gibbs-Duhem relations in the form: using Eqs 1.3 and 1.4, with the Gibbs-Duhem relations in the form: 1 2 1 2 1 1 * * 1 1 2 2 0 0 x x x x x Q x Q           (1.5) 1 2 1 2 1 1 * * 1 1 2 2 0 0 x x x x x Q x Q           (1.5) A relation between the concentration gradient and the temperature gradient at steady state is obtained as follows: A relation between the concentration gradient and the temperature gradient at steady state is obtained as follows:   * * 1 2 1 1 2 1 1 1 / x Q Q T x x x x T        (1.6)   * * 1 2 1 1 2 1 1 1 / x Q Q T x x x x T        (1.6) For a binary system, the conventional flux equation may be written as follows: For a binary system, the conventional flux equation may be written as follows:   1 1 1 2 1 1 2 m T m T T J D x x x D x S x x T T                  (1.7) (1.7) where m  is the molar density (mole/m3), D is the molecular diffusion coefficient, T T TS  is the thermal factor, and T S is the Soret coefficient. For steady state ( 1 2 0 J J   ), from Eq. 1.7, one can obtain another relation between the composition and temperature gradients: where m  is the molar density (mole/m3), D is the molecular diffusion coefficient, T T TS  is the thermal factor, and T S is the Soret coefficient. For steady state ( 1 2 0 J J   ), from Eq. 1.4.1 Theoretical models for thermal diffusion factor 1.7, one can obtain another relation between the composition and temperature gradients: 1 1 2 T x T x x T     (1.8) When Eq. 1.8 is compared with Eq. 1.6, a new expression for the thermal diffusion factor in terms of the net heat of transport and chemical potential for a binary system is introduced as follows:   * * 2 1 1 1 1 / T Q Q x x       (1.9)   * * 2 1 1 1 1 / T Q Q x x       (1.9)   * * 2 1 1 1 1 / T Q Q x x         * * 2 1 1 1 1 / T Q Q x x       (1.9) Eq. 1.9 is a general expression for the thermal diffusion factor. The thermal diffusion factor can be calculated through two main theoretical models: the phenomenological approach (or thermostatic approach) and the kinetic approach, which have been the basis of modeling the thermal diffusion factor in binary mixtures. Both approaches are based on the postulates in phenomenological theory of non-equilibrium thermodynamics. The theory of non-equilibrium thermodynamics is extended from equilibrium thermodynamics by introducing the following four postulates (Curie’s postulates), Bird et al. (2002): 1. The equilibrium thermodynamics relations apply to systems that are not in equilibrium, provided that the gradients are not too large (Quasi-equilibrium postulate). 2. All fluxes in the system may be written as linear relations involving all the forces (Linear postulate). 3. No coupling of fluxes and forces occurs if the difference in tensorial order of the flux and force is an odd number (Curie’s postulate). 4. In the absence of magnetic fields, the matrix of the coefficients in the flux-force relations is symmetric (Onsager’s reciprocal relations). Both the Haase model and the Kempers model use the thermostatic approach. The Haase model (1950) is based on the phenomenological theory, and the net heat of transport is interpolated with the partial molar enthalpy. 1.4.1 Theoretical models for thermal diffusion factor Based on this assumption the net heat of transport for a binary mixture is given by: 6 * * 1 2 2 1 2 1 1 1 2 2 2 1 - M M H H Q Q M x M x M M          (1.10) (1.10) where i H is the partial molar enthalpy of component i, and i M is the molecular weight of component i. By substituting Eq. 1.10 into Eq. 1.9, the thermal diffusion factor is expressed as: where i H is the partial molar enthalpy of component i, and i M is the molecular weight of component i. By substituting Eq. 1.10 into Eq. 1.9, the thermal diffusion factor is expressed as:     1 2 2 1 1 1 2 2 1 1 1 / H T M H M H M x M x x x        (1.11)     1 2 2 1 1 1 2 2 1 1 1 / H T M H M H M x M x x x        (1.11)    1 2 2 1 1 1 2 2 1 1 1 / M x M x x x     (1.11) The thermal diffusion factor for an ideal fluid at standard state is given as: The thermal diffusion factor for an ideal fluid at standard state is given as: The thermal diffusion factor for an ideal fluid at standard state is given as:   1 2 2 1 1 1 2 2 o o o T M H M H RT M x M x     (1.12)   1 2 2 1 1 1 2 2 o o o T M H M H RT M x M x     (1.12)   1 1 2 2 T RT M x M x   For an ideal fluid, the following relation   1 / i i x x RT     pertains, where R is the gas constant. The Haase model can be re-written as: For an ideal fluid, the following relation   1 / i i x x RT     pertains, where R is the gas constant. 1.4.1 Theoretical models for thermal diffusion factor The Haase model can be re-written as:           1 1 2 2 1 2 2 2 1 1 1 1 2 2 1 1 1 / o o o T H T RT M x M x M H H M H H M x M x x x             (1.13) (1.13) In 1989, Kempers proposed a thermodynamics theory of the thermal diffusion factor. This model was based on a statistical description of a two-bulb system. He obtained an expression for T by maximizing the partition function in the canonical ensemble of an idealized dual subsystem in a steady state; the net heat of transport and the thermal diffusion factor (for binary mixture) in Kempers model are expressed as: In 1989, Kempers proposed a thermodynamics theory of the thermal diffusion factor. This model was based on a statistical description of a two-bulb system. He obtained an expression for T by maximizing the partition function in the canonical ensemble of an idealized dual subsystem in a steady state; the net heat of transport and the thermal diffusion factor (for binary mixture) in Kempers model are expressed as: * * 1 2 2 1 2 1 1 1 2 2 2 1 VV H H Q Q V x V x V V           * * 1 2 2 1 2 1 1 1 2 2 2 1 VV H H Q Q V x V x V V           (1.14) (1.14)    1 2 2 1 1 1 1 1 2 2 / K T V H V H x x V x V x        (1.15) where iV is the partial molar volume of component i.    1 2 2 1 1 1 1 1 2 2 / K T V H V H x x V x V x        (1.15) where iV is the partial molar volume of component i. where iV is the partial molar volume of component i. where iV is the partial molar volume of component i. 1.4.1 Theoretical models for thermal diffusion factor Both models (Haase and Kempers) have been tested previously for a few hydrocarbon mixtures using the Soave-Redlich-Kwong Equation of State (SRK-EoS), Soave (1972), and Peng-Robinson Equation of State, (Peng and Robinson, 1976). It is found that the comparisons of theoretical results with experimental data were found to be qualitative. In the kinetic approach, the heat of transport is interpolated with the activation energy of molecular motion, which can be obtained from the viscosity of the mixture. Several expressions for the thermal diffusion factor can be obtained. For example, Dougherty and Drickamer (1955) obtained T  in terms of the activation energies for molecular movements, given as:       * * 1 2 2 1 2 2 1 1 1 1 1 1 1 2 2 / / / 2 DD T M V M V U V U V x x M x M x          (1.16) (1.16) where * i U  is the activation energy for molecular movement of component i, and is a function of viscosity and thermodynamic properties of the pure component. The comparison between the theoretical results for DD T  with the experimental data has been shown that the Dougherty-Drickamer model does not perform better than the other two models as shown above. And this model suffers from some shortcomings; for instance, thermodynamic properties of the components were obtained from the approximate Scatchard-Hildebrand theory (Hildebrand and Scott, 1950). It is known that Scatchard-Hildebrand theory is unreliable in dealing with non-ideal mixtures under different conditions. 8 8 A new model is based on the method of irreversible thermodynamics suggested by Shukla and Firoozabadi (1998) which is based on the Dougherty-Drickamer model. They use more accurate thermodynamic properties of mixtures obtained from the volume- translated Peng-Robinson Equation of State. By following the physical interpretation of Dougherty and Drickamer (1955), it is not possible to directly evaluate the net heat of transport. 1.4.1 Theoretical models for thermal diffusion factor Therefore, the probable energy, L W , supplied to fill a hole left by a detached molecule is given by: 1 1 2 2 L H H W xW x W   (1.19) (1.19) 1 1 2 2 L H H W xW x W   with 1 1 1 2 2 2 H H U W U W     1 1 1 2 2 2 H H U W U W     (1.20) (1.20) 2 2 2 H W   where i U is the partial molar internal energy of component i, and i is the ratio of the energy of vaporization, Vap i U  , to the energy of viscous flow (or defined as the energy of activation for viscous flow) , visc i U  (Glasstone et al., 1941) 9 9 i  can be evaluated in terms of the partial molar volume and mole fraction as follows:   1 1 1 2 2 / V xV x V       1 1 1 1 2 2 2 2 1 1 2 2 / / V xV x V V xV x V       (1.21)   1 1 1 1 2 2 / V xV x V   (1.21)   1 1 2 2 / xV x V  (1.21)   (1.   2 2 1 1 2 2 / V xV x V   (1.21) (   2 2 1 1 2 2 / V xV x V   with the relation (satisfying the Gibbs-Duhem relation): with the relation (satisfying the Gibbs-Duhem relation): 1 1 2 2 1 x x     (1.22) 1 1 2 2 1 x x     2 1  (1.22) (1.22) 1 1 2 2 1 x x     2 2 1  (1.22) using Eqs. 1.17-1.23 along with Eq. 1.9 the thermal diffusion factor is defined as: using Eqs. 1.17-1.23 along with Eq. 1.9 the thermal diffusion factor is defined as: using Eqs. 1.17-1.23 along with Eq. 1.4.1 Theoretical models for thermal diffusion factor Shukla and Firoozabadi relate it to the energy of detaching a molecule from its neighbours in the region of the mixture ( i H W ), and to the energy given up in that region when one molecule fills a hole ( L W ), as follows: * 1 1 1 H L Q W W    (1.17) * 2 2 2 H L Q W W    (1.18) (1.17) * 1 1 1 H L Q W W    (1.18) * 2 2 2 H L Q W W    where i is the volume fraction of molecules moving into a hole left by a molecule of type i in the mixture. Note that molecules may have different sizes and shapes. And the distribution of molecules to occupy holes in the mixture is completely random. Therefore, the probable energy, L W , supplied to fill a hole left by a detached molecule is given by: where i is the volume fraction of molecules moving into a hole left by a molecule of type i in the mixture. Note that molecules may have different sizes and shapes. And the distribution of molecules to occupy holes in the mixture is completely random. 1 C1 is methane, C3 is propane, C4 is butane, C7 is heptane, nC12 is n-dodecane, and C16 is Hecdecane also called Hexadecane 1.4.1 Theoretical models for thermal diffusion factor 1.9 the thermal diffusion factor is defined as:           1 1 2 2 2 1 1 1 1 2 2 2 1 1 1 1 1 1 2 2 / / / / / / F T U U V V xU x U x x x x V x V x                  (1.23) (1.23) Eq. 1.23 represents the thermal diffusion factor for a binary mixture in the Shukla- Firoozabadi model, and it is an explicit function of partial molar internal energies and volumes, energy of vaporization and viscous flow, and chemical potential. Shukla and Firoozabadi (1998) applied this model to hydrocarbon systems, i.e., 1C1/C3, C1/C4, C7/nC12, and C7/C16; non-polar non-hydrocarbon systems, i.e., Ar/CO2, N2/CO2, H2/N2, and H2/CO2; hydrocarbon and non-hydrocarbon systems, i.e., C1/N2 and C1/CO2. They found that comparisons of theoretical results with experimental data show a good agreement. In particular, the predicted sign of the thermal diffusion factor is consistent with experimental observations in the mixtures. In their work, the equilibrium properties of mixtures were obtained from the volume translated Peng-Robinson Equation of State (vtPR-EoS), and the values of iof different components are assumed to be equal for all fluids presented. From the viscosities of pure components, the values iappear to vary 10 from 3.5 to 5; Shukla and Firoozabadi adopted the universal values for i ( 1 2 4, 4     ) for all fluids presented. Firoozabadi et al. (2000), and Ghorayeb and Firoozabadi (2000) has developed their model from binary to multicomponent mixtures. They proposed the following expression for the net heat of transport for the ith component in an n-component mixture: * 1 1 1,........,                    n j j i i i n j i j j j j x U U V Q i n x V (1.24) (1.24) The Firoozabadi model is considered to be the first model dealing with thermodiffusion in multicomponent mixtures. Pan et al. (2007) compared different multicomponent thermodiffusion models and concluded that the Firoozabadi model was the best in general. This model will be explained in detail in Chapter 2. 1.4.1 Theoretical models for thermal diffusion factor As far as we know, few simulations dealing with thermodiffusion in multicomponent mixtures have been reported. For now, only two models are available, which have been developed by Kempers (1989 and 2001) and Firoozabadi et al., (2000). 1.4.2 Experimental methods for thermal diffusion factor The Soret effect can be measured by using different techniques which have been divided into groups (Platten, 2006). The first group of techniques are the convection free systems and the second group of techniques use convective coupling. In the first group, at steady state and in absence of convection, the thermodiffusion is balanced by molecular diffusion resulting in zero diffusion flux ( 0 iJ  ) as shown in the following equations:  1 2 (1 )     i T x D x x T (1.25)   1 2 (1 )       i m i T J D x D x x T (1.25) 11 11   1 1 1        i T T T x D S T D x x T (1.26) (1.26)   1 1 1        i T T T x D S T D x x T   1 1 1      T S T D x x T The first group include three different techniques to measure the thermodiffusion or Soret effect: The first group include three different techniques to measure the thermodiffusion or Soret effect: (i) Thermal Diffusion Forced Rayleigh Scattering (TDFRS). (ii) Standard Soret Cell (SSC). (iii) Laser Beam Deflection Technique (LBD). In the TDFRS technique, (Kohler and Muller, 2002; Wiegand and Kohler, 2002; Wiegand, 2004; Polyakov et al., 2006), two laser beams intersect within the sample, which is a fluid mixture in a glass container, and create a holographic interference grating. At the intersection of the two laser beams, interference fringes are created. And by putting some chemically inert dye in the mixture, energy is absorbed from the light field and the sample is heated with the spatial periodicity of the grating. Typically, the amplitude of the temperature grating is of the order of 10 to 100 μK . The temperature gradients within this thermal grating give rise via the Soret effect to a build-up of a concentration modulation with the same spatial periodicity. Both the temperature and the concentration grating are accompanied by a refractive index grating. The Soret coefficient can be measured from the resulting experimental data. The SSC, also called a thermal diffusion cell (Costeseque et al., 2004), consists of two horizontal rigid plane plates (e.g., copper or stainless steel). 1.4.2 Experimental methods for thermal diffusion factor Both plates are maintained at different temperatures in order to create a vertical temperature gradient. The system usually is heated from above in order to avoid free convection. Both the Soret coefficient and molecular diffusion coefficient can be obtained by measuring the concentration changes caused by the temperature gradient. The LBD use the same type of cell as described in above. The main difference being that two opposite lateral walls are made of glass of good optical quality. Kolodner et al. 12 (1988) and Zhang et al. (1996) applied this technique to measure the Soret effect for a binary mixture of water-ethanol. In the second group of techniques (Convection-coupled), the idea is to study the change of the velocity field under the effect of thermodiffusion. Therefore, the important parameter is the solutal contribution to the buoyancy force. A Thermogravitational Column (TGC) is an example of the second group method (Bou-Ali et al., 1998, Dutrieux et al., 2002, Leahy-Dios et al., 2005 and Haugen and Firoozabadi, 2007). The TGC method usually consists of two vertical concentric cylinders at two different temperatures in such a way as to create a horizontal temperature gradient. The liquid mixture is placed in a small gap between two vertical walls. Since the temperature gradient is horizontal, under the action of the Soret effect one component is displaced to the cold side and the other to the hot side. Due to the convection, the one at the cold side is transported to the bottom and the one that goes to the hot side is transported to the top of the cell. Thus, the combined effect of thermodiffusion and convection finally creates a vertical mass fraction gradient. The Soret coefficient can be obtained by measuring the concentration difference between the bottom and the top. The separation rate in this method is defined as the concentration difference between the top and the bottom cell. Platten et al. (2003) showed that the molecular separation or the difference in mass fraction between the top and bottom in a thermogravitational column can be substantially increased by inclining the column by an angle. In addition, the TGC can be used for porous media (Costesque et al., 1994 and Costeseque and Loubet, 2003) and ternary mixtures, (Leahy-Dios et al., 2005). Costeseque et al. (2004) conducted experiments in both a free fluid and a porous medium with a vertical temperature gradient. Porosity The porosity of a porous media, which is defined as the ratio of the volume of the pores (void spaces) to the total bulk volume of the media, is given by: p P s V V V   (1.27) p P s V V V   (1.27) (1.27) where  is the porosity, p V is the void volume or pore volume, and s V is the solid volume or matrix volume. where  is the porosity, p V is the void volume or pore volume, and s V is the solid volume or matrix volume. 1.4.2 Experimental methods for thermal diffusion factor It was found that, when the thermal conductivities of the fluid and solid matrix are of the same magnitude, the Soret coefficients do not differ significantly with respect to the case of the free fluid. Several researchers have published values of the Soret coefficient for organic molecules, polymers, and even electrolyte solutions. In particular, in 1999, research groups from different universities, who met at Fontainebleau, France, started a ground-based measurement campaign. The goal was to establish a reliable database of the Soret coefficient for three binary mixtures (Fontainebleau mixtures) composed of n-dodecane (nC12), Isobutylbenzene (IBB), and 13 1,2,3,4-tetrahydrophtalene (THN). As well, there was a clear desire to establish benchmark values for the Soret, thermal diffusion, and molecular diffusion coefficients so that they could be used to compare earth based results with results obtained through other means such as experiments in microgravity or numerical simulation. The values obtained from independent researchers were in strong agreement with each other, (Platten et al., 2003). 1.5.1 Fundamental properties of porous media A porous medium can be defined as a multiphase material, which is a solid containing void spaces (pores), either connected or unconnected. In petroleum engineering, a porous medium, also called reservoir rock, stores crude oil and natural gas. Permeability The permeability of a porous media can be defined as a measure of the connectivity of pore spaces. Mathematically, it is defined as the constant of proportionality in Darcy’s law. A micro scale measurement of grain size distribution shows that different grain sizes 14 and shapes affect permeability. A traditional unit for permeability is the Darcy (D), or more commonly the millidarcy (mD) (1 Darcy=9.86923x10−13m2). and shapes affect permeability. A traditional unit for permeability is the Darcy (D), or more commonly the millidarcy (mD) (1 Darcy=9.86923x10−13m2). Tortuosity The tortuosity () is a fundamental property of the porous media and is given as a ratio of effective average path (actual path), Le, of a fluid particle and the corresponding straight and shortest external distance (length between two points, L): eL L  (1.28) (1.28) Also the tortuosity refers to the link between diffusion coefficients in free layers and in the porous media, (Nield and Bejan, 2006; Bear, 1972; de Marsily, 1986), such as: Also the tortuosity refers to the link between diffusion coefficients in free layers and in the porous media, (Nield and Bejan, 2006; Bear, 1972; de Marsily, 1986), such as: * * 2 * * 2 or or T T T T D D D D D D D D         (1.29) * * 2 * * 2 or or T T T T D D D D D D D D         (1.29) where D is the diffusion coefficient in a free liquid, * D is the diffusion coefficient in a porous medium, T D is the thermodiffusion in a free liquid, and * T D the thermodiffusion in a porous medium. The value of the thermodiffusion coefficient is also affected by the solid matrix. The Soret coefficient should have the same value in a porous medium and in a free liquid, based on the argument that since both coefficients D and T D are of the same nature, the corrections should be the same and therefore their ratio should be unaffected by the porous medium, Platten and Costeseque (2004). where D is the diffusion coefficient in a free liquid, * D is the diffusion coefficient in a porous medium, T D is the thermodiffusion in a free liquid, and * T D the thermodiffusion in a porous medium. The value of the thermodiffusion coefficient is also affected by the solid matrix. The Soret coefficient should have the same value in a porous medium and in a free liquid, based on the argument that since both coefficients D and T D are of the same nature, the corrections should be the same and therefore their ratio should be unaffected by the porous medium, Platten and Costeseque (2004). 15 1.5.2 Diffusion flux 1.5.2 Diffusion flux Before explaining thermodiffusion, one needs to define the diffusion flux and several basic concepts. Two types of diffusion fluxes are often used in thermodiffusion research. First is the molar diffusion flux with a mole average velocity given by:   mol i mi i a J v v    (1.30)   mol i a v v  (1.30)   mol i mi i a J v v      mol i mi i a J v v    (1.30) where iJ is the molar diffusion flux of component i, mol av is an arbitrary reference velocity (mole average reference), mi  is the molar density of component i, and iv is the velocity of component i. mol av is defined by: where iJ is the molar diffusion flux of component i, mol av is an arbitrary reference velocity (mole average reference), mi  is the molar density of component i, and iv is the velocity of component i. mol av is defined by: n mol a i i i v x v  (1.31) n mol a i i i v x v  (1.31) where ix is the mole fraction of component i and n is number of components in the mixture. In a similar way, the mass diffusion flux with respect to the velocity mass av is define by: where ix is the mole fraction of component i and n is number of components in the mixture. In a similar way, the mass diffusion flux with respect to the velocity mass av is define by:   mass i i i a j v v    (1.32)   mass i i i a j v v      mass i i i a j v v    (1.32) where ij is the mass diffusion flux of component i, mass av is an arbitrary reference velocity (mass average reference), and i is mass density of component i. mass av is defined by: 1 n mass a i i i v v    (1.33) (1.33) 1 n mass a i i i v v    1 i i i v   (1.33) where i is the mass fraction of component i. where i is the mass fraction of component i. where i is the mass fraction of component i. 1.5.2 Diffusion flux The conventional diffusion fluxes equation can be expressed as follows: 1 1 n mol mol i m ij j Ti j J D x D T                1 1 n mass mass i ij j Ti j j D D T                 1 1 n mol mol i m ij j Ti j J D x D T                (1.36) 1 1 n mass mass i ij j Ti j j D D T                 (1.37) (1.36) (1.37) where is the mass density of the mixture, m is the molar density of the mixture, mol ij D and mass ij D are the molecular diffusion coefficient matrices (Fick’s diffusion coefficient) corresponding to iJ  and ij, respectively, mol Ti D and mass Ti D are the thermal diffusion coefficient matrices corresponding to iJ  and ij, respectively, T is the temperature, jx  is the vector of mole fraction gradients, j   is vector of mass fraction gradients and T  is the temperature gradient. The pressure diffusion contribution is not considered in Eqs 1.36 and 1.37, because its magnitude is about three orders smaller than that of thermodiffusion. In Eq. 1.36, , , , and mol mol i j ij Ti J x D D   are vectors, given by: where is the mass density of the mixture, m is the molar density of the mixture, mol ij D and mass ij D are the molecular diffusion coefficient matrices (Fick’s diffusion coefficient) corresponding to iJ  and ij, respectively, mol Ti D and mass Ti D are the thermal diffusion coefficient matrices corresponding to iJ  and ij, respectively, T is the temperature, jx  is the vector of mole fraction gradients, j   is vector of mass fraction gradients and T  is the temperature gradient. The pressure diffusion contribution is not considered in Eqs 1.36 and 1.37, because its magnitude is about three orders smaller than that of thermodiffusion. In Eq. 1.5.2 Diffusion flux where i is the mass fraction of component i. 16 In the absence of convection, when a steady-state condition is reached, the diffusion flux will vanish, and for n-components mixture, two relationships from the definitions of diffusion fluxes, iJ and ij can be found: In the absence of convection, when a steady-state condition is reached, the diffusion flux will vanish, and for n-components mixture, two relationships from the definitions of diffusion fluxes, iJ and ij can be found: 1 0 n i i J    (1.34) 1 0 n i i j    (1.35) 1 0 n i i J    (1.34) (1.35) From the Eqs. 1.34 and 1.35, it can be concluded that only n-1 of the n diffusion fluxes are independent. The conventional diffusion fluxes equation can be expressed as follows: From the Eqs. 1.34 and 1.35, it can be concluded that only n-1 of the n diffusion fluxes are independent. The conventional diffusion fluxes equation can be expressed as follows: From the Eqs. 1.34 and 1.35, it can be concluded that only n-1 of the n diffusion fluxes are independent. 1.5.2 Diffusion flux 1.36, , , , and mol mol i j ij Ti J x D D   are vectors, given by: 17 1 11 12 1 1 1 21 22 2 1 1 1 1 1 2 1 1 1 1 2 1 1 1 , , x x x and x mol mol mol n mol mol mol mol n i ij mol mol mol n n n n n T T j Ti n T n J D D D J D D D J D J D D D D D D D                                                                                   (1.38) For a binary mixture, Eqs 1.36 and 1.37 are given by: 1 11 12 1 1 1 21 22 2 1 1 1 1 1 2 1 1 , , mol mol mol n mol mol mol mol n i ij mol mol mol n n n n n J D D D J D D D J D J D D D                                              (1.38) or a binary mixture, Eqs 1.36 and 1.37 are given by: For a binary mixture, Eqs 1.36 and 1.37 are given by:   11 1 1 mol mol m T D x D T     (1.39)   1 11 1 1 mol mol m T J D x D T       (1.39)   1 11 1 1 mass mass T j D D T        (1.40)   1 11 1 1 mol mol m T J D x D T       (1.39)   1 11 1 1 mol mol m T J D x D T       (1.39)   11 1 1 mass mass T D D T       (1   1 11 1 1 mass mass T j D D T        (1.40) (1.40) Two important quantities, the Soret coefficient ( T S ) and thermal diffusion factor ( T ) have been defined as: Two important quantities, the Soret coefficient ( T S ) and thermal diffusion factor ( T ) have been defined as: 1 2 1 2 T T T D D S Dx x D   (1.41) T T T D TS T D   (1.42) 1 2 1 2 T T T D D S Dx x D   T T T D TS T D   (1.41) (1.42) where D is the molecular diffusion coefficient and T D is the thermodiffusion coefficient. 1.5.2 Diffusion flux By assuming the following relationship between mol D and mass D or mol T D and mass T D where D is the molecular diffusion coefficient and T D is the thermodiffusion coefficient. By assuming the following relationship between mol D and mass D or mol T D and mass T D 11 11 mol mass D D D   (1.43) (1.43) 11 11 mol mass D D D   18 1 1 1 2 1 2 mol mass T T T D D D x x    (1.44) 1 1 1 2 1 2 mol mass T T T D D D x x    (1.44) 1 1 1 2 1 2 mol mass T T T D D D x x    (1.44) Eqs. 1.39 and 1.40 can be expressed by: Eqs. 1.39 and 1.40 can be expressed by: Eqs. 1.39 and 1.40 can be expressed by: Eqs. 1.39 and 1.40 can be expressed by: Eqs. 1.39 and 1.40 can be expressed by:   1 1 1 2 m T J D x D x x T       (1.45)   1 1 1 2 T j D D T         (1.46)   1 1 1 2 m T J D x D x x T       (1.45)   1 1 1 2 T j D D T         (1.46)   1 1 1 2 m T J D x D x x T         1 1 1 2 T j D D T         (1.45)   1 1 1 2 m T J D x D x x T       (1.46) Eqs. 1.45 and 1.46 have been more frequently adopted in the literature to express 1J  and 1j. Eqs. 1.45 and 1.46 have been more frequently adopted in the literature to express 1J  and 1j. Eqs. 1.45 and 1.46 have been more frequently adopted in the literature to express 1J  and 1j. 19 2.1 Introduction In the non-equilibrium thermodynamics, (de Groot and Mazur, 1984), the rate of entropy production,, can be written as: 2 1 1 1 1 1 1 1 : v n n r k T k q k k k j j k k j k k H J J T J F R A T M T M T T                                    (2.1) (2.1) where q J  is the total heat flux, kJ  is diffusion flux of component k, T is the temperature, k H is the partial molar enthalpy of component k, k M is the molecular weight of component k, k is the chemical potential of component k, is the fluid stress tensor or viscous pressure tensor, k F  is the external force of component k, vis the average velocity ( n i i=1 v= v    ix ), j R is the chemical reaction rate, jA is the chemical affinity of the reactions, and r is the total number of chemical reactions. where q J  is the total heat flux, kJ  is diffusion flux of component k, T is the temperature, k H is the partial molar enthalpy of component k, k M is the molecular weight of component k, k is the chemical potential of component k, is the fluid stress tensor or viscous pressure tensor, k F  is the external force of component k, vis the average velocity ( n i i=1 v= v    ix ), j R is the chemical reaction rate, jA is the chemical affinity of the reactions, and r is the total number of chemical reactions. In Eq. 2.1, there are four thermodynamic driving forces: the gradient of temperature, the gradient of chemical potential and external force, the gradient of velocity, and the chemical reaction rate. 2.1 Introduction A new heat flux expression, * q J  , can be introduced, which is the linear combination of the heat flux and internal diffusion mass fluxes: * 1 n k q q k k k H J J J M       (2.2) * 1 n k q q k k k H J J J M       (2.2) 20 In a mixture consisting of n components, where the only external force is the gravity, and by assuming that there is no heat generation, viscous dissipation, and chemical reaction, the term K F is eliminated because the total summation of kJ is zero, 1 ( 0) n k k J     ; the entropy production rate is simplified as: In a mixture consisting of n components, where the only external force is the gravity, and by assuming that there is no heat generation, viscous dissipation, and chemical reaction, the term K F is eliminated because the total summation of kJ is zero, 1 ( 0) n k k J     ; the entropy production rate is simplified as: entropy production rate is simplified as: 1 * 2 1 1 1 n k n q k T k k n J T J T T M M                      (2.3) (2.3) The phenomenological equation for heat flux and diffusion flux can be given as: The phenomenological equation for heat flux and diffusion flux can be given as: 1 * 2 1 1 1 n T k T n q qq qk k k n J L T L T T M M                   (2.4) 1 2 1 1 1 , 1,2,........ 2.1 Introduction By applying the following relations, the Gibbs-Duhem expression and the relations among chemical potential, the fugacity and the partial molar volume are: 1 1 1 1 n T n j T j j n m P x x                  1 1 1 1 n T n j T j j n m P x x                  (2.8) (2.8) 21 , , , , ln 1,2,......., , 1,2,......., -1 j x T P j i i j j x T P f RT i n j n x x         (2.9) , , , 1,2,.........., j i i i x T i n T P V V i n P n         (2.10) (2.9) (2.10) where jx is the mole fraction of component j, and m is the molar density of the mixture; , , i R V f and iV are the universal gas constant, the total volume, the fugacity of component i, and the partial molar volume of component i, respectively. By using an equation of state, iV and if can be obtained. The diffusion flux, Eq. 2.5, can be written in a new form: , , 1 1 1 2 1 1 1 1 1 1 1 ln 1 + , 1,........, 1 l T P n n n i i n n iq j j n n jk j ii i i i ik l k j l i i n n ii ii j l x n n i i n n ik j j k k j ii k m M x M x L M x M x f RL M x T J L x M x M x RL T L M x M x M x L x V V P i n RTL M                                              (2.11) (2.11) The diffusion flux vector, 1 1 ( ,.... 2.1 Introduction -1 n T k T n i iq ik k k n J L T L i n T T M M                    (2.5) 1 * 2 1 1 1 n T k T n q qq qk k k n J L T L T T M M                   (2.4) (2.4) 1 2 1 1 1 , 1,2,........ -1 n T k T n i iq ik k k n J L T L i n T T M M                    (2.5) (2.5) where , , , and qq qk iq ik L L L L are the phenomenological coefficients, (Onsager, 1931). From the fourth postulate, Onsager’s reciprocal relation, the following relations exist: 1,2,.........., 1 , qi iq L L i n    (2.6) , 1,2,.........., 1 , ik ki L L i k n    (2.7) 1,2,.........., 1 , qi iq L L i n    , 1,2,.........., 1 , ik ki L L i k n    (2.6) , 1,2,.........., 1 i k n   (2 , 1,2,.........., 1 , ik ki L L i k n    (2.7) , 1,2,.........., 1 i k n   (2.7) The focus here is on diffusion flux, iJ  , because it is the aim of this work. 2.1 Introduction 1 j T P n n i i l l n n lk l ij in in ik k l ii l j x M x M x M x f D a d L i j n L M x              (2.13) (2.13) 22 , 1,....., 1 i Ti T in in k D a d M i n T    (2.14) 1 1 1 1 1 , 1,....., 1 n n i i n n pi in in ik j j k k j ii k m M x M x D a d L x V V i n RTL M                   (2.15) , 1,....., 1 i Ti T in in k D a d M i n T    (2.14) (2.14) 1 1 1 1 1 , 1,....., 1 n n i i n n pi in in ik j j k k j ii k m M x M x D a d L x V V i n RTL M                   (2.15) (2.15) lk  is the Kronecker delta function, 1 lk   for l=k and 0 lk   for l k  , i M is the molecular weight of component i, n M is the molecular weight of the reference component n, and M is the average molecular weight of the mixture ( 1 n i i i M M x   ), the coefficients , , and in in Ti a d k are given by: lk  is the Kronecker delta function, 1 lk   for l=k and 0 lk   for l k  , i M is the molecular weight of component i, n M is the molecular weight of the reference component n, and M is the average molecular weight of the mixture ( 1 n i i i M M x   ), the coefficients , , and in in Ti a d k are given by: 2 i n in M M a M  (2.16) 2 2 2 ii in m i n i n RL M d x x M M   (2.17) iq i n i n Ti Ti i n ii L x x M M k x x RTML    (2.18) (2.16) (2.17) iq i n i n Ti Ti i n ii L x x M M k x x RTML    (2.18) (2.18) ii RTML Comparing Eq. 2.1 Introduction ) n J J J     is written in short form: ( ) m T P J D x D T D P       (2.12) ( ) m T P J D x D T D P       (2.12) ( ) m T P J D x D T D P       (2.12) ( ) m T P J D x D T D P       (2.12) where     1 1 1 1 , ,....., , ,....., ij T T Tn P P pn D D D D D D D D          and   1 1 ,....... n x x x    , the molecular or Fick’s diffusion coefficients, ij D (m2/s), the thermodiffusion coefficients, Ti D (m2/s/K) , and the pressure diffusion coefficients, Pi D (m2/s/Pa) can be given based on Eq. 2.11 as: where     1 1 1 1 , ,....., , ,....., ij T T Tn P P pn D D D D D D D D          and   1 1 ,....... n x x x    , the molecular or Fick’s diffusion coefficients, ij D (m2/s), the thermodiffusion coefficients, Ti D (m2/s/K) , and the pressure diffusion coefficients, Pi D (m2/s/Pa) can be given based on Eq. 2.11 as: , , 1 1 1 1 ln , , 1,..... 2.1 Introduction 2.18 and Eq. 2.14, one can get the following formulation Comparing Eq. 2.18 and Eq. 2.14, one can get the following formulation , 1,2,..........., 1 m Ti i n Ti ii D TM M i n L RM      (2.19) (2.19) where Ti k is the thermal diffusion ratio of component i, and Ti  is the thermal diffusion factor of component i. The pressure diffusion is usually ignored in experimental approaches; because the magnitude of the pressure diffusion is about three orders smaller 23 than that of thermodiffusion. Therefore, the pressure in the mixture is assumed constant. Thus, the diffusion expression (Eq. 2.11) can be re-written as: than that of thermodiffusion. Therefore, the pressure in the mixture is assumed constant. Thus, the diffusion expression (Eq. 2.11) can be re-written as: , , 2 1 1 1 1 1 1 , l T P i i n n iq ii ii i n n n j j n n jk j i i i i n n ik l k j l ii j l x M x M x L T RL T RL J M x M x f M x M x M x L x L M x                                 (2.18) (2.18) Another method of expressing the heat flux in Eq. 2.2, q J  , is based on combining the concept of irreversible thermodynamics and molecular kinetic theory involving the heat of transport (Tichacek et al., 1956, and Denbigh, 1951): 1 n k q k k k Q J J M     (2.19) 1 n k q k k k Q J J M     (2.19) where k Q is the heat of transport of component k. Therefore, the expression for * q J  becomes: where k Q is the heat of transport of component k. 2.1 Introduction Therefore, the expression for * q J  becomes: * * 1 1 n n k k k q k k k k k k Q H Q J J J M M           (2.20) * * 1 1 n n k k k q k k k k k k Q H Q J J J M M           (2.20) where * k Q is the net heat of transport of component k. Consequently, Eq. 2.3 can be re- written in terms of the net heat of transport: where * k Q is the net heat of transport of component k. Consequently, Eq. 2.3 can be re- written in terms of the net heat of transport: * * 1 1 1 n k n k n T k k k n k n Q Q T J T M M T M M                                (2.21) (2.21) Using Eq. 2.21, one can write the phenomenological equation for diffusion flux (Eq. 2.5) in the following form: Using Eq. 2.21, one can write the phenomenological equation for diffusion flux (Eq. 2.5) in the following form: 24 * * 1 1 1 , 1,.........., -1 n k n k n i ik T k k n k n Q Q T J L i n T M M T M M                               (2.22) (2.22) omparing Eq. 2.22 with Eq. 2.5, the following formulation can be derived: Comparing Eq. 2.22 with Eq. 2.1 Introduction 2.5, the following formulation can be derived: * * 1 1 , 1,.........., -1 n k n iq ik k k n Q Q L L i n M M             (2.23) (2.23) As introduced in Section 1.4.1, the net heat of transport of component i can be expressed by: As introduced in Section 1.4.1, the net heat of transport of component i can be expressed by: * 1 1 , 1,.........., n j j i i i n j i j j j j x U U V Q i n x V                    (2.24) (2.24) 2.2 Equation of State 2.2 Equation of State It is a prerequisite to know reliable thermodynamic properties for the theoretical research on thermodiffusion. The thermodynamic properties are difficult to obtain fully from the literature and some of the properties are very difficult to measure accurately. It should be kept in mind that in all of the models mentioned in Chapter 1 some thermodynamic properties, including         1 1 x  , i U , i H and iV , are required. These values, together with another important thermodynamic property density, need to be derived from the equation of state (EOS). Therefore, for a certain mixture of interest the accuracy of the model not only relies on the model, but also on the EOS of choice as well as the numerical method utilized in the calculation. another important thermodynamic property density, need to be derived from the equation of state (EOS). Therefore, for a certain mixture of interest the accuracy of the model not only relies on the model, but also on the EOS of choice as well as the numerical method utilized in the calculation. 25 Two equations of state have been used in this research; the first is Peng-Robinson Equation of State (PR-EoS), (Peng and Robinson, 1976). It is one of the most popular cubic equations of state, and it is commonly used in the petroleum industry for hydrocarbon mixtures due to its simplicity and accuracy. The other equation of state is the volume translated Peng-Robinson Equation of State (vt-PR-EoS). In this equation of state, the molar volume in PR-EoS has been replaced by a corrected volume term. More details of these two equations of state will be introduced in the next sections. Two equations of state have been used in this research; the first is Peng-Robinson Equation of State (PR-EoS), (Peng and Robinson, 1976). It is one of the most popular cubic equations of state, and it is commonly used in the petroleum industry for hydrocarbon mixtures due to its simplicity and accuracy. The other equation of state is the volume translated Peng-Robinson Equation of State (vt-PR-EoS). In this equation of state, the molar volume in PR-EoS has been replaced by a corrected volume term. More details of these two equations of state will be introduced in the next sections. 2.2.1 Peng-Robinson Equation of State (PR-EoS) Peng and Robinson (1976) suggested an equation of the form:     ( ) p p p p p a T RT P V b V V b b V b       (2.25)     ( ) p p p p p a T RT P V b V V b b V b       (2.25) Where P is the pressure, V is the molar volume, p a is the attraction parameter (temperature dependent), R is the universal gas constant, and p b is the co-volume parameter which refers to the volume occupied by molecules under an infinitely high pressure. Where P is the pressure, V is the molar volume, p a is the attraction parameter (temperature dependent), R is the universal gas constant, and p b is the co-volume parameter which refers to the volume occupied by molecules under an infinitely high pressure. The compressibility factor Z can be expressed as: PV Z RT  (2.26) (2.26) Based on the PR-EoS, Eq. 2.24 can be re-written: Based on the PR-EoS, Eq. 2.24 can be re-written:       ( ) p p p p p a T V V Z V b RT V V b b V b       (2.27) 26 where p a and p b are component-dependent parameters; p b is independent of temperature and p a is temperature and acentric factor dependent (ω) . Both parameters can be calculated from critical pressure (Pc) and critical temperature (Tc) by: where p a and p b are component-dependent parameters; p b is independent of temperature and p a is temperature and acentric factor dependent (ω) . 2.2.2 The volume translated Peng-Robinson Equation of State (vtPR-EoS) The volume translation method has been proposed to modify the original PR-EoS by Peneloux et al. (1982) and Jhaverl and Youngren (1988). In this method, the predicated molar volume from the original PR-EoS is corrected by introducing a shift parameter, vis , to obtain a new molar volume (corrected molar volume), which is expressed by this relation: corrected PR EoS i vi pi V V x s b    (2.35) corrected PR EoS i vi pi V V x s b    (2.35) The shift parameters will be calculated by matching the pure molar liquid volume from PR-EoS to experimental data when a reduced temperature is equal to 0.7 ( 0.7 rT  ). 2.2.1 Peng-Robinson Equation of State (PR-EoS) Both parameters can be calculated from critical pressure (Pc) and critical temperature (Tc) by:     2 2 2 1/2 0.45724 1 1 c p r c R T a T P     (2.28) 0.0778 c p c RT b P  (2.29)     2 2 2 1/2 0.45724 1 1 c p r c R T a T P     (2.28) (2.28) 0.0778 c p c RT b P  (2.29) (2.29) where rT is the reduced temperature ( / r c T T T  ), is a function of the acentric factor given by: where rT is the reduced temperature ( / r c T T T  ), is a function of the acentric factor given by: y 2 0.37464 1.5422 0.26992 0 < < 0.5    ω ω ω (2.30) 2 3 0.3796 1.485 0.1644 +0.01667 0.5 < < 2    ω ω ω ω (2.31) 2 0.37464 1.5422 0.26992 0 < < 0.5    ω ω ω (2.30) 2 0.37464 1.5422 0.26992 0 < < 0.5    ω ω ω 2 3 0.3796 1.485 0.1644 +0.01667 0.5 < < 2    ω ω ω ω (2.31) (2.31) For mixtures, the parameters p a and p b can be obtained according to certain mixing rules. The following mixing rule has been established for hydrocarbon mixtures: For mixtures, the parameters p a and p b can be obtained according to certain mixing rules. The follo ing mi ing r le has been established for h drocarbon mi t res: For mixtures, the parameters p a and p b can be obtained according to certain mixing rules. For mixtures, the parameters p a and p b can be obtained according to certain mixing rules. The following mixing rule has been established for hydrocarbon mixtures: 0.5 1 1 n n p i j ij i j a x x a    (2.32) (2.32) (2.33) (2.34) 27 where pi a and pi b are the pure component parameters, ix is the mole fraction, and ijk is the interaction parameter between the ith and jth components. In this work, it should be noted that for simplicity, the binary interaction parameters for hydrocarbon mixtures are assumed to be zero in all the Equation of State. 2.3 Governing equations in porous medium The purpose of this section is to introduce the governing partial differential equations that describe the mixture fluid flow in a reservoir where the porous matrix, thermo-solutal and gravitational driving force control the flow behaviour. To represent flow behaviour in porous media, suitable boundary and initial conditions must be considered and selected, and appropriate governing equations derived. Therefore, mass continuity, momentum conservation and energy conservation equations are introduced. 28 The mass continuity equation is given by: The molar diffusion flux is subjected to a linear relationship of the driving forces of temperature and concentration gradients and it can be expressed by:     1 * * 1 ij n mi m Ti j J D X D T                    (2.39) (2.39) 29 For binary mixtures, 1x X    and 1 mi m J J    , and for ternary mixtures,           2 1 x x X    For binary mixtures, 1x X    and 1 mi m J J    , and for ternary mixtures,           2 1 x x X and 1 2 m mi m J J J            . * D , and * T D are the molecular diffusion and thermodiffusion coefficients and 1 2 m mi m J J J           . * D , and * T D are the molecular diffusion and thermodiffusion coefficients and 1 2 m mi m J J J           . * D , and * T D are the molecular diffusion and thermodiffusion coefficients of the fluid mixture in a porous medium, respectively. of the fluid mixture in a porous medium, respectively. The mass continuity equation is given by: The mass continuity equation is given by:   0 m m t       V  (2.36)   0 m m t       V  (2.36) t where m is the molar density of the mixture. ui vj wk    V     is the average of the fluid mixture velocity vector, u, v and w are the velocity components of x, y and z directions, respectively. v has been given various names, by different authors, such as seepage velocity, superficial velocity, and Darcy velocity. The term Darcy velocity is selected for this work; taking an average of the fluid velocity over a volume element consisting of fluid only yields the intrinsic average velocityV  , which is related to the Darcy velocity, by the Dupuit-Forchheimer relationship, (Nield and Bejan, 2006), as: v  = V   (2.37) v  = V   v  = V   (2.37) For multicomponent mixture, the continuity equation of component i is given as: For multicomponent mixture, the continuity equation of component i is given as:   ( ) , 1,2,...... -1 m i m i mi x x J i n t        V   (2.38) (2.38) t where mi J  is the molar diffusion flux of the ith component; ix is the mole fraction of the component i; mi m ix    is the molar density of the ith component. 2.3.2 Darcy’s law The porous matrix is assumed homogeneous and isotropic. Therefore, the Darcy’s law is applied. This is an empirical law formulated by the French engineer H. Darcy, (Darcy, 1856), and it is applicable to creeping flow, which generally describes the dynamics of most flows through porous media; it is written as: ( ) P g     κ v  (2.40) ( ) P g     κ v  (2.40) where κ is the permeability of a porous medium; is the dynamic viscosity of the fluid mixture, is the mass density of the fluid mixture, and g is the gravitational acceleration vector. By substituting the Darcy relation (Eq. 2.40) into the mass conservation equation (Eq. 2.36) the pressure can be derived from the obtained differential equation, which leads to the following equation for pressure: where κ is the permeability of a porous medium; is the dynamic viscosity of the fluid mixture, is the mass density of the fluid mixture, and g is the gravitational acceleration vector. By substituting the Darcy relation (Eq. 2.40) into the mass conservation equation (Eq. 2.36) the pressure can be derived from the obtained differential equation, which leads to the following equation for pressure: 0 m m x m y m z P g t x x P P g g y y z z                                                                    κ κ κ (2.41) (2.41) 30 2.3.3 The thermal energy conservation equation The thermal energy conservation equation for the matrix or solid phase can be expressed as follows, (Nield and Bejan, 2006):      2 1 1 s p s s s T C k T t               2 1 1 s p s s s T C k T t          (2.42) (2.42) where sT is the temperature of the matrix or solid, For the fluid mixture inside the porous medium, it can be expressed as: where sT is the temperature of the matrix or solid, For the fluid mixture inside the porous medium, it can be expressed as:     2 fl fl fl fl fl fl V ( ) p p T C C T k T t            (2.43) (2.43) where fl T is the temperature of the fluid mixture. Since the velocity of the matrix is zero, and there is no viscous dissipation and heat generation in either porous medium or the fluid mixture. And since in addition, thermal equilibrium was assumed between the solid and the liquid phases and occurs very quickly. The temperature of the matrix is assumed to be equal to the temperature of the fluid mixture  s fl T T T   . By adding, Eqs. 2.42 and 2.43, the energy conservation equation can be written as follows:     2 fl e v ( ) p p e T C C T k T t          (2.44)    2 fl e v ( ) p p e T C T k T t         (2.44 (2.44) where   p e C  is the effective volumetric heat capacity and ke is the effective thermal conductivity of the system. These effective physical parameters are related to the fluid properties and the solid matrix properties as follows: where   p e C  is the effective volumetric heat capacity and ke is the effective thermal conductivity of the system. Chapter 3 In this chapter, the validation of a new thermodiffusion cell designed to measure the molecular diffusion coefficient and the Soret coefficient of hydrocarbon binary systems under high pressure in a porous medium is presented. The experimental apparatus and the procedures are explained in this chapter. The results of the experiment are compared with the benchmark values and the theoretical model, which is developed in this chapter. 2.3.3 The thermal energy conservation equation These effective physical parameters are related to the fluid properties and the solid matrix properties as follows:       fl (1 ) p p p e s C C C        (2.45)       fl (1 ) p p p e s C C C        (2.45) 31 fl (1 ) e s k k k      (2.46) (2.46) fl (1 ) e s k k k      fl (1 ) e s k k k      (2.46) fl (1 ) e s k k k      where  fl p C  is the fluid volumetric heat capacity,   p s C  is the matrix volumetric heat capacity, fl k the fluid thermal conductivity, and sk is the matrix or porous medium thermal conductivity. where  fl p C  is the fluid volumetric heat capacity,   p s C  is the matrix volumetric heat capacity, fl k the fluid thermal conductivity, and sk is the matrix or porous medium thermal conductivity. 32 3.1 Introduction Several experimental methods were developed on the ground and in microgravity to measure both molecular diffusion and the Soret coefficients of binary mixtures at atmospheric pressure (Bou-Ali et al., 2003, Wittko and Kohler, 2003, Van Vaerenbergh and Legros, 1998), in order to provide a good understanding of separation processes and molecular interaction. Recently, the oil industry has shown a great interest in studying transport properties. Since conditions under which crude oil is found underground imply high pressure (HP) and diffusion in a porous medium, it is considered important to analyze the influence of the pressure and the interaction with a porous medium on the transport properties of liquid mixtures. This is done in order to achieve a better reliability of the algorithms used for the simulation of crude oil flow in the oil fields. Nevertheless, thermodiffusion data under reservoir conditions, i.e. at high pressures, are very scarce and not very recent [Rutherford and Drickamer (1954), Rutherford and Roof (1959)]. More recently, one may cite the high pressure measurements of the thermodiffusion coefficients made with a thermogravitational column (Urteaga et al. 2006, and Urteaga et al. 2008). This lack of HP measurements has led to the development of the Soret Coefficient for Crude Oil 33 (SCCO) project (Van Vaerenbergh et al., 2009). The main objective of this project is to perform microgravity measurements of thermodiffusion of various mixtures under HP conditions. Additionally, in the frame of this project, a ground HP experimental device, to measure thermodiffusion and molecular diffusion in binary mixtures, was designed. 3.2.1 Experimental description of the apparatus Figure 3.1 shows the experimental set up. The thermodiffusion cell is a cylinder, which is closed by two caps and connected to two thermoregulator baths that impose a thermal gradient along the axial direction of the cylinder. To avoid convection, a monolithic porous medium, which is silica (for silica porous medium, 0.45  and 20  κ mD), nearly fills the cell. Figure 3.3 shows the porous medium cylinder. At each extremity of the porous medium, a dead-volume allows the passage of a laser beam through sapphire windows. The cell, connected to the circuit of the filling and placed under the desired pressure, is installed in a Mach-Zehnder interferometer, as shown in Figure 3.1. The inner diameter of the cell is d = 10 mm. The thickness of each dead-volume is 6.4 mm and the length of the porous medium in the middle of the cell is 32.2 mm, (Figure 3.2). The thermodiffusion cell is designed to test a liquid mixture for a range of pressure between 1 and 1000 bar, and for a range of temperature between 5 oC and 40 oC. Two thermocouples (type K) are located in the middle of the dead-volumes in order to measure the temperature. A constant frequency red He-Ne laser, (with wavelength 0 632.8 nm, linearly polarized and output 5.0 mW) is used as a light source. The cell is covered by an insulator, in order to prevent any heat transfer from the cell wall, and the capillary tube between the cell and the manometer is insulated with foam. The following steps explain the procedure for filling the cell; the cell can be set in horizontal or vertical positions, while the cell filling is always made in a vertical position from the bottom: 34 1. The valve (V1) between fluid reservoir and volumetric pump is closed. Valves V3 and V4 are opened; 1. The valve (V1) between fluid reservoir and volumetric pump is closed. Valves V3 and V4 are opened; 1. The valve (V1) between fluid reservoir and volumetric pump is closed. Valves V3 and V4 are opened; 2. A vacuum is made in the cell and in the volumetric pump; then valve V4 is closed; 2. A vacuum is made in the cell and in the volumetric pump; then valve V4 is closed; 3. 3.2.1 Experimental description of the apparatus The vacuum valve (V2, which is a three-way valve) between the vacuum pump d h ll i l d h h i di d 2. A vacuum is made in the cell and in the volumetric pump; then valve V4 is closed; 3. The vacuum valve (V2, which is a three-way valve) between the vacuum pump and the cell is closed; then the vacuum pump is disconnected; 3. The vacuum valve (V2, which is a three-way valve) between the vacuum pump and the cell is closed; then the vacuum pump is disconnected; 4. The valve (V1) between the fluid reservoir and volumetric pump is opened. Due to the difference of pressure, the cell and the volumetric pump are filled; 5. The valves V2 and V4 are opened. A total of 100 cc of mixture to be studied circulates through the cell (under visual observation through the sapphire windows in the dead volumes, to make sure that there are no bubbles inside the cell); 6. Upon determining that the cell is totally filled, valve V4 is closed; 7. Finally, the desired pressure is achieved with a volumetric pump by pushing liquid into the cell; then valve V3 is closed and the volumetric pump is disconnected from the circuit. The cell is positioned in a Mach-Zehnder interferometer in order to detect the variation of the refractive index between the hot and cold dead-volumes. A monochrome CCD camera (1004x1004 pixels) collects the interferograms generated by the interference of the two beams passing through the dead-volumes of the cell. A rotation of the beam- splitter BS2 is necessary to obtain delocalised fringes in front of the monochrome CCD camera. Figure 3.4 shows the fringe direction, which depends on the cell position. When the cell is in the horizontal position, fringes are in the vertical direction, and vice versa. The interferograms grabbed by the CCD camera are recorded on a computer with the aid of custom-made software. For more details see Appendix A. 35 35 Figure 3.1: Experimental setup. M1, M2 and M3 are mirrors; BS1 and BS2 are beam-splitters; V1, V2, V3 and V4 are valves used for the filling of the cell. 3.2.1 Experimental description of the apparatus The phase difference can be given as:    2 1 Φ t d r t d r         w k k w k k k (3.1) (3.1) where w is the pulsation of the two beams; 2 k is the wave number of the beam with refractive index n2 at the hot side of the cell with thicknessd ; 1 k is the wave number of the beam with refractive index n1 at the cold side (same thickness); k is the wave number of the beams in air; r corresponds to the distance covered in the air by the two beams when the interferometer is adjusted at the optical contact, and  is the supplementary path difference covered by the beam that crosses the cold dead-volume considering the rotation of the beam splitter BS2. The phase difference at point is simplified as follows: where w is the pulsation of the two beams; 2 k is the wave number of the beam with refractive index n2 at the hot side of the cell with thicknessd ; 1 k is the wave number of the beam with refractive index n1 at the cold side (same thickness); k is the wave number of the beams in air; r corresponds to the distance covered in the air by the two beams when the interferometer is adjusted at the optical contact, and  is the supplementary path difference covered by the beam that crosses the cold dead-volume considering the rotation of the beam splitter BS2. 3.2.1 Experimental description of the apparatus CCD Volumetric pump M2 M3 BS1 BS2 Porous medium Vacuum pump Fluid P Convergent lens, f' = 2 cm L A S E R M1 V2 V1 V3 V4 Hot side Cold side Point  Convergent lens, f' = 2 cm Porous medium V4 Hot side Figure 3.1: Experimental setup. M1, M2 and M3 are mirrors; BS1 and BS2 are beam-splitters; V1, V2, V3 and V4 are valves used for the filling of the cell. 36 Figure 3.2: Cell design and geometrical configuration. Cell with insulator Cell with out insulator Cap Cap and the insulator Cell with insulator Cell with out insulator Cell with out insulator Cell with insulator Cell with out insulator Cap Cap and the insulator Cap Figure 3.2: Cell design and geometrical configuration. p p Figure 3.2: Cell design and geometrical configuration. 37 Figure 3.3: Photo of the porous medium used. Figure 3.3: Photo of the porous medium used. (a) (b) Figure 3.4: Interferogram recorded with a) the cell in horizontal position and b) the cell in vertical position. (a) (b) Figure 3.4: Interferogram recorded with a) the cell in horizontal position and b) the cell in vertical position. (a) (b) (b) (a) Figure 3.4: Interferogram recorded with a) the cell in horizontal position and b) the cell in vertical position. 38 The phase difference () between the two laser beams at the point of the interference field is represented by the difference between the phase of the beam that the crosses dead- volume at the hot side and the one that crosses the dead-volume at its cold side. 3.2.1 Experimental description of the apparatus The phase difference at point is simplified as follows:   2 1 Φ d     k k k (3.2)   2 1 Φ d     k k k   2 1 Φ d     k k k (3.2) 1 k and 2 k are given in terms of the wave length ( 0 ) in the vacuum as follows: 1 k and 2 k are given in terms of the wave length ( 0 ) in the vacuum as follows: 1 1 0 2 n    k , and 2 2 0 2 n    k (3.3) Relation (3.2) becomes: 1 1 0 2 n    k , and 2 2 0 2 n    k (3.3) Relation (3.2) becomes:   2 1 0 2 Φ n n d      k   2 1 0 2 Φ n n d      k (3.4) (3.4) Initially, the two baths of the thermoregulators are set at the same temperature (low temperature). The refractive index is the same in the two dead-volumes. Then, from Eq. 3.4, the phase difference depends only on the rotation of BS2. The right side of the cell is Initially, the two baths of the thermoregulators are set at the same temperature (low temperature). The refractive index is the same in the two dead-volumes. Then, from Eq. 3.4, the phase difference depends only on the rotation of BS2. The right side of the cell is 39 heated (T2) and the other side is kept at a low temperature (T1) (assuming that the cell is in a horizontal position). And the top side of the cell is heated (if the cell is in a vertical position); therefore, 2 1 T T  . 2 1 T T T    is the temperature difference between the hot side and the cold side of the cell. The phase difference for each orientation of BS2, is constant; Eq. 3.2.1 Experimental description of the apparatus 3.4 can be re-written as: 2 1 0 2 ΔΦ (Δ Δ ) d n n     (3.5) 2 1 0 2 ΔΦ (Δ Δ ) d n n     (3.5) where is the total variation of the phase difference between the laser beams having crossed the hot side and clod side, and 1 2 n n n      represents the total variation of the refractive index between the hot side and the cold side. Eq. 3.5 can be re-written as: where is the total variation of the phase difference between the laser beams having crossed the hot side and clod side, and 1 2 n n n      represents the total variation of the refractive index between the hot side and the cold side. Eq. 3.5 can be re-written as: 0 2 ΔΦ Δ d n    (3.6) 0 2 Δ d n    (3 (3.6) 0 n  For binary systems, the variation of refractive index is: For binary systems, the variation of refractive index is: c c n T T n n          (3.7) c c n T T n n          (3.7) where c is the concentration difference of the densest component between the hot and the cold sides. Coefficients T n  / and c n  / are the so-called contrast factors (Wittko and Köhler, 2003). At this initial stage, the thermal kinetics provides a significant change in the magnitude of phase difference [Zhang et al. (1996), Urteaga et al. (2006)]. Hence, their effects are completely decoupled. At the beginning of the experiment, the thermal gradient is dominant, so Eq. 3.7 can be simplified: where c is the concentration difference of the densest component between the hot and the cold sides. Coefficients T n  / and c n  / are the so-called contrast factors (Wittko and Köhler, 2003). At this initial stage, the thermal kinetics provides a significant change in the magnitude of phase difference [Zhang et al. (1996), Urteaga et al. (2006)]. Hence, their effects are completely decoupled. At the beginning of the experiment, the thermal gradient is dominant, so Eq. 3.2.1 Experimental description of the apparatus 3.7 can be simplified: (3.8) T  40 when / 0 n T    , the phase difference at point (Figure 3.1) is going to be increased (Eq. 3.6), and produces a scrolling of fringes. Once the thermal gradient is established, the second term in Eq. 3.7 becomes dominant due to the variation of the concentration promoted by the Soret effect. If / 0 n c   , and the system presents a positive Soret coefficient (the densest component goes toward the cold side; therefore Δ 0 c  ), the phase difference at point will be increased with time. Therefore, for systems with a positive Soret coefficient, the mass diffusion must produce a scrolling of fringes in the same direction as the effect of the temperature. 3.2.2 Theoretical relations During the second stage, when the concentration variation becomes dominant, the concentration difference of the densest component as a function of time is given by Mialdun and Shevtsova (2008), as follows:                        odd n r T t n n T c c S c , 2 2 2 0 0 exp 1 8 1 1   (3.9)                        odd n r T t n n T c c S c , 2 2 2 0 0 exp 1 8 1 1   (3.9) (3.9) The two unknown parameters in the above equation are the Soret coefficient T S and relaxation time r; 0c is the initial concentration of the densest component; t is the time. The relaxation time, where the diffusion takes place in the porous medium, is given by Mialdun and Shevtsova (2008), as follows: * 2 2 D L r    (3.10) (3.10) where L is the length of diffusion inside the porous medium (L=32.2 mm), and D* is the molecular diffusion coefficient in the porous medium, which is related to the molecular diffusion coefficient in the free liquid as: where L is the length of diffusion inside the porous medium (L=32.2 mm), and D* is the molecular diffusion coefficient in the porous medium, which is related to the molecular diffusion coefficient in the free liquid as: 41 (3.11) where D is the molecular diffusion coefficient in the free liquid, which is a function of the temperature, and composition of the binary mixture.  is the tortuosity of the molecular diffusion of the porous medium. 3.2.3 Analysis approach The phase difference is deduced from the analysis of the interferogram on a single line converted in terms of intensity (gray levels, see Figure 3.5). Light intensity is fitted by:   2 ( ) cos I x a bx c   (3.12) Figure 3.5: Gray level and adjustment of the first 150 pixels of the line 500 of an interferogram (THN/nC12 mixture 50 wt%; Tmean = 25 °C; o 3 C   T ; time = 512 s). 0 20 40 60 80 100 120 140 160 180 200 0 50 100 150 pixel level of gray yexp ycal   2 ( ) cos I x a bx c   (3.12) Fi 3 5 G l l d dj t t f th fi t 150 i l f th li 500 f i t f 0 20 40 60 80 100 120 140 160 180 200 0 50 100 150 pixel level of gray yexp ycal pixel Figure 3.5: Gray level and adjustment of the first 150 pixels of the line 500 of an interferogram (THN/nC12 mixture 50 wt%; Tmean = 25 °C; o 3 C   T ; time = 512 s). The chosen line of pixels is positively oriented from the cold side to the hot side. a, b and care computed by the method of least squares. / 2 c at x = 0. As an adjustment constraint, the condition is imposed that the values of c must be bounded between 2 /  and 2 /  . For a continuous and uniform scrolling of fringes, the parameterc, therefore, presents discontinuities. By counting the number of discontinuities, the number 42 of scrolled fringes can be evaluated. For each scrolling of one fringe, the phase difference between the two laser beams varies by 2. Then, the phase difference evolution of the second kinetic is fitted by:   2 2 2 , 8 1 ( ) 1 exp n odd t A n Bt C n               (3.13) (3.13) A, B and C are computed by using the method of least squares. In Eq. 3.13, three terms are retained in the sum leading to a sufficient accuracy (in the sum, n ranges 1 to3). 3.2.3 Analysis approach From Eqs 3.6 and 3.7, the concentration difference can be given as: 0 2       c n d c (3.14) 0 2       c n d c (3.14) Comparing Eqs 3.9, 3.10, 3.11, 3.13 and 3.14, the following formulations can be derived: Comparing Eqs 3.9, 3.10, 3.11, 3.13 and 3.14, the following formulations can be derived:   0 0 0 2 1         T A S n d c c T c 2 2 2      L B D   0 0 0 2 1         T A S n d c c T c (3.15) 2 2 2      L B D (3.16)   0 0 0 2 1         T A S n d c c T c 2 2 2      L B D   0 0 0 2 1         T A S n d c c T c (3.15) (3.15) (3.16) 3.3.1 Strategy for the validation of the cell The systems of the benchmark of Fontainebleau are the three binary mixtures of n- dodecane (nC12), isobutylbenzene (IBB) and 1,2,3,4-tetrahydronaphtalene (THN) for a concentration of 50 wt% at a temperature of 25 °C and at atmospheric pressure. The benchmark of the thermodiffusion coefficient DT, molecular diffusion coefficient D and 43 Soret coefficient ST and the values of the contrast factor   T P c n , /   measured for the three different mixtures are shown in Table 3.1, (Platten et al., 2003). Table 3.1: Benchmark values for thermodiffusion coefficient DT, molecular diffusion coefficient D and Soret coefficient T S and contrast factors   T P c n , /   at 1 atm and T = 25 °C. Mixture (50-50 wt%) T D (10-12 m2s-1K-1) D (10-10 m2s-1) T S (10-3 K-1)   T P c n , /   IBB-nC12 3.7  0.2 9.5  0.4 3.9  0.1 0.0628  0.0004 THN-nC12 5.9  0.3 6.21  0.06 9.5  0.5 0.1170  0.0004 THN-IBB 2.8  0.1 8.5  0.6 3.3  0.3 0.0544  0.0004 .1: Benchmark values for thermodiffusion coefficient DT, molecular diffusion coefficient and Soret coefficient T S and contrast factors   T P c n , /   at 1 atm and T = 25 °C. From Eqs. 3.13, 3.14 and 3.15, it is possible to predict which system would present the strongest values of the Soret coefficient and contrast factor, and which would give the strongest amplitude variation for the phase difference between the two laser beams for the second kinetic due to the mass diffusion. For this reason, the interferogram analysis method was tested and the validation of the cell was made on the THN-nC12 system. The two other mixtures presented weaker values for Soret coefficient and contrast factor. Then, the sensitivity of the analysis method was tested on the IBB-nC12 system. Since the benchmark was measured at atmospheric pressure, the pressure in the cell was kept at the atmospheric value for all experiments in this work. A recent numerical simulation done on the geometry of the cell has shown that in the horizontal position the characteristic times to reach the steady states are different in dead-volumes and in the porous medium, (Melnikov et al., 2009). 3.3.1 Strategy for the validation of the cell An interaction at interfaces between the dead- volumes and the porous medium, which would affect the diffusion in the cell, was suspected. For this reason, it was decided to validate the cell in the vertical position with top heating in order to avoid convection. 44 3.3.2.1 IBB-nC12 system The thermodiffusion cell is filled with the binary mixture of IBB (50 wt%) and nC12 (50 wt%) at the mean temperature of 25 oC and atmospheric pressure. The cell is heated from the top, and the difference of the temperature between the two thermoregulator baths is equal to T    5 oC (this difference is between the temperatures inside the two caps). Figure 3.6 shows the temperature distribution versus time in the dead-volumes that was measured with thermocouples in one of the experiments. It can be seen from this figure that the temperature difference is less that 5 oC, because the measurement of the temperature difference is taken at the centre the dead volumes. The program simultaneously acquires the relevant parameters of the cell, namely, pressure and temperature of the hot and cold dead- volumes via the NI-USB-9162 data acquisition card. Figure 3.6: Temperature versus time in dead-volumes for the system IBB-nC12 (50 wt%, Tmean= 25 °C, 5 T   oC, 1atm, vertical position). Figure 3.6: Temperature versus time in dead-volumes for the system IBB-nC12 (50 wt%, Tmean= 25 °C, 5 T   oC, 1atm, vertical position). 45 In Figure 3.7, the phase difference between the beam that crossed the hot dead-volume and the beam that crossed the cold dead-volume is plotted. It can be seen that a fast variation is obtained due to the thermal effect (first kinetic); after that a weak variation is observed due to the concentration effect (second kinetic). For both kinetics, the variation of the phase difference has the same sign, which indicates a positive sign for the Soret coefficient. Figure 3.8 shows the details only of the second kinetic of the phase difference for two experiments in the same configuration but with different durations of experiment, i.e. 5-day and 7-day. The initial value of the phase difference in the second experiment is less than in the first one, because the measured difference of temperature in the cell is lower in this experiment ( o 3 C T   for the 7 days experiment and o 2 C T   for the 5 days experiment, in which are measured between the two dead-volumes). Error bars in Figures 3.7 and 3.8 are not represented the error in the measurement, but they show that the phase difference is measured in a period of time. 3.3.2.1 IBB-nC12 system 46 220 225 230 235 240 245 250 255 260 265 0 100000 200000 300000 400000 500000 600000 time (s) phase difference (rad) 7 days fit 7 days 5 days fit 5 days Figure 3.7: Phase difference versus time (nC12-IBB, 50 wt%., Tmean = 25 °C, T  5 oC, 1 atm, vertical position), Figure 3.8: Second kinetic of the phase difference evolution for different durations of experiments (IBB-nC12, 50 wt%, T  5 oC, 1 atm, vertical position). 0 50 100 150 200 250 300 0 1 2 3 4 5 6 7 time (days) phase difference (rad) First kinetic due to the thermal effect Second kinetic due to the concentration effect 265 Figure 3.7: Phase difference versus time (nC12-IBB, 50 wt%., Tmean = 25 °C, T  5 oC, 1 atm, vertical position), 0 50 100 150 200 250 300 0 1 2 3 4 5 6 7 time (days) phase difference (rad) First kinetic due to the thermal effect Second kinetic due to the concentration effect 0 50 100 150 200 250 300 0 1 2 3 4 5 6 7 time (days) phase difference (rad) First kinetic due to the thermal effect Second kinetic due to the concentration effect phase difference (rad) 260 265 Figure 3.7: Phase difference versus time (nC12-IBB, 50 wt%., Tmean = 25 °C, T  5 oC, 1 atm, vertical position), time (days) Figure 3.7: Phase difference versus time (nC12-IBB, 50 wt%., Tmean = 25 °C, T  5 oC, 1 atm, vertical position), 220 225 230 235 240 245 250 255 260 265 0 100000 200000 300000 400000 500000 600000 time (s) phase difference (rad) 7 days fit 7 days 5 days fit 5 days gure 3.7: Phase difference versus time (nC12-IBB, 50 wt%., Tmean = 25 °C, T  5 oC, 1 atm, vertical position), Figure 3.8: Second kinetic of the phase difference evolution for different durations of experiments (IBB-nC12, 50 wt%, T  5 oC, 1 atm, vertical position). 220 225 230 235 240 245 250 255 260 265 0 100000 200000 300000 400000 500000 600000 time (s) phase difference (rad) 7 days fit 7 days 5 days fit 5 days Figure 3.8: Second kinetic of the phase difference evolution for different durations of experiments (IBB-nC12, 50 wt%, T  5 oC, 1 atm, vertical position). 3.3.2.1 IBB-nC12 system 47 In Table 3.2, the results of the adjustment of the two kinetics from Eq. 3.13 are reported. Parameters A, B and C are adjusted by using the method of least squares. Once these values are estimated, the value of tortuosity  for the porous medium is determined. For both experiments, the same value of  was found and it is near 1.4. Furthermore, this value is in good agreement with values that have been proposed by previous researchers, Platten et al. (2003). The difference between this work value of D and the value of the Benchmark of Fontainebleau is less than 1% for both experiments (7 days and 5 days). The same value of parameter B has been found for both experiments (as shown in Table 3.2); the same value of D was found, based on Eq. 3.16. A positive sign was found for the Soret coefficient, which is consistent with the literature data. For the experiment of 7 day’s duration, a very good agreement was found with the benchmark values and it is much better than that of the 5 day’s experiment. The measurement of ST was based on Eq. 3.15, where the contrast factor ( / n c  ) for the IBB-nC12 mixture has been taken from Wittko and Kohler (2003). To reduce the difference, an increase in the experiment duration (more than 7 days) is here recommended. Table 3.2: Measured difference of temperature T, adjustment parameters A, B and C, molecular diffusion coefficient in free liquid D, Soret coefficient ST and (IBB-nC12 50 wt%, Tmean = 25 °C, o 5 C T   , 1 atm). Duration of the experiment T  (°C) A B (10-6 s-1) C D 10 2 -1 (10 m .s )  Difference with benchmark ST (10-3 K-1) Difference with benchmark 7 days 3 18.05 3.80 243.47 9.47 -0.316% 3.95 1.28% 5 days 2 17.91 3.80 224.53 9.47 -0.34% 5.7 31.57% Table 3.2: Measured difference of temperature T, adjustment parameters A, B and C, molecular diffusion coefficient in free liquid D, Soret coefficient ST and (IBB-nC12 50 wt%, Tmean = 25 °C, o 5 C T   , 1 atm). A new experiment was performed, with a temperature difference of o 10 C T   between the and the mean temperature Tmean = 25 °C was kept the same as in the previous experiments. 3.3.2.1 IBB-nC12 system Figure 3.9 displays temperature distribution of the dead-volumes versus time that was measured with the thermocouples. In Figure 3.10, the phase difference between the laser beam that crosses the hot dead-volume and the laser beam that crosses the cold dead-volume is plotted. In Figure 3.11, the details of the second kinetic are presented. 48 Table 3.3 shows the results of the adjustment of the second kinetics with Eq. 3.13. An excellent agreement is found between measured D and T S values and the benchmark values as shown in Table 3.3. The value of the tortuosity is almost the same as in the previous experiments ( 1.4  ). The average difference of temperature between dead- volumes was measured ( T  4.2 oC). 20 21 22 23 24 25 26 27 28 29 0 100000 200000 300000 400000 500000 600000 700000 time (s) temperature in dead-volumes ( oC) Thot/top Tcold/bottom Figure 3.9: Temperature versus time in dead-volumes for the system IBB-nC12 (50 wt%, Tmean= 25 °C, o 10 C T   , 1atm, vertical position). 20 21 22 23 24 25 26 27 28 29 0 100000 200000 300000 400000 500000 600000 700000 time (s) temperature in dead-volumes ( oC) Thot/top Tcold/bottom Figure 3.9: Temperature versus time in dead-volumes for the system IBB-nC12 (50 wt%, Tmean= 25 °C, o 10 C T   , 1atm, vertical position). Figure 3.9: Temperature versus time in dead-volumes for the system IBB-nC12 (50 wt%, Tmean= 25 °C, o 10 C T   , 1atm, vertical position). 49 0 100 200 300 400 500 600 0 1 2 3 4 5 6 7 time (days) phase difference (rad) Figure 3.10: Phase difference versus time (nC12-IBB, 50 wt%, Tmean = 25 °C, T   5 oC, 1 atm, vertical position). 0 100 200 300 400 500 600 0 1 2 3 4 5 6 7 time (days) phase difference (rad) Figure 3.10: Phase difference versus time (nC12-IBB, 50 wt%, Tmean = 25 °C, T   5 oC, 1 a vertical position). 0 100 200 300 400 500 600 0 1 2 3 4 5 6 7 time (days) phase difference (rad) Figure 3.10: Phase difference versus time (nC12-IBB, 50 wt%, Tmean = 25 °C, T   5 oC, 1 atm, vertical position). 3.3.2.1 IBB-nC12 system 450 455 460 465 470 475 480 485 490 495 500 0 100000 200000 300000 400000 500000 600000 700000 time (s) phase difference (rad) experimental adjustment Figure 3.11: Second kinetic of the phase difference evolution for different durations of experiments (IBB-nC12, 50 wt%, o 10 C T   , 1 atm, vertical position ). 450 455 460 465 470 475 480 485 490 495 500 0 100000 200000 300000 400000 500000 600000 700000 time (s) phase difference (rad) experimental adjustment Figure 3.11: Second kinetic of the phase difference evolution for different durations of experiments (IBB-nC12, 50 wt%, o 10 C T   , 1 atm, vertical position ). 50 Table 3.3: Adjustment parameters A, B and C, molecular diffusion coefficient in free liquid D, Soret coefficient ST (IBB-nC12 50 wt%, o o 25 C, 10 C mean T T    , 1 atm). Duration of the experiment A B (10-6 s-1) C D 10 2 -1 (10 m .s )  Difference with benchmark ST (10-3 K-1) Difference with benchmark 7 days 30.90 3.96 257.63 9.49 -0.105% 4.1 4.878% 3.3: Adjustment parameters A, B and C, molecular diffusion coefficient in free liquid D, 3.3.2.2 THN-nC12 system The cell was filled with a new system of THN (50 wt%) and nC12 (50 wt%) at the mean temperatures Tmean = 25 °C and atmospheric pressure. The cell was heated from the top, and the temperature difference between the two baths was set to o 5 C T   . Figure 3.12 shows the details of the second kinetic of the phase difference between the beams that cross the dead-volumes. 220 230 240 250 260 270 280 290 300 310 0 100000 200000 300000 400000 500000 600000 time (s) phase difference (rad) experimental adjustment Figure 3.12: Second kinetic of the phase difference evolution for different durations of experiments (THN-nC12, 50 wt%, o 5 C T   , 1 atm, vertical position ). 220 230 240 250 260 270 280 290 300 310 0 100000 200000 300000 400000 500000 600000 time (s) phase difference (rad) experimental adjustment Figure 3.12: Second kinetic of the phase difference evolution for different durations of experiments (THN-nC12, 50 wt%, o 5 C T   , 1 atm, vertical position ). 51 51 The same procedure was applied for the second kinetic and the results of the adjustment of the second kinetic from Eq. 3.13 are shown in Table 3.4. Once again, a very good agreement is found between these experimental data and the benchmark values. The relative difference between the molecular diffusion coefficient and the value given in literature remains smaller than 1%. The value of the tortuosity is close to the previous one ( 1.4  ). Table 3.4: Adjustment parameters A, B and C, molecular diffusion coefficient in free liquid D, Soret coefficient ST (THN-nC12 50 wt%., Tmean = 25 °C, o 5 C T   , 1 atm). Duration of the experiment A B (10-6 s-1) C D 10 2 1 (10 . ) m s   Difference with benchmark ST (10-3 K-1) Difference with benchmark 6 days 94.09 2.70 223.59 6.20 -0.162 11.0 13.63% 3.3.3 Experimental cell set-up in the horizontal position A set of experiments was performed for the cell at the horizontal position. The IBB-nC12 (50-50 wt%) was tested at the mean temperature Tmean = 25 °C and atmospheric pressure. The cell was heated from the left side. When the difference of the temperature between the two thermoregulator baths was equal to o 5 C T   , a transient diffraction pattern grew up, causing the interference fringes to move quickly at the beginning of the experiment, (see Figure 3.13). The phenomenon of diffraction pattern is not perceptible when the set-up is in vertical position. The reason behind this phenomenon is that the convection is established quickly in the dead-volumes when the cell is in horizontal position; thus, the procedure of analysis of interferograms becomes difficult. 52 (a) (b) (c) Figure 3.13: Interferogram (IBB-nC12, 50 wt%, Tmean = 25 °C, o 5 C T   , 1 atm, horizontal position) images time recorded for the cell: a) 0 s, b) 1 min and c) 10 min. a) b) c) (c) c) (a) a) (b) b) (b) (a) (c) Figure 3.13: Interferogram (IBB-nC12, 50 wt%, Tmean = 25 °C, o 5 C T   , 1 atm, horizontal position) images time recorded for the cell: a) 0 s, b) 1 min and c) 10 min. Figure 3.14 shows the details of the second kinetic of the phase difference between the beams that cross dead-volumes. The results of the adjustment of the second kinetic from Eq. 3.13 are shown in Table 3.5. Figure 3.14 shows the details of the second kinetic of the phase difference between the beams that cross dead-volumes. The results of the adjustment of the second kinetic from Eq. 3.13 are shown in Table 3.5. 215 220 225 230 235 240 245 250 255 0 100000 200000 300000 400000 time (s) phase difference (rad) experimental adjustment Figure 3.14: Second kinetic of the phase difference evolution for different durations of experiments (IBB-nC12, 50 wt%, o 5 C T   , 1 atm, horizontal position ). 215 220 225 230 235 240 245 250 255 0 100000 200000 300000 400000 time (s) phase difference (rad) experimental adjustment Figure 3.14: Second kinetic of the phase difference evolution for different durations of experiments (IBB-nC12, 50 wt%, o 5 C T   , 1 atm, horizontal position ). 3.3.3 Experimental cell set-up in the horizontal position 53 Table 3.5: Adjustment parameters A, B and C, molecular diffusion coefficient in free liquid D, Soret coefficient ST (IBB-nC12, 50 wt%., Tmean = 25 °C, o 5 C T   , 1 atm, horizontal position). Duration of the experiment T (°C) A B (10-6 s-1) C D (10-10 m2s-1) Difference with benchmark ST (10-3 K-1) Difference with benchmark 4 days 3 34.25 3.98 219.55 9.54 0.42% 7.34 46.86% A good agreement for the molecular diffusion coefficient with the benchmark values was found, while there was a significant discrepancy between the Soret coefficient of this experiment and the benchmark value (error is equal to 46.86%). We recommend performing all the experiments in the future at vertical position by applying a vertical temperature gradient. 3.4.2 Mathematical model As introduced in section 1.3.2, the mass diffusion flux in an n-component fluid can be expressed as follows: 1 , 1 n i ij j T i j j D D T                 (3.17) 1 , 1 n i ij j T i j j D D T                 (3.17) where ij is the mass diffusion flux of the ith component,  is the density, j is the mass fraction of component j, T is the temperature. ij D and , T i D are the molecular diffusion and thermodiffusion coefficients of the fluid mixture, respectively. Both thermodiffusion and molecular diffusion coefficients, as introduced in Chapter 2, are described through the theory of non-equilibrium thermodynamics. In an n-component fluid, the molecular diffusion and thermodiffusion coefficients can be expressed by: 1 1 1 1 , 2 ln j n n l l n n lk l ij ik k l n n j j x T iq T i x M x M f R D L x M M x L D T                       (3.18) 1 1 1 1 , 2 ln j n n l l n n lk l ij ik k l n n j j x T iq T i x M x M f R D L x M M x L D T                       (3.18) (3.18) The thermodynamic properties of the fluid mixtures at equilibrium have been calculated using the volume-translated Peng-Robinson Equation of State (vt-PR-EoS). After the diffusion coefficient values ( , and ij T D D ) are calculated, these values were implemented in a porous medium model, applying the two-dimensional governing equation of the porous medium as introduced in section 2.3. The thermodynamic properties of the fluid mixtures at equilibrium have been calculated using the volume-translated Peng-Robinson Equation of State (vt-PR-EoS). 3.4.1 Model description A two-dimensional vertical porous medium, with a horizontal length (W) of 10 mm and height (H) of 32.2 mm is used. Two layers are added to the porous medium to represent the dead-volumes: one at the top of the porous medium and the second at the bottom, (see Figure 3.15). The porous medium is heated from the top at 27.5 oC and the bottom wall maintained at 22.5 oC, while lateral walls are assumed to be adiabatic. Silica is used as a porous material, and it is saturated with the fluid mixture. The properties of the porous medium and of the fluid mixtures are given in Table 3.6. The binary mixtures filling the vertical porous medium consist of nC12-THN, nC12-IBB and THN-IBB for a concentration of 50 wt%. In this work, all simulations are conducted at atmospheric pressure. 54 , 0, =0 h i T T J v   0, =0 iJ u  0, =0 iJ u  , 0, =0 c i T T J v   Figure 3.15: Schematic diagram of the porous medium and the boundary conditions. g H W 45 mm y x , 0, =0 c i T T J v   Figure 3.15: Schematic diagram of the porous medium and the boundary conditions. Table 3.6: Physical properties of the porous material and fluid mixture, Tmean=25 oC and 1 atm. Name Value Unit Porous medium height H 32.2 mm Porous medium horizontal length W 10.0 mm Top wall temperature Th 27.5 oC bottom wall temperature Tc 22.5 oC Pressure at the centre of the cavity 0.1013 MPa Silica density 2220 -3 kg.m Silica heat capacity 745 -1 -1 J.kg .K Silica thermal conductivity 1.38 -1 -1 W.m .K Silica porosity 0.45 Silica porous medium permeability 20 mD Silica porous medium tortuosity 1.4 - THN-nC12 mixture compositions 50-50 (% mass fr.) THN-nC12 mixture heat capacity 1890.55 -1 -1 J.kg .K THN-nC12 mixture thermal conductivity 0.132 -1 -1 W.m .K THN-IBB mixture compositions 50-50 (% mass fr.) THN-IBB mixture heat capacity 1722.90 -1 -1 J.kg .K THN-IBB mixture thermal conductivity 0.1260 -1 -1 W.m .K IBB-nC12 mixture compositions 50-50 (% mass fr.) IBB-nC12 mixture heat capacity 1979.98 -1 -1 J.kg .K IBB-nC12 mixture thermal conductivity 0.128 -1 -1 W.m .K le 3.6: Physical properties of the porous material and fluid mixture, Tmean=25 oC and 1 atm 55 3.4.2 Mathematical model After the diffusion coefficient values ( , and ij T D D ) are calculated, these values were implemented in a porous medium model, applying the two-dimensional governing equation of the porous medium as introduced in section 2.3. The porous medium governing equations are solved numerically subject to the boundary conditions of zero mass flux at the rigid wall, zero velocity (only the normal components) 56 at the rigid wall, fixed temperatures at the top and bottom walls but isothermal at lateral walls. The boundary conditions are shown in Figure 3.15 and the numerical solution procedure will be explained in detail in Chapter 5. 3.4.4 Comparison of experimental data with numerical model In this section, numerical results of three binary mixtures of THN, IBB and nC12 are presented. In addition, a comparison between the experimental data and the theoretical values is given in Table 3.8. Figure 3.16 shows the temperature contours along the vertical direction; the temperature gradient is only in the vertical direction. The numerical results of the THN-nC12 mixture are presented in details; the THN-IBB and IBB-nC12 mixture results are only given through the coefficient values ( , , and T T D D S ) in Table 3.7. Figure 3.16: Temperature distribution along the vertical position. Temp. Figure 3.16: Temperature distribution along the vertical position. 57 Figures 3.18 and 3.19 display the composition variation along the vertical position in the centre of the cavity for THN and nC12 components in the THN-nC12 mixture. It can be seen that due to the Soret effect, the THN concentration (heavier component) migrates to the cold side, while the lighter component (nC12) moves towards the hot side. Also, two dead-volumes are included: one at the top side and another at the bottom side (the permeability and the porosity are assumed to be high value in order to mimic the dead- volumes). The concentration of THN and nC12 deviates somewhat from linearity in the dead-volume regions, because the convection effect is significant in these regions. The same behaviour was found for the other mixture components (THN-IBB and IBB-nC12 mixtures). Figures 3.18 and 3.19 display the composition variation along the vertical position in the centre of the cavity for THN and nC12 components in the THN-nC12 mixture. It can be seen that due to the Soret effect, the THN concentration (heavier component) migrates to the cold side, while the lighter component (nC12) moves towards the hot side. Also, two dead-volumes are included: one at the top side and another at the bottom side (the permeability and the porosity are assumed to be high value in order to mimic the dead- volumes). The concentration of THN and nC12 deviates somewhat from linearity in the dead-volume regions, because the convection effect is significant in these regions. The same behaviour was found for the other mixture components (THN-IBB and IBB-nC12 mixtures). Figure 3.19 represents density variation in the centre of the cavity along the vertical position for three mixtures: THN-nC12, THN-IBB, and IBB-nC12. 3.4.4 Comparison of experimental data with numerical model The data in this figure are at 5 o T C   and at 50 wt%. The density variation along the vertical position is linear, as expected, since the temperature variation is linear and the pressure is assumed constant inside the cavity. 58 Figure 3.17: THN mass fraction distributions in the centre of the cavity along the vertical position, THN-nC12 mixture. 0.492 0.493 0.494 0.495 0.496 0.497 0.498 0.499 0.5 0.501 0.502 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 Height [m] THN mass fraction [-] Figure 3.17: THN mass fraction distributions in the centre of the cavity along the vertical position, THN-nC12 mixture. Figure 3.18: nC12 mass fraction distributions in the centre of the cavity along the vertical position, THN-nC12 mixture. 0.498 0.499 0.5 0.501 0.502 0.503 0.504 0.505 0.506 0.507 0.508 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 Height [m] nC12 mass fraction [-] 0.492 0.493 0.494 0.495 0.496 0.497 0.498 0.499 0.5 0.501 0.502 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 Height [m] THN mass fraction [-] Figure 3.17: THN mass fraction distributions in the centre of the cavity along the vertical position, THN-nC12 mixture. 0.492 0.493 0.494 0.495 0.496 0.497 0.498 0.499 0.5 0.501 0.502 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 Height [m] THN mass fraction [-] Figure 3.17: THN mass fraction distributions in the centre of the cavity along the vertical position, THN-nC12 mixture. 0 498 0.499 0.5 0.501 0.502 0.503 0.504 0.505 0.506 0.507 0.508 nC12 mass fraction [-] 0.492 0.493 0.494 0.495 0.496 0.497 0.498 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 Height [m] THN mass fraction Figure 3.17: THN mass fraction distributions in the centre of the cavity along the vertical position, THN-nC12 mixture. Figure 3.18: nC12 mass fraction distributions in the centre of the cavity along the vertical position, THN-nC12 mixture. 0.498 0.499 0.5 0.501 0.502 0.503 0.504 0.505 0.506 0.507 0.508 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 Height [m] nC12 mass fraction [-] Figure 3.18: nC12 mass fraction distributions in the centre of the cavity along the vertical position, THN-nC12 mixture. 3.4.4 Comparison of experimental data with numerical model 0.498 0.499 0.5 0.501 0.502 0.503 0.504 0.505 0.506 0.507 0.508 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 Height [m] nC12 mass fraction [-] Figure 3.18: nC12 mass fraction distributions in the centre of the cavity along the vertical position, THN-nC12 mixture. 59 Figure 3.19: Density distribution for three binary mixtures in the centre cavity a direction. 780 785 790 795 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 Height [m] Density [kg/m 3] IBB-nC12 895 900 905 910 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 Height [m] Density [kg/m 3] THN-IBB 795 800 805 810 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 Height [m] Density [kg/m 3] THN-nC12 895 900 905 910 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 H i ht [ ] Density [kg/m 3] THN-IBB 795 800 805 810 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 Height [m] Density [kg/m 3] THN-nC12 795 800 805 810 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 H i h [ ] Density [kg/m 3] THN-nC12 8 8 Density [kg/m 3] Density [kg/m 3] Figure 3 19: Density distribution for three binary mixtures in the centre cavity along the verti 780 785 790 795 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 Height [m] Density [kg/m 3] IBB-nC12 Height [m] Figure 3.19: Density distribution for three binary mixtures in the centre cavity along the vertical direction. 60 In Table 3.7, the molecular diffusion coefficient, Thermodiffusion coefficient, and Soret coefficient in a porous medium are calculated. Table 3.8 shows a comparison between this work results (which consists of two parts; experimental and numerical) and benchmark values. It can be see that a good agreement was found between the theoretical approach, which is based on the Shukla and Firoozabadi model, and the experimental approach. For instance, the percentage error for the THN-nC12 mixture between the experimental data and the theoretical values for and T D S are equal to 2.09% and 18%, respectively. It is also found that the IBB-nC12 mixture presents a weaker value of the Soret coefficient ( T S 4.95x10-3 K-1) with regard to the THN-nC12 mixture ( T S 9.025x10-3 K-1), and this observation has been noticed in the experimental data and benchmark value. 3.4.4 Comparison of experimental data with numerical model .7: Model in vertical position, the coefficient values for porous medium, Tmean= 25 oC and 1 atm. Table 3.7: Model in vertical position, the coefficient values for porous medium, Tmean= 25 oC an 1 atm. Mixture (50-50 wt%) * 10 2 -1 (10 m s ) . D  (Theo.) * 12 2 -1 (10 m s K) . T D  (Theo.) 3 -1 (10 K ) T S  (Theo.) THN-nC12 3.10 2.20 9.02 THN-IBB 5.55 1.56 2.85 IBB-nC12 4.48 1.62 4.95 Table 3.8: Theoretical and experimental molecular diffusion and Soret coefficients for three binary mixtures, Tmean= 25 oC and 1 atm (for 1.4 tortuosity 1.4). Mixture THN-nC12 (50-50 wt%) THN-IBB (50-50 wt%) IBB-nC12 (50-50 wt%) 10 2 -1 /10 m .s D  3 -1 /10 K T S  10 2 -1 /10 m .s D  3 -1 /10 K T S  10 2 -1 /10 m .s D  3 -1 /10 K T S  Theoretical 6.076 9.020 10.87 2.85 8.780 4.95 Experimental 6.20 11.0 -------- ------- 9.47 0.02  3.95 0.15  Benchmark 6.21 0.06  9.5 0.5  8.5 0.6  3.3 0.3  9.5 0.4  3.9 0.1  Error Theo. -Exp. 2.09% 18.0% ------ ------- 7.28% -25.31% Error Theo.-Bench 2.12% 5.05% -19.45% 13.63% 7.57% -26.92% Error Exp.-Bench. -0.162% 13.63% -------- -------- -0.316% 1.28% Table 3.8: Theoretical and experimental molecular diffusion and Soret coefficients for thr binary mixtures, Tmean= 25 oC and 1 atm (for 1.4 tortuosity 1.4). 61 3.5 Summary In this work, a new thermodiffusion cell designed for high pressure was validated at atmospheric pressure. The cell was installed in a Mach-Zehnder interferometer, with each beam crossing one of the dead-volumes. Two mixtures of the Benchmark of Fontainebleau have been tested in the vertical position (THN-nC12 and IBB-nC12 at 50 wt% and Tmean =25 °C). From the analysis of the second kinetic of the evolution of the phase difference between the laser beam that passes through the hot dead-volume and the laser beam that passes through the cold dead-volume, the diffusion coefficient in free liquid, D, and Soret coefficient, T S , of the two mixtures and the tortuosity  for the porous medium were determined. For both mixtures, an excellent agreement was found between the molecular diffusion coefficient of this work and the benchmark values, and the percentage error was less than 1%. In addition, it was found that the difference between the Soret coefficients for this work and the benchmark data is acceptable for the experiments of duration up to seven days. To improve the determination of T S with the cell in a vertical position, it is suggested to increase the value of the imposed temperature gradient and increasing the time of the experiment (about two weeks in order to reach the steady state condition). The value of the tortuosity of the porous medium is close to 1.4, which is consistent with the literature data. Also it was found that the theoretical results are in good agreement with the experimental data. When the cell was set in the horizontal position, convection in dead-volumes became possible. For a difference of the temperature applied between thermoregulator baths ( o 5 C T   ) a diffraction pattern came up and at the beginning of the experiment, and the procedure of analysis of interferograms became difficult. 62 Chapter 4 In this chapter, the densities of three binary mixtures of n-dodecane (nC12), isobutylbenzene (IBB) and 1,2,3,4-tetrahydronaphtalene (THN) for pressures varying from 0.1 to 20 MPa at an average temperature of 25 °C were measured. By a derivative method, the thermal expansion and concentration expansion coefficients for binary mixtures of equal mass fraction have been determined. In addition viscosities have been measured and compared with theoretical estimates. In order to accurately predict the thermal expansion and concentration expansion coefficients, the densities of the binary mixtures were calculated using Peng-Robinson and volume translated Peng-Robinson Equations of State. 4.1 Introduction In recent years, due to limited energy resources, the characterization of petroleum reservoirs has gained a lot of interest. In this context especially knowledge of transport properties of hydrocarbon mixtures such as linear alkanes and organic ring compounds is very important (Montel, 1998 and Van Vaerenbergh et al., 2005). Different techniques exist to determine the thermal diffusive properties of liquid mixtures (Platten, 2006). The thermogravitational columns have been validated in a benchmark test for three binary hydrocarbon mixtures (Platten et al., 2003). With this technique, the value of the thermodiffusion coefficient of a binary at the steady-state is given by Blanco et al., (2006):   4 0 0 504 1 T T c m ga D z            4 0 0 504 1 T T c m ga D z            4 0 0 504 1 T T c m ga D z          (4.1) 63 where g is the acceleration of gravity, T  the thermal expansion coefficient of the mixture, c the concentration expansion coefficient of the mixture, m  the dynamic viscosity of the mixture, a the “gap” (distance between the inner cylinder and the exterior cylinder of the column where the fluid of study is), 0  the initial mass fraction of the densest component in the binary mixture and /z the vertical gradient of density  in the column at the steady-state. The parameters , , T T   and m  are therefore thermophysical properties needed for the determination of the thermodiffusion coefficient. The systems of the benchmark of Fontainebleau, (Platten et al., 2003), are composed of binaries THN and nC12, THN and IBB and IBB and nC12 (mass fraction of 50% and mean temperature of 25 °C). Measurements of thermodiffusion coefficient under high pressure are in progress (Urteaga et al., 2008), but thermophysical properties and molecular and thermodiffusion coefficients are known for these systems only at atmospheric pressure. The aim of this work is to provide these properties at high pressure. In this work, the measurements for the thermal expansion and concentration expansion coefficients and for the viscosity of the binaries of the benchmark of Fontainebleau for pressures varying from 0.1 to 20 MPa at 25 °C are presented. 4.1 Introduction The predicted coefficients from the Peng- Robinson, and volume translated Peng-Robinson Equations of State are compared with the measured values. The Lohrenz-Bray-Clark model was used for the prediction of the viscosities. 4.2 Experimental Setup The THN (99%), nC12 (99%) and IBB (99%) were purchased from Sigma-Aldrich. The properties of the three components are given in Table 4.1, where i ω is the acentric factor and vi s is a shift parameters as they are introduced in sections 2.2.1 and 2.2.2. 64 Table 4.1: Pure component parameters for PR-EoS and vt-PR-EoS. PR & vt-PR Component C T (K) C P (MPa) i ω vi s IBB 650.15 3.0398 0.3811 -0.0805 THN 720.15 3.6200 0.3278 0.0146 nC12 658.20 1.8239 0.5734 0.0985 Table 4.1: Pure component parameters for PR-EoS and vt-PR-EoS. 4.2.1 Apparatus 4.2 leads to a simple relation, where density is linearly related to the square of the measured period of oscillation: 2 ( , ) ( , ) ( , ) T p A T p B T p     (4.3) 2 ( , ) ( , ) ( , ) T p A T p B T p     (4.3) 65 where 2 ( , ) 4 ( , ) ( , ) ( , ) o C A T p V T p m B T p V T p    (4.4) where 2 ( , ) 4 ( , ) ( , ) ( , ) o C A T p V T p m B T p V T p    (4.4) where 2 ( , ) 4 ( , ) ( , ) ( , ) o C A T p V T p m B T p V T p    where (4.4) The parameters ( , ) A T p and ( , ) B T p are determined theoretically from measuring the period of oscillation for two reference substances of known density (e.g. distilled water and ethanol) as calibration samples over the whole range of pressure and temperature. Taking into account the accuracy of measurements of the temperature, the pressure, and the period of oscillation measurement for two reference substance and the studied systems, the overall experimental uncertainty in the reported density values is estimated to be ± 5.0x10-4 3 g/cm . This uncertainty is similar to those that are reported in previous studies by Comunas et al. (2008), and Miyake et al. (2008). The parameters ( , ) A T p and ( , ) B T p are determined theoretically from measuring the period of oscillation for two reference substances of known density (e.g. distilled water and ethanol) as calibration samples over the whole range of pressure and temperature. Taking into account the accuracy of measurements of the temperature, the pressure, and the period of oscillation measurement for two reference substance and the studied systems, the overall experimental uncertainty in the reported density values is estimated to be ± 5.0x10-4 3 g/cm . This uncertainty is similar to those that are reported in previous studies by Comunas et al. (2008), and Miyake et al. (2008). 4.2.1 Apparatus Densities of the binary mixtures were measured with a high pressure-high temperature vibrating tube densimeter DMA HPM (Anton-Paar). An mPDS-2000V3 evaluation unit was connected to DAM HPM in order to read out the measurement data. The complete experimental assembly has been described in detail by Lagourette et al. (1992), (see Figure B.1 Appendix B). The measurement principle is based on the determination of the oscillation period of the vibrating U-tube densimeter which contains the sample (fluid mixture); the oscillation period, in seconds, can be defined by: ( , ) ( , ) ( , ) 2 o m T p V T p T p C      (4.2) (4.2) where o m is the mass of the U-tube empty, V is the internal volume of the tube, is the sample density, and C is the spring constant ( 1 . N m). Re-arrangement Eq. 4.2 leads to a simple relation, where density is linearly related to the square of the measured period of oscillation: where o m is the mass of the U-tube empty, V is the internal volume of the tube, is the sample density, and C is the spring constant ( 1 . N m). Re-arrangement Eq. 4.2.1 Apparatus A falling body high pressure high temperature viscometer (HPHTV-100 semi-automatic Stony Brook Scientific) was used to measure the viscosity of the compressed liquids. In this apparatus, a stainless steel cylinder falls vertically through a fluid of unknown viscosity at given conditions of temperature and pressure. A measurement of the falling body terminal velocity allows a determination of the fluid viscosity. The viscosity is a function of the falling body time, the density difference between the cylinder and the fluid. A toluene has been used as calibrating fluid, (Vieira et al., 1997). Decane has been used to verify the calibration (Oliveira et al., 1992 and Kashiwagi and Makita 1995). Each measurement of the falling time was repeated three times at thermal and mechanical equilibrium, and it is reproducible to better than 1%. The final value is an average of these measurements. The total uncertainty of the obtained viscosity values was estimated to be within 2%, which is comparable to the values estimated by other authors for similar devices, as has been discussed in previous papers (Et-Tahir et al., 1995 and Leahy-Dios and Firoozabadi 2007). Viscosity values at 0.1, 4, and 10 MPa are given in Table 4.5. The 66 viscosity at 20 MPa was not measured experimentally, because of the 14 MPa limitation of the viscosimeter. viscosity at 20 MPa was not measured experimentally, because of the 14 MPa limitation of the viscosimeter. 4.2.2 Methods 4.2.2 Methods The thermal expansion coefficient is given by: The thermal expansion coefficient is given by: , 0 1 T P T              (4.5) , 0 1 T P T              (4.5) where 0 is the density of the mixture at the temperature 0T and   T  /  is the derivative of the density with temperature at 0T and at a mass fraction of 50%. Figure 4.1 shows the experimental data of densities for five temperatures centred on 25 °C and for pressures varying from 0.1 MPa to 20 MPa for the mixture of IBB and nC12. where 0 is the density of the mixture at the temperature 0T and   T  /  is the derivative of the density with temperature at 0T and at a mass fraction of 50%. Figure 4.1 shows the experimental data of densities for five temperatures centred on 25 °C and for pressures varying from 0.1 MPa to 20 MPa for the mixture of IBB and nC12. The results were fitted by a linear function using the method of least squares. Considering the uncertainty of the measurements and the sensitivity of the method of least squares adjustment, the uncertainty of T  is estimated to be ± 0.310-4 K-1 for the three systems at all pressures (0.1-20 MPa). It is found that the results for 0.1 MPa are in an agreement with those published by Leahy-Dios and Firoozabadi (2007) for the same systems at atmospheric pressure. The concentration expansion coefficient is given by: The concentration expansion coefficient is given by: , 0 1 c P T              (4.6) , 0 1 c P T              (4.6) where  is the mass fraction of the densest component. The temperature is now fixed and the density on a small interval of concentrations centred at 0 0.5  is measured. The approach for the determination of c  is the same as the one used for the measurement of 67 the T . 4.2.2 Methods Figure 4.2 shows the densities for five mass fractions of the densest component for the mixture of IBB-nC12 and are presented at different pressures. the T . Figure 4.2 shows the densities for five mass fractions of the densest component for the mixture of IBB-nC12 and are presented at different pressures. Figure 4.1: Density of IBB-nC12 mixture as a function of the temperature and for different pressures (0.1-20 MPa). 0.7850 0.7900 0.7950 0.8000 0.8050 0.8100 10 15 20 25 30 35 40 T [oC] Density [g/cm ] 0.1 MPa 2 MPa 4 MPa 6 MPa 8 MPa 10 MPa 12 MPa 14 MPa 16 MPa 18 MPa 20 MPa Figure 4.1: Density of IBB-nC12 mixture as a function of the temperature and for different pressures (0.1-20 MPa). The uncertainty of c  is estimated to be ± 0.009 for IBB-nC12 mixture, ± 0.01 for THN- nC12 mixture and ±0.008 for THN-IBB mixture. The results of 0.1 MPa are in agreement with those published in the literature for the same systems at atmospheric pressure, Leahy-Dios and Firoozabadi (2007). Figure 4.3 and Figure 4.4 show the densities of THN-nC12 mixture with temperature and mass fraction at different pressures, respectively. Figure 4.5 and Figure 4.6 show the densities of THN-IBB mixture with temperature and mass fraction at different pressures, respectively. A similar observation for mixture IBB-nC12 is found for the other two mixtures. 68 Figure 4.2: Density of IBB-nC12 mixture at 25 °C as function of the mass fraction of the densest component and for different pressures (0.1-20 MPa). 0.7850 0.7900 0.7950 0.8000 0.8050 0.8100 0.46 0.47 0.48 0.49 0.5 0.51 0.52 0.53 0.54 Mass Fraction [-] Density [g/cm3] 0.1 MPa 2 MPa 4 MPa 6 MPa 8 MPa 10 MPa 12 MPa 14 MPa 16 MPa 18 MPa 20 MPa 0.7850 0.7900 0.7950 0.8000 0.8050 0.8100 0.46 0.47 0.48 0.49 0.5 0.51 0.52 0.53 0.54 Mass Fraction [-] Density [g/cm3] 0.1 MPa 2 MPa 4 MPa 6 MPa 8 MPa 10 MPa 12 MPa 14 MPa 16 MPa 18 MPa 20 MPa Figure 4.2: Density of IBB-nC12 mixture at 25 °C as function of the mass fraction of the densest component and for different pressures (0.1-20 MPa). Figure 4.3: Density of THN-nC12 mixture at 25 °C as a function of the temperature and for different pressures (0.1-20 MPa). 4.2.2 Methods 0.8350 0.8400 0.8450 0.8500 0.8550 0.8600 10 15 20 25 30 35 40 T [°C] Density [g/cm 3] 0.1 MPa 2 MPa 4 MPa 6 MPa 8 MPa 10 MPa 12 MPa 14 MPa 16 MPa 18 MPa 20 MPa 0.8350 0.8400 0.8450 0.8500 0.8550 0.8600 10 15 20 25 30 35 40 T [°C] Density [g/cm 3] 0.1 MPa 2 MPa 4 MPa 6 MPa 8 MPa 10 MPa 12 MPa 14 MPa 16 MPa 18 MPa 20 MPa Figure 4.3: Density of THN-nC12 mixture at 25 °C as a function of the temperature and for different pressures (0.1-20 MPa). 69 Figure 4.4: Density of the THN-nC12 mixture at 25 °C as function of the mass fraction of the densest component and for different pressures (0.1-20 MPa). 0.8350 0.8400 0.8450 0.8500 0.8550 0.8600 0.46 0.47 0.48 0.49 0.5 0.51 0.52 0.53 0.54 Mass fraction [-] Density [g/cm3] 0.1 MPa 2 MPa 4 MPa 6 MPa 8 MPa 10 MPa 12 Mpa 14 MPa 16 MPa 18 MPa 20 MPa 0.8350 0.8400 0.8450 0.8500 0.8550 0.8600 0.46 0.47 0.48 0.49 0.5 0.51 0.52 0.53 0.54 Mass fraction [-] Density [g/cm3] 0.1 MPa 2 MPa 4 MPa 6 MPa 8 MPa 10 MPa 12 Mpa 14 MPa 16 MPa 18 MPa 20 MPa Figure 4.4: Density of the THN-nC12 mixture at 25 °C as function of the mass fraction of the densest component and for different pressures (0.1-20 MPa). Figure 4.5: Density of the THN-IBB mixture at 25 °C as a function of the temperature and for different pressures (0.1-20 MPa). 0.8975 0.9025 0.9075 0.9125 0.9175 0.9225 10 15 20 25 30 35 40 T [ oC] Density [g/cm 3] 0.1 MPa 2 MPa 4 MPa 6 MPa 8 MPa 10 MPa 12 MPa 14 MPa 16 MPa 18 MPa 20 MPa 0.8975 0.9025 0.9075 0.9125 0.9175 0.9225 10 15 20 25 30 35 40 T [ oC] Density [g/cm 3] 0.1 MPa 2 MPa 4 MPa 6 MPa 8 MPa 10 MPa 12 MPa 14 MPa 16 MPa 18 MPa 20 MPa Figure 4.5: Density of the THN-IBB mixture at 25 °C as a function of the temperature and for different pressures (0.1-20 MPa). 70 Figure 4.6: Density of THN-IBB mixture at 25 °C as function of the mass fraction of the densest component and for different pressures (0.1-20 MPa). 4.2.2 Methods 0.8975 0.9025 0.9075 0.9125 0.9175 0.9225 0.46 0.47 0.48 0.49 0.5 0.51 0.52 0.53 0.54 Mass fraction [-] Density [g/cm3] 0.1 MPa 2 MPa 4 MPa 6 MPa 8 MPa 10 MPa 12 MPa 14 MPa 16 MPa 18 MPa 20 MPa Figure 4.6: Density of THN-IBB mixture at 25 °C as function of the mass fraction of the densest component and for different pressures (0.1-20 MPa). 4.3 Theoretical Approach For the evaluation of the density, the compressibility factor of the mixtures has been calculated with different equations of state. The compressibility factor Z in Peng- Robinson Equation of State (PR-EoS), as introduced in section 2.2, can be written as follows:       ( ) p p p p p a T V V Z V b RT V V b b V b       (4.7) (4.7) The pure component parameters p a (attraction parameter) and p b (co-volume) can be calculated from critical properties and also depend on the temperature and acentric factor, as shown in Eqs. 2.27 and 2.28. The pure component parameters p a (attraction parameter) and p b (co-volume) can be calculated from critical properties and also depend on the temperature and acentric factor, as shown in Eqs. 2.27 and 2.28. 71 71 It has been found that the density prediction appears to be more accurate when the volume translated Peng-Robinson Equation of State (vt-PR-EoS) is used. The vt-PR-EoS is proposed by Péneloux et al. (1982). The shift parameters vis (Table 4.1) were calculated by Jhaverl and Youngren (1988). The viscosity of the fluid mixtures () in cP is obtained with a method proposed by Lohrenz et al. (1964), which was based on predicting the viscosity of reservoir fluids from their compositions, as follows:   4 4 10          (4.8)   4 4 10          (4.8) where  and  are given by: where  and  are given by: (4.9) (4.10) and 2 3 4 0.1023 0.023364 0.05833 0.40758 0.0093324 r r r r           (4.11) 2 3 4 0.1023 0.023364 0.05833 0.40758 0.0093324 r r r r           (4.11) (4.11) where i is the viscosity, ci T is the critical temperature, ci P is the critical pressure, and i M is the molecular weight of the ith component.  is a variable related to the reduced 72 72 density with a fourth order polynomials. / r c    is the reduced density of the mixture. is the density of the liquid mixture calculated using the P-R-EoS and c  is the pseudocritical density of the mixture. 4.4 Results and Discussion The measured mixture densities have been compared with densities which were calculated using PR-EoS and vt-PR-EoS for pressures varying from 0.1 MPa to 20 MPa. Comparison is given in Table 4.2; the full results of the experimental measurement of the densities for the three binary mixtures are shown in Appendix B. As one may notice, the vt-PR-EoS proves to be more efficient for predicting the densities than the PR-EoS. For the vt-PR-EoS the disagreement with the experimental densities is the same for the three binaries. PR-EoS gives good results only for the THN-IBB system (mixture of aromatics), which is logical, given that cubic equations are not suitable for long alkanes. It is noticed that the performances of the equations of state are independent of the pressure, presumably because the temperature is not very high. The thermal expansion and concentration expansion coefficients have been calculated (using PR-EoS and vt-PR-EoS) from the densities calculated for temperatures centred on 25 °C and for mass fractions centred on 50%, and with the same methodology used for experimental data. The comparison between the measured and the calculated thermal expansion coefficients and the concentration expansion coefficients for different pressures (from 0.1 to 20 MPa) are given in Table 4.3 and Table 4.4, respectively. From these tables, the comparisons with measured values show that vt-PR-EoS has better agreement with experiments than the PR-EoS. 73 Table 4.2: Theoretical and experimental densities for binary mixtures (50 wt% and 25 °C) Exp. PR-EoS vt-PR-EoS P (MPa) 3 (g.cm )   -3 (g.cm )  difference -3 (g.cm )  difference IBB-nC12 0.1 0.7917 0.7518 5.04% 0.7669 3.13% 4 0.7945 0.7543 5.06% 0.7695 3.15% 10 0.7986 0.7579 5.10% 0.7733 3.17% 20 0.8051 0.7632 5.20% 0.7787 3.28% THN-nC12 0.1 0.8407 0.7745 7.87% 0.8221 2.21% 4 0.8434 0.7767 7.91% 0.8246 2.23% 10 0.8471 0.78 7.92% 0.8282 2.23% 20 0.8530 0.7847 8.01% 0.8336 2.27% THN-IBB 0.1 0.9036 0.9016 0.22% 0.8745 3.22% 4 0.9061 0.9039 0.24% 0.8767 3.24% 10 0.9101 0.9073 0.31% 0.8799 3.32% 20 0.9161 0.9122 0.43% 0.8846 3.44% Table 4.3: Theoretical and experimental thermal expansion coefficient for binary mixtures (50 wt% and 25 °C). Exp. 4.4 Results and Discussion PR-Eos vt-PR-EoS P (MPa) 4 1 .10 (K ) T B  4 1 .10 (K ) T B  difference 4 1 .10 (K ) T B  difference IBB-nC12 0.1 9.3 6.06 34.8% 6.18 33.5% 4 8.9 5.73 35.7% 5.94 33.3% 10 9.0 5.29 41.2% 5.64 37.3% 20 8.6 4.70 45.4% 5.24 39.1% THN-nC12 0.1 8.6 5.56 35.4% 5.90 31.4% 4 8.3 5.28 36.3% 5.60 32.5% 10 8.4 4.92 41.5% 5.19 38.2% 20 8.0 4.42 44.7% 4.62 42.3% THN-IBB 0.1 8.8 5.76 34.6% 5.59 36.5% 4 8.5 5.53 35.0% 5.34 37.2% 10 8.5 5.21 38.7% 5.01 41.1% 20 8.1 4.76 41.3% 4.54 44.0% ble 4.3: Theoretical and experimental thermal expansion coefficient for binary mixtures (50 wt% and 25 °C). 74 e 4.4: Theoretical and experimental concentration expansion coefficient for binary mixt (50 wt% and 25 °C). Exp. PR-EoS vt-PR-EoS P (MPa) c  c  difference c  difference IBB-nC12 0.1 0.13 0.266 -104.6% 0.104 20.0% 4 0.13 0.265 -103.8% 0.103 20.8% 10 0.13 0.265 -103.8% 0.101 22.3% 20 0.12 0.264 -120.0% 0.010 91.7% THN-nC12 0.1 0.27 0.335 -24.1% 0.256 5.2% 4 0.27 0.334 -23.7% 0.254 5.9% 10 0.26 0.332 -27.7% 0.252 3.1% 20 0.26 0.329 -26.5% 0.249 4.2% THN-IBB 0.1 0.13 0.070 46.2% 0.153 -17.7% 4 0.13 0.070 46.2% 0.153 -17.7% 10 0.13 0.068 47.7% 0.152 -16.9% 20 0.12 0.067 44.2% 0.151 -25.8% Table 4.4: Theoretical and experimental concentration expansion coefficient for binary mixtures (50 wt% and 25 °C). Using the Lohrenz-Bray-Clark model for estimating the viscosity of the mixtures (Eq. 4.8) and the calculated densities (PR-EoS and vt-PR-EoS), it was possible to evaluate viscosities for each binary mixture and from each equation of state. The comparison between the measured and the calculated viscosities for pressures varying from 0.1 to 20 MPa is given in Table 4.5. The viscosity values at 0.1, 4 and 10 MPa are given, but the viscosity at 20 MPa was not measured experimentally, because of the 14 MPa limitation of the viscosimeter. The results reveal that the Lohrenz-Bray-Clark model, combined with the vt-PR-EoS, gives better agreement with measured data than the PR-EoS for the prediction of the viscosity of the three binaries. The percentage error was found to range from 11% to 18% when the vt-PR-EoS was used and from 14% to 37.5% for PR-EoS. 75 able 4.5: Theoretical and experimental viscosities for binary mixtures (50 wt% and 25 °C Exp. 4.4 Results and Discussion PR-EoS vt-PR-EoS P (MPa) (cP)  (cP)  difference (cP)  difference IBB-nC12 0.1 1.09 0.764 29.9% 0.902 17.2% 4 1.14 0.786 31.1% 0.930 18.4% 10 1.22 0.818 33.0% 0.970 20.5% 20 -- 0.866 -- 1.033 -- THN-nC12 0.1 1.46 0.965 33.9% 1.691 -15.8% 4 1.53 0.989 35.4% 1.746 -14.1% 10 1.64 1.026 37.4% 1.827 -11.4% 20 -- 1.082 -- 1.954 -- THN-IBB 0.1 1.33 1.540 -15.8% 1.156 13.1% 4 1.37 1.580 -15.3% 1.182 13.7% 10 1.43 1.638 -14.5% 1.221 14.6% 20 -- 1.731 -- 1.282 -- 4.5: Theoretical and experimental viscosities for binary mixtures (50 wt% and 25 °C). Chapter 5 In this chapter, a three-dimensional porous cavity is filled with two ternary mixtures. The first mixture consists of methane (C1), n-butane (nC4) and n-dodecane (nC12) at a pressure of 35.0 MPa and the second ternary mixture is nC12, THN, and IBB at 1 atmospheric pressure. Theses are used to investigate numerically the flow interaction due to the presence of thermodiffusion and buoyancy forces. A lateral heating condition is applied. The molecular diffusion and thermodiffusion coefficients are functions of temperature, concentration and viscosity of mixture components. 4.5 Summary The thermal expansion and concentration expansion coefficients and the viscosity of mixtures are necessary properties for the determination of the thermodiffusion coefficient. The densities of binaries of nC12, IBB and THN for pressures going from 0.1 to 20 MPa for temperatures centred on 25 °C and for concentrations centred on 50% have been measured. By a derivative method, the thermal expansion and concentration expansion coefficients at 25 °C and 50% mass fraction were determined. Viscosities were directly measured using high pressure high temperature viscometer (HPHTV-100). In order to accurately predict the thermal and concentration expansion coefficients, an attempt was made first to calculate the densities of the binaries using PR-EoS and vt-PR-EoS. The comparisons with measured densities show that vt-PR-EoS has better agreement with experiments than the PR-EoS. From calculated densities, the thermal expansion and concentration expansion coefficients were evaluated. The combination of the model of Lohrenz-Bray-Clark for the viscosity of liquid mixtures, and the densities calculated with the two equations of state proved to be inefficient for the prediction of the viscosities of the binaries. 76 Chapter 5 Soret Effect for a Ternary Mixture in a Porous Cavity: Modeling with Variable Diffusion Coefficients and Viscosity 5.1 Introduction The thermodiffusion process can occur in both liquid and gaseous mixtures. Studies based on the thermodynamics of irreversible processes have shown that thermodiffusion in liquids, along with the effect of natural convection, can in fact greatly influence the composition distribution in hydrocarbon reservoirs. The Ludwig-Soret effect is commonly represented by the thermodiffusion coefficient, especially for multicomponent mixtures. The theoretical study of this effect is usually carried out based on non-equilibrium thermodynamics, de Groot and Mazur (1984). Recently, a large amount of research has been done on thermodiffusion in porous media by Haugen and Firoozabadi (2005), and Platten (2005). It is known that convection has a significant influence on the accuracy of Soret coefficient measurements. Utilization of porous media may help in reducing the convection-induced distortion. Costeseque et al. (2004) conducted diffusion experiments in both free and porous media in a Soret cell. They reported that the molecular diffusion and thermodiffusion coefficients in porous media were related to those in clear fluid via the tortuosity. However the Soret coefficient 77 77 (the ratio of molecular diffusion coefficient to thermodiffusion coefficient for binary mixtures) is identical for both configurations. Riley and Firoozabadi (1998) presented a model to investigate the effects of natural convection and diffusion (thermal, pressure and fickian) on a single-phase binary mixture of methane and n-butane in a horizontal cross-sectional reservoir in the presence of a prescribed linear temperature field. The compositional distribution in the reservoir under both horizontal and lateral heating conditions was carefully examined. It was found that increasing the permeability increased the horizontal compositional variation. A local maximum and/or minimum value exists in the compositional gradient as a function of the permeability. Delware et al. (2004) studied these phenomena for a binary system in a square cavity. The energy equation is solved simultaneously allowing the observing of temperature variation in the model. Various thermal boundary conditions are examined. Their results revealed that in the lateral heating case the Soret effect is found to be weak, whereas in the bottom heating case the Soret effect is more pronounced. Firoozabadi et al. (2000) developed a model for thermal diffusion factors in multicomponent non-ideal mixtures. This model was based on the thermodynamics of irreversible processes where the effects of both equilibrium and non-equilibrium properties are incorporated. The equilibrium properties, such as partial internal energies and fugacities, were estimated using the volume-translated Peng-Robinson equation of state. 5.1 Introduction On the other hand, the non-equilibrium properties, such as viscosity, were accounted for by incorporating the energy of viscous flow. This model has been used by Jiang et al. (2008) to examine the thermodiffusion convection of a water-ethanol mixture in porous medium under high pressure. To the best of our knowledge, the only thermodiffusion measurement for hydrocarbon ternary mixtures is reported by Leahy-Dios et al., (2005). In this work, an experiment was done by using the thermogravitational column method, and they measured the thermodiffusion coefficient for two different ternary mixtures: one of the mixtures consisted of normal alkane (nC12) and two aromatics (IBB and THN), and the second 78 mixture consisted of two normal alkanes (octane and decane) and one aromatic (1- methylnaphthalene). Their results revealed that in the ternary mixture of octane-decane-1- methylnaphthalene, a sign of the thermodiffusion coefficient for decane is changed as the composition changes, despite the fact that the two normal alkanes are similar. In this work, attempts are made in simulating the thermosolutal convection of two ternary mixtures. In addition, a comparison is made between the experimental data of the ternary mixture (Leahy-Dios et al., 2005) and the theoretical results using the Firoozabadi model with two equations of state (PR-EoS and vt-PR-EoS). 5. 3 Model Description The porous cavity has a horizontal length of 32 mm, a width of 10 mm, and a vertical height of 10 mm, as shown in Figure 5.1. The porous material is Al2O3 powder; and the cavity is saturated with the ternary fluid mixture. Physical properties of the liquid mixtures and the porous medium are given in Table 5.1. The left wall of the cavity is kept at a temperature of Tc=10 oC and the right wall at Th=50 oC. Due to the Soret effect, component separation will happen in the porous cavity even under the gravity condition; and the system will gradually reach the steady state. The pressure in the cavity is maintained at 35.0 MPa for C1-nC4-nC12 mixture and 1 atm for nC12-IBB-THN mixture. Figure 5.1: Schematic diagram of the horizontal porous cavity and boundary conditions. , 0, V 0 c mi T T J     W H L x y z 0, 0, V 0 q mi J J     0, 0, V 0 q mi J J     0 0 , , V h mi T T J     0, 0, V 0 q mi J J     Figure 5.1: Schematic diagram of the horizontal porous cavity and boundary conditions 79 Table 5.1: Physical properties of the mixture and porous material, Tave =303 K. Name Value Unit Porous cavity length L 32 mm Porous cavity width W 10.0 mm Porous cavity height H 10.0 mm Left wall temperature T1 10 oC Right wall temperature T2 50 oC Pressure at the centre of the cavity 35.0, 0.1013 MPa Al2O3 density 3983.6 kg/m3 Al2O3 heat capacity 786.2745 J/kg/K Al2O3 thermal conductivity 43 W/m/K Al2O3 porosity 0.4 Al2O3 porous medium permeability 100 ~ 107 mD Al2O3 porous medium tortuosity 1.3 C1-nC4-nC12 mixture compositions 50-20-30 (% mole fr.) C1-nC4-nC12 mixture heat capacity 2355.4 J/kg/K C1-nC4-nC12 mixture thermal conductivity 0.1158 W/m/K nC12-THN-IBB mixture compositions 33.3-33.3-33.4 (% mass fr.) nC12-THN-IBB mixture heat capacity 1885.9 J/kg/K nC12-THN-IBB mixture thermal conductivity 0.123 W/m/K Table 5.1: Physical properties of the mixture and porous material, Tave =303 K. 5.4 Governing Equations and Boundary Conditions The mass continuity equation is shown as:   0 m m t       V  (5.1)   0 m m t       V    0 m m t       V  (5.1) (5.1) For a multicomponent mixture, the continuity equation of component i is given as:   ( ) , 1,2,...... - mi mi mi J i n t        V     ( ) , 1,2,...... -1 mi mi mi J i n t        V   (5.2) (5.2) 80 where mi J  is the molar diffusion flux of the ith component. The mole flux is subjected to a linear relationship to the driving forces of temperature and concentration gradient, and it can be expressed by:     1 * * 1 n mi m ij Ti j J D X D T                    (5.3) (5.3) where T is the temperature. For binary mixtures, 1x X    and 1 mi m J J    , and for ternary           2 1 x x X and 1 2 m mi m J J J            . * D and * T D are the molecular diffusion and mixtures,           2 1 x x X and 1 2 m mi m J J J            . * D and * T D are the molecular diffusion and mixtures,           2 1 x x X and 1 2 m mi m J J J           . 5.4 Governing Equations and Boundary Conditions 5.1, 81 the pressure can be solved from the obtained differential equation, which leads to the following equation for pressure: the pressure can be solved from the obtained differential equation, which leads to the following equation for pressure:   0 κ κ κ x y z P m g m t x x P P g g m m y y z z                                                                    (5.6) (5.6) The thermal energy conservation equation is expressed as follows: The thermal energy conservation equation is expressed as follows:   p e 2 p fl e ( ) V ( ) C T C T k T t             p e 2 p fl e ( ) V ( ) C T C T k T t           (5.7) (5.7) where   p e C  is the effective volumetric heat capacity of the system and ke is the effective thermal conductivity of the system. These effective physical parameters are related to the fluid properties and the solid matrix properties as follows: where   p e C  is the effective volumetric heat capacity of the system and ke is the effective thermal conductivity of the system. 5.4 Governing Equations and Boundary Conditions These effective physical parameters are related to the fluid properties and the solid matrix properties as follows:       fl (1 ) p p p e s C C C        (5.8)       fl (1 ) p p p e s C C C        (5.8) fl s (1 ) ek k k      (5.9) fl s (1 ) ek k k      (5.9) The boundary conditions in this model are: (1) zero mass flux through all walls; (2) no- slip walls; and (3) lateral heating condition on two side walls and other walls are adiabatic. Figure 5.1 depicts the boundary conditions. 5.4 Governing Equations and Boundary Conditions * D and * T D are the molecular diffusion and thermodiffusion coefficients of the fluid mixture in the porous medium, respectively, which are related to the molecular diffusion coefficient and thermodiffusion coefficient in free liquid as: thermodiffusion coefficients of the fluid mixture in the porous medium, respectively, which are related to the molecular diffusion coefficient and thermodiffusion coefficient in free liquid as:     * * 2 2 1 1 , ij ij Ti Ti M T D D D D             (5.4) (5.4) where D and T D are the molecular and thermodiffusion coefficients, which are functions of the temperature and composition of the fluid mixture. M  , and T  are tortuosities for the molecular diffusion and thermodiffusion coefficients in the porous cavity, respectively. Based on Costeseque’s findings (2004), the molecular diffusion and thermodiffusion tortuosities were set equal to the tortuosity of the porous medium. The porous matrix is assumed homogeneous and isotropic. Therefore the Darcy equation is applied: ( ) P g      κ V  (5.5) ( ) P g      κ V  (5.5) where κ and are the permeability and the porosity of the porous medium, respectively. By substituting the Darcy relation, Eq. 5.5, into the mass conservation equation, Eq. 5.4 Molecular Diffusion and Thermodiffusion Model The thermodiffusion can be described through the theory of non-equilibrium thermodynamics, (de Groot and Mazur, 1984). In an n-component fluid, by neglecting the 82 viscous pressure tensor and reaction, and assuming a mechanical equilibrium in the system, linear relations can be established between the mass and heat fluxes and their driving forces, respectively. These relations, as explained in Chapter 2, are called Onsager Equations and can be expressed by: 1 ' 2 1 1 1 n T k T n q qq qk k k n J L T L T T M M                  (5.10) 1 2 1 1 1 n T k T n i iq ik k k n J L T L T T M M                  (5.11) 1 ' 2 1 1 1 n T k T n q qq qk k k n J L T L T T M M                  (5.10) (5.10) 1 2 1 1 1 n T k T n i iq ik k k n J L T L T T M M                  (5.11) (5.11) where q J is the heat flux, Lqq, Lqk, Liq and Lik are the phenomenological coefficients called Onsager coefficients, k  is the chemical potential of component k. Through Eqs. 5.3 and 5.11, one can get the following formulation, (Firoozabadi et al., 2000): where q J is the heat flux, Lqq, Lqk, Liq and Lik are the phenomenological coefficients called Onsager coefficients, k  is the chemical potential of component k. Through Eqs. 5.4 Molecular Diffusion and Thermodiffusion Model 5.3 and 5.11, one can get the following formulation, (Firoozabadi et al., 2000): , , 1 1 1 1 2 ln j l j x T T i n n n n l l lk ij ik k l m n n j iq m f x x M x M R D L x M M L D T                       (5.12) (5.12) where xj is the mole fraction of component j, Mj is the molecular weight of component j, 1 n j j j M M x   is the molecular weight of the mixture, fj is the fugacity of component j, R is the gas constant, Lik and Liq are Onsager coefficients, and jk  is the Kronecker delta ( jk  =1, if j=k and jk  =0, if j k). where xj is the mole fraction of component j, Mj is the molecular weight of component j, 1 n j j j M M x   is the molecular weight of the mixture, fj is the fugacity of component j, R where xj is the mole fraction of component j, Mj is the molecular weight of component j, is the gas constant, Lik and Liq are Onsager coefficients, and jk  is the Kronecker delta ( jk  =1, if j=k and jk  =0, if j k). All diffusion coefficients are defined as functions of thermodynamic properties of the mixture and Onsager coefficients. As shown from Eq. 5.12, both thermodiffusion and molecular diffusion coefficients can be given in terms of phenomenological coefficients. 83 According to the theory of non-equilibrium thermodynamics, a relationship between iq L and ik L is found by introducing the net heat of transport * k Q . 5.4 Molecular Diffusion and Thermodiffusion Model This relation is given by: * * 1 1 n k n iq ik k k n Q Q L L M M            (5.13) (5.13) In the Firoozabadi model the net heat of transport is given by: In the Firoozabadi model the net heat of transport is given by: * 1 1 n j j i i i n j i j j j j x U U V Q V x                  (5.14) (5.14) where i U  is the residual partial molar internal energy of the component i; iV is the partial molar volume of component i; iis the ratio of vaporization energy (also called cohesive energy) to energy of viscous flow of component i   / vap visc i i i U U   . By following Eyring’s viscosity theory (Glasstone et al., 1941), the value of i for each component can be obtained from plots of ln m against 1/ T for pure components. In this work, the same value of 4.0 for iof all components in hydrocarbon mixtures (non- associating mixtures) is assigned, as suggested by Shukla and Firoozabadi (1998). where i U  is the residual partial molar internal energy of the component i; iV is the partial molar volume of component i; iis the ratio of vaporization energy (also called cohesive energy) to energy of viscous flow of component i   / vap visc i i i U U   . By following Eyring’s viscosity theory (Glasstone et al., 1941), the value of i for each component can be obtained from plots of ln m against 1/ T for pure components. In this work, the same value of 4.0 for iof all components in hydrocarbon mixtures (non- associating mixtures) is assigned, as suggested by Shukla and Firoozabadi (1998). Besides the net heat of transport (Eq. 5.14), molecular or Fick’s diffusion coefficients are also needed to calculate thermodiffusion coefficients. The diffusion coefficients in a dilute binary mixture have to be evaluated before calculating Fick’s diffusion coefficients. 5.4 Molecular Diffusion and Thermodiffusion Model In this work, the expression given by Hayduk and Minhas (1982) is used for hydrocarbon binary mixture, (Taylor and Krishna, 1993): 1 (10.2/ 0.791) 0 8 0.71 1.47 13.3 10 V i ij j D V T       1 (10.2/ 0.791) 0 8 0.71 1.47 13.3 10 V i ij j D V T       (5.15) 84 where 0 ij D is the binary infinite dilution diffusion coefficient of solute i in solvent j , [cm2/s]; Vi is the molar volume of component i at its normal boiling point, [cm3/mol]; j  is the viscosity of pure component j, [MPa.s], and T is the mixture temperature, [K]. For a multicomponent mixture, the Maxwell-Stephan diffusion coefficient, ij D , is calculated based on the binary coefficients, 0 ij D . 5.4 Molecular Diffusion and Thermodiffusion Model Taylor and Krishna (1993) have suggested the following formula:   /2 0 1 , ( ) ( ) k j i n x x x o o o ij ji ij ik jk k k i j D D D D D     (5.16) (5.16) From the multicomponent mass transfer theory, the mass flux is given by: From the multicomponent mass transfer theory, the mass flux is given by: From the multicomponent mass transfer theory, the mass flux is given by: 1 m m J B X      (5.17) 1 m m J B X      (5.17) 1 m m J B X      1 m m J B X      (5.17) Here the matrix B is defined by: Here the matrix B is defined by: 1 1 1 ij i ij in n i k ij k in ik k i x i j B D D x x i j B D D                           (5.18) 1 1 1 ij i ij in n i k ij k in ik k i x i j B D D x x i j B D D                           (5.18) and the elements of matrix are: and the elements of matrix are: and the elements of matrix are: 1 i ij i j i f x f x     (5.19) 1 i ij i j i f x f x     (5.19) From Fick’s law, the mass flux can be written as: 85 m m J D X     m m J D X     (5.20) Comparing Eq. 5.20 with Eq. 5.17, one can have: Comparing Eq. 5.20 with Eq. 5.17, one can have: Comparing Eq. 5.20 with Eq. 5.17, one can have: (5.21) 1 D B   Therefore, the diffusion coefficient, ij D , can be determined based on the elements in B and  matrices with the an equation of state and other physical properties. 5.4 Molecular Diffusion and Thermodiffusion Model Therefore, the diffusion coefficient, ij D , can be determined based on the elements in B and  matrices with the an equation of state and other physical properties. With the Firoozabadi model for multicomponent mixtures and an equation of state, after specifying the mixture components and knowing the parameters of each pure component, the molecular diffusion coefficient and the thermodiffusion coefficient can be calculated by following these steps: 1. calculate thermodynamic properties of the mixture (partial molar volume, fugacity, internal energy, etc) using the equation of state. In this work, PR-EoS and vt-PR-EoS are used; 2. calculate the B (Bij) and ( ij ) matrices and then molecular diffusion coefficients; 3. calculate the Onsager coefficients (Lik) from molecular diffusion coefficients following Eq. 5.12; 4. calculate the Onsager coefficients (Liq) using Eq. 5.13; 4. calculate the Onsager coefficients (Liq) using Eq. 5.13; 5. finally, calculate the thermodiffusion coefficient and all mass flux terms. In this work, the viscosity of the fluid mixture () is obtained with a method proposed by Lohrenz et al. (1964), as explained in detail in section 4.3, and given by * 4 4 ( 10 ) /         (5.22) 86 5.5 Numerical Solution Procedure Equations 5.1, 5.2, 5.5, 5.6 and 5.7 are solved numerically by using the control volume method, subject to the boundary conditions of zero flux at the rigid wall. All the walls are assumed to be solid walls, so three zero components of the velocity are maintained. The second-order centred scheme is used in the space discretization, and a semi-implicit first-order scheme is used for the temporal integration. With respect to the non-linear convection terms, the power-law scheme is applied in order to achieve higher accuracy for the combined convection and diffusion cases. The obtained linear system of algebraic equations is solved at each time step using a bi-conjugated gradient iteration method with a given convergence criterion, which has been confirmed over many tests for the required accuracy. At the initial time step, the velocities were set to zero in the computational domain where initial pressure and concentration are specified. The convergence criterion is set for three parameters, the pressure, temperature and composition. 5.5 Numerical Solution Procedure The relative errors between internal iteration and any two successive time steps are calculated as follows: For internal iteration at each time step, k, 1 k, k, 1 1 1 1 1 s s ny nx nz ijl ijl s i j l ijl nx ny nz                (5.23) (5.23) For convergence checking between two successive time steps after the convergence of the internal iteration, For convergence checking between two successive time steps after the convergence of the internal iteration, k, 1 k 1, 0 k 1, 0 1 1 1 1 s s ny nx nz ijl ijl s i j l ijl nx ny nz                 (5.24) (5.24) where θ represents the pressure, temperature, and composition, respectively, i, j, and l represent mesh indices along x, y, and z directions of the porous cavity; k denotes the time step; s is the indicator of inner iterations; s0 is the indicator of the converged inner iteration at the last time step and nx, ny, and nz represent the mesh number in x, y, and z where θ represents the pressure, temperature, and composition, respectively, i, j, and l represent mesh indices along x, y, and z directions of the porous cavity; k denotes the time step; s is the indicator of inner iterations; s0 is the indicator of the converged inner iteration at the last time step and nx, ny, and nz represent the mesh number in x, y, and z 87 directions, respectively. The values of pressure, temperature and composition are defined in the centre of each control volume, but the velocities are defined on the surface of each control volume, or grid cell. The solution procedure begins by assuming initial pressure, temperature and concentration values in the mixture. The fluid is considered to be weakly compressible, and the Peng-Robinson EoS is used to calculate the fluid thermodynamic properties. In fact, the density, and molecular diffusion and thermodiffusion coefficients are functions of the temperature and species compositions. They are evaluated for each control volume. The thermal conductivity is assumed constant in the analysis. The solution procedure begins by assuming initial pressure, temperature and concentration values in the mixture. 5.5 Numerical Solution Procedure The fluid is considered to be weakly compressible, and the Peng-Robinson EoS is used to calculate the fluid thermodynamic properties. In fact, the density, and molecular diffusion and thermodiffusion coefficients are functions of the temperature and species compositions. They are evaluated for each control volume. The thermal conductivity is assumed constant in the analysis. To get an optimum number of grids, mesh sensitivity analysis was examined for this work model. Performing a mesh sensitivity analysis is an integral part of producing accurate, time-efficient and cost-effective results. The mesh sensitivity is characterized by the average Nusselt number ( Nu ), as shown in Figure 5.2 and Table 5.2, which is defined at both lateral walls (hot and cold walls) of the porous medium as follows: 1 wall L T Nu dydz WH T x            (5.25) 1 wall L T Nu dydz WH T x            1 wall L T Nu dydz WH T x            (5.25) (5.25) The average Nusselt number is equivalent to the non-dimensional heat flux averaged over the wall surface of the cavity. Figure 5.2 shows the relation between the average Nusselt number and different types of mesh. From this Figure, the best mesh can be chosen and adopted in the simulations. As can be seen, there is no significant difference in the average Nusselt number values between mesh type 4 and mesh type 8, so any mesh between them can be selected. The mesh type 6 (50x50x50 control volume) is selected. 88 Figure 5.2: Nusselt number with mesh size. 1.005 1.01 1.015 1.02 1 2 3 4 5 6 7 8 Mesh size Type Average Nusselt number Figure 5.2: Nusselt number with mesh size. 1.005 1.01 1.015 1.02 1 2 3 4 5 6 7 8 Mesh size Type Average Nusselt number Average Nusselt number Mesh size Type Figure 5.2: Nusselt number with mesh size. Table 5.2: Mesh size sensitivity. Type 3D Mesh number Nu 1 10x10x10 1.008881 2 16x16x16 1.011856 3 20x20x20 1.013047 4 30x30x30 1.013677 5 40x40x40 1.014065 6 50x50x50 1.014326 7 60x60x60 1.014515 8 70x70x70 1.014656 89 5.6.1 Comparison between experimental data and numerical calculation The only experimental data for a ternary mixture available in the literature is given by Leahy-Dios et al. (2005). Table 5.3 shows the comparison between our numerical result and the experimental data for a ternary mixture of nC12-THN-IBB. It is noticed that by using vt-PR-EoS for evaluating the thermodynamic properties, which are used in the Firoozabadi model, one obtains better agreement with the experimental data. It is found that the percentage errors for 1 2 3 , and T T T D D D , when the vt-PR-EoS is used, are 12.63%, 23.11% and 49.65%, respectively. Furthermore, the mixture component’s behaviour also is identical, for both experimental data and numerical values have the same sign for thermodiffusion coefficients. It can be concluded that the two components (nC12 and THN) migrate, one to the hot side and the other to the cold side, while the IBB was distributed randomly within the cavity, as its thermodiffusion coefficient value is small, and this confirmed that its separation remains small. Table 5.3: The thermodiffusion coefficients (experimental and theoretical) of nC12-THN-IBB ternary mixture (33.3-33.3-33.4 wt%) at 25 oC and 1 atm. Method 12 2 -1 , 12 (10 m .s K) T nC D  12 2 -1 , (10 m .s K) T THN D  12 2 -1 , (10 m .s K) T IBB D  Experimental -1.021 0.33 0.874 0.43 0.147 Theo. PR-EoS -1.523 1.262 0.261 Error 49.16% 44.39% 77.55% Theo.vt-PR-EoS -1.150 1.076 0.074 Error 12.63% 23.11% 49.65% Table 5.3: The thermodiffusion coefficients (experimental and theoretical) of nC12-T ternary mixture (33.3-33.3-33.4 wt%) at 25 oC and 1 atm. The density meter (DMA 5000-Anton Paar with an accuracy of 6 3 1x10 g/cm   and temperature fluctuation o 0.001 C  ) has been used to measure the density of the nC12- THN-IBB mixture at different temperatures and compared with the numerical results in order to validate the equation of state (vt-PR-Eos). Figure 5.3 shows the variation of the density against the temperature for both the numerical model and the experimental data. 90 An excellent agreement was found between our numerical model and the experimental data, and the percentage error was less than 3.0%. An excellent agreement was found between our numerical model and the experimental data, and the percentage error was less than 3.0%. Figure 5.3: Density of nC12-IBB-THN mixture vs. temperature. 5.6.1 Comparison between experimental data and numerical calculation 0.8000 0.8200 0.8400 0.8600 0.8800 10 15 20 25 30 35 40 45 50 T [oC] Density [g/cm3] Exp. vt-PR-EoS Density [g/cm3] Figure 5.3: Density of nC12-IBB-THN mixture vs. temperature. 5.6.2 Permeability effect on composition distribution As is known, the ternary mixture in this simulation consists of 50% methane (C1, component 1), 20% n-butane (nC4, component 2) and 30% n-dodecane (nC12, component 3). The compositional separation in the mixture is investigated for permeability ranging from 102 mD to 106 mD. It is found that the effect of permeability on separation is very strong, (see Figures 5.4 and 5.5), where the distribution of methane (C1) and n-butane (nC4) are illustrated along the vertical (z=0~H, x=L/2, y=W/2) and horizontal (x=0~L, z=H/2, y=W/2) directions. It is clear that the separation of mixture components in the horizontal direction decreases continuously as the permeability increases from 102 mD to 106 mD. When the permeability is 102 mD, the separation happens mainly along the horizontal direction, (see Figures 5.4a and 5.5a); while in the vertical direction it is very weak, (see Figures 5.4b and 5.5b). 91 Figure 5.4: Methane (C1) mole fraction distributions along the centre of the cavity. 0.496 0.498 0.500 0.502 0.504 0.000 0.002 0.004 0.006 0.008 0.010 Height H [m] C1 mole fraction [-] κ=100 md κ=1000 md κ=10000 md κ=100000 md κ=1000000 md 0.47 0.48 0.49 0.50 0.51 0.52 0.53 0.000 0.004 0.008 0.012 0.016 0.020 0.024 0.028 0.032 Length L [m] C1 mole fraction [-] κ=100 md κ=1000 md κ=10000 md κ=100000 md κ=1000000md (a) Horizontal distribution (b) Vertical distribution 0.47 0.48 0.49 0.50 0.51 0.52 0.53 0.000 0.004 0.008 0.012 0.016 0.020 0.024 0.028 0.032 Length L [m] C1 mole fraction [-] κ=100 md κ=1000 md κ=10000 md κ=100000 md κ=1000000md (a) Horizontal distribution Figure 5.4: Methane (C1) mole fraction distributions along the centre of the cavity. 0.496 0.498 0.500 0.502 0.504 0.000 0.002 0.004 0.006 0.008 0.010 Height H [m] C1 mole fraction [-] κ=100 md κ=1000 md κ=10000 md κ=100000 md κ=1000000 md (b) Vertical distribution C1 mole fraction [-] Figure 5.4: Methane (C1) mole fraction distributions along the centre of the cavity. 92 0.1990 0.1992 0.1994 0.1996 0.1998 0.2000 0.2002 0.2004 0.2006 0.000 0.004 0.008 0.012 0.016 0.020 0.024 0.028 0.032 Length L [m] nC4 mole fraction [-] κ=100 md κ=1000 md κ=10000 md κ=100000 md κ=1000000 md (a) Horizontal distribution Figure 5.5: n-butane (nC4) mole fraction distributions along the centre of the cavity. 5.6.2 Permeability effect on composition distribution 0.1990 0.1992 0.1994 0.1996 0.1998 0.2000 0.2002 0.2004 0.2006 0.000 0.004 0.008 0.012 0.016 0.020 0.024 0.028 0.032 Length L [m] nC4 mole fraction [-] κ=100 md κ=1000 md κ=10000 md κ=100000 md κ=1000000 md 0.19990 0.19992 0.19994 0.19996 0.19998 0.20000 0.20002 0.20004 0.20006 0.20008 0.20010 0.000 0.002 0.004 0.006 0.008 0.010 Height H [m] nC4 mole fraction [-] κ=100 md κ=1000 md κ=10000 md κ=100000 md κ=1000000 md (b) Vertical distribution (a) Horizontal distribution (a) Horizontal distribution Figure 5.5: n-butane (nC4) mole fraction distributions along the centre of the cavity. 0.19990 0.19992 0.19994 0.19996 0.19998 0.20000 0.20002 0.20004 0.20006 0.20008 0.20010 0.000 0.002 0.004 0.006 0.008 0.010 Height H [m] nC4 mole fraction [-] κ=100 md κ=1000 md κ=10000 md κ=100000 md κ=1000000 md (b) Vertical distribution Figure 5.5: n-butane (nC4) mole fraction distributions along the centre of the cavity. 93 Figure 5.6 displays the C1 and nC4 composition distribution in the centre x-z plane for different permeabilities ( κ = 10, 102, 104, and 106 mD). An almost linear contour of methane and n-butane along the horizontal direction at 10, and 102 mD, is found. At those low permeabilities, the convection effect is greatly suppressed and the thermodiffusion effect is dominant, (see Figures 5.6a and 5.6b). As the permeability increases to 104 mD, the buoyancy convection begins to play a role causing a mixing in the cavity; therefore, the separations of methane and n-butane show a weakening in the horizontal direction, and curved lines are found in the vertical direction, (see Figure 5.6c). As the permeability continues to increase beyond 105 mD, the Soret effect is greatly weakened and the dominant effect of convection makes the separation almost impossible, (see Figure 5.6d). Figures 5.6a and 5.6b also illustrate the direction of the component separation due to thermodiffusion at low permeabilities. Methane, being the light component, moves towards the hot wall; and n-butane, being heavier than methane, moves towards the cold wall. Figure 5.7 shows the nC12 and THN composition distribution in the centre x-z plane for different permeabilities (κ = 102, 104, 105 and 107 mD). In this Figure, the trend of nC12 and THN is similar to those in Figure 5.6. 5.6.2 Permeability effect on composition distribution As can be seen, the nC12 component migrates toward a hot side, being a lighter component and has a negative thermodiffusion coefficient, and the THN migrates to the cold side which is the heavier component and has a positive thermodiffusion coefficient, (see Figure 5.7a and 5.7b). As already noticed, the permeability has a significant effect on the separation of mixture components. At low permeability the Soret effect is significant; while at high permeability convection becomes dominant. To further examine this phenomenon, a variable known as the separation ratio, q, is introduced: max max min min /(1 ) /(1 ) x x q x x    (5.26) max max min min /(1 ) /(1 ) x x q x x    (5.26) where max x and min x are the maximum and minimum concentrations of a solute component in the porous cavity, respectively. where max x and min x are the maximum and minimum concentrations of a solute component in the porous cavity, respectively. 94 The permeability impact on separation in the porous cavity can be shown with the separation ratio versus the permeability relation, given in Figures 5.8 and 5.9 for mixtures C1-nC4-nC12 and nC12-THN-IBB, respectively. From Figure 5.8, three distinct zones can be identified: (I) the permeability is below 102 mD. In this zone, the Soret effect is completely dominant. The separation ratio remains constant, and is about 1.248 for methane and 1.007 for n-butane; (II) the permeability is between 102 mD to 105 mD. In this zone both Soret and convection play important roles and as a result of this combined effect the separation ratio gradually reduces as the permeability increases; (III) the permeability is higher than 105 mD. In this zone the convection is the dominant mechanism, therefore, the separation ratio decreases rapidly towards 1.0, which indicates that the components are largely mixed in the cavity and no noticeable separation can be achieved. The permeability impact on separation in the porous cavity can be shown with the separation ratio versus the permeability relation, given in Figures 5.8 and 5.9 for mixtures C1-nC4-nC12 and nC12-THN-IBB, respectively. From Figure 5.8, three distinct zones can be identified: (I) the permeability is below 102 mD. In this zone, the Soret effect is completely dominant. 5.6.2 Permeability effect on composition distribution The separation ratio remains constant, and is about 1.248 for methane and 1.007 for n-butane; (II) the permeability is between 102 mD to 105 mD. In this zone both Soret and convection play important roles and as a result of this combined effect the separation ratio gradually reduces as the permeability increases; (III) the permeability is higher than 105 mD. In this zone the convection is the dominant mechanism, therefore, the separation ratio decreases rapidly towards 1.0, which indicates that the components are largely mixed in the cavity and no noticeable separation can be achieved. Three zones are noticed in Figure 3.9 (similar behaviour to the one in Figure 3.8). In the first zone (I), the thermodiffusion is dominant and the separation ratio is constant (q=1.31 for THN component and q=1.45 for nC12 component), where the permeability value is between 0.01 to 30 mD. The permeability value for the second zone (II) is between 30 and 105 mD; both thermodiffusion and convection effect are found. In the third zone (III), the convection plays a significant roll, and the permeability in this zone is between 105 to 107 mD. This theoretical analysis can be very useful in many ways. For example, in the case of measuring diffusion coefficients in a porous cavity, an optimal design of the porous cavity will be the key to determine if the experiments can be feasibly performed. A too low permeability cavity will require a long time for experiments to establish the steady state; therefore, it is ineffective or even infeasible. A too high permeability cavity, on the other hand, will fail to suppress the negative effect of convection. If the mixture of methane (50%), n-butane (20%) and n-dodecane (30%) is to be measured, the optimal permeability would be around 102 mD. 95 Benano-Melly et al. (2001) found that the maximum value for the separation ratio, q=qmax , existed for the permeability value, κ κm  given by: 120 κ M m m T D g TH       (5.27) 120 κ M m m T D g TH       (5.27) (5.27) This analytical expression predicts that the maximum separation of methane will occur at κm =320 mD whereas our numerical calculation showed that the maximum separation ratio occurred at κm =350 mD (qmax=1.2509). 5.6.2 Permeability effect on composition distribution This discrepancy between the analytical and the numerical modeling is justified by the fact that in our case the solutal buoyancy is included and both the thermodiffusion and molecular diffusion coefficients are functions of temperature and fluid mixture. 96 Figure 5.6: C1/nC4 compositional distributions in the centre x-z plane of the C1-nC4-nC12 ternary mixture. (a) κ =10 mD (b) κ =102 mD (c) κ =104 mD (d) κ =106 mD (a) κ =10 mD (b) κ =102 mD Figure 5.6: C1/nC4 compositional distributions in the centre x-z plane of the C1-nC4-nC12 (c) κ =104 mD (d) κ =106 mD (d) κ =106 mD (c) κ =104 mD (d) κ =106 mD Figure 5.6: C1/nC4 compositional distributions in the centre x-z plane of the C1-nC4-nC12 ternary mixture. 97 Figure 5.7: nC12/THN compositional distribution in the centre x-z plane of the nC12-THN-IBB ternary mixture. 0 .2 4 8 4 0 .2 5 7 8 0 .2 6 7 1 0 .2 7 6 4 0 .2 8 5 8 0 .2 9 5 1 0 .3 0 4 5 0 .3 1 3 8 0.2740 0.2765 0.2790 0.2814 0.2839 0.2864 0 .3 8 9 2 0 .3 8 1 5 0 .3 7 3 8 0 .3 6 6 0 0 .3 5 8 3 0 .3 5 0 6 0 .3 4 2 8 0 .3 3 5 1 0.3685 0.3663 0.3640 0.3618 0.3596 0.3573 0.2814 0.2808 0.2812 0.2810 0.2801 0.2818 0.2811 0.2811 0.2811 0.2811 0.3619 0.3620 0.3622 0.3623 0.3625 0.3617 0.3630 0.3622 0.3622 0.3622 0.3622 (a) κ =102 mD (b) κ =104 mD (c) κ =105 mD (d) κ =107 mD 0.2740 0.2765 0.2790 0.2814 0.2839 0.2864 0.3685 0.3663 0.3640 0.3618 0.3596 0.3573 (b) κ =104 mD (b) κ =104 mD 0.2814 0.2808 0.2812 0.2810 0.2801 0.2818 0.2811 0.2811 0.2811 0.2811 0.3619 0.3620 0.3622 0.3623 0.3625 0.3617 0.3630 (c) κ =105 mD 0.3622 0.3622 0.3622 0.3622 (d) κ =107 mD (c) κ =105 mD (d) κ =107 mD Figure 5.7: nC12/THN compositional distribution in the centre x-z plane of the nC12-THN-IBB ternary mixture. 98 Figure 5.8: Permeability vs. separation ratio of C1-nC4-nC12. Figure 5.9: Permeability vs. separation ratio of the nC12-THN-IBB ternary mixture. 5.6.2 Permeability effect on composition distribution 1 1.05 1.1 1.15 1.2 1.25 1.3 0.001 0.01 0.1 1 10 100 1000 10000 100000 1000000 Permeability [mD] Separation Ratio [-] Methane n-butane I II III 1 1.1 1.2 1.3 1.4 1.5 0.1 1 10 100 1000 10000 100000 1000000 10000000 Permeability [mD] Separation Ratio [-] n-dodecane THN I II III 1 1.05 1.1 1.15 1.2 1.25 1.3 0.001 0.01 0.1 1 10 100 1000 10000 100000 1000000 Permeability [mD] Separation Ratio [-] Methane n-butane I II III Figure 5.8: Permeability vs. separation ratio of C1-nC4-nC12. Figure 5.8: Permeability vs. separation ratio of C1-nC4-nC12. Figure 5.9: Permeability vs. separation ratio of the nC12-THN-IBB ternary mixture. 1 1.1 1.2 1.3 1.4 1.5 0.1 1 10 100 1000 10000 100000 1000000 10000000 Permeability [mD] Separation Ratio [-] n-dodecane THN I II III Figure 5.9: Permeability vs. separation ratio of the nC12-THN-IBB ternary mixture. 99 5.6.3 Viscosity effect on diffusion coefficients All the calculations in previous sections assume that the viscosities of the mixture components are constants and are evaluated at the average temperature (Tav=303 K). In this section the viscosity variation due to the temperature will be considered for each component in order to investigate the viscosity effect on , T D D and the compositional calculation. Figure 5.10 shows the variation of the n-dodecane and n-butane viscosity as a function of the temperature. It can be seen that the viscosity of n-dodecane (nC12, carrier fluid) changes significantly with the temperature. When the temperature varies between Tc=283 K and Th=323 K, the viscosity of n-dodecane varies between 1.85x10-3 Pa.s and 9.45x10-4 Pa.s, while for n-butane no major variation is observed. As for the methane, it was found that the viscosity does not vary with temperature, which is similar to the n- butane behaviour. The viscosity of each component at three different temperatures, which are the cold temperature cT , the hot temperature hT and the average temperature av T , was estimated. For each temperature condition the calculation was repeated taking into consideration the new estimated viscosity. The variation of molecular diffusion and thermodiffusion coefficients versus the horizontal direction are shown in Figures 5.11 and 5.12, when the permeability is set equal to 102 mD. It is found that the thermodiffusion is affected by the change in viscosity. The deviation of the methane thermodiffusion coefficient is about 10% (see Figure5.11a), while the deviation of the n-butane thermodiffusion coefficient is about 4.5%, (see Figure 5.11b). Figure 5.12 illustrates the variation of the molecular diffusion coefficient along the horizontal centre line direction for methane when the permeability is set equal to 102 mD. Results reveal that again the molecular diffusion coefficient based on the viscosity at Tc and Th deviates about 9% from the one at Tav. A Similar variation is observed for the n- butane component. 100 Figure 5.10: Viscosity variation with temperature of n-dodecane and n-butane. 0.00E+00 4.00E-04 8.00E-04 1.20E-03 1.60E-03 2.00E-03 283.15 288.15 293.15 298.15 303.15 308.15 313.15 318.15 323.15 Temperature [K] Viscosity [kg/m/s] nC12 nC4 0.00E+00 4.00E-04 8.00E-04 1.20E-03 1.60E-03 2.00E-03 283.15 288.15 293.15 298.15 303.15 308.15 313.15 318.15 323.15 Temperature [K] Viscosity [kg/m/s] nC12 nC4 Viscosity [kg/m/s] Temperature [K] Figure 5.10: Viscosity variation with temperature of n-dodecane and n-butane. 101 Figure 5.11: Thermodiffusion coefficient distributions for methane and n-butane along the horizontal direction (κ =102 mD). 5.6.3 Viscosity effect on diffusion coefficients 5.00E-13 6.00E-13 7.00E-13 8.00E-13 9.00E-13 1.00E-12 1.10E-12 1.20E-12 1.30E-12 0.000 0.004 0.008 0.012 0.016 0.020 0.024 0.028 0.032 Length L [m] Thermal diffusion coef. DT2 [m2/s/K] η at Tc η at Tav η at Th -8.50E-12 -8.00E-12 -7.50E-12 -7.00E-12 -6.50E-12 -6.00E-12 -5.50E-12 -5.00E-12 0.000 0.004 0.008 0.012 0.016 0.020 0.024 0.028 0.032 Length L [m] Thermal diffusion coef. DT1 [m2/s/K] η at Tc η at Tav η at Th (a) Methane (b) n-Butane -8.50E-12 -8.00E-12 -7.50E-12 -7.00E-12 -6.50E-12 -6.00E-12 -5.50E-12 -5.00E-12 0.000 0.004 0.008 0.012 0.016 0.020 0.024 0.028 0.032 Length L [m] Thermal diffusion coef. DT1 [m2/s/K] η at Tc η at Tav η at Th (a) Methane Figure 5.11: Thermodiffusion coefficient distributions for methane and n-butane along the horizontal direction (κ =102 mD). 5.00E-13 6.00E-13 7.00E-13 8.00E-13 9.00E-13 1.00E-12 1.10E-12 1.20E-12 1.30E-12 0.000 0.004 0.008 0.012 0.016 0.020 0.024 0.028 0.032 Length L [m] Thermal diffusion coef. DT2 [m2/s/K] η at Tc η at Tav η at Th (b) n-Butane Figure 5.11: Thermodiffusion coefficient distributions for methane and n-butane along the horizontal direction (κ =102 mD). 102 Figure 5.12: Variation of the molecular diffusion coefficient of methane along the horizontal cavity (κ =102 mD). 3.80E-09 4.00E-09 4.20E-09 4.40E-09 4.60E-09 4.80E-09 5.00E-09 5.20E-09 5.40E-09 0 0.004 0.008 0.012 0.016 0.02 0.024 0.028 0.032 Length L [m] Molecular diffusion coef. D11 [m2/s] η at Tc η at Tav η at Th Figure 5.12: Variation of the molecular diffusion coefficient of methane along the horizontal cavity (κ =102 mD). 5.8 Summary The thermosolutal convections of two ternary mixtures, C1-nC4-nC12 and nC12-THN- IBB in a porous cavity subjected to, respectively, 35.0 MPa and 1 atm pressure, and a horizontal temperature gradient are investigated over a wide range of permeability. The model was based on the non-equilibrium thermodynamics theory, and diffusion coefficients were calculated with time and space dependent fluid properties and compositions. The component concentration distribution and the separation ratio were used to examine the behaviour of the thermodiffusion and buoyancy-driven convection. The impact of permeability variation on the component separation results at steady state is numerically investigated. It was found that for permeability below 300 mD the thermodiffusion (or Soret coefficient) for both mixtures is dominant; and above this level, 103 buoyancy convection became the dominant mechanism. The viscosity is found to influence the evaluation of the molecular and thermodiffusion coefficients. Furthermore, the results showed that with variable viscosity the flow due to thermodiffusion is affected. The comparison between numerical result and the experimental data for a ternary mixture of THN-IBB-nC12 is carried out. It is noticed that by using vt-PR-EoS for evaluating the thermodynamic properties, which are used in the Firoozabadi model, one obtains better agreement with the experimental data. It is found that the percentage errors for 1 2 3 , and T T T D D D , when the vt-PR-EoS is used, are 12.63%, 23.11% and 9.65%, respectively. Furthermore, the mixture component’s behaviours also are identical, for both experimental data and numerical values have the same sign for thermodiffusion coefficients. The two components (nC12 and THN) migrate, one to the hot side and the other to the cold side, while the IBB was distributed randomly within the, as its thermodiffusion coefficient value is small, and this confirmed that its separation remains small. Finally, the density of the nC12-THN-IBB mixture at different temperatures was compared with the numerical results in order to validate the equation of state (vt-PR-Eos). The results showed an excellent agreement between numerical model and the experimental data, and the percentage error was less than 3.%. 104 Chapter 6 Three-Dimensional Study of Permeability Effect on Convection in Heterogeneous Porous Medium Filled with a Ternary Hydrocarbon Mixture In this chapter, the effect of permeability in the heterogeneous porous medium on fluid transport is studied with consideration of thermodiffusion (Soret effect) and molecular diffusion. A ternary mixture of nC12-THN-IBB in a porous medium subjected to lateral heating is numerically investigated at atmospheric pressure. Employing a single phase model and Darcy’s law, the continuity and the energy equations are solved numerically, using the finite volume method (a combination of a commercial Computational Fluid Dynamics software, Fluent, and our FORTRAN Code). Various permeability ratios ( / 10, 100, 1000 f s  κ κ and 10000) are examined in this study, and they cover a wide range of oil reservoirs. The temperature, fluid flow and solute fields are discussed in detail in order to show the sub-domain’s effect on fluid transport, especially when thermodiffusion is taken into consideration. 6.1 Introduction This work focuses on fluid transport in a heterogeneous porous medium, where molecular diffusion and thermodiffusion or Soret effect have been taken into consideration. This work is considered an extension to our previous work (Chapter 5), in which molecular and thermodiffusion in a homogeneous porous medium are examined. It was shown that the permeability has a significant effect on the separation of the mixture components. At low permeability the thermodiffusion effect was significant; whereas at high permeability convection becomes dominant and the concentration of mixture components becomes uniform. 105 There are many applications related to fluid flow through a heterogeneous or fractured porous medium, such as in the areas of geosciences, oil reservoirs, ground-water hydrology and nuclear waste storage, (Tsang and Birkholzer et al. 1999, Jianchun et al. 2003, Wu et al. 2006). The research on fluid flow in fractured porous reservoirs dates back to the 1960’s. Barenblatt et al. (1960) and Warren and Root (1963) presented the concept of dual-porosity within petroleum reservoirs to investigate the fractures of the porous media. They assumed interconnected set fractures which are supplied with fluid from numerous small matrix blocks between them, and they called this system an idealized model. Kazemi et al. (1976) were the first to incorporate the dual-porosity concept into a numerical model, with application to fluid flow on a large scale. One porosity is associated with the porous matrix and the other associated with the fracture. They also extended the dual-porosity model to a dual-porosity/dual-permeability model, where the simulation of fractured reservoirs involves discretization of the solution domain into two continua, one the domain representing the primary matrix, and a secondary domain representing fractured formulations. Saghir and Islam (1999) numerically investigated the effect of convection in a dual- porosity/dual-permeability porous medium by using salty and fresh water. They applied the Brinkman momentum balance equation and simultaneously solved the mass and energy equations using a two-dimensional model. Ghorayeb and Firoozabadi (2000) studied the effect of the fracture parameters on the fluid compositional variation, including the fracture aperture (or fracture permeability), fracture intensity, and fracture connectivity. Numerical results revealed that for a high fracture aperture, a pronounced convective motion within the fracture takes place, whereas the composition is only affected beyond a certain fracture aperture. They also examined the effect of connected and discrete fractures on compositional variation: connected fractures influence the compositional variation much more than discrete fractures. 6.1 Introduction Their results also indicated that the main effects on the compositional variation are due to the surrounding fractures. A model for flow interaction between a fracture and the rest of the porous medium has been presented by Alboin et al. (2002). In this model the fracture is an interface dividing 106 the domain of calculation into sub-domains. Fractures in a porous medium were considered individually with higher permeability than the surrounding rock. The phenomenon of three-dimensional variable-density flow in fractured porous media was studied by Graf and Therrien (2005). Two cases were tested: in the first case, the fracture was discretized by inclined two-dimensional elements, while in the second case, the fracture consisted of horizontal and vertical elements. They found that the variable- density flow in a porous matrix with a 45o inclined fracture shows two different convention cells that form at different times. Both cells grew with time and migrated downwards into the aquifer. They also found that the high permeability fractures appear as a barrier to convection. Jiang et al. (2006) carried out a mathematical simulation to illustrate the thermodiffusion phenomena of binary mixtures in the two-dimensional heterogeneous porous medium, which was laterally heated while filled with a binary mixture of methane and n-butane. The thermodiffusion process, the concentration distribution, and the separation ratio due to the temperature gradient and natural convection flow were investigated. The numerical model was based on a vertical porous medium, where the permeability in the lower region of the porous medium was kept constant, while varying the permeability in the upper region of the porous medium. They found that as the permeability increases in the upper domain, the convection flow dominates in the upper region, and the opposite results were obtained when the permeability was lower. Thus, they observed that the heterogeneity of the porous medium has a strong effect on the convective flow pattern. In addition, they argued that the Soret effect varied drastically in the upper region of the model in accordance with the variation of the permeability. In this present work, an attempt is made to simulate the thermosolutal convection of a ternary mixture of nC12-THN-IBB in the presence of thermodiffusion in a non- homogeneous porous medium; also, in the present study, the geometry, which was used in the previous chapter, is scaled up by a factor of 1000. 107 6.2 Model Description A rectangular prism of heterogeneous porous medium, with a horizontal length L of 32 m, thickness W of 10 m and height H of 10 m is used (see Figure 6.1). The permeability of the matrix (main domain) is lower than the permeability of the sub-domains, and these sub-domains are assumed to be in equilibrium with the rest of the porous medium (matrix). In order to examine the effect of the sub-domains on the fluid flow and mixture composition, two different configurations of a saturated heterogeneous porous medium are examined here, (see Figure 6.2). The width of the sub-domain is assumed to be 2 m. Physical properties of the ternary mixture and its components are given in Tables 6.1 and 6.2, respectively. The walls are assumed to be non-reacting, solid, impermeable, with lateral heating in the x direction, where the left wall of the heterogeneous porous medium is kept at a temperature o 10 C cT  and the right wall at o 50 C hT  ; and the remaining walls are assumed to be adiabatic; the pressure in the whole rectangular prism is maintained at 1 atm. The fluid is assumed to be compressible, with no chemical reaction, no interactive forces between the porous medium particles and the fluid mixture. Figure 6.1: Schematic diagram of the rectangular porous medium and boundary conditions. , 0, =0 c i T T J u   x y z 0, 0 iJ w   0, 0 iJ w   0 0 , , h i T T J u    W H L g 0, 0 iJ v   Figure 6.1: Schematic diagram of the rectangular porous medium and boundary conditions. g g g p y 108 Figure 6.2: Heterogeneous porous medium configurations, x-z plane. b) Configuration 2 a) Configuration 1 κ f f  κ f f  κ f f  κ , s s  κ , s s  κ , s s  κ , s s  κ , f f  κ , s s  κ , s s  κ , s s  κ , s s  a) Configuration 1 κ , f f  κ , s s  κ , s s  κ , s s  κ , s s  Figure 6.2: Heterogeneous porous medium configurations, x-z plane. 6.2 Model Description b) Configuration 2 a) Configuration 1 κ f f  κ f f  κ f f  κ , s s  κ , s s  κ , s s  κ , s s  κ , s s  κ , s s  a) Configuration 1 a) Configuration 1 b) Configuration 2 κ f f  κ f f  κ f f  κ , s s  κ , s s  κ , s s  κ , s s  b) Configuration 2 Figure 6.2: Heterogeneous porous medium configurations, x-z plane. Figure 6.2: Heterogeneous porous medium configurations, x-z plane. 109 Table 6.1: Physical properties of the fluid mixture and porous material at T0=303.15 K and p0=1 atm. Name Value Unit porous medium density 3983.6 -3 kg.m porous medium heat capacity 786.2745 -1 -1 J.kg .K porous medium thermal conductivity 43 -1 -1 W.m .K nC12-THN-IBB mixture compositions 33.3-33.3-33.4 or 0.281-0.362-0.357 (% mass fr.) (% mole fr.) Heat capacity of mixture 1885.9 -1 -1 J.kg .K Molecular weight of nC12 170.33 -1 kg.kmol Molecular weight of THN 132.20 -1 kg.kmol Molecular weight of IBB 134.22 -1 kg.kmol Thermal conductivity of mixture 0.123 -1 -1 W.m .K 11 12 21 22 D D D D       -10 -10 -10 -09 7.52x10 9.06x10 3.00x10 1.11x10         2 -1 m .s * * ,1 ,2 T T D D     -7 -7 - 4.41x10 2.84x10     -1 -1 kg.s .m Table 6.2: Physical properties of the mixture components at T0=303.15 K. Component  K cT   Pa cP   -3 kg.m    -1 -1 J.kg .K p C   -1 1 W.m .K k  nC12H26 658.20 1.82x106 741.86 2220.97 0.1368 THN 720.15 3.62x106 963.48 1662.63 0.1293 IBB 650.15 3.04x106 844.95 1820.52 0.1205 Table 6.2: Physical properties of the mixture components at T0=303.15 K. 110 6.3 Governing Equations The following are the governing equations, which were introduced in Chapter 2. 6.3.1 Conservation of mass The molecular diffusion coefficients can be defined as follows, (Taylor and Krishna 1993): 111  1 * ij D D A B     1 * ij D D A B    (6.4) where 1 n j i ii j in n ij i j i x M x M A D M D M                1 1 ij i ij j in n M M A x D M D M             1 ii i i n i M M B x x M M          ij i j n M M B x M M           where (6.5) (6.6) (6.7) ij i j n M M B x M M           (6.8) (6.8) Where ij D is the Maxwell-Stefan coefficient, M is the molecular weight of the mixture n Where ij D is the Maxwell-Stefan coefficient, M is the molecular weight of the mixture ( 1 n i i i M M x   ), and i M is the molecular weight of the component i. 6.3.1 Conservation of mass The differential mass continuity equation for the entire mixture is given as follows       0 u v w t x y z                 (6.1) (6.1) where  is the density of the mixture fluid, u ,v , and w are the velocity components in x, y and z directions, respectively. The mass continuity equation for species i is: where  is the density of the mixture fluid, u ,v , and w are the velocity components in x, y and z directions, respectively. The mass continuity equation for species i is:   1 ( 1,2,....... 1, 0 ) n i i i i ij i n t j            V    (6.2) (6.2) where ij is the diffusive mass flux of species i, i is the mass fraction of the component i. The diffusive mass flux can be expressed as follows, (Taylor and Krishna 1993): where ij is the diffusive mass flux of species i, i is the mass fraction of the component i. The diffusive mass flux can be expressed as follows, (Taylor and Krishna 1993): 1 * * , 1 n i ij j T i j T j D D T           (6.3) (6.3) where T is temperature, * ij D is the binary molecular diffusion coefficient of the fluid mixture in the porous medium, * T D is the thermodiffusion coefficient in the porous medium of component i. In this study, the thermodiffusion coefficients are assumed constant, and have been evaluated with time and space dependent fluid properties based on the non-equilibrium thermodynamics theory (as explained in the previous chapter). The molecular diffusion coefficients can be defined as follows, (Taylor and Krishna 1993): where T is temperature, * ij D is the binary molecular diffusion coefficient of the fluid mixture in the porous medium, * T D is the thermodiffusion coefficient in the porous medium of component i. In this study, the thermodiffusion coefficients are assumed constant, and have been evaluated with time and space dependent fluid properties based on the non-equilibrium thermodynamics theory (as explained in the previous chapter). 6.3.2 Conservation of momentum 6.1, the pressure differential equation becomes the following: 0 x y z P P P g g g t x x y y z z                                                  κ κ κ (6.13) (6.13) onservation of energy 6.3.2 Conservation of momentum In porous media, Darcy’s equation can be used and is valid, when v is sufficiently small (i.e. the Reynolds number of the flow, based on a typical pore diameter, is of the order of unity or smaller), as explained by Nield and Bejan (2006). Darcy’s equation can be expressed as follows: ( ) P g      κ V   (6.9) (  112 The Boussinesq approximation is assumed to be valid for the range of temperature and composition in this study, so that the density of fluid the mixture is expressed as follows:     1 0 0 0 1 1 n T i i i i T T                      (6.10) (6.10) where 0 1 T T              0 1 i i T               0 1 T T              (6.11) 0 1 i i T               (6.12) 0 1 T T              0 1 i i T               (6.11) (6.12) T is the thermal expansion coefficient due to temperature change, and i   is the compositional expansion coefficient of component i due to the concentration gradient. The volume-translated Peng-Robinson Equation of State (vt-PR-EoS), (Peneloux et al., 1982 and Jhaverl and Youngren, 1988), is used to calculate the thermal expansion coefficient and the volumetric concentration expansion coefficient. By substituting the Darcy relation (6.9) into the mass conservation Eq. 6.3.3 Conservation of energy Since the velocity of the matrix is zero, and there is no heat generation in either porous medium or the fluid mixture, and since in addition, thermal equilibrium was assumed between the solid and the liquid phases and occurs very quickly, the temperature of the 113 matrix is assumed to be equal to the temperature of the fluid mixture. The energy conservation equation can be written as follows: matrix is assumed to be equal to the temperature of the fluid mixture. The energy conservation equation can be written as follows:     2 ( ) p p e T C C T k T t          fl e V  (6.14) (6.14) where   p e C  is the effective volumetric heat capacity and ke is the effective thermal conductivity of the system. These effective physical parameters are related to the fluid properties and the solid matrix properties, as shown in Chapter 2. where   p e C  is the effective volumetric heat capacity and ke is the effective thermal conductivity of the system. These effective physical parameters are related to the fluid properties and the solid matrix properties, as shown in Chapter 2. 6.4 Boundary Conditions The boundary conditions used in the model, also shown in Figure 6.1, are as follows: 1. The lateral walls have constant temperatures, and other walls are assumed to be adiabatic 1. The lateral walls have constant temperatures, and other walls are assumed to be adiabatic 0, x L  0 , c h x x L T T T T     (6.15) 0, x L  0 , c h x x L T T T T     (6.15) 0, W y  0 T y    (6.16) 0, H z  0 T z    (6.17) (6.15) 0, W y  0 T y    (6.16) 0, H z  0 T z    (6.17) 0, W y  0 T y    0 T y    (6.16) (6.16) 0, H z  0 T z    (6.17) (6.17) 2. There is neither fluid flow nor diffusion flux through the boundaries 2. There is neither fluid flow nor diffusion flux through the boundaries 0, x L  V. 0, 0 x N J    0, x L  V. 0, 0 x N J    (6.18) 0, x L  V. 0, 0 x N J    (6.18) 0, W y  V. 0, 0 y N J    0, W y  V. 0, 0 y N J    (6.19) 0, W y  V. 0, 0 y N J    (6.19) 0, W y  V. 0, 0 y N J    (6.19) (6.19) 0, H z  V. 0, 0 z N J    (6.20) 0, H z  V. 0, 0 z N J    (6.20) 0, H z  V. 0, 0 z N J    0, H z  V. 0, 0 z N J    V. 0, 0 z N J    (6.20) (6.20) 114 where N is the unit normal vector. The initial conditions are 0 1 atm, p  and 0T 303.15 K. The conditions at the matrix and sub-domain interface are assumed to be continuous for the normal component of the diffusion flux, pressure, and mole fraction. 6.4 Boundary Conditions The Darcy’s law is used to calculate the velocity (V  ). 6.5 Numerical Solution Procedure Equations 6.1, 6.2, 6.13 and 6.14 are solved numerically by using the control volume technique (Fluent User’s Guide 2006), subject to the boundary conditions as depicted in Figure 6.1. The SIMPLE algorithm is implemented for solving the governing equations. It uses a relationship between velocity and pressure correction to obtain the pressure field. The second-order upwind scheme is used in the discretization of the governing equations. The resulting linear system of algebraic equations is solved at each time step using a bi- conjugate gradient iteration method with a given convergence criterion, which has been confirmed over many tests for the required accuracy. The numerical procedure derived from the SIMPLE algorithm is summarized by Anderson (1995) as: Equations 6.1, 6.2, 6.13 and 6.14 are solved numerically by using the control volume technique (Fluent User’s Guide 2006), subject to the boundary conditions as depicted in Figure 6.1. The SIMPLE algorithm is implemented for solving the governing equations. It uses a relationship between velocity and pressure correction to obtain the pressure field. 1. Guess the pressure field * P , and start the iterative solution with the guessed pressure. 2. Solve the momentum equations for the three velocity components (u, v, and w) using the * P values. The velocities obtained may be symbolized * * * , u v and w due to . P 3. Solve the pressure-correction equation; because the values of * * , u v and w were obtained from the guessed values, therefore, an equation for pressure correction is needed using the continuity equation, so that the corrected pressure is given by * P P P   (6.21) * P P P   (6.21) (6.21) The same method can be applied to obtain the corrected velocity components The same method can be applied to obtain the corrected velocity components The same method can be applied to obtain the corrected velocity components 115 * * * u u u v v v w w w          (6.22) (6.22) where the velocity correction , and u v w   may be obtained from P. 4. Solve the other conservation equation using the corrected values of , and u v w and P . 4. Solve the other conservation equation using the corrected values of , and u v w and P . 5. 6.5 Numerical Solution Procedure Set , and u v w and P obtained from step 2 and step 3 as new values for * * * * , , u v w and P and return to step 2. Repeat this process until a convergence criterion is reached. At the initial time step, the velocities were set to zero in the computational domain, where initial pressure, temperature, and concentration are specified. The criterion of convergence is set for the unknown parameters including pressure, temperature, velocities, and concentration. Convergence is reached when the iterations of the system continue until an error value of 10-6 is found between two successive iterations. More details of the numerical procedure may be found in Patankar (1980), and Versteeg and Malalasekera (2007). After discretization, the conservation equation for a general variable (), at any control volume or cell (and a general nodal point is identified by P) can be written as P P nb nb nb a a b      (6.23) P P nb nb nb a a b      (6.23) where Pa is the centre coefficient, nb a represents the influence coefficients for neighboring cells, and b is the contribution of the constant part of the source term and of the boundary conditions. The residual, R  , or the error between iterations is defined as 116 P P nb nb cells P nb P P cells P a b a R a        (6.24) P P nb nb cells P nb P P cells P a b a R a        (6.24) The values of pressure, temperature and composition are defined in the centre, whereas the velocities are defined on the surface of each control volume, or grid cell. Different mesh sizes are tested in order to adopt the proper mesh size in a three-dimensional porous medium. A mesh size of 80 × 40 × 40 control volume has been adopted. 6.6 Results and Discussion In this work, lateral heating in a heterogeneous porous medium filled with a hydrocarbon ternary mixture has been simulated. Porosity is an important parameter when simulating the transport phenomena in porous media. Bulk volume and porosity are typically larger in the matrix than in the sub-domains, while permeability of sub-domains is typically much larger than that of the matrix. The permeability ( s κ ) and porosity ( s ) in the primary domain are assumed 10 mD, and 0.4, respectively, whereas the permeability of sub-domains ( f κ ) ranges from 10 to 10000 mD with a porosity ( f) of 0.2. Different configurations were adopted, but for simplicity, the results of only two configurations are presented in this chapter, (see Figure 6.2). The width of the sub-domain in all the configurations is assumed to be 2 m. 6.6.1 Variation of concentration with permeability In this section, the composition of a hydrocarbon ternary mixture of nC12-THN-IBB is investigated for different ranges of / f s κ κ and for two configurations, which are shown in Figure 6.2. Sub-domains have been created by putting a layer in the porous medium with a different porosity and corresponding permeability to mimic the heterogeneity. The concentration of the first component (nC12) and second component (THN) against the 117 permeabilities ratio are examined. Figures 6.3 to 6.10 illustrate the mole fraction at the centre of the geometry (x-z plane). Figure 6.3 shows the mole fraction of nC12 along the horizontal direction (x-direction) for configuration1 (Conf.1) at three different locations along the vertical direction (z = 2.5 m, z = 5 m, and z = 7.5 m). There is no noticeable effect from the sub-domains on the concentration, because the permeability of the sub-domains is equal to that of the matrix ( f s   κ κ 10 mD). It is observed that the lighter component (nC12) concentration migrates to the hot side (has a negative thermodiffusion coefficient); this is consistent with our previous work in a homogeneous porous medium (Jaber et al. 2008). A higher concentration of nC12 is found near the bottom edge of the porous medium. Furthermore, it is observed that the nC12 concentration is decreased at the top edge because of the role of thermodiffusion. The nC12 concentration is repeated in Figure 6.4 for configuration2 (Conf.2). The same findings as above have been noticed, but the minimum and maximum values of the concentration are not the same, due to orientation of the sub-domains. For instance, at location z = 7.5 m, the values of the nC12 (Conf.1) are between 0.25 and 0.294, (see Figure 6.3), while for Conf.2, the values are between 0.245 and 0.293, (see Figure 6.4). Figures 6.5 and 6.6 show the THN concentration at three different elevations (z = 2.5 m, z = 5 m, and z = 7.5 m) along the horizontal direction for Conf.1 and 2, respectively. The results shown in these Figures are for f s   κ κ 10 mD. It is noticeable that THN (the heavier component) migrates to the cold side (has a positive thermodiffusion coefficient); also, it is observed that the THN accumulates near the top edge of the porous medium. 6.6.1 Variation of concentration with permeability For instance, the THN concentration at z = 7.5 m (near the top edge) varies from 0.342 at the hot side to 0.386 at the cold side, while for z = 2.5 m (near the bottom edge) it varies from 0.33 to 0.375 (Figure 6.5). It can be concluded from Figures 6.3 to 6.6 that the nC12 and THN concentrations vary nearly linearly along horizontal direction, when f s   κ κ 10 mD. 118 Figure 6.3: n-dodecane (nC12) mole fraction along the horizontal direction ( / 10 /10 f s  κ κ ), Conf.1. 0.24 0.25 0.26 0.27 0.28 0.29 0.3 0.31 0.32 0 4 8 12 16 20 24 28 32 X [m] Dodecane mole Fraction [-] z=2.5 m z=5 m z=7.5 m 0.24 0.25 0.26 0.27 0.28 0.29 0.3 0.31 0.32 0 4 8 12 16 20 24 28 32 X [m] Dodecane mole Fraction [-] z=2.5 m z=5 m z=7.5 m Figure 6.3: n-dodecane (nC12) mole fraction along the horizontal direction ( / 10 /10 f s  κ κ ), Conf.1. Figure 6.4: n-dodecane (nC12) mole fraction along the horizontal direction ( / 10 /10 f s  κ κ ), Conf.2. 0.24 0.25 0.26 0.27 0.28 0.29 0.3 0.31 0.32 0 4 8 12 16 20 24 28 32 X [m] Dodecane mole fraction [-] z=2.5 m z=5 m z=7.5 m 0.24 0.25 0.26 0.27 0.28 0.29 0.3 0.31 0.32 0 4 8 12 16 20 24 28 32 X [m] Dodecane mole fraction [-] z=2.5 m z=5 m z=7.5 m Figure 6.4: n-dodecane (nC12) mole fraction along the horizontal direction ( / 10 /10 f s  κ κ ), Conf.2. 119 Figure 6.5: THN mole fraction along the horizontal direction ( / 10 /10 f s  κ κ ), Conf.1. 0.33 0.34 0.35 0.36 0.37 0.38 0.39 0 4 8 12 16 20 24 28 32 X [m] THN mole fraction [-] z=2.5 m z=5 m z=7.5 m Figure 6.5: THN mole fraction along the horizontal direction ( / 10 /10 f s  κ κ ), Conf.1. 6.6.1 Variation of concentration with permeability 0.33 0.34 0.35 0.36 0.37 0.38 0.39 0 4 8 12 16 20 24 28 32 X [m] THN mole fraction [-] z=2.5 m z=5 m z=7.5 m THN mole fraction [-] Figure 6.5: THN mole fraction along the horizontal direction ( / 10 /10 f s  κ κ ), Conf.1 Figure 6.6: THN mole fraction along the horizontal direction ( / 10 /10 f s  κ κ ), Conf.2. 0.33 0.34 0.35 0.36 0.37 0.38 0.39 0 4 8 12 16 20 24 28 32 X [m] THN mole fraction [-] z=2.5 m z=5 m z=7.5 m 0.33 0.34 0.35 0.36 0.37 0.38 0.39 0 4 8 12 16 20 24 28 32 X [m] THN mole fraction [-] z=2.5 m z=5 m z=7.5 m Figure 6.6: THN mole fraction along the horizontal direction ( / 10 /10 f s  κ κ ), Conf.2. 120 Figures 6.7 and 6.8 show the concentration distribution of nC12 for / 100 f s  κ κ Figures 6.7 and 6.8 show the concentration distribution of nC12 for / 10000 /10 f s  κ κ . It can be seen that the sub-domains have a significant influence on the compositional variation of the mixture components. By comparing Figure 6.3 with Figure 6.7 (both Figures for Conf.1), the concentration at z = 5 m becomes larger near the cold side than at other elevations (z = 2.5 m and 7.5 m), which is due to the convection inside the sub- domain. In addition, in the sub-domain zones, the mixing is enhanced, because of the enhanced flow rate, (Figure 6.7). For Conf.2, in Figure 6.8, the concentration in the sub- domain regions (three vertical sub-domains) is not linear when compared to the concentration in Figure 6.4. The same scenario has been noticed for the second component (THN), as shown in Figures 6.9 and 6.10. Figures 6.7 and 6.8 show the concentration distribution of nC12 for / 10000 /10 f s  κ κ . It can be seen that the sub-domains have a significant influence on the compositional variation of the mixture components. By comparing Figure 6.3 with Figure 6.7 (both Figures for Conf.1), the concentration at z = 5 m becomes larger near the cold side than at other elevations (z = 2.5 m and 7.5 m), which is due to the convection inside the sub- domain. 6.6.1 Variation of concentration with permeability In addition, in the sub-domain zones, the mixing is enhanced, because of the enhanced flow rate, (Figure 6.7). For Conf.2, in Figure 6.8, the concentration in the sub- domain regions (three vertical sub-domains) is not linear when compared to the concentration in Figure 6.4. The same scenario has been noticed for the second component (THN), as shown in Figures 6.9 and 6.10. In general, the lighter and the heavier components’ behaviour within the ternary mixture is similar to the one in a binary mixture (Ghorayeb et al., 2003), where the lighter component migrates to hot side and increase with depth. 121 Figure 6.7: n-dodecane (nC12) mole fraction along the horizontal direction ( / 10000 /10 f s  κ κ ), Conf.1. 0.24 0.25 0.26 0.27 0.28 0.29 0.3 0.31 0.32 0 4 8 12 16 20 24 28 32 X [m] Dodecane mole fraction [-] z=2.5 m z=5 m z=7.5 m 0.24 0.25 0.26 0.27 0.28 0.29 0.3 0.31 0.32 0 4 8 12 16 20 24 28 32 X [m] Dodecane mole fraction [-] z=2.5 m z=5 m z=7.5 m Figure 6.7: n-dodecane (nC12) mole fraction along the horizontal direction ( / 10000 /10 f s  κ κ ), Conf.1. Figure 6.8: n-dodecane (nC12) mole fraction along the horizontal direction ( / 10000 /10 f s  κ κ ), Conf.2. 0.24 0.25 0.26 0.27 0.28 0.29 0.3 0.31 0.32 0 4 8 12 16 20 24 28 32 X [m] Dodecane mole fraction [-] z=2.5 m z=5 m z=7.5 m Figure 6.8: n-dodecane (nC12) mole fraction along the horizontal direction ( / 10000 /10 f s  κ κ ), Conf.2. 0.24 0.25 0.26 0.27 0.28 0.29 0.3 0.31 0.32 0 4 8 12 16 20 24 28 32 X [m] Dodecane mole fraction [-] z=2.5 m z=5 m z=7.5 m Figure 6.8: n-dodecane (nC12) mole fraction along the horizontal direction ( / 10000 /10 f s  κ κ ), Conf.2. 122 Figure 6.9: THN mole fraction along the horizontal direction ( / 10000 /10 f s  κ κ ), Conf.1. 0.33 0.34 0.35 0.36 0.37 0.38 0.39 0 4 8 12 16 20 24 28 32 X [m] THN mole fraction [-] z=2.5 m z=5 m z=7.5 m Figure 6.9: THN mole fraction along the horizontal direction ( / 10000 /10 f s  κ κ ), Conf.1. 6.6.2 Comparison of flow patterns The flow patterns in the centre of the porous medium for the x-z plane at different elevations (z = 2.5 m, z = 5 m, and z = 7.5 m) are plotted in Figures 6.11-6.14. This may help to illustrate the sub-domains’ effect on the fluid behaviour inside the heterogeneous porous media. Figure 6.11 shows the magnitude of velocity for the 10 f s   κ κ mD case. It is found that the value of velocity is minimum at z = 5 m, while it is identical for z = 2.5 m and 7.5 m. This case is identical to the case without sub-domain because the The flow patterns in the centre of the porous medium for the x-z plane at different elevations (z = 2.5 m, z = 5 m, and z = 7.5 m) are plotted in Figures 6.11-6.14. This may help to illustrate the sub-domains’ effect on the fluid behaviour inside the heterogeneous porous media. Figure 6.11 shows the magnitude of velocity for the 10 f s   κ κ mD case. The flow patterns in the centre of the porous medium for the x-z plane at different elevations (z = 2.5 m, z = 5 m, and z = 7.5 m) are plotted in Figures 6.11-6.14. This may help to illustrate the sub-domains’ effect on the fluid behaviour inside the heterogeneous porous media. Figure 6.11 shows the magnitude of velocity for the 10 f s   κ κ mD case. It is found that the value of velocity is minimum at z = 5 m, while it is identical for z = 2.5 m and 7.5 m. This case is identical to the case without sub-domain because the permeability of the matrix and sub-domain are the same. As a result, the presence of the sub-domains cannot be seen to be influential in this Figure. The same behaviour has been observed for Conf.2, (see Figure 6.12). Figures 6.13 and 6.14 present the magnitude of velocity for / 10000 /10. f s  κ κ In Figure 6.13, the fluid velocity for z = 5 m (Conf.1), which is located at the centre of the sub- domain zone along the horizontal direction, is higher than that at z = 2.5 m and 7.5 m. The fluid velocity at z = 2.5 m and 7.5 m are almost similar. 6.6.1 Variation of concentration with permeability 0.33 0.34 0.35 0.36 0.37 0.38 0.39 0 4 8 12 16 20 24 28 32 X [m] THN mole fraction [-] z=2.5 m z=5 m z=7.5 m THN mole fraction [-] Figure 6.9: THN mole fraction along the horizontal direction ( / 10000 /10 f s  κ κ ), Conf.1. Figure 6.10: THN mole fraction along the horizontal direction ( / 10000 /10 f s  κ κ ), Conf.2. 0.33 0.34 0.35 0.36 0.37 0.38 0.39 0 4 8 12 16 20 24 28 32 X [m] THN mole fraction [-] z=2.5 m z=5 m z=7.5 m 0.33 0.34 0.35 0.36 0.37 0.38 0.39 0 4 8 12 16 20 24 28 32 X [m] THN mole fraction [-] z=2.5 m z=5 m z=7.5 m Figure 6.10: THN mole fraction along the horizontal direction ( / 10000 /10 f s  κ κ ), Conf.2. Figure 6.10: THN mole fraction along the horizontal direction ( / 10000 /10 f s  κ κ ), Conf.2. 123 123 6.6.2 Comparison of flow patterns Also it can be seen that there are two peaks in the middle, which are inside the vertical sub-domain. In the first one, the flow enters the sub-domain zone and in the second one, the flow leaves the sub-domain zone. Figure 6.14 shows the flow pattern for Conf.2. It is observed that in the sub-domain zones, the flow becomes stronger and much higher than the one in the matrix; this is attributed to the high permeability in the sub-domain zones. In summary, the buoyancy-driven convection is stronger in the sub-domains because the permeability is higher, and the fluid can move easily. It is evident that the acceleration of the fluid in the sub-domain zones is much higher. 124 Figure 6.11: The magnitude of velocity along the horizontal direction ( / 10 /10 f s  κ κ ), Conf.1. 0.0E+00 1.0E-10 2.0E-10 3.0E-10 4.0E-10 5.0E-10 6.0E-10 7.0E-10 0 4 8 12 16 20 24 28 32 X [m] V [m/s] z=2.5 m z=5 m z=7.5 m 0.0E+00 1.0E-10 2.0E-10 3.0E-10 4.0E-10 5.0E-10 6.0E-10 7.0E-10 0 4 8 12 16 20 24 28 32 X [m] V [m/s] z=2.5 m z=5 m z=7.5 m Figure 6.11: The magnitude of velocity along the horizontal direction ( / 10 /10 f s  κ κ ), Conf.1. Figure 6.12: The magnitude of velocity along the horizontal direction ( / 10 /10 f s  κ κ ), Conf.2. 0.0E+00 1.0E-10 2.0E-10 3.0E-10 4.0E-10 5.0E-10 6.0E-10 7.0E-10 8.0E-10 0 4 8 12 16 20 24 28 32 X [m] V [m/s] z=2.5 m z=5 m z=7.5 m 0.0E+00 1.0E-10 2.0E-10 3.0E-10 4.0E-10 5.0E-10 6.0E-10 7.0E-10 8.0E-10 0 4 8 12 16 20 24 28 32 X [m] V [m/s] z=2.5 m z=5 m z=7.5 m Figure 6.12: The magnitude of velocity along the horizontal direction ( / 10 /10 f s  κ κ ), Conf.2. 125 Figure 6.13: The magnitude of velocity along the horizontal direction ( / 10000 /10 f s  κ κ ), Conf.1. 2 This result has already been presented in Jaber et al. (2008a). 6.6.2 Comparison of flow patterns 0.0E+00 5.0E-10 1.0E-09 1.5E-09 2.0E-09 2.5E-09 3.0E-09 3.5E-09 4.0E-09 0 4 8 12 16 20 24 28 32 X [m] V [m/s] z=2.5 m z=5 m z=7.5 m 0.0E+00 5.0E-10 1.0E-09 1.5E-09 2.0E-09 2.5E-09 3.0E-09 3.5E-09 4.0E-09 0 4 8 12 16 20 24 28 32 X [m] V [m/s] z=2.5 m z=5 m z=7.5 m Figure 6.13: The magnitude of velocity along the horizontal direction ( / 10000 /10 f s  κ κ ), Conf.1. 0.0E+00 2.0E-09 4.0E-09 6.0E-09 8.0E-09 1.0E-08 1.2E-08 0 4 8 12 16 20 24 28 32 X [m] V [m/s] z=2.5 m z=5 m z=7.5 m Figure 6.14: The magnitude of velocity along the horizontal direction ( / 10000 /10 f s  κ κ ), Conf.2. 0.0E+00 2.0E-09 4.0E-09 6.0E-09 8.0E-09 1.0E-08 1.2E-08 0 4 8 12 16 20 24 28 32 X [m] V [m/s] z=2.5 m z=5 m z=7.5 m Figure 6.14: The magnitude of velocity along the horizontal direction ( / 10000 /10 f s  κ κ ), Conf.2. 126 6.6.3 Separation ratio The separation ratio parameter may play a role in understanding the mass transfer process, so that the convection effect on the thermodiffusion process is investigated for different permeability ratios in terms of the separation ratio. The permeability has a significant effect on the separation of mixture components. At low permeability the Soret effect is significant; while at high permeability convection becomes dominant. To further examine this phenomenon, and to understand how the thermodiffusion convection coupling works, a variable known as the separation ratio, q, is introduced. The separation ratio of each component can be defined as follows, as introduced in section 5.6.2: max max min min /(1 ) /(1 ) x x q x x    (6.25) max max min min /(1 ) /(1 ) x x q x x    (6.25) where max x and min x are the maximum and minimum mole fractions of component i. In this study, the trend of the separation ratio is identical to the one presented in Chapter 52. The separation ratio, q, can be plotted against the permeability ratio as shown in Figures 6.15 and 6.16 for Conf. 1 and 2, respectively. In each Figure, the separation ratio for component 1 (nC12) and component 2 (THN) is shown. In these Figures, the permeability values of the primary domain and sub-domains vary between 10 mD and 10000 mD, which represent the values in the oil reservoir rocks (Bear 1972). Three different regions can be identified in these two Figures. From Figure 6.15 for Conf.1, Region (I) is for the permeability ratio of 10 ( / 100 /10 κ κ f s  ). In this region, the separation ratio is almost constant; the separation ratio of nC12 (the lighter component) is 1.2 and higher than that of the THN (the heavier component), which is 1.055. In this region the Soret effect or thermodiffusion is dominant. Region (II) is for a permeability ratio in a range from 10 to 100. In this region both the Soret effect and convection play a role. In Region (III), the Soret effect gradually decreases, as the convection increases. 127 In general, it is found that the separation ratio value for Conf.2 is higher than that for Conf.1 due to the orientation of sub-domains. 6.6.3 Separation ratio For instance, the separation ratio of the lighter component (nC12) is between 1.48 and 1.43 for Conf.2, and is between 1.22 and 1.19 for Conf.1. Figure 6.15: Separation ratio as a function of the permeability ratio, Conf.1. 1 1.05 1.1 1.15 1.2 1.25 1.3 1 10 100 1000 Permeability Ratio [-] Separation Ratio [-] n-dodecane THN I II III II III Permeability Ratio [-] Figure 6.15: Separation ratio as a function of the permeability ratio, Conf.1. 128 Figure 6.16: Separation ratio as a function of the permeability ratio, Conf.2. 1.25 1.3 1.35 1.4 1.45 1.5 1 10 100 1000 Permeability Ratio [-] Separation Ratio [-] n-dodecane THN I II III Separation Ratio [-] II Permeability Ratio [-] Figure 6.16: Separation ratio as a function of the permeability ratio, Conf.2. 6.7 Summary In this work, a three-dimensional model has been numerically simulated to illustrate the interaction between thermodiffusion and buoyancy-driven convection in a single phase hydrocarbon ternary mixture (nC12-THN-IBB) inside a heterogeneous porous medium. A horizontal temperature gradient was applied in a porous medium under atmospheric pressure. In the analysis, the impact of the permeability ratio on the composition of the mixture components, velocity in the porous medium, and the separation ratio was investigated. It was found that the heterogeneous porous medium has a significant influence on the composition of the mixture components. It was also found that the sub- domains have a strong effect on the convective patterns, and the fluid flow in the sub- domain zones is much higher than that in the matrix domain. Furthermore, the separation ratio of Conf.2 is higher than that for Conf.1 due to the orientation of the sub-domains. 129 7.1 Conclusions The Thermodiffusion phenomenon (or Soret effect) is a coupling between a temperature gradient and its resulting mass flux in a multicomponent system. As an important non- equilibrium process, this phenomenon appears in a wide range of physical and chemical processes in nature, including studies of oil reservoir analysis, and fluid separation. In this work, an extensive numerical modelling and estimation of the diffusion coefficient of hydrocarbon mixtures was complemented with an experimental approach. Based on the theoretical and experimental work carried out, the following conclusions can be drawn: In this work, an extensive numerical modelling and estimation of the diffusion coefficient of hydrocarbon mixtures was complemented with an experimental approach. Based on the theoretical and experimental work carried out, the following conclusions can be drawn: 1. In thermodiffusion models, equilibrium thermodynamic quantities are widely used. Therefore, it is a prerequisite to know reliable thermodynamic properties for the theoretical research on thermodiffusion. An irreversible thermodynamics model, which combines Onsager’s theory with the theory of rate and equation of state, yielding the Firoozabadi model, appear to be appropriate for thermodiffusion estimation for multicomponent hydrocarbon mixtures, and it is derived based on the four postulates in non-equilibrium thermodynamics. Two equations of state, the Peng-Robinson Equation of State (PR-EoS) and the volume translated Peng Robinson Equation of State (vt-PR-EoS), were adopted to estimate the thermodynamic properties of mixtures. 1. In thermodiffusion models, equilibrium thermodynamic quantities are widely used. Therefore, it is a prerequisite to know reliable thermodynamic properties for the theoretical research on thermodiffusion. An irreversible thermodynamics model, which combines Onsager’s theory with the theory of rate and equation of state, yielding the Firoozabadi model, appear to be appropriate for thermodiffusion estimation for multicomponent hydrocarbon mixtures, and it is derived based on the four postulates in non-equilibrium thermodynamics. Two equations of state, the Peng-Robinson Equation of State (PR-EoS) and the volume translated Peng Robinson Equation of State (vt-PR-EoS), were adopted to estimate the thermodynamic properties of mixtures. 2. A new thermodiffusion cell designed for high pressure was validated at atmospheric pressure. The cell was installed in a Mach-Zehnder interferometer. Two mixtures of the Benchmark of Fontainebleau were tested in the vertical position (nC12-IBB and nC12-THN at 50% mass fraction and Tmean = 25 °C). 7.1 Conclusions From the analysis of the 130 second kinetic of the evolution of the phase difference between the laser beams that passed through the hot and the cold dead volumes, the diffusion coefficient in free liquid, D, and Soret coefficient, T S , of the two mixtures and the tortuosity  for the porous medium were determined. For both mixtures, an excellent agreement was found between the molecular diffusion coefficient of this work and the benchmark values, and the percentage error was less than 1%. Also, it was found that the difference between the Soret coefficients for this work and the benchmark data was acceptable for the experiments of duration up to seven days. To improve the determination of T S with the cell in a vertical position, increasing the value of the imposed temperature gradient and increasing the time of the experiment (about two weeks in order to reach the steady state condition) were suggested. Furthermore, it was concluded that, when the cell was set in the horizontal position, convection in dead-volumes became possible. For a difference of the temperature applied between thermoregulator baths ( o 5 C T   ), a diffraction pattern came up at the beginning of the experiment, and the procedure of analysis of interferograms became difficult. 3. The measurements for the thermal expansion and concentration expansion coefficients and for the viscosity of the binaries of nC12, IBB and THN for pressures varying from 0.1 to 20 MPa at 25 °C and concentrations centred on 50% were measured. The densities of binaries under the same conditions were measured as well. In addition, viscosities were directly measured using a high pressure high temperature viscometer (HPHTV-100). The comparisons with measured densities show that vt-PR-EoS has a better agreement with experiments than the PR-EoS. The combination of the model of Lohrenz-Bray-Clark for the viscosity of liquid mixtures, and the densities calculated with the two equations of state, proved to be inefficient for the prediction of the viscosities of the binaries. 4. Thermodiffusion plays a significant role in the variation of the composition mixture components in a porous medium reservoir, such as in oil reservoirs. The interaction between the thermodiffusion phenomenon and buoyancy driven convection in a porous medium reservoir filled with a ternary hydrocarbon mixture was numerically 131 investigated. 7.1 Conclusions Two different ternary mixtures, a C1-nC4-nC12 at 35 MPa and nC12- THN-IBB at 1 atm pressure, were investigated for different permeabilites ranging from 10 to 107 mD. The model was based on the non-equilibrium thermodynamics theory, and diffusion coefficients were calculated with time- and space-dependent fluid properties and compositions. The component concentration distribution and the separation ratio were used to examine the behaviour of the thermodiffusion and buoyancy-driven convection inside the cavity. It was found that, for permeability below 300 mD, the thermodiffusion for both mixtures was dominant; and above this level, buoyancy convection became the dominant mechanism. The viscosity was found to influence the evaluation of the molecular and thermodiffusion coefficients. The result showed that, with variable viscosity, the flow due to thermodiffusion was affected. Throughout, the comparison between the numerical results and the experimental data for a ternary mixture of THN-IBB-nC12, using vt-PR-EoS for evaluating the thermodynamic properties, yields better agreement with the experimental data. It was found that the percentage errors for 1 2 3 , and T T T D D D , when the vt-PR-EoS was used, were 12.63%, 23.11% and 49.65%, respectively. Furthermore, the mixture component’s behaviour also was found to be identical, for both experimental data and numerical values, and was found to have the same sign for thermodiffusion coefficients. The two components (nC12 and THN) migrated, one to the hot side and the other to the cold side, while the IBB was distributed randomly within the cavity, as its thermodiffusion coefficient value is small, and this confirmed that its separation remains small. Finally, the density of the nC12-THN-IBB mixture at different temperatures was compared with the numerical results in order to validate the equation of state (vt-PR-Eos). The results showed an excellent agreement between the numerical model and the experimental data, with the percentage error being less than 3%. 5. A three-dimensional model was numerically simulated to illustrate the interaction between thermodiffusion and buoyancy-driven convection in a hydrocarbon ternary 132 mixture (nC12-THN-IBB) inside a heterogeneous porous medium. A horizontal temperature gradient was applied in a porous medium under atmospheric pressure. Employing a single phase model and Darcy’s law, the continuity and the energy equations were solved numerically, using the finite volume method. The permeability of the matrix (main domain,κs 10 mD) was kept constant, while the permeability of the sub-domain, , f κ was varied. 7.1 Conclusions Different permeability ratios ( / κ κ f s  10, 100, 1000 and 10000) were examined in this study, which covered a wide range of oil reservoirs. The impact of the permeability ratio on the composition of the mixture components, velocity in the porous medium, and the separation ratio was investigated. It was found that the heterogeneous porous medium had a significant influence on the composition of the mixture components. Moreover, it was found that the sub-domains had a strong effect on the convective patterns, and the fluid flow in the sub-domain zones was much higher than that in the matrix domain. Furthermore, the separation ratio of Conf.2 was higher than that for Conf.1 due to the orientation of the sub-domains. 7.2 Contributions The main goal of this work was to investigate experimentally and numerically the fluid transport of hydrocarbon mixtures inside porous media with consideration of thermodiffusion and molecular diffusion. The following are the contributions of this work:  A FORTRAN program was developed to solve numerically the thermo-solutal convection for multicomponent mixtures inside porous media by applying the Firoozabadi model.  A new experimental technique based on optical digital interferometry for measuring Soret and molecular coefficients in porous media was carried out. A publication related to this contribution is Jaber et al. (2009).  The measurements for the thermal expansion and concentration expansion coefficients and for the viscosity of the binaries of the benchmark of 133 The predicted coefficients from the Peng-Robinson, and volume translated Peng- Robinson Equations of State were compared with the measured values. A publication related to this contribution is Batalle, et al. (2009). The predicted coefficients from the Peng-Robinson, and volume translated Pen  The Soret effect of two ternary mixtures in a homogenous porous medium cavity was studied for different permeabilities. A publication related to this contribution is Jaber and Saghir (2006), Jaber et al. (2008a), and Jaber et al. (2008b).  An attempt was made to simulate the thermo-solutal convection of a hydrocarbon ternary mixture in the presence of thermodiffusion in a non-homogenous porous medium. A publication related to this contribution is Jaber and Saghir (2008) and Jaber and Saghir (2010). 7.3 Recommendations There are still some related open issues that need to be studied, so future work may be carried out to address the following:  The measurements of thermodiffusion and molecular diffusion coefficients for multicomponent hydrocarbon fluid mixtures;  Further development of a theoretical model for multicomponent systems;  The measurements of the Soret coefficients at high pressure for the three binary mixtures. The values of the contrast factor, , ( / )P T n c   , at high pressure, which are needed to evaluate the Soret coefficient, must be measured because they are not present in the literature. 134 References Alboin, C., Jaffre, J., Robert, J. E. and Serres, C. (2002): “Modeling Fractures as Interfaces for Flow and Transport in Porous Media” in Fluid Flow and Transport in Porous Media: Mathematical and Numerical Treatment, Z. Chen and R.E. Ewing eds., Contemporary Mathematics 295, American Mathematical Society, pp. 13-24. Anderson, J. D. (1995): “Computational Fluid Dynamics: The Basics with Applications” McGraw-Hill, New York. Barenblatt, G. I., Zheltov, I. P. and Kochina, I. N. (1960): “Basic Concepts in the Theory of Seepage of Homogeneous Liquids in Fissured Rocks” PMM, Applied Mathematic and Mechanic, vol. 24, pp. 1286-1303. Bataller, H., Miqueu, C., Plantier, F., Daridon, J., Jaber, T. J., Abbasi, A. and Saghir, M. Z., (2009): “Comparison between Experimental and Theoretical Estimations of the Thermal Expansion, Concentration Expansion Coefficients and Viscosity for Binary mixtures under pressure up to 20 MPa” Journal of Chemicals engineering data, vol. 54, no.6, pp. 1710-1715. Bear, J. (1972): “Dynamic of Fluid in Porous Media” Elsevier, New York. Benano-Melly, L.B., Caltagirone, J.-P, Faissat, B., Montel, F. and Costeseque, P. (2001): “Modeling Soret Coefficient Measurement Experiments in Porous Media Considering Thermal and Solutal Convection” International Journal of Heat and Mass Transfer, vol. 44, pp. 1285-1297. Bird, R. B., Stewart, W.E and Lightfoot, E. N. (2002): “Transport Phenomena” 2nd Ed, John Wiley and sons, p. 765. Blanco, P., Bou-Ali, M. M., PLatten, J. L., Madariaga, J. A., Urteaga, P., and Santamaria, C. (2006): “The Thermodiffusion Coefficient for Organic Mixtures of n- alkanes n-Ci-nC18 (i=5,…,13): Influence of the Chain Length” Thermodiffusion: Basic & Applications, Bou-Ali, M. M. and Platten, J. K., Eds. Mondragon Unibertsitateko Zerbitzu Editorial, Mondragon, Spain, pp. 427-438. Bou-Ali, M. M., Ecenarro, O., Madariaga, J. A., Santamaria, C. M. and Valencia, J. J. (1998): “Thermogravitational Measurement of the Soret Coefficient of Liquid Mixtures” Journal of Physics: Condensed Matter, vol. 10, pp. 3321-3331. 135 Bou-Ali, M.M., Valencia, J.J., Madariaga, J.A., Santamaria, C., Ecenarro, O., and Dutrieux, J.F., (2003): “Determination of the Thermodiffusion Coefficient in Three Binary Organic Liquid Mixtures by the Thermogravitational Method” Philosophical Magazine, vol. 83, no. 17-18, pp. 2011-2015. Comunas, M.J.P., Bazile, J.P., Baylaucq, A., and Boned, C. (2008): “Density of Diethyl Adipate Using a New Vibrating Tube Densimeter from (293.15 to 403.15) K and up to 140 MPa Calibration and Measurements” Journal of Chemical Engineering and Data, vol. 53, no. 4. pp. 986-994. Costeseque P., Pollak, T., Platten J. K., and Marcoux M. References (2004): “Simultaneous Evaluation of Soret and Fick Coefficients in a Free and a Packed Vertical Gradient Soret Cell” 6th International Meeting on Thermodiffusion (IMT6), Varenna, Italy. Costeseque, P., Pollak, T., Platten, J. K., and Marcoux, M. (2004): “Transient-state Method for Coupled Evaluation of Soret and Fick Coefficients, and Related Tortuosity Factors, Using Free and Porous Packed Thermodiffusion Cells: Application to CuSO4 Aqueous Solution (0.25m)” European Physical Journal, vol. 15, no. 3, pp. 249–253. Costeseque, P., El Maataoui, M., and Riviere, M. (1994): “Selective Enrichments Induced by Thermogravitational Diffusion in Porous Medium on Hydrocarbon Species of Crude Oil and Specific Case of Paraffin Isomers” Entropy, vol. 94, no. pp 94-100. Costeseque, P. and Loubet, J-C. (2003): “Measuring the Soret Coefficient of Binary Hydrocarbon Mixture in Packed Thermogravitational Columns” Philosophical Magazine, vol. 83, pp. 2017-2022. Darcy, H. (1856): Les fontaines publiques de la ville de Dijon, V. Dalmont, Paris, pp 305-401. Daubert, T. E. and Danner, R. P. (1989): “Physical and Thermodynamic Properties of Pure Chemicals: Data Compilation” Hemisphere Publishing Corporation, New York. Daubert, T. E. and Danner, R. P. (1989): Physical and Thermodynamic Properties of Pure Chemicals: Data Compilation” Hemisphere Publishing Corporation, New York. de Groot, S. R. (1945): “ L’Effect Soret” Amsterdam. de Groot, S.R and Mazur, P. (1984): “Non-Equilibrium Thermodynamics” Dover P bli ti I N Y k p p g p , de Groot, S. R. (1945): “ L’Effect Soret” Amsterdam. de Groot, S. R. (1945): “ L’Effect Soret” Amsterdam. de Groot, S.R and Mazur, P. (1984): “Non-Equilibrium Thermodynamics” Dover Publication Inc., New York. Delware, F., Chacha, M, Ghorayeb K, and Saghir, M.Z. (2004): “Compositional Variation Considering Diffusion and Convection for Binary Mixture in Porous Media” Journal of Porous Media, vol. 7, no. 2, pp. 1-19. 136 de Marsily, G. (1986): “Quantitative Hydrogeology for Engineers” Academic Press, San Diego. Denbigh, K. G. (1951): “The Thermodynamics of the Steady State” Wiley, New York. Dougherty, E. L., and Drickamer H. G. (1955): “A Theory of Thermal Diffusion in Liquids” Journal of Chemical Physics, vol. 23, no. 2, pp. 295-309. Dutrieux, J. F, Platten, J. K., Chavepeyer, G., and Bou-Ali, M. M. (2002): “On the Measurement of Positive Soret Coefficients” Journal of Physical Chemistry B, vol. 106, no. 23, pp. 6104–6114. de Marsily, G. (1986): “Quantitative Hydrogeology for Engineers” Academic Press, San Diego. de Marsily, G. (1986): “Quantitative Hydrogeology for Engineers” Academic Press, San Diego. Denbigh, K. G. References (1951): “The Thermodynamics of the Steady State” Wiley, New York. Dougherty, E. L., and Drickamer H. G. (1955): “A Theory of Thermal Diffusion in Liquids” Journal of Chemical Physics, vol. 23, no. 2, pp. 295-309. Dutrieux, J. F, Platten, J. K., Chavepeyer, G., and Bou-Ali, M. M. (2002): “On the Measurement of Positive Soret Coefficients” Journal of Physical Chemistry B, vol. 106, no. 23, pp. 6104–6114. Et-Tahir, A. Boned, C., Lagourette, B., and Xans, P. (1995): “Determination of the Viscosity of Various Hydrocarbons and Mixtures of Hydrocarbons versus Temperature and Pressure” International Journal of Thermophysics, vol. 6, pp. 1309-1334. Fick, A. (1855): “On Liquid Diffusion” Philosophical Magazine, vol. 10, no. 63, pp. 30- 39. Firoozabadi, A. (1999): “Thermodynamics of Hydrocarbon Reservoirs” McGraw-Hill, New York. Firoozabadi, A., Ghoryeb, K., and Shukla, K. (2000): “Theoretical Model of Thermal Diffusion Factors in Multicomponent Mixtures” American Institute of Chemical Engineers Journal, vol. 46, no. 5, pp. 892-900. FLUENT 6.3 User’s Guide, 2006. Ghoryeb, K, and Firoozabadi, A. (2000): “Molecular, Pressure, and Thermal Diffusion in Nonideal Multicomponent Mixtures” American Institute of Chemical Engineers Journal, vol. 46, no. 5, pp. 883-891. Ghorayeb, K., and Firoozabadi, A. (2000): “Numerical Study of Natural Convection Diffusion in Fractured Porous Media” Society of Petroleum Engineers Journal, vol. 5, no.1, pp. 12-20. Ghorayeb, K., Firoozabadi, A., and Toshiyuki, A. (2003): “Interpretation of the Unusual Fluid Distribution in the Yufutsu Gas-Condensate Field” Society of Petroleum Engineers Journal, vol. 8, no. 2, pp. 114-123. Glasstone S, Laidler, K.J, and Eyring, H. (`1941): “The Theory of Rate Processes: the Kinetics of Chemical Reaction, Viscosity, Diffusion and Electrochemical Phenomena” New York, McGraw-Hill, McGraw-Hill, New York. 137 Graf, T., and Therrien, R. (2005): “Variable-density Groundwater Flow and Solute Transport in Porous Media Containing Non-uniform Discrete Fractures” Advance in water Resources, vol. 28, pp. 1351-1367. Haase, R. (1950): Z. Phys. Chem., vol. 196, p. 219 Haugen, K. B., and Firoozabadi, A. (2007): “Transient Separation of Multicomponent Liquid Mixtures in Thermogravitational Columns” Journal of Chemical Physics, vol. 127, pp. 154507(1-9). Haugen, K. B., and Firoozabadi, A. (2005): “On Measurement of Thermal Diffusion Coefficients in Multicomponent Mixtures” Journal of Chemical Physics, vol. 122, pp. 14516 (1-7). Hayduk, W. and Minhas, B. S. (1982): “Correlation for Predictions of Molecular Diffusivities in Liquids” Canadian Journal of Chemical Engineering, vol. 60, pp. 295- 299. Hildebrand, J. H., and Scott, R. L. References (1950): “The Solubility of Nonelectrolytes” 3rd Ed, Reinhold Publishing Co., New York. Israfilov, H., Jannataliyev, R., Safarov, J., Shahverdiyev, A., and Hassel, E. (2009): “The ( , , p T  ) Properties and Apparent Molar Volumes V of LiNO3 + C2H5OH” Acta Chimica Slovenica, vol. 56, no. 1, pp. 95-108. Jaber, T. J. and Saghir, M. Z. (2006): “Numerical Analysis of Ludwig-Soret Effect of one Binary Mixture and Two Ternary Mixtures in an Al2O3 Porous Cavity” ASME International Mechanical Engineering Congress and Exposition, Nov. 5-10, Chicago, Illinois, USA. Jaber, T. J., Yan, Yu and Saghir, M. Z. (2008a): “Soret Effect for a Ternary Mixture in Porous Cavity: Modeling with Variable Diffusion Coefficients and Viscosity” Journal of Fluids Engineering, vol. 30, pp. 081703-1. Jaber, T. J., Yan, Yu and Saghir M. Z. (2008b): “Comparison between Theoretical Model and Experimental data of Thermodiffusion Coefficients for Ternary Hydrocarbon Mixtures of Methane, n-Butane and n-Dodecane” International Meeting on Thermal Diffusion (IMT8), June 9-13, Bonn-Bad Godesberg, Germany. Jaber, T. J. and Saghir, M. Z. (2008): “Numerical Study of Permeability Effect on Convection in Fractured Porous Media Filled with Hydrocarbon Ternary Mixture” 138 ICHMT International Symposium on Advances in Computational Heat Transfer, May 11- 16, Marrakech, Morocco. Jaber, T. J., Bataller, H. and Saghir, M. Z. (2009): “Measurement of Thermodiffusion Coefficient for Binary Hydrocarbon Fluid Mixtures in Porous Medium: Experimental and Numerical Results” ASME International Mechanical Engineering Congress and Exposition, Nov. 13-19, Lake Buena Vista, Florida, USA. Jaber, T. J., Bataller, H. and Saghir, M. Z. (2009): “Measurement of Thermodiffusion Coefficient for Binary Hydrocarbon Fluid Mixtures in Porous Medium: Experimental and Numerical Results” ASME International Mechanical Engineering Congress and Exposition, Nov. 13-19, Lake Buena Vista, Florida, USA. Jaber, T. J. and Saghir, M. Z. (2010): “Three-dimensional Study of Permeability Effect on Convection in Heterogeneous Porous Medium Filled with a Ternary Hydrocarbon Mixture” Journal of porous medium, accepted, No. 1511-JPM, in press. Jhaver, B. S. and Youngren, G. K. (1988): “Three-Parameter Modification of the Peng- Robinson Equation of State to Improve Volumetric Prediction” Journal of SPE Reservoir Engineering, vol. 3, pp. 1033-1040. Jiang, C. G., Saghir, M. Z., and Kawaji M. (2006): “Numerical Analysis of Thermal- Solutal Convection in Heterogeneous Porous Media” Journal of Applied Mechanics, vol. 73, pp. 21-25. Jiang, C.G., Jaber, T. J., Batalle, H., and Saghir, M. Z. References (2007): “Simulation of Ludwig-Soret effect of a Water-ethanol Mixture in a Cavity Filled with Aluminum Oxide Powder under High Pressure” International Journal of Thermal Sciences, vol. 47, no. 2, pp. 126-135. Jossi, J. A., Stiel, L. I., and Thodos, G. (1962): “The Viscosity of Pure Substances in the Dense Gaseous and Liquid Phases” American Institute of Chemical Engineers Journal, vol. 8, no. 1, pp. 59-63. Kanti, M., Zhou, H., Ye, S., Boned, C., Lagourette, B., Saint-Guirons, H., Xans, P., and Montel, F. (1989): “Viscosity of Liquid Hydrocarbons, Mixtures and Petroleum cuts, as a Function of Pressure and Temperature” Journal of Chemical Physics, vol. 93, pp. 3860-3864. Kazemi, H. L., Merrill, S., Porterfild, K. L., and Zeman, P. R. (1976): “Numerical Simulation of Water-Oil Flow in Naturally Fractured Reservoirs” Society of Petroleum Engineers Journal, vol. 16, no. 6, pp. 317-326. Kempers, L.J.T.M. (1989): “A Thermodynamic Theory of the Soret Effect in a Multicomponent liquid” Journal of Chemical Physics. vol. 90, pp. 6541-6548. 139 Kempers, L.J.T.M. (2001): “A Comprehensive Thermodynamic Theory of the Soret Effect in a Multicomponent Gas, Liquid, or Solid” Journal of Chemical Physics, vol. 115, no. 14, pp.6330–6341. Kempers, L.J.T.M. (2001): “A Comprehensive Thermodynamic Theory of the Soret Effect in a Multicomponent Gas, Liquid, or Solid” Journal of Chemical Physics, vol. 115, no. 14, pp.6330–6341. Kohler, W., and Muller, B. (1995): “Soret and Mass diffusion Coefficients of Toluene/n-hexane Mixtures” Journal of Chemical Physics, vol. 103, pp. 4367-4370. Kolodner, P., Williams, H., and Moe, C., (1988): “Optical Measurement of the Soret Coefficient of Ethanol/water Solution” Journal of Chemical Physics, vol. 88, no. 10, pp. 6512-6524. Lagourette, B., Boned, C., Saint-Guirons, H., Xans, P., and Zhou, H. (1992): “Densimeter Calibration Method Versus Temperature and Pressure” Journal of Measurement Science and Technology, vol. 3, pp. 699-703. Leahy-Dios, A., Bou-Ali, M. M., Platten, J. K., and Firoozabadi A. (2005): “Measurements of Molecular and Thermal Diffusion Coefficients in Ternary Mixture” Journal of Chemical Physics, vol. 122, no. 23, pp. 1-12. Leahy-Dios, A., and Firoozabadi, A. (2007): “Molecular and Thermal Diffusion Coefficients of Alkane-Alkane and Alkane-Aromatic Binary Mixtures: Effect of Shape and Size of Molecules” Journal of Physical Chemistry B, vol.111, no. 1, pp. 191-198. Liu, Jianchun, Bodvarsson, G.S., and Wu, Yu-Shu (2003): “Analysis of Flow Behaviour in Fractured Lithophysal Reservoirs” Journal of Contaminant Hydrology, vol. 62-63, pp. 189–211. Lohrenz, J., Bray, B. G., and Clark, C. R. References (1964): “Calculating Viscosities of Reservoir Fluids from Their Compositions” Journal of Petroleum Technology, vol. 16, no. 10, pp. 1171–1176. Ludwig, C. (1856): “Diffusion zwischen ungleich erwärmten Otern gleich zusammengesetzter Lösunger Sitzber” Akad. Wiss. Wien, Math. Naturw. Kl, vol. 20, p. 539. Montel, F. (1998): “The Role of Thermodynamics in Modeling Species Distributions of Hydrocarbon in Petroleum Reservoirs” Entropy, vol. 34, no. 214, pp. 7-10. Nield, D. A. and Bejan, A. (2006): “Convection in Porous Media” Springer, 3rd edition. Miyake, Y., Baylaucq, A., Plantier, F., Bessieres, D., Ushiki, H., and Boned, C. (2008): “High-pressure (up to 140 MPa) Density and Derivative Properties of Some 140 (pentyl-, hexyl-, and heptyl-) Amines Between (293.15 and 353.15) K” Journal of Chemical Thermodynamics, vol. 40, pp. 836-845. (pentyl-, hexyl-, and heptyl-) Amines Between (293.15 and 353.15) K” Journal of Chemical Thermodynamics, vol. 40, pp. 836-845. Melnikov D.E., Shevtsova V.M., Mialdum A., and Bataller H. (2009): “Role of Liquid-Porous Interface in Soret Separation” Fourth International Conference on Thermal Engineering: Theory and Applications, January 12-14, Abu Dhabi, UAE. Mialdun, A., and Shevtsova, V.M. (2008): “Development of Optical Digital Interferometry Technique for Measurement of Thermodiffusion Coefficients” International Journal Heat and Mass Transfer, vol. 51, no. 11-12, pp.3164–3178. Melnikov D.E., Shevtsova V.M., Mialdum A., and Bataller H. (2009): “Role of Liquid-Porous Interface in Soret Separation” Fourth International Conference on Thermal Engineering: Theory and Applications, January 12-14, Abu Dhabi, UAE. Mialdun, A., and Shevtsova, V.M. (2008): “Development of Optical Digital Interferometry Technique for Measurement of Thermodiffusion Coefficients” International Journal Heat and Mass Transfer, vol. 51, no. 11-12, pp.3164–3178. Oliveira, C. M. B. P., and Wakeham W. A. (1992): “Vibrating-wire Viscometers for Liquids at High pressures” International Journal of Thermophysics, vol. 13, pp. 773-790. Onsager, L. (1931): “Reciprocal Relations in Irreversible Processes” Physical Review, vol. 37, pp. 405-426. Kashiwagi, H., and Makita, T. (1982): “Viscosity of Twelve Hydrocarbon Liquids in the Temperature Range 298–348 K at Pressures up to 110 MPa” International Journal of Thermophysics, vol. 3, pp. 289-305. Pan, S., Yan, Y., Jaber, T. J., Kawaji, M., and Saghir, M. Z., (2007):“The Evaluation of Thermal Diffusion Models for Ternary Hydrocarbon Mixture” Journal Non- Equilibrium Thermodynamics, vol. 32, no. 3, pp. 1-9. Patankar, S. V. (2006): “Numerical Heat Transfer and Fluid Flow” New York: McGraw-Hill. Peneloux, A., Rauzy, E., and Freze, R. (1982): “A Consistent Correction for Redlich- Kwong-Soave Volumes” Journal of Fluid Phase Equilibria, vol. 8, no. 1, pp. 7–23. References Peng, D. Y. and Robinson, D. B. (1976): “A New Two-constant Equation of State” Industrial Engineering Chemistry Fundamentals, vol. 15, no. 1, pp. 59-64. Kwong-Soave Volumes” Journal of Fluid Phase Equilibria, vol. 8, no. 1, pp. 7–23. Peng, D. Y. and Robinson, D. B. (1976): “A New Two-constant Equation of State” Industrial Engineering Chemistry Fundamentals, vol. 15, no. 1, pp. 59-64. Platten, J. K. (2006): “The Soret Effect: a Review of Recent Experimental Results” Journal of Applied Mechanics, vol. 73, pp. 5-15. Platten, J. K., Bou-Ali, M. M., and Dutrieux, J. F. (2004): “Thermal Diffusion in Ternary Systems: an Experimental Study” In Proceeding of the 6th International Meeting of Thermodiffuiosn (IMT6), Varenna, Italy. Platten, J. K., and Costeseque, P. (2004): “The Soret Coefficient in Porous Media” Journal of Porous Media, vol. 7, no. 4, pp. 317-329. 141 Platten, J. K., Bou-Ali, M. M., and Dutrieux, J. F. (2003): “Enhance Molecular Separation in Inclined Thermogravitational Columns” Journal of Physical Chemistry B, vol. 107, no. 42, pp. 11763-11767. Platten, J. K., Bou-Ali M. M., Costeseque, P., Dutrieux J. F., Kohler W., Leppla C., Wiegand S., and Wittko G. (2003): “Benchmark Values for Soret, Thermal Diffusion and Diffusion Coefficients of Three Binary Organic Liquid Mixtures” Philosophical Magazine, vol. 83, pp.1965-1971. Polyakov, P., Luettmer-Strathmann, J., and Wiegand, S. (2006): “Study of the Thermal Diffusion Behaviour of Alkane/Benzene Mixture by Thermal Diffusion Forced Rayleigh Scattering Experiments and Lattice Model Calculations” Journal of Physical Chemistry B, vol. 110, n. 51 pp. 26215-26224. Riley, M. F. and Firoozabadi, A. (1998): “Compositional Variation in Hydrocarbon Reservoirs with Natural Convection and Diffusion” American Institute of Chemical Engineers Journal, vol. 44, no. 2, pp. 452-464. Rutherford, W. M. and Drickamer, H. G. (1954): “Theory of Thermal Diffusion in Liquids and the Use of Pressure to Investigate the Theory” Journal of Chemical Physics, vol. 22, no. 7, pp.1157-1165. Rutherford, W.M., and Roof, J.G. (1959): “Thermal Diffusion in Methane-n-Butane Mixtures in the Critical Region” Journal of Physical Chemistry, vol. 63, pp. 1506-1511. Saghir, M. Z. and Islam, M. R. (1999): “Double Diffusive Convection in Dual- Permeability, Dual-Porosity Porous Media” International Journal of Heat and Mass Transfer, vol. 42, no. 3, pp. 437-454. Scatchard, G. (1931): Chemical Reviews, vol. 8, p. 321. Shukla, K., and Firoozabadi, A. (1998): “A New Model of Thermal Diffusion Coefficients in Binary Hydrocarbon Mixtures” Journal Industrial & Engineering Chemistry Research, vol. 37, pp. 3331-3342. Soave, G. References (1972): “Equilibrium Constants from a Modified Redlich-Kwong Equation of State” Journal of Chemical Engineering Science, vol. 27, pp. 1197-1203. Soret, C. (1879): “Sur l’état d’équilibre que prend au point de vue de sa concentration une solution saline primitivement homogène dont deux parties sont portées à des 142 températures différentes” Archives des Sciences Physiques et Naturelles de Genève, vol. 2, pp. 48-61. Soret, C. (1880): “Influence de la temperature sur la distribution des sels dans leurs solutions” Compte-Rendu de l’Academie des Sciences, Paris, vol. 91, pp. 289-291. Taylor, R., and Krishna, R. (1993): “Multicomponent Mass Transfer” New York, Wiley. Tichacek, L. J., Kmak, W. L., and Drickamer, H. G. (1956): “Thermal Diffusion in Liquids; the Effect of Non-Ideality and Association” Journal Physics Chemistry, vol. 60, pp. 660-665. Tsang, Y.W., and Birkholzer, J.T. (1999): “Predictions and Observations of the Thermal–hydrological Conditions in the Single Heater Test” Journal of Contaminant Hydrology, vol. 38, no. 1–3, pp. 385–425. Urteaga, P., Plantier, F., Bou-Ali, M.M., and Bataller, H. (2008): “Thermodiffusion Coefficient (DT) for Binary Hydrocarbon Mixtures at High Pressures, Thermal Nonequilibrium” Lecture Notes of the 8th International Meeting on Thermodiffusion, edited by S.Wiegand, W. Köhler, J.K.G. Dhont, Schriften des Forschungszentrums Jülich, Germany, pp. 77-82. Urteaga, P., Bou-Ali, M.M, and Blanco, P. (2008): “Thermogravitational Technique at High Pressure for Liquid Mixtures, Thermal Nonequilibrium” Lecture Notes of the 8th International Meeting on Thermodiffusion, edited by S.Wiegand, W. Köhler, J.K.G. Dhont, Schriften des Forschungszentrums Jülich, Germany, pp. 71-76. High Pressure for Liquid Mixtures, Thermal Nonequilibrium” Lecture Notes of the 8th International Meeting on Thermodiffusion, edited by S.Wiegand, W. Köhler, J.K.G. Dhont, Schriften des Forschungszentrums Jülich, Germany, pp. 71-76. Urteaga, P., Bou-Ali, M.M., Platten, J.K., Madariaga, J.A., and Santamaria, C., in M.M. Bou-Ali and Platten, J.K. (Ed.) (2006): “Thermodiffusion: Basics and Applications” Mondragon Unibertsitateko Zerbitzu Editoriala: Mondragon, Spain. Van Vaerenbergh, S., and Legros, J.C. (1998): “Soret Coefficients of Organic Solutions Measured in the Microgravity SCM Experiment and by the Flow and Benard Cells” Journal of Physical Chemistry, vol. 102, pp. 4426-4431. Van Vaerenbergh S., Shapiro A., Galliero G., Montel F., Legros J., Caltagirone J., Daridon J., and Saghir M.Z. (2005): “Multicomponent Processes in Crudes” European Space Agency Special Publication, vol. 1290, pp. 202-213. Urteaga, P., Bou-Ali, M.M., Platten, J.K., Madariaga, J.A., and Santamaria, C., in M.M. Bou-Ali and Platten, J.K. (Ed.) (2006): “Thermodiffusion: Basics and Applications” Mondragon Unibertsitateko Zerbitzu Editoriala: Mondragon, Spain. References Urteaga, P., Bou-Ali, M.M., Platten, J.K., Madariaga, J.A., and Santamaria, C., in M.M. Bou-Ali and Platten, J.K. (Ed.) (2006): “Thermodiffusion: Basics and Applications” Mondragon Unibertsitateko Zerbitzu Editoriala: Mondragon, Spain. Van Vaerenbergh, S., and Legros, J.C. (1998): “Soret Coefficients of Organic Solutions Measured in the Microgravity SCM Experiment and by the Flow and Benard Cells” Journal of Physical Chemistry, vol. 102, pp. 4426-4431. Van Vaerenbergh, S., and Legros, J.C. (1998): “Soret Coefficients of Organic Solutions Measured in the Microgravity SCM Experiment and by the Flow and Benard Cells” Journal of Physical Chemistry, vol. 102, pp. 4426-4431. Van Vaerenbergh S., Shapiro A., Galliero G., Montel F., Legros J., Caltagirone J., Daridon J., and Saghir M.Z. (2005): “Multicomponent Processes in Crudes” European Space Agency Special Publication, vol. 1290, pp. 202-213. 143 Van Vaerenbergh S., Srinivasan S., and Saghir M.Z. (2009): “Thermodiffusion in Multicomponent Hydrocarbon Mixtures: Experimental Investigations and Computational Analysis” Journal of Chemical Physics, vol. 131, pp. 114505-1-114505-8. Vieira dos Santos, F.J. and Nieto de Castro, C. A. (1997): “Viscosity of Toluene and Benzene under High Pressure” International Journal of Thermophysics, vol. 18, no. 2, pp. 367-378. Versteeg, H. K., and Malalasekera W. (2007): “An Introduction to Computational Fluid Dynamics, the Finite Volume Method” 2nd edition, Pearson. Warren, J. E., and Root, P. J. (1963): “The Behaviour of Naturally Fractured Reservoirs” Society of Petroleum Engineers Journal, vol. 3, no. 3, pp. 245-255. Wiegand, S. (2004): “Thermal Diffusion in Liquid Mixtures and Polymer Solutions” Journal of Physics-condensed Matter, vol. 16, no. 10:R357–R379. Wiegand, S., and Köhler, W. (2002):“Thermal Nonequilibrium Phenomena in Fluid Mixtures” (Lecture Notes in Physics), Springer-Verlag, edited by W. Köhler and S. Wiegand, vol. 584, pp. 189-210. Wittko, G., and Köhler, W. (2003):“Precise Determination of the Soret, Thermal diffusion and Mass Diffusion Coefficients of Binary Mixtures of n-Dodecane, Isobutylbenzene and 1,2,3,4-Tetrahydronaphthalene by Holographic Grating Technique” Philosophical Magazine, vol. 83, no. 17-18, pp. 1973-1987. Wu, Y.S., Mukhopadhyay, S., Zhang, K., and Bodvarsson, G.S. (2006): “A Mountain-Scale Thermal-Hydrologic Model for Simulating Fluid Flow and Heat Transfer in Unsaturated Fractured Rock” Journal of Contaminant Hydrology, vol. 86, pp. 128-159. Zhang, K.J., Briggs, M.E., Gammon, R.W., and Sengers, J.V. (1996): “Optical Measurement of the Soret Coefficient and the Diffusion Coefficient of Liquid Mixtures” Journal of Chemical Physics, vol. 104, no. 17, pp. 6881-6892. 144 Schematically, the program Refr_In software performs the following operations: 1. Initialization of the link with the CCD camera; the COHU CCD Camera 7700 was used in this project (1004x1004 pixels with 10 bit depth at 30 FPS). 1. Initialization of the link with the CCD camera; the COHU CCD Camera 7700 was used in this project (1004x1004 pixels with 10 bit depth at 30 FPS). 2. Acquisition of sequential images at constant, selectable time steps. 3. Showing of the acquired image (on/off) to allow visual check of the interferograms and choice of the Region of Interest (ROI). 3. Showing of the acquired image (on/off) to allow visual check of the interferograms and choice of the Region of Interest (ROI). 4. Recording (on/off) of the pixel values of the entire image in tiff file. 4. Recording (on/off) of the pixel values of the entire image in tiff file. 5. Selection of the ROI in the form of a vertical (perpendicular to grating pattern) line over the image area. 6. Low-pass filtering (on/off) of the pixel values in the ROI to give better fit results. 6. Low-pass filtering (on/off) of the pixel values in the ROI to give better fit results. 7. Normalization (on/off) of the pixel values in the ROI to give better fit results. 7. Normalization (on/off) of the pixel values in the ROI to give better fit results. 8. Recording (on/off) of the pixel values of the ROI in txt file. 8. Recording (on/off) of the pixel values of the ROI in txt file. 9. Initialization of the fitting parameters. 9. Initialization of the fitting parameters. 10. Fitting of the pixel values of the ROI to get the phase value of the sinusoidal pattern. 10. Fitting of the pixel values of the ROI to get the phase value of the sinusoidal pattern. 11. Plotting the data of the pixel values in the ROI, and the values given by fit process in a graph format to allow visual check of the fit process. 11. Plotting the data of the pixel values in the ROI, and the values given by fit process in a graph format to allow visual check of the fit process. 12. Acquisition of the values of the pressure, and temperatures of the hot and cold dead-volumes via NI-USB-9162 data acquisition card. 12. Acquisition of the values of the pressure, and temperatures of the hot and cold dead-volumes via NI-USB-9162 data acquisition card. 13. Custom-Made Software Package Custom-made software in the LabView environment allows the image acquisition and the extraction of useful quantities, see Figure A.1. The program simultaneously acquires the relevant parameters of the cell, namely, pressure and temperature of the hot and cold dead- volumes. Schematically, the program Refr_In software performs the following operations: Schematically, the program Refr_In software performs the following operations: Schematically, the program Refr_In software performs the following operations: It has been chosen to investigate just one vertical line out of the entire image, because this is enough for a good characterization of the sinusoidal modulation. The intensity of the laser and the exposure time of the CCD camera are tuned in a preliminary set-up phase, so that the maximum grey level within an image is in the order of 240-250 not to saturate the image. Other parameters of the CCD camera are adjusted in order to exploit the CCD dynamic at its best. After selection of the Region of Interest (ROI) in the form of a vertical line on which all maths will be performed, a low-pass filter is applied to the pixel values of the ROI to eliminate most of the noise. Eventually ROI pixel values are normalized so that all peaks are at about unit height. This is sometimes necessary because of non-uniformities of the laser beam due to dust and other spurious in the beam path. The consequence of these non-uniformities is an increase in the convergence time and a decrease in the accuracy of the following fitting procedure through a sinusoidal function. Schematically, the program Refr_In software performs the following operations: Plotting the data of the pressure in a graph format. 13. Plotting the data of the pressure in a graph format. 13. Plotting the data of the pressure in a graph format. 14. Plotting the data of the phase in a graph format. 14. Plotting the data of the phase in a graph format. 14. Plotting the data of the phase in a graph format. 15. Recording the data of phase, pressure, and temperatures of the hot and cold dead- volumes as a function of time. 15. Recording the data of phase, pressure, and temperatures of the hot and cold dead- volumes as a function of time. 145 Typical, interferograms consist of a vertical intensity grating, which is captured by the CCD camera and converted in a matrix of grey levels. The CCD camera utilized for these experiments is an 8-bit camera, so that images are digitized in grey levels ranging from 0 to 255. It has been chosen to investigate just one vertical line out of the entire image, because this is enough for a good characterization of the sinusoidal modulation. The intensity of the laser and the exposure time of the CCD camera are tuned in a preliminary set-up phase, so that the maximum grey level within an image is in the order of 240-250 not to saturate the image. Other parameters of the CCD camera are adjusted in order to exploit the CCD dynamic at its best. After selection of the Region of Interest (ROI) in the form of a vertical line on which all maths will be performed, a low-pass filter is applied to the pixel values of the ROI to eliminate most of the noise. Eventually ROI pixel values are normalized so that all peaks are at about unit height. This is sometimes necessary because of non-uniformities of the laser beam due to dust and other spurious in the beam path. The consequence of these non-uniformities is an increase in the convergence time and a decrease in the accuracy of the following fitting procedure through a sinusoidal function. Typical, interferograms consist of a vertical intensity grating, which is captured by the CCD camera and converted in a matrix of grey levels. The CCD camera utilized for these experiments is an 8-bit camera, so that images are digitized in grey levels ranging from 0 to 255. The fitting is operated on filtered and normalized ROI pixel values trough the equation: The fitting is operated on filtered and normalized ROI pixel values trough the equation: 2 ( ) cos ( ) I x a bx c   2 ( ) cos ( ) I x a bx c   (A.1) At the beginning of the experiment the user must provide initial values for these parameters. Typical values are: a = 1, and b = 0.01-0.05. The half of the phase, c, is completely unknown in principle and is the output of the fitting procedure. The fitting then utilizes the output parameters of the preceding image as initial values for the fitting of the image under analysis. This has two important consequences; first, convergence is very fast since a and b should not vary during the entire experiment, and second, the value of c(which here is not limited between 0 and , like typically done) does not need unwrapping procedures and is directly displayed in the User Interface. 146 Figure A.1: Front-screen of the LabView-Refr_In software. Figure A.1: Front-screen of the LabView-Refr_In software. 147 Appendix B Appendix B The measurement value of the Density of the three binary Mixtures Figure B.1: Experimental setup of DMA HPM, Israfilov et al. (2009): 1 flash for the probe; 2,7, 16, and 17 valves; 3 and 11 fitting; 4 pressure intensifier; 5 pressure sensor HP-1; 6 pressure sensor P-10; 8 valve for closing of system during experiment; 10 vacuum indicator; 12 visual window; 13 vibration tube; interface module; 18 thermostat F32-ME; 19 vacuum pump; 20 thermos for cooling. vibration U-tube mPDS-2000V3 control unit mPDS-2000V3 control unit Figure B.1: Experimental setup of DMA HPM, Israfilov et al. (2009): 1 flash for the probe; 2,7, 16, and 17 valves; 3 and 11 fitting; 4 pressure intensifier; 5 pressure sensor HP-1; 6 pressure sensor P-10; 8 valve for closing of system during experiment; 10 vacuum indicator; 12 visual window; 13 vibration tube; interface module; 18 thermostat F32-ME; 19 vacuum pump; 20 thermos for cooling. 148 Table B.1: Experimental densities of the binary IBB-nC12 (50 wt% mass fraction) as function of the temperature and the pressure. 2 ( ) cos ( ) I x a bx c   P (MPa) T (°C) (g.cm-3) 0.1 20.0 0.7954 0.1 22.5 0.7936 0.1 25.0 0.7917 0.1 27.5 0.7899 0.1 30.0 0.7881 2 20.0 0.7967 2 22.5 0.7949 2 25.0 0.7931 2 27.5 0.7912 2 30.0 0.7896 4 20.0 0.7981 4 22.5 0.7963 4 25.0 0.7945 4 27.5 0.7926 4 30.0 0.7911 6 20.0 0.7995 6 22.5 0.7977 6 25.0 0.7959 6 27.5 0.7940 6 30.0 0.7925 8 20.0 0.8011 8 22.5 0.7991 8 25.0 0.7973 8 27.5 0.7954 8 30.0 0.7940 10 20.0 0.8025 10 22.5 0.8005 10 25.0 0.7986 10 27.5 0.7968 10 30.0 0.7954 12 20.0 0.8037 12 22.5 0.8018 12 25.0 0.7999 12 27.5 0.7981 12 30.0 0.7967 14 20.0 0.8050 14 22.5 0.8031 14 25.0 0.8013 14 27.5 0.7994 14 30.0 0.7981 16 20.0 0.8063 16 22.5 0.8043 16 25.0 0.8026 149 16 27.5 0.8007 16 30.0 0.7994 18 20.0 0.8076 18 22.5 0.8056 18 25.0 0.8038 18 27.5 0.8020 18 30.0 0.8008 20 20.0 0.8089 20 22.5 0.8068 20 25.0 0.8051 20 27.5 0.8033 20 30.0 0.8020 150 Table B.2: Experimental densities of the binary THN-nC12 (50 wt% mass fraction) as function of the temperature and the pressure. P (MPa) T (°C) (g.cm-3) 0.1 20.0 0.8444 0.1 22.5 0.8424 0.1 25.0 0.8407 0.1 27.5 0.8388 0.1 30.0 0.8373 2 20.0 0.8457 2 22.5 0.8436 2 25.0 0.8420 2 27.5 0.8400 2 30.0 0.8385 4 20.0 0.8468 4 22.5 0.8449 4 25.0 0.8434 4 27.5 0.8413 4 30.0 0.8399 6 20.0 0.8480 6 22.5 0.8460 6 25.0 0.8447 6 27.5 0.8425 6 30.0 0.8411 8 20.0 0.8495 8 22.5 0.8474 8 25.0 0.8459 8 27.5 0.8438 8 30.0 0.8424 10 20.0 0.8508 10 22.5 0.8486 10 25.0 0.8471 10 22.5 0.8498 12 25.0 0.8484 12 27.5 0.8463 12 30.0 0.8449 14 20.0 0.8531 14 22.5 0.8510 14 25.0 0.8495 14 27.5 0.8475 14 30.0 0.8461 16 20.0 0.8542 16 22.5 0.8521 16 25.0 0.8507 16 27.5 0.8488 16 30.0 0.8475 151 18 20.0 0.8554 18 22.5 0.8533 18 25.0 0.8519 18 27.5 0.8499 18 30.0 0.8487 20 20.0 0.8566 20 22.5 0.8544 20 25.0 0.8530 20 27.5 0.8510 20 30.0 0.8498 152 Table B.3: Experimental densities of the binary THN-IBB (50 wt% mass fraction) as function of the temperature and the pressure. 2 ( ) cos ( ) I x a bx c   P (MPa) T (°C) (g.cm-3) 0.1 20.0 0.9079 0.1 22.5 0.9057 0.1 25.0 0.9036 0.1 27.5 0.9015 0.1 30.0 0.9000 2 20.0 0.9091 2 22.5 0.9068 2 25.0 0.9047 2 27.5 0.9029 2 30.0 0.9013 4 20.0 0.9103 4 22.5 0.9081 4 25.0 0.9061 4 27.5 0.9042 4 30.0 0.9026 6 20.0 0.9116 6 22.5 0.9093 6 25.0 0.9075 6 27.5 0.9054 6 30.0 0.9040 8 20.0 0.9130 8 22.5 0.9106 8 25.0 0.9088 8 27.5 0.9068 8 30.0 0.9053 10 20.0 0.9143 10 22.5 0.9119 10 25.0 0.9101 10 27.5 0.9080 10 30.0 0.9065 12 20.0 0.9154 12 22.5 0.9131 12 25.0 0.9113 12 27.5 0.9092 12 30.0 0.9078 14 20.0 0.9166 14 22.5 0.9143 14 25.0 0.9125 14 27.5 0.9104 14 30.0 0.9090 16 20.0 0.9177 16 22.5 0.9154 16 25.0 0.9137 153 16 27.5 0.9116 16 30.0 0.9103 18 20.0 0.9188 18 22.5 0.9166 18 25.0 0.9149 18 27.5 0.9128 18 30.0 0.9114 20 20.0 0.9201 20 22.5 0.9177 20 25.0 0.9161 20 27.5 0.9140 20 30.0 0.9126 154 Table B.4: Experimental densities of the binary IBB-nC12 (T0=25 oC) as function of the mass fraction of the densest component and the pressure. P (MPa) 1  (g.cm-3) 0.1 0.48 0.7895 0.1 0.49 0.7904 0.1 0.50 0.7917 0.1 0.51 0.7927 0.1 0.52 0.7936 2 0.48 0.7909 2 0.49 0.7920 2 0.50 0.7931 2 0.51 0.7941 2 0.52 0.7952 4 0.48 0.7924 4 0.49 0.7935 4 0.50 0.7945 4 0.51 0.7957 4 0.52 0.7966 6 0.48 0.7939 6 0.49 0.7949 6 0.50 0.7959 6 0.51 0.7970 6 0.52 0.7979 8 0.48 0.7953 8 0.49 0.7963 8 0.50 0.7973 8 0.51 0.7984 8 0.52 0.7993 10 0.48 0.7966 10 0.49 0.7974 10 0.50 0.7986 10 0.51 0.7997 10 0.52 0.8006 12 0.48 0.7979 12 0.49 0.7987 12 0.50 0.7999 12 0.51 0.8010 12 0.52 0.8019 14 0.48 0.7992 14 0.49 0.8001 14 0.50 0.8013 14 0.51 0.8023 14 0.52 0.8032 16 0.48 0.8005 16 0.49 0.80131 6 0.50 0.8026 16 0.51 0.8035 16 0.52 0.8045 18 0.48 0.8017 155 18 0.49 0.8026 18 0.50 0.8038 18 0.51 0.8047 18 0.52 0.8057 20 0.48 0.8031 20 0.49 0.8039 20 0.50 0.8051 20 0.51 0.8060 20 0.52 0.8070 156 Table B.5: Experimental densities of the binary THN-nC12 (T0=25 oC) as function of the mass fraction of the densest component and the pressure. 2 ( ) cos ( ) I x a bx c   P (MPa) 1  (g.cm-3) 0.1 0.48 0.8357 0.1 0.49 0.8384 0.1 0.50 0.8407 0.1 0.51 0.8427 0.1 0.52 0.8448 2 0.48 0.8369 2 0.49 0.8397 2 0.50 0.8420 2 0.51 0.8440 2 0.52 0.8461 4 0.48 0.8383 4 0.49 0.8410 4 0.50 0.8434 4 0.51 0.8453 4 0.52 0.8475 6 0.48 0.8396 6 0.49 0.8422 6 0.50 0.8447 6 0.51 0.8465 6 0.52 0.8487 8 0.48 0.8409 8 0.49 0.8435 8 0.50 0.8459 8 0.51 0.8478 8 0.52 0.8499 10 0.48 0.8421 10 0.49 0.8447 10 0.50 0.8471 10 0.51 0.8488 10 0.52 0.8511 12 0.48 0.8433 12 0.49 0.8459 12 0.50 0.8484 12 0.51 0.8503 12 0.52 0.8522 14 0.48 0.8446 14 0.49 0.8471 14 0.50 0.8495 14 0.51 0.8515 14 0.52 0.8535 16 0.48 0.8457 16 0.49 0.8483 16 0.50 0.8507 16 0.51 0.8527 16 0.52 0.8546 18 0.48 0.8469 157 18 0.49 0.8493 18 0.50 0.8519 18 0.51 0.8538 18 0.52 0.8557 20 0.48 0.8481 20 0.49 0.8506 20 0.50 0.8530 20 0.51 0.8550 20 0.52 0.8570 158 Table B.6: Experimental densities of the binary THN-IBB (T0=25 oC) as function of the mass fraction of the densest component and the pressure. P (MPa) 1  (g.cm-3) 0.1 0.48 0.9013 0.1 0.49 0.9025 0.1 0.50 0.9036 0.1 0.51 0.9050 0.1 0.52 0.9060 2 0.48 0.9026 2 0.49 0.9038 2 0.50 0.9047 2 0.51 0.9062 2 0.52 0.9073 4 0.48 0.9040 4 0.49 0.9051 4 0.50 0.9061 4 0.51 0.9076 4 0.52 0.9088 6 0.48 0.9053 6 0.49 0.9064 6 0.50 0.9075 6 0.51 0.9088 6 0.52 0.9100 8 0.48 0.9066 8 0.49 0.9077 8 0.50 0.9088 8 0.51 0.9101 8 0.52 0.9112 10 0.48 0.9078 10 0.49 0.9090 10 0.50 0.9101 10 0.51 0.9112 10 0.52 0.9124 12 0.48 0.9090 12 0.49 0.9102 12 0.50 0.9113 12 0.51 0.9125 12 0.52 0.9136 14 0.48 0.9102 14 0.49 0.9115 14 0.50 0.9125 14 0.51 0.9137 14 0.52 0.9148 16 0.48 0.9114 16 0.49 0.9126 16 0.50 0.9137 16 0.51 0.9150 16 0.52 0.9160 18 0.48 0.9126 159 18 0.49 0.9137 18 0.50 0.9149 18 0.51 0.9161 18 0.52 0.9171 20 0.48 0.9138 20 0.49 0.9149 20 0.50 0.9161 20 0.51 0.9172 20 0.52 0.9182 160 3 Cp value is obtained from National Institute of standards and Technology (NIST) Appendix C Physical and Thermodynamic Properties of Pure Hydrocarbon Components Table C.1: Pure hydrocarbon components parameters which used in this work Tmean=25 oC, (Daubert and Danner 1989). Pure component cT (K) cP (Pa) M (kg/kmol) ω (-)  (kg.m-3) p C (J.kg-1.K-1) k (W.m-1.K-1) CH4 (C1) 190.58 4.60x106 16.043 0.0108 161.71 2913.293 0.0790 nC4H10 (nC4) 425.18 3.79x106 58.123 0.1993 572.92 2435.07 0.1048 THN (C10H12) 720.15 3.62x106 132.205 0.3811 967.48 1645.41 0.1300 IBB (C10H14) 650.15 3.04x106 134.221 0.3278 849.08 1801.57 0.1220 nC12H26 (nC12) 658.20 1.82x106 170.338 0.5734 745.34 2206.40 0.1368 161
https://openalex.org/W3012037888	https://www.nature.com/articles/srep25429.pdf	English	null	Correction: Corrigendum: Quantum-confinement and Structural Anisotropy result in Electrically-Tunable Dirac Cone in Few-layer Black Phosphorous	Scientific reports	2,016	cc-by	222	www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Corrigendum: Quantum- confinement and Structural Anisotropy result in Electrically- Tunable Dirac Cone in Few-layer Black Phosphorous Kapildeb Dolui & Su Ying Quek Scientific Reports 5:11699; doi: 10.1038/srep11699; published online 01 July 2015; updated 09 May 2016 This Article contains an error in Figure 2: the arrow depicting the electric field should point right to left. The correct Figure 2 appears below as Figure 1. Figure 1. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Figure 1. Figure 1. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Scientific Reports \| 6:25429 \| DOI: 10.1038/srep25429
https://openalex.org/W3135804596	https://re.eltech.ru/jour/article/download/486/527	Russian	null	Study of the Backscatter Radiation Pattern of a Quadrupole Antenna with a High-Impedance Ground Plane of Large Electrical Sizes	Izvestiâ vysših učebnyh zavedenij Rossii. Radioèlektronika	2,021	cc-by	7,685	Исследование диаграммы обратного излучения квадрупольной антенны с высокоимпедансным экраном больших электрических размеров Е. Р. Гафаров, Ю. П. Саломатов Е. Р. Гафаров, Ю. П. Саломатов Сибирский федеральный университет, Красноярск, Россия egafarov@sfu-kras.ru Е. Р. Гафаров, Ю. П. Саломатов Сибирский федеральный университет, Красноярск, Россия egafarov@sfu-kras.ru Сибирский федеральный университет, Красноярск, Россия Сибирский федеральный университет, Красноярск, Россия egafarov@sfu-kras.ru Оригинальная статья Оригинальная статья https://doi.org/10.32603/1993-8985-2021-24-1-6-14 Исследование диаграммы обратного излучения квадрупольной антенны с высокоимпедансным экраном больших электрических размеров Study of the Backscatter Radiation Pattern of a Quadrupole Antenna © Гафаров Е. Р., Саломатов Ю. П., 2021 Контент доступен по лицензии Creative Commons Attribution 4.0 License This work is licensed under a Creative Commons Attribution 4.0 License Электродинамика, микроволновая техника, антенны Электродинамика, микроволновая техника, антенны УДК 621.396.67.012.12 Известия вузов России. Радиоэлектроника. 2021. Т. 24, № 1. С. 6–14 Известия вузов России. Радиоэлектроника. 2021. Т. 24, № 1. С. 6–14 Известия вузов России. Радиоэлектроника. 2021. Т. 24, № 1. С. 6–14 Известия вузов России. Радиоэлектроника. 2021. Т. 24, № 1. С. 6–14 Journal of the Russian Universities. Radioelectronics. 2021, vol. 24, no. 1, pp. 6 Известия вузов России. Радиоэлектроника. 2021. Т. 24, № 1. С. 6–14 Journal of the Russian Universities. Radioelectronics. 2021, vol. 24, no. 1, pp. 6–14 у д р , Journal of the Russian Universities. Radioelectronics. 2021, vol. 24, no. 1, pp. 6–14 Abstract It was established that higher gain slopes can be obtained using different types of ground planes; however, lower backscatter radiation values are achievable only using high-impedance struc- tures. It was observed that the same slope of the radiation pattern (about 1 dB/°) for GNSS lower frequencies can be obtained at different 0 = 12λ , R and, presumably, at 0 20λ . Conclusion. A high-impedance ground plane with a diameter of 0 = 12λ R is preferable for a quadrupole antenna at low GNSS frequencies. A further increase in the ground plane size will insignificantly improve its characteristics. Keywords: GNSS antenna, high impedance ground screen, conventional ground screen, backward radiation pattern rds: GNSS antenna, high impedance ground screen, conventional ground screen, backward radiation patt For citation: Gafarov E. R., Salomatov Yu. P. Study of the Backscatter Radiation Pattern of a Quadrupole Antenna with a High-Impedance Ground Plane of Large Electrical Sizes. Journal of the Russian Universities. Radioelectronics. 2021, vol. 24, no. 1, pp. 6–14. doi: 10.32603/1993-8985-2021-24-1-6-14 For citation: Gafarov E. R., Salomatov Yu. P. Study of the Backscatter Radiation Pattern of a Quadrupole Antenna with a High-Impedance Ground Plane of Large Electrical Sizes. Journal of the Russian Universities. Radioelectronics. 2021, vol. 24, no. 1, pp. 6–14. doi: 10.32603/1993-8985-2021-24-1-6-14 Conflict of interest. The authors declare no conflict of interest. Abstract Introduction. The multipath resistance of GNSS antennas is largely determined by the gain slope of the ampli- tude radiation pattern at sliding angles (angles close to the horizon). The gain slope of the antenna radiation pattern is determined by the size of its ground plane. This article investigates the dependence between the gain slope and ground plane diameter R of a quadrupole antenna. Aim. To analyse the impact of the diameter of conventional and high-impedance ground planes on the backscat- ter radiation pattern of a quadrupole antenna at sliding angles. Materials and methods. Computer simulations were carried out in CAD CST Studio Suite using the methods of finite element analysis (FEM), finite difference time domain (FDTD) and template based post-processing. finite element analysis (FEM), finite difference time domain (FDTD) and template based post-processing. Results. Quadrupole antennas with a capacitive high-impedance ground plane and a conventional flat ground plane were simulated. The dependence of the average gain slope at sliding angles on the diameter of the ground plane was determined at low   fн and upper   fв GNSS frequencies. The analysis of the down/up ratio, the roll- off gain and the multipath ratio for R = 1…20 of the wavelength of capacitive high-impedance and ground planes conventional flat was performed. It was established that higher gain slopes can be obtained using different types of ground planes; however, lower backscatter radiation values are achievable only using high-impedance struc- tures. It was observed that the same slope of the radiation pattern (about 1 dB/°) for GNSS lower frequencies Results. Quadrupole antennas with a capacitive high-impedance ground plane and a conventional flat ground plane were simulated. The dependence of the average gain slope at sliding angles on the diameter of the ground plane was determined at low   fн and upper   fв GNSS frequencies. The analysis of the down/up ratio, the roll- Results. Quadrupole antennas with a capacitive high-impedance ground plane and a conventional flat ground plane were simulated. The dependence of the average gain slope at sliding angles on the diameter of the ground plane was determined at low   fн and upper   fв GNSS frequencies. The analysis of the down/up ratio, the roll- off gain and the multipath ratio for R = 1…20 of the wavelength of capacitive high-impedance and ground planes conventional flat was performed. Аннотация 6–14 Известия вузов России. Радиоэлектроника. 2021. Т. 24, № 1. С. 6–14 Journal of the Russian Universities. Radioelectronics. 2021, vol. 24, no. 1, pp. 6–14 Electrodynamics, Microwave Engineering, Antennas Study of the Backscatter Radiation Pattern of a Quadrupole Antenna with a High-Impedance Ground Plane of Large Electrical Sizes Evgeniy R. Gafarov, Yury P. Salomatov Siberian Federal University, Krasnoyarsk, Russia egafarov@sfu-kras.ru Аннотация ц Введение. Устойчивость антенн глобальных навигационных спутниковых систем (ГНСС) к многолучевой интерференции во многом определяется крутизной амплитудной диаграммы направленности (ДН) в обла- сти скользящих углов (углов, близких к горизонту). Крутизна ДН антенны определяется размером ее экрана. В статье представлено исследование зависимости крутизны амплитудной ДН от диаметра экрана квадру- польной антенны R. ледования. Анализ влияния диаметра обычного и высокоимпедансного экранов на ДН и диа- ратного излучения (ДОИ) квадрупольной антенны, в том числе в области скользящих углов. Цель исследования. Анализ влияния диаметра обычного и высокоимпедансного экранов на ДН и диа- грамму обратного излучения (ДОИ) квадрупольной антенны, в том числе в области скользящих углов. Материалы и методы. Численные исследования проведены в САПР СВЧ (CST Studio Suite) методом конеч- ных элементов (finite element method – FEM) и методом конечных разностей во временной области (FDTD), а также методами постобработки результатов. р р у Результаты. Проведено моделирование квадрупольной антенны с емкостным высокоимпедансным и плоским проводящим экранами. Установлено наличие зависимости средней крутизны ДН на скользящих углах от диаметра экрана на нижней   fн и верхней   fв частотах ГНСС. В ходе исследования выполнен анализ ДН, отношения назад/вперед (down/up или DU), коэффициента усиления в направлении на горизонт (горизонтальное усиление – ГУ) и коэффициента многолучевости (MR) для диаметра R = 1…20 длин волн вы- сокоимпедансного и проводящего экранов. Выявлено, что с целью получения высокой крутизны ДН на скользящих углах возможно применение различных типов экранов, но низкий уровень ДОИ достижим только с применением высокоимпедансной структуры. Показано, что одну и ту же крутизну амплитудной ДН (около 1 дБ/°) для нижних частот (НЧ) ГНСС возможно получить при разных диаметрах экрана 0 = 12λ R и, предположительно, 0 20λ . Заключение. Высокоимпедансный экран решетки вертикальных стержней диаметром 0 = 12λ R является предпочтительным для квадрупольной антенны на НЧ ГНСС. Дальнейшее увеличение экрана может лишь незначительно улучшать его характеристики. Ключевые слова: антенна ГНСС, высокоимпедансный экран, проводящий экран, диаграмма обратного излучения Для цитирования: Гафаров Е. Р., Саломатов Ю. П. Исследование диаграммы обратного излучения квадру- польной антенны с высокоимпедансным экраном больших электрических размеров // Изв. вузов России. Радиоэлектроника. 2021. Т. 24, № 1. С. 614. doi: 10.32603/1993-8985-2021-24-1-6-14 Конфликт интересов. Авторы заявляют об отсутствии конфликта интересов. Конфликт интересов. Авторы заявляют об отсутствии конфликта интересов. Статья поступила в редакцию 17.11.2020; принята к публикации после рецензирования 20.12.2020; опубликована онлайн 25.02.2021 6 Известия вузов России. Радиоэлектроника. 2021. Т. 24, № 1. С. 6–14 Journal of the Russian Universities. Radioelectronics. 2021, vol. 24, no. 1, pp. Submitted 17.11.2020; accepted 20.12.2020; published online 25.02.2021 High-impedance surface formed by a choke ring (a) and a rod array (б). Frequency dependence of reactance of rod array (в) Частотная зависимость импеданса поверхно- сти решетки стержней (рис. 1, в) имеет гиперболи- ческую форму с резонансной частотой с. f Ниж- нюю частоту диапазона ГНСС н 1160 МГц f  вы- бирают вблизи с. f На верхней частоте диапазона в 1610 МГц f  импеданс поверхности уменьша- ется. Для повышения импеданса на вf исполь- зуют высокоимпедансные поверхности с несколь- кими резонансами поверхностного сопротивления [9], но для электрически больших экранов второй резонанс не является обязательным [8]. В [10] по- казано, что высокоимпедансная поверхность в виде решетки стержней обладает меньшей на 30 % крутизной частотной зависимости поверхност- ного сопротивления по сравнению с choke ring и может использоваться на всех частотах ГНСС. Кроме того, известно [11], что при достаточно вы- соком импедансе структуры уровень ДН слабона- правленного источника в направлениях, близких к касательным к экрану, практически не зависит от импеданса и определяется только размером экрана. При этом условии ДОИ перестает зависеть от импеданса экрана для любых углов возвыше- ния, что снижает требования к сопротивлению им- педансной поверхности. ДОИ состоит в использовании обычного плоского проводящего экрана, расположенного за антенной [3, 4]. Однако проводящие экраны могут искажать как фазовую, так и амплитудную ДН, поэтому в ГНСС экраны такого типа не нашли широкого применения. Наибольший интерес представляет анализ высокоимпедансных экранов, уменьшение ДОИ в которых сопровождается повышением ста- бильности фазового центра и увеличением кросс- поляризационной развязки [5]. Важное свойство высокоимпедансных поверхностей состоит в уменьшении амплитуды поверхностных волн бла- годаря формированию искусственного реактив- ного импеданса из проводящих областей. Высокоимпедансные экраны для антенн ГНСС в виде структур гофрированного фланца (choke ring) известны с конца XX в. Наличие канавок структуры choke ring (рис. 1, а) формирует поверх- ность с высоким сопротивлением, показанную штриховой линией. В последнее время вместо choke ring применяется штыревая высокоимпе- дансная структура (рис. 1, б), более простая в из- готовлении [6]. Основы функционирования такого экрана подобны структуре choke ring. Как экран типа choke ring, так и решетка стержней являются емкостными высокоимпеданс- ными поверхностями. Электрические размеры обоих экранов аналогичны. Шаг решетки стерж- ней и расстояние между кольцами структуры choke ring одинаковы и составляют 8; P  вы- сота обеих высокоимпедансных поверхностей равна четверти длины волны. Диаметр стержней много меньше длины волны. Параметры высо- коимпедансных экранов изучены в [7, 8]. Неоднократно отмечалась важность резкого перепада коэффициента усиления (КУ) в области скользящих углов для антенн ГНСС [5, 10]. Submitted 17.11.2020; accepted 20.12.2020; published online 25.02.2021 – по типу: проводящие (ground plane – GP) и вы- сокоимпедансные (high impedance structure – HIS), а также их комбинацию – полупрозрачные экраны; – по типу: проводящие (ground plane – GP) и вы- сокоимпедансные (high impedance structure – HIS), а также их комбинацию – полупрозрачные экраны; Введение. Экран антенны спутниковой радио- навигации является основным элементом, опре- деляющим свойства диаграммы направленности (ДН) под скользящими углами к горизонту и спо- собность антенны к подавлению отражений от рельефа подстилающей поверхности (так называ- емой многолучевой интерференции). Совместно эти факторы определяют точность и устойчи- вость работы системы позиционирования [1, 2]. – по форме: плоские, вогнутые и выпуклые (3D); – по размеру: малые (не превосходящие длины волны излучения λ) и большие (более λ). Свойства антенны в рабочем направлении (в так называемой верхней полусфере) описываются ДН, а в противоположном направлении (в нижней полусфере) – диаграммой обратного излучения (ДОИ). Наиболее простой способ уменьшения Экраны высокоточных антенн глобальных навигационных спутниковых систем (ГНСС) можно классифицировать различным образом: Исследование диаграммы обратного излучения квадрупольной антенны с высокоимпедансным экраном больших электрических размеров Study of the Backscatter Radiation Pattern of a Quadrupole Antenna with a High-Impedance Ground Plane of Large Electrical Sizes Известия вузов России. Радиоэлектроника. 2021. Т. 24, № 1. С. 6–14 Известия вузов России. Радиоэлектроника. 2021. Т. 24, № 1. С. 6–14 у д р , Journal of the Russian Universities. Radioelectronics. 2021, vol. 24, no. 1, pp. 6–14 у р , Journal of the Russian Universities. Radioelectronics. 2021, vol. 24, no. 1, pp. 6–14 Journal of the Russian Universities. Radioelectronics. 2021, vol. 24, no. 1, pp. 6–14 а б в Рис. 1. Высокоимпедансная поверхность, сформированная гофрированным фланцем (а) и решеткой стержней с треугольной сеткой (б). Частотная зависимость реактивного сопротивления решетки стержней (в) Fig. 1. High-impedance surface formed by a choke ring (a) and a rod array (б). Frequency dependence of reactance of rod array (в) Импедансная поверхность 4  P Импедансная поверхность 4  P P P f нf 1160 МГц вf 1610 МГц s ImZ сf Рис. 1. Высокоимпедансная поверхность, сформированная гофрированным фланцем (а) и решеткой стержней с треугольной сеткой (б). Частотная зависимость реактивного сопротивления решетки стержней (в) Fig. 1. High-impedance surface formed by a choke ring (a) and a rod array (б). Frequency dependence of reactance of rod array (в) Рис. 1. Высокоимпедансная поверхность, сформированная гофрированным фланцем (а) и решеткой стержне с треугольной сеткой (б). Частотная зависимость реактивного сопротивления решетки стержней (в) Fig. 1. Известия вузов России. Радиоэлектроника. 2021. Т. 24, № 1. С. 6–14 Journal of the Russian Universities. Radioelectronics. 2021, vol. 24, no. 1, pp. 6–14 ствуют значения угла  90 0 90 .   Подроб- ное описание антенны представлено в [13]. Методы исследования. Численное моделиро- вание проведено при помощи пакета САПР CST Studio Suite [12] методом конечных элементов (finite element method – FEM) и методом конечных разностей во временно́й области (FDTD) с исполь- зованием постобработки результатов моделирова- ния (Template based Post-processing). Квадрупольная антенна имеет широкую ДН, но также и высокий уровень ДОИ. Сохранение широко- угольной ДН на частотах приема навигационных спутников и одновременное уменьшение ДОИ воз- можно с применением высокоимпедансного экрана. Submitted 17.11.2020; accepted 20.12.2020; published online 25.02.2021 По- этому в настоящей статье представлены резуль- таты исследований высокоимпедансных и прово- дящих экранов как малых, так и больших электри- ческих размеров в совокупности со слабонаправ- ленной антенной для оценки ее характеристик направленности. 8 Исследование диаграммы обратного излучения квадрупольной антенны с высокоимпедансным экраном больших электрических размеров Study of the Backscatter Radiation Pattern of a Quadrupole Antenna with a High-Impedance Ground Plane of Large Electrical Sizes Известия вузов России. Радиоэлектроника. 2021. Т. 24, № 1. С. 6–14 Journal of the Russian Universities. Radioelectronics. 2021, vol. 24, no. 1, pp. 6–14 Известия вузов России. Радиоэлектроника. 2021. Т. 24, № 1. С. 6–14 Journal of the Russian Universities. Radioelectronics. 2021, vol. 24, no. 1, pp. 6–14 Результаты исследований. Далее постобработ- кой данных для скользящих углов   80 100   получены вторичные результаты: отношение ДН и ДОИ в нижней полусфере   90 180 90    DU, в направлениях, зеркальных относительно скользящего угла 90 :       θ 180 – θ θ F DU F   и средняя крутизна ДН в области скользящих углов:      100 80 θ 1 20 F F F                 с шагом 1 .  б Рис. 2. Модель квадрупольной антенны (а) и ее диаграмма направленности (б) Fig. 2. Quadrupole antenna model (а) and it's radiation pattern (б) а нf вf 90 120 ,   60 30 0 –2 –4 –6 –8 F, дБ Рис. 3. Квадрупольная антенна с высокоимпедансной поверхностью решетки стержней Fig. 3. Quadrupole antenna with high-impedance surface of rod array Импедансная поверхность 0 R  Квадрупольная антенна б Рис. 2. Модель квадрупольной антенны (а) и ее диаграмма направленности (б) Fig. 2. Quadrupole antenna model (а) and it's radiation pattern (б) а нf вf 90 120 ,   60 30 0 –2 –4 –6 –8 F, дБ б Рис. 2. Модель квадрупольной антенны (а) и ее диаграмма направленности (б) Fig. 2. Quadrupole antenna model (а) and it's radiation pattern (б) а нf вf 90 120 ,   60 30 0 –2 –4 –6 –8 F, дБ а     θ 180 – θ θ F DU F       θ 180 – θ θ F DU F   и средняя крутизна ДН в области скользящих углов:      100 80 θ 1 20 F F F                 б нf вf 90 120 ,   60 30 0 –2 –4 –6 –8 дБ с шагом 1 .  Квадрупольная антенна б Рис. 2. Модель квадрупольной антенны (а) и ее диаграмма направленности (б) Fig. 2. Quadrupole antenna model (а) and it's radiation pattern (б) Рис. 3. Квадрупольная антенна с высокоимпедансной поверхностью решетки стержней Fig. 3. Quadrupole antenna with high-impedance surface of rod array Рис. 3. Квадрупольная антенна с высокоимпедансной поверхностью решетки стержней Fig. 2. Результаты исследований. Анализ поля квадрупольной антенны с экраном большого электрического размера. Проанализиро- вана антенная система (рис. 3), состоящая из слабо- направленной квадрупольной антенны и высокоим- педансного экрана диаметром 0 1...20, R  где 0 0 c f  (c – скорость распространения электро- магнитной энергии в вакууме; 0 1400 MГц f  – средняя частота диапазона ГНСС). Чтобы исклю- чить зависимость поверхностного импеданса от направления тока в плоскости экрана, стержни вы- сокоимпедансной поверхности расположены на треугольной сетке. Проведено сравнение с квадру- польной антенной на плоском проводящем экране того же диаметра. Квадрупольная антенна. Для исследования с двумя типами экранов использована квадрупольная антенна, так как ее ДН является достаточно широ- кой как на нижних, так и на верхних частотах ГНСС. Квадрупольная антенна представляет собой антенные элементы в виде четырех монополей (рис. 2, а), запитываемых делителем мощности. Де- литель обеспечивает равное деление мощности между монополями со сдвигом фаз между ними 90º с целью формирования поля круговой поляризации. Делитель выполнен в двухслойной реализации для уменьшения размеров антенны. Экран двух слоев делителя и монополей является общим. Размеры антенны 96 × 96 × 45 мм. Антенна является всеси- стемной, т. е. работает во всех диапазонах частот ГНСС. Типичные ДН на нижней   н f и верхней   вf частотах ГНСС приведены на рис. 2, б (θ – угол возвышения, верхней полусфере соответ- Численное моделирование антенной системы выполнено в среде CST Studio Suite во временно́й области на прямоугольной сетке (Hexahedral). На первом этапе исследования методом численного моделирования получены ДН. Далее постобработ- кой данных для скользящих углов   80 100   получены вторичные результаты: отношение ДН и ДОИ в нижней полусфере   90 180 90    DU, в направлениях, зеркальных относительно скользящего угла 90 :   Исследование диаграммы обратного излучения квадрупольной антенны с высокоимпедансным экраном больших электрических размеров Study of the Backscatter Radiation Pattern of a Quadrupole Antenna with a High-Impedance Ground Plane of Large Electrical Sizes 9 (θ – угол возвышения, верхней полусфере соответ- первом этапе исследования методом численного моделирования получены ДН. Результаты исследований. Quadrupole antenna model (а) and it's radiation pattern (б) 9 Исследование диаграммы обратного излучения квадрупольной антенны с высокоимпедансным экраном больших электрических размеров Study of the Backscatter Radiation Pattern of a Quadrupole Antenna with a High-Impedance Ground Plane of Large Electrical Sizes Исследование диаграммы обратного излучения квадрупольной антенны с высокоимпедансным экраном больших электрических размеров Study of the Backscatter Radiation Pattern of a Quadrupole Antenna with a High-Impedance Ground Plane of Large Electrical Sizes Известия вузов России. Радиоэлектроника. 2021. Т. 24, № 1. С. 6–14 Journal of the Russian Universities. Radioelectronics. 2021, vol. 24, no. 1, pp. 6–14 Известия вузов России. Радиоэлектроника. 2021. Т. 24, № 1. С. 6–14 Journal of the Russian Universities. Radioelectronics. 2021, vol. 24, no. 1, pp. 6–14 зывает, что при малых размерах экрана   10 R  крутизна быстро возрастает с увеличением раз- мера экрана, а при больших размерах   10 R  изменяется незначительно. Значения на нижней и верхней границах частотного диапазона ГНСС в области малых R  близки, следовательно, запас емкостного импеданса решетки стержней достато- чен во всем диапазоне частот. С увеличением диа- метра экрана первый минимум ДН (рис. 5, б) вна- чале перемещается к значению 100 ,   а затем уменьшается. Этим можно объяснить всплеск за- висимости при значении диаметра около 12 (рис. 4, а), где первый минимум ДН приходится на значение угла 100 .   Из частотной зависимости  θ DU (рис. 6) следует преимущество экрана диаметра 12 на н. f На вf первый минимум ДН Одним из параметров, характеризующих устойчивость антенны ГНСС к многолучевой ин- терференции, является средний коэффициент мно- голучевости (multipath ratio – MR), определенный как отношение среднего КУ в верхней полусфере 0 90 G  к среднему КУ в нижней полусфере 90 180 : G  0 90 90 180 . G MR G      Коэффициент многолучевости также получен в результате постобработки. Исследование проведено на верхней в н f с   и нижней н в f с   граничных часто- тах ГНСС. Характеристики представлены в зави- симости от диаметра экрана R, нормированного на длину волны , R  для верхней   в  и нижней   н  частот диапазона ГНСС. Ввиду симмет- рии ДН квадрупольной антенны характеристики приведены для углов 0 180 .   F, дБ Рис. 5. Результаты исследований. Radiation pattern of the quadrupole antennas: а – HIS в 0 1.15 f f  ; б – HIS н 0 0.83 f f  ; в – GP в 0 1.15 f f  –40 в 0 1.15 f f  а б в 0 30 90 ,   60 120 150 –10 F, дБ –20 –30 –40 0 12 R  12 1 4 HIS н 0 0.83 f f  0 30 90 ,   60 120 150 –10 F, дБ –20 –30 –40 0 20 R  12 1 4 GP в 0 1.15 f f  б Рис. 4. Зависимость средней крутизны ДН от размера экрана для квадрупольной антенны с HIS (а) и GP (б) Fig. 4. Dependence of the radiation pattern average steepness on the size for a quadrupole antenna with the high impedance structure screen (а) and the ground plane screen (б) 0 4 8 12 16 20 0 R  0.1 0.3 0.5 0.7 0.9 , дБ F  в 0 1.15 f f  н 0 0.83 f f  0 4 8 12 16 20 0 R  0.1 0.2 0.3 0.4 0.5 , дБ F  в 0 1.15 f f  н 0 0.83 f f  0.6 а 0 4 8 12 16 20 0 R  0.1 0.3 0.5 0.7 0.9 , дБ F  в 0 1.15 f f  н 0 0.83 f f  а б 0 4 8 12 16 20 0 R  0.1 0.2 0.3 0.4 0.5 , дБ F  в 0 1.15 f f  н 0 0.83 f f  0.6 а Рис. 4. Зависимость средней крутизны ДН от размера экрана для квадрупольной антенны с HIS (а) и GP (б) Fig. 4. Dependence of the radiation pattern average steepness on the size for a quadrupole antenna with the high impedance structure screen (а) and the ground plane screen (б) Рис. 4. Зависимость средней крутизны ДН от размера экрана для квадрупольной антенны с HIS (а) и GP (б) Fig. 4. Dependence of the radiation pattern average steepness on the size for a quadrupole antenna with the high impedance structure screen (а) and the ground plane screen (б) Рис. 4. Зависимость средней крутизны ДН от размера экрана для квадрупольной антенны с HIS (а) и GP (б) Fig. 4. Результаты исследований. ДН квадрупольных антенн: а – HIS в 0 1.15 f f  ; б – HIS н 0 0.83 f f  ; в – GP в 0 1.15 f f  Fig. 5. Radiation pattern of the quadrupole antennas: а – HIS в 0 1.15 f f  ; б – HIS н 0 0.83 f f  ; в – GP в 0 1.15 f f  0 30 90 ,   60 120 150 –10 F, дБ –20 –30 –40 0 20 R  12 1 4 HIS в 0 1.15 f f  а б в 0 30 90 ,   60 120 150 –10 F, дБ –20 –30 –40 0 12 R  12 1 4 HIS н 0 0.83 f f  0 30 90 ,   60 120 150 –10 F, дБ –20 –30 –40 0 20 R  12 1 4 GP в 0 1.15 f f  Обсуждение. Анализ зависимости средней крутизны ДН для скользящих направлений от диа- метра высокоимпедансного экрана (рис. 4, а) пока- Исследование диаграммы обратного излучения квадрупольной антенны с высокоимпедансным экраном больших электрических размеров Study of the Backscatter Radiation Pattern of a Quadrupole Antenna with a High-Impedance Ground Plane of Large Electrical Sizes 10 б Рис. 4. Зависимость средней крутизны ДН от размера экрана для квадрупольной антенны с HIS (а) и GP (б) Fig. 4. Dependence of the radiation pattern average steepness on the size for a quadrupole antenna with the high impedance structure screen (а) and the ground plane screen (б) 0 4 8 12 16 20 0 R  0.1 0.3 0.5 0.7 0.9 , дБ F  в 0 1.15 f f  н 0 0.83 f f  0 4 8 12 16 20 0 R  0.1 0.2 0.3 0.4 0.5 , дБ F  в 0 1.15 f f  н 0 0.83 f f  0.6 а Рис. 5. ДН квадрупольных антенн: а – HIS в 0 1.15 f f  ; б – HIS н 0 0.83 f f  ; в – GP в 0 1.15 f f  Fig. 5. Результаты исследований. Dependence of the radiation pattern average steepness on the size for a quadrupole antenna with the high impedance structure screen (а) and the ground plane screen (б) Исследование диаграммы обратного излучения квадрупольной антенны с высокоимпедансным экраном больших электрических размеров Study of the Backscatter Radiation Pattern of a Quadrupole Antenna with a High-Impedance Ground Plane of Large Electrical Sizes Известия вузов России. Радиоэлектроника. 2021. Т. 24, № 1. С. 6–14 Известия вузов России. Радиоэлектроника. 2021. Т. 24, № 1. С. 6–14 Известия вузов России. Радиоэлектроника. 2021. Т. 24, № 1. С. 6–14 Journal of the Russian Universities. Radioelectronics. 2021, vol. 24, no. 1, pp. 6–14 Journal of the Russian Universities. Radioelectronics. 2021, vol. 24, no. 1, pp. 6–14 DU, дБ 0 15 45 ,   30 60 75 –15 –30 0 1 R  12 20 4 в 0 1.15 f f  Рис. 6. Отношение ДН и ДОИ квадрупольной антенны с HIS: а – н 0 0.83 f f  ; б – в 0 1.15 f f  Fig. 6. Radiation pattern vs backward radiation pattern ratio for the quadrupole antenna with HIS: а – н 0 0.83 f f  ; б – в 0 1.15 f f  DU, дБ 0 15 45 ,   30 60 75 –15 –30 0 1 R  12 20 4 н 0 0.83 f f  а б DU, дБ 0 15 45 ,   30 60 75 –15 –30 0 1 R  12 20 4 н 0 0.83 f f  а а Рис. 6. Отношение ДН и ДОИ квадрупольной антенны с HIS: а – н 0 0.83 f f  ; б – в 0 1.15 f f  Fig. 6. Radiation pattern vs backward radiation pattern ratio for the quadrupole antenna with HIS: а – н 0 0.83 f f  ; б – в 0 1.15 f f  сфере ДН квадрупольной антенны над проводя- щим экраном при 1 R  имеет значительную из- резанность, сформированную противофазными токами кромки экрана (рис. 5, в). Этот факт делает затруднительным применение проводящих экра- нов больших электрических размеров для квадру- польной антенны. Поэтому характеристики усиле- ния при скользящих углах 90 G  и DU для ан- тенны над проводящим экраном не приведены. Результаты исследований. В отличие от квадрупольной антенны применение плоского проводящего экрана большого электри- ческого размера для патч-антенн возможно вслед- ствие малых значений 90 G  [14, 15]. (рис. 5, а) при 20 R  также приходится на зна- чение угла 100 .   Причем в этом случае наблю- даются наилучшие DU и крутизна ДН. Важно отметить фактическое отсутствие воз- действия высокоимпедансного экрана на ДН квад- рупольной антенны вплоть до угла 70 .   Для скользящих углов вид ДН значительно изменяется. Как видно из зависимостей коэффициента усиле- ния при скользящих углах 90 G  от диаметра высокоимпедансного экрана (рис. 7), ход кривых существенно зависит от диаметра и при 4 5 R  составляет менее –20 дБ как на верх- ней, так и на нижней частотах диапазона ГНСС. Крутизна ДН квадрупольной антенны над про- водящим экраном (рис. 5, б) на н f и вf изменя- ется аналогично антенне с высокоимпедансным экраном. Однако в последнем случае значения крутизны ДН при 20 R  почти двукратно пре- вышают значения для антенны с обычным прово- дящим экраном. Кроме того, за пределами сколь- зящих углов как в нижней, так и верхней полу- Для квадрупольной антенны с проводящим экраном увеличение R  не сопровождается зна- чительным изменением MR (рис. 8). В случае вы- сокоимпедансного экрана это отношение быстро возрастает и максимально в области значений 8, R  затем имеет место небольшой спад из-за роста первого бокового лепестка ДН (рис. 5). Рис. 7. Коэффициент усиления квадрупольной антенны с HIS на скользящих углах 0 4 8 12 16 R  –10 –14 –18 –22 90 , дБ G  в 0 1.15 f f  н 0 0.83 f f  Исследование диаграммы обратного излучения квадрупольной антенны с высокоимпедансным экраном больших электрических размеров Study of the Backscatter Radiation Pattern of a Quadrupole Antenna with a High-Impedance Ground Plane of Large Electrical Sizes 11 Рис. 7. Коэффициент усиления квадрупольной антенны с HIS на скользящих углах Fig. 7. The gain of the quadrupole antenna with HIS at sliding angles 0 4 8 12 16 R  –10 –14 –18 –22 90 , дБ G  в 0 1.15 f f  н 0 0.83 f f  Рис. 8. Средний коэффициент многолучевости исследуемых антенн Fig. 8. Результаты исследований. Average multipath of the antennas under study 0 4 8 12 16 0 2R  5 10 15 20 , дБ MR 25 HIS GP в 0 1.15 f f   в 0 0.83 f f   Рис. 8. Средний коэффициент многолучевости 0 4 8 12 16 0 2R  5 10 15 20 , дБ MR 25 HIS GP в 0 1.15 f f   в 0 0.83 f f   н 0 0.83 f f  Рис. 7. Коэффициент усиления квадрупольной антенны с HIS на скользящих углах 90 , д  Рис. 7. Коэффициент усиления квадрупольной антенны с HIS на скользящих углах 90  Рис. 8. Средний коэффициент многолучевости исследуемых антенн Рис. 8. Средний коэффициент многолучевости исследуемых антенн Fig. 7. The gain of the quadrupole antenna with HIS at sliding angles Fig. 8. Average multipath of the antennas under study 11 Исследование диаграммы обратного излучения квадрупольной антенн с высокоимпедансным экраном больших электрических размеров Study of the Backscatter Radiation Pattern of a Quadrupole Antenna with a High-Impedance Ground Plane of Large Electrical Sizes Известия вузов России. Радиоэлектроника. 2021. Т. 24, № 1. С. 6–14 Journal of the Russian Universities. Radioelectronics. 2021, vol. 24, no. 1, pp. 6–14 чам позиционирования ограничены, так как значи- тельное сужение ДН приводит к опасности срыва слежения за сигналами пригоризонтных спутни- ков. В этой связи с целью увеличения коэффици- ента усиления при скользящих углах с сохране- нием диаметра экрана, а значит и крутизны ДН в этой области возможно использование трехмер- ного (3D) экрана со спадом образующей высо- коимпедансной поверхности. В общем случае при- менение высокоимпедансного экрана диаметром более 12 R  возможно, если отсутствует габа- ритное ограничение. Заключение. Приведенные результаты иссле- дования показывают, что высокоимпедансный экран диаметром 12 R  является предпочти- тельным для квадрупольной антенны. Дальнейшее увеличение экрана лишь незначительно улучшает ее характеристики в области скользящих углов. Тем не менее, как отмечено в [16], подавление от- раженного сигнала из нижней полусферы при вы- соких углах к плоскости экрана прямо пропорцио- нально уровню ДН источника в направлении на экран. Возможности улучшения ДОИ за счет управления шириной ДН для приложений к зада- Список литературы 10. Tatarnikov D. V. Topcon Full Wave GNSS Refer- ence Station Antenna with Convex Impedance Ground Plane. URL: https://insidegnss.com/wp-content/up- loads/2011/10/Topcon_PN-A5_antenna_white_paper.pdf (дата обращения 14.10.2020) 1. Татарников Д. В. Экраны антенн высокоточной геодезии по сигналам глобальных навигационных спутниковых систем. Ч. 1: Идеально проводящие и им- педансные экраны // Антенны. 2008. № 4 (131). С. 6–19. 2. Mitigation of Multipath through the Use of an Arti- ficial Magnetic Conductor for Precision GPS Surveying An- tennas / W. E. McKinzie, R. B. Hurtado, B. K. Klimczak, J. D. Dutton // IEEE Ant. and Prop. Intern. Symp. San Antonio, USA, 16–21 June 2002. Proc. Vol. 4. Piscataway: IEEE, 2002. P. 640–643. doi: 10.1109/APS.2002.1017065 11. Periodic Structures, High Impedance and Semitrans- parent Surfaces in Antennas for Centimeter and Millimeter Precision of Positioning with the Global Navigation Satellite Systems / D. V. Tatarnikov, A. V. Astakhov, A. P. Stepanenko, A. A. Generalov // J. of Physics: Conf. Series. 2018. Vol. 1092. Ac. № 012150. 4 p. doi: 10.1088/1742-6596/1092/1/012150. 3. Multiband Three Layer GNSS Microstrip Antenna / E. R. Gafarov, A. A. Erokhin, A. V. Stankovsky, Y. P. Sa- lomatov // Intern. Siberian Conf. on Control and Com- munications (SIBCON). Tomsk, Russia, 18–20 Apr. 2019. Piscataway: IEEE, 2019. Ac. № 18756269. 4 p. doi: 10.1109/SIBCON.2019.8729623 12. CST STUDIO SUITE 2020 – моделирование трех- мерных электромагнитных структур / ООО "Евроинтех". Люберцы, Моск. обл. URL: http://www.eu- rointech.ru/eda /microwave_design/cst/CST-STUDIO- SUITE.phtml (дата обращения 09.02.2021) 13. Gafarov E. R., Erokhin A. A., Salomatov Y. P. Broad- band Four-Fed Circularly Polarized Quadrupole Antenna // Intern. Siberian Conf. on Control and Communications (SIBCON). Astana, Kazakhstan, June 29–30, 2017. Pisca- taway: IEEE, 2017. Ac. № 17082594. doi: 10.1109/SIBCON. 2017.7998468 4. Multipath Mitigation by Conventional Antennas with Ground Planes and Passive Vertical Structures / D. V. Tatar- nikov, V. Fillipov, I. Soutiaguine, A. Astakhov, A. Stepanenko, P. Shamatulsky // GPS Solutions. 2005. Vol. 9, iss. 3. P. 194–201. 5. Springer Handbook of Global Navigation Satellite Sys- tems / ed. by P. J. G. Teunissen, O. Montenbruck. Berlin: Springer, 2016. 1206 p. doi: 10.1007/978-3-319-42928-1_4. 14. On Site Validation in GNSS Stations for Multipath and Interference Mitigation Structure / A. Dessantis, L. Dicecca, L. D'Agristina, V. Crino, P. Gabellini, P. Russo, F. A. Fernandez, A. Castro, A. Quiles, R. Weiler // 7th ESA Workshop on Satellite Navigation Technologies and European Workshop on GNSS Signals and Signal Processing., Noordwijk, Netherlands, 3–5 Dec. 2014. Piscataway: IEEE, 2014. Ac. 1. Татарников Д. В. Экраны антенн высокоточной геодезии по сигналам глобальных навигационных спутниковых систем. Ч. 1: Идеально проводящие и им- педансные экраны // Антенны. 2008. № 4 (131). С. 6–19. 1. Татарников Д. В. Экраны антенн высокоточной геодезии по сигналам глобальных навигационных спутниковых систем. Ч. 1: Идеально проводящие и им- педансные экраны // Антенны. 2008. № 4 (131). С. 6–19. 2. Mitigation of Multipath through the Use of an Arti- ficial Magnetic Conductor for Precision GPS Surveying An- tennas / W. E. McKinzie, R. B. Hurtado, B. K. Klimczak, J. D. Dutton // IEEE Ant. and Prop. Intern. Symp. San Antonio, USA, 16–21 June 2002. Proc. Vol. 4. Piscataway: IEEE, 2002. P. 640–643. doi: 10.1109/APS.2002.1017065 3. Multiband Three Layer GNSS Microstrip Antenna / E. R. Gafarov, A. A. Erokhin, A. V. Stankovsky, Y. P. Sa- lomatov // Intern. Siberian Conf. on Control and Com- munications (SIBCON). Tomsk, Russia, 18–20 Apr. 2019. Piscataway: IEEE, 2019. Ac. № 18756269. 4 p. doi: 10.1109/SIBCON.2019.8729623 4. Multipath Mitigation by Conventional Antennas with Ground Planes and Passive Vertical Structures / D. V. Tatar- nikov, V. Fillipov, I. Soutiaguine, A. Astakhov, A. Stepanenko, P. Shamatulsky // GPS Solutions. 2005. Vol. 9, iss. 3. P. 194–201. 5. Springer Handbook of Global Navigation Satellite Sys- tems / ed. by P. J. G. Teunissen, O. Montenbruck. Berlin: Springer, 2016. 1206 p. doi: 10.1007/978-3-319-42928-1_4. 6. Leick A., Rapoport L., Tatarnikov D. GPS Satellite Surveying. 4th ed. New York: Wiley, 2015. 836 p. 7. Tatarnikov D. V, Astakhov A. V. Large Impedance Ground Plane Antennas for mm-accuracy of GNSS Position- ing in Real Time // Progress in Electromagnetics Research Symp. Stockholm, Sweden, Aug. 12–15, 2013. Proc. P. 1825–1829. 8. Sievenpiper D. F. High Impedance Electromagnetic Исследование диаграммы обратного излучения квадрупольной антенны с высокоимпедансным экраном больших электрических размеров Study of the Backscatter Radiation Pattern of a Quadrupole Antenna with a High-Impedance Ground Plane of Large Electrical Sizes Список литературы С. 6–14 Journal of the Russian Universities. Radioelectronics. 2021, vol. 24, no. 1, pp. 6–14 Journal of the Russian Universities. Radioelectronics. 2021, vol. 24, no. 1, pp. 6–14 Информация об авторах Гафаров Евгений Раисович – магистр по направлению "Радиотехника" (2009), аспирант, старший пре- подаватель кафедры радиотехники Сибирского федерального университета. Автор 30 научных работ. Сфера научных интересов – антенны и СВЧ-устройства; антенны ГНСС и антенные решетки. Адрес: Сибирский федеральный университет, пр. Свободный, д. 79, Красноярск, 660041, Россия E-mail: egafarov@sfu-kras.ru https://orcid.org/0000-0002-7810-7196 Сибирский федеральный университет, пр. Свободный, д. 79, Красноярск, 660041, Россия egafarov@sfu-kras.ru Саломатов Юрий Петрович – кандидат технических наук (1982), профессор (2013) кафедры радиотех- ники Сибирского федерального университета. Автор 240 научных работ. Сфера научных интересов – ФАР; ЦФАР; квазиоптические антенны и антенные решетки. Ц р Адрес: Сибирский федеральный университет, пр. Свободный, д. 79, Красноярск, 660041, Россия E-mail: ysalomatov@sfu-kras.ru https://orcid.org/0000-0003-4309-226X Список литературы № 14947811 doi: 10.1109/NAVITEC.2014.7045137 14. On Site Validation in GNSS Stations for Multipath and Interference Mitigation Structure / A. Dessantis, L. Dicecca, L. D'Agristina, V. Crino, P. Gabellini, P. Russo, F. A. Fernandez, A. Castro, A. Quiles, R. Weiler // 7th ESA Workshop on Satellite Navigation Technologies and European Workshop on GNSS Signals and Signal Processing., Noordwijk, Netherlands, 3–5 Dec. 2014. Piscataway: IEEE, 2014. Ac. № 14947811 doi: 10.1109/NAVITEC.2014.7045137 6. Leick A., Rapoport L., Tatarnikov D. GPS Satellite Surveying. 4th ed. New York: Wiley, 2015. 836 p. 7. Tatarnikov D. V, Astakhov A. V. Large Impedance Ground Plane Antennas for mm-accuracy of GNSS Position- ing in Real Time // Progress in Electromagnetics Research Symp. Stockholm, Sweden, Aug. 12–15, 2013. Proc. P. 1825–1829. 15. Broadband Capacitively Coupled Stacked Patch Antenna for GNSS Applications / D. Li, P. Guo, Q. Dai, Y. Fu / IEEE Antennas and Wireless Propagation Lett. 2012. Vol. 11. P. 701–704. doi: 10.1109/LAWP.2012.2205129 8. Sievenpiper D. F. High Impedance Electromagnetic Surfaces: Ph. D. dissertation / University of California. Los Angeles, 1999. 150 p. 15. Broadband Capacitively Coupled Stacked Patch Antenna for GNSS Applications / D. Li, P. Guo, Q. Dai, Y. Fu / IEEE Antennas and Wireless Propagation Lett. 2012. Vol. 11. P. 701–704. doi: 10.1109/LAWP.2012.2205129 9. Modifications to GPS Reference Station Antennas to Reduce Multipath / A. Kerkhoff, R. B. Harris, C. P. Pe- tersen, A. Pickard // Proc. of the 23d Intern. Technical Meeting of the Satellite Division of the Institute of Naviga- tion. Sept. Portland, USA, 21–24, 2010. P. 866–878. 9. Modifications to GPS Reference Station Antennas to Reduce Multipath / A. Kerkhoff, R. B. Harris, C. P. Pe- tersen, A. Pickard // Proc. of the 23d Intern. Technical Meeting of the Satellite Division of the Institute of Naviga- tion. Sept. Portland, USA, 21–24, 2010. P. 866–878. 16. Tatarnikov D. V. Semi-Transparent Ground Planes excited by Magnetic Line Current // IEEE Anten. and Propag. 2012. Vol. 60, iss. 6. P. 2843–2852. doi: 10.1109/TAP.2012.2194636 12 следование диаграммы обратного излучения квадрупольной антенны с высокоимпедансным экраном больших электрических размеров Study of the Backscatter Radiation Pattern of a Quadrupole Antenna with a High-Impedance Ground Plane of Large Electrical Sizes Известия вузов России. Радиоэлектроника. 2021. Т. 24, № 1. С. 6–14 J l f th R i U i iti R di l t i 2021 l 24 1 6 14 Известия вузов России. Радиоэлектроника. 2021. Т. 24, № 1. References 10. Tatarnikov D. V. Topcon Full Wave GNSS Refer- ence Station Antenna with Convex Impedance Ground Plane. Available at: https://insidegnss.com/wp-content/up- loads/2011/10/Topcon_PN-A5_antenna_white_paper.pdf (accessed 14.10.2020) 1. Tatarnikov D. V. Antenna ground planes for high- precision positioning with the global navigation satellites signals. P. 1. Ideal conducting and impedance ground planes. Antennas. 2008, no. 4 (131), pp. 6–19. (In Russ.) 10. Tatarnikov D. V. Topcon Full Wave GNSS Refer- ence Station Antenna with Convex Impedance Ground Plane. Available at: https://insidegnss.com/wp-content/up- loads/2011/10/Topcon_PN-A5_antenna_white_paper.pdf (accessed 14.10.2020) 2. McKinzie W. E., Hurtado R. B., Klimczak B. K., Dut- ton J. D. Mitigation of Multipath through the Use of an Ar- tificial Magnetic Conductor for Precision GPS Surveying Antennas. IEEE Ant. and Prop. Intern. Symp. 16–21 June 2002, San Antonio, USA. Proc. Vol. 4. Piscataway: IEEE, 2002. P. 640–643. doi: 10.1109/APS.2002.1017065 2. McKinzie W. E., Hurtado R. B., Klimczak B. K., Dut- ton J. D. Mitigation of Multipath through the Use of an Ar- tificial Magnetic Conductor for Precision GPS Surveying Antennas. IEEE Ant. and Prop. Intern. Symp. 16–21 June 2002, San Antonio, USA. Proc. Vol. 4. Piscataway: IEEE, 2002. P. 640–643. doi: 10.1109/APS.2002.1017065 11. Tatarnikov D. V., Astakhov A. V., Stepanenko A. P., Generalov A. A. Periodic Structures, High Impedance and Semitransparent Surfaces in Antennas for Centimeter and Millimeter Precision of Positioning with the Global Navigation Satellite Systems. J. of Physics: Conf. Series. 2018, vol. 1092, ac. no. 012150, 4 p. doi: 10.1088/1742- 6596/1092/1/012150 3. Gafarov E. R., Erokhin A. A., Stankovsky A. V., Salo- matov Y. P. Multiband Three Layer GNSS Microstrip An- tenna. Intern. Siberian Conf. on Control and Communi- cations (SIBCON). 18–20 April 2019, Tomsk, Russia. Pis- cataway: IEEE, 2019, Ac. no. 18756269, 4 p. doi: 10.1109/SIBCON.2019.8729623 12. CST MICROWAVE STUDIO 2016. Work flow & Solver Overview. CST MICROWAVE STUDIO, 2016, 124 p. Available at: https://manualzz.com /doc/28231181/cst- microwave-studio (accessed 2021/09/02) 13. Gafarov E. R., Erokhin A. A., Salomatov Y. P. Broad- band Four-Fed Circularly Polarized Quadrupole Antenna. Intern. Siberian Conf. on Control and Communications (SIBCON). June 29–30, 2017, Astana, Kazakhstan. Pisca- taway, IEEE, 2017, ac. no. 17082594. doi: 10.1109/SIBCON. 2017.7998468 4. Tatarnikov D. V., Fillipov V., Soutiaguine I., Asta- khov A., Stepanenko A., Shamatulsky P. Multipath Mitigation by Conventional Antennas with Ground Planes and Passive Vertical Structures. GPS Solutions. 2005, vol. 9, iss. 3, pp. 194–201. 5. Springer Handbook of Global Navigation Satellite Systems. Ed. by P. J. G. Teunissen, O. Montenbruck. References С. 6–14 Известия вузов России. Радиоэлектроника. 2021. Т. 24, № 1. С. 6–14 у р , Journal of the Russian Universities. Radioelectronics. 2021, vol. 24, no. 1, pp. 6–14 References Berlin, Springer, 2016, 1206 p. doi: 10.1007/978-3-319-42928-1_4. 5. Springer Handbook of Global Navigation Satellite Systems. Ed. by P. J. G. Teunissen, O. Montenbruck. Berlin, Springer, 2016, 1206 p. doi: 10.1007/978-3-319-42928-1_4. 14. Dessantis A., Dicecca L., D'Agristina L., Crino V., Gabellini P., Russo P., Fernandez F. A., Castro A., Quiles A., Weiler R. On Site Validation in GNSS Stations for Multipath and Interference Mitigation Structure. 7th ESA Workshop on Satellite Navigation Technologies and European Work- shop on GNSS Signals and Signal Processing. 3–5 Dec. 2014, Noordwijk, Netherlands. Piscataway, IEEE, 2014, ac. no. 14947811. doi: 10.1109/NAVITEC.2014.7045137 6. Leick A., Rapoport L., Tatarnikov D. GPS Satellite Surveying. 4th ed. New York, Wiley, 2015, 836 p. 6. Leick A., Rapoport L., Tatarnikov D. GPS Satellite Surveying. 4th ed. New York, Wiley, 2015, 836 p. 7. Tatarnikov D. V, Astakhov A. V. Large Impedance Ground Plane Antennas for mm-accuracy of GNSS Position- ing in Real Time. Progress in Electromagnetics Research Symp. Aug. 12–15, 2013, Stockholm, Sweden. Proc., pp. 1825–1829. 15. Li D., Guo P., Dai Q., Fu Y. Broadband Capacitively Coupled Stacked Patch Antenna for GNSS Applications. IEEE Antennas and Wireless Propagation Lett. 2012, vol. 11, pp. 701–704. doi: 10.1109/LAWP.2012.2205129 8. Sievenpiper D. F. High Impedance Electromagnetic Surfaces. Ph. D. dissertation, University of California. Los Angeles, 1999, 150 p. 8. Sievenpiper D. F. High Impedance Electromagnetic Surfaces. Ph. D. dissertation, University of California. Los Angeles, 1999, 150 p. 16. Tatarnikov D. V. Semi-Transparent Ground Planes excited by Magnetic Line Current. IEEE Anten. and Propag. 2012, vol. 60, iss. 6, pp. 2843–2852. doi: 10.1109/TAP.2012.2194636 16. Tatarnikov D. V. Semi-Transparent Ground Planes excited by Magnetic Line Current. IEEE Anten. and Propag. 2012, vol. 60, iss. 6, pp. 2843–2852. doi: 10.1109/TAP.2012.2194636 9. Kerkhoff A., Harris R. B., Petersen C. P., Pickard A. Modifications to GPS Reference Station Antennas to Re- duce Multipath. Proc. of the 23d Intern. Technical Meeting of the Satellite Division of the Institute of Navigation. Sep- tember 21–24, 2010. Portland, USA, pp. 866–878. 13 Исследование диаграммы обратного излучения квадрупольной антенны с высокоимпедансным экраном больших электрических размеров Study of the Backscatter Radiation Pattern of a Quadrupole Antenna with a High-Impedance Ground Plane of Large Electrical Sizes Исследование диаграммы обратного излучения квадрупольной антенны с высокоимпедансным экраном больших электрических размеров Study of the Backscatter Radiation Pattern of a Quadrupole Antenna with a High-Impedance Ground Plane of Large Electrical Sizes Известия вузов России. Радиоэлектроника. 2021. Т. 24, № 1. Information about the authors Evgeniy R. Gafarov, Master’s degree in Radio Engineering (2009), postgraduate, senior lecturer of Radio Engi- neering Department of the Siberian Federal University (SFU). The author of 30 scientific publications. Area of exper- tise: antennas and microwave devices; GNSS antennas and antenna arrays. Evgeniy R. Gafarov, Master’s degree in Radio Engineering (2009), postgraduate, senior lecturer of Radio Engi- neering Department of the Siberian Federal University (SFU). The author of 30 scientific publications. Area of exper- tise: antennas and microwave devices; GNSS antennas and antenna arrays. Address: Siberian Federal University, 79 Svobodny Ave., Krasnoyarsk 660041, Russia E il f @ f k Address: Siberian Federal University, 79 Svobodny Ave., Krasnoyarsk 660041, Russia E-mail: egafarov@sfu-kras.ru g @ https://orcid.org/0000-0002-7810-7196 Yury P. Salomatov, Cand. Sci. (Eng.) (1982), Professor (2013) of Department of Radio Engineering of the Si- berian Federal University. The author of 240 scientific publications. Area of expertise: phased arrays; digital phased arrays; quasi-optical antennas and antenna arrays. Yury P. Salomatov, Cand. Sci. (Eng.) (1982), Professor (2013) of Department of Radio Engineering of the Si berian Federal University. The author of 240 scientific publications. Area of expertise: phased arrays; digital phased arrays; quasi-optical antennas and antenna arrays. Address: Siberian Federal University, 79 Svobodny Ave., Krasnoyarsk 660041, Russia E il l t @ f k y q p y Address: Siberian Federal University, 79 Svobodny Ave., Krasnoyarsk 660041, Russia E-mail: ysalomatov@sfu-kras.ru https://orcid org/0000 0003 4309 226X Address: Siberian Federal University, 79 Svobodny Ave., Krasnoyarsk 660041, Russia E-mail: ysalomatov@sfu-kras.ru Address: Siberian Federal University, 79 Svobodny Ave., Krasnoyarsk 660041, Russia E-mail: ysalomatov@sfu-kras.ru Исследование диаграммы обратного излучения квадрупольной антенны с высокоимпедансным экраном больших электрических размеров Study of the Backscatter Radiation Pattern of a Quadrupole Antenna with a High-Impedance Ground Plane of Large Electrical Sizes 14 Исследование диаграммы обратного излучения квадрупольной антенны с высокоимпедансным экраном больших электрических размеров Study of the Backscatter Radiation Pattern of a Quadrupole Antenna with a High-Impedance Ground Plane of Large Electrical Sizes
https://openalex.org/W2935281165	https://europepmc.org/articles/pmc6480218?pdf=render	English	null	Use of a Single Wireless IMU for the Segmentation and Automatic Analysis of Activities Performed in the 3-m Timed Up & Go Test	Sensors	2,019	cc-by	11,711	Received: 15 February 2019; Accepted: 19 March 2019; Published: 6 April 2019 Abstract: Falls represent a major public health problem in the elderly population. The Timed Up & Go test (TU & Go) is the most used tool to measure this risk of falling, which offers a unique parameter in seconds that represents the dynamic balance. However, it is not determined in which activity the subject presents greater difﬁculties. For this, a feature-based segmentation method using a single wireless Inertial Measurement Unit (IMU) is proposed in order to analyze data of the inertial sensors to provide a complete report on risks of falls. Twenty-ﬁve young subjects and 12 older adults were measured to validate the method proposed with an IMU in the back and with video recording. The measurement system showed similar data compared to the conventional test video recorded, with a Pearson correlation coefﬁcient of 0.9884 and a mean error of 0.17 ± 0.13 s for young subjects, as well as a correlation coefﬁcient of 0.9878 and a mean error of 0.2 ± 0.22 s for older adults. Our methodology allows for identifying all the TU & Go sub–tasks with a single IMU automatically providing information about variables such as: duration of sub–tasks, standing and sitting accelerations, rotation velocity of turning, number of steps during walking and turns, and the inclination degrees of the trunk during standing and sitting. Keywords: timed up & go test; activity segmentation; inertial sensors sensors sensors sensors Sensors 2019, 19, 1647; doi:10.3390/s19071647 Use of a Single Wireless IMU for the Segmentation and Automatic Analysis of Activities Performed in the 3-m Timed Up & Go Test Paulina Ortega-Bastidas 1, Pablo Aqueveque 2,* , Britam Gómez 2, Francisco Saavedra 2 and Roberto Cano-de-la-Cuerda 3 Paulina Ortega-Bastidas 1, Pablo Aqueveque 2,* , Britam Gómez 2, Francisco Saavedra 2 and Roberto Cano-de-la-Cuerda 3 Paulina Ortega-Bastidas 1, Pablo Aqueveque 2,* , Britam Gómez 2, Francisco Saavedra 2 and Roberto Cano-de-la-Cuerda 3 1 Kinesiology Department, Faculty of Medicine, Universidad de Concepción, 4030000 Concepcion, Chile; portegab@udec.cl 1 Kinesiology Department, Faculty of Medicine, Universidad de Concepción, 4030000 Concepcion, Chile portegab@udec.cl 2 Electrical Engineering Department, Faculty of Engineering, Universidad de Concepción, 219 Edmundo Larenas St., 4030000 Concepción, Chile; britam.gomez@biomedica.udec.cl (B.G.); frsaaved@gmail.com (F.S.) 3 Physiotherapy, Occupational Therapy, Rehabilitation and Physical Medicine Department, Universidad Rey Juan Carlos, 28922 Madrid, Spain; roberto.cano@urjc.es * Correspondence: pablo.aqueveque@biomedica.udec.cl; Tel.: +56-41-2661262 * Correspondence: pablo.aqueveque@biomedica.udec.cl; Tel.: +56-41-2661262 1. Introduction The number of people over 60 years of age is rapidly increasing worldwide. The main reasons for this demographic change are the increase in life expectancy and the fall in the birth rate [1]. This has become a public health problem, since aging is generally associated with a decrease in physical and psychological capacity, as well as an increase in the risk of disability, dependence and a number of comorbidities [1,2]. One of the consequences generated by aging is the increased risk of falls, which have been deﬁned as accidental events in which the person falls after losing control of the center of gravity and no effort is made to restore the balance, or it is inefﬁcient [3]. Falls represent a major public health problem in the elderly population. Approximately one third of the population over 65 years old has experienced at least one fall per year. In addition, this frequency increases by 50% in individuals older than 85 years. Between 20% and 30% of falls result in an injury that requires medical attention, constituting the leading cause of death or non-fatal Sensors 2019, 19, 1647; doi:10.3390/s19071647 www.mdpi.com/journal/sensors www.mdpi.com/journal/sensors Sensors 2019, 19, 1647 2 of 22 injury in older adults [4–6]. Therefore, the early detection of the decreased function of the elderly is important to initiate precociously the preventive measures that allow for maintaining their functional independence [7]. Normally, rehabilitation professionals perform evaluations through observation, as well as through the application of scales or assessment instruments, which provide a certain level of objectivity to the evaluations. There is a large number of tests and scales that allow for evaluating and assessing the static balance, dynamic balance and gait of healthy subjects or with motor problems [8], the Timed Up & Go test (TU & Go) being the most used worldwide. This test measures the dynamic balance and functional mobility in older adults, as well as in the neurological population [9–11]. The TU & Go is a simple test that can be performed anywhere, and consists of the subject getting up from a chair from the sitting to the bipedal position, walking three meters, turn, returning and sitting on the chair again, as illustrated in Figure 1. The variable measured is the total time taken by the test and then the score assigned in seconds is observed, which is correlated with the risk of falls [9,12]. Figure 1. 1. Introduction Activities performed in the 3-m TU & Go test. 1 = standing, 2 = ﬁrst walk, 3 = 3-m turning, 4 = second walk, 5 = pre-sitting turning, and 6 = sitting. Figure 1. Activities performed in the 3-m TU & Go test. 1 = standing, 2 = ﬁrst walk, 3 = 3-m turning, 4 = second walk, 5 = pre-sitting turning, and 6 = sitting. Some of the advantages of the TU & Go test is the simplicity in its application and its short duration. In addition, it requires little equipment and allows the possibility that people with a functional impairment can perform the test. However, one of the limitations is that the TU & Go, although it provides relevant information about the risk of falls, is not capable of determining subjects with greater difﬁculties objectively. Barry et al. [13] mentioned that a limitation in the predictive value of the test could be explained by the fact that it is a unique test that evaluates the overall balance and mobility, which can be improved by combining it with technological tools for the movement analysis, such as optoelectronic laboratories with passive reﬂective markers, considered the gold standard instrument for analysis of human movement, or several alternatives, like wireless motion sensors such as inertial sensors or inertial measurement units (IMU). Optoelectronic laboratories, despite providing accurate measurements, are expensive and their application takes a long time, since training and experience is required to interpret the results. In addition, in several countries, there are rural or remote locations with no resources for these advanced technological evaluations systems. In recent years, a variety of evidence has been observed regarding the development of different devices that use inertial sensors, applications and/or smartphones as a low-cost alternative to optoelectronic systems, which have allowed for speciﬁcally visualizing the phases in which subjects could present greater problems with the consequent probability to fall during the application of the TU Sensors 2019, 19, 1647 3 of 22 & Go test [14,15]. Different authors [7,8,11] indicate that the phases of the TU & Go test correspond to: the transition from sitting position to standing, walking towards the turning mark, turning, walking back to the chair, turning and the sit back. This has been shown to increase the predictive value of the test in relation to the risk of falls and the phases in which subjects present greater difﬁculties. 1. Introduction Thus, it has been demonstrated that it is possible to implement an automatic segmentation of these phases or activities through feature-based algorithms [11,16], complex algorithms based on machine learning [7,17] or by principal components analysis (PCA) [18]—as techniques based in Wavelet decomposition [19]—using inertial sensors. Feature-based algorithms have the advantage of being simple to implement, but their performance is diminished due to the great variability that exists in the morphology of the signals they use to perform the segmentation—angular velocity and acceleration—those that depend on the environment and the execution time of the activities to be identiﬁed. Algorithms based on machine learning have a good response to the great variability of the characteristics present in the signals to be processed, but they are complex in their implementation—like those that use principal components analysis—and, to guarantee the above, they depend on a large amount of data where the mentioned variabilities are presented. One way to reduce the disadvantages of feature-based methods is using the orientation data from Inertial Measurement Units (IMU) [14,20]. This orientation angles have been used in different parts of the body for the segmentation of activities during the execution of the test, since they have low variability in their inter-subject characteristics, allowing for extracting characteristics of each phase independently [21,22]. In relation to the above, this study presents an automatic segmentation methodology that uses a feature-based algorithm to identify the typical sub-tasks carried out during a three-meter TU & Go test in two groups of subjects (young and older adults) using the orientation angles of a single wireless IMU on the back (L3–L4, approximately) to analyze independently the data of the inertial sensors, in order to provide a complete report on the risk of falls and promote the use of low–cost technological objective elements and simple use in hospitals or rehabilitation centers of rural or remote locations. 2.1. Design and Setting 2.1. Design and Setting A study with a descriptive design is presented, in which experimental tests were performed to analyze the segmentation of the TU & Go test stages, contrasted with the measurements obtained using a wireless IMU on the lower–back. A total of 25 healthy young subjects (18 men and 7 women) between 25 and 33 years old, and 12 elderly subjects (7 men and 5 women) between 59 and 93 years old were recruited in the city of Concepcion, Chile. Exclusion criteria were the diagnosis of a neurological, vestibular, musculoskeletal or systemic disease that could alter the ability to walk; the diagnosis of any cardiovascular, respiratory or metabolic disease or other conditions that could interfere with the present study; having undergone surgery on the trunk and lower limbs at least two years prior to the present study, the use of assistive devices for walking and the presence of serious visual alterations that could alter the gait pattern. The measurements were carried out at the Biomedical Engineering laboratory and in the facilities of the Kinesiology department of the Universidad de Concepción. Prior to the measurements, the test was explained to the participants and two trials without data collection were performed to check the understanding of each participant. The authorization was requested through informed consent, which was approved by the Biosecurity, Bioethical and Ethical Committee of the University of Concepción (Number 3180551). 2.2. IMU Sensor IMU sensor develop in the biomedical engineering laboratory of the Universidad de Concepción, Chile. Figure 2. IMU sensor develop in the biomedical engineering laboratory of the Universidad de Concepción, Chile. 2.2. IMU Sensor A homemade IMU—developed at the laboratory of Biomedical Engineering of the Universidad de Concepción—was used. Sensors 2019, 19, 1647 4 of 22 The chip sensor utilized [23] has a three-axis accelerometer, a three-axis gyroscope and a three-axis magnetometer (see Table 1), as well as an embedded internal processor able to fuse the magnetic and the inertial data using an extended Kalman ﬁlter to accurately deliver the orientation in quaternions, to avoid singularities presents in the Euler and Navigation representations. Then, the orientation data is obtained in angle representation with an accuracy of ±1 deg. The data is sampled at 100 Hz with a low cost 32–bit microcontroller with an Advance RISC (reduce instruction set computer) Machine (ARM) Cortex-M0+ processor and sent to a software application via Bluetooth 3.0 up to a maximum distance of 20 m without risk of occlusion. The entire system is powered by a 500-mAh LiPo battery, which gives 10-h of autonomy. Table 1. Characteristics of the inertial and magnetic sensors of the IMU used. Sensor Axis Range Bandwidth Resolution Output Rate Accelerometer XYZ ±16 G 62.5 Hz 14 bits (≈1.95 mG) 100 Hz Gyroscope XYZ ±2000 dps 32 Hz 16 bits (≈0.061 dps) 100 Hz Magnetometer XY ±1300 [µT] 10 Hz 13 bits (≈317 [ηT]) 20 Hz - Z ±2500 [µT] 10 Hz 15 bits (≈152 [ηT]) 20 Hz Figure 2 shows the implemented sensor and its disposition on the subjects. Figure 2. IMU sensor develop in the biomedical engineering laboratory of the Universidad de Concepción, Chile. Table 1. Characteristics of the inertial and magnetic sensors of the IMU used. Sensor Axis Range Bandwidth Resolution Output Rate Accelerometer XYZ ±16 G 62.5 Hz 14 bits (≈1.95 mG) 100 Hz Gyroscope XYZ ±2000 dps 32 Hz 16 bits (≈0.061 dps) 100 Hz Magnetometer XY ±1300 [µT] 10 Hz 13 bits (≈317 [ηT]) 20 Hz - Z ±2500 [µT] 10 Hz 15 bits (≈152 [ηT]) 20 Hz Fi 2 h th i l t d d it di iti th bj t Table 1. Characteristics of the inertial and magnetic sensors of the IMU used. Figure 2 shows the implemented sensor and its disposition on the subjects. Figure 2 shows the implemented sensor and its disposition on the subjects. g e s o s e p e e e se so s spos o o e s jec s Figure 2. 2.4. Segmentation Algorithm For the segmentation of standing, walking, turning and sitting activities, an algorithm was designed. This algorithm processed inclination (Pitch) and rotation (Yaw) signals of an inertial sensor placed on the back of a subject at L3–L4, approximately. 2.3. Test Procedure The developed sensor is positioned on the back at the height of L3–L4 for young and elderly subjects. It has been shown that the use of a single IMU in that position allows for the detection of all gait events, biomechanical elements of the pelvis and other spatial and temporal kinematics factor [24–26]. The TU & Go test was performed following the recommendations of [22]. Three meters away from the chair, a cone was used to mark the location where the patients had to make the turn. Before carrying out the tests, the procedure was explained with a demonstration, to resolve and clarify doubts. Each participant was recorded at 60 fps using a GOPRO HERO 7 high resolution digital video camera (GoPro, Inc., San Mateo, CA, USA) using lineal FOV (Field of View) mode to reduce the image distortion. Three TU & Go test repetitions were performed by the older adults group, using for the analysis the performance of the higher time of the test carried out. For the young group, only one test was carried out. Figure 3 shows the set up used for the measurements. 5 of 22 Sensors 2019, 19, 1647 Figure 3. Set up used for the acquisition data during 3-m TU & Go test. Figure 3. Set up used for the acquisition data during 3-m TU & Go test. 2.4.1. Standing/Sitting Events Identiﬁcation To determine the events of standing and sitting, the Pitch signal was used. This corresponds to the inclination actions during the activities to be identiﬁed. When a subject is standing or sitting, he tends to make a slight forward inclination with respect to the resting position until it regains its initial inclination (see Figure 4), which can even be observed in subjects with reduced mobility [27]. (a) Standing (b) Sitting (c) Inclination angle during standing and sitting Figure 4. Identiﬁcation of Standing/Sitting events using Pitch. (b) Sitting (a) Standing (c) Inclination angle during standing and sitting Figure 4. Identiﬁcation of Standing/Sitting events using Pitch. Figure 4. Identiﬁcation of Standing/Sitting events using Pitch. Sensors 2019, 19, 1647 6 of 22 In order to condition the Pitch signal, this is smoothed by an average ﬁlter of order N = 5 (see Equation (1)) and normalized by the absolute maximum of the smooth signal, as seen in Equation (2): In order to condition the Pitch signal, this is smoothed by an average ﬁlter of order N = 5 (see Equation (1)) and normalized by the absolute maximum of the smooth signal, as seen in Equation (2): Pitchsmooth(n) = 1 N [Pitch(n) + Pitch(n + 1) + ... + Pitch(n + N −1)], (1) (1) Pitchnorm(n) = Pitchsmooth(n) max(\|Pitchsmooth\|). (2) (2) Then, the Pitch signal is processed using a local maximum detector, ﬁnding Tpeak1 and Tpeak2, that corresponds to the maximum angle of inclination in the standing and sitting events, respectively (see Figure 5). Then, the Pitch signal is processed using a local maximum detector, ﬁnding Tpeak1 and Tpeak2, that corresponds to the maximum angle of inclination in the standing and sitting events, respectively ( Fi 5) Figure 5. Peaks identiﬁed for the search of start and end of standing and sitting actions. Figure 5. Peaks identiﬁed for the search of start and end of standing and sitting actions. Then, the difference between samples to the left of Tpeak1 is calculated, as indicated in Equation (3), and to the right of Tpeak1, as indicated in Equation (4), to start the search for the standing action, were i is a sample iterator. 2.4.1. Standing/Sitting Events Identiﬁcation Thus, the start and end of standing action are obtained using Equations (5) and (6), respectively, with a factor of 0.05 to determine the threshold slope stop, which was found experimentally: ∆1 = \|Pitchnorm(Tpeak1 −i −0.1) −Pitchnorm(Tpeak1 −i)\| < 0.05, i = 0, 0.1, 0.2, . . . , (3) ∆2 = \|Pitchnorm(Tpeak1 + i + 0.1) −Pitchnorm(Tpeak1 + i)\| < 0.05, i = 0, 0.1, 0.2, . . . , (4) standingi = Tpeak1 −i, (5) standingf = Tpeak1 + i. (6) (3) (5) (6) Similarly, through the same methodology, the sitting action is sought using the difference between samples to the left of Tpeak2, as indicated in Equation (7), and to the right of Tpeak2, as indicated in Equation (8). Then, the start and end of standing action are obtained using the Equations (9) and (10), respectively: ∆3 = \|Pitchnorm(Tpeak2 −i −0.1) −Pitchnorm(Tpeak2 −i)\| < 0.05, i = 0, 0.1, 0.2, ..., (7) ∆4 = \|Pitchnorm(Tpeak2 + i + 0.1) −Pitchnorm(Tpeak2 + i)\| < 0.05, i = 0, 0.1, 0.2, ..., (8) ∆3 = \|Pitchnorm(Tpeak2 −i −0.1) −Pitchnorm(Tpeak2 −i)\| < 0.05, i = 0, 0.1, 0.2, ..., (7) ∆4 = \|Pitchnorm(Tpeak2 + i + 0.1) −Pitchnorm(Tpeak2 + i)\| < 0.05, i = 0, 0.1, 0.2, ..., (8) sittingi = Tpeak2 −i, (9) sittingf = Tpeak2 + i. (10) (7) (8) (9) (10) Figure 6 shows the ﬁnal result of the search method and Figure 7 shows the proposed standing/sitting activities identiﬁcation method. Sensors 2019, 19, 1647 7 of 22 7 of 22 Figure 6. Result of the proposed method for the search of the beginning (standingi) and the end (standingf ) of the Standing event and for the search of the beginning (sittingi) and the end (sittingf ) of the Sitting event. Figure 6. Result of the proposed method for the search of the beginning (standingi) and the end Figure 6. Result of the proposed method for the search of the beginning (standingi) and the end (standingf ) of the Standing event and for the search of the beginning (sittingi) and the end (sittingf ) of the Sitting event. Figure 6. Result of the proposed method for the search of the beginning (standingi) and the end (standingf ) of the Standing event and for the search of the beginning (sittingi) and the end (sittingf ) of the Sitting event. Figure 7. 2.4.1. Standing/Sitting Events Identiﬁcation Automatic feature-based segmentation algorithm for the standing and sitting activities in the 3–m TU & Go test. Figure 7. Automatic feature-based segmentation algorithm for the standing and sitting activities in the 3–m TU & Go test. 2.4.2. Turning Events Identiﬁcation To determine the turns around the 3-m mark and prior to sitting, Yaw is used, which corresponds to changes in the orientation of the sensor during turns. When a subject performs the TU & Go test, it is subjected to a circuit that forces it to make turns of 180 degrees approximately, which can be unequivocally measured by the sensor in the back, since, during turns, the sensor also changes its orientation next to it (see Figure 8). 8 of 22 Sensors 2019, 19, 1647 (a) Turning (b) Rotation angles during turning Figure 8 Turning events identiﬁcation using Yaw (a) Turning Figure 8. Turning events identiﬁcation using Yaw. Figure 8. Turning events identiﬁcation using Yaw. Figure 8. Turning events identiﬁcation using Yaw. To condition the orientation change signal, as for the inclination signal, this is smoothed by an average ﬁlter of order N = 5 (see Equation (11)) and normalized by the absolute maximum of the signal, as seen in Equation (12): Yawsmooth(n) = 1 N [Yaw(n) + Yaw(n + 1) + ... + Yaw(n + N −1)], (11) Yawnorm(n) = Yawsmooth(n) max(\|Yawsmooth\|). (12) Yawsmooth(n) = 1 N [Yaw(n) + Yaw(n + 1) + ... + Yaw(n + N −1)], (11) (11) Yawnorm(n) = Yawsmooth(n) max(\|Yawsmooth\|). (12) (12) Then, the rotation signal is derived and processed to identify the maximum value max(d/dt) and the minimum value min(d/dt), which are useful to identify the start and end points of the ﬁrst turn and the second turn, respectively, as seen in Figure 9. Figure 9. Maximum and minimum of the rotation signal derived to start the search for turning events. Thus, through a sliding window of 0.1 s or 10 samples (Equations (13) and (14)), the start of the 3-m tuning is searched by calculating the mean of the samples in the window to the left of max(d/dt) and the end of the 3-m tuning calculating the mean of the samples in the window to the right of max(d/dt) using Equations (15) and (16), respectively: Figure 9. Maximum and minimum of the rotation signal derived to start the search for turning events. Figure 9. Maximum and minimum of the rotation signal derived to start the search for turning events. 2.4.2. Turning Events Identiﬁcation Thus, through a sliding window of 0.1 s or 10 samples (Equations (13) and (14)), the start of the 3-m tuning is searched by calculating the mean of the samples in the window to the left of max(d/dt) and the end of the 3-m tuning calculating the mean of the samples in the window to the right of max(d/dt) using Equations (15) and (16), respectively: Thus, through a sliding window of 0.1 s or 10 samples (Equations (13) and (14)), the start of the 3-m tuning is searched by calculating the mean of the samples in the window to the left of max(d/dt) and the end of the 3-m tuning calculating the mean of the samples in the window to the right of max(d/dt) using Equations (15) and (16), respectively: Sensors 2019, 19, 1647 Sensors 2019, 19, 1647 W1 = [Tmax(d/dt) −10 −i, Tmax(d/dt) −i], (13) W2 = [Tmax(d/dt) + i, Tmax(d/dt) + 10 + i], (14) W1 > 0.02, i = 0, 0.01, 0.02, 0.03, ..., (15) W2 < 0.9, i = 0, 0.01, 0.02, 0.03, ... (16) (13) (15) W2 < 0.9, i = 0, 0.01, 0.02, 0.03, ... (16) (16) Finally, the start and the end of the 3-m turning are obtained through Equations (17) and (18), respectively: Finally, the start and the end of the 3-m turning are obtained through Equations (17) and (18), respectively: ( ) (17) (18) turn 1i = min(W1), (17) turn 1f = max(W2). (18) (17) (17) (18) Then, using a sliding window of 0.1 s or 10 samples (Equations (19) and (20)), the start of the pre-sitting tuning is sought by calculating the mean of the samples in the window to the left of min(d/dt) and the end of the pre-sitting tuning calculating the mean of the samples in the window to the right of min(d/dt) using Equations (21) and (22), respectively: W3 = [Tmin(d/dt) −10 −i, Tmin(d/dt) −i], (19) W4 = [Tmin(d/dt) + i, Tmin(d/dt) + 10 + i], (20) W3 < 0.9, i = 0, 0.01, 0.02, 0.03, ..., (21) W4 > 0.02, i = 0, 0.01, 0.02, 0.03, ... 2.4.2. Turning Events Identiﬁcation (22) (19) (20) (21) (22) Similarly, the start and the end of the pre-sitting turning are obtained through Equations (23) and (24), respectively: Similarly, the start and the end of the pre-sitting turning are obtained through Equations (23) and (24), respectively: turn 2i = min(W3), (23) turn 2f = max(W4). (24) (23) (24) Figure 10 shows the ﬁnal result of the search method and Figure 11 indicates the proposed turning activities’ identiﬁcation method. Figure 10. Result of the proposed method for the search of the beginning (turn1i) and the end (turn1f ) of the 3-m turning event and for the search of the beginning (turn2i) and the end (turn2f ) of the pre-sitting event. Figure 10. Result of the proposed method for the search of the beginning (turn1i) and the end (turn1f ) of the 3-m turning event and for the search of the beginning (turn2i) and the end (turn2f ) of the pre-sitting event. 10 of 22 Sensors 2019, 19, 1647 Figure 11. Automatic feature-based segmentation algorithm for the turning actions in the 3-m TU & Go test. Figure 11. Automatic feature-based segmentation algorithm for the turning actions in the 3-m TU & Go test. 3. Results The data were analyzed using MATLAB R2017b (The MathWorks, Inc., Natick, MA, USA) to obtain the results presented in this section. The video recording was analyzed using the Wondershare Filmora version 8.4.0 video editor (Wondershare Software Ltd., Shenzhen, China). 3.1. IMU Measurements Validation versus the Standard Clinical Procedure Results from the measurements obtained by the proposed methodology versus the typical visual clinical procedure in young subjects. (a) Pearson correlation between IMU and the video record analysis; (b) Bland–Altman plot between IMU and the video record analysis. The measurement system with the proposed segmentation algorithm and methodology could Table 2. Results from the measurements obtained by the proposed methodology versus the typical visual clinical procedure in young subjects. Subject Height [cm] Weight [kg] Age [years] Gender Total Video Time s Total IMU Time s Subject 1 179 93 25 male 10.46 10.26 Subject 2 168 70 25 male 10.73 10.56 Subject 3 176 81 28 male 10.66 10.52 Subject 4 164 68 26 female 8.60 8.24 Subject 5 160 68 25 female 8.63 8.43 Subject 6 162 70 29 male 8.00 7.92 Subject 7 168 80 26 female 10.40 9.83 Subject 8 152 48 25 female 10.16 9.29 Subject 9 195 90 26 female 10.30 10.01 Subject 10 190 80 29 male 10.66 10.63 Subject 11 169 68 29 male 10.60 10.62 Subject 12 153 77 27 male 10.60 10.61 Subject 13 160 73 33 female 10.93 10.76 Subject 14 172 81 31 female 10.26 10.09 Subject 15 168 70 26 male 10.50 10.38 Subject 16 173 79 28 male 10.46 10.40 Subject 17 170 73 25 male 10.50 10.36 Subject 18 163 60 32 male 8.86 8.83 Subject 19 175 70 28 male 8.83 8.72 Subject 20 171 68 33 male 9.06 8.98 Subject 21 165 70 32 male 8.36 8.18 Subject 22 179 73 26 male 10.10 9.94 Subject 23 185 79 26 male 9.16 8.99 Subject 24 190 83 30 male 10.33 10.33 Subject 25 160 58 29 male 10.30 9.99 (a) (b) Figure 12. Results from the measurements obtained by the proposed methodology versus the typical visual clinical procedure in young subjects. (a) Pearson correlation between IMU and the video record analysis; (b) Bland–Altman plot between IMU and the video record analysis. ect Height [cm] Weight [kg] Age [years] Gender Total Video Time s Total IMU Time s (b) (a) (a) (b) Figure 12. Results from the measurements obtained by the proposed methodology versus the typical visual clinical procedure in young subjects. (a) Pearson correlation between IMU and the video record analysis; (b) Bland–Altman plot between IMU and the video record analysis. 3.1. IMU Measurements Validation versus the Standard Clinical Procedure To evaluate the performance of the proposed methodology in relation to the typical visual clinical procedure, the total time from each TU & Go test captured in videos for young population was tabulated obtained as the average of the times observed frame by frame by two different evaluators to compensate the bias introduced by the subjectivity present in the real practice. This was used to observe the measurements error and the Pearson correlation coefﬁcient with respect to the typical clinical application to assess the strength of the association between the total time from each TU & Go test captured by video and the IMU measurements. The results are tabulated in Table 2 and shown in Figure 12. 11 of 22 Sensors 2019, 19, 1647 Table 2. Results from the measurements obtained by the proposed methodology versus the typical visual clinical procedure in young subjects. Table 2. Results from the measurements obtained by the proposed methodology versus the typical visual clinical procedure in young subjects. Subject Height [cm] Weight [kg] Age [years] Gender Total Video Time s Total IMU Time s Subject 1 179 93 25 male 10.46 10.26 Subject 2 168 70 25 male 10.73 10.56 Subject 3 176 81 28 male 10.66 10.52 Subject 4 164 68 26 female 8.60 8.24 Subject 5 160 68 25 female 8.63 8.43 Subject 6 162 70 29 male 8.00 7.92 Subject 7 168 80 26 female 10.40 9.83 Subject 8 152 48 25 female 10.16 9.29 Subject 9 195 90 26 female 10.30 10.01 Subject 10 190 80 29 male 10.66 10.63 Subject 11 169 68 29 male 10.60 10.62 Subject 12 153 77 27 male 10.60 10.61 Subject 13 160 73 33 female 10.93 10.76 Subject 14 172 81 31 female 10.26 10.09 Subject 15 168 70 26 male 10.50 10.38 Subject 16 173 79 28 male 10.46 10.40 Subject 17 170 73 25 male 10.50 10.36 Subject 18 163 60 32 male 8.86 8.83 Subject 19 175 70 28 male 8.83 8.72 Subject 20 171 68 33 male 9.06 8.98 Subject 21 165 70 32 male 8.36 8.18 Subject 22 179 73 26 male 10.10 9.94 Subject 23 185 79 26 male 9.16 8.99 Subject 24 190 83 30 male 10.33 10.33 Subject 25 160 58 29 male 10.30 9.99 (a) (b) Figure 12. 3.1. IMU Measurements Validation versus the Standard Clinical Procedure The measurement system with the proposed segmentation algorithm and methodology could deliver data similar to those obtained by the observational clinical application of the TU & Go test measured in the videos for young population (see Figure 12). Here, a Pearson correlation coefﬁcient of 0.9884 (see Figure 12a) and a mean error of 0.17 ± 0.13 s was observed, as shown in Figure 12b. In addition, the proposed methodology in the older adults was used to evaluate the performance in the target population of this procedure. In addition, two different evaluators analyzed the videos of the tests carried out by each subject independently, the results of Table 3 and Figure 13 being obtained. In addition, the proposed methodology in the older adults was used to evaluate the performance in the target population of this procedure. In addition, two different evaluators analyzed the videos of the tests carried out by each subject independently, the results of Table 3 and Figure 13 being obtained. 12 of 22 Sensors 2019, 19, 1647 Table 3. Results from the measurements obtained by the proposed methodology versus the typical visual clinical procedure in the older adults group, where rof = risk of falling, high = high risk of falling, low = low risk of falling and no = no risk of falling. The classiﬁcation of the risk of falling of the subjects was obtained by following the manual of the ministry of health (MINSAL) for the Chilean population [28]. Subject Height [cm] Weight [kg] Age [years] Gender Total Video Time s (rof) Total IMU Time s (rof) Subject 1 168 75 60 male 11.00 (low) 10.51 (low) Subject 2 168 72 60 male 9.63 (no) 8.98 (no) Subject 3 156 90 63 male 10.73 (low) 10.57 (low) Subject 4 170 68 65 male 9.76 (no) 9.59 (no) Subject 5 179 64 71 male 12.00 (low) 11.95 (low) Subject 6 157 62 60 female 13.76 (low) 13.13 (low) Subject 7 178 93 67 male 9.36 (no) 9.27 (no) Subject 8 160 51 63 female 9.03 (no) 8.87 (no) Subject 9 145 70 59 female 10.43 (low) 9.92 (no) Subject 10 173 84 59 male 10.60 (low) 10.23 (low) Subject 11 160 68 59 female 9.33 (low) 9.44 (low) Subject 12 157 61 93 female 43 (high) 42.99 (high) (a) (b) Figure 13. 3.1. IMU Measurements Validation versus the Standard Clinical Procedure Results from the measurements obtained by the proposed methodology versus the typical visual clinical procedure in the older adults group. (a) Pearson correlation between IMU and the video record analysis without the high risk of falling subject; (b) Bland–Altman plot between IMU and the video record analysis. t Height [cm] Weight [kg] Age [years] Gender Total Video Time s (rof) Total IMU Time s (rof) (a) (b) (a) (b) Figure 13. Results from the measurements obtained by the proposed methodology versus the typical visual clinical procedure in the older adults group. (a) Pearson correlation between IMU and the video record analysis without the high risk of falling subject; (b) Bland–Altman plot between IMU and the video record analysis. The measurement system with the proposed methodology has a similar performance to that obtained for healthy young subjects, with a Pearson correlation coefﬁcient of 0.9878 (see Figure 13a) and a mean error of 0.20 ± 0.22 s as shown in Figure 13b. Moreover, the proposed methodology was capable of correctly classifying the 92% of the subjects measured according to their risk of falling, taking into account the total time of the test assessed by the IMU, including subjects with a high risk of falling (see subject 12 of Table 3). 3.2. Activities Segmentation Analysis of the TU & Go Test To evaluate the performance of the proposed segmentation algorithm, the recording videos of the tests of each subject were analyzed and the time of each stage was tabulated to compare it with the times obtained when processing the inclination data (Pitch) and the sensor rotation data (Yaw). Figure 14 shows the correlation for the activity segmentation time for the young subjects, showing that the minimum correlation is obtained in the identiﬁcation of the transition between the end of the standing and the start of the ﬁrst walk with a Pearson correlation coefﬁcient of 0.8138 (see Figure 14a). The best correlation occurs in the identiﬁcation times of the transition between the end of the 3-m turning and the start of the second walk with a Pearson correlation coefﬁcient of 0.9854 (see Figure 14d). 13 of 22 Sensors 2019, 19, 1647 The above indicates that the measurement system has a high degree of agreement with respect to a visual segmentation. (a) (b) (c) (d) (e) (f) (g) Figure 14. Pearson correlation coefﬁcient for the segmentation time for each sub task of the TU & Go test in young subjects. (a) end of the standing/start of the ﬁrst walk; (b) end of the ﬁrst walk/start of the 3-m turn; (c) end of the 3-m turn/start of the second walk; (d) end of the second walk/start of the pre-sitting turn; (e) end of the pre-sitting turn; (f) start of the sitting; (g) end of the sitting. (b) (c) (a) (b) (a) (c) (d) (e) (f) (d) (f) (e) (g) (g) Figure 14. Pearson correlation coefﬁcient for the segmentation time for each sub task of the TU & Go test in young subjects. (a) end of the standing/start of the ﬁrst walk; (b) end of the ﬁrst walk/start of the 3-m turn; (c) end of the 3-m turn/start of the second walk; (d) end of the second walk/start of the pre-sitting turn; (e) end of the pre-sitting turn; (f) start of the sitting; (g) end of the sitting. Figure 14. Pearson correlation coefﬁcient for the segmentation time for each sub task of the TU & Go test in young subjects. 3.2. Activities Segmentation Analysis of the TU & Go Test (a) end of the standing/start of the ﬁrst walk; (b) end of the ﬁrst walk/start of the 3-m turn; (c) end of the 3-m turn/start of the second walk; (d) end of the second walk/start of the pre-sitting turn; (e) end of the pre-sitting turn; (f) start of the sitting; (g) end of the sitting. Figure 15 shows the measurement errors between the proposed segmentation methodology for each sub-task during the TU & Go test in the young subjects compared to the video analysis. In Figure 15a, it is observed that the segmentation algorithm is capable of identifying the transition between the end of the standing and the start of the ﬁrst walk with an average error of −0.02 s. Figure 15b shows that the transition between the end of the ﬁrst walk and the start of the 3-m turning is identiﬁed with an average error of 0.36 s. Figure 15c shows that the transition between the end of the 3-m turning and the start of the second walk is identiﬁed with an average error of 0.11 s. Figure 15d shows that the transition between the end of the second walk and the start of the pre-sitting turn is identiﬁed with an average error of 0.25 s. The sub-task of the end of the pre-sitting turning is identiﬁed with an average error of 0.16 s (see Figure 15e). Regarding the sitting sub-task, the algorithm identiﬁes the start with an average error of 0.18 s (see Figure 15f) and the end with an average error of 0.24 s (see Figure 15g). 14 of 22 14 of 22 Sensors 2019, 19, 1647 (a) (b) (c) (d) (e) (f) (g) Figure 15. Error plots for the segmentation time for each sub task of the TU & Go test in young subjects. The segmented lines correspond to the mean error. (a) end of the standing/start of the ﬁrst walk; (b) end of the ﬁrst walk/start of the 3-m turn; (c) end of the 3-m turn/start of the second walk; (d) end of the second walk/start of the pre-sitting turn; (e) end of the pre-sitting turn; (f) start of the sitting; (g) end of the sitting. (b) (a) (c) (b) (c) (a) (d) (f) (e) (d) (f) (e) (g) (g) Figure 15. Error plots for the segmentation time for each sub task of the TU & Go test in young subjects. 3.2. Activities Segmentation Analysis of the TU & Go Test The segmented lines correspond to the mean error. (a) end of the standing/start of the ﬁrst walk; (b) end of the ﬁrst walk/start of the 3-m turn; (c) end of the 3-m turn/start of the second walk; (d) end of the second walk/start of the pre-sitting turn; (e) end of the pre-sitting turn; (f) start of the sitting; (g) end of the sitting. Figure 15. Error plots for the segmentation time for each sub task of the TU & Go test in young subjects. The segmented lines correspond to the mean error. (a) end of the standing/start of the ﬁrst walk; (b) end of the ﬁrst walk/start of the 3-m turn; (c) end of the 3-m turn/start of the second walk; (d) end of the second walk/start of the pre-sitting turn; (e) end of the pre-sitting turn; (f) start of the sitting; (g) end of the sitting. Figure 16 shows the measurement errors between the proposed segmentation methodology for each sub-task during the TU & Go test in the older adults group compared to the video analysis. In Figure 16a, it is observed that the segmentation algorithm is capable of identifying the transition between the end of the standing and the start of the ﬁrst walk with an average error of 0.07 s. Figure 16b shows that the transition between the end of the ﬁrst walk and the start of the 3-m turning is identiﬁed with an average error of 0.29 s. Figure 16c shows that the transition between the end of the 3-m turning and the start of the second walk is identiﬁed with an average error of 0.43 s. Figure 16d shows that the transition between the end of the second walk and the start of the pre-sitting turn is identiﬁed with an average error of 0.63 s. The sub-task of the end of the pre-sitting turning is identiﬁed with an average error of 0.21 s (see Figure 16e). Regarding the sitting sub-task, the algorithm identiﬁes the start with an average error of 0.25 s (see Figure 16f) and the end with an average error of 0.26 s (see Figure 16g). 15 of 22 15 of 22 Sensors 2019, 19, 1647 (a) (b) (c) (d) (e) (f) (g) Figure 16. Error plots for the segmentation time for each sub task of the TU & Go test in the older adults group. 3.2. Activities Segmentation Analysis of the TU & Go Test The segmented lines correspond to the mean error. (a) end of the standing/start of the ﬁrst walk; (b) end of the ﬁrst walk/start of the 3-m turn; (c) end of the 3-m turn/start of the second walk; (d) end of the second walk/start of the pre-sitting turn; (e) end of the pre-sitting turn; (f) start of the sitting; (g) end of the sitting. (b) (a) (c) (b) (a) (d) (e) (f) (f) (d) (e) (g) (g) Figure 16. Error plots for the segmentation time for each sub task of the TU & Go test in the older adults group. The segmented lines correspond to the mean error. (a) end of the standing/start of the ﬁrst walk; (b) end of the ﬁrst walk/start of the 3-m turn; (c) end of the 3-m turn/start of the second walk; (d) end of the second walk/start of the pre-sitting turn; (e) end of the pre-sitting turn; (f) start of the sitting; (g) end of the sitting. Figure 16. Error plots for the segmentation time for each sub task of the TU & Go test in the older adults group. The segmented lines correspond to the mean error. (a) end of the standing/start of the ﬁrst walk; (b) end of the ﬁrst walk/start of the 3-m turn; (c) end of the 3-m turn/start of the second walk; (d) end of the second walk/start of the pre-sitting turn; (e) end of the pre-sitting turn; (f) start of the sitting; (g) end of the sitting. 3.3. Characterization of the Signals Acquired from the Stages of the TU & Go Test After the segmentation, each action performed by the subjects is characterized independently, and the following variables are obtained: duration of each sub-task, standing acceleration (Acc. Su), sitting acceleration (Acc. Sd), rotation velocity of the 3-m turning (Vel. T1), rotation velocity of the pre-sitting turning (Vel. T2), number of steps during the ﬁrst walk (W1), number of steps during the second walk (W2), number of steps during the 3-m turning (T1), number of steps during the pre-sitting turning (T2), inclination degrees of the trunk during standing (Pitch Su) and sitting (Pitch Sd) as shown in Figure 17. The time duration of the sub-tasks performed by the young subjects are shown in Figure 18 and in Table 4. In Table 5, the parameters extracted from the signals of angular velocity and acceleration of the inertial sensors of the IMU are observed. Figure 18a illustrates that the standing times obtained by the segmentation algorithm have a distribution close to the values observed by the video recordings, with an average error of −0.08 ± 0.15 s. Figure 18b presents one of the most different distributions in the times obtained for the ﬁrst walk with an average error of 0.42 ± 0.20 s. Figure 18c presents a less dispersed distribution for the 3-m turning times than the tabulated ones from the video recording, estimated with an error of −0.19 ± 0.21 s. Regarding to the second walk times (see Figure 18d) and sitting times (see Figure 18f), an estimation close to the tabulated values are observed from the video analysis, with an error of Sensors 2019, 19, 1647 16 of 22 16 of 22 0.17 ± 0.11 s and −0.01 ± 0.19 s, respectively. Finally, the pre-sitting turning times are estimated with an error of −0.08 ± 0.11 s, despite the slight difference in the distributions observed in Figure 18e. Figure 17. Characteristics measured after the segmentation of the activities carried out in the 3-m TU & Go test. The measured characteristics correspond to the signals of vertical acceleration (AccZ) and sagittal angular velocity (GyrY). Figure 17. Characteristics measured after the segmentation of the activities carried out in the 3-m TU & Go test. The measured characteristics correspond to the signals of vertical acceleration (AccZ) and sagittal angular velocity (GyrY). (a) (b) (c) (d) (e) (f) Figure 18. 3.3. Characterization of the Signals Acquired from the Stages of the TU & Go Test Average time duration and error from the IMU activities segmentation algorithm compared to the video analysis of the young subjects. Table 4. Average time duration and error from the IMU activities segmentation algorithm compared to the video analysis of the young subjects. Standing First Walk 3-m Turning Second Walk Pre-Sitting Turning Sitting Video duration s 1.16 ± 0.15 2.51 ± 0.35 1.65 ± 0.25 2.39 ± 0.36 1.03 ± 0.14 1.48 ± 0.17 IMU duration s 1.24 ± 0.07 2.10 ± 0.45 1.85 ± 0.16 2.22 ± 0.37 1.10 ± 0.06 1.48 ± 0.15 IMU error estimation s −0.08 ± 0.15 0.42 ± 0.20 −0.19 ± 0.21 0.17 ± 0.11 −0.08 ± 0.11 −0.01 ± 0.19 Table 5. Characteristics obtained after the implemented segmentation of the activities performed by the young subjects during the TU & Go test. Acc. Su [m/s2] Acc. Sd [m/s2] Vel. T1 [deg/s] Vel. T2 [deg/s] Steps W1 Steps W2 Steps T1 Steps T2 Pitch Su [deg] Pitch Sd [deg] Minimum 3.9102 4.459 126.3125 181.6875 2 3 2 1 2.2979 3.236 Maximum 9.1532 7.497 240.6875 264.5625 7 6 5 3 8.9954 9.9904 Mean 6.49 ± 1.42 6.09 ± 0.87 156.25 ± 28.49 213.62 ± 22.31 4.29 ± 1.29 4.22 ± 1.05 3.55 ± 0.89 1.88 ± 0.80 5.35 ± 1.89 6.25 ± 1.82 On the other hand, the time duration of the sub-tasks performed by the older adults group are shown in Figure 19 and in Table 6. In Table 7, the parameters extracted from the signals of angular velocity and acceleration of the inertial sensors of the IMU are observed. Table 5. Characteristics obtained after the implemented segmentation of the activities performed by the young subjects during the TU & Go test. On the other hand, the time duration of the sub-tasks performed by the older adults group are shown in Figure 19 and in Table 6. In Table 7, the parameters extracted from the signals of angular velocity and acceleration of the inertial sensors of the IMU are observed. On the other hand, the time duration of the sub-tasks performed by the older adults group are shown in Figure 19 and in Table 6. In Table 7, the parameters extracted from the signals of angular velocity and acceleration of the inertial sensors of the IMU are observed. 3.3. Characterization of the Signals Acquired from the Stages of the TU & Go Test On the other hand, the time duration of the sub-tasks performed by the older adults group are shown in Figure 19 and in Table 6. In Table 7, the parameters extracted from the signals of angular velocity and acceleration of the inertial sensors of the IMU are observed. (a) (b) (c) (d) (e) (f) Figure 19. Distribution plots of the duration times obtained with the segmentation algorithm compared with the video analysis in the older adults group. (a) duration distribution for standing time; (b) duration distribution for the ﬁrst walk time; (c) duration distribution for the 3-m turning; (d) duration distribution for the second walk time; (e) duration distribution for the pre-sitting turning time; (f) duration distribution for sitting time. (b) (c) (a) (b) (c) (a) (d) (e) (f) (d) (f) (e) Figure 19. Distribution plots of the duration times obtained with the segmentation algorithm compared with the video analysis in the older adults group. (a) duration distribution for standing time; (b) duration distribution for the ﬁrst walk time; (c) duration distribution for the 3-m turning; (d) duration distribution for the second walk time; (e) duration distribution for the pre-sitting turning time; (f) duration distribution for sitting time. Figure 19. Distribution plots of the duration times obtained with the segmentation algorithm compared with the video analysis in the older adults group. (a) duration distribution for standing time; (b) duration distribution for the ﬁrst walk time; (c) duration distribution for the 3-m turning; (d) duration distribution for the second walk time; (e) duration distribution for the pre-sitting turning time; (f) duration distribution for sitting time. Table 6. Average time duration and error from the IMU activities segmentation algorithm compared to the video analysis of the older adults group. Table 6. Average time duration and error from the IMU activities segmentation algorithm compared to the video analysis of the older adults group. Table 6. Average time duration and error from the IMU activities segmentation algorithm compared to the video analysis of the older adults group. 3.3. Characterization of the Signals Acquired from the Stages of the TU & Go Test Distribution plots of the duration times obtained with the segmentation algorithm compared with the video analysis in the young subjects. (a) duration distribution for standing time; (b) duration distribution for the ﬁrst walk time; (c) duration distribution for the 3-m turning; (d) duration distribution for the second walk time; (e) duration distribution for the pre-sitting turning time; (f) duration distribution for sitting time. (b) (a) (c) (a) (b) (c) (d) (e) (f) (d) (f) (e) Figure 18. Distribution plots of the duration times obtained with the segmentation algorithm compared with the video analysis in the young subjects. (a) duration distribution for standing time; (b) duration distribution for the ﬁrst walk time; (c) duration distribution for the 3-m turning; (d) duration distribution for the second walk time; (e) duration distribution for the pre-sitting turning time; (f) duration distribution for sitting time. 17 of 22 Sensors 2019, 19, 1647 Table 4. Average time duration and error from the IMU activities segmentation algorithm compared to the video analysis of the young subjects. Standing First Walk 3-m Turning Second Walk Pre-Sitting Turning Sitting Video duration s 1.16 ± 0.15 2.51 ± 0.35 1.65 ± 0.25 2.39 ± 0.36 1.03 ± 0.14 1.48 ± 0.17 IMU duration s 1.24 ± 0.07 2.10 ± 0.45 1.85 ± 0.16 2.22 ± 0.37 1.10 ± 0.06 1.48 ± 0.15 IMU error estimation s −0.08 ± 0.15 0.42 ± 0.20 −0.19 ± 0.21 0.17 ± 0.11 −0.08 ± 0.11 −0.01 ± 0.19 Table 5. Characteristics obtained after the implemented segmentation of the activities performed by the young subjects during the TU & Go test. Acc. Su [m/s2] Acc. Sd [m/s2] Vel. T1 [deg/s] Vel. T2 [deg/s] Steps W1 Steps W2 Steps T1 Steps T2 Pitch Su [deg] Pitch Sd [deg] Minimum 3.9102 4.459 126.3125 181.6875 2 3 2 1 2.2979 3.236 Maximum 9.1532 7.497 240.6875 264.5625 7 6 5 3 8.9954 9.9904 Mean 6.49 ± 1.42 6.09 ± 0.87 156.25 ± 28.49 213.62 ± 22.31 4.29 ± 1.29 4.22 ± 1.05 3.55 ± 0.89 1.88 ± 0.80 5.35 ± 1.89 6.25 ± 1.82 On the other hand, the time duration of the sub-tasks performed by the older adults group are shown in Figure 19 and in Table 6. In Table 7, the parameters extracted from the signals of angular velocity and acceleration of the inertial sensors of the IMU are observed. Table 4. 4. Discussion In this study, a segmentation of the TU & Go test activities using a single wireless IMU was performed in two different age groups. For this, a comparison between the measurements obtained by the typical observational analysis and the proposed methodology, the segmentation of the TU & Go test activities and the characterization of the inertial signals acquired from the TU & Go test stages were carried out with interesting results. Effective treatment, speciﬁcally for gait disturbances and for risk of falls assessment, requires reliable tools. Tridimensional motion capturing systems are the gold standard of gait assessment, but, due to the space and time requirements and the high cost of the equipment, its use in clinical practice is far from routine [29]. In addition, a limited number of consecutive strides can be measured and they require camera and markers that may limit their use in the clinical practice. In this context, IMUs may be used to assess gait performance and risk of falls. Moreover, these technologies present several advantages such as they are portable, low-cost and lightweight; they are good at measuring acceleration and turns, they are suitable for measuring brief, high speed events and they can be used indoors and outdoors regardless of lighting conditions; ﬁnally, they can continuously evaluate over long periods of time. However, they must answer to their usefulness as a clinical tool [30]. Thus, these technological devices must be assessed in terms of clinical feasibility and psychometric properties [31,32]. The proposal model obtained a high correlation respect to video recording (see Figure 12), allowing for identifying all the events with only a single wireless IMU which present a low variability in characteristics inter-subjects. It allows for extracting characteristics of each phases independently, in comparison with other works that use a body ﬁxed sensor array [11,16]. In general, during the proof of concept of the proposed methodology in young subjects, a high degree of concordance was obtained with respect to the segmentation performed by video, which is demonstrated in the correlation coefﬁcients of Figure 14. In the case of the group of older adults, the correlation analysis was omitted because the results could be unrepresentative due to the low number of subjects and the atypical data present in the subject with a high risk of falls (see subject 12 of Table 3). 3.3. Characterization of the Signals Acquired from the Stages of the TU & Go Test Standing First Walk 3-m Turning Second Walk Pre-Sitting Turning Sitting Video duration s 1.41 ± 0.71 3.49 ± 3.26 2.23 ± 1.82 3.75 ± 3.75 1.32 ± 0.14 1.65 ± 0.56 IMU duration s 1.36 ± 0.50 3.25 ± 3.70 2.08 ± 1.30 3.56 ± 3.13 1.46 ± 1.30 1.64 ± 0.62 IMU error estimation s 0.05 ± 0.30 0.23 ± 0.61 0.14 ± 0.62 0.19 ± 0.78 −0.01 ± 0.27 0.01 ± 0.29 Standing First Walk 3-m Turning Second Walk Pre-Sitting Turning Sitting Video duration s 1.41 ± 0.71 3.49 ± 3.26 2.23 ± 1.82 3.75 ± 3.75 1.32 ± 0.14 1.65 ± 0.56 IMU duration s 1.36 ± 0.50 3.25 ± 3.70 2.08 ± 1.30 3.56 ± 3.13 1.46 ± 1.30 1.64 ± 0.62 IMU error estimation s 0.05 ± 0.30 0.23 ± 0.61 0.14 ± 0.62 0.19 ± 0.78 −0.01 ± 0.27 0.01 ± 0.29 Sensors 2019, 19, 1647 18 of 22 18 of 22 Table 7. Characteristics obtained after the implemented segmentation of the activities performed by the older adults group during the TU & Go test. In Figure 19, it is observed that the proposed method delivers similar distributions to those measured by video, but less variable, which is due to the present subjectivity when discriminating the transition from one sub-task to another visually. The atypical data in all the sub-images of Figure 19 are due to the subjectivity with a high risk of falling measured, demonstrating that the algorithm and the measurement methodology used allows identifying it as well as the visual method, but automatically. In general, the data presented in Table 6 indicate that the older adults group delay more than the young subjects in perform each sub-task (see Table 4), maintaining a similar error with respect to that observed in video. From Tables 5 and 7, it can be observed that the parameters that most differ between the two populations measured—young and older adults—are the maximum speed of both turnings, being lower in the elderly subjects than in young subjects. The largest number of steps measured for each walking sub-task of Table 7 belong to subject 12 with a high risk of falls and which is also possible to measure automatically using a simple local peak detection algorithm on the acceleration signal once the segmentation is performed. 4. Discussion The worst correlation obtained was found in the identiﬁcation of the moment in which the person ﬁnishes the sit-to-stand transition, due to the difﬁculty to identify in a visual way the exact point of the end of Sensors 2019, 19, 1647 19 of 22 19 of 22 the standing and the beginning of the ﬁrst walk sub-task. However, the proposed analysis algorithm was capable of identifying the exact transition point with an average maximum error of 0.36 s (see Figure 15b) for young subjects and a maximum average error of 0.63 s for older adults (see Figure 16d). The segmentation presented in the present study allows for improving objectivity to the clinical practice in the evaluation of the performance of the patients. By observing the standing/sitting phases using Pitch, it is possible to determine the inclination of the trunk during sit to walk transfer in young subjects (see Table 5) and older adults subjects (see Table 7. This is relevant because, as mentioned by Pozaic et al., the transition from sit to walk is the event with the highest number of falls in the elderly population [33]. On the other hand, the analysis of the turning phases through Yaw will allow for observing the degrees and the time that the subject takes to carry out this activity (Figure 18c,e). The ability to turn safely is relevant to functional independence and is considered another difﬁcult task with a high risk of falling [34,35]. Although the conventional TU & Go test is a clinical tool that allows for measuring the risk of falling, it uses a single global parameter to estimate it. However, the stage that could be interfering in the execution of the task in less time is not speciﬁcally discriminate. The current methodology provides information about variables such as: standing acceleration (Acc. Su), sitting acceleration (Acc. Sd), rotation velocity of the 3-m turning (Vel. T1), rotation velocity of the pre-sitting turning (Vel. T1), number of steps during the ﬁrst walk (Steps W1), number of steps during the second walk (Steps W2), number of steps during the 3-m turning (Steps T1), number of steps during the pre-sitting turning (Steps T2), inclination degrees of the trunk during standing (Pitch Su) and sitting (Pitch Sd) (see Table 5). Other authors [36–39] have explored the use of IMU technology for gait assessment during the TU & Go test. Author Contributions: Conceptualization, P.O.B., B.G., P.A. and R.C.d.l.C.; Methodology, P.O.B., B.G. and R.C.d.l.C.; Software, B.G. and F.S.; Data curation, P.O.B. and B.G.; Formal analysis, B.G.; Resources, P.O.B., B.G., F.S. and P.A.; Writing—original draft, P.O.B. and B.G.; Writing—review and editing, P.O.B. and B.G.; Supervision, P.A. and R.C.d.l.C. 4. Discussion However, this approach allows for segmenting the transitions between each sub-task in an exact and automatic way, using a simple algorithm and a low–cost movement sensor, allowing for extracting characteristics of each one of them due to the positioning of the IMU in the young and older adults groups. The above can be extrapolated when performing tests of 6 and 10 m to extract more parameters of the gait during the ﬁrst and second walk. This study presents some limitations. Firstly, the segmentation of each event during the analysis of the videos was performed in an observational way. Second, it is not possible to determine how the algorithm would perform in other populations such as people with neurological disorders. However, the whole sample recruited was determined as the starting point to be capable of validating the algorithm and the sensors used in pathological population in future research. Clinical validation studies of these devices should be carried out in populations with speciﬁc characteristics related to gait and balance impairments. Conﬂicts of Interest: The authors declare no conﬂict of interest. Funding: This research was funded by FONDECYT Posdoctorado n◦3180551. 5. Conclusions In conclusion, an IMU located on the back may detect main gait events and spatial-temporal kinematic factors during the TU & Go test, with excellent correlation with the conventional visual clinical procedure in young and older adults. Thus, a user-friendly technological tool to health-care professionals may offer objective measurements for the segmentation of the activities as standing, walking, turning and sitting related to critical events related with the risk of falls. Author Contributions: Conceptualization, P.O.B., B.G., P.A. and R.C.d.l.C.; Methodology, P.O.B., B.G. and R.C.d.l.C.; Software, B.G. and F.S.; Data curation, P.O.B. and B.G.; Formal analysis, B.G.; Resources, P.O.B., B.G., F.S. and P.A.; Writing—original draft, P.O.B. and B.G.; Writing—review and editing, P.O.B. and B.G.; Supervision, P.A. and R.C.d.l.C. Funding: This research was funded by FONDECYT Posdoctorado n◦3180551. Funding: This research was funded by FONDECYT Posdoctorado n◦3180551. Funding: This research was funded by FONDECYT Posdoctorado n◦3180551. Conﬂicts of Interest: The authors declare no conﬂict of interest. 20 of 22 20 of 22 Sensors 2019, 19, 1647 References 1. Jeon, M.Y.; Jeong, H.; Petrofsky, J.; Lee, H.; Yim, J. Effects of a randomized controlled recurrent fall prevention program on risk factors for falls in frail elderly living at home in rural communities. Med. Sci. Monit. 2014, 20, 2283. 2. Kingston, A.; Wohland, P.; Wittenberg, R.; Robinson, L.; Brayne, C.; Matthews, F.E.; Jagger, C.; Green, E.; Gao, L.; Barnes, R.; et al. Is late-life dependency increasing or not? A comparison of the Cognitive Function and Ageing Studies (CFAS). Lancet 2017, 390, 1676–1684. [CrossRef] . Sharif, S.I.; Al-Harbi, A.B.; Al-Shihabi, A.M.; Al-Daour, D.S.; Sharif, R.S. Falls in the elderly: Assessmen prevalence and risk factors. Pharm. Pract. 2018, 16, 1206. [CrossRef] [PubMed] 4. Talarska, D.; Strugała, M.; Szewczyczak, M.; Tobis, S.; Michalak, M.; Wróblewska, I.; Wieczorowska-Tobis, K. Is independence of older adults safe considering the risk of falls? BMC Geriatr. 2017, 17, 66. [CrossRef] [PubMed] 5. Qiu, H.; Rehman, R.Z.U.; Yu, X.; Xiong, S. Application of Wearable Inertial Sensors and a New Test Battery for Distinguishing Retrospective Fallers from Non-fallers among Community-dwelling Older People. Sci. Rep. 2018, 8, 16349. [CrossRef] [PubMed] 6. Sun, D.Q.; Huang, J.; Varadhan, R.; Agrawal, Y. Race and fall risk: data from the National Health and Aging Trends Study (NHATS). Age Ageing 2016, 45, 120–127. [CrossRef] [PubMed] 7. Hellmers, S.; Izadpanah, B.; Dasenbrock, L.; Diekmann, R.; Bauer, J.; Hein, A.; Fudickar, S. Towards an Automated Unsupervised Mobility Assessment for Older People Based on Inertial TUG Measurements. Sensors 2018, 18, 3310. [CrossRef] 8. Park, S.H. Tools for assessing fall risk in the elderly: A systematic review and meta-analysis. Agi Exp. Res. 2018, 30, 1–16. [CrossRef] [PubMed] 9. Zasadzka, E.; Borowicz, A.M.; Roszak, M.; Pawlaczyk, M. Assessment of the risk of falling with the use of timed up and go test in the elderly with lower extremity osteoarthritis. Clin. Interv. Aging 2015, 10, 1289. [CrossRef] [PubMed] 10. Persson, C.U.; Danielsson, A.; Sunnerhagen, K.S.; Grimby-Ekman, A.; Hansson, P.O. Timed Up & Go as a measure for longitudinal change in mobility after stroke–Postural Stroke Study in Gothenburg (POSTGOT). J. Neuroeng. Rehabil. 2014, 11, 83. 11. Nguyen, H.; Lebel, K.; Boissy, P.; Bogard, S.; Goubault, E.; Duval, C. Auto detection and segmentation of daily living activities during a Timed Up and Go task in people with Parkinson’s disease using multiple inertial sensors. J. Neuroeng. Rehabil. 2017, 14, 26. [CrossRef] [PubMed] 12. Podsiadlo, D.; Richardson, S. References The timed “Up & Go”: A test of basic functional mobility for frail elderly persons. J. Am. Geriatr. Soc. 1991, 39, 142–148. [PubMed] 13. Barry, E.; Galvin, R.; Keogh, C.; Horgan, F.; Fahey, T. Is the Timed Up and Go test a useful predictor of risk of falls in community dwelling older adults: A systematic review and meta-analysis. BMC Geriatr. 2014, 14, 14. [CrossRef] [PubMed] 14. Galán-Mercant, A.; Barón-López, F.J.; Labajos-Manzanares, M.T.; Cuesta-Vargas, A.I. Reliability and criterion-related validity with a smartphone used in timed-up-and-go test. BioMed. Eng. Online 2014, 13, 156. [CrossRef] 15. Kleiner, A.F.R.; Paciﬁci, I.; Vagnini, A.; Camerota, F.; Celletti, C.; Stocchi, F.; De Pandis, M.F.; Galli, M. Timed Up and Go evaluation with wearable devices: Validation in Parkinson’s disease. J. Bodyw. Mov. Ther. 2018, 22, 390–395. [CrossRef] 16. Weiss, A.; Mirelman, A.; Buchman, A.S.; Bennett, D.A.; Hausdorff, J.M. Using a body-ﬁxed sensor to identify subclinical gait difﬁculties in older adults with IADL disability: Maximizing the output of the timed up and go. PLoS ONE 2013, 8, e68885. [CrossRef] 17. Nguyen, H.; Lebel, K.; Bogard, S.; Goubault, E.; Boissy, P.; Duval, C. Using inertial sensors to automatically detect and segment activities of daily living in people with Parkinson’s disease. IEEE Trans. Neural Syst. Rehabil. Eng. 2018, 26, 197–204. [CrossRef] 18. Tanaka, N.; Zakaria, N.A.; Kibinge, N.K.; Kanaya, S.; Tamura, T.; Yoshida, M. Fall-risk classiﬁcation of the timed up-and-go test with principle component analysis. Int. J. Neurorehabilit. 2014, 1, 106. 21 of 22 Sensors 2019, 19, 1647 19. Soangra, R.; Lockhart, T.E.; Van de Berge, N. An approach for identifying gait events using wavelet denoising technique and single wireless IMU. Proc. Hum. Factors Ergon. Soc. Annu. Meet. 2011, 55, 1990–1994. [CrossRef] 20. Lebel, K.; Nguyen, H.; Duval, C.; Plamondon, R.; Boissy, P. Capturing the cranio-caudal signature of a turn with inertial measurement systems: methods, parameters robustness and reliability. Front. Bioeng. Biotechnol. 2017, 5, 51. [CrossRef] [PubMed] 21. Vervoort, D.; Vuillerme, N.; Kosse, N.; Hortobágyi, T.; Lamoth, C.J. Multivariate analyses and classiﬁcation of inertial sensor data to identify aging effects on the Timed-Up-and-Go test. PLoS ONE 2016, 11, e0155984. [CrossRef] 22. Salarian, A.; Horak, F.B.; Zampieri, C.; Carlson-Kuhta, P.; Nutt, J.G.; Aminian, K. iTUG, a sensitive and reliable measure of mobility. IEEE Trans. Neural Syst. Rehabil. Eng. 2010, 18, 303–310. [CrossRef] 23. Sensortec, B. Intelligent 9-Axis Absolute Orientation Sensor; BNO055 Datasheet; 2014. Available online: https://easyeda.com/gerrychen/BNO055_Project-aPDdNKIiS (accessed on 5 February 2019). References 24. Kose, A.; Peruzzi, A.; Cereatti, A.; Laudani, L.; Della Croce, U. Detection of heel strikes and toe-offs during gait using a single inertial measurement unit attached to the waist. In Proceedings of the Second National Congress of Biongineering, Turin, Italy, 8–10 July 2010; Volume 233. 25. Weinberg, H. Using the ADXL202 in Pedometer and Personal Navigation Applications; Analog Devices AN-602 Application Note; 2002; Volume 2, pp. 1–6. Available online: http://www.bdtic.com/download/adi/ 513772624an602.pdf (accessed on 5 February 2019). 26. Sejdi´c, E.; Lowry, K.A.; Bellanca, J.; Perera, S.; Redfern, M.S.; Brach, J.S. Extraction of stride events from gait accelerometry during treadmill walking. IEEE J. Transl. Eng. Health Med. 2016, 4, 1–11. [CrossRef] 27. Pham, M.H.; Warmerdam, E.; Elshehabi, M.; Schlenstedt, C.; Bergeest, L.M.; Heller, M.; Haertner, L.; Ferreira, J.; Berg, D.; Schmidt, G.; et al. Validation of a lower back “wearable”-based sit-to-stand and stand-to-sit algorithm for patients with Parkinson’s disease and older adults in a home-like environment. Front. Neurol. 2018, 9, 652. [CrossRef] 28. Mancilla, E.; Valenzuela, J.; Escobar, M. Timed up and go right and left unipodal stance results in Chilean older people with different degrees of disability. Rev. Med. Chile 2015, 143, 39–46. 29. Gor-Garcia-Fogeda, M.D.; de la Cuerda, R.C.; Tejada, M.C.; Alguacil-Diego, I.M.; Molina-Rueda, F. Observational gait assessments in people with neurological disorders: A systematic review. Arch. Phys. Med. Rehabil. 2016, 97, 131–140. [CrossRef] 30. Linares-Del Rey, M.; Vela-Desojo, L.; Cano-de la Cuerda, R. Mobile phone applications in Parkinson’s disease: A systematic review. Neurología 2019, 34, 38–54. (In English) [CrossRef] 31. Hobart, J.C.; Lamping, D.L.; Thompson, A.J. Evaluating neurological outcome measures: the bare essentials. J. Neurol. Neurosurg. Psychiatry 1996, 60, 127. [CrossRef] 32. Andresen, E.M. Criteria for assessing the tools of disability outcomes research. Arch. Phys. Med. Rehabil. 2000, 81, S15–S20. [CrossRef] 33. Pozaic, T.; Lindemann, U.; Grebe, A.K.; Stork, W. Sit-to-stand transition reveals acute fall risk in activities of daily living. IEEE J. Transl. Eng. Health Med. 2016, 4, 1–11. [CrossRef] 34. Chan, W.N.; Tsang, W.W. The performance of stroke survivors in turning-while-walking while carrying out a concurrent cognitive task compared with controls. PLoS ONE 2017, 12, e0189800. [CrossRef] 35. Mancini, M.; Schlueter, H.; El-Gohary, M.; Mattek, N.; Duncan, C.; Kaye, J.; Horak, F.B. Continuous monitoring of turning mobility and its association to falls and cognitive function: A pilot study. J. Gerontol. Ser. A Biomed. Sci. Med. Sci. 2016, 71, 1102–1108. [CrossRef] 36. c⃝2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). References Zampieri, C.; Salarian, A.; Carlson-Kuhta, P.; Nutt, J.G.; Horak, F.B. Assessing mobility at home in people with early Parkinson’s disease using an instrumented Timed Up and Go test. Parkinsonism Relat. Disord. 2011, 17, 277–280. [CrossRef] 37. Beyea, J.; McGibbon, C.A.; Sexton, A.; Noble, J.; O’Connell, C. Convergent validity of a wearable sensor system for measuring sub-task performance during the timed Up-and-Go Test. Sensors 2017, 17, 934. [CrossRef] Sensors 2019, 19, 1647 22 of 22 38. Pedrana, A.; Comotti, D.; Locatelli, P.; Traversi, G. Development of a telemedicine-oriented gait analysis system based on inertial sensors. In Proceedings of the 2018 7th International Conference on Modern Circuits and Systems Technologies (MOCAST), Thessaloniki, Greece, 7–9 May 2018; pp. 1–4. 38. Pedrana, A.; Comotti, D.; Locatelli, P.; Traversi, G. Development of a telemedicine-oriented gait analysis system based on inertial sensors. In Proceedings of the 2018 7th International Conference on Modern Circuits and Systems Technologies (MOCAST), Thessaloniki, Greece, 7–9 May 2018; pp. 1–4. 39. Reinfelder, S.; Hauer, R.; Barth, J.; Klucken, J.; Eskoﬁer, B.M. Timed Up-and-Go phase segmentation in Parkinson’s disease patients using unobtrusive inertial sensors. In Proceedings of the 2015 37th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC), Milano, Italy, 25–29 August 2015; pp. 5171–5174. 39. Reinfelder, S.; Hauer, R.; Barth, J.; Klucken, J.; Eskoﬁer, B.M. Timed Up-and-Go phase segmentation in Parkinson’s disease patients using unobtrusive inertial sensors. In Proceedings of the 2015 37th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC), Milano, Italy, 25–29 August 2015; pp. 5171–5174. c⃝2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). c⃝2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W4304789114	https://www.frontiersin.org/articles/10.3389/fmicb.2022.998287/pdf	English	null	Rethinking treatment paradigms for the deployment of SARS-CoV-2 antiviral drugs on the shifting landscape of new variants	Frontiers in microbiology	2,022	cc-by	3,457	TYPE Opinion PUBLISHED 12 October 2022 DOI 10.3389/fmicb.2022.998287 TYPE Opinion PUBLISHED 12 October 2022 DOI 10.3389/fmicb.2022.998287 TYPE Opinion PUBLISHED 12 October 2022 DOI 10.3389/fmicb.2022.998287 Rethinking treatment paradigms for the deployment of SARS-CoV-2 antiviral drugs on the shifting landscape of new variants OPEN ACCESS EDITED BY Yushun Wan, Chongqing Medical University, China REVIEWED BY Masaud Shah, Ajou University, South Korea CORRESPONDENCE Alexandra Calmy Alexandra.calmy@hcuge.ch SPECIALTY SECTION This article was submitted to Virology, a section of the journal Frontiers in Microbiology RECEIVED 19 July 2022 ACCEPTED 26 September 2022 PUBLISHED 12 October 2022 CITATION Hentzien M, Owen A, Strub-Wourgaft N, Pérez-Casas C, Trøseid M and Calmy A (2022) Rethinking treatment paradigms for the deployment of SARS-CoV-2 antiviral drugs on the shifting landscape of new variants. Front. Microbiol. 13:998287. doi: 10.3389/fmicb.2022.998287 Maxime Hentzien1, Andrew Owen2, Nathalie Strub-Wourgaft3, Carmen Pérez-Casas4, Marius Trøseid5 and Alexandra Calmy1 1HIV/AIDS Unit, Infectious Diseases Division, Geneva University Hospitals, Geneva, Switzerland, 2Centre of Excellence in Long-acting Therapeutics (CELT), University of Liverpool, Liverpool, United Kingdom, 3COVID Response and Pandemic Preparedness Director, Drugs for Neglected Diseases Initiative (DNDi), Geneva, Switzerland, 4Strategy, Unitaid, Geneva, Switzerland, 5Section of Clinical Immunology and Infectious Diseases, Oslo University Hospital, Oslo, Norway Hentzien M, Owen A, Strub-Wourgaft N, Pérez-Casas C, Trøseid M and Calmy A (2022) Rethinking treatment paradigms for the deployment of SARS-CoV-2 antiviral drugs on the shifting landscape of new variants. Front. Microbiol. 13:998287. doi: 10.3389/fmicb.2022.998287 COVID-19, direct-acting antivirals, immunocompromised, resistance, variant emergence, monoclonal antibodies, omicron, variants Protecting emerging treatment options Sabin et al., 2022). The emergence of resistance mutation thus impacting treatment eﬃcacy is more likely if a patient has been exposed to speciﬁc antiviral drugs. In addition, it remains unclear if the small percent rebound occurrence (2%) observed with nirmatrelvir/r in the EPIC-HR (Evaluation of Protease Inhibition for COVID-19 in High-Risk Patients) trial, performed in the delta variant era, is underestimating a risk (Boucau et al., 2022; Rubin, 2022) that would be particularly of concern in patients harboring an impaired immune system and in the omicron era. In one recent case series, one out of 7 patients who had a virologic rebound also had an immunosuppressing condition (Boucau et al., 2022). Another recent case series (Coulson et al., 2022) revealed that all three patients with viral rebound were highly immunocompromised. This potentially raises concerns about the need of longer antiviral courses, especially in these patients. Sabin et al., 2022). The emergence of resistance mutation thus impacting treatment eﬃcacy is more likely if a patient has been exposed to speciﬁc antiviral drugs. In addition, it remains unclear if the small percent rebound occurrence (2%) observed with nirmatrelvir/r in the EPIC-HR (Evaluation of Protease Inhibition for COVID-19 in High-Risk Patients) trial, performed in the delta variant era, is underestimating a risk (Boucau et al., 2022; Rubin, 2022) that would be particularly of concern in patients harboring an impaired immune system and in the omicron era. In one recent case series, one out of 7 patients who had a virologic rebound also had an immunosuppressing condition (Boucau et al., 2022). Another recent case series (Coulson et al., 2022) revealed that all three patients with viral rebound were highly immunocompromised. This potentially raises concerns about the need of longer antiviral courses, especially in these patients. Several crucial issues warrant urgent attention to optimize the use of these emerging treatment options (Figure 1). First, as proven to be transformational for HIV, rapid, aﬀordable access to early antiviral treatment to slow the tide of new variants is critical to eﬀective “test-and-treat” strategies to protect the most fragile patients and avoid a severe and/or persistent infection. After more than 2 years of pandemic, progress has been slow (Hasan et al., 2022) and public health attention has recently been attracted by the low-proﬁle agreement during the (World Trade Organization, 2022) in Geneva in May 2022 (Financial Times, 2022). Protecting emerging treatment options Together with vaccination, early diagnosis and treatment have the ability to reduce disease worsening, to reduce transmission and to constrain variability in viral sequences (United Kingdom Scientiﬁc Advisory Group for Emergencies, 2021). Preclinical data have clearly demonstrated that virological eﬃcacy is higher for combinations of existing antiviral drugs than single agents (Abdelnabi et al., 2021; Jeong et al., 2022; Li et al., 2022). To achieve the goal of changing the treatment guidelines in SARS-CoV-2-infected immunocompromised individuals, independent and academic clinical trials for drug combinations should be considered as an urgent, unmet research priority. Today, collaboration with industry to allow early access to antiviral drugs to be combined has been an objective still to be achieved (Bloomberg (Europe Edition), 2022). Certain potent monoclonal antibodies, such as bebtelovimab, cannot even be accessed for research or for routine care outside of the USA (Hentzien et al., 2022). Second, although the combined eﬀect of omicron and increasing vaccine deployment in some regions has shifted the demand response from hospital to outpatient care, considerable uncertainty exists about who is now at risk for severe omicron disease (Skarbinski et al., 2022). While the risk/beneﬁt ratio across at-risk subpopulations has unquestionably changed in vaccinated populations, gains made can only be preserved if those at highest risk are rapidly diagnosed and receive treatment in less than one week. Third, high levels of antiviral eﬃcacy will be critically important, especially in immunocompromised patients who are grossly underrepresented in registrational trials (John and John, 2020; Trøseid et al., 2022). Causes of immunosuppression are diverse (including organ/stem cell transplants, cancer, immunosuppressive medications or uncontrolled HIV) and these patients represent a signiﬁcant proportion of the population, e.g., 7 million adults in the USA (Harpaz et al., 2016), but also in low- and middle-income countries due to the high prevalence of uncontrolled HIV. Overall, the mortality risk with omicron is still unclear, but protection of those who cannot be eﬀectively vaccinated or protected by a prior SARS- CoV-2 infection remains imperative (Overvad et al., 2022). Importantly, in regions where HIV is highly prevalent, there is a clear need and opportunity to reinforce HIV epidemic control by prompt diagnosis and sustained viral suppression with antiretrovirals, key factors to also enable the control of SARS COV-2 spread in this group (Msomi et al., 2021; Meiring et al., 2022). Introduction Monoclonal antibodies targeting the anti-SARS-CoV-2 spike (S) protein are prescribed in high-income countries to prevent severe disease in at-risk patients. Although studies report eﬃcacy as between 50–85% (Weinreich et al., 2021; Gupta et al., 2022; Montgomery et al., 2022), global access is currently largely inequitable (Wiltz et al., 2022). Multivariant omicron (B.1.1.529) and subvariant (BA.2 followed by BA.4 and BA.5) dominance has challenged the treatment landscape for mild-to-moderate disease, introducing considerable uncertainty on the eﬃcacy of monoclonal antibodies (Cao et al., 2022; Yamasoba et al., 2022) and leading to changes to initial recommendations for some of them (United States Food Drug Administration, 2022). Contemporaneously, oral, direct-acting antivirals with a reported eﬃcacy ranging from 30% (molnupiravir) (Jayk Bernal et al., 2022) to 89–90% (nirmatrelvir/ritonavir) (United States Food Drug Administration, 2021) have recently received conditional or emergency approval in some countries and been recommended in international guidelines such as the World Health Organization guidelines (World Health Organization, 2022). S-217622, also known as ensitrelvir, a 3CL protease inhibitor that has been shown to signiﬁcantly reduce the infectious viral load (Mukae et al., 2022a,b), is currently in phase 3 trials and waiting for emergency approval in Japan (Otake, 2022) and should be submitted soon in China (Notice Regarding the Initiation of the Submission of Preparation Materials for a New Drug Application for S-217622, a Therapeutic Drug for COVID-19, in China, 2022). The main purpose of this opinion paper is to highlight the possible strategies to optimize and protect current and future therapeutic options to treat the most vulnerable patients. COPYRIGHT © 2022 Hentzien, Owen, Strub-Wourgaft, Pérez-Casas, Trøseid and Calmy. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 01 01 Frontiers in Microbiology frontiersin.org Hentzien et al. 10.3389/fmicb.2022.998287 Author contributions single agent (United States Food Drug Administration, 2021). This is particularly the case where concentrations achieved are close to the therapeutic eﬃcacy threshold or in the case of low compliance. MH and AC wrote the ﬁrst manuscript draft. All authors critically reviewed the manuscript, validated the ﬁnal version, and agreed to be accountable for the content of the work. It is incumbent upon the international research community and the pharmaceutical industry to pool knowledge and provide the critical information that the World Health Organization and country-level authorities so urgently require, as well as early diagnosis and increased access to vaccines and antiviral therapy. The resistance risk for existing drugs has been woefully understudied throughout development, making it extremely challenging to rationalize during policy development. Looking beyond eﬃcacy, drug combinations will unquestionably reduce the rate at which resistance and new variants impacting treatment options emerge and could be made available and accessible to those in need if timely eﬀorts are made. Conﬂict of interest The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. In conclusion, we call for combination therapies to be tested in adequately powered clinical trials in the target population of immunocompromised patients, both in wealthy and in low- income countries where HIV-driven immunosuppression is prevalent. If higher eﬃcacy is conﬁrmed, the diversity of possible combinations will enable the tailoring of therapeutic options to individual patient needs (e.g., avoiding drug-drug interactions in transplant patients) as well as their speciﬁc regional context (e.g., oral-only combinations). Acknowledgments We thank Rosemary Sudan for revision of the English. in a potentiation of antiviral eﬃcacy in a SARS-CoV-2 hamster infection model. eBioMedicine 72, 103595. doi: 10.1016/j.ebiom.2021.103595 Expert opinion Treatment optimization has been truly transformational for other viral diseases [e.g., HIV/hepatitis C virus (Cohen et al., 2011)] and was only achieved when antiviral drug combinations became the mainstay. With few drugs currently available, the opportunity must be seized prior to the emergence of resistance to drugs deployed widely as monotherapies. Combinations of polymerase inhibitors and polymerase/protease inhibitors have proven highly successful for other viruses and in animal models for SARS-CoV-2 (Abdelnabi et al., 2021; Jeong et al., 2022). Thus, as drugs that are appropriate to combine are available, there is no good reason not to study them clinically. In addition to the opportunities that combinations present for a more potent antiviral response (individual beneﬁt), there can be no doubt that the rate at which resistance emerges will also be reduced (public health beneﬁt). Higher potency will result in a lower variability in sequences through a lower degree of replication. In addition, the probability of the occurrence of multiple mutations to drive resistance to multiple antivirals simultaneously is much lower than for a Although there are many other causes for variant emergence (host jump or adaptation, vaccine exposure, to name the most frequent), data conﬁrm that immunocompromised patients with long-term SARS-CoV-2 replication are particularly susceptible to resistance and transmissible variant emergence (Clark et al., 2021; Destras et al., 2022; Quaranta et al., 2022; Frontiers in Microbiology frontiersin.org 02 Hentzien et al. 10.3389/fmicb.2022.998287 FIGURE 1 Potential impact of SARS-CoV-2 antiviral drugs optimization in protecting available antivirals in the shifting landscape of new variants. FIGURE 1 Potential impact of SARS-CoV-2 antiviral drugs optimization in protecting available antivirals in the shifting landscape of new variant Abdelnabi, R., Foo, C. S., Kaptein, S. J. F., Zhang, X., Do, T. N. D., Langendries, L., et al. (2021). The combined treatment of Molnupiravir and Favipiravir results Publisher’s note gov.uk/government/publications/nervtag-antiviral-drug-resistance-and-the-use- of-directly-acting-antiviral-drugs-daas-for-covid-19-8-december-2021/nervtag- antiviral-drug-resistance-and-the-use-of-directly-acting-antiviral-drugs-daas- for-covid-19-8-december-2021 (accessed February 11, 2022). United Kingdom Scientiﬁc Advisory Group for Emergencies (2021). NERVTAG: Antiviral drug resistance and the use of directly acting antiviral drugs (DAAs) for COVID-19, 8 December 2021 [Internet]. Available online at: https://www. gov.uk/government/publications/nervtag-antiviral-drug-resistance-and-the-use- of-directly-acting-antiviral-drugs-daas-for-covid-19-8-december-2021/nervtag- antiviral-drug-resistance-and-the-use-of-directly-acting-antiviral-drugs-daas- for-covid-19-8-december-2021 (accessed February 11, 2022). Hentzien, M., Autran, B., Piroth, L., Yazdanpanah, Y., and Calmy, A. (2022). A monoclonal antibody stands out against omicron subvariants: a call to action for a wider access to bebtelovimab. Lancet Infect. Dis. 22, 1278. doi: 10.1016/S1473-3099(22)00495-9 Jayk Bernal, A., Gomes da Silva, M. M., Musungaie, D. B., Kovalchuk, E., Gonzalez, A., Delos Reyes, V., et al. (2022). Molnupiravir for oral treatment of Covid-19 in nonhospitalized patients. N. Engl. J. Med. 386, 509–520. doi: 10.1056/NEJMoa2116044 United States Food and Drug Administration (2021). Coronavirus (COVID-19) Update: FDA Authorizes First Oral Antiviral for Treatment of COVID-19. FDA. Available online at: https://www.fda.gov/news-events/press-announcements/ coronavirus-covid-19-update-fda-authorizes-ﬁrst-oral-antiviral-treatment- covid-19 (accessed February 11, 2022). United States Food and Drug Administration (2021). Coronavirus (COVID-19) Update: FDA Authorizes First Oral Antiviral for Treatment of COVID-19. FDA. Available online at: https://www.fda.gov/news-events/press-announcements/ coronavirus-covid-19-update-fda-authorizes-ﬁrst-oral-antiviral-treatment- covid-19 (accessed February 11, 2022). Jeong, J. H., Chokkakula, S., Min, S. C., Kim, B. K., Choi, W.-S., Oh, S., et al. (2022). Combination therapy with nirmatrelvir and molnupiravir improves the survival of SARS-CoV-2 infected mice. BioRxiv. doi: 10.1101/2022.06.27.497875 John, N. A., and John, J. E. (2020). Implications of COVID-19 infections in sickle cell disease. Pan. Afr. Med. J. 36, 81. doi: 10.11604/pamj.2020.36.158.24011 United States Food and Drug Administration (2022). FDA updates Sotrovimab emergency use authorization. FDA. Available online at: https://www.fda.gov/ drugs/drug-safety-and-availability/fda-updates-sotrovimab-emergency-use- authorization (accessed May 15, 2022). Li, P., Wang, Y., Lavrijsen, M., Lamers, M. M., de Vries, A. C., Rottier, R. J., et al. (2022). SARS-CoV-2 Omicron variant is highly sensitive to molnupiravir, nirmatrelvir, and the combination. Cell Res. 32, 322–324. doi: 10.1038/s41422-022-00618-w Weinreich, D. M., Sivapalasingam, S., Norton, T., Ali, S., Gao, H., Bhore, R., et al. (2021). REGEN-COV antibody combination and outcomes in outpatients with Covid-19. New Engl. J. Med. 38, e8. doi: 10.1056/NEJMoa2035002 Weinreich, D. M., Sivapalasingam, S., Norton, T., Ali, S., Gao, H., Bhore, R., et al. (2021). REGEN-COV antibody combination and outcomes in outpatients with Covid-19. New Engl. J. Med. 38, e8. doi: 10.1056/NEJMoa2035002 Meiring, S., Tempia, S., Bhiman, J. N., Buys, A., Kleynhans, J., Makhasi, M., et al. (2022). Prolonged shedding of SARS-CoV-2 at high viral loads amongst hospitalised immunocompromised persons living with HIV, South Africa. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. References Abdelnabi, R., Foo, C. S., Kaptein, S. J. F., Zhang, X., Do, T. N. D., Langendries, L., et al. (2021). The combined treatment of Molnupiravir and Favipiravir results frontiersin.org Frontiers in Microbiology 03 Hentzien et al. Hentzien et al. 10.3389/fmicb.2022.998287 10.3389/fmicb.2022.998287 Bloomberg (Europe Edition) (2022). Pﬁzer’s Grip on Paxlovid Thwarts Research on Covid Treatment. Bloomberg.com. Available online at: https://www. bloomberg.com/news/articles/2022-05-18/pﬁzer-s-tight-paxlovid-rein-stymies- drug-combination-research (accessed July 14, 2022). Mukae, H., Yotsuyanagi, H., Ohmagari, N., Doi, Y., Sakaguchi, H., Sonoyama, T., et al. (2022b). Eﬃcacy and safety of ensitrelvir in patients with mild-to-moderate COVID-19: the phase 2b part of a randomized, placebo-controlled, phase 2/3 study. MedRxiv [Preprint]. doi: 10.1101/2022.06.22.22276792 Boucau, J., Uddin, R., Marino, C., Regan, J., Flynn, J. P., Choudhary, M. C., et al. (2022). Characterization of virologic rebound following nirmatrelvir-ritonavir treatment for COVID-19. Clin. Infect. Dis. doi: 10.1101/2022.05.24.22275326 Notice Regarding the Initiation of the Submission of Preparation Materials for a New Drug Application for S-217622, a Therapeutic Drug for COVID-19, in China. (2022). [Internet] 4 July. Available online at: https://www.shionogi.com/global/en/ news/2022/07/e220704.html (accessed July 7, 2022). Cao, Y., Yisimayi, A., Jian, F., Song, W., Xiao, T., Wang, L., et al. (2022). BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection. Nature. 608, 593–602. doi: 10.1101/2022.04.30.489997 Otake, T. (2022). Japanese panel postpones verdict on Shionogi’s COVID pill until July. The Japan Times. 23 June. Available online at: https://www.japantimes. co.jp/news/2022/06/23/national/science-health/shionogi-covid19-pill-verdict- delay/ (accessed July 7, 2022). Clark, S. A., Clark, L. E., Pan, J., Coscia, A., McKay, L. G. A., Shankar, S., et al. (2021). SARS-CoV-2 evolution in an immunocompromised host reveals shared neutralization escape mechanisms. Cell. 184, 2605–2617.e18. doi: 10.1016/j.cell.2021.03.027 Overvad, M., Koch, A., Jespersen, B., Gustafsson, F., Krause, T. G., Hansen, C. H., et al. (2022). Outcomes following SARS-CoV-2 infection in individuals with and without solid organ transplantation; a Danish nationwide cohort study. Am. J. Transplant. doi: 10.1111/ajt.17142. [Epub ahead of print]. Cohen, M. S., Chen, Y. Q., McCauley, M., Gamble, T., Hosseinipour, M. C., Kumarasamy, N., et al. (2011). Prevention of HIV-1 infection with early antiretroviral therapy. N. Engl. J. Med. 365, 493–505. Yamasoba, D., Kosugi, Y., Kimura, I., Fujita, S., Uriu, K., Ito, J., et al. (2022). Neutralisation sensitivity of SARS-CoV-2 omicron subvariants to therapeutic monoclonal antibodies. Lancet Infect. Dis. 22, 942–943. doi: 10.1016/S1473-3099(22)00365-6 Publisher’s note doi: 10.1056/NEJMoa1105243 Quaranta, E. G., Fusaro, A., Giussani, E., D’Amico, V., Varotto, M., Pagliari, M., et al. (2022). SARS-CoV-2 intra-host evolution during prolonged infection in an immunocompromised patient. Int. J. Infect. Dis. 122, 444–448. doi: 10.1016/j.ijid.2022.06.023 Coulson, J. M., Adams, A., Gray, L. A., and Evans, A. (2022). COVID-19 “Rebound” associated with nirmatrelvir/ritonavir pre-hospital therapy. J. Infect. 85, 436–480 doi: 10.1016/j.jinf.2022.06.011 Rubin, R. (2022). From positive to negative to positive again-the mystery of why COVID-19 rebounds in some patients who take paxlovid. JAMA 327, 2380–2382. doi: 10.1001/jama.2022.9925 Destras, G., Bal, A., Simon, B., Lina, B., and Josset, L. (2022). Sotrovimab drives SARS-CoV-2 omicron variant evolution in immunocompromised patients. Lancet Microbe. 3, e559. doi: 10.1016/S2666-5247(22)00120-3 Sabin, A. P., Richmond, C. S., and Kenny, P. A. (2022). Emergence and onward transmission of a SARS-CoV-2 E484K variant among household contacts of a bamlanivimab-treated patient. Diagn. Microbiol. Infect. Dis. 103, 115656. doi: 10.1016/j.diagmicrobio.2022.115656 Financial Times (2022). WTO agrees partial patent waiver for Covid-19 vaccines [Internet]. June 17. Available online at: https://www.swissinfo.ch/eng/business/ wto-agrees-partial-patent-waiver-for-covid-19-vaccines/47681524 (accessed July 7, 2022). Skarbinski, J., Wood, M. S., Chervo, T. C., Schapiro, J. M., Elkin, E. P., Valice, E., et al. (2022). Risk of severe clinical outcomes among persons with SARS-CoV-2 infection with diﬀering levels of vaccination during widespread Omicron (B.1.1.529) and Delta (B.1.617.2) variant circulation in Northern California: A retrospective cohort study. Lancet Reg. Health Am. 12, 100297. doi: 10.1016/j.lana.2022.100297 Gupta, A., Gonzalez-Rojas, Y., Juarez, E., Crespo Casal, M., Moya, J., Rodrigues Falci, D., et al. (2022). Eﬀect of sotrovimab on hospitalization or death among high- risk patients with mild to moderate COVID-19: a randomized clinical trial. JAMA 327, 1236–1246. doi: 10.1001/jama.2022.2832 Harpaz, R., Dahl, R. M., and Dooling, K. L. (2016). Prevalence of immunosuppression among US adults, 2013. JAMA 316, 2547. doi: 10.1001/jama.2016.16477 Trøseid, M., Hentzien, M., Ader, F., Cardoso, S. W., Arribas, J. R., Molina, J.-M., et al. (2022). Immunocompromized patients have been neglected in Covid-19 trials: A call for action. Clin. Microbiol. Infect. 28, 1182–1183. doi: 10.1016/j.cmi.2022. 05.005 Hasan, Q., Elfakki, E., Fahmy, K., Mere, O., Ghoniem, A., Langar, H., et al. (2022). Inequities in the deployment of COVID-19 vaccine in the WHO Eastern Mediterranean Region, 2020-2021. BMJ Glob. Health. 7 (Suppl 4), e008139. United Kingdom Scientiﬁc Advisory Group for Emergencies (2021). NERVTAG: Antiviral drug resistance and the use of directly acting antiviral drugs (DAAs) for COVID-19, 8 December 2021 [Internet]. Available online at: https://www. Publisher’s note Clin. Infect. Dis. 116, S25. doi: 10.1093/cid/ciac077 Wiltz, J. L., Feehan, A. K., Molinari, N. M., Ladva, C. N., Truman, B. I., Hall, J., et al. (2022). Racial and ethnic disparities in receipt of medications for treatment of COVID-19 — United States, March 2020–August 2021. Morb. Mortal. Wkly. Rep. 71, 96–102. doi: 10.15585/mmwr.mm7103e1 Montgomery, H., Hobbs, F. D. R., Padilla, F., Arbetter, D., Templeton, A., Seegobin, S., et al. (2022). Eﬃcacy and safety of intramuscular administration of tixagevimab–cilgavimab for early outpatient treatment of COVID-19 (TACKLE): a phase 3, randomised, double-blind, placebo-controlled trial. Lancet Respir. Med. 10, 985–996. doi: 10.1016/S2213-2600(22)00180-1 World Health Organization (2022). Therapeutics and COVID-19: living guideline Available online at: https://app.magicapp.org/#/guideline/nBkO1E (accessed July 18, 2022). World Trade Organization (2022). Draft texts on WTO response to pandemic, IP response sent to ministers for decision [Internet]. 10 June. Available online at: https://www.wto.org/english/news_e/news22_e/covid_10jun22_e.htm (accessed July 7, 2022). Msomi, N., Lessells, R., Mlisana, K., and de Oliveira, T. (2021). Africa: tackle HIV and COVID-19 together. Nature 600, 33–36. doi: 10.1038/d41586-021-03546-8 Mukae, H., Yotsuyanagi, H., Ohmagari, N., Doi, Y., Imamura, T., Sonoyama, T., et al. (2022a). A randomized phase 2/3 study of ensitrelvir, a novel oral SARS- CoV-2 3C-like protease inhibitor, in japanese patients with mild-to-moderate COVID-19 or asymptomatic SARS-CoV-2 infection: results of the phase 2a part. Antimicrob. Agents Chemother. doi: 10.1128/aac.00697-22. [Epub ahead of print]. Yamasoba, D., Kosugi, Y., Kimura, I., Fujita, S., Uriu, K., Ito, J., et al. (2022). Neutralisation sensitivity of SARS-CoV-2 omicron subvariants to therapeutic monoclonal antibodies. Lancet Infect. Dis. 22, 942–943. doi: 10.1016/S1473-3099(22)00365-6 04 Frontiers in Microbiology frontiersin.org
https://openalex.org/W3113574166	http://revistas.ubiobio.cl/index.php/RU/article/download/4143/56	Spanish; Castilian	null	Permeabilidad del espacio indígena. Discursos de propietarios Mapuche sobre la expansión urbana en el periurbano de Temuco, Araucanía-Chile	Urbano	2,020	cc-by	8,419	PERMEABILIDAD DEL ESPACIO INDÍGENA DISCURSOS DE PROPIETARIOS MAPUCHE SOBRE LA EXPANSIÓN URBANA EN EL PERIURBANO DE TEMUCO, ARAUCANÍA-CHILE1 PERMEABILITY OF THE INDIGENOUS SPACE. DISCOURSES OF MAPUCHE LANDOWNERS ON URBAN EXPANSION IN PERIURBANIAN TEMUCO, ARAUCANÍA-CHILE Recibido: 27-05-2020 Aceptado: 13-11-2020 Recibido: 27-05-2020 Aceptado: 13-11-2020 Recibido: 27-05-2020 Aceptado: 13-11-2020 ERIC ITURRIAGA GUTIÉRREZ 2 FÉLIX ROJO MENDOZA 3 MIGUEL ESCALONA ULLOA 4 Este trabajo se desarrolló en el marco del Fondecyt Regular Nº 1201255, “Los gustos espaciales en la producción de espacios urbanos dentro del Chile neoliberal: el caso de Temuco-Padre Las Casas e Iquique-Alto Hospicio”. 1 Magíster en Planificación y Gestión Territorial Municipio de Lonquimay, Araucanía, Chile Coordinador, Gestor Comunitario y AFI programa familias https://orcid.org/0000-0002-2614-6302 erichiturriaga@gmail.com 2 Doctor en Geografía Universidad Católica de Temuco, Temuco, Chile Profesor Asociado, Departamento de Sociología y Ciencia Política https://orcid.org/0000-0001-5794-5652 frojo@uct.cl 3 Doctor en Estudios Urbanos Universidad Católica de Temuco, Temuco, Chile Profesor Asistente Departamento de Ciencias Ambientales https://orcid.org/0000-0002-7597-4868 mescalon@uct.cl 4 DOI: https://doi.org/10.22320/07183607.2020.23.42.10 Las ciudades se caracterizan por ejercer una constante presión sobre el suelo periurbano rural. Las lógicas bajo las cuales opera el mercado inmobiliario y distintos otros agentes sumado a la flexibilidad de los instrumentos de planificación urbana que regulan el territorio, hace de los entornos urbanos espacios en permanente cambio. Temuco, una de las ciudades intermedias más importantes de Chile en cuanto al número de población, se ha desarrollado a partir de estas mismas lógicas. Sin embargo, y a diferencia de otras ciudades chilenas, la presencia de Áreas de Protección de Territorio Indígena (APTI), asociadas a comunidades mapuche, establece barreras legales que impiden el crecimiento convencional de la ciudad. De igual forma, es posible observar cómo en las últimas décadas, estas tierras han sido permeables a distintos usos, fuera de las dimensiones que supuestamente protege la ley. El presente trabajo explora los discursos de propietarios mapuche de suelo periurbano respecto a los cambios que estas áreas han experimentado en el último tiempo producto de la expansión de la ciudad. Para ello, se realizaron 20 entrevistas a propietarios mapuche de zonas periurbanas aledañas a Labranza, área urbana de Temuco, las cuales fueron analizadas bajo los parámetros de la Teoría Fundamentada. Entre los resultados obtenidos, destacan las estrategias de presión sobre este suelo provenientes de distintos agentes privados, la pérdida de sentido ancestral de la tierra por parte de algunas comunidades mapuche, que terminan vendiendo bajo distintos resquicios legales y la resistencia a la intromisión externa que aún persiste en mucho de ellos. Esto último evidencia que las estrategias de resistencia mapuche no sólo existen en territorios afectados por la intervención forestal, sino también en aquellos espacios que son tensionados por el rápido crecimiento de las ciudades. Palabras clave: extensión al medio rural, población indígena, planificación urbana, suburbios, urbanización. PERMEABILIDAD DEL ESPACIO INDÍGENA. DISCURSOS DE PROPIETARIOS MAPUCHE SOBRE LA EXPANSIÓN URBANA EN EL PERIURBANO DE TEMUCO, ARAUCANÍA-CHILE ERIC ITURRIAGA GUTIÉRREZ, FÉLIX ROJO MENDOZA, MIGUEL ESCALONA ULLOA REVISTA URBANO Nº 42 / NOVIEMBRE 2020 - ABRIL 2021 Las modalidades de dicha resistencia que hoy ejercen las comunidades mapuche se relacionan, entre otros ámbitos, con la expansión urbana sobre suelo indígena protegido legalmente. En este contexto, la ciudad de Temuco (Chile) ha estado históricamente circunscrita a un territorio rodeado por suelo mapuche protegido por el Estado, el cual configura un llamado “cinturón suicida”5, expresión que no sólo se utiliza metafóricamente para dar cuenta de los bolsones de pobreza y vulnerabilidad existentes durante la primera mitad del siglo XX (Foerster y Montecino, 1988), sino también como restricción geográfica para el desarrollo urbano futuro de la ciudad. A pesar de ello, y más allá de esta importante característica en términos de la posesión de la tierra circundante, en la actualidad es posible observar que este territorio protegido es cada vez más permeable a la urbanización. Las razones de aquello son fundamentalmente dos: por un lado, el creciente interés por la amenidad natural que, de forma paralela, es acompañado por un cambio de sentido asociado al declive de la vida urbana en términos subjetivos que experimentan muchos habitantes de las ciudades, fenómeno conocido como contraurbanización; y, por otro lado, la falta de vinculación reglamentaria del suelo en estas zonas. En referencia a esto último, es posible constatar un desacople entre los planes que regulan la ciudad, ya que mientras el Plan Regulador Comunal de Temuco (PRCT) vigente desde 2010 sólo se encarga de la zonificación de los distintos usos dentro de la ciudad, la ley indígena N°19.253 regula el destino de las tierras indígenas periféricas al área urbana, denominadas Áreas de Protección de Territorio Indígena (APTI) e identificadas en el artículo 15 de la ordenanza del Plan (Rojo, Alvarado, Olea y Salazar, 2020). La existencia de este periurbano protegido se vincula con el actuar del Estado chileno sobre suelo mapuche. El proceso de unificación territorial iniciado por el Estado en la segunda mitad de siglo XIX significó la movilización del ejército hacia el sur, razón por la cual las comunidades mapuche presentes en la Araucanía, tal como ocurrió en su momento con los españoles, establecieron una resistencia armada en defensa de su territorio (Viera, 2015). 5 En 1946 el Diario Austral utilizaba esta expresión para dar cuenta de los problemas que estas áreas representaban para el desarrollo urbano de Temuco. 6i ERIC ITURRIAGA GUTIÉRREZ 2 FÉLIX ROJO MENDOZA 3 MIGUEL ESCALONA ULLOA 4 Cities are characterized by exerting constant pressure on peri-urban rural land. The logics under which the real estate market and other different agents operate, together with the flexibility of urban planning instruments that regulate the territory, means urban space environments are permanently changing. Temuco, one of the most important intermediate cities in Chile in terms of population numbers, operates under the same logics. However, unlike other Chilean cities, the presence of Indigenous Territory Protection Areas (APTI) associated with Mapuche communities, establishes legal barriers that impede the conventional growth of the city. Likewise, it is possible to see how in recent decades these lands have been permeable to different uses, outside the dimensions supposedly protected by law. This work explores the discourses of Mapuche peri-urban landowners regarding the changes that these areas have recently undergone as a result of the city’s expansion. To do this, 20 interviews were conducted with Mapuche landowners from peri-urban areas around Labranza, an urban area of Temuco, which were analyzed under the parameters of the Grounded Theory. Among the results obtained, the following stand out: pressure strategies on this land from different private agents, the loss of ancestral sense of the land by some Mapuche communities that end up selling under different legal loopholes, and the resistance to external interference that still persists in many of them. The latter shows that there are Mapuche resistance strategies not only in territories affected by forestry intervention, but also in those spaces under stress from the rapid growth of cities. Keywords: extension to rural areas; indigenous population; urban planning; suburbs; urbanization Uno de los instrumentos que contribuyó a ello fue la ley promulgada el 4 de diciembre del año 1866, la cual ordenaba deslindar los terrenos pertenecientes al pueblo mapuche y otorgar un Título de Merced sobre predios rurales (Almonacid, 2009; Chihuailaf, 2014). Estos últimos estaban ubicados fuera de los límites de las ciudades que nacían en la Araucanía, las que por lo general tendían a ser habitadas por colonos chilenos, alemanes, suizos y franceses (Ferrando, 2012). I. INTRODUCCIÓN I. INTRODUCCIÓN Las acciones de resistencia por parte del pueblo mapuche se remontan a los actos fundacionales de asentamientos españoles ocurridos en 1552 al interior del Wallmapu/Araucanía. De esta manera, el conquistador español se transforma en el primer actor con el cual tuvieron que lidiar las comunidades indígenas, quien, frente a la imposibilidad de dominar el territorio araucano, se vio en la obligación de parlamentar y sellar algunos tratados de buena vecindad (Pinto, 2003). Transcurridos tres siglos, el Estado chileno se constituye en un segundo actor frente al que el pueblo mapuche establece resistencias; mismas que fracasarán a finales del siglo XIX, cuando comenzará, en consecuencia, el desplazamiento de gran parte de estas comunidades a sectores precordilleranos, lejos de los espacios de significación ancestral. En la actualidad, las formas de invasión del territorio mapuche parecen ser más sutiles, sin la imposición de la fuerza física que caracterizó a estos dos agentes mencionados. No obstante, se lleva a cabo, de igual modo, una incursión en estos territorios indígenas por parte de nuevos actores, razón por la cual las comunidades indígenas han tenido que adaptar distintas estrategias de resistencia. Si bien la ley que otorgaba Títulos de Merced tenía la intención de reparar los daños ocasionados por la incursión del Estado en la Araucanía, los propietarios mapuche fueron igualmente víctimas de numerosas ventas fraudulentas de suelo, cobros indebidos por deuda y otras estafas que mermaron su control respecto a las tierras entregadas (Pinto, 2003; López, Valenzuela y Carrasco, 2017). Con el fin de subsanar estos problemas, y a partir de un parlamento6 realizado en Nueva Imperial el año 1993, el presidente Patricio Aylwin reconoce la importancia de recuperar las tierras ancestrales, para lo cual crea la CONADI (Corporación Nacional de Desarrollo Indígena), que presenta entre sus funciones la compra y repartición de tierras a comunidades mapuche (López et al., 2017). Así, y a partir del artículo 13 de la Ley Indígena 19.253, se declara que estas tierras no podrán ser enajenadas, embargadas, gravadas, ni adquiridas por prescripción, salvo entre comunidades o personas de una misma etnia. 6 Los “parlamentos” son reuniones que el pueblo mapuche sostuvo primero con los españoles y luego con el Estado ch resolver los conflictos entre ambas partes. PERMEABILIDAD DEL ESPACIO INDÍGENA. DISCURSOS DE PROPIETARIOS MAPUCHE SOBRE LA EXPANSIÓN URBANA EN EL PERIURBANO DE TEMUCO, ARAUCANÍA-CHILE ERIC ITURRIAGA GUTIÉRREZ, FÉLIX ROJO MENDOZA, MIGUEL ESCALONA ULLOA REVISTA URBANO Nº 42 / NOVIEMBRE 2020 - ABRIL 2021 Una vez derrotado el pueblo mapuche, el Estado reclama las tierras como propias, para luego subastarlas a privados o entregárselas a inmigrantes no nacionales que comenzaron el proceso de colonización (Henríquez, 2013) y explotación de sus fértiles tierras, transformando a esta zona en el granero de Chile y, posteriormente, en uno de los íconos de la industria forestal (Escalona, 2020). Considerando lo indicado, los juegos por el control de este territorio tienen distintas aristas. Entre ellas, la intención por parte del sistema político de cambiar la actual prohibición de 5 En 1946 el Diario Austral utilizaba esta expresión para dar cuenta de los problemas que estas áreas representaban para el desarrollo urbano de Temuco.i son reuniones que el pueblo mapuche sostuvo primero con los españoles y luego con el Estado chileno con el fin de entre ambas partes. Ahora bien, los procesos de contraurbanización en Temuco enlazados, tanto al capital financiero, como a la actuación particular de distintas clases sociales, producto del gusto por la amenidad natural, se enfrentan a la presencia de restricciones para la expansión urbana convencional. El motivo de ello: un periurbano vinculado a Títulos de Merced indígenas (Figura 1); situación que introduce una serie de limitaciones para el desplazamiento (estacional, de segunda residencia) o migración (definitiva, asociada a la oferta del mercado inmobiliario) hacia el periurbano de la ciudad. venta de estas tierras7, lo cual abre la posibilidad de que algunas comunidades indígenas comiencen a interesarse en negociar sus tierras dentro del mercado de suelos. Producto de estos intereses, las consecuencias futuras sobre el área metropolitana funcional de Temuco serán significativas en cuanto a compactación y densificación de la mancha urbana (Rojo et al., 2020). Ahora bien, los procesos de contraurbanización en Temuco enlazados, tanto al capital financiero, como a la actuación particular de distintas clases sociales, producto del gusto por la amenidad natural, se enfrentan a la presencia de restricciones para la expansión urbana convencional. El motivo de ello: un periurbano vinculado a Títulos de Merced indígenas (Figura 1); situación que introduce una serie de limitaciones para el desplazamiento (estacional, de segunda residencia) o migración (definitiva, asociada a la oferta del mercado inmobiliario) hacia el periurbano de la ciudad. II. MARCO TEÓRICO Una parte importante de los trabajos referidos a la extensión de la ciudad sobre el suelo rural han sido abordados a partir de la noción de contraurbanización, esto es, el proceso bajo el cual la población se ha movido hacia el campo, desconcentrando físicamente los territorios (Mitchell, 2004). El debate en torno a este fenómeno tiende a relevar los motivos que llevan a concretar este desplazamiento residencial, entre de los cuales el gozo por los espacios naturales es el más mencionado. Por tal razón, la contraurbanización es también denominada “migración por amenidad”; expresión que pone énfasis en los movimientos de población vinculados a la percepción que las personas tienen respecto a que su calidad de vida será mejor en lugares alejados de la ciudad (Hidalgo, Borsdorf y Plaza, 2009; Janoschka, 2013; Vergara, Sánchez y Zunino, 2019). Independiente de la denominación específica, lo cierto es que este fenómeno de desplazamiento y creación de nuevos nodos de asentamientos humanos está transformando socialmente las áreas rurales, haciendo que la población nativa coexista con distintos tipos de habitantes neorrurales, estos últimos, con intereses variados (Méndez, 2014). Una causa que permite explicar las presiones del fenómeno de contraurbanización sobre territorios indígenas son las políticas urbanas tradicionales que han operado históricamente sobre las ciudades; estas se han caracterizado por establecer usos de suelo y categorías de ordenación que terminan por desarticular cualquier atisbo de organización del territorio indígena. En este marco, la presencia de las ciudades rememora la dominación política y el traspaso de un modelo cultural que instaló por distintas partes del mundo la sociedad occidental (Soja, 1996). En el caso chileno, la fundación de las ciudades en la antigua frontera araucana justificó una serie de acciones coloniales (De Ramón, 1992) que acabaron por promover un sentido común dominante, a partir del que el pueblo mapuche y su vivencia alejada de la ‘apretura’ de las ciudades fueron vistos como ‘primitivos o salvajes’ (De Ovalle, 1646). Esta construcción negativa del otro o, como la llama Spivak (1998), violencia epistémica, se fundamenta en el intento de dominar en nombre de una supremacía cultural (Bhabha, 2002). Dichas acciones de distinción colonial se han mantenido hasta nuestros días, reflejándose en la producción de suelo urbano que experimentan las comunidades mapuche ubicadas en las proximidades de las ciudades. PERMEABILIDAD DEL ESPACIO INDÍGENA. DISCURSOS DE PROPIETARIOS MAPUCHE SOBRE LA EXPANSIÓN URBANA EN EL PERIURBANO DE TEMUCO, ARAUCANÍA-CHILE ERIC ITURRIAGA GUTIÉRREZ, FÉLIX ROJO MENDOZA, MIGUEL ESCALONA ULLOA REVISTA URBANO Nº 42 / NOVIEMBRE 2020 - ABRIL 2021 El presente trabajo explora y describe los discursos de propietarios mapuche del periurbano de Temuco respecto a los cambios que ha experimentado este suelo, con el propósito de comprender los procesos de presión que han sufrido estos territorios por parte de diferentes actores, privados e institucionales, ligados al mercado inmobiliario y a la planificación urbana. En este sentido, se entiende por “comunidades mapuche” aquellos grupos que habitan las APTI ubicadas en el entono de la ciudad. En términos globales, la expansión constante de las políticas y planes urbanos sobre espacios indígenas ha provocado una serie de tensiones. Entre estas, destacan el juego simbólico de inclusión y exclusión que enfrentan muchos de los habitantes de estas zonas en América Latina, motivo por el cual deben pensar constantemente su etnicidad y las propias nociones de comunidad (Herrera, 2018); así también, los problemas de administración y gestión de estas tierras -fruto de la ausencia de títulos de propiedad y políticas de planificación, en países como Australia (Wilson et al., 2018)-; y, por último, el poder parcial que ejercen las autoridades tradicionales en estas áreas, debido al rol preponderante que, en concreto, tienen las instituciones gubernamentales de algunos países africanos en su gestión (Brandful, Osei y Asuama, 2020). 7 Parte importante de la consulta indígena del año 2019 estuvo centrada en los títulos de dominios y la posibilidad de las comunidades mapuche de arrendarlas o venderlas. PERMEABILIDAD DEL ESPACIO INDÍGENA. DISCURSOS DE PROPIETARIOS MAPUCHE SOBRE LA EXPANSIÓN URBANA EN EL PERIURBANO DE TEMUCO, ARAUCANÍA-CHILE ERIC ITURRIAGA GUTIÉRREZ, FÉLIX ROJO MENDOZA, MIGUEL ESCALONA ULLOA REVISTA URBANO Nº 42 / NOVIEMBRE 2020 - ABRIL 2021 Figura 1. Títulos de Merced en la comuna de Temuco. Fuente: Elaboración de los autores en base a información de CONADI (2018). Diseño cartográfico de Camila Salinas. aledañas a Labranza, área desacoplada del espacio urbano consolidado de Temuco, que surge como respuesta a la falta de suelo urbanizable en la ciudad para los sectores más pobres durante la década del noventa (Figura 1). comunidades que habitan en su entorno resulta un aspecto central, razón por la cual, tal de cómo ocurrió a fines del siglo XIX, el avance urbano niega la diferencia y la existencia de otras maneras de habitar el territorio constituidas desde la historicidad del pueblo mapuche (Lincopi, 2015; Mansilla e Imilan, 2020). Se estableció, entonces, la estrategia del muestreo teórico basado en la Teoría Fundamentada (Strauss y Corbin, 2002), para la selección final de 20 propietarios mapuches, todos los cuales pertenecían a distintos Títulos de Merced y comunidades indígenas (Tabla 1). Los criterios para el cumplimiento de esta selección fueron los siguientes: a) que los propietarios se encontraran cerca de zonas residenciales, para lo que se definieron tres macrozonas de búsqueda (Figura 2); b) que el propietario tuviera un dominio vigente de la propiedad (titularidad) o que esta se hallara en estado de sucesión de herencia; y c) que el terreno no se encontrara en calidad de suelo eriazo. A pesar de la relevancia del tema, no existen trabajos que aborden en profundidad la presión de distintos intereses sobre el territorio periurbano indígena de Temuco. Lo que se conoce sobre esta ciudad aborda la presencia desigual de grupos sociales en el espacio (Garín, Salvo y Bravo, 2009; Vergara, 2019), las disputas por el derecho a la ciudad y la vivienda a mediados del siglo pasado (Vergara, Gola y Huiliñir, 2015), o bien, los procesos de crecimiento urbano del último tiempo (Marchant et al., 2016; Rojo et al., 2019). Si bien existen algunos escritos que exponen el potencial de conflictividad que representan las presiones de expansión urbana sobre suelo indígena (Quiñones y Gálvaez, 2015; Rojo et al., 2019; Mansilla e Imilan, 2020), no profundizan en las aristas particulares que representan estos nudos críticos para el futuro. PERMEABILIDAD DEL ESPACIO INDÍGENA. DISCURSOS DE PROPIETARIOS MAPUCHE SOBRE LA EXPANSIÓN URBANA EN EL PERIURBANO DE TEMUCO, ARAUCANÍA-CHILE ERIC ITURRIAGA GUTIÉRREZ, FÉLIX ROJO MENDOZA, MIGUEL ESCALONA ULLOA REVISTA URBANO Nº 42 / NOVIEMBRE 2020 - ABRIL 2021 Durante julio del año 2019, a todos estos propietarios se les aplicó entrevistas semi-estructuradas que contenían preguntas relacionadas con dimensiones socio-espaciales, como el apego cultural a la tierra, el uso actual de la propiedad, las percepciones sobre el avance de la ciudad y el rol de la tierra para el desarrollo futuro de sus comunidades. 128 II. MARCO TEÓRICO Entre los aspectos que caracterizan esta producción espacial, la negación de la cualidad indígena de la tierra, territorio y territorialidad de las La ciudad de Temuco no es ajena a este proceso de contraurbanización. En efecto, y producto de la intensificación del capital financiero sobre las ciudades (Harvey, 2014; Méndez, 2018), ámbito bajo el cual se acumula el capital en la economía global actual (Engels, [1873] 2006; Smith, 2012), existe un creciente interés del mercado inmobiliario por urbanizar zonas periurbanas destinadas a grupos de alta renta (Rojo, Jara y Frick, 2019; Marchant, Frick y Vergara, 2016; Vergara, 2019). Asimismo, los gustos espaciales de amenidad que ofrecen estas áreas, que terminan por masificar la idea de una “casita en el campo”, determina que muchas personas estén pensando en desplazarse a estas zonas (Rojo, 2019). que terminan por masificar la idea de una “casita en el campo”, determina que muchas personas estén pensando en desplazarse a estas zonas (Rojo, 2019). Figura 1. Títulos de Merced en la comuna de Temuco. Fuente: Elaboración de los autores en base a información de CONADI (2018). Diseño cartográfico de Camila Salinas. III. METODOLOGÍA El resultado de esta estrategia fue la creación de tres categorías centrales: cotidianidad bajo presión; permeabilidad de agentes privados y fin de la territorialidad mapuche; y resistencia y proyecciones de lucha por la tierra. La información cualitativa proporcionada por las entrevistas fue complementada con la elaboración de cartografías, que permitieron expresar espacialmente las macrozonas de Labranza que en la actualidad están siendo permeadas por el capital financiero. Su digitalización y procesamiento fue realizado utilizando Sistemas de Información Geográfica (ArcGis 10.5) y empleando fuentes obtenidas de CONADI y del propio territorio de indagación (en lo que respecta a los usos del suelo actual). Se viene con todo, hoy día la inmobiliaria tiene la aprobación del municipio. Porque se está aplicando el plan regulador en la comunidad, no se han hecho las consultas correspondientes para llevar el convenio 169 (...) se han hecho decretos y miles de cosas. Pero nosotros no tenemos idea de lo que ellos hacen. (Mujer, 50 años, Zona oeste) III. METODOLOGÍA Para responder al objetivo del trabajo se decidió focalizar la búsqueda de propietarios mapuches en zonas periurbanas Las entrevistas fueron procesadas con el software ATLAS ti.8 y analizadas siguiendo los lineamientos de la comparación Macro zonas N° y nombre titulos de merced N° de personalidad juridica y nombre de comunidad indigena N° entrevistados MZ Norte 262-Ignacio Elgueta 88-Ignacio Elgueta 1 2102-Ignacio Elgueta 2 1 347-Jose Cheuquean 1505-Jose cheuquian 1 2115-Jose cheuquian 2 1 258-Nahuelguen 1656-Nahuelguen 2 346-Antonio Colines 1613-Antonio coline 2 MZ Oeste 347-Jose Cheuquean 1505-Jose cheuquian 1 2115-Jose cheuquian 2 1 258-Nahuelguen 1656-Nahuelguen 3 422-Antonio Huaiquilaf 1943-Antonio Huaiquilaf 2 MZ Este 360-Juan Huaiquinao 960-Juan Huaiquinao 2 259-Hueche Huenulaf 1694-Hueche Huenulaf 1 362-Antonio Huala 1775-Antonio Huala 2 TOTAL 8 11 20 Tabla 1. Descripción general de las comunidades indígenas contempladas en el estudio y número de entrevistas realizadas. Fuente: Elaboración de los autores. 129 PERMEABILIDAD DEL ESPACIO INDÍGENA. DISCURSOS DE PROPIE LA EXPANSIÓN URBANA EN EL PERIURBANO DE TEMUCO, ARAUC ERIC ITURRIAGA GUTIÉRREZ, FÉLIX ROJO MENDOZA, MIGUEL ESC REVISTA URBANO Nº 42 / NOVIEMBRE 2020 - ABRIL 2021 PÁG. 124 - 134 ISSN 0717 3997 / 0718 3607 Figura 2. Macrozonas aledañas a Labranza contempladas en la búsqueda y selección de entrevistados. Fuente: Elaboración de los autores en base a CONADI (2020). Figura 2. Macrozonas aledañas a Labranza contempladas en la búsqueda y selección de entrevistados. Fuente: Elaboración de los autores en base a CONADI (2020). zonas aledañas a Labranza contempladas en la búsqueda y selección de entrevistados. Fuente: Elaboración de los autores e 2020) En el contexto del periurbano de Temuco, la falta de suelo urbano en áreas consolidadas de la ciudad hace que el mercado inmobiliario movilice una serie de actividades sobre territorio indígena. Entre ellas, los propietarios mapuche reconocen, en primer lugar, una asociación entre el gobierno local que planifica y gestiona el uso del suelo en el periurbano y las empresas inmobiliarias que buscan ampliar la oferta de viviendas en estas áreas. Lo anterior se da, según el discurso de estos propietarios, obviando las normativas de protección que rigen sobre los pueblos indígenas en Chile: constante, principio que plantea la Teoría Fundamentada para ir explorando y descubriendo patrones latentes en el discurso social (Strauss y Corbin, 2002). PERMEABILIDAD DEL ESPACIO INDÍGENA. DISCURSOS DE PROPIETARIOS MAPUCHE SOBRE LA EXPANSIÓN URBANA EN EL PERIURBANO DE TEMUCO, ARAUCANÍA-CHILE ERIC ITURRIAGA GUTIÉRREZ, FÉLIX ROJO MENDOZA, MIGUEL ESCALONA ULLOA REVISTA URBANO Nº 42 / NOVIEMBRE 2020 - ABRIL 2021 Eso legalmente era una escombrera, pero el dueño ganaba más plata dejando la basura de Temuco, por lo que empezó a tirarnos de todo acá, la contaminación completa. Tuvimos que hacer una denuncia y pararon. Pero hoy se transformará en inmobiliaria. (Mujer, 50 años, Zona oeste) No nos pueden invadir o afectar nuestro vivir. Por ejemplo, allá arriba está una empresa que se llama San Pablo, y a la vista se ve inofensiva, pero atrás de esa empresa viven familias y este tipo, a veces, a las 5 de la mañana echa a andar un camión y despierta a todos, entonces ya hay una contaminación acústica. (Hombre, 57 años, Zona este) IV. RESULTADOS Sin embargo, esta actuación deliberada por parte de las autoridades municipales y el mercado inmobiliario tendría una dimensión de presión aplicada directamente sobre el territorio mapuche, a la que se suman aquellas empresas que se han instalado en los últimos años en el sector. Estas presiones generan molestias en la cotidianidad de las comunidades mapuche, que terminan por afectar la calidad de vida de las personas que habitan estas áreas: Tal como ya lo mencionaba Alonso de Ovalle (1646) en sus descripciones, la vivencia del espacio para el pueblo mapuche se caracteriza por una relación estrecha con (en) la naturaleza. Sin embargo, este tipo de relaciones con el espacio no resultaban propicias para llevar a cabo la explotación del Wallmapu/ Araucanía. Su uso común no era el más adecuado para acumular capital, o bien, instalar algún ciclo productivo (Escalona y Barton, 2020; Escalona 2020). Teniendo presente estos elementos, el análisis de los discursos actuales da cuenta del cambio en la vida cotidiana que ha ocurrido a lo largo de la historia regional, pero que se ha intensificado en aquellos espacios de ‘frontera’ entre la ciudad y las comunidades mapuche. La manifestación de permeabilidad del suelo indígena respecto al avance de la ciudad se expresa en la diversificación de actividades industriales y la intensificación del mercado inmobiliario en Labranza y sus entornos. Como consecuencia de lo anterior, pueden generarse problemas de conectividad social y reemplazos en los usos de suelo tradicionalmente presentes en el área (Gasic, 2018; Elorza, 2019). La cotidianidad bajo presión Las presiones del sector inmobiliario e industrial presente en la zona también se expresan a través de las consecuencias que su acción ha dejado en el territorio. Así, por ejemplo, se puede señalar la aparición de una serie de poblaciones habitadas por sectores de clases medias y bajas, construidas por empresas inmobiliarias, muchas de las cuales están planificadas sobre la base de la política de vivienda subsidiaria en Chile. Una primera categoría central reconocida en los discursos de propietarios se relaciona con las presiones que reciben permanentemente las comunidades mapuche, por parte de distintos agentes que quieren utilizar sus tierras. Se sabe que la presión que ejerce la ciudad sobre suelo periurbano genera una serie de conflictos, entre los cuales destacan las disputas entre el uso residencial y agrícola (Ávila, 2009; Hidalgo et al., 2009), la presión constante de la actividad inmobiliaria para ampliar el horizonte de actuación del capital financiero sobre suelo rural (Jiménez, Hidalgo, Campesino y Alvarado, 2018), y la propia historia e inercia del funcionamiento rural, que marca distinciones importantes con las formas de operar en la ciudad (Haller, 2017). Me encanta el campo y defiendo el campo. Cuido mis árboles nativos, elimino los árboles exóticos. Las poblaciones irrumpen a la comunidad, porque piensan que todo esto les pertenece. Salen a la hora que quieren a cazar, caminar, pescar. (Mujer, 42 años, Zona norte) Figura 3. Usos de suelo asociados a áreas mapuche del entorno de Labranza. Fuente: Elaboración de los autores. Figura 3. Usos de suelo asociados a áreas mapuche del entorno de Labranza. Fuente: Elaboración de los autores. Sé que algunas comunidades (mapuche) sí han salido afectadas, porque ahora tienen la población muy cerca de ellos. (Mujer, 24 años, Zona oeste) Sé que algunas comunidades (mapuche) sí han salido afectadas, porque ahora tienen la población muy cerca de ellos. (Mujer, 24 años, Zona oeste) vida suburbana, en el marco de un proceso de contraurbanización que afecta a zonas cercanas a la ciudad. Lo dicho se refleja en la construcción de villas con loteos irregulares de 50 o 60 casas cada una y en la inclusión de territorio rural mapuche como zona urbana dentro del Plan Regulador Comunal de Temuco, lo que termina por evidenciar que determinadas áreas del periurbano pueden proyectar usos distintos hacia el futuro, entre ellos, los relacionados con la amenidad (Figura 3). La cotidianidad bajo presión Es posible, de esta manera, reconocer una presión permanente de la ciudad y su avance hacia zonas periurbanas mapuche. Los propietarios mapuche reconocen que esta forma de operar no sólo proviene del sector privado, que presentaría motivos coherentes respecto a la posibilidad de maximizar capital a partir del suelo; es además respaldada por una cierta pasividad de la institucionalidad pública representada por el municipio, la cual incluso dejaría ciertas ambigüedades al momento de planificar la proyección de la ciudad en su entorno. Estas acciones han suplantado el habitar mapuche, que se caracterizaba por su vivencia en lofs8, desde donde gestionaban su subsistencia mediante prácticas de recolección, cultivo y trueque. A raíz de las múltiples presiones que las comunidades mapuche reciben, muchos de los dueños de estas tierras han comenzado a vender, encontrando para ello resquicios que logran superar la prohibición que por ley caracteriza a estas áreas. Es por ello que esta segunda categoría central da cuenta del sentido otorgado, en los discursos analizados, a la encrucijada de aquellos habitantes que, finalmente, acaban por desprenderse de sus tierras. Entre los “ajustes legales” para la venta de tierra destacan la permuta por otros suelos (no necesariamente en el periurbano de la ciudad), el loteo de esas tierras adquiridas, la reventa permanente en el tiempo, la organización de las nuevas comunidades mapuche y, por último, la exigencia de regularización de dicho suelo por parte del gobierno local. Algunos de esos aspectos se ven expresados en los siguientes extractos de entrevistas: 8 En las comunidades mapuche los lofs corresponden a unidades familiares en donde el lonco es el jefe. Permeabilidad de agentes privados y fin de la territorialidad mapuche Una parte importante del territorio mapuche protegido por ley experimenta actualmente formas de irrupción residencial, dada la escasez de suelo urbano en el espacio consolidado de la ciudad o al encarecimiento de este debido, precisamente, a la falta de áreas urbanizables. Esto no sólo se conecta con el aumento en la presencia de proyectos asociados al mercado inmobiliario, sino también con el creciente número de personas interesadas en la Fue como esas permutas, gente que vendía y compraba un pedazo más lejos, como que cambiaba más tierra de lo que tenía y después esa persona compraba el lote y después 8 En las comunidades mapuche los lofs corresponden a unidades familiares en donde el lonco es el jefe. loteaban lotes chicos. Entonces empezaban a vender, entonces la gente como nunca hubo título y después se organizaron como villa y ahí empezaron a regularizarse. (Hombre, 40 años, Zona este) me dicen “señora le damos 100 millones por una hectárea”, ahí igual uno lo piensa, porque la plata igual la necesito. Yo sé que muchos han vendido por necesidad, pero también sé que la venta de tierra mapuche es pan para hoy, hambre para mañana. (Mujer, 52 Años, Zona oeste) Acá hay más casas de las permitidas por terreno, hay 3, 4 casas juntas (…) entonces nosotros queremos regularizar eso, pero que lo haga la municipalidad, no nosotros, porque eso tiene un costo y nosotros no lo vamos a pagar, porque hemos pagado el ser rurales en este espacio, estamos como al medio. (Hombre, 55 años, Zona este) Creo que con Labranza no hay nada más que hacer, yo creo que van a seguir vendiendo casas, pero hay comunidades indígenas que son bien fuertes en su petitorio. Yo creo que por ahí va a haber un estancamiento. (Mujer, 47 Años, Zona Oeste). Entre las estrategias que utilizan algunas comunidades mapuche para resistir al poder de expansión por parte del mercado inmobiliario principalmente, sobresalen aquellas referidas a la asesoría legal que reciben de organizaciones que luchan por los derechos de este pueblo, así como la proyección del estigma que los ha seguido durante las últimas décadas. Respecto a esta última estrategia, los discursos de propietarios mapuche destacan que el rasgo cotidiano atribuido al mapuche de “terrorista” se utiliza como una forma de persuasión al momento de querer impedir el trabajo de las empresas inmobiliarias en suelo indígena. PERMEABILIDAD DEL ESPACIO INDÍGENA. DISCURSOS DE PROPIETARIOS MAPUCHE SOBRE LA EXPANSIÓN URBANA EN EL PERIURBANO DE TEMUCO, ARAUCANÍA-CHILE ERIC ITURRIAGA GUTIÉRREZ, FÉLIX ROJO MENDOZA, MIGUEL ESCALONA ULLOA REVISTA URBANO Nº 42 / NOVIEMBRE 2020 - ABRIL 2021 Lo expuesto muestra que para algunos propietarios la extensión de la ciudad sobre suelo indígena protegido abre alternativas para que las comunidades mapuche puedan aprovechar los supuestos beneficios de la urbanidad, entre ellos, los ligados con la consecución de la vivienda propia y la regularización de aquellas ya existentes. Y esto no implica únicamente la proyección de zonas residenciales de alta densidad, asociadas a la extensión de la mancha urbana, también involucra la consolidación de viviendas para segunda residencia (relacionadas con parcelas de agrado). Porque ellos hacen un estadio que con lo que es la ley indígena se podía hacer, pero se olvidaron que no podían intervenir en la vida normal de los vecinos, y ellos impedían el libre tránsito de todos. (Hombre, 55 Años, Zona este) Cabe notar que esta categoría central presente en el discurso de propietarios mapuche se articula a partir de una dimensión relevante para construir las estrategias de resistencia frente a la intromisión del mercado inmobiliario: la defensa del territorio como eje identitario del pueblo mapuche: Permeabilidad de agentes privados y fin de la territorialidad mapuche Por otro lado, el cambio del espíritu de la ley indígena no sólo está movilizando la permuta y venta de tierras a personas no indígenas, sino también al interior de las propias comunidades mapuche. Es así como el hecho que el Plan Regulador Comunal contemple como urbano al suelo mapuche protegido, es visualizado por algunos propietarios como un aspecto positivo. Y, frente a esta realidad, piden además ser tratados para todos los efectos como comunidades mapuche urbanas: Estamos quedándonos como urbano y porque hace 10 años lo somos…porque nosotros no queremos dejar de ser urbanos, pero queremos que nos traten como urbanos, pero vivimos como rurales. Entonces lo que queremos (…) es que nosotros no tenemos ninguna casa regularizada, porque nosotros no existimos en la municipalidad y si queremos postular a algún mejoramiento de vivienda, no podemos. (Hombre, 55 años, Zona este) Entonces al final dando bote, bote y bote, no nos quedó de otra que nosotros mismos hacerle presión a la empresa. Le paramos la obra y como hay un estigma de la quema de camiones, la gente se las ingenió y llenaban bidones con agua, para que pensaran que era combustible y por miedo empezaron a negociar con el gerente, el abogado y otra gente más. (Mujer, 27 Años, Zona norte) Resistencia y proyecciones de lucha por la tierra Nosotros no queremos vender en el futuro a ninguna inmobiliaria, no vender a ningún huinca (…) Así como los espacios de los menokos, los que vieron son los pocos que quedan (...) de hecho el que queda debería llamarse Menoko de Huetrolhue, porque está en Huetrolhue, es parte de nuestra cultura encontrarse con el Menoko, si ese se seca perdería la identidad (...) de hecho la gente de las poblaciones ve esto como campo, pero no como comunidad. (Mujer, 27 Años, Zona norte) Independiente del hecho que muchas comunidades mapuche han ofertado sus tierras dentro del mercado del suelo convencional, lo cierto es que de igual forma existen muchos propietarios que declaran la resistencia como una categoría central importante al momento de estructurar los discursos respecto a la expansión de la ciudad. Aunque no relativizan el alto valor que sus tierras tienen en estos momentos, a causa del crecimiento urbano de Temuco -y considerando la tentación que eso significa en el contexto de una población mapuche que presenta una serie de necesidades materiales-, ellos expresan que la resistencia dentro de las comunidades mapuche del sector es ciertamente fuerte: En este sentido, la pérdida de suelo mapuche implica que una parte importante de la espiritualidad de estas comunidades indígenas se diluye para siempre, rompiendo con ello prácticas ancestrales vinculadas a la tierra y lo que ésta les entrega. Ejemplo de lo anterior es el mencionado Menoko, el cual representa un sitio sagrado para La idea es poder resistir a esto, para mí, personalmente, es una resistencia. No podemos decir que la plata no atrae, porque si también se posiciona como un nuevo espacio de resistencia. No hay que olvidar que donde hay hegemonía, hay fuerzas contra- hegemónicas emergentes, con otras narrativas y reclamos. el pueblo mapuche por la diversidad de especies medicinales que allí se dan, y, por tanto, al valor que posee para la buena salud de la comunidad. Considerando los resultados, se desprende que el rol protector del territorio indígena asumido por el Estado durante el último siglo se desvanece en la práctica cuando se observa que las regulaciones del suelo operan en distintas direcciones y en diferentes escalas, configurando grises escenarios futuros, de la mano de la introducción de agentes externos a las comunidades mapuche. Resistencia y proyecciones de lucha por la tierra Y esta situación se vuelve aún más compleja cuando se discuten iniciativas que buscan modificar la Ley Indígena 19.253 en las dimensiones referidas al control y la protección territorial, lo cual se empalma con el interrumpido proceso de consulta indígena de 2019 que, entre otros aspectos, centraba muchas de las preguntas en temas relacionados con la propiedad de la tierra. Estos aspectos terminan por consolidar la idea -y su materialización en un futuro no muy lejano- de un periurbano indígena permeable a nuevos controladores. V. CONCLUSIONES El “cinturón suicida” con que alguna vez fue bautizado el periurbano de la ciudad, continúa siendo un espacio codiciado y adquirido por parte de agentes externos a las comunidades mapuche. Y, más allá de los resguardos que el Estado intentó establecer para la defensa de la tierra indígena de la Araucanía, existen hoy una serie de estrategias que han facilitado la ocupación de estas áreas, permitiendo que se extiendan los espacios susceptibles de ser incorporados al mercado del suelo que opera en Temuco. Los discursos de propietarios mapuche de zonas periurbanas de la ciudad reconocen que la permeabilidad de su territorio no sólo se debe a la presión constante que ejerce el mercado inmobiliario para la expansión de la ciudad, ni al desplazamiento de personas que pretenden vivenciar la vida de campo, y que visibilizan el citado proceso de contraurbanización en constante crecimiento al interior de la comuna. Más bien, dicha permeabilidad estaría principalmente vinculada a una dimensión central: la flexibilidad en los instrumentos que regulan y resguardan la propiedad de la tierra indígena. VI. REFERENCIAS BIBLIOGRÁFICAS Almonacid, F. (2009). El problema de la propiedad de la tierra en el sur de Chile (1850-1930). Historia, 1(42), 5-56. DOI: http://dx.doi.org/10.4067/S0717- 71942009000100001 Almonacid, F. (2009). El problema de la propiedad de la tierra en el sur de Chile (1850-1930). Historia, 1(42), 5-56. DOI: http://dx.doi.org/10.4067/S0717- 71942009000100001 Ávila, H. (2009). Periurbanización y espacios rurales en la periferia de las ciudades. Estudios Agrarios, 15(41), 93-123. Ávila, H. (2009). Periurbanización y espacios rurales en la periferia de las ciudades. Estudios Agrarios, 15(41), 93-123. Con estos antecedentes, el rol que cumple la flexibilización de los instrumentos de planificación urbana, según el discurso de los propietarios mapuche, representa quizás uno de los aspectos operativos claves para entender muchos de los cambios que ha experimentado el suelo indígena. Desde esta perspectiva, las tácticas de expulsión de las comunidades que limitan con la ciudad ya no están relacionadas con el uso de la coerción violenta, sino más bien con el establecimiento de ciertas acciones ilegales que son complementadas con lagunas regulatorias que permiten interpretaciones laxas de la actual normativa de protección que rige sobre estas áreas. Bajo este supuesto, sería el propio Estado, representado en el gobierno local de Temuco, el que está facilitando las condiciones para que el suelo indígena deje de existir. En esa línea, el Plan regulador se convierte en un instrumento de planificación útil para acceder a una parte importante de la tierra indígena que rodea a esta ciudad. Bhabha, H. (2002). El lugar de la cultura. Buenos Aires: Manantial. Brandful, P., Osei M. y Asuama, YA. (2020) Urban land use planning in Ghana: Navigating complex coalescence of land ownership and administration. Land use policy, (99), 1-10. DOI: https://doi.org/10.1016/j.landusepol.2020.105054 Brandful, P., Osei M. y Asuama, YA. (2020) Urban land use planning in Ghana: Navigating complex coalescence of land ownership and administration. Land use policy, (99), 1-10. DOI: https://doi.org/10.1016/j.landusepol.2020.105054 Chihuailaf, A. (2014) El Estado chileno y la región de la Frontera a fines del siglo XIX. Amérique Latine Histoire et Mémoire. Les Cahiers ALHIM, (28). DOI: https://doi. org/10.4000/alhim.5108 De Ovalle, A. (1646). Histórica relación del Reyno de Chile y de las misiones y ministerios que exercita en el la Compañía de Jesús. Impreso en Roma por Francisco Caballo. Recuperado de http://www.memoriachilena.gob.cl/602/ w3-article-8380.html De Ramón, A. (1992) Urbanización y dominación. Reflexión acerca del rol de las ciudades en América Latina (1535-1625). Boletín de historia y geografía (Universidad Católica Blas Cañas), (12), 5-31. De Ramón, A. VI. REFERENCIAS BIBLIOGRÁFICAS (1992) Urbanización y dominación. Reflexión acerca del rol de las ciudades en América Latina (1535-1625). Boletín de historia y geografía (Universidad Católica Blas Cañas), (12), 5-31. Diario Austral (1946). El cinturón suicida de Cautín estrangula su progreso. Diario Austral, 8 de noviembre de 1946, Temuco. Recuperado de http:// biblioteca.clacso.edu.ar/clacso/gt/20190613035537/Mov_indigenas_y_ autonomias.pdf A pesar de las presiones ejercidas sobre áreas mapuche, facilitadas por la actuación activa del Estado en cuanto a los criterios de planificación territorial, persisten resistencias en las comunidades indígenas, emulando con ello las innumerables luchas que han debido enfrentar a lo largo de su historia por el resguardo de sus tierras. La relevancia de esta categoría central descrita en el trabajo es que revela un tipo de dinámicas de resistencia que se da en otro tipo de territorio mapuche, distinto al que comúnmente ha estado centrada la literatura, la cual se relaciona fundamentalmente con las estrategias de defensa del pueblo mapuche en áreas rurales ocupadas por empresas forestales (Marimán, Valenzuela y Cortés, 2015; Pineda, 2014). De esta forma, el periurbano de Temuco Elorza A, (2019). Segregación residencial y estigmatización territorial. Representaciones y prácticas de los habitantes de territorios segregados. EURE, 45(135), 91-109. DOI: http://dx.doi.org/10.4067/S0250-71612019000200091 Engels, F. (2006). Contribución al problema de la vivienda. Madrid: Fundación de Estudios Socialistas Federico Engels. Escalona Ulloa, M. (2020). Transformaciones territoriales en Wallmapu/ Araucanía. Una ecología política histórica. En Escalona Ulloa, M., Muñoz- Pedreros, A. y Figueroa Hernández, D. (Eds.), Gobernanza ambiental. Reflexiones y debates desde La Araucanía (pp. 19-70). Santiago: RIL Editores. Escalona Ulloa, M. (2020). Transformaciones territoriales en Wallmapu/ Araucanía. Una ecología política histórica. En Escalona Ulloa, M., Muñoz- Pedreros, A. y Figueroa Hernández, D. (Eds.), Gobernanza ambiental. Reflexiones y debates desde La Araucanía (pp. 19-70). Santiago: RIL Editores. Méndez-Sastoque, M. (2014). Una tipología de los nuevos habitantes del campo: aportes para el estudio del fenómeno neorrural a partir del caso de Manizales, Colombia. Revista de economia e sociologia rural, 51(1), 31-48. Méndez-Sastoque, M. (2014). Una tipología de los nuevos habitantes del campo: aportes para el estudio del fenómeno neorrural a partir del caso de Manizales, Colombia. Revista de economia e sociologia rural, 51(1), 31-48. Escalona Ulloa, M. y Barton J. (2020). A ‘Landscapes of Power’ framework for historical political ecology: The production of cultural hegemony in Araucanía-Wallmapu. Area, 52(2), 445-454. Ferrando, R. (2012). Y así nació la frontera. Temuco: Ediciones Universidad Católica de Temuco, en Gobernanza Mitchell, C. (2004). PERMEABILIDAD DEL ESPACIO INDÍGENA. DISCURSOS DE PROPIETARIOS MAPUCHE SOBRE LA EXPANSIÓN URBANA EN EL PERIURBANO DE TEMUCO, ARAUCANÍA-CHILE ERIC ITURRIAGA GUTIÉRREZ, FÉLIX ROJO MENDOZA, MIGUEL ESCALONA ULLOA REVISTA URBANO Nº 42 / NOVIEMBRE 2020 - ABRIL 2021 Rojo, F., Jara, T. y Frick, J. (2019). Las urbanizaciones cerradas en la ciudad intermedia: El caso de Temuco, 2005-2014. Revista Bitácora Ubano-Territorial, 29(1), 79-90. DOI: http://dx.doi.org/10.15446/bitacora.v29n1.63192 Rojo, F., Jara, T. y Frick, J. (2019). Las urbanizaciones cerradas en la ciudad intermedia: El caso de Temuco, 2005-2014. Revista Bitácora Ubano-Territorial, 29(1), 79-90. DOI: http://dx.doi.org/10.15446/bitacora.v29n1.63192 Hidalgo, R., Borsdorf, A. y Plaza, F. (2009). Parcelas de agrado alrededor de Santiago y Valparaíso: ¿Migración por amenidad a la chilena? Revista Geografía Norte Grande, (44), 93-112. Smith, N (2012). La nueva frontera urbana. Ciudad revanchista y gentrificación. Madrid: Edición Madrid Traficantes de Sueños. Smith, N (2012). La nueva frontera urbana. Ciudad revanchista y gentrificación. Madrid: Edición Madrid Traficantes de Sueños. Janoschka, M. (2013). Nuevas geografías migratorias en América Latina: prácticas de ciudadanía en un destino de turismo residencial. Scripta Nova, Revista Electrónica de Geografía y Ciencias Sociales, 17(439). Spivak, G. (1998). ¿Puede hablar el sujeto subalterno? Orbis Tertius, 3(6), 175-235. Jiménez, V., Hidalgo, R., Campesino, A., y Alvarado, V. (2018). Normalización del modelo neoliberal de expansión residencial más allá del límite urbano en Chile y España. EURE, 44(132), 27-46. DOI: http://dx.doi.org/10.4067/s0250- 71612018000200027 Soja, E (1996). Thirdspace; journeys to Los Angeles and other real and imagined places. Malden: Blacwkell. Strauss, A. y Corbin J. (2002). Bases de la investigación cualitativa. Técnicas y procedimientos para desarrollar la Teoría Fundamentada. Antioquia: Editorial Universidad de Antioquia. Lincopi, C. (2015) La emergencia de la ciudad colonial en Ngülu Mapu: control social, desposesión e imaginarios urbanos. En Antileo Baeza L., Calfío Montalva M. y Huinca-Piutrin H. (Eds.) Awükan ka kuxankan zugu wajmapu new. Violencias coloniales en Wajmapu (pp.107-140). Temuco: Ediciones comunidad de historia mapuche. Lincopi, C. (2015) La emergencia de la ciudad colonial en Ngülu Mapu: control social, desposesión e imaginarios urbanos. En Antileo Baeza L., Calfío Montalva M. y Huinca-Piutrin H. (Eds.) Awükan ka kuxankan zugu wajmapu new. Violencias coloniales en Wajmapu (pp.107-140). Temuco: Ediciones comunidad de historia mapuche. Vergara, L., Gola, R. y Huiliñir, V. (2015). Los inicios de la insustentabilidad: problemas urbanos e institucionalidad en la ciudad de Temuco, 1955- 1970. Cuadernos de Vivienda y Urbanismo, 8(16), 264-281. DOI: https://dxdoi. org/10.11144/Javeriana.cvu8-16.iipu López M., Valenzuela A. y Carrasco C (2017). Propuesta Simbiótica Natural- Cultural en Territorio Mapuche de Arauco. Investigaciones Geográficas, (54), 61- 84. Recuperado de https://investigacionesgeograficas.uchile.cl/index.php/ IG/article/view/48042/50583 López M., Valenzuela A. y Carrasco C (2017). Propuesta Simbiótica Natural- Cultural en Territorio Mapuche de Arauco. VI. REFERENCIAS BIBLIOGRÁFICAS Making sense of counterurbanization. Journal of Rural Studies, 20, 15–34. DOI: https://doi.org/10.1016/S0743-0167(03)00031-7 Mitchell, C. (2004). Making sense of counterurbanization. Journal of Rural Studies, 20, 15–34. DOI: https://doi.org/10.1016/S0743-0167(03)00031-7 Foerster, R. y Montecino, S. (1988). Organizaciones, líderes y contiendas mapuches: (1900-1970). Santiago. Ediciones CEM. Pinto, J. (2003). La formación del Estado y la nación, y el pueblo mapuche. Santiago: Dibam. Pinto, J. (2003). La formación del Estado y la nación, y el pueblo mapuche. Santiago: Dibam. Gasic, I. (2018). Inversiones e intermediaciones financieras en el mercado del suelo urbano. Principales hallazgos a partir del estudio de transacciones de terrenos en Santiago de Chile, 2010-2015. EURE, 44(133), 29-50. DOI: http:// dx.doi.org/10.4067/s0250-71612018000300029 Quiñones, J. y Gálvez, J. (2015). Estimación y estructura de los ingresos de familias mapuches rurales de zonas periurbana de Temuco, Chile. Mundo agrario, 16(32). Recuperado de https://www.redalyc.org/articulo. oa?id=84541597007 Garín, A., Salvo, S. y Bravo, G. (2009). Segregación residencial y políticas de vivienda en Temuco. 1992-2002. Revista de Geografía Norte Grande, (44), 113-128. Pineda, C. (2014). Mapuche resistiendo al capital y al Estado. El caso de la Coordinadora Arauco Malleco en Chile. Latinoamérica. Revista de estudios Latinoamericanos, 59, 99-128. DOI: https://doi.org/10.1016/S1665- 8574(14)71727-2 Haller, A. (2017). Los impactos del crecimiento urbano en los campesinos andinos. Un estudio de percepción en la zona rural/ urbana de Huancayo, Perú. Espacio y Desarrollo, (29), 37-56. Rojo, F., Alvarado, V., Olea, J., Salazar, A. (2020) Definiendo el Temuco metropolitano: consideraciones para un nuevo modelo de urbanización extendida en la Araucanía. Revista AUS (27), 41-49. DOI: https://doi.org/10.4206/ aus.2020.n27-05 Harvey, D. (2014). Ciudades rebeldes: Del derecho de la ciudad a la revolución urbana. Madrid: Ediciones Akal. Henríquez, L. (2013). Cinco décadas de transformaciones en la Araucanía rural. POLIS revista latinoamericana, 12(34), 147-164. Rojo, F. (2019). Las dinámicas de clases en la producción de espacios urbanos: el caso de Temuco. Tesis para optar al grado de Doctor del Geografía. Instituto de Geografía. Pontificia Universidad Católica de Chile. Recuperado de https:// repositorio.uc.cl/handle/11534/23702 Rojo, F. (2019). Las dinámicas de clases en la producción de espacios urbanos: el caso de Temuco. Tesis para optar al grado de Doctor del Geografía. Instituto de Geografía. Pontificia Universidad Católica de Chile. Recuperado de https:// repositorio.uc.cl/handle/11534/23702 Herrera, M. (2018) Comunidades indígenas urbanas: disputas y negociación por el reconocimiento. Andamios, 15(36), 113-134. PERMEABILIDAD DEL ESPACIO INDÍGENA. DISCURSOS DE PROPIETARIOS MAPUCHE SOBRE LA EXPANSIÓN URBANA EN EL PERIURBANO DE TEMUCO, ARAUCANÍA-CHILE ERIC ITURRIAGA GUTIÉRREZ, FÉLIX ROJO MENDOZA, MIGUEL ESCALONA ULLOA REVISTA URBANO Nº 42 / NOVIEMBRE 2020 - ABRIL 2021 Investigaciones Geográficas, (54), 61- 84. Recuperado de https://investigacionesgeograficas.uchile.cl/index.php/ IG/article/view/48042/50583 Vergara, L. (2019). Medianización social y transformaciones residenciales recientes en ciudades de La Araucanía. Revista CUHSO, 29 (2), 36-60. DOI: http://dx.doi.org/10.7770/0719-2789.2019.cuhso.02.a03 Mansilla, P. e Imilan, W. (2020). Colonialidad del poder, desarrollo urbano y desposesión mapuche: urbanización de tierras mapuche en la Araucanía chilena. Scripta Nova, 24(630). Recuperado de https://revistes.ub.edu/index. php/ScriptaNova/article/view/21225/30806 Mansilla, P. e Imilan, W. (2020). Colonialidad del poder, desarrollo urbano y desposesión mapuche: urbanización de tierras mapuche en la Araucanía chilena. Scripta Nova, 24(630). Recuperado de https://revistes.ub.edu/index. php/ScriptaNova/article/view/21225/30806 Vergara, L., Sánchez, C. y Zunino, H. (2019). Migración por estilo de vida: ¿Creando comunidades diversas y cohesionadas? El caso de Los Riscos, Pucón, Chile. Revista Austral de Ciencias Sociales, (36), 47-67. DOI: http://dx.doi. org/10.7440/res55.2016.11 Marchant, C., Frick, J. P. y Vergara, L. (2016). Urban growth trends in midsize Chilean cities: the case of Temuco. Urbe, Revista Brasileira de Gestão Urbana, 8(3), 375-389. Marchant, C., Frick, J. P. y Vergara, L. (2016). Urban growth trends in midsize Chilean cities: the case of Temuco. Urbe, Revista Brasileira de Gestão Urbana, 8(3), 375-389. Viera, P. (2015). La reconstrucción del lof tradicional mapuche como alternativa contra hegemónica de organización social y productiva. Revista de geografía e interdiciplinaridade interespaco, 1(3). Recuperado de http://www. periodicoseletronicos.ufma.br/index.php/interespaco/article/view/4460 /2449 Marimán, J., Valenzuela, E. y Cortés, F. (2015). El nuevo ciclo de movilización mapuche en Chile: la emergencia de la CAM y el proyecto autonomista para una región plurinacional. Araucaria. Revista Iberoamericana de Filosofía, Política y Humanidades,17(34), 279-301. Marimán, J., Valenzuela, E. y Cortés, F. (2015). El nuevo ciclo de movilización mapuche en Chile: la emergencia de la CAM y el proyecto autonomista para una región plurinacional. Araucaria. Revista Iberoamericana de Filosofía, Política y Humanidades,17(34), 279-301. Wilson, R., Pearce, T., Jones, K., Fleischfresser, S., Davis, B., Jones, G. y Lieske. S. (2018). Indigenous Land Management in Peri-Urban Landscapes: An Australian Example. Society & Natural Resources, 31(3), 335-350. DOI: https://doi.org/10.10 80/08941920.2017.1383543 Méndez, R. (2018). La telaraña financiera. Una geografía de la financiarización y su crisis. Santiago de Chile: Colección Estudios Urbanos UC/RIL editores. Méndez, R. (2018). La telaraña financiera. Una geografía de la financiarización y su crisis. Santiago de Chile: Colección Estudios Urbanos UC/RIL editores.
https://openalex.org/W3123898963	https://europepmc.org/articles/pmc4440347?pdf=render	English	null	The architecture of embodied cue integration: insight from the â€œmotivation as cognitionâ€ perspective	Frontiers in psychology	2,015	cc-by	3,732	OPINION published: 21 May 2015 doi: 10.3389/fpsyg.2015.00658 The Malleable Nature of Embodied Cues in Judgment and Behavior At the core of embodied cognition research is the assumption that higher level processing is grounded in the organism’s lower level sensory and motor experiences (Barsalou, 1999, 2008; Meier et al., 2012; Winkielman et al., 2015b). Past research of perceptual multimodal cue integration has demonstrated that several mechanisms underlie perceptual integration (Treisman and Gelade, 1980; Zmigrod and Hommel, 2013). Based on embodied cognition theory, which indicates that activation automatically spreads from concepts driven by experiences in the physical world to their metaphorically-related social concepts (for reviews, Williams et al., 2009; Meier et al., 2012), it was proposed that to produce action, embodied cues associate between lower level and higher level cues. However, little is known about the factors that modulate this integration. This gap in the literature is of relevance because research of embodied cognition has demonstrated that perceptual symbols can lead to diﬀerent patterns of activation across diﬀerent contexts (Barsalou, 2008), which makes predictions about judgment and behavior diﬃcult. For example, the associations between physical warmth/coldness and psychological warmth/coldness across diﬀerent contexts yielded both assimilative eﬀects (e.g., physical warmth increases psychological warmth) (Williams and Bargh, 2008) and contrast eﬀects (e.g., physical coldness increases the need for social warmth) (Zhong and Leonardelli, 2008; Bargh and Shalev, 2012; Shalev and Bargh, 2014; Zhang and Risen, 2014). The architecture of embodied cue integration: insight from the “motivation as cognition” perspective Department of Education and the Zlotowski Center of Neuroscience, Ben-Gurion University of the Negev, Beer Sheva, Israel Keywords: concept activation, cue integration, embodied cognition, feature integration, goal systems theory, motivation as cognition, social cognition Reviewed by: Reviewed by: Guillaume T. Vallet, Centre de Recherche de l’Institut Universitaire de Gériatrie de Montréal, Canada Correspondence: Idit Shalev, shalevid@bgu.ac.il Correspondence: Idit Shalev, shalevid@bgu.ac.il Following the recent pragmatic turn in cognitive science, according to which cognitive processes and their underlying neural activity patterns should be studied primarily with respect to their roles in action generation (Glenberg et al., 2013), I argue that embodied cues are integrated according to their momentary functions within each individual’s system of goals. Conceptualized as cognitive representations of desired end-points that aﬀect evaluations, emotions and behaviors (Fishbach and Ferguson, 2007), goals serve as reference points toward which behavior is directed. I suggest that analyzing embodied cue integration from the “motivation as cognition” perspective (Kruglanski et al., 2002) may add to our understanding of which cues are perceived, what response is determined as appropriate in a given situation, and why diﬀerent judgments and behaviors may be elicited by the activation of similar sets of embodied cues. In the sections below, I will discuss three types of constraints that stem from the “motivation as cognition” perspective, including the motivational properties of embodied cue integration (Eitam and Higgins, 2010), the allocational properties of embodied cue integration (based on attentional resource-limitation, see Kahneman, 1973), and the structural properties of cognitive-interconnectedness and uniqueness (Kruglanski et al., 2002). Specialty section: This article was submitted to Cognition, a section of the journal Frontiers in Psychology Received: 22 November 2014 Accepted: 05 May 2015 Published: 21 May 2015 Edited by: Edited by: Nicolas Vermeulen, Université Catholique de Louvain, Belgium Edited by: Nicolas Vermeulen, Université Catholique de Louvain, Belgium The Motivational Properties of Embodied Cue Integration The second assumption of embodied cue integration is that the fundamental allocation property relies on limited mental resources. From that perspective, the allocation of cognitive resources has a functional purpose, namely, to minimize the extent to which mental resources are exploited in the creation of uniﬁed percepts. The property of limited resources is demonstrated, for instance, by motor ﬂuency eﬀects observed only when individuals are involved in monitoring situational constraints. For example, research showed that compared with rigid right-handers, ﬂexible right-handers recalled product orientations better and showed a preference for objects on which the handle was oriented in the direction of the hand used for grasping (Eelen et al., 2013). The ﬁrst assumption of embodied cue integration is that because numerous sensori-motor cues can serve as the material for multiple social inferences, a selection process is needed to determine which cues to integrate in a given situation to create meaning. I suggest that perceptual or conceptual saliency depends on whether a mental representation reﬂects the individual’s momentary goals, and what, if any, relationship those goals have with the salient cues in the immediate environment of the individual (Balcetis and Dunning, 2009; Balcetis et al., 2012). A similar line of thought was suggested by De Houwer (2009), indicating that associative learning eﬀects are determined not only by the direct experience of events but also by prior knowledge and instructions. Pursuing this logic, Eitam and Higgins (2010) suggest that whether, and the degree to which, a stimulated mental representation is activated reﬂects the relative weights of one or any combination of three sources of motivational relevance: value relevance, or the extent to which acting on a mental representation will bring about desired results and/or prevent undesired results; control relevance, or the eﬃcacy with which the activated representation makes things happen; and truth relevance, which establishes what is real. Thus, the relative extents to which these sources are relevant to the individual’s needs determines the level Another application for the limited resources eﬀect is demonstrated by the switching cost entailed in shifting attention from one modality to another (e.g., from audition to vision), indicating that the second stimulus is processed more slowly than it would have been had the two stimuli used the same modality (e.g., Spence et al., 2001). What Is the “Motivation as Cognition” Perspective? The “motivation as cognition” perspective assigned distinct functions to motivational and cognitive variables. A basic assumption is that motivation can ﬂuctuate from one moment to the next, thus determining the extent to which any kind of information (strategic and peripheral; conscious and unconscious) is processed (Kruglanski and Thompson, 1999). Mental representations of motivational networks comprise inter- connected goals and means that may be automatically activated simultaneously by diﬀerent cues, and as such, they may compete with each other for mental resources (Kruglanski et al., 2002). Likewise, according to this approach, several cognitive properties set the constraints within which the motivational properties may express themselves. Because both motivation and embodied cognition are types of cognition, this set of cognitive constraints may explain the way motivation inﬂuences embodied cue integration. In the sections below, I will discuss the constraints on cue integration, including the motivational properties of embodied cue integration (Eitam and Higgins, 2010), the allocational properties of embodied cue integration (based on attentional resource-limitation, see Kahneman, 1973), and the structural properties of cognitive-interconnectedness and uniqueness (Kruglanski et al., 2002). Citation: Shalev I (2015) The architecture of embodied cue integration: insight from the “motivation as cognition” perspective. Front. Psychol. 6:658. doi: 10.3389/fpsyg.2015.00658 May 2015 \| Volume 6 \| Article 658 Frontiers in Psychology \| www.frontiersin.org 1 The architecture of embodied cognition Shalev Shalev and duration of activation, regardless of the content of the representation. The three types of constraints, adopted from goal systems theory (Kruglanski et al., 2002), were invoked to explain the process of embodied cue integration. Indeed, recent ﬁndings of embodied cognition research have provided strong evidence for the eﬀect of motivational relevance on cue integration. For example, one study demonstrated the source of value relevance by showing that the adoption of approach-type postures (e.g., leaning forward) was associated with increases in neural activation characteristic of approach situations (Harmon-Jones et al., 2011). In another study, the performance of avoidance type movements (pushing a shopping cart as opposed to holding it) was associated with fewer reward-oriented consumer choices at the checkout counter (Van den Bergh et al., 2011). The source of control relevance was demonstrated by showing that embodied simulations of facial expressions were expressed for conceptual understanding only if they were relevant to solving the task at hand (Niedenthal et al., 2009), indicating that embodiments are not passive byproducts of conceptual processing (Winkielman et al., 2015a). Finally, the source of truth relevance was shown in a study where participants were asked to verify or deny that a certain object has a certain property (i.e., answer a question such as “Do cats have wings?”). The results showed that the speed of property veriﬁcation was related to the perceptual salience of the feature in question (Solomon and Barsalou, 2004). For example, property veriﬁcation was quicker the more conspicuous the property, presumably because such properties are easier to see in a recalled or simulated visual representation. Frontiers in Psychology \| www.frontiersin.org Conclusions This paper suggests that several constraints based on the “motivation as cognition” paradigm modulate the interrelations between perception, emotion and action, and in so doing, they inﬂuence embodied cue integration in both bottom- up and top-down manners. On the one hand, active goals inﬂuence the feasibility of relevant embodied cues. On the other hand, the perceiver’s likelihood of drawing a speciﬁc inference may be proportional to the strengths of the associations between contextual cues and sights and sounds encountered by the individual (Zaki, 2013). Based on this reasoning, I suggest that inferences are highly ﬂexible and context- dependent, and therefore, they vary in accordance with situational framing eﬀects (Loersch and Payne, 2011; Wiltshire et al., 2015). As with other psychological phenomena, individual diﬀerences (e.g., physical disability, mental health conditions) could increase the likelihood that speciﬁc motivational states will be associated with particular embodied cues. Likewise, the repetition of speciﬁc social inferences in response to similar sensori-motor contextual cues is possible and may depend on the strength of the association within unique cue conﬁgurations. The contribution of the embodied cue integration approach goes beyond explaining the variability of ﬁndings across diﬀerent contexts. By combining cognitive architecture, semantic metaphoric conﬁgurations and structural motivational properties, embodied cue integration oﬀers a possible path for integrating diﬀerent lines of thought in the ﬁeld of embodied cognition. The Structural Properties of Embodied Cue Integration The third assumption of embodied cue integration is that uniﬁed percept conﬁgurations are inﬂuenced by sensori-motor cue interconnections, including the form and associative strength of those interconnections. The strength of association between multi-modal units is positively related to the uniqueness of the interconnections (Kruglanski et al., 2002). This dynamic helps explain why speciﬁc embodied metaphors have stronger associative links than other metaphors with sensori-motor cues. A possible explanation could be that the repetition of speciﬁc social inferences across diﬀerent contexts in response to sensori-motor contextual cues occurs when the strength of the association is high or in populations where this motivation is accessible. For example, evidence that washing one’s hands also “washes away” feelings of guilt was found not only in a normal population (Zhong and Liljenquist, 2006; Lee and Schwarz, 2010, 2011), but also among patients with obsessive compulsive disorder in whom the association between contents related to physical and psychological cleanliness is stronger (Reuven et al., 2014). Likewise, research indicates that core metaphors (e.g., temperature, distance) are associated with multiple conceptual phrases (Schnall, 2014), suggesting possible variability in the strengths of the associations between sub-metaphors associated with the core metaphor. Likewise, individual and cultural diﬀerences also inﬂuence these associative strengths and may have an impact on the replicability of ﬁndings (Shalev and Bargh, 2014). Acknowledgments The research was partially supported by the Helmsley Charitable Trust through the Agricultural, Biological and Cognitive Robotics Center of Ben-Gurion University of the Negev. I thank Nicolas Vermeulen for handling the manuscript, Guillaume T. Vallet and reviewer for their helpful comments, and Joseph Tzelgov for his constructive comments on a previous version of this manuscript. Another structural application of embodied cue integration is the substitutability relations of cues associated with an identical mental representation. For example, studies of the metaphorical links between physical and social temperatures The Motivational Properties of Embodied Cue Integration The switching cost was also demonstrated by the perceptual simulation approach, indicating that verifying the properties of concepts in the auditory modality was slower after verifying a property in a diﬀerent modality than after verifying one in the same modality (Pecher et al., 2003). The limited resources assumption has several consequences. First, I suggest that the integration process is fundamentally May 2015 \| Volume 6 \| Article 658 Frontiers in Psychology \| www.frontiersin.org 2 The architecture of embodied cognition Shalev economic and that it operates automatically by activating samples of the interconnection between sounds, sights, and other sensory signals that were encoded in memory based on previous learning (Brunel et al., 2009; Zmigrod et al., 2009; Bargh and Morsella, 2010; Vallet et al., 2010). Evidence for automatic activation is based on the ideomotor theory, which assumes the existence of an automatic action–eﬀect integration mechanism that binds motor patterns and action eﬀect representation (Chartrand and Bargh, 1999; Zmigrod and Hommel, 2013). Second, as was recently proposed by Winkielman et al. (2015b), I argue that non conscious automatic signals, including ﬂuency and a sense of coherence, inform fundamental cognitive and social judgments (Winkielman and Schooler, 2011; Schwarz, 2015), thereby consuming fewer cognitive resources. (e.g., “warm smile,” “cold as ice”) showed that participants perceive others as “warmer” after they have held a warm rather than a cold cup of coﬀee (Williams and Bargh, 2008; IJzerman and Semin, 2009, 2010; Shalev and Bargh, 2011; Bargh and Shalev, 2012). Likewise, they experience a room as physically colder after having been socially rejected (Zhong and Leonardelli, 2008), indicating a possible substitutability between physical and semantic psychological concepts. Bargh, J. A., and Morsella, E. (2010). “Unconscious behavioral guidance systems,” in Then a Miracle Occurs: Focusing on Behavior in Social Psychological Theory and Research, eds C. R. Agnew, D. E. Carlston, W. G. Graziano, and J. R. Kelly (New York, NY: Oxford University Press), 89–118. doi: 10.1093/acprof:oso/9780195377798.003.0006 Bargh, J. A., and Shalev, I. (2012). The substitutability of physical and social warmth in daily life. Emotion 12, 154–162. doi: 10.1037/a0023527 Balcetis, E., and Dunning, D. (2009). Wishful seeing more desired objects are seen as closer. Psychol. Sci. 21, 147–152. doi: 10.1177/0956797609356283 Balcetis, E., Dunning, D., and Granot, Y. (2012). Subjective value determines initial dominance in binocular rivalry. J. Exp. Soc. Psychol. 48, 122–129. doi: 10.1016/j.jesp.2011.08.009 Bargh, J. A., and Morsella, E. (2010). “Unconscious behavioral guidance systems,” in Then a Miracle Occurs: Focusing on Behavior in Social Psychological Theory and Research, eds C. R. Agnew, D. E. Carlston, W. G. Graziano, and J. R. Kelly (New York, NY: Oxford University Press), 89–118. doi: 10.1093/acprof:oso/9780195377798.003.0006 Bargh, J. A., and Shalev, I. (2012). The substitutability of physical and social warmth in daily life. Emotion 12, 154–162. doi: 10.1037/a0023527 References (2013). Cue integration a common framework for social cognition and physical perception. Pers. Psychol. Sci. 8, 296–312. doi: 10.1177/1745691613475454 Kruglanski, A. W., Shah, J. Y., Fishbach, A., Friedman, R., Chun, W. Y., and Sleeth-Keppler, D. (2002). A theory of goal systems. Adv. Exp. Soc. Psychol. 34, 331–378. doi: 10.1016/S0065-2601(02)80008-9 Kruglanski, A. W., and Thompson, E. P. (1999). Persuasion by a single route: a view from the unimodel. Psychol. Inq. 10, 83–109. doi: 10.1207/S15327965PL 100201 Zhang, Y., and Risen, J. L. (2014). Embodied motivation: using a goal systems framework to understand the preference for social and physical warmth. J. Pers. Soc. Psychol. 107, 965. doi: 10.1037/a0038153 Lee, S. W. S., and Schwarz, N. (2010). Washing away postdecisional dissonance. Science 328, 709. doi: 10.1126/science.1186799 Zhong, C. B., and Leonardelli, G. J. (2008). Cold and lonely: does social exclusion feel literally cold? Psychol. Sci. 19, 838–842. doi: 10.1111/j.1467- 9280.2008.02165.x Lee, S. W. S., and Schwarz, N. (2011). Wiping the slate clean: psychological consequences of physical cleansing. Curr. Dir. Psychol. Sci. 20, 307–311. doi: 10.1177/0963721411422694 Zhong, C. B., and Liljenquist, K. (2006). Washing away your sins: threatened morality and physical cleansing. Science 313, 1451–1452. doi: 10.1126/science.1130726 Loersch, C., and Payne, B. K. (2011). The situated inference model an integrative account of the eﬀects of primes on perception, behavior, and motivation. Pers. Psychol. Sci. 6, 234–252. doi: 10.1177/1745691611406921 Zmigrod, S., and Hommel, B. (2013). Feature integration across multimodal perception and action: a review. Multisens. Res. 26, 143–157. doi: 10.1163/22134808-00002390 Meier, B. P., Schnall, S., Schwarz, N., and Bargh, J. A. (2012). Embodiment in social psychology. Top. Cogn. Sci. 4, 705–716. doi: 10.1111/j.1756-8765.2012.01212.x Zmigrod, S., Spapé, M., and Hommel, B. (2009). Intermodal event ﬁles: integrating features across vision, audition, taction, and action. Psych. Res. 73, 674–684. doi: 10.1007/s00426-008-0163-5 Niedenthal, P. M., Winkielman, P., Mondillon, L., and Vermeulen, N. (2009). Embodiment of emotional concepts: Evidence from EMG measures. J. Pers. Soc. Psychol. 96, 1120–1136. doi: 10.1037/a0015574 Conﬂict of Interest Statement: The author declares that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Pecher, D., Zeelenberg, R., and Barsalou, L. W. (2003). Verifying diﬀerent-modality properties for concepts produces switching costs. Psychol. Sci. 14, 119–124. doi: 10.1111/1467-9280.t01-1-01429 Reuven, O., Liberman, N., and Dar, R. (2014). The eﬀect of physical cleaning on threatened morality in individuals with obsessive-compulsive disorder. Clin. Psychol. Sci. 2, 224–229. References Motivation in mental accessibility: relevance of a representation (ROAR) as a new framework. Soc. Pers. Psychol. Comm. 4, 951–967. doi: 10.1111/j.1751-9004.2010.00309.x Williams, L. E., and Bargh, J. A. (2008). Experiencing physical warmth promotes interpersonal warmth. Science 322, 606–607. doi: 10.1126/science.1162548 Fishbach, A., and Ferguson, M. J. (2007). “The goal construct in social psychology,” in Social Psychology: Handbook of Basic Principles, eds A. W. Kruglanski and E. T. Higgins (New York, NY: Guilford), 490–515. Williams, L. E., Huang, J. Y., and Bargh, J. A. (2009). The scaﬀolded mind: higher mental processes are grounded in early experience of the physical world. Eur. J. Soc. Psychol. 39, 1257–1267. doi: 10.1002/ejsp.665 Glenberg, A. M., Witt, J. K., and Metcalfe, J. (2013). From the revolution to embodiment 25 years of cognitive psychology. Pers. Psychol. Sci. 8, 573–585. doi: 10.1177/1745691613498098 Wiltshire, T. J., Lobato, E. J., McConnell, D. S., and Fiore, S. M. (2015). Prospects for direct social perception: a multi-theoretical integration to further the science of social cognition. Front. Hum. Neurosci. 8:1007. doi: 10.3389/fnhum.2014.01007 Harmon-Jones, E., Gable, P. A., and Price, T. F. (2011). Leaning embodies desire: Evidence that leaning forward increases relative left frontal cortical activation to appetitive stimuli. Bio. Psychol. 87, 311–313. doi: 10.1016/j.biopsycho.2011.03.009 Winkielman, P., Niedenthal, P., Wielgosz, J., Eelen, J., and Kavanagh, L. C. (2015a). “Embodiment of cognition and emotion,” in APA Handbook of Personality and Social Psychology, Attitudes and Social Cognition, eds. M. Mikulincer, P. R. Shaver, E. Borgida, and J. A. Bargh (Washington, DC: American Psychological Association), 151–175. doi: 10.1037/14341-004 IJzerman, H., and Semin, G. R. (2009). The thermometer of social relations mapping social proximity on temperature. Psychol. Sci. 20, 1214–1220. doi: 10.1111/j.1467-9280.2009.02434.x IJzerman, H., and Semin, G. R. (2010). Temperature perceptions as a ground for social proximity. J. Exp. Soc. Psychol. 46, 867–873. doi: 10.1016/j.jesp.2010.07.015 Winkielman, P., and Schooler, J. W. (2011). Splitting consciousness: unconscious, conscious, and metaconscious processes in social cognition. Eur. Rev. Soc. Psychol. 22, 1–35. doi: 10.1080/10463283.2011.576580 Shalev, I., and Bargh, J. A. (2011). Use of priming-based interventions to facilitate psychological health: commentary to Kazdin and Blasé (2011). Pers. Psychol. Sci. 6, 488–492. doi: 10.1177/1745691611416993 Winkielman, P., Ziembowicz, M., and Nowak, A. (2015b). The coherent and ﬂuent mind: how uniﬁed consciousness is constructed from cross- modal inputs via integrated processing experiences. Front. Psychol. 6:83. doi: 10.3389/fpsyg.2015.00083 Kahneman, D. (1973). Attention and Eﬀort. Englewood Cliﬀs, NJ: Prentice-Hall. Kahneman, D. (1973). Attention and Eﬀort. Englewo Zaki, J. References Balcetis, E., and Dunning, D. (2009). Wishful seeing more desired objects are seen as closer. Psychol. Sci. 21, 147–152. doi: 10.1177/0956797609356283 Balcetis, E., Dunning, D., and Granot, Y. (2012). Subjective value determines initial dominance in binocular rivalry. J. Exp. Soc. Psychol. 48, 122–129. doi: 10.1016/j.jesp.2011.08.009 May 2015 \| Volume 6 \| Article 658 Frontiers in Psychology \| www.frontiersin.org 3 The architecture of embodied cognition Shalev Shalev Barsalou, L. W. (1999). Perceptual symbol systems. Behav. Brain Sci. 22, 577–660. doi: 10.1017/S0140525X99002149 Schwarz, N. (2015). “Metacognition,” in APA Handbook of Personality and Social Psychology: Attitudes and Social Cognition, eds E. Borgida and J. A. Bargh (Washington, DC: APA), 203–229. Barsalou, L. W. (2008). Grounded cognition. Annu. Rev. Psychol. 59, 617–645. doi: 10.1146/annurev.psych.59.103006.093639 Shalev, I., and Bargh, J. (2014). On the association between loneliness and physical warmth-seeking through bathing: reply to Donnellan et al. (2014) and three further replications of bargh and shalev (2012) study 1. Emotion. doi: 10.1037/emo0000046 Brunel, L., Labeye, E., Lesourd, M., and Versace, R. (2009). The sensory nature of episodic memory: sensory priming eﬀects due to memory trace activation. J. Exp. Psychol. Learn. Mem. Cogn. 35, 1081. doi: 10.1037/ a0015537 Solomon, K. O., and Barsalou, L. W. (2004). Perceptual simulation in property veriﬁcation. Mem. Cognit. 32, 244–259. doi: 10.3758/BF03196856 Chartrand, T. L., and Bargh, J. A. (1999). The chameleon eﬀect: the perception– behavior link and social interaction. J. Pers. Soc. Psychol. 76:893. doi: 10.1037/0022-3514.76.6.893 Spence, C., Nicholls, M. E., and Driver, J. (2001). The cost of expecting events in the wrong sensory modality. Percept. Psychophys. 63, 330–336. doi: 10.3758/BF03194473 De Houwer, J. (2009). The propositional approach to associative learning as an alternative for association formation models. Learn. Behav. 37, 1–20. doi: 10.3758/LB.37.1.1 Treisman, A. M., and Gelade, G. (1980). A feature-integration theory of attention. Cogn. Psychol. 12, 97–136. doi: 10.1016/0010-0285(80)90005-5 Eelen, J., Dewitte, S., and Warlop, L. (2013). Situated embodied cognition: monitoring orientation cues aﬀects product evaluation and choice. J. Consum. Psychol. 23, 424–433. doi: 10.1016/j.jcps.2013.04.004 Vallet, G., Brunel, L., and Versace, R. (2010). The perceptual nature of the cross- modal priming eﬀect: arguments in favor of a sensory-based conception of memory. Exp. Psychol. 57, 376. doi: 10.1027/1618-3169/a000045 Psychol. 23, 424–433. doi: 10.1016/j.jcps.2013.04.004 Van den Bergh, B., Schmitt, J., and Warlop, L. (2011). Embodied myopia. J. Mark. Res. 48, 1033–1044. doi: 10.1509/jmr.09.0503 Eitam, B., and Higgins, E. T. (2010). References doi: 10.1177/2167702613485565 Copyright © 2015 Shalev. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Schnall, S. (2014). “Are there basic metaphors?,” in The Power of Metaphor: Examining its Inﬂuence on Social Life, eds M. J. Landau, M. D. Robinson, and B. P. Meier (Washington, DC: American Psychological Association), 225–247. May 2015 \| Volume 6 \| Article 658 Frontiers in Psychology \| www.frontiersin.org 4
https://openalex.org/W2810333089	http://uco.edu.es/ucopress/ojs/index.php/ucoarte/article/download/11007/10132	Italian	null	FASHION COLLECTION: FROM ELEONORA DUSE TO JULIE PETERS-DESTERACT	UCOarte	2,017	cc-by	17,656	Palabras clave Colección, Arte, Moda, El siglo veinte, Colección Textil, Patrocinio, Donación, Historia de la moda y el vestuario, Historia del arte contemporáneo. COLECCIÓN DE MODA: DE ELEONORA DUSE A JULIE PETERS-DESTERACT. ELISA RISI Università di Bergamo. Italia Abstract This article analyzes a particular branch of the relationship that combines the art and fashion world with focus on the theme of textile collecting, operated by female characters. The area of textile collecting connects and unites the world of art and that of fashion. The study is based on the three-partition proposed by french historian, Julie Verlaine, who divides female art collecting practices into three main types: "collectionneuse", "commanditaire" and "donor". Using this subdivision, I intend to analyze the textile collecting - a phenomenon that is markedly developed by female protagonists - knowing that the analysis of each path denotes the difficulty of a rigid categorization and net boundaries: these practices show indicative, fluid and permeable typologies of collectionists. Resumen Este artículo analiza una rama particular de la relación que combina el mundo del arte y el mundo de la moda con el enfoque en el tema de la colección de textiles, operado por personajes femeninos. El área de la colección de textiles conecta y une el mundo del arte y el de la moda. El estudio se basa en la triple partición propuesta por la historiadora francesa Julie Verlaine, que divide las prácticas de colección de arte femenino en tres tipos principales: "collectionneuse", "commanditaire" y "donor". Utilizando esta subdivisión, intento analizar la colección textil -un fenómeno marcadamente desarrollado por protagonistas femeninos - sabiendo que el análisis de cada percurso denota la dificultad de una categorización rígida y límites netos: estas prácticas muestran tipologías de coleccionistas indicativas, fluidas y permeables. Ucoarte. Revista de Teoría e Historia del Arte, 6, 2017, pp.125-148. ISSN: 2255-1905 Elisa Risi Elisa Risi La storica francese Julie Verlaine suddivide le pratiche del collezionismo d’arte al femminile in tre tipologie principali che sono quella della “collectionneuse”, della “commanditaire” e della “donatrice”.1 La femme collectionneuse è colei che riunisce opere d’arte già esistenti; la femme commanditaire svolge il ruolo di mecenate e committente, richiedendo agli artisti opere per una destinazione specifica; la femme donatrice, infine, si caratterizza per l’atto della donazione di un’opera, di un insieme di elementi o di un’intera collezione ad una o più istituzioni.2 p Facendo mia la tripartizione di Julie Verlaine, mi accingo ad analizzare il collezionismo nell’ambito del tessile, fenomeno marcatamente al femminile, tenendo presente che l’analisi del percorso di ogni collezionista denota la difficoltà di una categorizzazione rigida.3 Lontano dall’essere marcate da confini netti, queste pratiche si presentano come tipologie indicative e fluide, permeabili, che hanno un senso solo nel momento in cui si adattano ai percorsi particolari di ogni singola donna, che spesso incarna tipologie e pratiche molteplici, che si intrecciano tra loro, dando luogo a casi particolarmente interessanti e certamente più ricchi. L’indagine sulle personalità femminili protagoniste di questo studio svela una serie di percorsi salienti che, a partire dalla fine del XIX secolo, delineano l’evoluzione del collezionismo tessile fino alla contemporaneità. La progressione cronologica di questi ritratti fornisce, quindi, una panoramica sulle pratiche e sulle forme differenti di questo fenomeno. q p p q Alla donna collezionista, mecenate e donatrice, è interessante aggiungere un’ulteriore tipologia che caratterizza questo universo: la stilista. Creatrice di collezioni tessili e di moda, questa figura completa e arricchisce il panorama delineato. Un aspetto importante che lega questi personaggi è il loro rapporto con grandi istituzioni museali, sotto varie forme, che vanno dalla donazione, al prestito per mostre ed esposizioni, fino al lavoro all’interno di un ente museale. Queste donne sono figure fondamentali in quanto garanti della salvaguardia del patrimonio artistico legato alla storia e all’universo del tessile e della moda. Anche se alcune protagoniste dello studio sono personalità che hanno segnato in modo indelebile sia le ultime decadi del XIX secolo sia la contemporaneità, il secolo chiamato in causa è soprattutto il Novecento, secolo di profondi mutamenti e di diritti acquisiti da parte delle donne, che si susseguono in parallelo alle importanti trasformazioni che interessano l’abbigliamento in quanto manifestazione della vita sociale, culturale e artistica di queste figure. 1 Per i termini in lingua francese cf. l’opera di J. Verlaine, Femmes collectionneuses d’art et mécènes de 1880 à nos jours, Malakoff Cedex, Hazan, 2013, op. cit., p. 19. 2 Ibidem. 3 Sulla difficoltà di categorizzare ciascuna collezionista d’arte vedere J. Verlaine in Femmes collectionneuses d’art et mécènes de 1880 à nos jours, op. cit., p. 19. 4 Per quanto riguarda la biografia di Eleonora Duse si veda E. A. Rheinhardt, Eleonora Duse, Roma, LIT EDIZIONI, 2015. 1 Per i termini in lingua francese cf. l’opera di J. Verlaine, Femmes collectionneuses d’art et mécènes de 1880 à n jours, Malakoff Cedex, Hazan, 2013, op. cit., p. 19. 2 Ibidem. 3 Sulla difficoltà di categorizzare ciascuna collezionista d’arte vedere J. Verlaine in Femmes collectionneuses d’art et mécènes de 1880 à nos jours, op. cit., p. 19. 4 Per quanto riguarda la biografia di Eleonora Duse si veda E. A. Rheinhardt, Eleonora Duse, Roma, LIT EDIZIONI, 2015. Key words Collection, Art, Fashion, The twentieth century, Textile Collection, Patronage, Donation, History of fashion and costume, History of contemporary art. D Ucoarte. Revista de Teoría e Historia del Arte, 6, 2017, pp.125-148. ISSN: 2255-1905 Elisa Risi Il pittore ottocentesco osserva i costumi dell’epoca e li riproduce sulla tela con grande precisione. Figura 1. James-Jacques-Joseph Tissot, Il ricevimento, 1886. Il pittore ottocentesco osserva i costumi dell’epoca e li riproduce sulla tela con grande precisione. Figura 1. James-Jacques-Joseph Tissot, Il ricevimento, 1886. Il pittore ottocentesco osserva i costumi dell’epoca e li riproduce sulla tela con grande precisione. in Francia ha inizio il periodo detto della Belle Epoque, in Inghilterra chiamato epoca edoardiana, che va dall’inizio del XX secolo sino allo scoppio della prima guerra mondiale e che si diffonde in gran parte dell’Europa. Il clima che si respira in questi anni è caratterizzato da una grande ostentazione e prodigalità.5 Rinomati creatori d’abiti come Charles Frederick Worth,6 Jacques Doucet e Jeanne Paquin, che avevano inaugurato saloni di haute couture durante il secolo precedente,7 aderiscono alla sensibilità dell’epoca e all’Art Nouveau, che aspira ad ottenere una bellezza caratterizzata da una combinazione di eleganza e opulenza. Le creazioni di questo periodo richiedono l’uso di lunghi corsetti, che al fine di ottenere l’effetto di una silhouette artificiale a S, distorcono molto la figura femminile: il corpo della donna, oltre che essere costretto in una forma innaturale, è anche impedito nel movimento8 (fig. 1). in Francia ha inizio il periodo detto della Belle Epoque, in Inghilterra chiamato epoca edoardiana, che va dall’inizio del XX secolo sino allo scoppio della prima guerra mondiale e che si diffonde in gran parte dell’Europa. Il clima che si respira in questi anni è caratterizzato da una grande ostentazione e prodigalità.5 Rinomati creatori d’abiti come Charles Frederick Worth,6 Jacques Doucet e Jeanne Paquin, che avevano inaugurato saloni di haute couture durante il secolo precedente,7 aderiscono alla sensibilità dell’epoca e all’Art Nouveau, che aspira ad ottenere una bellezza caratterizzata da una combinazione di eleganza e opulenza. Le creazioni di questo periodo richiedono l’uso di lunghi corsetti, che al fine di ottenere l’effetto di una silhouette artificiale a S, distorcono molto la figura femminile: il corpo della donna, oltre che essere costretto in una forma innaturale, è anche impedito nel movimento8 (fig. 1). ( g ) Nella moda dell’epoca si riflette lo spirito di questi anni, da cui Eleonora Duse si distacca. Le sue scelte, infatti, sono lontane da quelle della moda della Belle Epoque e dallo stile Art Nouveau e convergono invece verso uno stile nuovo, che si afferma pianamente con l’avvento della prima guerra mondiale. 5 Cf. J. Laver, A. de la Haye, A. Tucker, Moda e costume. Breve storia dall’antichità a oggi, Ginevra-Milano, Rizzoli- Skira, 2003, p. 231. 6 Cf. J. Laver, A. de la Haye, A. Tucker, Moda e costume, op. cit., p. 200, che dipinge Charles Frederick Worth come uno dei primi grandi creatori di moda, uno dei primi stilisti: “Charles Frederick Worth, che pur essendo inglese di nascita, in dieci anni era riuscito a diventare un dittatore della moda parigina, esigeva che fossero le signore a venire a trovarlo”. Lo storico francese Hippolyte Taine descrive la scena: “le dame, ansiose di farsi vestire da Worth, vengono a fargli visita nel suo salone. Quest’omino asciutto, nero, nervoso, le riceve vestito di una giacca di velluto, abbandonato su un divano con aria disinvolta, un sigaro fra le labbra. Dice loro: ‘Cammini! Si volti! Bene! Torni fra una settimana, e le preparerò una toilette che vada bene per lei’. Non sono loro a scegliere, è lui. E loro non chiedono di meglio di lasciarlo fare, e perfino per questo occorre che gli siano state presentate da qualcuno”. 7 Sulla nascita e la celebrazione della figura del creatore di moda o stilista, cf. note 19, 20 e 21 di questo studio. 8 Come si evince dalla collezione del Kyoto Costume Institute, La Moda. Storia dal XVIII al XX secolo, Köln, Taschen, 2003, p. 332. 5 Cf. J. Laver, A. de la Haye, A. Tucker, Moda e costume. Breve storia dall’antichità a oggi, Ginevra-Milano, Rizzoli- kira, 2003, p. 231. 6 Cf J L A d l H A T k M d i 200 h di i Ch l F d i k W h 8 Come si evince dalla collezione del Kyoto Costume Institute, La Moda. Storia dal XVIII al XX secolo, Köl Taschen, 2003, p. 332. Elisa Risi g Eleonora Duse, una tra le più grandi attrici del teatro moderno, incarna fra le prime figure della femme commanditaire, mecenate e committente presso grandi artisti sia di abiti ad uso personale sia di capi per le scene teatrali; come vedremo nel corso dello studio sarà soprattutto Mariano Fortuny ad essere il suo stilista prediletto. La collezione dell’attrice è significativa poiché simboleggia e si fa portavoce della sua personalità forte e carismatica. Nata nel 1858 a Vigevano da una famiglia di attori, Eleonora Duse trascorre un’infanzia nomade, recitando sin da bambina. Si impone sulle scene teatrali nazionali con un tipo di recitazione febbrile e di forte impatto emotivo e visivo, lontano dai consueti canoni recitativi dell'epoca, conquistando vasta fama e ammirazione. Sono anche le coraggiose scelte di repertorio a renderla famosa, le pièces contemporanee di autori come Dumas, Ibsen, Gor’kij, che portano sulla ribalta vizi e valori borghesi.4 La sua collezione tessile è costellata di abiti che commissiona e affida alle mani di talentuosi artisti contemporanei, e che sono, anche nel caso dell’uso privato, vere e proprie opere d’arte. La maggior parte dei capi sono attribuiti all’atelier di Mariano Fortuny, interprete ideale del gusto e della personalità dell’attrice, che si afferma sia come artista che come stilista di moda agli inizi del Novecento. Intorno al 1895 126 Colección de moda: de Eleonora Duse a Julie Peters-Desteract. Colección de moda: de Eleonora Duse a Julie Peters-Desteract. in Francia ha inizio il periodo detto della Belle Epoque, in Inghilterra chiamato epoca edoardiana, che va dall’inizio del XX secolo sino allo scoppio della prima guerra mondiale e che si diffonde in gran parte dell’Europa. Il clima che si respira in questi anni è caratterizzato da una grande ostentazione e prodigalità.5 Rinomati creatori d’abiti come Charles Frederick Worth,6 Jacques Doucet e Jeanne Paquin, che avevano inaugurato saloni di haute couture durante il secolo precedente,7 aderiscono alla sensibilità dell’epoca e all’Art Nouveau, che aspira ad ottenere una bellezza caratterizzata da una combinazione di eleganza e opulenza. Le creazioni di questo periodo richiedono l’uso di lunghi corsetti, che al fine di ottenere l’effetto di una silhouette artificiale a S, distorcono molto la figura femminile: il corpo della donna, oltre che essere costretto in una forma innaturale, è anche impedito nel movimento8 (fig. 1). Nella moda dell’epoca si riflette lo spirito di questi anni da cui Eleonora Duse si distacca Figura 1. James-Jacques-Joseph Tissot, Il ricevimento, 1886. Elisa Risi È Paul Poiret a lanciare per primo una nuova linea di abiti che non richiede l’uso del corsetto: nel 1906 crea lo stile ellenico, fatto di capi dal taglio dritto, caratterizzati da una linea ampia, privi di corsetto e con la vita alta. In questo modo, 127 Elisa Risi Figura 2. Paul Poiret, abiti da sera in copertina di Art Goût Beauté, 1923. Figura 2. Paul Poiret, abiti da sera in copertina di Art Goût Beauté, 1923. Figura 2. Paul Poiret, abiti da sera in copertina di Art Goût Beauté, 1923. Figura 2. Paul Poiret, abiti da sera in copertina di Art Goût Beauté, 1923. lo stilista francese sposta il baricentro della figura dalla cintura alle spalle (sin dal Rinascimento, con poche eccezioni, il corsetto stringeva il punto vita e determinava la linea degli abiti e questo elemento era caratterizzante dell’abbigliamento delle donne occidentali). Lo stilista cerca e crea una nuova forma di bellezza9 (fig. 2). lo stilista francese sposta il baricentro della figura dalla cintura alle spalle (sin dal Rinascimento, con poche eccezioni, il corsetto stringeva il punto vita e determinava la linea degli abiti e questo elemento era caratterizzante dell’abbigliamento delle donne occidentali). Lo stilista cerca e crea una nuova forma di bellezza9 (fig. 2). ( g ) Le sue creazioni sono abbellite ispirandosi allo stile esotico e rivelano la nostalgia di terre straniere, atteggiamento che caratterizza questo periodo del ventesimo secolo. All’inizio del Novecento la pittura orientale, la pubblicazione della traduzione de Le mille e una notte, il debutto dei Ballets Russes a Parigi nel 1909 e l’attenzione sempre più marcata verso il Giappone (che apre le sue porte all’Occidente alla fine del diciannovesimo secolo), contribuiscono alla nascita e allo sviluppo di un grande “desiderio” d’Oriente10 (fig. 3). pp g g L’Orientalismo e il Giapponismo hanno un forte impatto su vari settori dell’arte e della letteratura, di conseguenza anche su quello della moda. Jean Philippe Worth, figlio del noto Charles Frederick Worth, affianca già dal 1874 il padre nella sartoria di famiglia; responsabile di tutta la parte creativa dal 1895, segue queste nuove tendenze e diviene tra le firme più conosciute della haute couture, annoverando tra le sue clienti celebri attrici, come Eleonora Duse appunto, ma anche Sarah Bernhardt.11 Anche in altri paesi europei, oltre alla Francia, c’è una ricerca rivolta al nuovo e all’esotico. 9 Ibidem. 10 Cf. Dalla collezione del Kyoto Costume Institute, La Moda, op. cit., p. 333. 11 Cf. C. Chiarelli (a cura di), Donne protagoniste nel Novecento, Livorno, Sillabe, 2013, pp. 27 e 29. 12 Ivi, op. cit., p. 12. Elisa Risi Mariano Fortuny, spagnolo di nascita, interpreta audacemente queste tendenze e crea un nuovo stile del vestirsi. Jean Philippe Worth e in particolar modo Fortuny, sono tra i creatori prediletti da Eleonora Duse; il rapporto che si instaura, oltre che essere di committenza è caratterizzato da un sentimento di affetto e di amicizia. Gli abiti che Eleonora Duse affida ai propri creatori si inscrivono nella storia dello stile e della moda di questo periodo storico. Tuttavia, l’opera di committenza tende anche a valorizzare la sua personalità e rende gli abiti creazioni uniche. La maggior parte dei capi di questa collezione non sono costumi di scena, sono infatti “tuniche dal ductus essenziale, che per quanto da lei presumibilmente indossate nella quotidianità, lasciano tuttavia immaginare una gestualità eloquente e solenne” 12 (fig. 4). 128 Colección de moda: de Eleonora Duse a Julie Peters-Desteract. Quest’abito, di attribuzione Fortuny, è di proprietà della Galleria del Costume di Palazzo Pitti di Firenze. Eleonora Ilaria Bullogh, divenuta poi suor Mary Mark, nipote della celebre attrice (secondogenita di Enrichetta, unica figlia di Eleonora Duse), è colei che, negli anni e con estrema cura, ha assicurato la pregevole eredità ad importanti istituzioni museali e fondazioni. Gli abiti e gli accessori, che costituiscono una collezione di grande valore, sono stati donati oltre che alla Galleria del Costume di Firenze, anche al Victoria & Albert Museum di Londra e alla Fondazione Giorgio Cini di Venezia, accompagnati da vario materiale d’archivio.13 L’attrice colleziona lungo tutta la vita abiti e accessori di alto livello artistico, frutto della commissione affidata ai suoi creatori prediletti, e segni che riflettono la sua personalità, rispecchiando il gusto e la sensibilità per i tessuti preziosi. Come lei, vi sono altre donne che si distinguono per l’attenzione posta al mondo del tessile e per la collezione di capi pregiati. Gli abiti, per tutte queste personalità, assumono il valore di opera d’arte, arrivando ad essere parte integrante della loro vita. p g Rosa Genoni nasce nel 1867, vive nella stessa epoca di Eleonora Duse, ma rispetto a lei si situa in una posizione differente se non opposta poiché incarna il ruolo di stilista di moda. p pp p La nascita e la legittimazione di questa nuova figura risale alla seconda metà dell’Ottocento. La Rivoluzione Francese del 1789 porta al crollo del tradizionale ordinamento gerarchico della società francese e vede la nascita di una nuova classe sociale: la borghesia. J p p 18 “[…] Nel 1858, la scrittura del saggio De la Mode (ad opera di Théophile Gautier) accoglie e sviluppa queste considerazioni in particolare alla luce della convinzione, come già aveva avuto modo di sostenere Balzac e soprattutto come dirà Baudelaire, che il sistema della moda ottocentesco testimonia più di ogni altra manifestazione della cultura, del carattere e dello spirito del secolo. Il 1858 è peraltro l’anno che sancisce, grazie all’invenzione di Worth, non solo una vera e propria rivoluzione nel processo di creazione dell’abito, ma un nuovo sistema della moda […] d’ora in poi bipolare” F. Franchi, I veli della modernità, Bergamo, Edizioni Sestante, 2001, op. cit., p. 22. 14 Cf. Dalla collezione del Kyoto Costume Institute, La Moda, op. cit., p. 150. 15 Sull’evoluzione del mondo e dell’industria della moda nel XIX secolo cf. Dalla collezione del Kyoto ostume Institute, La Moda, op. cit., p. 150. 16 Come sostiene A. Sangiovanni in Le parole e le figure: storia dei media in Italia dall'età liberale alla Seconda Guerra ondiale, Roma, Donzelli Editore, 2012, p. 20. 13 Per quanto riguarda la trasmissione dell’eredità di Eleonora Duse e le donazioni ad istituzioni museal fondazioni, vedere il testo di C. Chiarelli (a cura di), Donne protagoniste nel Novecento, op. cit., pp. 26 e 29. p 17 Cf. J. Laver, A. de la Haye, A. Tucker, Moda e costume, op. cit., p. 200. 13 Per quanto riguarda la trasmissione dell’eredità di Eleonora Duse e le donazioni ad istituzioni museali e fondazioni, vedere il testo di C. Chiarelli (a cura di), Donne protagoniste nel Novecento, op. cit., pp. 26 e 29. 14 Cf. Dalla collezione del Kyoto Costume Institute, La Moda, op. cit., p. 150. 15 Sull’evoluzione del mondo e dell’industria della moda nel XIX secolo cf. Dalla collezione del Kyoto Costume Institute, La Moda, op. cit., p. 150. 16 Come sostiene A. Sangiovanni in Le parole e le figure: storia dei media in Italia dall'età liberale alla Seconda Guerra mondiale, Roma, Donzelli Editore, 2012, p. 20. 17 Cf. J. Laver, A. de la Haye, A. Tucker, Moda e costume, op. cit., p. 200. 18 “[…] Nel 1858, la scrittura del saggio De la Mode (ad opera di Théophile Gautier) accoglie e sviluppa queste considerazioni in particolare alla luce della convinzione, come già aveva avuto modo di sostenere Balzac e soprattutto come dirà Baudelaire, che il sistema della moda ottocentesco testimonia più di ogni altra manifestazione della cultura, del carattere e dello spirito del secolo. Il 1858 è peraltro l’anno che sancisce, grazie all’invenzione di Worth, non solo una vera e propria rivoluzione nel processo di creazione dell’abito, ma un nuovo sistema della moda […] d’ora in poi bipolare” F. Franchi, I veli della modernità, Bergamo, Edizioni Sestante, 2001, op. cit., p. 22. 15 Sull’evoluzione del mondo e dell’industria della moda nel XIX secolo cf. Dalla collezione del Kyoto Costume Institute, La Moda, op. cit., p. 150. 16 Come sostiene A. Sangiovanni in Le parole e le figure: storia dei media in Italia dall'età liberale alla Seconda Guerra mondiale, Roma, Donzelli Editore, 2012, p. 20. 19 “La paradossale inversione di tendenza che […] vede la celebrazione pubblica del grande creatore di moda e al contempo la desacralizzazione del letterato e in particolare dell’artista, è il presupposto da cui prende le mosse il saggio gautiano […] Gautier intende […] richiamare l’artista moderno alle sue responsabilità”: ad essere portavoce della modernità “del carattere e dello spirito del secolo” opera in cui il sistema della moda ottocentesco eccelle, come sostiene F. Franchi ne I veli della modernità, op. cit., pp. 22-23. g 21 Ivi, op. cit., p. 22. , , p , pp 20 Come sostiene Bruno de Rossella ne La mode (Paris, Imprimerie Nationale, 1980) e ribadisce ulteriormente F. Franchi ne I veli della modernità, op. cit., pp. 22-23, il prestigio di cui gode il creatore di moda deriva dalla necessità della borghesia di legittimare una figura nuova, quale è quella del borghese nella società e del sarto nel mondo dell’arte dell’epoca. Il creatore di moda, o stilista, e la sua celebrazione pubblica diviene una forma di legittimazione sociale che attesta il potere crescente della borghesia. 23 Cf. Dalla collezione del Kyoto Costume Institute, La Moda, op. cit., p. 150. p 22 Ibidem. Elisa Risi Tuttavia, fino al secondo Impero la nobiltà gode di un ripristinato potere ed è l’imperatrice Eugenia, moglie di Napoleone III, che si impone come guida nel mondo della moda. Con la Terza Repubblica, a partire dal 1870, ha luogo un nuovo sconvolgimento dell’ordine sociale che porta all’affermazione della borghesia come classe sociale dominante. Le figure di riferimento nel mondo della moda diventano, di conseguenza, le signore della ricca borghesia, le attrici famose e le demi-mondaines, che si affermano come vere e proprie icone in grado di influenzare profondamente le tendenze di questo settore.14 Nella seconda metà del XIX secolo diventano fruitrici della haute couture. Quest’epoca è caratterizzata da un progressivo allargamento della fascia di popolazione che può permettersi di interessarsi di moda. Infatti, negli anni Cinquanta dell’Ottocento, in Francia, sorgono i primi grandi magazzini, che contribuiscono, grazie all’offerta di una grande varietà di prodotti a prezzi vantaggiosi, alla diffusione massiccia di abiti e accessori di moda.15 Il primo grande magazzino, il Bon Marché di Parigi, nasce nel 1852 ed è sintomatico del fenomeno dell’industrializzazione della moda: molteplici fattori, la rivoluzione industriale e la modernizzazione in primo luogo, ma anche l’ampio uso della pubblicità, provocano il cambiamento delle forme del commercio, in particolare di quello al dettaglio, che inizia ad essere indirizzato alle classi medie, soprattutto per quanto riguarda l’abbigliamento, con la crescente diffusione dell’abito confezionato industrialmente, in serie16 (nelle epoche precedenti gli abiti erano disegnati e confezionati da persone di una classe relativamente umile, che andavano di casa in casa per vestire le gentildonne, ed erano per la maggior parte donne a loro volta).17 Risale alla seconda metà del XIX secolo anche lo sviluppo della figura dello stilista, che si afferma pienamente nel 185818 con la definitiva elevazione della moda a 129 Elisa Risi Figura 3. Paul Poiret, costume da uomo e da donna, rispettivamente 1914, 1913. Figura 3. Paul Poiret, costume da uomo e da donna, rispettivamente 1914, 1913. 19 “La paradossale inversione di tendenza che […] vede la celebrazione pubblica del grande creatore di moda e al contempo la desacralizzazione del letterato e in particolare dell’artista, è il presupposto da cui prende le mosse il saggio gautiano […] Gautier intende […] richiamare l’artista moderno alle sue responsabilità”: ad essere portavoce della modernità “del carattere e dello spirito del secolo” opera in cui il sistema della moda ottocentesco eccelle, come sostiene F. Franchi ne I veli della modernità, op. cit., pp. 22-23. 20 Come sostiene Bruno de Rossella ne La mode (Paris, Imprimerie Nationale, 1980) e ribadisce ulteriormente F. Franchi ne I veli della modernità, op. cit., pp. 22-23, il prestigio di cui gode il creatore di moda deriva dalla necessità della borghesia di legittimare una figura nuova, quale è quella del borghese nella società e del sarto nel mondo dell’arte dell’epoca. Il creatore di moda, o stilista, e la sua celebrazione pubblica diviene una forma di legittimazione sociale che attesta il potere crescente della borghesia. 21 Ivi, op. cit., p. 22. Figura 3. Paul Poiret, costume da uomo e da donna, rispettivamente 1914, 1913. creazione artistica: il sarto viene considerato un artista.19 A partire da questo momento, la promozione sociale di questa figura culminerà nella sua vera e propria celebrazione pubblica.20 Nasce, dunque, un “nuovo sistema della moda”21 sancito da una rivoluzione nel processo di produzione dell’abito e caratterizzato dalla bipolarità che oppone una produzione di massa ad una confezione elitaria di lusso e su misura ad opera dei primi grandi stilisti di moda.22 Tutto ciò, in concomitanza con altri progressi del secolo, come la nascita delle esposizioni universali, la prima delle quali si tiene a Londra nel 1851, le nuove possibilità di trasporto grazie alla ferrovia e alla nave a vapore, fa sì che si produca un vivace movimento internazionale di prodotti e merci. Nascono inoltre, in questo periodo, molte riviste di moda, che contribuiscono all’affermazione e al successo mondiale della moda parigina, che nel XIX secolo acquisisce e consolida il suo primato nel campo della moda.23 creazione artistica: il sarto viene considerato un artista.19 A partire da questo momento, la promozione sociale di questa figura culminerà nella sua vera e propria celebrazione pubblica.20 Nasce, dunque, un “nuovo sistema della moda”21 sancito da una rivoluzione nel processo di produzione dell’abito e caratterizzato dalla bipolarità che oppone una produzione di massa ad una confezione elitaria di lusso e su misura ad opera dei primi grandi stilisti di moda.22 Tutto ciò, in concomitanza con altri progressi del secolo, come la nascita delle esposizioni universali, la prima delle quali si tiene a Londra nel 1851, le nuove possibilità di trasporto grazie alla ferrovia e alla nave a vapore, fa sì che si produca un vivace movimento internazionale di prodotti e merci. Nascono inoltre, in questo periodo, molte riviste di moda, che contribuiscono all’affermazione e al successo mondiale della moda parigina, che nel XIX secolo acquisisce e consolida il suo primato nel campo della moda.23 All’inizio del XX secolo Rosa Genoni si adopera per costruire le basi di una nascente moda italiana, cercando di riscattarla dalla subordinazione alla Francia e cercando ispirazione in modelli dell’arte italiana. “Considerata già nel 1907 l’ideatrice della moda italiana dai più influenti quotidiani del tempo, Rosa Genoni è sicuramente uno dei personaggi italiani più 130 Colección de moda: de Eleonora Duse a Julie Peters-Desteract. Colección de moda: de Eleonora Duse a Julie Peters-Desteract. Figura 4. Mariano Fortuny (attr.) Manifattura veneziana, sopravveste, circa 1915 Figura 4. 25 Come sostiene Cristina Tajani, assessore alle Politiche per il Lavoro, Moda e Design di Milano in occasione della retrospettiva dedicata a Rosa Genoni a Milano presso il Palazzo Castiglioni (tenutasi dal 17 al 25 settembre 2015). ) 26 Cf. a proposito la biografia di Rosa Genoni redatta da Aurora Fiorentini all’interno di C. Chiarelli (a cura di), Donne protagoniste nel Novecento, op. cit., p. 17. 24 Citazione attinta dal volume di C. Chiarelli (a cura di), Donne protagoniste nel Novecento, op. cit., p. 16. Colección de moda: de Eleonora Duse a Julie Peters-Desteract. 24 Citazione attinta dal volume di C. Chiarelli (a cura di), Donne protagoniste nel Novecento, op. cit., p. 16. 25 Come sostiene Cristina Tajani, assessore alle Politiche per il Lavoro, Moda e Design di Milano in occasione della retrospettiva dedicata a Rosa Genoni a Milano presso il Palazzo Castiglioni (tenutasi dal 17 al 25 settembre 2015). 26 Cf a proposito la biografia di Rosa Genoni redatta da Aurora Fiorentini all’interno di C Chiarelli (a cura Figura 3. Paul Poiret, costume da uomo e da donna, rispettivamente 1914, 1913. Mariano Fortuny (attr.) Manifattura veneziana, sopravveste, circa 1915 24 Citazione attinta dal volume di C. Chiarelli (a cura di), Donne protagoniste nel Novecento, op. cit., p. 16. 25 Come sostiene Cristina Tajani, assessore alle Politiche per il Lavoro, Moda e Design di Milano in occasione della retrospettiva dedicata a Rosa Genoni a Milano presso il Palazzo Castiglioni (tenutasi dal 17 al 25 settembre 2015). 26 Cf. a proposito la biografia di Rosa Genoni redatta da Aurora Fiorentini all’interno di C. Chiarelli (a cura di), Donne protagoniste nel Novecento, op. cit., p. 17. Figura 4. Mariano Fortuny (attr.) Manifattura veneziana, sopravveste, circa 1915 In questo contesto riveste la prima cattedra di Storia del Costume italiana (che terrà per venticinque anni, fino al 1930, quando lascerà l’incarico per non giurare fedeltà al Fascismo).27 In questi anni la sua personalità e le sue scelte creative lavorative si impongono con successo tanto da sancire la nascita di una moda italiana finalmente svincolata dal primato francese e caratterizzata da una linea innovativa proprio perché ispirata ai capolavori dell’arte nazionale. Per l’innovazione delle proposte, all’Esposizione Universale di Milano del 1906, nella Sezione Abbigliamenti Femminili, Rosa Genoni ottiene il Gran Premio per la sezione Arti Decorative da parte della giuria internazionale dell’Expo, che premia i suoi abiti ispirati alla tradizione dell'arte pittorica rinascimentale28 (fig. 5). creatrice di moda italiana e all’impegno politico, Rosa Genoni affianca l’impegno pedagogico e didattico, collaborando con la Scuola Professionale Femminile dell’Umanitaria (nota istituzione di servizi sociali lombarda) dove diventa dirigente della Sezione Sartoria e docente. In questo contesto riveste la prima cattedra di Storia del Costume italiana (che terrà per venticinque anni, fino al 1930, quando lascerà l’incarico per non giurare fedeltà al Fascismo).27 In questi anni la sua personalità e le sue scelte creative lavorative si impongono con successo tanto da sancire la nascita di una moda italiana finalmente svincolata dal primato francese e caratterizzata da una linea innovativa proprio perché ispirata ai capolavori dell’arte nazionale. Per l’innovazione delle proposte, all’Esposizione Universale di Milano del 1906, nella Sezione Abbigliamenti Femminili, Rosa Genoni ottiene il Gran Premio per la sezione Arti Decorative da parte della giuria internazionale dell’Expo, che premia i suoi abiti ispirati alla tradizione dell'arte pittorica rinascimentale28 (fig. 5). Rosa Genoni è unanimemente considerata come la progenitrice della moda Made in Italy,29 figura ricca e complessa, sarà impegnata per tutta la vita oltre che nell’innovazione tessile, anche nell’attività politica e sociale. Il suo prezioso lascito per i futuri stilisti della moda italiana, può essere individuato nella rete di rimandi, influenze e richiami al mondo dell’arte italiana che si trovano riposti ad impreziosire i suoi abiti. p p “Entrambe tese a riformare la natura, a tra-vestire il reale, moda e arte si prestano al tal punto reciproco aiuto che la loro relazione si costituisce come indissolubile”30 e tesa a marcare il nascente sistema della moda. Il legame tra questi due universi si consolida a partire dall’epoca moderna. 27 Ivi, op. cit., p. 18. 28 Ibidem. 29 Come sostiene Cristina Tajani, assessore alle Politiche per il Lavoro, Moda e Design di Milano in occasione della retrospettiva dedicata a Rosa Genoni a Milano presso il Palazzo Castiglioni (tenutasi dal 17 al 25 settembre 2015). 30 Citazione di F. Franchi, I veli della modernità, op. cit., p. 37. Figura 4. Mariano Fortuny (attr.) Manifattura veneziana, sopravveste, circa 1915 importanti nel campo del costume e della moda di inizio secolo”.24 Questa donna si impone infatti come prima grande stilista italiana, capace di instaurare un dialogo profondo tra il mondo della moda, del tessile e quello dell’arte. Le collezioni di abiti da lei create si distinguono per uno stile leggero e innovativo al tempo stesso, uno stile che esclude gli eccessi e che lega la creatività a punti di riferimento precisi e sicuri, sia nelle forme sia nella ricerca dei tessuti.25 La stilista si distingue inoltre per l’attività politica e sociale che caratterizza tutta la sua vita, battendosi per i diritti delle donne sin dal 1884, anno in cui inizia a frequentare i circoli operai socialisti di Milano. In seguito al primo soggiorno parigino, avvenuto in questi anni nel ruolo di inviata del Partito Operaio Italiano, decide di fermarsi nella capitale della moda francese per perfezionare la lingua e le tecniche di cucito e di ricamo (nel 1884 aveva ottenuto la qualifica di “maestra” presso la sartoria milanese Dall’Oro). Rientrata a Milano, nel 1893 si trova impegnata sul fronte delle rivendicazioni contro lo sfruttamento del lavoro femminile ed entra a far parte della Lega promotrice degli Interessi Femminili; dopo questa presa di posizione morale e politica stringe rapporti con i nomi più celebri del femminismo di sinistra e grazie a ciò, nello stesso anno, viene invitata al Congresso Internazionale Socialista- Laburista di Zurigo.26 Parallelamente al suo impegno politico, che si intensifica negli anni con la militanza all’interno del Movimento Femminile Socialista, Rosa Genoni si afferma nel panorama internazionale dell’industria della moda: dopo svariati soggiorni a Parigi, a partire dal 1903 sino al 1914 gestisce con grande successo, prima nel ruolo di première poi in quello di direttrice, la Maison Haardt e Figli in Corso Vittorio Emanuele 28, esclusivo atelier milanese in cui aveva iniziato a lavorare nel 1895. A partire dal 1905 al ruolo saliente di prima 131 Elisa Risi creatrice di moda italiana e all’impegno politico, Rosa Genoni affianca l’impegno pedagogico e didattico, collaborando con la Scuola Professionale Femminile dell’Umanitaria (nota istituzione di servizi sociali lombarda) dove diventa dirigente della Sezione Sartoria e docente. 28 Ibidem. 27 Ivi, op. cit., p. 18. ) 30 Citazione di F. Franchi, I veli della modernità, op. cit., p. 37. Figura 4. Mariano Fortuny (attr.) Manifattura veneziana, sopravveste, circa 1915 In questo contesto riveste la prima cattedra di Storia del Costume italiana (che terrà per venticinque anni, fino al 1930, quando lascerà l’incarico per non giurare fedeltà al Fascismo).27 In questi anni la sua personalità e le sue scelte creative lavorative si impongono con successo tanto da sancire la nascita di una moda italiana finalmente svincolata dal primato francese e caratterizzata da una linea innovativa proprio perché ispirata ai capolavori dell’arte nazionale. Per l’innovazione delle proposte, all’Esposizione Universale di Milano del 1906, nella Sezione Abbigliamenti Femminili, Rosa Genoni ottiene il Gran Premio per la sezione Arti Decorative da parte della giuria internazionale dell’Expo, che premia i suoi abiti ispirati alla tradizione dell'arte pittorica rinascimentale28 (fig. 5). G i i id l i i d ll d d i Figura 5. Rosa Genoni, manto da corte Pisanello, 1906. Figura 5. Rosa Genoni, manto da corte Pisanello, 1906. Figura 5. Rosa Genoni, manto da corte Pisanello, 1906. Figura 5. Rosa Genoni, manto da corte Pisanello, 1906. creatrice di moda italiana e all’impegno politico, Rosa Genoni affianca l’impegno pedagogico e didattico, collaborando con la Scuola Professionale Femminile dell’Umanitaria (nota istituzione di servizi sociali lombarda) dove diventa dirigente della Sezione Sartoria e docente. In questo contesto riveste la prima cattedra di Storia del Costume italiana (che terrà per venticinque anni, fino al 1930, quando lascerà l’incarico per non giurare fedeltà al Fascismo).27 In questi anni la sua personalità e le sue scelte creative lavorative si impongono con successo tanto da sancire la nascita di una moda italiana finalmente svincolata dal primato francese e caratterizzata da una linea innovativa proprio perché ispirata ai capolavori dell’arte nazionale. Per l’innovazione delle proposte, all’Esposizione Universale di Milano del 1906, nella Sezione Abbigliamenti Femminili, Rosa Genoni ottiene il Gran Premio per la sezione Arti Decorative da parte della giuria internazionale dell’Expo, che premia i suoi abiti ispirati alla tradizione dell'arte pittorica rinascimentale28 (fig. 5). creatrice di moda italiana e all’impegno politico, Rosa Genoni affianca l’impegno pedagogico e didattico, collaborando con la Scuola Professionale Femminile dell’Umanitaria (nota istituzione di servizi sociali lombarda) dove diventa dirigente della Sezione Sartoria e docente. Figura 4. Mariano Fortuny (attr.) Manifattura veneziana, sopravveste, circa 1915 La nuova classe sociale dominante, la borghesia, non solo celebra pubblicamente la figura del sarto e dello stilista di moda come figure emblematiche e funzionali alla legittimazione di questa nuova classe sociale, bensì elegge la moda come segno 132 Colección de moda: de Eleonora Duse a Julie Peters-Desteract. della modernità poiché portatrice degli elementi chiave che la caratterizzano: la tensione verso il cambiamento e la trasformazione perpetua, l’aleatorio e la dinamicità del mutamento. L’importanza del legame che si sviluppa in quest’epoca tra l’arte e la moda si evince dalle riflessioni di celebri figure della modernità a partire da Charles Baudelaire e Théophile Gautier, fino a Stéphane Mallarmé e Marcel Proust.31 “Eleggere la moda a produttrice di ideali equivale a negare l’esistenza di un modello perfetto, e quindi a celebrare il transitorio nel quale è contenuta la bellezza, ma anche lo spirito dell’epoca”.32 La moda, in particolare la moda femminile, sperimenta in questo preciso momento storico, segnato dai fenomeni della mercificazione e della riproduzione, la perdita dell’originale a scapito della proliferazione e della molteplicità delle copie. La moda abbassa e allarga il suo bacino di attrazione e ciò rende possibile tanto per le grandi signore borghesi e per le duchesse, quanto per le attrici e le donne della classe media, di attingere a questo nuovo sistema.33 , g q Le riviste di moda come Vogue (1892, New York) e la Gazzette du Bon Ton (1912-1915, Parigi) stabiliscono un metodo per informare il mondo sulle evoluzioni della haute couture. Le immagini svolgono un ruolo fondamentale in queste riviste, un ruolo che diviene presto predominante, anche grazie al lavoro talentuoso di nuovi artisti, in particolare di Paul Iribe e di Georges Lepape. Quest’epoca è conosciuta come l’età d’oro dell’illustrazione di moda. Tra gli stilisti, Paul Poiret è il primo ad utilizzare il catalogo di moda come strumento che permette ai singoli creatori di mostrare il proprio lavoro e pubblica Les Robes de Paul Poiret by Paul Iribe (1908) e Les Choses de Paul Poiret (1911), illustrati da Georges Lepape.34 Una conseguenza del ruolo decisivo delle riviste è quella di portare molti compratori e giornalisti di moda a recarsi nella capitale francese per tenersi aggiornati, Parigi in questo momento storico è il centro nevralgico per il mondo della moda: nel 1910 viene fondata la Chambre Syndacale de la Couture Parisienne35 con lo scopo di divulgare e centralizzare la preparazione delle collezioni. y p p 37 La relazione tra le avanguardie storiche della storia dell’arte e il mondo della moda è trattata nel tes Dalla collezione del Kyoto Costume Institute, La Moda, op. cit., p. 334. 31 Per approfondire il legame tra letteratura e moda cf. F. Franchi, I veli della modernità, op. cit., p. 37. 32 Citazione di F. Franchi, I veli della modernità, op. cit., p. 37. 33 Ivi, p. 38. 34 Per quanto riguarda lo sviluppo delle riviste e dell’illustrazione di moda cavallo tra i due secoli cf. Dalla collezione del Kyoto Costume Institute, La Moda, op. cit., p. 333. 35 Ibidem. 36 La prima guerra mondiale, come tutte le altre, produce sul mondo e sulle attività della moda un effetto drastico, “nefasto” e fino alla fine del conflitto si registrano rare novità in questo campo, come sostengono J. Laver, A. de la Haye, A. Tucker in Moda e costume, op. cit., p. 266. 37 La relazione tra le avanguardie storiche della storia dell’arte e il mondo della moda è trattata nel testo Dalla collezione del Kyoto Costume Institute, La Moda, op. cit., p. 334. , p 34 Per quanto riguarda lo sviluppo delle riviste e dell’illustrazione di moda cavallo tra i due secoli cf. Dalla llezione del Kyoto Costume Institute, La Moda, op. cit., p. 333. 35 Ibid 36 La prima guerra mondiale, come tutte le altre, produce sul mondo e sulle attività della moda un effetto drastico, “nefasto” e fino alla fine del conflitto si registrano rare novità in questo campo, come sostengono J. Laver, A. de la Haye, A. Tucker in Moda e costume, op. cit., p. 266. 31 Per approfondire il legame tra letteratura e moda cf. F. Franchi, I veli della modernità, op. cit., p. 37. 32 Citazione di F. Franchi, I veli della modernità, op. cit., p. 37. 33 I i 38 33 Ivi, p. 38. Figura 4. Mariano Fortuny (attr.) Manifattura veneziana, sopravveste, circa 1915 “Per lei creare un abito era un modo per esprimere la sua arte, non un mezzo per avviare una tendenza della moda. I suoi vividi dipinti astratti decorano i tessuti e i suoi abiti […]”.39 Da questo legame nascono i celebri abiti simultanei, caratterizzati da colori luminosi e da una forma e un taglio molto semplici, che mettono in particolare risalto le composizioni geometriche e il cromatismo (fig. 6). p g ( g ) Gli abiti creati sono di tendenza astratta e presentano l’utilizzo di stoffe stampate che richiamano fedelmente il suo stile pittorico. Si può evincere come questa illustre donna si inscriva contemporaneamente in più tipologie e pratiche del collezionismo tessile al femminile. Sonia Delaunay è stilista e artista, creatrice di collezioni che sono al tempo stesso sia emblematiche per la storia della moda, in quanto influiranno in modo determinante sugli stilisti delle successive generazioni, sia determinanti per il patrimonio della storia dell’arte. g p p L’influenza delle avanguardie storiche segna profondamente anche il percorso di Elsa Schiaparelli che debutta nel mondo della moda come creatrice di abbigliamento sportivo e da spiaggia. La stilista allarga la sua attività fino ad includere abbigliamento di città e abiti da sera, diventando rapidamente una delle firme più celebri della haute couture.40 È il suo rapporto con gli artisti delle avanguardie storiche, in particolar modo del Dadaismo e del Surrealismo, che le permette di creare uno stile inimitabile, creativo e aperto alle contaminazioni artistiche che nascono dal legame che unisce i due universi, moda e arte. Elsa Schiaparelli (1890-1973) nasce a Roma, dove trascorre l’infanzia, da una famiglia di intellettuali di origine piemontese; sin da giovane viaggia moltissimo. Si stabilisce dapprima a New York e successivamente a Parigi, dove, incoraggiata dal celebre stilista Paul Poiret, nel 1927 fonda la sua prima boutique in rue Cambon e si lancia nel mondo della creazione di moda. Nel 1933 inaugura un nuovo atelier a Londra e nel 1935 sposta la boutique parigina in Place Vendôme.41 Negli anni Trenta domina la moda con una grande intuitività e con un acume che le permettono di precorrere i tempi in modo brillante. 38 A. Cocchi, La moda di Sonia Delaunay, articolo consultato il 18 novembre 2015 e pubblicato nel blog Geometrie Fluide (http://www.geometriefluide.com/pagina.asp?cat=avanguardie-900&prod=sonia-dealunay- moda), di seguito la bibliografia del testo: V. Maugeri A. Paffumi, Storia della moda e del costume, Firenze, Calderini Editore, 2005; L. Kybalovà, O. Herbenovà, M. Lamarovà, Enciclopedia illustrata del costume, La Spezia, F.lli Melita Editore, 1988; C. Giorgetti, Manuale di Storia del Costume e della Moda, Firenze, Cantini Gruppo D'Adamo Editore; E. Morini, Storia della moda XVIII-XX secolo, Ginevra-Milano, Skira editore, 2006; F. Podreider, Guida alla raccolta di stoffe di Rosa Genoni, Podreider, Dattiloscritto, Archivio Storico della Società Umanitaria di Milano; C. Bertelli, M. Robilant, F. Filippi, L. Lecci, P. Valenti, La storia dell'arte. Vol. 5 Novecento e oltre, Milano, Torino, Edizioni scolastiche Bruno Mondadori, 2010; G. Dorfles, A. Vettese, Arti visive. Il Novecento. Protagonisti e movimenti, Bergamo, Atlas editore, 1999; AA.VV, La nuova Enciclopedia dell'Arte Garzanti, Garzanti editore, 1986. Figura 4. Mariano Fortuny (attr.) Manifattura veneziana, sopravveste, circa 1915 Tuttavia l’avvento della prima guerra mondiale cambia e blocca drasticamente le attività della haute couture. Durante la guerra l’industria della moda asserve ai bisogni pratici e produce abiti confezionati in serie dai tagli semplici e adatti al drastico cambiamento apportato dal primo conflitto mondiale.36 Quando nel 1919 la moda torna a riprendere le sue attività, il legame con il mondo dell’arte si fa intenso e sempre più prolifico, compenetrandosi con gli spunti e le aperture apportate dai movimenti avanguardistici che si succedono nel panorama artistico europeo. Negli anni Venti la relazione tra moda e arte raggiunge un’intimità senza precedenti. Gli stilisti lavorano a fianco degli artisti per trovare ispirazione. Le Avanguardie Storiche sostengono che l’ambiente vitale nella sua totalità, compreso l’abbigliamento, deve essere armonioso in sé, come una singola manifestazione artistica.37 Il disegno e le tecniche artistiche d’avanguardia si innestano nel mondo della moda e arricchiscono il settore dell’abbigliamento. La pittrice Sonia Delaunay, creando una gamma di disegni per i tessuti, è la prima donna, esponente d’avanguardia, che applica le sue creazioni al mondo della moda. Nasce nel 1885 in Ucraina, svolge la sua formazione a San Pietroburgo e nel 1906 si sposta a Parigi. Sposa Robert Delaunay e approfondisce insieme a lui gli studi sul colore e sulla luce. Attraverso questa ricerca e sperimentazione i coniugi arrivano all’invenzione di una corrente artistica astratta: l’Orfismo. A partire dal 1911 la pittrice sperimenta queste ricerche in vari campi dell’arte applicata. La prima opera d’arte applicata astratta è un copriletto realizzato con la tecnica del patchwork, formato da pezzi di stoffa di 133 Elisa Risi forme geometriche e colori diversi cuciti insieme in una grande composizione. Il suo atelier, che porta il nome di “Laboratorio Simultaneo”, vede nascere le sue prime creazioni di sartoria intrise delle tecniche artistiche che la contraddistinguono. Sonia Delaunay crea non solo abiti ma anche accessori vari come sciarpe, borse, cappelli, con disegni geometrici e colori basati sulla legge ottica dei contrasti simultanei.38 Questo tipo di effetto ottico (che ha luogo soprattutto accostando i colori complementari) si ottiene quando si accostano tinte tra loro opposte, che se avvicinate aumentano la propria luminosità. Da questo momento e per tutti gli anni Venti la pittrice si dedica alla creazione nel campo della moda. Contemporaneamente a questa attività continua a dipingere considerando la pittura e la moda due campi strettamente connessi. y , , p , p 41 Per la biografia di Elsa Schiaparelli cf. Archivi della Moda del Novecento, Ministero dei beni e delle attività culturali e del turismo, direzione generale per gli archivi, in collaborazione con ANAI, approfondimento: Schiaparelli Elsa. p 42 Sul rapporto tra Elsa Schiaparelli, l’arte e la tecnologia cf. Dalla collezione del Kyoto Costume Institute, La Moda, op. cit., p. 483. y , , p , p 40 Per il contesto storico e della cultura della moda in cui si innesta Elsa Schiaparelli cf. Dalla collezione d Kyoto Costume Institute, La Moda, op. cit., p. 335. g , , ; , p z , 39 Dalla collezione del Kyoto Costume Institute, La Moda, op. cit., p. 467. Figura 4. Mariano Fortuny (attr.) Manifattura veneziana, sopravveste, circa 1915 Determinante è l’attenzione che pone lungo tutta la sua carriera ai nuovi sviluppi delle arti e della tecnologia.42 Tra gli anni Venti e il 1954 (anno di chiusura dell’atelier) Elsa Schiaparelli collabora a fianco di Christian Bérard, Jean Cocteau, Salvador Dalì, Francis Picabia, Man Ray, Marchel Duchamp, creando 134 Colección de moda: de Eleonora Duse a Julie Peters-Desteract. Figura 6. Sonia Delaunay, mantello realizzato per Gloria Swanson, 1923-24, capo esposto alla mostra Color Moves: The Art and Fashion of Sonia Delaunay realizzata presso il Cooper-Hewitt, National Design Museum, 2011, foto di Wolfgang Woessner. Figura 6. Sonia Delaunay, mantello realizzato per Gloria Swanson, 1923-24, capo esposto alla mostra Color Moves: The Art and Fashion of Sonia Delaunay realizzata presso il Cooper-Hewitt, National Design Museum, 2011, foto di Wolfgang Woessner. collezioni illuminate dall’estro di questi artisti. Sono linee uniche per i tagli, i colori, la sperimentazione di tessuti inediti e l’uso di nuovi materiali. L’arte diviene non solo fonte di ispirazione, ma parte integrante dei suoi modelli. La frequentazione degli artisti a lei contemporanei determina uno stile fortemente influenzato dall’arte del XX secolo, tanto che nel 1932 The New Yorker scrive “Un vestito Schiapparelli è come un dipinto moderno”.43 Tra le innovazioni apportate da questa stilista-artista della moda, importante è l’invenzione della sfilata moderna ossia l’idea di sviluppare le collezioni attorno ad un unico tema e di spettacolarizzarle.44 Nella scelta dei materiali sperimenta per la prima volta il rayon, il vinile, il cellophane e per i tessuti si distingue per l’utilizzo del tweed, il tessuto escorce d’arbre e le fibre artificiali.45 collezioni illuminate dall’estro di questi artisti. Sono linee uniche per i tagli, i colori, la sperimentazione di tessuti inediti e l’uso di nuovi materiali. L’arte diviene non solo fonte di ispirazione, ma parte integrante dei suoi modelli. 47 Per le informazioni sulla mostra Elsa Schiaparelli e Miuccia Prada. Impossible conversations presso il Costume Institute del Metropolitan Museum of Art di New York e la riapertura della maison Schiaparelli a Parigi, nel 2012, cf. Treccani, la Cultura Italiana, Enciclopedia, Schiaparelli Elsa, articolo consultato il 24 novembre 2015 (http://www.treccani.it/). 44 Per Elsa Schiaparelli come ideatrice della sfilata moderna cf. nella sezione enciclopedia, Vogue Italia, Elsa Schiaparelli, articolo consultato il 18 novembre 2015 (http://www.vogue.it/encyclo/stilisti/s/elsa-schiaparelli). 45 Cf. Archivi della Moda del Novecento, Ministero dei beni e delle attività culturali e del turismo, direzione generale per gli archivi, in collaborazione con ANAI, approfondimento: Schiaparelli Elsa. 46 Ivi, p. 490. 43 Citazione nell’articolo di M. Capella, Arte e moda: un incontro surreale, p. 24, consultato il 18 novembre 2015 (http://artemoda.unibg.it/allegati/aam_t2_pubblicazioni_allegati/8/b613cdf727b37f3f8ca79d7a3cdd602e08 899f98.pdf). 43 Citazione nell’articolo di M. Capella, Arte e moda: un incontro surreale, p. 24, consultato il 18 novembre 2015 (http://artemoda.unibg.it/allegati/aam_t2_pubblicazioni_allegati/8/b613cdf727b37f3f8ca79d7a3cdd602e08 899f98.pdf). 44 Per Elsa Schiaparelli come ideatrice della sfilata moderna cf. nella sezione enciclopedia, Vogue Italia, Elsa Schiaparelli, articolo consultato il 18 novembre 2015 (http://www.vogue.it/encyclo/stilisti/s/elsa-schiaparelli). 45 Cf. Archivi della Moda del Novecento, Ministero dei beni e delle attività culturali e del turismo, direzione generale per gli archivi, in collaborazione con ANAI, approfondimento: Schiaparelli Elsa. 46 Ivi, p. 490. 47 Per le informazioni sulla mostra Elsa Schiaparelli e Miuccia Prada. Impossible conversations presso il Costume Institute del Metropolitan Museum of Art di New York e la riapertura della maison Schiaparelli a Parigi, nel 2012, cf. Treccani, la Cultura Italiana, Enciclopedia, Schiaparelli Elsa, articolo consultato il 24 novembre 2015 (http://www.treccani.it/). Elisa Risi Riprendendo l’indagine sulle forme del collezionismo tessile e di moda al femminile, si può notare come le pratiche attraverso le quali si sviluppa questo fenomeno non sono caratterizzate da tipologie rigide e categoriche. Ogni donna presenta un percorso che è particolare e che può presentare differenti e molteplici forme di attualizzazione. p p p p Nell’epoca moderna l’abito assume un’importanza fondamentale nella cultura occidentale, tanto da diventare la seconda pelle dell’uomo, un elemento essenziale sia poiché riveste il compito di rendere visibile il corpo (che non ha significato nella sua nudità naturale) sia perché è indice dell’instabilità e della transitorietà, che sono gli elementi caratterizzanti la moda e lo spirito dell’epoca.48 “Le vêtement [..] est devenu pour l’homme une sorte de peau dont il ne se sépare sans aucun prétexte et qui lui adhère comme le pelage à l’animal, à ce point que la forme réelle du corps est de nos jours tout à fait tombée en oubli […]”.49 Per comprendere l’importanza capitale dell’abito ottocentesco nella nuova società borghese moderna occorre inoltre precisare che esistono “[…] due diverse mode, l’una, quella maschile, destinata a neutralizzare il corpo, a paralizzarlo in una forma pochissimo soggetta a oscillazioni, l’altra, quella femminile, tesa ad esibirlo, a manipolarlo e a sottoporlo ad ogni sorta di fluttuazione”.50 Ne consegue che il corpo di moda femminile assimila e si carica di significati anche sociali, primo tra tutti quello di compensare la neutralità dell’abito maschile e di assolvere una funzione nuova che mira alla rappresentazione della potenza e della ricchezza della borghesia.51 Questa funzione di rappresentazione e di legittimazione dello status sociale che assolve la moda femminile si radica in profondità e persiste a lungo nella società dell’alta borghesia novecentesca. g Negli anni Trenta sono ancora stiliste come Elsa Schiapparelli, Gabrielle Chanel e Madeleine Vionnet a rappresentare l’avanguardia della moda. In questi stessi anni la fotografia diventa sempre più importante per le riviste. A partire dagli anni Trenta fa la sua prima apparizione la fotografia a colori e le principali immagini delle riviste di moda diventano le fotografie, piuttosto che i disegni o i dipinti. Lo scoppio della seconda guerra mondiale nel 1939 danneggia in modo significativo la scena della haute couture parigina, molte case di moda sono costrette a chiudere e le poche rimaste aperte devono affrontare problemi come la scarsità di materiali e la fuga dei clienti. , p , p 51 P. Perrot sostiene che nella società moderna borghese l’esilio del corpo maschile investe le donne di una nuova funzione :“[…] signifier par procuration dans la splendeur inchangée de leurs toilettes et dans l’opulence de leur chairs le statut social, la puissance pécuniaire du père, du mari, de l’amant pour qu’il s’agit d’amasser la richesse sans plus l’exhiber directement” citazione dal testo P. Perrot, Les dessus et les dessous de la bourgeoisie, Paris, Fayard, 1981, op. cit., p. 63. p , p , p 49 Nel testo di F. Franchi, I veli della modernità, op. cit., p. 25 si trova questa citazione di T. Gautier che rafforza la riflessione dell’abito ottocentesco come seconda pelle che riveste e dona significato al corpo borghese. 52 Per l’analisi dell’ascesa della moda americana durante e dopo la seconda guerra mondiale cf. Dalla collezione del Kyoto Costume Institute, La Moda, op. cit., pp. 335-336. 53 Ivi, p. 336. 50 Ivi, op. cit., p. 27. 48 Secondo il pensiero di F. Franchi ne I veli della modernità, op. cit., p. 25. Figura 4. Mariano Fortuny (attr.) Manifattura veneziana, sopravveste, circa 1915 La frequentazione degli artisti a lei contemporanei determina uno stile fortemente influenzato dall’arte del XX secolo, tanto che nel 1932 The New Yorker scrive “Un vestito Schiapparelli è come un dipinto moderno”.43 Tra le innovazioni apportate da questa stilista-artista della moda, importante è l’invenzione della sfilata moderna ossia l’idea di sviluppare le collezioni attorno ad un unico tema e di spettacolarizzarle.44 Nella scelta dei materiali sperimenta per la prima volta il rayon, il vinile, il cellophane e per i tessuti si distingue per l’utilizzo del tweed, il tessuto escorce d’arbre e le fibre artificiali.45 In questo momento storico gli artisti utilizzano oggetti utilitaristici quotidiani come soggetti delle proprie opere, Elsa Schiaparelli è la prima che porta questi oggetti nella haute couture. Dopo la seconda guerra mondiale si sposta da New York, dove trascorre il periodo bellico, a Parigi. I suoi tentativi di riprendere l’attività falliscono e nel 1954 si ritira.46 Nel 2012, presso il Costume Institute del Metropolitan Museum of Art di New York, ha luogo la mostra Elsa Schiaparelli e Miuccia Prada. Impossible conversations. Nello stesso anno la maison ha riaperto la storica boutique di Parigi in cui la stilista esponeva le sue linee di alta moda, dando nuova vita al marchio italiano.47 La sua intera carriera è stata segnata dallo sforzo costante volto ad integrare arte e moda (fig. 7). 135 Elisa Risi Durante il periodo bellico lo Stato impone un rigido sistema di razionamento che porta come conseguenza lo sviluppo della gonna più corta e, al contempo, della moda militare. I materiali con cui si producono i cappelli non sono razionalizzati e questo porta alla creazione di modelli ampi, turbanti, e delle scarpe con la zeppa di sughero, molto diffuse a causa della carenza di pellame. Il declino della moda francese comporta l’ascesa della moda americana. Gli Stati Uniti, che prima della guerra erano stati clienti assidui della haute couture parigina, sviluppano una propria industria dell’abbigliamento.52 Durante la seconda guerra mondiale si affermano come protagonisti del sistema della moda, non tanto nel settore della haute couture, quanto invece nel settore dell’abbigliamento informale, da usare tutti i giorni: dell’abito prêt-à-porter, pronto-da- indossare.53 Sulla scia di questo nuovo stile americano di successo, dopo la Liberazione, nel 1944 Parigi si rimette al lavoro e grandi stilisti francesi come Christian Dior ricominciano a presentare collezioni. A partire dal secondo dopoguerra nasce e si afferma in modo definitivo y 53 Ivi, p. 336. 136 Colección de moda: de Eleonora Duse a Julie Peters-Desteract. Figura 7. Giorgio De Chirico, Vogue (edizione americana), 1° gennaio 1937; Elsa Schiaparelli, abito da sera, 1939. Figura 7. Giorgio De Chirico, Vogue (edizione americana), 1° gennaio 1937; Elsa Schiaparelli, abito da sera, 1939. anche l’alta moda italiana. Ken Scott è uno dei grandi stilisti emergenti in questo periodo. Il decennio degli anni Cinquanta determina la sua affermazione a livello internazionale e a partire dal 1962, anno di creazione del marchio Ken Scott, lo stilista affida a Susan Nevelson l’esclusività per l’ideazione dei disegni e delle fantasie degli abiti prodotti. La storia e gli sviluppi della moda portano lo studio a focalizzare l’attenzione su questa donna, pittrice e creatrice di collezioni che, grazie agli incredibili disegni e alle fantasie sorprendenti, hanno rivoluzionato quest’universo. Con lei, il rapporto tra il mondo della moda e quello dell’arte si rafforza e giunge ad intrecciarsi in modo profondo. Susan Nevelson nasce nel 1924 a Boston nel New England. Sposa il figlio della scultrice Louise Nevelson e dal 1951 si sposta e vive tra Parigi e Firenze. Il soggiorno in queste due città artistiche è fondamentale per la sua formazione autodidatta come pittrice e disegnatrice. Tre anni dopo ritorna a New York dove lavora creando disegni per varie ditte tessili newyorkesi. Soggiorna e lavora in Svizzera sempre nell’ambito dell’abbigliamento. 54 Per la biografia di Susan Nevelson cf. notizie biografiche di Kim Coston nel volume C. Chiarelli (a cura di), Donne protagoniste nel Novecento, op. cit., pp. 74-75. , pp 56 Per le notizie sulla mostra La metamorfosi italiana 1943-1968 presso il Guggenheim Museum di New York (1994) cf. notizie di Kim Coston nel volume C. Chiarelli (a cura di), Donne protagoniste nel Novecento, op. cit., p. 76. 57 Il libro più famoso di Anna Rontani è Caleidoscopio sul mare, Firenze, Editore Pacini Fazzi, 1995. Con questo libro vince il premio Fiorino d’Oro di Firenze. 55 Ivi, pp. 75-76. Elisa Risi Nel 1960 si stabilisce definitivamente a Firenze come disegnatrice indipendente: dal suo studio invia le nuove creazioni a New York, in Svizzera e a Ken Scott, al quale la lega un’amicizia di lunga data. Nel 1955 lo stilista americano fonda il marchio Falconetto con il quale si lancia con successo nel mondo dell’alta moda. Nel 1962 la pittrice accetta il contratto di esclusività dei suoi disegni per il marchio nascente: Ken Scott.54 Il contratto consolida il loro stretto, quasi simbiotico rapporto, che li legherà fino alla morte di lui, nel 1991. Le creazioni di Susan Nevelson sono emblematiche della personalità 137 Elisa Risi ardente e vivace dell’artista e il suo talento si manifesta nella produzione di stampe colorate che divengono un contrassegno e un pilastro del marchio Ken Scott. L’energia dei fiori stampati e le sfilate dal taglio teatrale organizzate dallo stilista stupiscono giornalisti di moda e acquirenti internazionali, portando la firma ad un grande successo. Ken Scott e Susan Nevelson hanno contribuito a lanciare la sfida italiana alla supremazia francese, ponendo le basi per il sistema della moda italiano. Susan Nevelson è l’ideatrice delle fantasie e la musa ispiratrice della casa di moda Ken Scott, anche se il suo contributo alla moda italiana spesso non è adeguatamente riconosciuto. Le stampe dell’artista sono apparse su Vogue, Bazaar e su altri mezzi d’informazione eminenti, e sono state indossate da celebri clienti, modelle e attrici, da Brigitte Bardot a Audrey Hepburn, da Sofia Loren a Mina, da Peggy Guggenheim a Twiggy.55 Nel 1994 il Guggenheim Museum di New York ha celebrato il design italiano con una mostra intitolata La metamorfosi italiana 1943-1968. Su tre delle cinque creazioni di Ken Scott figurano le stampe della pittrice, che ha partecipato all’evento in qualità di rappresentante del marchio.56 Ad oggi Susan Nevelson rimane la forza vitale della casa di moda, incarnandone lo spirito attraverso i suoi disegni e la sua arte (fig. 8). A i di S l i i l l di ll fi Figura 8. Susan Nevelson, abito da sera Gufi, 1968. Figura 8. Susan Nevelson, abito da sera Gufi, 1968. Figura 8. Susan Nevelson, abito da sera Gufi, 1968. Figura 8. Susan Nevelson, abito da sera Gufi, 1968. , pp 56 Per le notizie sulla mostra La metamorfosi italiana 1943-1968 presso il Guggenheim Museum di New York (1994) cf. notizie di Kim Coston nel volume C. Chiarelli (a cura di), Donne protagoniste nel Novecento, op. cit., p. 76. 57 Il libro più famoso di Anna Rontani è Caleidoscopio sul mare, Firenze, Editore Pacini Fazzi, 1995. Con questo libro vince il premio Fiorino d’Oro di Firenze Figura 8. Susan Nevelson, abito da sera Gufi, 1968. Figura 8. Susan Nevelson, abito da sera Gufi, 1968. ardente e vivace dell’artista e il suo talento si manifesta nella produzione di stampe colorate che divengono un contrassegno e un pilastro del marchio Ken Scott. L’energia dei fiori stampati e le sfilate dal taglio teatrale organizzate dallo stilista stupiscono giornalisti di moda e acquirenti internazionali, portando la firma ad un grande successo. Ken Scott e Susan Nevelson hanno contribuito a lanciare la sfida italiana alla supremazia francese, ponendo le basi per il sistema della moda italiano. Susan Nevelson è l’ideatrice delle fantasie e la musa ispiratrice della casa di moda Ken Scott, anche se il suo contributo alla moda italiana spesso non è adeguatamente riconosciuto. Le stampe dell’artista sono apparse su Vogue, Bazaar e su altri mezzi d’informazione eminenti, e sono state indossate da celebri clienti, modelle e attrici, da Brigitte Bardot a Audrey Hepburn, da Sofia Loren a Mina, da Peggy Guggenheim a Twiggy.55 Nel 1994 il Guggenheim Museum di New York ha celebrato il design italiano con una mostra intitolata La metamorfosi italiana 1943-1968. Su tre delle cinque creazioni di Ken Scott figurano le stampe della pittrice, che ha partecipato all’evento in qualità di rappresentante del marchio.56 Ad oggi Susan Nevelson rimane la forza vitale della casa di moda, incarnandone lo spirito attraverso i suoi disegni e la sua arte (fig. 8). A d d ardente e vivace dell’artista e il suo talento si manifesta nella produzione di stampe colorate che divengono un contrassegno e un pilastro del marchio Ken Scott. L’energia dei fiori stampati e le sfilate dal taglio teatrale organizzate dallo stilista stupiscono giornalisti di moda e acquirenti internazionali, portando la firma ad un grande successo. Ken Scott e Susan Nevelson hanno contribuito a lanciare la sfida italiana alla supremazia francese, ponendo le basi per il sistema della moda italiano. Susan Nevelson è l’ideatrice delle fantasie e la musa ispiratrice della casa di moda Ken Scott, anche se il suo contributo alla moda italiana spesso non è adeguatamente riconosciuto. Le stampe dell’artista sono apparse su Vogue, Bazaar e su altri mezzi d’informazione eminenti, e sono state indossate da celebri clienti, modelle e attrici, da Brigitte Bardot a Audrey Hepburn, da Sofia Loren a Mina, da Peggy Guggenheim a Twiggy.55 Nel 1994 il Guggenheim Museum di New York ha celebrato il design italiano con una mostra intitolata La metamorfosi italiana 1943-1968. Figura 8. Susan Nevelson, abito da sera Gufi, 1968. Su tre delle cinque creazioni di Ken Scott figurano le stampe della pittrice, che ha partecipato all’evento in qualità di rappresentante del marchio.56 Ad oggi Susan Nevelson rimane la forza vitale della casa di moda, incarnandone lo spirito attraverso i suoi disegni e la sua arte (fig. 8). p g ( g ) Anna Rontani, coetanea di Susan Nevelson, si situa nel solco di quelle figure emblematiche per il sistema della moda italiano che vede, dopo la nascita con stiliste del calibro di Rosa Genoni, l’affermazione definitiva e il successo internazionale a partire dal secondo dopoguerra. Anna Rontani è mecenate dei grandi stilisti italiani emergenti in questo periodo e personaggio emblematico della vita mondana che scorre a fianco e a sostegno del mondo della moda. Altro elemento che la accomuna alla contessa è la presenza di parte della sua collezione alla Galleria di Palazzo Pitti di Firenze. “Patrimonio tessile” è un termine che ben si presta all’analisi di questa collezione. Anna Rontani nasce nel 1924 a Massarosa, in provincia di Lucca, è una scrittrice57 e filosofa, viaggiatrice e compagna del nobile Giovanni 138 Colección de moda: de Eleonora Duse a Julie Peters-Desteract. Figura 9. Abito di adolescente, India, Cachemire, XIX secolo, fotografia scattata da Elisa Risi presso Museo Guimet, Parigi, venerdì 16 ottobre 2015. Il capo è di lana e seta, ricamato con inserti e fili d’oro nelle maniche e nelle giunture. Figura 9. Abito di adolescente, India, Cachemire, XIX secolo, fotografia scattata da Elisa Risi presso Museo Guimet, Parigi, venerdì 16 ottobre 2015. Il capo è di lana e seta, ricamato con inserti e fili d’oro nelle maniche e nelle giunture. Falchini con cui condivide molte passioni tra cui quella del viaggio e lo stile di vita intenso e mondano caratterizzato da feste, concerti, ricevimenti, stile che mantiene anche dopo la morte di lui.58 Il fenomeno del collezionismo segna la personalità e la vita di questa donna. Anna Rontani si documenta e segue appassionatamente le evoluzioni della moda italiana, e spesso, a partire dall’analisi delle riviste di moda, segnala e commissiona gli abiti che desidera a case di moda di fiducia. Ama anche la haute couture francese, che commissiona alla fidata sartoria delle Sorelle Sorgentini di Viareggio, specializzate e abili nella riproduzione di modelli dei couturiers francesi. La scelta di materiali pregiati e di tessuti preziosi caratterizza il gusto e la raffinatezza del suo stile. 58 Per la biografia di Anna Rontani cf. notizie biografiche di Elisa Masiero nel volume C. Chiarelli (a cura di), Donne protagoniste nel Novecento, op. cit., pp. 114-115. 59 Ivi, pp. 115, 116. Figura 8. Susan Nevelson, abito da sera Gufi, 1968. Sempre aggiornata sulle novità della moda, oltre alle Sorelle Sorgentini, commissiona abiti anche a Elda Pavan e Jole Venziani, stiliste italiane emergenti in questo periodo storico, a Renato Balestra e a stilisti internazionali come il tunisino Loris Azzaro.59 Accanto al ruolo di mecenate Anna Rontani si distingue per essere al contempo collezionista di abiti e di viaggi. Alla parete del suo studio tiene un planisfero sul quale sono marcati, attraverso bandiere colorate, tutti i luoghi che ha visitato e in cui ha vissuto. Per tutta la vita, tiene diari sulle esperienze che vive e sugli abiti, i modelli e i tessuti che ama. Per ogni occasione importante commissiona un capo speciale e questo comporta l’arricchimento della sua collezione. Il collezionismo in questo caso assolve la funzione di specchio della personalità, della vita e delle esperienze di questa donna, che conserva con estrema cura tutti i suoi abiti, dalla prima giovinezza sino agli anni Cinquanta e oltre. Inoltre, se si analizza la sua collezione tessile si può leggere la storia e l’evoluzione della moda italiana ed occidentale a lei contemporanea. Memoria e testimonianza, riflesso e ritratto, questi abiti costituiscono quindi una collezione leggibile come le pagine dei diari che caratterizzano l’attività di questa donna. Anna Rontani fa del collezionismo uno stile di vita: collezione di immagini sono i diari ricchi di fotografie di abiti, di memorie di viaggi e di momenti salienti della propria vita; 139 Elisa Risi Figura 10. Abiti indiani, provenienti dal lascito di Krishnâ Riboud al Museo Guimet di Parigi parte della sua collezione. Figura 10. Abiti indiani, provenienti dal lascito di Krishnâ Riboud al Museo Guimet di Parigi parte della sua collezione. collezione di capi e creazioni tessili di notevole livello artistico è il suo guardaroba. Tutto ciò si concatena e diventa uno strumento per leggere ed interpretare, svelare, la vita di questa donna. Cecilia Matteucci Lavarini è una figura speculare ad Anna Rontani: a lei si oppone per la consapevolezza e la coscienza che la portano a creare un’importante collezione di abiti di alta moda. Anna Rontani non si autodefinisce mai con il termine collezionista e non designa il proprio guardaroba come collezione, tuttavia, analizzando retrospettivamente il suo lascito emerge una collezione vera e propria ed un attitudine al collezionare, che permettono di leggere nitidamente sia la personalità di questa donna, sia la storia della moda italiana post bellica. 60 Citazione attinta dal volume C. Chiarelli (a cura di), Donne protagoniste nel Novecento, op. cit., p. 87. 61 Ivi, op. cit., p. 86. 61 Ivi, op. cit., p. 86. Figura 8. Susan Nevelson, abito da sera Gufi, 1968. Per l’imponenza e per il valore che rappresentano per la storia della moda e del costume, i suoi capi vengono spesso richiesti da enti museali e importanti istituzioni artistiche per mostre ed esposizioni.64 Cecilia Matteucci Lavarini instaura un legame affettivo con ogni capo che entra a far parte della sua collezione, investe i suoi abiti, i suoi accessori e i gioielli di un valore particolare ed unico. Questo personaggio vive appieno il fenomeno del collezionismo e la casa-museo di Bologna ne è la dimostrazione. Non soltanto emblema della femme collectionneuse, ma anche donatrice, dichiara da molti anni che la destinazione finale della sua collezione deve essere il museo: “Vorrei che la gente che va a vedere i miei abiti li possa amare come li ho amati io, considerandoli come tante opere d’arte, davanti alle quali la disgrazia più grande è perdere il desiderio di conoscere”.65 Fedele alla sua volontà e a questa prospettiva dona alla Galleria del Costume di Palazzo Pitti di Firenze una serie di abiti fondamentali e rappresentativi del percorso e delle evoluzioni della moda dagli anni Venti del Novecento sino alle creazioni contemporanee del XXI secolo. Krishnâ Riboud si distingue in questo elaborato per essere la figura più rappresentativa Figura 11. Paul Poiret, manto, 1920 circa. L’abito presentato è attribuito al celebre stilista parigino Paul Poiret. Il capo è confezionato in velluto di seta stampato con motivo a rose stilizzate. Figura 11. Paul Poiret, manto, 1920 circa. L’abito presentato è attribuito al celebre stilista parigino Paul Poiret. Il capo è confezionato in velluto di seta stampato con motivo a rose stilizzate. Figura 11. Paul Poiret, manto, 1920 circa. L’abito presentato è attribuito al celebre stilista parigino Paul Poiret. Il capo è confezionato in velluto di seta stampato con motivo a rose stilizzate. Figura 11. Paul Poiret, manto, 1920 circa. L’abito presentato è attribuito al celebre stilista parigino Paul Poiret. Il capo è confezionato in velluto di seta stampato con motivo a rose stilizzate. unico. Ho il dono o il talento di riuscire a portare l’originale e l’importabile”.62 È questo approccio particolare e anticonvenzionale al mondo della moda che determina la molteplicità degli stili e la varietà delle celebri firme che rientrano nella sua collezione. 62 Ivi, op. cit., p. 87. 63 Per le notizie relative alla collezione tessile di Cecilia Matteucci Lavarini cf. il testo di Alessandro Guasti nel volume C. Chiarelli (a cura di), Donne protagoniste nel Novecento, op. cit., pp. 87-88. 64 Recentemente Cecilia Matteucci Lavarini ha prestato molti pezzi per la mostra Diana Vreeland after Diana Vreeland a Venezia e Schiaparelli and Prada: Impossible Conversations a New York, entrambe nel 2012. Anni prima, la mostra Interpretazioni personali nella storia dell’abbigliamento, alla Galleria del Costume di Palazzo Pitti a Firenze, espose una parte del suo guardaroba che comprendeva Lagerfeld per Chanel, Dior, Balmain, Galliano. 65 Citazione tratta da articolo di S. Deganello, Who is Cecilia Matteucci Lavarini?, consultato il 19 novembre 2015 (http://www.rocaille.it/who-is-cecilia-matteucci-lavarini/). Figura 8. Susan Nevelson, abito da sera Gufi, 1968. Gli abiti e gli accessori appartengono a nomi illustri come Balenciaga, Chanel, Courrèges, Dior, Fendi, Galliano, Gucci, Lanvin, Martin Margiela, McQueen, Siant Lauren, Schiaparelli, Versace, Louis Vuitton.63 La collezione conta circa 3000 pezzi, di cui fanno parte non solo abiti moderni ma anche capi antichi e molti accessori e gioielli. Per l’imponenza e per il valore che rappresentano per la storia della moda e del costume, i suoi capi vengono spesso richiesti da enti museali e importanti istituzioni artistiche per mostre ed esposizioni.64 Cecilia Matteucci Lavarini instaura un legame affettivo con ogni capo che entra a far parte della sua collezione, investe i suoi abiti, i suoi accessori e i gioielli di un valore particolare ed unico. Questo personaggio vive appieno il fenomeno del collezionismo e la casa-museo di Bologna ne è la dimostrazione. Non soltanto emblema della femme collectionneuse, ma anche donatrice, dichiara da molti anni che la destinazione finale della sua collezione deve essere il museo: “Vorrei che la gente che va a vedere i miei abiti li possa amare come li ho amati io, considerandoli come tante opere d’arte, davanti alle quali la disgrazia più grande è perdere il desiderio di conoscere”.65 Fedele alla sua volontà e a questa prospettiva dona alla Galleria del Costume di Palazzo Pitti di Firenze una serie di abiti fondamentali e rappresentativi del percorso e delle evoluzioni della moda dagli anni Venti del Novecento sino alle creazioni contemporanee del XXI secolo. unico. Ho il dono o il talento di riuscire a portare l’originale e l’importabile”.62 È questo approccio particolare e anticonvenzionale al mondo della moda che determina la molteplicità degli stili e la varietà delle celebri firme che rientrano nella sua collezione. Gli abiti e gli accessori appartengono a nomi illustri come Balenciaga, Chanel, Courrèges, Dior, Fendi, Galliano, Gucci, Lanvin, Martin Margiela, McQueen, Siant Lauren, Schiaparelli, Versace, Louis Vuitton.63 La collezione conta circa 3000 pezzi, di cui fanno parte non solo abiti moderni ma anche capi antichi e molti accessori e gioielli. Per l’imponenza e per il valore che rappresentano per la storia della moda e del costume, i suoi capi vengono spesso richiesti da enti museali e importanti istituzioni artistiche per mostre ed esposizioni.64 Cecilia Matteucci Lavarini instaura un legame affettivo con ogni capo che entra a far parte della sua collezione, investe i suoi abiti, i suoi accessori e i gioielli di un valore particolare ed unico. Figura 8. Susan Nevelson, abito da sera Gufi, 1968. Tanto Anna Rontani, dunque, agisce istintivamente, quanto Cecilia Matteucci Lavarini agisce coscienziosamente, ponendosi per questo agli antipodi della posizione di Anna Rontani. Cecilia Matteucci Lavarini si considera e si autodefinisce collezionista. “La mia collezione è il mio autoritratto, non l’avere, ma l’essere, è un solitario lavoro a tempo pieno, realizzato pensando sempre alla destinazione finale, il museo”.60 Dalle sue parole si evince, dunque, oltre che la passione e l’amore per il mondo dell’alta moda, anche la consapevolezza del valore che essa assume per lei, essendo il riflesso della sua personalità. La dichiarazione è fondamentale anche perché afferma la volontà di consegnare al futuro le creazioni tessili, realizzate nel tempo dalle più rinomate firme di stilisti francesi e italiani, con l’obiettivo esplicito di rendere visibili gli anni di ricerca che sono alla base di questa collezione. “La moda è il presente, la novità, è curiosità, ma al tempo stesso è conoscere il passato. Per chi ne possiede l’abilità, significa saperne intravedere quello che sarà il domani”.61 La cura nella scelta e la ricerca sono motivate dall’impulso ad accrescere e perfezionare le proprie acquisizioni, al fine di creare una collezione in cui si ritrovi, oltre al proprio stile, anche gli sviluppi e la storia dell’evoluzione della moda italiana ed internazionale. Cecilia Matteucci Lavarini frequenta le maggiori sfilate di moda e le case d’asta più rinomate, come Drouot e Christie’s, viaggiando per tutta Europa alla ricerca dell’unicità e della particolarità. “La moda mi dà la possibilità narcisisticamente appagante di interpretare su di me epoche e costumi, di indossare travolgenti ed inimitabili pezzi storici di couture. Amo differenziarmi da tutti nel vestire, cerco sempre qualcosa che sia bello, prezioso, anche di difficile portabilità, purché 140 Colección de moda: de Eleonora Duse a Julie Peters-Desteract. Colección de moda: de Eleonora Duse a Julie Peters-Desteract. unico. Ho il dono o il talento di riuscire a portare l’originale e l’importabile”.62 È questo approccio particolare e anticonvenzionale al mondo della moda che determina la molteplicità degli stili e la varietà delle celebri firme che rientrano nella sua collezione. Gli abiti e gli accessori appartengono a nomi illustri come Balenciaga, Chanel, Courrèges, Dior, Fendi, Galliano, Gucci, Lanvin, Martin Margiela, McQueen, Siant Lauren, Schiaparelli, Versace, Louis Vuitton.63 La collezione conta circa 3000 pezzi, di cui fanno parte non solo abiti moderni ma anche capi antichi e molti accessori e gioielli. 62 Ivi, op. cit., p. 87. , p , p 63 Per le notizie relative alla collezione tessile di Cecilia Matteucci Lavarini cf. il testo di Alessandro Gua nel volume C. Chiarelli (a cura di), Donne protagoniste nel Novecento, op. cit., pp. 87-88. la mostra Interpretazioni personali nella storia dell’abbigliamento, alla Galleria del Costume di Palazzo Pitti a Firenze, espose una parte del suo guardaroba che comprendeva Lagerfeld per Chanel, Dior, Balmain, Galliano. 65 Citazione tratta da articolo di S. Deganello, Who is Cecilia Matteucci Lavarini?, consultato il 19 novembre 2015 (http://www.rocaille.it/who-is-cecilia-matteucci-lavarini/). p p g p g p , , , 65 Citazione tratta da articolo di S. Deganello, Who is Cecilia Matteucci Lavarini?, consultato il 19 novembre 2015 (http://www.rocaille.it/who-is-cecilia-matteucci-lavarini/). Figura 11. Paul Poiret, manto, 1920 circa. L’abito presentato è attribuito al celebre stilista parigino Paul Poiret. Il capo è confezionato in velluto di seta stampato con motivo a rose stilizzate. ( ), p g , p , pp 64 Recentemente Cecilia Matteucci Lavarini ha prestato molti pezzi per la mostra Diana Vreeland after Diana reeland a Venezia e Schiaparelli and Prada: Impossible Conversations a New York, entrambe nel 2012. Anni prima, mostra Interpretazioni personali nella storia dell’abbigliamento, alla Galleria del Costume di Palazzo Pitti a Firenze, pose una parte del suo guardaroba che comprendeva Lagerfeld per Chanel, Dior, Balmain, Galliano. 65 Cit i t tt d ti l di S D ll Wh i C ili M tt i L i i? lt t il 19 b Figura 8. Susan Nevelson, abito da sera Gufi, 1968. Questo personaggio vive appieno il fenomeno del collezionismo e la casa-museo di Bologna ne è la dimostrazione. Non soltanto emblema della femme collectionneuse, ma anche donatrice, dichiara da molti anni che la destinazione finale della sua collezione deve essere il museo: “Vorrei che la gente che va a vedere i miei abiti li possa amare come li ho amati io, considerandoli come tante opere d’arte, davanti alle quali la disgrazia più grande è perdere il desiderio di conoscere”.65 Fedele alla sua volontà e a questa prospettiva dona alla Galleria del Costume di Palazzo Pitti di Firenze una serie di abiti fondamentali e rappresentativi del percorso e delle evoluzioni della moda dagli anni Venti del Novecento sino alle creazioni contemporanee del XXI secolo. p Krishnâ Riboud si distingue in questo elaborato per essere la figura più rappresentativa della donna donatrice. Svolge un ruolo fondamentale in quanto collezionista nel settore del tessile e per la donazione dell’intera collezione al Museo Guimet di Parigi, arricchendone il 141 Elisa Risi Figura 12. Maria Monaci Gallenga, mantello, 1920 circa. Capo confezionato interamente in velluto di seta nero stampato a motivi di grande rapporto, di ispirazione turca. Figura 12. Maria Monaci Gallenga, mantello, 1920 circa. Capo confezionato interamente in velluto di seta nero stampato a motivi di grande rapporto, di ispirazione turca. patrimonio tanto da renderlo il museo europeo con la più importante sezione destinata all’Arte Tessile Asiatica. Personaggio emblematico per il contributo dato allo sviluppo del legame tra il mondo dell’arte e quello del tessile, Krishnâ Riboud si dedica soprattutto ai tessuti e agli abiti tradizionali delle regioni del continente asiatico. Le sue origini sono indiane, nasce nella regione del Bengala, a Dacca nel 1926. Dopo la perdita prematura del padre viene allevata dalla famiglia materna a Calcutta ed in particolare dal prozio Rabindranath Tagore. Nella famiglia, come a Santiniketan, sede dell’università fondata da Tagore, La Visva Bharati University, si respira uno spirito cosmopolita.66 Questo tipo di educazione e di apertura la porta a proseguire gli studi negli Stati Uniti sostenuta da una borsa accordata dalla Mayling Soong Foundation.67 Insieme al marito, Jean Riboud, intesse profonde ed intense relazioni con gli artisti, gli scrittori e gli intellettuali newyorkesi più importanti del mondo contemporaneo. Nel 1951 Jean Riboud si sposta a Parigi con lei per adempire alla carica di presidente della multinazionale Schlumberger Limited. f p p p g 67 Fondazione istituita presso lo Wellesley College di Boston. L'idea della Fondazione Mayling Soong si sviluppa da una discussione nata per trovare un modo adatto per marcare il 25° anniversario della laurea presso lo Wellesley College della Signora Mayling Soong, moglie del presidente cinese Tchiang Kai-chek. Lo scopo della Fondazione è quello di rafforzare e sviluppare il lavoro offerto dall'Università per l'arte, la geografia, la storia, la filosofia e le religioni orientali. La Fondazione si insedia in carica all’università di Wellesley nell’ottobre 1942. Promuove una serie di conferenze e lo scambio di studiosi tra Oriente ed Occidente. 66 Per la biografia di Krishnâ Riboud cf. notizie biografiche nell’articolo di Jean-François Jarrige, Krishnā Riboud (1926-2000) all’interno della rivista Arts asiatiques, Paris, Année 2000, Volume 55, Numéro 1, pp. 162- 163; e nel testo di Aurelie Samuel, curatrice incaricata della Collezione Riboud, Collectionner les textiles : constituer une collection de référence, l'exemple de Krishnâ Riboud, visionato personalmente presso il Museo Guimet, Parigi. 68 Sul mecenatismo dei coniugi Riboud cf. articolo di Jean-François Jarrige, Krishnā Riboud (1926-2000), op. cit., pp. 162-163; e testo di Aurelie Samuel, Collectionner les textiles : constituer une collection de référence, l'exemple de Krishnâ Riboud, visionato personalmente presso il Museo Guimet, Parigi. Guimet, Arts asiatiques, Paris, Année 2004, Volume 59, Numéro 1, pp. 154 157. 70 Io stessa ho potuto consultare i cataloghi, prendere visione dei testi e dei tessuti della collezione Riboud accedendo alla Biblioteca e alla Galleria Jean e Krishnâ Riboud che hanno sede a Parigi, al Museo Guimet. 69 Per le notizie sulla fondazione dell’A.E.D.T.A cf. articolo di V. Lefèvre, La collection Krishnā Riboud au musée Guimet, Arts asiatiques, Paris, Année 2004, Volume 59, Numéro 1, pp. 154-157. 70 Io stessa ho potuto consultare i cataloghi, prendere visione dei testi e dei tessuti della collezione Riboud accedendo alla Biblioteca e alla Galleria Jean e Krishnâ Riboud che hanno sede a Parigi, al Museo Guimet. 69 Per le notizie sulla fondazione dell’A.E.D.T.A cf. articolo di V. Lefèvre, La collection Krishnā Riboud au musée Guimet, Arts asiatiques, Paris, Année 2004, Volume 59, Numéro 1, pp. 154-157. Figura 8. Susan Nevelson, abito da sera Gufi, 1968. La coppia, con grande eleganza e discrezione, approfitta del tenore di vita agiato per sostenere artisti, ricercatori e pionieri nel mondo delle arti.68 Krishnâ Riboud ritorna sovente in India. Fedele alla tradizione e alla discendenza dei Tagore si interessa alle arti e alle tradizioni popolari del Paese natale. Agli inizi degli anni Cinquanta con l’acquisto dei primi Sari Baluchari inizia a riunire una propria collezione attestante la ricchezza e la varietà delle tecniche tessili e dei tessuti indiani. Il Sari Baluchari è un tipo di sari, un indumento indossato dalle donne in tutta l'India e Bangladesh. È nato in Bengala ed è stato tradizionalmente prodotto principalmente in Murshidabad anche 142 Colección de moda: de Eleonora Duse a Julie Peters-Desteract. Colección de moda: de Eleonora Duse a Julie Peters-Desteract. Figura 13. Immagine tratta dal sito ufficiale di Julie Peters- Desteract (http://www.juliepeters-art.com). Figura 13. Immagine tratta dal sito ufficiale di Julie Peters- Desteract (http://www.juliepeters-art.com). Figura 13. Immagine tratta dal sito ufficiale di Julie Peters- Desteract (http://www.juliepeters-art.com). Fondando questo organo Krishnâ Riboud riesce nell’obiettivo di unire la propria passione e natura di collezionista ad un percorso e ad una ricerca scientifica, rendendo la propria collezione accessibile sia a ricercatori e studiosi sia al grande pubblico, grazie ad eventi espositivi e mostre.70 Spinta dalla volontà di condivisione di un 143 Elisa Risi Elisa Risi Figura 14. Immagine tratta dal sito ufficiale di Julie Peters- Desteract (http://www.juliepeters-art.com). Figura 14. Immagine tratta dal sito ufficiale di Julie Peters- Desteract (http://www.juliepeters-art.com). patrimonio artistico universale la Riboud lega la propria collezione, la più importante collezione privata al mondo in questo ambito, al Museo Guimet.71 Nel 1990 una prima donazione di 150 tessuti provenienti dal fondo dell’A.E.D.T.A arricchisce il patrimonio del Museo parigino, fino ad allora relativamente povero nella sezione tessile asiatica. Krishnâ Riboud per tutta la sua vita si fa garante del legame che unisce le più grandi istituzioni museali all’universo del tessile rappresentato dalla sua grande collezione e dall’impegno costante e tenace nella ricerca e nello sviluppo delle collezioni sia dell’A.E.D.T.A sia del Museo Guimet. Ad attestare il suo impegno per la promozione delle relazioni tra Francia e India e il suo ruolo chiave per il patrimonio artistico universale nel 1996 viene insignita della Legione d’Onore. Dopo la sua scomparsa nel 2000, risale al 2003 la donazione della quasi totalità della collezione, 4000 tessuti circa, al Museo Guimet72 (fig. 10). , , ( g ) Anna Piaggi (1931-2012) è collezionista di alta moda e personaggio di spicco in questo settore per il suo ruolo di giornalista. “Amo l’atmosfera, gli abiti […]. La mia natura è sempre stata attratta dal superficiale”. “Il mio cappello è qualcosa di personale. Contiene l’anima, il sentimento e le sensazioni che muovono questo piccolo mondo”.73 Queste dichiarazioni svelano il particolare rapporto che questa donna intrattiene con il fenomeno del collezionismo. Anna Piaggi è un amante degli abiti e degli accessori di moda che seppur nella loro natura effimera e transitoria, “superficiale”, per usare le sue parole, sono rappresentativi dell’epoca in cui vive. La moda per lei è il testimone del presente fuggevole e della contemporaneità e per questo assume un valore profondo che va ad investire, attraverso un legame non solo materiale ma affettivo, ogni capo e accessorio che entra a far parte della sua collezione. 73 Le dichiarazioni trascritte sono di Anna Piaggi e sono attinte dalla presentazione della mostra Hat-Ology organizzata dal Costume Moda Immagine di Milano e dedicata ai cappelli di Anna Piaggi, curati dal suo cappellaio di fiducia, Stephen Jones. La mostra si è svolta presso il Palazzo Morando di Milano dal 22 settembre al 30 novembre 2013. 71 Per la donazione risalente al 1990 della collezione Riboud al Museo Guimet cf. V. Lefèvre, La collection Krishnā Riboud au musée Guimet, op. cit., pp. 154-157. 72 Ibidem Figura 13. Immagine tratta dal sito ufficiale di Julie Peters- Desteract (http://www.juliepeters-art.com). se attualmente Bishnupur e le zone circostanti del Bengala Occidentale sono gli unici luoghi dove vengono prodotti gli autentici Sari Baluchari. La produzione richiede più di una settimana di lavoro e il Sari Baluchari gode dello status di Geographical Indication (GI) in India e rientra nel patrimonio culturale e artistico dell’Asia (fig. 9). se attualmente Bishnupur e le zone circostanti del Bengala Occidentale sono gli unici luoghi dove vengono prodotti gli autentici Sari Baluchari. La produzione richiede più di una settimana di lavoro e il Sari Baluchari gode dello status di Geographical Indication (GI) in India e rientra nel patrimonio culturale e artistico dell’Asia (fig. 9). p ( g ) Sostenuta dal marito e dall’amore per le arti, sarà sempre impegnata a promuovere in modo vicendevole il patrimonio artistico orientale in occidente e l’arte occidentale in India. A partire dal 1964 instaura una relazione privilegiata con il Museo Guimet: in questo ambito e da questa collaborazione prende avvio un vasto programma di ricerca sui tessuti antichi dell’Asia centrale. La particolarità e la novità dello studio condotto da Krishnâ Riboud sta nel focalizzare le ricerche sulle differenti tecniche utilizzate per le varie realizzazioni tessili asiatiche. Si tratta di un approccio innovativo che permette di aprire le prospettive anche sul contesto ricco che permea questi saperi, sviluppando le ricerche sociali e culturali sul popolo indiano e asiatico in generale. Fino ad allora le grandi collezioni di tessuti dell’Asia centrale del Museo Guimet, del Museo nazionale di New Delhi e del British Museum erano state costituite da studi fatti quasi esclusivamente da una prospettiva iconografica e della storia dell’arte. Di conseguenza, fino alla metà del XX secolo si avevano flebili conoscenze su tutte le tecniche di tessitura asiatiche. Nel 1979 Krishnâ Riboud, consapevole dell’importanza di questo approccio scientifico al mondo tessile asiatico, crea l’Association pour l'Étude et la Documentation des Textiles d'Asie (A.E.D.T.A).69 L’Associazione, situata nell’Avenue de Breteuil a Parigi, sviluppa un notevole centro di documentazione e di ricerca scientifica, dotato di una biblioteca e di una fototeca e diviene un modello riconosciuto per lo studio tecnico delle collezioni tessili. 74 Per la biografia di Anna Piaggi e la carriera professionale cf. il volume C. Chiarelli (a cura di), Donne protagoniste nel Novecento, op. cit., pp. 64-65. 75 Citazione del New York Times ripresa da E. Bellino nell’articolo Anna Piaggi frasi e moda consultato il 19 novembre 2015 (http://www.theladycracy.it/2014/09/27/anna-piaggi-frasi-e-moda/). 76 Per il valore di patrimonio artistico e culturale attribuito ai capi della collezione di Anna Piaggi cf. le notizie presenti nel testo di C. Chiarelli nel volume C. Chiarelli (a cura di), Donne protagoniste nel Novecento, op. cit., p. 65. 77 Ibidem. Figura 13. Immagine tratta dal sito ufficiale di Julie Peters- Desteract (http://www.juliepeters-art.com). Il legame con il mondo tessile emerge soprattutto nella sua carriera lavorativa, che la vede impegnata a formare e sviluppare il mestiere di redattrice di moda. Grazie alla conoscenza della storia del costume, alla sua abilità espositiva e alla sua preparazione, diviene 144 Colección de moda: de Eleonora Duse a Julie Peters-Desteract. la prima donna ad impegnarsi professionalmente e ad emergere a livello internazionale come giornalista e scrittrice di moda. Insieme al marito Alfa Castaldi (sposato nel 1962) uno dei fotografi italiani più importanti e con la collega Anna Riva, getta le basi di questo nuovo mestiere. All’inizio degli anni Sessanta diventa fashion editor presso Arianna, una delle prime riviste femminili di Mondadori e successivamente fa da editor presso la Condé Nast. Il successo internazionale di Anna Piaggi è sancito dall’impegno professionale per Vogue, rivista con cui collabora dal 1988 nella rubrica intitolata D.P. Doppie Pagine di Anna Piaggi. Il volume Fashion Algebra edito da Thames & Hudson del 1999 raccoglie questi articoli scritti per Vogue Italia in un linguaggio surreale e onirico che rispecchia in modo sottile le sue intuizioni e il suo stile. Alla fine degli anni Ottanta è opinionista prima per Panorama e poi per L’Espresso; è questo il periodo in cui lo stilista Karl Lagerfeld le dedica il libro Anna Chronique edito nel 1986 da Longanesi.74 Anna Piaggi ribalta il concetto della moda. Con lei acquista forza ed espressione lo stile Made in Italy ed è la prima ad utilizzare il vintage, prima ancora che questo concetto nasca e si sviluppi. Arriva a possedere migliaia di abiti, scarpe e cappelli, che includono capi d’abbigliamento degli ultimi duecento anni. Anna Piaggi si distingue anche per essere un’interprete efficace, una musa e un’artista per molti stilisti a lei contemporanei. In tutta la sua carriera, più di cinquant’anni, scrive migliaia di editoriali e a lei vengono dedicati libri e mostre a livello internazionale. Il suo stile si ricorda per essere eccentrico e stravagante, sempre diverso e destabilizzante. Oltre agli abiti di epoche e stili lontani, tipico di questa donna è anche il trucco estremamente appariscente: trucco bianco con gote tinte di rosso, contorno occhi azzurro o nero, labbra rosse e ciuffo turchino. L’obiettivo è per lei quello di diventare un’opera d’arte che possa lasciare il segno: “Lavorava con gli abiti come i pittori con i tubetti di colore. 79 Le informazioni sull’artista contemporanea Julie Peters-Desteract sono attinte dal suo sito ufficiale (http://www.juliepeters-art.com consultato il 19 novembre 2015) e da molteplici interviste e incontri con l’artista, avvenuti in prima persona, durante il periodo che ho trascorso in Francia nei mesi estivi e nell’autunno dell’anno 78 Citazione dell’artista attinta dal suo sito ufficiale di cui scrivo di seguito il riferimento: http://www.juliepeters- art.com. consultato il 19 novembre 2015. Julie Peters-Desteract realizza il proprio sito ufficiale interamente in lingua inglese, ciò è sintomatico dello stile di vita dell’artista che la porta a spostarsi perennemente e a viaggiare e vivere in tutto il mondo. Figura 13. Immagine tratta dal sito ufficiale di Julie Peters- Desteract (http://www.juliepeters-art.com). Tra un secolo a nessuno importerà nulla della moda commerciale: vorranno sapere invece chi era quella donna” scrive il New York Times.75 Parte della collezione di Anna Piaggi si trova alla Galleria del Costume di Firenze. Nel 2009 l’Ufficio Esportazione della Soprintendenza per i beni artistici ed etnoantropologici sospende l’esportazione di alcuni dei suoi abiti destinati ad esser venduti all’asta da Christie’s a Londra. Dopo esser stati sottoposti ad accertamenti, il Ministero per i Beni e le attività culturali decide di acquistarli, destinandoli alla Galleria di Palazzo Pitti di Firenze. Dopo un intenso lavoro di restauro gli abiti sono stati salvati e sono conservati presso quest’importante istituzione museale.76 La natura della collezione si inserisce in modo interessante in questo elaborato poiché comprende abiti creati dai primi grandi stilisti di moda dell’epoca moderna, come Paul Poiret, sino ad arrivare a capi creati da stiliste del calibro di Maria Monaci Gallenga ed Elsa Schiaparelli77 (fig. 11-12). p ( g ) Se si scorre la collezione di Anna Piaggi si può leggere, nei nomi degli stilisti di riferimento e nello stile degli abiti, l’evoluzione della storia della moda moderna occidentale. g Nella panoramica che delinea le pratiche del collezionismo tessile al femminile questo studio si propone di approfondire la figura della donna stilista, artista e creatrici di collezioni. l h d d l Nella panoramica che delinea le pratiche del collezionismo tessile al femminile questo studio si propone di approfondire la figura della donna stilista, artista e creatrici di collezioni. L’artista contemporanea Julie Peters-Desteract chiude questo studio. Nata in Francia nel 1975, ottiene il titolo accademico presso l’Ecole des Beaux-Arts a Marsiglia nel 2001 dopo aver conseguito la laurea in Textiles presso l’Ecole Superieure des arts Appliqués Duppéré a Parigi. La sua attività artistica, che si dipana attraverso forme molteplici e multimediali, si sviluppa parallelamente all’insegnamento dell’arte e alla presenza attiva nelle scuole. La sua arte la porta ad essere in continuo movimento e a viaggiare e vivere in tutto il mondo. Si presenta come una figura interessante per questo studio per la sua dedizione al mondo del tessile e per la sua convinzione nella necessità di preservare le tecniche di tintura e di ricamo 145 Elisa Risi antiche che si stanno andando perdendo nella contemporaneità. Il percorso artistico la porta nel 1994 a condurre degli studi storici in questo ambito. Figura 13. Immagine tratta dal sito ufficiale di Julie Peters- Desteract (http://www.juliepeters-art.com). Partendo dal presupposto che i materiali tessili si sviluppano con la nascita dell’umanità e sono presenti in ogni cultura, constata che esistono miti, pratiche cerimoniali e credenze legate ad indumenti, così come esistono tutta una serie di abilità tecniche incredibilmente elaborate che coronano il mondo del tessile. Nonostante ciò, spesso questo sapere viene considerato una sotto-categoria nella gerarchia del mondo dell’arte. Spinta dal desiderio di legittimare questa forma d’arte e dalla volontà di preservare le tecniche tessili antiche Julie Peters-Desteract trasforma questa ambizione in progetti artistici che si sviluppano in tutto il mondo, in particolare in Asia, presso popolazioni le cui tecniche tessili e la cui cultura in generale sono in via di estinzione. Come afferma lei stessa “I learnt ancient gestures while embroidering. Some of these gestures are on the verge of extinction. I have a sense that an important part of our identity is disappearing. I think we must preserve and remember these gestures”.78 Il progetto artistico Guizhou en marche si sviluppa nell’omonima provincia sud-occidentale della Cina. Questo territorio è sede di molti gruppi etnici. Tra questi i Miao sono una minoranza etnica proveniente dal nord della Cina e stabilitasi successivamente nelle zone montuose del Guizhou. L’etnia Miao è molto diversa da quella Han (il gruppo etnico maggioritario cinese), possiede infatti una lingua, delle credenze e una forma d’arte completamente differenti ed è nota per l’arte dei suoi rinomati manufatti tessili tinti con la tecnica del Batik ad indaco. I Miao sono un popolo senza scrittura, i loro miti fondatori vengono trasmessi sin dall’origine attraverso le proprie creazioni tessili che diventano un elemento sacro in cui risiede la loro storia, la cultura e la loro memoria. Praticando una forma d’arte che risale alla tradizione più antica di questo popolo, i Miao creano indumenti incredibilmente lavorati e di notevole livello artistico. All’origine del processo la disegnatrice (sono da sempre e sempre le donne che portano avanti questa tradizione tessile) bagna il “La Dao” letteralmente il coltello di cera (simile ad una penna stilografica) nella cera vergine calda e l'applica sulla stoffa. La cera all'interno del coltello rimane liquida solamente per qualche secondo, il coltello ha quindi bisogno di essere intinto frequentemente nella cera calda. I disegni sono eseguiti a mano. L'iconografia e le mitologie che decorano questi tessuti hanno attraversato intere generazioni di donne. I simboli più comuni sono la farfalla madre, il dragone, l'uccello, il pesce, il tamburo. Figura 13. Immagine tratta dal sito ufficiale di Julie Peters- Desteract (http://www.juliepeters-art.com). Alcuni simboli sono più decorativi come ad esempio i fiori, gli altri sono invece pregni di significati che rimandano alla ricca tradizione e alla storia dei Miao. La presenza umana all’interno dei disegni operati sulle stoffe è molto tarda. Quando il disegno è finito, la stoffa viene legata a delle canne di bambù, viene spruzzata con acqua per migliorare la capacità di assorbimento del colore ed infine viene immersa nella tinta a base di indaco per 30 minuti. Quando la stoffa viene tolta per la prima volta dalla tintura, il colore è leggermente giallo. Dopo alcuni minuti si trasforma in un blu leggero. La stoffa ha bisogno di circa 30 minuti affinché il colore si sviluppi pienamente, dopo di che è pronta per essere immersa nuovamente nella tinta per altri 30 minuti. Il blu profondo tipico della tintura ad indaco viene ottenuto dopo 10 immersioni nella tinta (per un tempo totale di 10 ore). Il passaggio finale prevede l’immersione della stoffa in acqua bollente, in questo modo la cera fonde e si libera dalla stoffa. Il disegno bianco (protetto dalla cera) appare in seguito ed è valorizzato dallo sfondo blu profondo che caratterizza il Batik ad indaco dei Miao. Da questa collaborazione quasi decennale tra l’artista e questo gruppo etnico sono nati vari progetti artistici ed esposizioni museali e, durante gli anni, questa donna ha dato luogo ad una modesta ma interessante collezione personale di tessuti provenienti oltre che dal Guizhou anche da altre regioni ed etnie della Cina e del continente asiatico.79 Julie Peters-Desteract è un personaggio antiche che si stanno andando perdendo nella contemporaneità. Il percorso artistico la porta nel 1994 a condurre degli studi storici in questo ambito. Partendo dal presupposto che i materiali tessili si sviluppano con la nascita dell’umanità e sono presenti in ogni cultura, constata che esistono miti, pratiche cerimoniali e credenze legate ad indumenti, così come esistono tutta una serie di abilità tecniche incredibilmente elaborate che coronano il mondo del tessile. Nonostante ciò, spesso questo sapere viene considerato una sotto-categoria nella gerarchia del mondo dell’arte. Spinta dal desiderio di legittimare questa forma d’arte e dalla volontà di preservare le tecniche tessili antiche Julie Peters-Desteract trasforma questa ambizione in progetti artistici che si sviluppano in tutto il mondo, in particolare in Asia, presso popolazioni le cui tecniche tessili e la cui cultura in generale sono in via di estinzione. Figura 13. Immagine tratta dal sito ufficiale di Julie Peters- Desteract (http://www.juliepeters-art.com). Come afferma lei stessa “I learnt ancient gestures while embroidering. Some of these gestures are on the verge of extinction. I have a sense that an important part of our identity is disappearing. I think we must preserve and remember these gestures”.78 Il progetto artistico Guizhou en marche si sviluppa nell’omonima provincia sud-occidentale della Cina. Questo territorio è sede di molti gruppi etnici. Tra questi i Miao sono una minoranza etnica proveniente dal nord della Cina e stabilitasi successivamente nelle zone montuose del Guizhou. L’etnia Miao è molto diversa da quella Han (il gruppo etnico maggioritario cinese), possiede infatti una lingua, delle credenze e una forma d’arte completamente differenti ed è nota per l’arte dei suoi rinomati manufatti tessili tinti con la tecnica del Batik ad indaco. I Miao sono un popolo senza scrittura, i loro miti fondatori vengono trasmessi sin dall’origine attraverso le proprie creazioni tessili che diventano un elemento sacro in cui risiede la loro storia, la cultura e la loro memoria. Praticando una forma d’arte che risale alla tradizione più antica di questo popolo, i Miao creano indumenti incredibilmente lavorati e di notevole livello artistico. All’origine del processo la disegnatrice (sono da sempre e sempre le donne che portano avanti questa tradizione tessile) bagna il “La Dao” letteralmente il coltello di cera (simile ad una penna stilografica) nella cera vergine calda e l'applica sulla stoffa. La cera all'interno del coltello rimane liquida solamente per qualche secondo, il coltello ha quindi bisogno di essere intinto frequentemente nella cera calda. I disegni sono eseguiti a mano. L'iconografia e le mitologie che decorano questi tessuti hanno attraversato intere generazioni di donne. I simboli più comuni sono la farfalla madre, il dragone, l'uccello, il pesce, il tamburo. Alcuni simboli sono più decorativi come ad esempio i fiori, gli altri sono invece pregni di significati che rimandano alla ricca tradizione e alla storia dei Miao. La presenza umana all’interno dei disegni operati sulle stoffe è molto tarda. Quando il disegno è finito, la stoffa viene legata a delle canne di bambù, viene spruzzata con acqua per migliorare la capacità di assorbimento del colore ed infine viene immersa nella tinta a base di indaco per 30 minuti. Quando la stoffa viene tolta per la prima volta dalla tintura, il colore è leggermente giallo. Dopo alcuni minuti si trasforma in un blu leggero. corrente (esperienza che ho scelto di fare a seguito della partecipazione positiva, all’inizio dell’A.A. 2015/2016, al progetto Erasmus promosso dall’Università degli Studi di Bergamo). Colección de moda: de Eleonora Duse a Julie Peters-Desteract. rilevante per questo studio, in quanto, fin dagli esordi della proprio percorso artistico, eleva l’ambito del tessile a suo campo di ricerca e di creazione prediletto. Artista del tessile e collezionista, è emblema del rapporto che nella contemporaneità lega profondamente tra loro i due mondi in oggetto. La relazione tra l’universo artistico e quello della moda avviene sovente grazie al fenomeno del collezionismo, soglia che permette la commistione di questi domini e che porta ad un arricchimento considerevole per entrambi (fig. 13-14-15). Storia della moda e del costume: Barthes R., Il senso della moda. Forme e significati dell’abbigliamento, a cura di Marrone G., Torino, Einaudi, 2006. Dalla collezione del Kyoto Costume Institute, La Moda, storia dal XVIII al XX secolo, Köl Taschen, 2003. Laver J., Moda e costume: breve storia dall'antichità a oggi, Milano, Rizzoli, 2003. Sangiovanni A., Le parole e le figure: storia dei media in Italia dall'età liberale alla Seconda Guerra mondiale, Roma, Donzelli Editore, 2012. Collezionismo tessile: Chiarelli C. (a cura di), Donne protagoniste nel Novecento/ Women in the spotlight in the twentieth century, Livorno, Sillabe, 2013. Samuel A., Collectionner les textiles : constituer une collection de référence, l'exemple de Krishnâ Riboud, Paris, Musée Guimet, 2012. Jarrige J.-F., Krishnā Riboud (1926-2000), all’interno della rivista “Arts asiatiques”, Paris, Année 2000, Volume 55, Numéro 1. Lefèvre V., La collection Krishnā Riboud au musée Guimet, all’interno della rivista “Arts asiatiques”, Paris, Année 2004, Volume 59, Numéro 1. Letteratura francese e critica letteraria: Franchi F., I veli della modernità: saperi letterari sulla moda nell'Ottocento francese, Bergamo, Bergamo University Press, Edizioni Sestante, 2001. Gautier T., De la mode, Paris, Poulet-Malassis et De Broise, 1858. Perrot P Les dessus et les dessous de la bourgeoisie Paris Fayard 1981 Gautier T., De la mode, Paris, Poulet-Malassis et De Broise, 1858. Gautier T., De la mode, Paris, Poulet-Malassis et De Broise, 1858. P P L d l d d l b i i P i F d 1981 Perrot P., Les dessus et les dessous de la bourgeoisie, Paris, Fayard, 1981. Perrot P., Les dessus et les dessous de la bourgeoisie, Paris, Fayard, 1981. Figura 13. Immagine tratta dal sito ufficiale di Julie Peters- Desteract (http://www.juliepeters-art.com). La stoffa ha bisogno di circa 30 minuti affinché il colore si sviluppi pienamente, dopo di che è pronta per essere immersa nuovamente nella tinta per altri 30 minuti. Il blu profondo tipico della tintura ad indaco viene ottenuto dopo 10 immersioni nella tinta (per un tempo totale di 10 ore). Il passaggio finale prevede l’immersione della stoffa in acqua bollente, in questo modo la cera fonde e si libera dalla stoffa. Il disegno bianco (protetto dalla cera) appare in seguito ed è valorizzato dallo sfondo blu profondo che caratterizza il Batik ad indaco dei Miao. Da questa collaborazione quasi decennale tra l’artista e questo gruppo etnico sono nati vari progetti artistici ed esposizioni museali e, durante gli anni, questa donna ha dato luogo ad una modesta ma interessante collezione personale di tessuti provenienti oltre che dal Guizhou anche da altre regioni ed etnie della Cina e del continente asiatico 79 Julie Peters Desteract è un personaggio 146 rilevante per questo studio, in quanto, fin dagli esordi della proprio percorso artistico, eleva l’ambito del tessile a suo campo di ricerca e di creazione prediletto. Artista del tessile e collezionista, è emblema del rapporto che nella contemporaneità lega profondamente tra loro i due mondi in oggetto. La relazione tra l’universo artistico e quello della moda avviene sovente grazie al fenomeno del collezionismo, soglia che permette la commistione di questi domini e che porta ad un arricchimento considerevole per entrambi (fig. 13-14-15). rilevante per questo studio, in quanto, fin dagli esordi della proprio percorso artistico, eleva l’ambito del tessile a suo campo di ricerca e di creazione prediletto. Artista del tessile e collezionista, è emblema del rapporto che nella contemporaneità lega profondamente tra loro i due mondi in oggetto. La relazione tra l’universo artistico e quello della moda avviene sovente grazie al fenomeno del collezionismo, soglia che permette la commistione di questi domini e che porta ad un arricchimento considerevole per entrambi (fig. 13-14-15). 147 Elisa Risi Sitografia http://www.comune.milano.it/wps/portal/ist/it/news/primopiano/tutte_notizie/lavoro_ sviluppo_ricerca/rosa_genoni_omaggio [Intervista a Cristina Tajani, 01/03/2016]. Collezionismo: De Toni E., L’immaginario del collezionismo, Bergamo, Bergamo University Press, Edizioni Sestante, 2010. Grazioli E La collezione come forma d’arte Milano Johan & Levi 2012 , g z , g , g y , Sestante, 2010. Grazioli E., La collezione come forma d’arte, Milano, Johan & Levi, 2012. , Grazioli E., La collezione come forma d’arte, Milano, Johan & Levi, 2012. Collezionismo d’arte: Verlaine J., Femmes collectionneuses d'art et mécènes, de 1880 à nos jours, Paris, Hazan, 2013. Collezionismo tessile: http://www.moda.san.beniculturali.it/wordpress/ [Archivi della Moda del Novecento, Ministero dei beni e delle attività culturali e del turismo, direzione generale per gli archivi, in collaborazione con ANAI, 01/03/2016]. http://artemoda.unibg.it/allegati/aam_t2_pubblicazioni_allegati/8/b613cdf727b37f3f8ca7 9d7a3cdd602e08899f98.pdf [Capella M., Arte e moda: un incontro surreale, 18/11/2015]. http://www.rocaille.it/who-is-cecilia-matteucci-lavarini/ [Deganello S., Who is Cecilia Matteucci Lavarini?, 19/11/2015]. http://www.juliepeters-art.com [Sito ufficiale di Julie Peters-Desteract, 19/11/2015]. http://www.comune.milano.it/wps/portal/ist/it/news/primopiano/tutte_notizie/lavoro sviluppo_ricerca/rosa_genoni_omaggio [Intervista a Cristina Tajani, 01/03/2016]. http://www.vogue.it/encyclo/stilisti/s/elsa-schiaparelli [Sezione enciclopedia, Vogue Italia, Elsa Schiaparelli, 18/11/2015]. http://www.vogue.it/encyclo/stilisti/s/elsa-schiaparelli [Sezione enciclopedia, Vogue Italia, Elsa Schiaparelli, 18/11/2015]. http://www.treccani.it/ [Treccani, la Cultura Italiana, Enciclopedia, Schiaparelli Elsa, 24/11/2015]. http://www.treccani.it/ [Treccani, la Cultura Italiana, Enciclopedia, Schiaparelli Elsa, 24/11/2015]. 148 Colección de moda: de Eleonora Duse a Julie Peters-Desteract. 149
https://openalex.org/W4212898635	https://orbi.uliege.be/bitstream/2268/260333/1/EGU21-2660-print.pdf	English	null	Aragonite is calcite’s best friend at the seafloor	Research Square (Research Square)	2,021	cc-by	516	EGU21-2660, updated on 26 May 2021 https://doi.org/10.5194/egusphere-egu21-2660 EGU General Assembly 2021 © Author(s) 2021. This work is distributed under the Creative Commons Attribution 4.0 License. Reference: Bingeman, C.W., Varner, J.E., Martin, W.P., 1953. The Effect of the Addition of Organic Materials on the Decomposition of an Organic Soil. Soil Science Society of America Journal 17, 34-38. Aragonite is calcite’s best friend at the seafloor Olivier Sulpis1, Priyanka Agrawal1, Mariette Wolthers1, Guy Munhoven Jack Middelburg1 1Department of Earth Sciences, Utrecht University, The Netherlands (o.j.t.sulpis@uu.nl) 2Département d’Astrophysique, Géophysique et Océanographie, Université de Liège 3School of Life Science, University of Lincoln, United Kingdom Olivier Sulpis1, Priyanka Agrawal1, Mariette Wolthers1, Guy Munhoven2 Jack Middelburg1 1Department of Earth Sciences, Utrecht University, The Netherlands (o.j.t.sulpis@uu.nl) 2Département d’Astrophysique, Géophysique et Océanographie, Université de Liège 3School of Life Science, University of Lincoln, United Kingdom Olivier Sulpis1, Priyanka Agrawal1, Mariette Wolthers1, Guy Munhoven2, Matthew Walker3, and Jack Middelburg1 1Department of Earth Sciences, Utrecht University, The Netherlands (o.j.t.sulpis@uu.nl) 2Département d’Astrophysique, Géophysique et Océanographie, Université de Liège 3School of Life Science, University of Lincoln, United Kingdom Olivier Sulpis1, Priyanka Agrawal1, Mariette Wolthers1, Guy Munhoven2 Jack Middelburg1 1Department of Earth Sciences, Utrecht University, The Netherlands (o.j.t.sulpis@uu.nl) 2Département d’Astrophysique, Géophysique et Océanographie, Université de Liège 3School of Life Science, University of Lincoln, United Kingdom Aragonite is about 50% more soluble than calcite in seawater and its pelagic production is dominated by pteropods. Moreover, it could account for a large fraction of marine CaCO3 export. The aragonite compensation depth (ACD, the depth at which accumulation is balanced by dissolution) is generally very close to the aragonite saturation depth, i.e. within a few hundred metres. Conversely, the calcite compensation depth (CCD) can be 1-2 kilometres deeper than the calcite saturation depth. That aragonite disappears shallower than calcite in marine sediments is coherent with aragonite’s greater solubility, but why is the calcite lysocline, i.e. the distance between its compensation and saturation depths, much thicker than its aragonite equivalent? Here, we suggest that at the seafloor, the addition of a soluble CaCO3 phase (aragonite) results in the preservation of a predeposited stable CaCO3 phase (calcite), and term this a negative priming action. In soil science, priming action refers to the increase in soil organic matter decomposition rate that follows the addition of fresh organic matter, supposedly resulting from a globally increased microbial activity (Bingeman et al., 1953). Using a new 3D model of CaCO3 dissolution at the grain scale, we show that a conceptually similar phenomenon could occur at the seafloor, in which the dissolution of an aragonite pteropod at the sediment-water interface buffers the porewaters and causes the preservation of surrounding calcite. Since aragonite-producing organisms are particularly vulnerable to ocean acidification, we expect an increasing calcite to aragonite ratio in the CaCO3 flux reaching the seafloor as we go further in the Anthropocene. This could, in turn, hinder the proposed aragonite negative priming action, and favour chemical erosion of calcite sediments. Reference: Bingeman, C.W., Varner, J.E., Martin, W.P., 1953. The Effect of the Addition of Organic Materials on the Decomposition of an Organic Soil. Soil Science Society of America Journal 17, 34-38.
https://openalex.org/W2161537292	https://cgejournal.biomedcentral.com/track/pdf/10.1186/2052-6687-1-2	English	null	Approaches to canine health surveillance	Canine genetics and epidemiology	2,014	cc-by	12,523	REVIEW Open Access © 2014 O'Neill et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Approaches to canine health surveillance Dan G O’Neill1, David B Church2, Paul D McGreevy3, Peter C Thomson3 and Dave C Brodbelt1 Abstract Effective canine health surveillance systems can be used to monitor disease in the general population, prioritise disorders for strategic control and focus clinical research, and to evaluate the success of these measures. The key attributes for optimal data collection systems that support canine disease surveillance are representativeness of the general population, validity of disorder data and sustainability. Limitations in these areas present as selection bias, misclassification bias and discontinuation of the system respectively. Canine health data sources are reviewed to identify their strengths and weaknesses for supporting effective canine health surveillance. Insurance data benefit from large and well-defined denominator populations but are limited by selection bias relating to the clinical events claimed and animals covered. Veterinary referral clinical data offer good reliability for diagnoses but are limited by referral bias for the disorders and animals included. Primary-care practice data have the advantage of excellent representation of the general dog population and recording at the point of care by veterinary professionals but may encounter misclassification problems and technical difficulties related to management and analysis of large datasets. Questionnaire surveys offer speed and low cost but may suffer from low response rates, poor data validation, recall bias and ill-defined denominator population information. Canine health scheme data benefit from well-characterised disorder and animal data but reflect selection bias during the voluntary submissions process. Formal UK passive surveillance systems are limited by chronic under-reporting and selection bias. It is concluded that active collection systems using secondary health data provide the optimal resource for canine health surveillance. Keywords: Surveillance, Epidemiology, Canine, Data source, Primary-care practice, Referral practice, Insurance, Questionnaire, Health scheme, Cancer registry, Disorder O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 Correspondence: doneill@rvc.ac.uk 1Veterinary Epidemiology, Economics and Public Health, The Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Herts AL9 7T, UK Full list of author information is available at the end of the article Introduction Disease surveillance describes the monitoring of population health to ascertain the existence and changes in disease levels in combination with an appropriate mitigation plan once disease levels become inordinate [1-3] and is now an established veterinary activity for disease control [4]. Effective canine health surveillance provides information that supports disorder prioritisation, improved disorder management, focussed clinical research, advice on breed standards reform and regulation to improve animal welfare [5]. Optimal data sources for canine disease surveillance require representativeness of the general population, a well- defined denominator population, validity of disorder diag- nosis data and sustainability. Limitations in these areas present as selection bias, misclassification bias and discon- tinuation of the system respectively [4]. Surveillance data may be formatted as unstructured (free-form text), semi-structured (non-standardised lists) or structured data (standardised coding) [17]. Unstructured data are problematic for large-scale studies because of colloquial language, non-standard abbreviations and misspellings [18,19] but may provide contextual informa- tion that is otherwise unavailable [20]. Analytic methods for free-form text are progressing [21]. Structured data entry using standardised coding terminologies including the VeNom codes [22] and the AAHA (American Animal Hospital Association) Diagnostic Terms [23] may improve analytic efficiency [24,25]. Obstacles to effective data collection, analysis and interpretation are common to both human and animal surveillance. These include ethical constraints [26], data warehousing [27], setting valid case definitions [28], data quality and missing data [29,30], appropriate coding sys- tems [31], bias [32], generalisability [33], participation [34] linking data sources [35], financial cost [36], clinical cod- ing [37], developing syndromic surveillance [38] and im- pact assessment [39]. Veterinary surveillance methods can benefit from experiences gained during the development of human methods [40]. Further opportunities for improved veterinary surveillance methods stem from the advent of Big Data techniques for data management, analysis and accessibility [41], and developments in the science of natural language processing (NLP) [42,43]. Amongst other epidemiological applications, health surveillance data can be analysed to derive disorder prevalence proportion (proportion of animals affected) and incidence risk (proportion of previously healthy animals that become diseased over a specified period) estimates, perform risk factor (attributes associated with disease occurence) studies and examine survival in affected dogs [1]. Prevalence data are currently available on only 1% of inherited disorders affecting popular UK dog breeds [6]. A deficiency of disorder prevalence information relating to UK dogs has been identified as a major constraint to effective reforms to purebred dog health [7-9]. Lay summary and health records about dogs, have to be collected. This should be done in a standardised way where the same terms are used to describe diseases and symptoms and laboratory tests. The collection of disease information is described as being health surveillance or disease monitoring. To be effective, systems for doing this should be representative of the whole population. Methods for doing this are now developing but until recently the only ways to collect such information were by using records from pet insurance companies or from veterinary referral clinics. Problems can exist with such data as they can have a selection or refer- ral bias and not truly represent the picture at the popula- tion level or include information that is incorrect due to disease misclassification. This review describes the his- tory and development of health surveillance systems in canine medicine and what their strengths and weaknesses are. It also describes some of the new ways this is now being taken forward to collect high quality health data to support clinical and genetic studies. An ability to identify at a population level how many dogs, within breeds or across all breeds develop certain diseases, either over a fixed time period (e.g. each year) or as a proportion of the total population is very important. This helps establish whether some animals and breeds are particularly susceptible to a disease or whether conditions are becoming more or less common. The percentage of affected dogs in a population is called the disease prevalence and the number of new cases of disease in a year is called the disease incidence. These are critical measurements for studying patterns of health and disease and form a branch of medicine called epidemi- ology. Veterinary epidemiology has been slow to develop but is now recognised as being critical for improving ca- nine health and welfare. To do this effectively, information * Correspondence: doneill@rvc.ac.uk 1Veterinary Epidemiology, Economics and Public Health, The Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Herts AL9 7T, UK Full list of author information is available at the end of the article Page 2 of 13 O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 for dogs include pet insurance, referral practice and primary-care practice records [14-16]. Introduction Novel epidemiological information on disorders in dogs can assist with welfare prioritisation of disorders for appropriate focus of research efforts and breeding programs [6,10]. Increased awareness by veterinarians of disorder frequency and survival can improve diagnostic protocols, optimise case management and enhance prognostic advice given to clients [11]. Diverse data sources have been used to support canine disorder surveillance but there is an absence of a universally-accepted standard for ‘good practice’ in veterinary surveillance methodology [44]. This review is intended to evaluate current sources of canine health data and to identify their strengths and weaknesses as surveillance sources in order to assist with interpretation of results from studies based on these data. Collection processes for surveillance data may be passive or active [4]. Passive collection occurs at the discretion of the owner or veterinarian, whose willingness or ability to participate can limit the validity of the emergent data [12]. Passive systems typically suffer from incomplete reporting, selection bias and frequently lack a defined denominator population [13]. Active collection describes systematic data collection methods, usually from a defined population, location and timespan, and can be relatively timely, complete and accurate [13]. Referral practice clinical records Although technically easy to analyse [25] and their large size lending statistical power to gain meaningful results even for uncommon breeds [54,55], insurance data have some important limitations. Diagnostic term validity may vary between disorders depending on the ease of clinical diagnosis, the veterinarian’s clinical acumen and the veterinary practice’s facilities. For example, diagnosis validation for atopic dermatitis claims showed high agreement with veterinary medical records that claim dogs had allergic skin disease (97.6%) but only moderate agreement for full atopic dermatitis diagnostic criteria (40.9-84.2%) [15,56]. The Veterinary Medical Data Base (VMDB) holds 7 million standardised abstracted records from 26 veterinary schools in the US and Canada [75] with a coding system that records diagnostic terms using either pathophysiologic, histologic or descriptive terminologies [76]. VMDB data mining is based on discrete factors including breed, age, sex and diagnostic code and non-associated institutions are charged for data searches. The large study population lends high statistical power to VMDB analyses, enabling exploration of rare disorders or disorder-within-breed studies [12] such as thyroid cancer, 0.2% prevalence [77], discospondylitis, 0.2%, [78], bronchiectasis, 0.05%, [79] and leptospirosis, 0.04% [80]. Other prevalence studies that used VMDB data have investigated cataract [81], glaucoma [81] and cardiac tumours [82]. However, VMDB studies are limited by inconsistencies in data completeness and quality, and by the mixing of referral data with some primary-care data [15]. Only nine of the contributing universities use the structured SNOMED coding system that links clinical care events to terms selected from a comprehensive list of disorder concepts and descriptions [83,84]. The currency of the VMDB data is low, given that just 14 universities have uploaded data since the year 2000 [12,75]. Insured dogs may poorly represent the wider national dog population [15]. Insurance coverage varies with breed and purebred status [49] and life-cover may end when dogs reach 10 years of age [57]. Younger animals were heavily overrepresented in a UK insured population [58], requiring age-standardisation of results for generalisation [59]. Insured animals may receive more-frequent veterinary visits and undergo more medical procedures than non-insured animals [15]. A UK study using primary-care practice electronic patient record (EPR) data showed that insured dogs had over twice the odds of a diagnosis of chronic kidney disease compared with uninsured dogs [11]. Insurance status may even affect mortality by impacting on euthanasia decisions [15]. O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 Page 3 of 13 Insurance data research has lead to many useful publications on dogs covering specific disorders and overall morbidity and mortality (Table 1). Good understanding and interpretation of limiting factors are important when considering insurance data for canine health surveillance. Proposed studies should be considered on an individual basis or possibly even abandoned in the case of unavailable or inaccurate data [15]. Swedish insurance studies have benefited from an open approach to data sharing and strong research collaboration between Agria Insurance and academic colleagues that could be mirrored in other countries [55]. events and the denominator insured animals across large populations of dogs [15]. Validation studies between Agria Insurance demographic data and veterinary clinical records showed high agreement for dog breed (95%) and sex (99%), fair agreement for diagnosis (84%) but only moderate agreement for year of birth (66%), suggesting that insurance data are of adequate quality for research purposes [25]. Location information within insurance records allows spatial analysis of geographic risk factors. Post-code data have been used to show associations between the incidence of canine atopic dermatitis and average annual rainfall levels, proximity to a veterinary dermatologist, country sector and increased human population [53]. Pet insurance databases Animal insurance databases have been increasingly used for epidemiological research since the 1970s [45]. A literature review in 2009 of publications based on dog insurance data identified 16 Swedish studies using Agria Insurance data (http://www.agriavet.co.uk/) and three UK studies using PetProtect Insurance data (http://www.petprotect.co.uk/) [15]. About 20 pet insurance providers [46] insure an estimated 34.0-40.3% of UK dogs [46,47]. In Sweden, 68.4% of dogs are insured, with 61.0% of these insured dogs being covered by Agria Pet Insurance [48] alone [49]. Estimates suggest that just 4% of dogs in Canada [50] and 0.3-3.0% of dogs in America [51,52] are insured. Data used to support health surveillance may be primary or secondary. Primary data are collected specifically for the research while secondary data have been pre-collected by a third party for some other reason. Primary data collection offers better control of the types and quality of the data and may be easier to validate. However, secondary data sources may offer more efficient collection, reduced costs, larger sample size, better representativeness and reduced bias [14]. Secondary sources of health data For research purposes, insurance databases benefit from holding information on both the numerator clinical Referral practice clinical records Referral data spanning 1995–2010 from the University of California-Davis Veterinary Medical Teaching Hospital were analysed to report purebred dog predispositions to 24 inherited disorders [85]. The authors considered that more intensive evaluation within breeds with published disorder predisposition and increased willingness of owners of pure- bred dogs to spend heavily on clinical investigations may lead to over-representation of some disorders in specific breeds and in purebreds more generally [86]. Insurance data include only clinical events that are non-excluded and where the cost exceeds the deductible excess. Exclusions have tended to increase over time and vary by policy, breed and the medical history of individual animals [15,60]. Insurance claim levels may also vary between breeds. For example, death claims with an associated diagnosis were received for just 50% of insured crossbred dogs compared with over 80% of insured Bernese Mountain Dogs and Cavalier King Charles Spaniels [15]. Cohort insurance studies to monitor health status within individual animals over time can be problematic because repeat-disorder claim levels are affected by changing insurance status and dynamic exclusions applied over time based on claim history [61]. Despite the promise of good reliability for diagnoses from referral clinical data, referral biases towards complicated cases requiring more specialised care and towards locations closer to specialist centres limit the generalisability of study results [12]. Clients and animals that are referred are filtered by diagnostic work-ups, insurance status and financial considerations [15,87]. Referral bias is likely to Page 4 of 13 Page 4 of 13 O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 Table 1 Selected published and findings studies on dog health based on insurance data Topic & main conclusion Insurer Country Table 1 Selected published and findings studies on dog health based on insurance data Topic & main conclusion Insurer Country Reference German Shepherd Dog, predisposed to immune-mediated diseases Agriaa Sweden Vilson et al. [62] Atopic dermatitis, offspring of bitches fed non-commercial diet during lactation protected Agriaa Sweden Nodtvedt et al. (2007) [53] Bone tumours, Irish Wolfhound, St. Bernard, and Leonberger at increased risk Agriaa Sweden Egenvall et al. (2007) [63] Cancer, skin and soft tissue tumours had highest prevalence PetProtectb UK Dobson et al. (2002) [58] Demography, insured dogs similar to the general dog population Agriaa Sweden Sallander (2001) [64] Diabetes mellitus, highest incidence in Australian Terrier, Samoyed, Swedish Elkhound and Swedish Lapphund Agriaa Sweden Fall et al. Referral practice clinical records (2007) [65] Dystocia/caesarean section, Scottish terrier at increased risk Agriaa Sweden Bergstrom (2006) [66] Heart disease, Irish Wolfhound, Cavalier King Charles Spaniel and Great Dane showed highest mortality Agriaa Sweden Egenvall et al. (2006) [67] Intervertebral disc degeneration, Miniature Dachshund, Standard Dachshund and Doberman Pinscher had highest incidence Agriaa Sweden Bergknut et al. (2012) [68] Lymphoma, Bull mastiff, Bulldog and Boxer had high incidence PetProtectb UK Edwards et al. (2003) [69] Mammary tumours, highest incidence in English Springer Spaniel, Doberman and Boxer Agriaa Sweden Egenvall et al. (2005b) [70] Morbidity and mortality, marked breed differences in survival Agriaa Sweden Bonnett and Egenvall (2010) [55] Morbidity and mortality (1995–1996), insurance data useful for epidemiological studies Agriaa Sweden Egenvall et al. (2000a) [60] Mortality, wide breed differences in survival Agriaa Sweden Egenvall et al. (2000b) [71] Mortality, >40% of deaths from trauma, tumours and locomotor disorders Agriaa Sweden Bonnett et al. (1997) [72] Mortality (1995–2000), Irish Wolfhound and Great Dane had highest mortality Agriaa Sweden Egenvall et al. (2005a) [57] Mortality (1995–2000), 62% of deaths from tumour, trauma, locomotor, heart and neurological disorders Agriaa Sweden Bonnett et al. (2005) [73] Pyometra, increased risk Rough Collies, Rottweilers, Cavalier King Charles Spaniels, Golden Retrievers, Bernese Mountain Dogs, and English Cocker Spaniels Agriaa Sweden Egenvall et al. (2001) [74] ahttp://www.agriavet.co.uk bhttp://www.petprotect.co.uk. ahttp://www.agriavet.co.uk bhttp://www.petprotect.co.uk. Electronic recording of clinical data is now central to human and animal healthcare [92,93]. Data collected from the 90% of UK veterinary practices that use electronic practice management systems (PMSs) can contribute enormously to clinical research [94-96]. The ‘Independent Inquiry into Dog Breeding’ report cited primary-care practice electronic clinical data using standardised coding of diagnoses as the optimal data source for reliable prevalence estimation [7]. However, early attempts at large-scale electronic surveillance struggled to cope with the large volumes of clinical data collected [19] and initial veterinary PMSs did not en- force structured coding systems [97], although there is now evidence that practising veterinarians accept a clinical rationale for standardised data recording [98,99]. vary between disorders and to compromise the validity of prevalence studies that compare multiple disorders [12]. An ill-defined denominator population contain- ing few healthy animals further limits referral data for prevalence estimation [88]. Referral clinical datasets may be less reliable for generalisable prevalence estimation and may be best reserved to test hypotheses relating to specific causal mechanisms [89]. Referral practice clinical records vary between disorders and to compromise the validity of prevalence studies that compare multiple disorders [12]. An ill-defined denominator population contain- ing few healthy animals further limits referral data for prevalence estimation [88]. Referral clinical datasets may be less reliable for generalisable prevalence estimation and may be best reserved to test hypotheses relating to specific causal mechanisms [89]. ahttp://www.agriavet.co.uk bhttp://www.petprotect.co.uk. Primary-care practice clinical records Analysis of primary-care practice data benefits from the cumulative clinical experience of general practitioners to offer unique insights into companion animal health [24] and can support an evidence-based approach that is relevant to primary-care practitioners [90]. In the US, the National Companion Animal Study (NCAS) spanned 1992–1995 and analysed coded clinical data from 31,484 dogs treated at 52 first-opinion clinics [5]. Clinical diagnostic terms were recorded onto paper by attending clinicians before codification to a standardised nomenclature (PetTerms; developed dynamically during the study) and electronic transfer to a proprietary PMS [100]. An early example of primary-care practice surveillance (1998–2001) used manual paper-based data collection by veterinary students undergoing extramural studies to describe overall reasons for veterinary presentation and the prevalence of dermatological diagnoses [91]. This study concluded that, although practicable for short-term and highly focused studies, clinical research using paper-based records was highly labour-intensive and unsustainable for long-term studies. The first of three published NCAS studies described age, breed, sex, diet and body condition score, and reported Page 5 of 13 O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 prevalence estimates for the most common disorders diagnosed [5]. However, the study was compromised by limiting the denominator population to just those animals with at least one coded diagnosis (36.3% of unique animal records), potential transcription error during the paper-to-electronic transfer of data and the absence of a prior-standardised coding system. Two further NCAS publications on obesity in cats [101] and dogs [102] demonstrated the potential to augment secondary EPR data with additional primary data collection on diet and body condition score for enhanced investigations [103]. The NCAS studies highlighted the importance to sustainable surveillance of standardised coding, direct recording of electronic data by clinicians, inclusion of all clinical care events and electronic integration between PMSs and research databases. electronic laboratory reports from over 18,000 private veterinary practices [117]. Resultant publications on dog health have covered vaccine safety [19,107,118,119], tick infestation [108] and toxic exposure [115]. However, NCASP surveillance was limited by confidentiality issues, delayed dissemination of results and difficulties in managing such large volumes of data [117]. It is reported that NCASP has been discontinued [120]. VetCompass (Veterinary Companion Animal Surveillance System) was developed at the Royal Veterinary College (RVC) in collaboration with the University of Sydney for companion animal surveillance using primary-care practice clinical data. Primary-care practice clinical records A pilot phase, spanning 2007–2009, preceded implementation of the full UK project from Septem- ber 2009 onwards. VetCompass holds clinical data on over 275,000 dogs from 189 UK practices (August 2013) (http://www.rvc.ac.uk/VetCompass/Index.cfm). Attending clinicians record VeNom code [22] summary diagnosis terms during episodes of clinical care [121]. Clinical data are automatically uploaded weekly to the VetCompass database [122]. Published VetCompass studies have covered pharmacotherapeutics [122], demography [123] and specific disorders [11,121] of dogs. Current VetCompass projects aim to prioritise the welfare impact of common disorders in dogs, to evaluate the longitudinal course of canine mitral valve disease and to pilot the linkage of pedigree data to clinical health records in collaboration with the UK Kennel Club (KC) [47]. VetCompass has been developed in Australia and preliminary project work is underway in Spain, Germany and New Zealand. Realisation of the full surveillance potential of VetCompass has been constrained by limitations in automated information extraction from large datasets but current work to apply NLP methods offers promise [124]. Eight hundred Banfield Pet Hospitals (http://www. banfield.com/) have generated clinical data on over 2.2 million dogs across 43 states in the USA [104]. Surveillance based on Banfield clinical data benefits from the use of a single PMS with daily uploads of standardised EPRs to a single computer server [105]. Collaborative studies using Banfield Pet Hospital data have reported on canine disorders including nematode parasitism, demodicosis, pancreatitis and atopic dermatitis (Table 2) while internal Banfield studies have been published online as ‘State of Pet Health’ reports [104]. Eight hundred Banfield Pet Hospitals (http://www. banfield.com/) have generated clinical data on over 2.2 million dogs across 43 states in the USA [104]. Surveillance based on Banfield clinical data benefits from the use of a single PMS with daily uploads of standardised EPRs to a single computer server [105]. Collaborative studies using Banfield Pet Hospital data have reported on canine disorders including nematode parasitism, demodicosis, pancreatitis and atopic dermatitis (Table 2) while internal Banfield studies have been published online as ‘State of Pet Health’ reports [104]. The National Companion Animal Surveillance Program (NCASP) was developed at Purdue University in 2003 with a $1.2 million grant from the Centers for Disease Control and Prevention (CDC) to provide near real-time syndromic surveillance of pet animals as sentinels for bioterrorism, emerging zoonoses, toxic chemical exposures and for veterinary drug and vaccine pharmacovigilance [116]. Primary-care practice clinical records Banfield EPR data were linked with Antech Diagnostics The National Companion Animal Surveillance Program (NCASP) was developed at Purdue University in 2003 with a $1.2 million grant from the Centers for Disease Control and Prevention (CDC) to provide near real-time syndromic surveillance of pet animals as sentinels for bioterrorism, emerging zoonoses, toxic chemical exposures and for veterinary drug and vaccine pharmacovigilance [116]. Banfield EPR data were linked with Antech Diagnostics Table 2 Selected publications and findings on dog health based on Banfield Pet Hospital clinical data Topic Study period Reference Gonadectomy, is a risk factor for obesity 1998-2010 Lefebvre et al. [106] Vaccine-associated adverse events, multiple vaccines doses administered per visit increased risk 2002-2003 Moore et al. [107] Tick infestation, younger, male and sexually intact dogs at increased risk 2002-2004 Raghavan et al. [108] Periodontal disease, associated with cardiovascular-related conditions 2002-2006 Glickman et al. [109] Periodontal disease, positive association with the incidence of azotaemic CKD 2002-2008 Glickman et al. [110] Nematode parasitism, age, body weight, sex, breed and geographic region were risk factors 2003-2006 Mohamed et al. [111] Tick infestation, systematic monitoring of veterinary and human medical data can improve detection in tick activity 2006-2007 Rhea et al. [103] Pancreatitis, prevalence of 23 per 10,000 patients 2006 Lewis [112] Demodicosis, American Staffordshire Terrier, Staffordshire Bull Terrier and Chinese Shar-pei at highest risk 2006 Plant et al. [105] Atopic dermatitis, 1.7% prevalence 2007 Lund [113] Castration, 64% prevalence 2007 Trevejo et al. [114] Environmental monitoring, methods using veterinary records require further development 2006 Maciejewski [115] and findings on dog health based on Banfield Pet Hospital clinical data O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 Page 6 of 13 The Small Animal Veterinary Surveillance Network (SAVSNET) was launched in 2008 at the University of Liverpool as a pilot project collecting data from UK veterinary diagnostic laboratories and veterinary practices [125] before becoming a registered charity in 2012 and entering a partnership with the British Small Animal Veterinary Association (BSAVA) [126]. A SAVSNET study of antibacterial prescribing patterns identified the importance of data validation for automated search strategies of primary-care practice data by showing substantial variation between the positive predictive value of four diagnoses, abscess (82%), diarrhoea (91%), cystitis (100%) and coughing (90%) [127]. Veterinary cancer registries Cancer registries systematically collect and analyse cancer data and are considered key to human cancer control, with 449 registries covering 21% of the world human population [135]. However, veterinary cancer registries are uncommon, often short-lived and suffer from poor communication and collaboration [136]. The VMDB began in 1964 as a hospital-based cancer registry [75,137] and has published studies in dogs that covered cutaneous mast cell tumours [138], pros- tate carcinoma [139], cardiac tumours [82] and osteosar- coma [140]. Referral bias limits generalisability from VMDB study results to the wider dog population [12]. The Norwegian Canine Cancer Registry (NCCR) was established in 1990 and has reported results from studies that investigated mammary tumours in bitches [141,142]. The Danish Veterinary Cancer Registry (DVCR) was established in 2005 as an online registry for passive veterinarian upload of clinical information [137]. Published DVCR studies have included cancer frequency in dogs in Denmark [143] and canine mast cell tumours [144]. The Animal Tumour Registry of Vicenza and Venice (Italy) was established in 2005 and used data on 2,509 samples submitted by 164 veterinary clinics to report cancer incidence in dogs. A denominator dog population was estimated using a telephone survey [145]. The Animal Tumour Registry of Genoa (Italy) used data from 6,743 canine tumour biopsies submitted between 1985 and 2002 to report cancer incidence in dogs. A denominator population was estimated using a capture-recapture methodology [146]. These studies acknowledged limitation from poor denominator population enumeration and used differing methods to estimate missing values. The telephone survey for population estimation benefitted from relative speed and additional collection of other useful demographic data. The Centre for Evidence-based Veterinary Medicine (CEVM) was established in 2009 at the University of Nottingham [130]. The CEVM aims to promote the use of reliable and relevant science (or evidence) in clinical decision-making between veterinary surgeons and the owners of the animals. To facilitate this, the CEVM has created a small network of sentinel practices who they work very closely with, to look at the complexity of consultations, the reliability and limitations of EPRs and identify important areas for future research for veterinarians, owners and their animals. To-date, primary-care practice EPR data have been an under-used surveillance resource [5]. Studies using EPR data may encounter misclassification problems [127] and technical difficulties related to management and analysis of large datasets [120]. Veterinary cancer registries Additionally, primary-care veterinary data mainly feature disorders that either prompted a veterinary-care visit or were detected during an otherwise veterinary examination and thus may miss that proportion of the overall disorder burden of dogs that does not receive veterinary attention. Just 44.1% of the true illness events in dogs are severe or persistent enough to lead to veterinary attention [131]. However, primary- care EPR research boasts increasingly large datasets for achieving good precision within study output [132] and the investigation of rare events [133], good prospects of generalisation to the wider dog popula- tion from the 70% of UK dogs that are registered with a veterinary practice [33,46] and clinical rele- vance of the emergent results to practising veterinar- ians [127]. Cohort data collection can facilitate survival and co-morbidity analyses for chronic diseases [11,134]. Ongoing developments in database management, analytic techniques, standardised coding and collaborative re- search design should enhance the surveillance role for primary-care practice EPR research [13]. Although useful for some risk factor studies, cancer registry clinical data are limited for disease surveillance in dogs by variable reporting, referral bias, denominator popu- lation enumeration problems and geographical variation [12,147]. While under-reporting might generally be ex- pected, cancer registries may also over-report certain neo- plasias because of screening programs, research focus, new diagnostic modalities or free histopathology. Inconsistent inclusion criteria, nomenclature and classification schemes have limited comparisons of results across schemes. The use of standardised coding and diagnostic systems, data ex- traction directly from PMSs and cross-linking with path- ology laboratory systems would enhance the application of veterinary cancer registry data for disease surveillance. Primary-care practice clinical records A moderate negative impact from using opt-in consent was indicated by the 2.6% of clients who declined to participate [127], suggesting the relevance of appropriate consent protocols within project design [128]. Syndromic surveillance results are also posted on the project website [129]. Veterinary cancer registries Canine health schemes Over 120 dog breeds have at least one DNA test available [157]. The results of formal DNA screening schemes co-ordinated by the KC are published online, providing surveillance data that can be linked with KC pedigree information on phenotype and parentage [158]. These data have been analysed to estimate the mutation prevalence for primary lens luxation among affected breeds [159]. However, selection bias arising from systematic avoidance of or intensive testing of known affected lines have limited generalisation from these studies to the wider dog population [160]. Information collected directly from the dog-owning public may improve representativeness of the overall dog population. The Pet Food Manufacturers Association (PFMA) 2012 survey used data from 2,159 face-to-face interviews to report demographic estimates for the UK dog population [149]. However, the lack of reporting on response rates, selection criteria, statistical weightings and confidence intervals limit the validity of the PFMA survey results. A UK general public telephone survey investigating dog ownership achieved a response rate 37% from a total of 1,656 calls and reported that 23.92% of households owned at least one dog [46]. A random-digit dialling telephone survey in Ireland generated 1,250 completed responses from 105,803 calls (1.2%) to describe dog and cat demography but was limited by the low completion rate [150]. Brain stem auditory evoked response (BAER) testing distinguishes bilaterally and unilaterally hearing-impaired dogs from non-affected animals [161]. BAER data have been used to report prevalence estimates for deafness in Dalmatians [162], Border Collies [163] and Australian Cattle Dogs [164] as well as across multiple breeds [165]. These studies benefitted from well-defined case inclusion criteria and large study sizes but the voluntary submission process may have affected the presentation probabilities for known deaf individuals. Veterinary practice questionnaires can collect data from either the practice teams or their clients. A questionnaire circulated by email and post to 2,763 UK veterinary prac- tices was used to report the number of practice-registered dogs but was limited by a low response rate (3.7%) [46]. A questionnaire distributed at UK veterinary clinics and dog shows investigating inter-dog aggression reported a completion rate of 3,897 from 14,566 distributed ques- tionnaires (26.8%) [151]. An Australian study distributed questionnaires at veterinary clinics and pet shops to investi- gate owners’ attitudes towards obesity in dogs and reported a 36.5% response rate [152]. Integration of owner-recorded data offers the potential to complement veterinarian-derived data within broader study designs. Questionnaire-based data collection Registered breeders represent a knowledgeable and important sub-population of dog-owners. The KC/BSAVA Page 7 of 13 O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 of generally decreased volunteerism, over-surveying, increasingly complicated surveys and conversion from landline to mobile phone use [155]. That said, increasing internet access has enhanced the promise for internet-based questionnaires [156] and careful study design can mitigate some of these drawbacks. Study designs that include nested questionnaires can benefit from focused primary data collection. UK health survey of purebred dogs collected information from breeders on overall dog health, breeding and mortality as well as puppy birth defects. Of approximately 56,363 questionnaires forwarded to breed club members, 13,759 useable forms were returned (24%), representing 36,006 live dogs [148]. This large survey provided a useful resource of information on purebred dog health and mortality but generalisation was limited by the low response rate, absence of veterinary validation of reported disorders and inclusion of only those dogs owned by breed club members. Future studies aimed at breeder groups could benefit from linkage to veterinary diagnoses and greater prior involvement of breed clubs. Canine health schemes The BVA (British Veterinary Association)/KC hip dysplasia (HD) and elbow dysplasia (ED) schemes hold data on over 100,000 radiographs assessed since 1984. Dogs evaluated under these schemes must be permanently identified by microchip or tattoo [166] and breed median scores are published to assist breeding decisions [167]. Linkage between HD and ED schemes results and KC pedigree data has allowed pedigree evaluation and generation of estimated breeding values (EBVs) [168] for HD [169] and ED [170] in Labrador Retrievers. However, selection bias resulting from predominant inclusion of registered purebreds and the unlikelihood of submissions from dogs that are clinically affected or have obviously affected radiographs limit generalisation and may bias prevalence estimates downwards [171]. Questionnaire surveys are common methods for active collection of primary veterinary epidemiological data [153] and benefit from relative speed, repeatability, low cost and the capture of information on multiple risk factors and confounders. However, potential drawbacks associated with the use of questionnaires include low response rates, loss of information on temporality, difficulties with data validation, recall and non-responder bias, and ill-defined denominator populations [154]. Participation in questionnaire-based studies have been declining by about 1% annually from 1970 to 2003 because The BVA/KC/ISDS (International Sheepdog Society) eye scheme holds eye test data spanning over 30 years of testing for 11 hereditary eye conditions in over 50 breeds [172]. These data have been analysed to estimate the incidence [173] and inheritability [174] of multifocal retinal dysplasia in the Golden Retriever and the incidence of cataracts among Labrador Retrievers [175]. Eye scheme data for research are limited by misclassification bias from Page 8 of 13 O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 Table 3 Advantages and limitations to data sources used for health surveillance in dogs Data source Advantages Limitations Applications Pet insurance databases Large size Difficult to validate Agria Pet Insurance data analysis in Sweden [15] Defined denominator Questionable representativeness of the general population Pet Protect insurance data analysis in UK [58,69] High reliability for breed and sex Loss of data on low-cost or excluded disorders Coded diagnoses Referral practice clinical records Good diagnostic reliability? Referral bias Veterinary Medical Data Base (VMDB) [75] Coded diagnoses? Poorly defined denominator Large databases Poorly representative Primary-care practice clinical records Large databases Diagnostic reliability? Banfield Pet Hospital [104] Highly representative? Canine health schemes Technical complexities NCAS [5] Coded diagnoses Only events with veterinary care NCASP [116] Defined denominator VetCompass [47] Generalisability SAVSNET [129] CEVM [130] Veterinary cancer registries Human registries common Referral bias Veterinary Medical Data Base (VMDB) [75] Good diagnostic reliability Poorly defined denominator Danish Veterinary Cancer Registry [137]. Poorly representative Questionnaire-based data collection Relatively inexpensive Response rate The KC/BSAVA UK health survey of purebred dogs [148]. Flexible Difficult to validate Can nest within other study designs Loss of information on temporality Canine health schemes Large databases Poorly representative BVA/KC hip dysplasia and elbow dysplasia scheme [194] Diagnostic reliability Selection bias BVA/KC elbow dysplasia scheme [195] Linkage to KC pedigree data The BVA/KC/ISDS eye scheme [172]. Permanent animal identification Other companion animal surveillance systems in the UK Relatively inexpensive Under-reporting SARSS [176] Poorly defined denominator Selection bias DACTARI [186] Poor generalisability CICADA [189] Other companion animal surveillance systems in the UK SARSS, Suspected Adverse Reaction Surveillance Scheme Other companion animal surveillance systems in the UK SARSS, Suspected Adverse Reaction Surveillance Scheme Veterinary pharmacovigilance in the UK is monitored by the Veterinary Medicines Directorate (VMD) via SARSS using passive reporting of adverse events to veterinary medicines in both animals and humans [176]. The number of adverse events reported per year for dogs increased from 653 to 1,615 between the years 2003 to 2011 [177-185]. The 2011 result [182] equates to an average of only one report per year for every 10 of the 17,260 registered home-practising veterinary surgeons in the UK [95]. This diagnosis difficulties for ocular disorders and selection bias from testing only a small proportion of KC-registered purebreds and very few non-registered dogs [173]. Canine health schemes generally harvest primary data from voluntary owner submissions and often benefit from well-characterised data, permanent animal identification (microchip, tattoo), openly published results and linkage with genetic databases [166]. However, intrinsic selection bias from passive collection processes and questionable representation of the overall dog popula- tion limit the generalisability of study results. Page 9 of 13 O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 the epidemiological importance of permanent identification of individual animals [192,193]. suggests heavy under-reporting and likely selection bias with resultant questionable reliability of SARRS data for general surveillance purposes. Competing interests ll h f h All authors of this review are involved in the development of the VetCompass Surveillance System which collects and studies primary-care veterinary clinical data [47]. Conclusions Increasing demand for veterinary surveillance is constrained by decreasing availability of human and financial resources [191]. Credible canine health surveillance requires a reliable data source with a well-defined denominator population, evidence of representativeness and validity and appropriate study design [4]. Active data collection is preferred for veterinary surveillance because of reduced selection bias and known selection probability for each epidemiological unit that permit estimation of absolute, rather than relative, risk values [2]. Secondary data are becoming increasingly important for companion animal surveillance because of their collection efficiency, reduced costs, larger sample size, better representativeness and reduced bias [14]. Integration between data sources can facilitate knowledge extraction and interpretability within individual studies and underlines DACTARI, The Dog and Cat Travel and Risk Information DACTARI, The Dog and Cat Travel and Risk Information The DACTARI surveillance scheme was launched in 2003 by the Department for Environment Food and Rural Affairs with involvement from BVA and BSAVA as a national voluntary reporting scheme to monitor exotic diseases events in dogs and cats in Great Britain [186]. The scheme focuses on four disorders: leishmaniosis, babesiosis, ehrlichiosis and dirofilariasis. However, from 2001 to 2011, only 109 reports were received for these four diseases in dogs. Given that Bristol University reported 257 cases of canine leishmaniosis in the UK between 2005 and 2007 [187], this suggests heavy under-reporting to DAC- TARI of the true exotic disease burden. CICADA, The Companion Animal Disease Survey The CICADA survey is coordinated by MSD Animal Health (http://www.msd-animal-health.co.uk/) as a UK web-based disease surveillance scheme with voluntary online submissions of animal health data by veterinarians and professional organisations. The scheme aims to report disease trends, recent outbreaks and current hot spots [188]. Between October 2011 and June 2012, only 93 insti- tutions (laboratories, universities or veterinary practices) submitted information to CICADA [189], suggesting high selection bias and limited generalisability to the wider animal population. Abbreviations AAHA A i Abbreviations AAHA: American Animal Hospital Association; BSAVA: British Small Animal Veterinary Association; BVA: British Veterinary Association; CDC: Centers for Disease Control and Prevention; CEVM: The Centre for Evidence-based Veter- inary Medicine; CICADA: Companion Animal Disease Survey; DACTARI: Dog and Cat Travel and Risk Information; DVCR: Danish Veterinary Cancer Registry; EBV: Estimated breeding value; ED: Elbow dysplasia; EPR: Electronic patient record; HD: Hip dysplasia; ISDS: International Sheepdog Society; KC: The Kennel Club; NCAS: National Companion Animal Study; NCASP: National Companion Animal Surveillance Program; NCCR: Norwegian Canine Cancer Registry; PFMA: Pet Food Manufacturers Association; PMS: Practice management systems; RVC: Royal Veterinary College; SARSS: Suspected Adverse Reaction Surveillance Scheme; SAVSNET: Small Animal Veterinary Surveillance Network; VetCompass: Veterinary Companion Animal Surveillance System; VMD: Veterinary Medicines Directorate; VMDB: Veterinary Medical Database. Rather than being useful for general surveillance, passive reporting systems may better suited for generating alarm signals that trigger more intensive targeted surveillance [19]. Passive collection may suffer from chronic under-reporting, inadequately defined denominator populations, selection bias and poor generalisation, and such systems are difficult to sustain in the long-term [190]. Canine health schemes Many data sources have been recruited for canine health surveillance, each with distinct prevailing advan- tages and limitations (Table 3). Insurance data benefit from large and well-defined denominator populations but are limited by selection bias relating to the clinical events claimed and animals covered. Veterinary referral clinical data offer good reliability for diagnoses but are limited by referral bias for the disorders and animals referred. Primary-care practice EPR data benefit from strong alignment with the general dog population and veterinary validation but encounter technical difficulties related to the management and analysis of large datasets. Veterinary cancer registries offer good diagnostic reli- ability but may have ill-defined denominator populations and poor representativeness. Questionnaire surveys are relatively inexpensive and collect primary data but suffer from issues relating to validation and response rates. Canine health scheme data benefit from well-characterised disorder and animal data but are subject to selection bias from the voluntary submission process. Formal UK passive surveil- lance systems are limited by chronic under-reporting and selection bias. It is concluded that active collection systems using secondary health data currently provide the optimal single resource for canine health surveillance and that linking multiple data sources can substantially amplify the research potential. References Cambridge: University of Cambridge; 2010. 8. Rooney N, Sargan D: Pedigree dog breeding in the UK, a major welfare concern?. West Sussex, RSPCA: Horsham; 2008. 9. APGAW: A healthier future for pedigree dogs. London: The Associate 7. Bateson P: Independent inquiry into dog breeding. Cambridge: University of Cambridge; 2010. 34. Galea S, Tracy M: Participation Rates in Epidemiologic Studies. Ann Epidemiol 2007, 17:643–653. 8. Rooney N, Sargan D: Pedigree dog breeding in the UK, a major welfare concern?. West Sussex, RSPCA: Horsham; 2008. 35. Schnitzer PG, Slusher P, Van Tuinen M: Child maltreatment in Missouri, Combining data for public health surveillance. Am J Prev Med 2004, 27:379–384. 9. APGAW: A healthier future for pedigree dogs. London: The Associate Parliamentary Group for Animal Welfare; 2009. 10. McGreevy PD: Breeding for quality of life. Anim Welf 2007, 16:125–128. 11 O’N ill DG Elli J Ch h DB M G PD Th PC B db l DC 36. Crompton P: The National Programme for Information Technology - an overview. J Vis Comm Med 2007, 30:72–77. 11. O’Neill DG, Elliott J, Church DB, McGreevy PD, Thomson PC, Brodbelt DC: Chronic kidney disease in dogs in UK veterinary practices, prevalence, risk factors, and survival. J Vet Intern Med 2013, 27:814–821. 37. de Lusignan S: The barriers to clinical coding in general practic A literature review. Inform Health Soc Care 2005, 30:89–97. 12. Bartlett PC, Van Buren JW, Neterer M, Zhou C: Disease surveillance and referral bias in the veterinary medical database. Preventive Veterinary Medicine 2010, 94:264–271. 38. Mandl KD, Overhage JM, Wagner MM, Lober WB, Sebastiani P, Mostashari F, Pavlin JA, Gesteland PH, Treadwell T, Koski E, Hutwagner L, Buckeridge DL, Aller RD, Grannis S: Implementing syndromic surveillance, a practical guide informed by the early experience. J Am Med Inform Assoc 2004, 11:141–150. 13. Moore GE, Lund E: Disease reporting and surveillance, where do companion animal diseases fit in? Vet Clin N Am Small Anim Pract 2009, 39:225–240. 39. Chaudhry B, Wang J, Wu S, Maglione M, Mojica W, Roth E, Morton SC, Shekelle PG: Systematic review, impact of health information technology on quality, efficiency, and costs of medical care. Ann Intern Med 2006, 144:742–752. 14. Sorensen HT, Sabroe S, Olsen J: A framework for evaluation of secondary data sources for epidemiological research. Int J Epidemiol 1996, 25:435–442. 15. O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 Acknowledgements F di b h 22. The VeNom Coding Group: In VeNom Veterinary Nomenclature. Edited by Group TVC; 2013. http://www.venomcoding.org/VeNom/Welcome.html, VeNom Coding Group. Funding support by the Royal Society for the Prevention of Cruelty to Animals (RSPCA) for this study is acknowledged. Editing assistance from Cate Webb is gratefully acknowledged. 23. AAHA: AAHA Diagnostic Terms. American Animal Hospital Association; 2013 https://www.aahanet.org/Library/Diagnostic.aspx Received: 30 October 2013 Accepted: 14 January 2014 Published: 16 April 2014 Received: 30 October 2013 Accepted: 14 January 2014 Published: 16 April 2014 27. Wisniewski MF, Kieszkowski P, Zagorski BM, Trick WE, Sommers M, Weinstein RA: Development of a clinical data warehouse for hospital infection control. J Am Med Inform Assoc 2003, 10:454–462. Authors’ contribution DON, is the primary author for the current work and was involved in the conception, design, data collection and drafting of the manuscript. DC, contributed to the conception and design of the work, and helped to draft and revise the manuscript. PM, contributed to the conception and design of the work, and helped to draft and revise the manuscript. PT, contributed to the conception and design of the work, and helped to draft and revise the manuscript. DB, was involved in the conception, design, data collection, drafting and revising of the manuscript. All authors read and approved the final manuscript. Page 10 of 13 References 1. Thrusfield M: Veterinary Epidemiology. Oxford: Blackwell Publishing; 2007:610. 1. Thrusfield M: Veterinary Epidemiology. Oxford: Blackwell Publishing; 2007:610. 28. Koo D, Wharton M, Birkhead G: Case Definitions for Infectious Conditions Under Public Health Surveillance, US Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention. Atlanta: Centers for Disease Control and Prevention; 1997. y y 2. Salman MD: Animal disease surveillance and survey systems, methods and applications. Ames Iowa: Iowa State Press, Blackwell; 2003. 222 p. 2. Salman MD: Animal disease surveillance and survey systems, methods and applications. Ames Iowa: Iowa State Press, Blackwell; 2003. 222 p. 2. Salman MD: Animal disease surveillance and survey systems, meth 2. Salman MD: Animal disease surveillance and survey systems, methods and applications. Ames Iowa: Iowa State Press, Blackwell; 2003. 222 p. 3. Salman MD: Surveillance tools and strategies for animal diseases in a shifting climate context. Animal health research reviews; 2013. 3. Salman MD: Surveillance tools and strategies for animal diseases in a shifting climate context. Animal health research reviews; 2013. 29. Andresen EM, Catlin TK, Wyrwich KW, Jackson-Thompson J: Retest reliability of surveillance questions on health related quality of life. J Epidemiol Community Health 2003, 57:339–343. 4. Doherr MG, Audigé L: Monitoring and surveillance for rare health-related events, a review from the veterinary perspective. Philosophical transactions of the Royal Society of London Series B, Biological sciences 2001, 356:1097–1106. 30. Buhi ER, Goodson P, Neilands TB: Out of Sight, Not Out of Mind, Strategies for Handling Missing Data. Am J Health Behav 2008, 32:83–92. 5. Lund EM, Armstrong PJ, Kirk CA, Kolar LM, Klausner JS: Health status and population characteristics of dogs and cats examined at private veterinary practices in the United States. J Am Vet Med Assoc 1999, 214:1336–1341. 31. Bergen DC, Beghi E, Medina MT: Revising the ICD-10 codes for epilepsy and seizures. Epilepsia 2012, 53:3–5. 32. Haut ER, Pronovost PJ: SUrveillance bias in outcomes reporting. JAMA 2011, 305:2462–2463. 6. Collins LM, Asher L, Summers J, McGreevy P: Getting priorities straight, risk assessment and decision-making in the improvement of inherited disorders in pedigree dogs. Vet J 2011, 189:147–154. 33. Nattinger AB, McAuliffe TL, Schapira MM: Generalizability of the surveillance, epidemiology, and end results registry population, Factors relevant to epidemiologic and health care research. J Clin Epidemiol 1997, 50:939–945. disorders in pedigree dogs. Vet J 2011, 189:147–154. 7. Bateson P: Independent inquiry into dog breeding. Author details 1 24. Faunt K, Lund E, Novak W: The power of practice, harnessing patient outcomes for clinical decision making. Vet Clin N Am Small Anim Pract 2007, 37:521–532. 1Veterinary Epidemiology, Economics and Public Health, The Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Herts AL9 7T, UK. 2Small Animal Medicine and Surgery Group, The Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Herts AL9 7TA, UK. 3Faculty of Veterinary Science, The University of Sydney, R.M.C. Gunn Building (B19), Sydney, NSW 2006, Australia. 25. Egenvall A, Bonnett BN, Olson P, Hedhammar Å: Validation of computerized Swedish dog and cat insurance data against veterinary practice records. Prev Vet Med 1998, 36:51–65. 26. Welsh S, Hassiotis A, O’Mahoney G, Deahl M: Big brother is watching you–the ethical implications of electronic surveillance measures in the elderly with dementia and in adults with learning difficulties. Aging Mental Health 2003, 7:372–375. Received: 30 October 2013 Accepted: 14 January 2014 Published: 16 April 2014 O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 Veterinary Record 2013, 173:116. 86. Dotson MJ, Hyatt EM: Understanding dog-human companionship. J Business Res 2008, 61:457–466. 63. Egenvall A, Nødtvedt A, Hv E: Bone tumors in a population of 400 000 insured Swedish dogs up to 10 y of age, incidence and survival. Canadian J Veterinary Res 2007, 71:292–299. 87. Ward MP, Kelman M: Companion animal disease surveillance, A new solution to an old problem? Spatial Spatio-temporal Epidemiol 2011, 2:147–157. 64. Sallander M, Hedhammar Å, Rundgren M, Lindberg JE: Demographic data of a population of insured Swedish dogs measured in a questionnaire study. Acta Veterinaria Scandinavica 2001, 42:71–80. 88. Tobias KM, Rohrbach BW: Association of breed with the diagnosis of congenital portosystemic shunts in dogs, 2,400 cases (1980–2002). J Am Veterinary Med Assoc 2003, 223:1636–1639. 89. Green LW, Glasgow RE: Evaluating the relevance, generalization, and applicability of research. Evaluation Health Prof 2006, 29:126–153. 65. Fall T, Hamlin HH, Hedhammar Å, Kämpe O, Egenvall A: Diabetes mellitus in a population of 180,000 insured dogs, incidence, survival, and breed distribution. J Veterinary Internal Med 2007, 21:1209–1216. 90. Holmes MA, Ramey DW: An introduction to evidence-based veterinary medicine. Veterinary Clinics of North America, Equine Practice 2007, 23:191–200. 66. Bergstrom A, Nødtvedt A, Lagerstedt AS, Egenvall A: Incidence and breed predilection for dystocia and risk factors for cesarean section in a Swedish population of insured dogs. Veterinary Surg 2006, 35:786–791. 91. Hill PB, Lo A, Eden CA, Huntley S, Morey V, Ramsey S, Richardson C, Smith DJ, Sutton C, Taylor MD, Thorpe E, Tidmarsh R, Williams V: Survey of the prevalence, diagnosis and treatment of dermatological conditions in small animals in general practice. Veterinary Record 2006, 158:533–539. 67. Egenvall A, Bonnett BN, Haggstrom J: Heart disease as a cause of death in insured Swedish dogs younger than 10 years of age. J Veterinary Internal Med 2006, 20:894–903. 92. Chassin MR, Galvin RW: The urgent need to improve health care quality, Institute of Medicine national roundtable on health care quality. JAMA, The Journal of the American Medical Association 1998, 280:1000–1005. 68. Bergknut N, Egenvall A, Hagman R, Gustås P, Hazewinkel HAW, Meij BP, Lagerstedt A: Incidence of intervertebral disk degeneration–related diseases and associated mortality rates in dogs. J Am Veterinary Med Assoc 2012, 240:1300–1309. 93. Adams VJ, Waldner CL, Campbell JR: Analysis of a practice management computer software program for owner compliance with recall reminders. O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 Ioannidis JPA: Why most published research findings are false. PLoS Med 2005, 2:e124. 55. Bonnett BN, Egenvall A: Age patterns of disease and death in insured Swedish dogs, cats and horses. J Comp Pathol 2010, 142:S33–S38. 79. Hawkins EC, Basseches J, Berry CR, Stebbins ME, Ferris KK: Demographic, clinical, and radiographic features of bronchiectasis in dogs, 316 cases (1988–2000). J Am Veterinary Med Assoc 2003, 223:1628–1635. 56. Nødtvedt A, Bergvall K, Emanuelson U, Egenvall A: Canine atopic dermatitis, validation of recorded diagnosis against practice records in 335 insured Swedish dogs. Acta Veterinaria Scandinavica 2006, 48:8. 80. Ward MP: Prevalence of and risk factors for leptospirosis among dogs in the United States and Canada, 677 cases (1970–1998). J Am Veterinary Med Assoc 2002, 220:53–58. 57. Egenvall A, Bonnett BN, Hedhammar A, Olson P: Mortality in over 350,000 insured Swedish dogs from 1995–2000, II. breed-specific age and survival patterns and relative risk for causes of death. Acta Veterinaria Scandinavica 2005, 46:121–136. 81. Gelatt KN, MacKay EO: Prevalence of the breed-related glaucomas in pure-bred dogs in North America. Veterinary Ophthalmol 2004, 7:97–111. 82. Ware WA, Hopper DL: Cardiac Tumors in Dogs, 1982–1995. J Veterinary Internal Med 1999, 13:95–103. 58. Dobson JM, Samuel S, Milstein H, Rogers K, Wood JLN: Canine neoplasia in the UK, estimates of incidence rates from a population of insured dogs. J Small Animal Pract 2002, 43:240–246. 83. Zaninelli M, Campagnoli A, Reyes M, Rojas V: The O3-Vet project, Integration of a standard nomenclature of clinical terms in a veterinary electronic medical record for veterinary hospitals. Comput Methods Programs Biomed 2012, 108:760–772. 59. Thrusfield MV: Demographic characteristics of the canine and feline populations of the UK in 1986. J Small Animal Pract 1989, 30:76–80. 84. SNOMED CT: Systematized Nomenclature of Medicine-Clinical Terms. International Health Terminology Standards Development Organisation; 2011. http://www.ihtsdo.org/snomed-ct/ 60. Egenvall A, Bonnett BN, Olson P, Hedhammar Å: Gender, age, breed and distribution of morbidity and mortality in insured dogs in Sweden during 1995 and 1996. Veterinary Record 2000, 146:519–525. 61. Dixon S: Pet insurance, understanding the current state of play. Veterinary Business J 2012, 116:6–9. 85. Bellumori TP, Famula TR, Bannasch DL, Belanger JM, Oberbauer AM: Prevalence of inherited disorders among mixed-breed and purebred dogs, 27,254 cases (1995–2010). J Am Veterinary Med Assoc 2013, 242:1549–1555. 62. Vilson Å, Bonnett B, Hansson-Hamlin H, Hedhammar Å: Disease patterns in 32,486 insured German Shepherd Dogs in Sweden, 1995–2006. O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 46. Asher L, Buckland E, Phylactopoulos CL, Whiting M, Abeyesinghe S, Wathes C: Estimation of the number and demographics of companion dogs in the UK. BMC Veterinary Res 2011, 7:74. 80,000 insured female dogs in Sweden from 1995 to 2002. Prev Veterinary Med 2005, 69:109–127. 71. Egenvall A, Bonnett BN, Shoukri M, Olson P, Hedhammar Å, Dohoo I: Age pattern of mortality in eight breeds of insured dogs in Sweden. Prev Veterinary Med 2000, 46:1–14. 47. VetCompass: VetCompass, Health surveillance for UK companion animals. London: RVC Electronic Media Unit; 2013. http://www.rvc.ac.uk/ VetCOMPASS/. 72. Bonnett BN, Egenvall A, Olson P, Hedhammar Å: Mortality in insured Swedish dogs, rates and causes of death in various breeds. Veterinary Record 1997, 141:40–44. 48. Agria Pet Insurance: Agria Pet Insurance. Agria Pet Insurance; 2013. http:// www.agriapet.co.uk/. 73. Bonnett BN, Egenvall A, Hedhammar Å, Olson P: Mortality in over 350,000 insured Swedish dogs from 1995–2000, I. breed-, gender-, age- and cause-specific rates. Acta Veterinaria Scandinavica 2005, 46:105–120. 49. Egenvall A: Survey of the Swedish dog population, age, gender, breed, location and enrolment in animal insurance. Acta Veterinaria Scandinavica 1999, 40:231–240. 50. Perrin T: The business of urban animals survey, the facts and statistics on companion animals in Canada. The Canadian Veterinary Journal [La Revue Veterinaire Canadienne] 2009, 50:48–52. 74. Egenvall A, Hagman R, Bonnett BN, Hedhammar A, Olson P, Lagerstedt A: Breed risk of pyometra in insured dogs in Sweden. J Veterinary Internal Med 2001, 15:530–538. 75. VMDB: The Veterinary Medical Database. VMDB; 2013. http://www.vmdb. org/vmdbinfo.html 51. Embrace Pet Insurance: Facts & Statistics on the US Pet Insurance Industry. Embrace Pet Insurance; 2012. http://www.embracepetinsurance.com/about- us/press/pet-insurance-statistics.aspx. 76. Fleming JM, Creevy KE, Promislow DEL: Mortality in North American dogs from 1984 to 2004, an investigation into age-, size-, and breed-related causes of death. J Veterinary Internal Med 2011, 25:187–198. 52. American Pet Products Association: Industry Statistics & Trends. APPA; 2012. http://americanpetproducts.org/press_industrytrends.asp 53. Nødtvedt A, Guitian J, Egenvall A, Emanuelson U, Pfeiffer DU: The spatial distribution of atopic dermatitis cases in a population of insured Swedish dogs. Prev Veterinary Med 2007, 78:210–222. 77. Wucherer KL, Wilke V: Thyroid cancer in dogs, an update based on 638 cases (1995–2005). J Am Animal Hospital Assoc 2010, 46:249–254. 78. Burkert BA, Kerwin SC, Hosgood GL, Pechman RD, Fontenelle JP: Signalment and clinical features of diskospondylitis in dogs, 513 cases (1980–2001). J Am Veterinary Med Assoc 2005, 227:268–275. 54. References Egenvall A, Nødtvedt A, Penell J, Gunnarsson L, Bonnett BN: Insurance data for research in companion animals, benefits and limitations. Acta Vet Scand 2009, 51:42. 40. de Lusignan S, Krause P: The Hayes principles, learning from the national pilot of information technology and core generalisable theory in informatics. Inform Primary Care 2010, 18:73–77. 16. Buring JE: Primary data collection, what should well-trained epidemiology doctoral students be able to do? Epidemiology 2008, 19:347–349. 41. Howe D, Costanzo M, Fey P, Gojobori T, Hannick L, Hide W, Hill DP, Kania R, Schaeffer M, St Pierre S, Twigger S, White O, Yon RS: Big Data, the future of biocuration. Nature 2008, 455:47–50. 17. Resnik P, Niv M, Nossal M, Kapit A, Toren R: Communication of clinically relevant information in electronic health records, a comparison between structured data and unrestricted physician language; 2008. Fall. 42. Melton GB, Hripcsak G: Automated detection of adverse events using natural language processing of discharge summaries. J Am Med Inform Assoc 2005, 12:448–457. 18. Zhou B: Keyword Search on Large-Scale Structured, Semi-Structured, and Unstructured Data. In Handbook of Data Intensive Computing. Edited by Furht B, Escalante A. New York: Springer; 2011:733–751. 43. Chapman WW, Christensen LM, Wagner MM, Haug PJ, Ivanov O, Dowling JN, Olszewski RT: Classifying free-text triage chief complaints into syn- dromic categories with natural language processing. Artif Intell Med 2005, 33:31–40. 19. Moore GE, Frana TS, Guptill LF, Ward MP, Lewis HB, Glickman LT: Postmarketing surveillance for dog and cat vaccines, new resources in changing times. J Am Vet Med Assoc 2005, 227:1066–1069. 20. Liao KP, Cai T, Gainer V, Goryachev S, Zeng-treitler Q, Raychaudhuri S, Szolovits P, Churchill S, Murphy S, Kohane I, Karlson EW, Plenge RM: Electronic medical records for discovery research in rheumatoid arthritis. Arthritis Care Res 2010, 62:1120–1127. 44. Woolhouse MEJ, Fèvre EM, Handel I, Heller J, Tildesley MJ, Parkin T, Reid SWJ: Guide to good practice for quantitative veterinary epidemiology. 2011:35. 21. Lam K, Parkin T, Riggs C, Morgan K: Use of free text clinical records in identifying syndromes and analysing health data. Vet Rec 2007, 161:547–551. 45. Greenhall JE: Causes of insurance claims paid. Modern Veterinary Pract 1979, 60:853. Page 11 of 13 O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 120. Brady S, Norris JM, Kelman M, Ward MP: Canine parvovirus in Australia, the role of socio-economic factors in disease clusters. Veterinary J 2012, 193:522–528. 96. Powell J, Buchan I: Electronic health records should support clinical research. J Med Internet Res 2005, 7:e4. 97. Hornof WJ, Brentson PR, Self JA, Ballance DW: Development of a complete electronic medical record in an academic institution. J Am Veterinary Med Assoc 2001, 218:1771–1775. 121. Kearsley-Fleet L, O’Neill DG, Volk HA, Church DB, Brodbelt DC: Prevalence and risk factors for canine epilepsy of unknown origin in the UK. Veterinary Record 2013, 172:338. 98. Estberg L, Case JT, Walters RF, Cardiff RD, Galuppo LD: Word search performance for diagnoses of equine surgical colics in free-text electronic patient records. Prev Veterinary Med 1998, 34:161–174. 122. O’Neill DG, Hendricks A, Summers J, Brodbelt DC: Primary care veterinary usage of systemic glucocorticoids in cats and dogs in three UK practices. J Small Animal Pract 2012, 53:217–222. 99. Crowe DC: An appeal for better automated medical records. J Am Veterinary Med Assoc 2003, 223:1566–1567. 123. O’Neill DG, Church DB, McGreevy PD, Thomson PC, Brodbelt DC: Longevity and mortality of owned dogs in England. Veterinary J 2013, 198:638–643. 100. Lund EM, Klausner JS, Ellis LB, Whitney JR: PetTerms, a standardized nomenclature for companion animal practice. Online J Veterinary Res 1998, 2:64–86. 124. Wang X, Chase H, Markatou M, Hripcsak G, Friedman C: Selecting information in electronic health records for knowledge acquisition. J Biomed Informatics 2010, 43:595–601. 101. Lund EM, Armstrong PJ, Kirk CA, Klausner JS: Prevalence and risk factors for obesity in adult cats from private US veterinary practices. Int J Appl Res Veterinary Med 2005, 3:88–96. 125. Radford A: National surveillance of small animal disease in the UK. Veterinary Record 2010, 166:471–472. 102. Lund EM, Armstrong PJ, Kirk CA, Klausner JS: Prevalence and risk factors for obesity in adult dogs from private US veterinary practices. Int J Appl Res Veterinary Med 2006, 4:177–186. y 126. BSAVA: SAVSNET The next stage. BSAVA Companion 2013:4–5. 126. BSAVA: SAVSNET The next stage. BSAVA Com 127. Radford AD, Noble PJ, Coyne KP, Gaskell RM, Jones PH, Bryan JGE, Setzkorn C, Tierney Á, Dawson S: Antibacterial prescribing patterns in small animal veterinary practice identified via SAVSNET, the small animal veterinary surveillance network. Veterinary Record 2011, 169:310. 103. O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 Rhea SK, Glickman SW, Waller A, Ising A, Maillard JM, Lund EM, Glickman LT: Evaluation of routinely collected veterinary and human health data for surveillance of human tick-borne diseases in North Carolina. Vector Borne and Zoonotic Diseases 2011, 11:9–14. 104. Anon: State of Pet Health 2013 Report. Banfield: Pet Hospital; 2013. 128. Pulley J, Clayton E, Bernard GR, Roden DM, Masys DR: Principles of human subjects protections applied in an opt-out, de-identified biobank. Clinical Translational Sci 2010, 3:42–48. 105. Plant JD, Lund EM, Yang M: A case–control study of the risk factors for canine juvenile-onset generalized demodicosis in the USA. Veterinary Dermatology 2011, 22:95–99. 129. SAVSNET: SAVSNET. University of Liverpool; 2012. http://www.liv.ac.uk/ SAVSNET/ 106. Lefebvre SL, Yang M, Wang M, Elliott DA, Buff PR, Lund EM: Effect of age at gonadectomy on the probability of dogs becoming overweight. J Am Veterinary Med Assoc 2013, 243:236–243. 130. CEVM: Centre for Evidence-based Veterinary Medicine. Nottingham: University of Nottingham; 2013. 107. Moore GE, Guptill LF, Ward MP, Glickman NW, Faunt KK, Lewi 107. Moore GE, Guptill LF, Ward MP, Glickman NW, Faunt KK, Lewis HB, Glickman LT: Adverse events diagnosed within three days of vaccine administration in dogs. J Am Veterinary Med Assoc 2005, 227:1102–1108. 131. Clements D, Handel I, Rose E, Querry D, Pugh C, Ollier W, Morgan K, Kennedy L, Sampson J, Summers K, de Bronsvoort BM: Dogslife, a web-based longitu- dinal study of Labrador Retriever health in the UK. BMC Veterinary Res 2013, 9:13. g J Am Veterinary Med Assoc 2005, 227:1102–1108. 132. Nakagawa S, Cuthill IC: Effect size, confidence interval and statistical significance, a practical guide for biologists. Biological Rev 2007, 82:591–605. 108. Raghavan M, Glickman N, Moore G, Caldanaro R, Lewis H, Glickman L: Prevalence of and risk factors for canine tick infestation in the United States, 2002–2004. Vector Borne and Zoonotic Diseases 2007, 7:65–75. 133. Schneeweiss S, Avorn J: A review of uses of health care utilization databases for epidemiologic research on therapeutics. J Clin Epidemiol 2005, 58:323–337. 109. Glickman LT, Glickman NW, Moore GE, Goldstein GS, Lewis HB: Evaluation of the risk of endocarditis and other cardiovascular events on the basis of the severity of periodontal disease in dogs. J Am Veterinary Med Assoc 2009, 234:486–494. 134. David M, Ware R, Donald M, Alati R: Assessing generalisability through the use of disease registers, findings from a diabetes cohort study. Br Med J Open 2011, 1:1–7. 110. O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 Glickman LT: Association between chronic azotemic kidney disease and the severity of periodontal disease in dogs. Prev Veterinary Med 2011, 99:193–200. 135. Nødtvedt A, Berke O, Bonnett BN, Brønden L: Current status of canine cancer registration - report from an international workshop. Veterinary Comparative Oncol 2012, 10:95–101. 111. Mohamed AS, Moore GE, Glickman LT: Prevalence of intestinal nematode parasitism among pet dogs in the United States (2003–2006). J Am Veterinary Med Assoc 2009, 234:631–637. 136. Parkin DM: The evolution of the population-based cancer registry. Nature Rev Cancer 2006, 6:603–612. 112. Lewis HB: Risk factors for canine pancreatitis. DataSavant November/ December, 17–22. ; 2007. 137. Brønden LB, Flagstad A, Kristensen AT: Veterinary cancer registries in companion animal cancer, a review. Veterinary Comparative Oncol 2007, 5:133–144. 113. Lund E: Epidemiology of canine atopic dermatitis. Veterinary Focus 2011, 21:32–33. 138. Michels GM, Knapp DW, DeNicola DB, Glickman N, Bonney P: Prognosis following surgical excision of canine cutaneous mast cell tumors with histopathologically tumor-free versus nontumor-free margins, a retrospective study of 31 cases. J Am Animal Hospital Assoc 2002, 38:458–466. 114. Trevejo R, Yang M, Lund EM: Epidemiology of surgical castration of dogs and cats in the United States. J Am Veterinary Med Assoc 2011, 238:898–904. 115. Maciejewski R, Glickman N, Moore G, Zheng C, Tyner B, Cleveland W, Ebert D, Glickman L: Companion animals as sentinels for community exposure to industrial chemicals, the Fairburn, GA, propyl mercaptan case study. Public Health Reports 2008, 123:333–342. 139. Cornell KK, Bostwick DG, Cooley DM, Hall G, Harvey HJ, Hendrick MJ, Pauli, BU, Render JA, Stoica G, Sweet DC, Waters DJ: Clinical and pathologic aspects of spontaneous canine prostate carcinoma, a retrospective analysis of 76 cases. Prostate 2000, 45:173–183. 140. Ru G, Terracini B, Glickman LT: Host related risk factors for canine osteosarcoma. Veterinary J 1998, 156:31–39. 116. Glickman L, Glickman N: The National Companion Animal Surveillance System NCASP. Purdue University; 2012. http://www.slideserve.com/noleta/the- national-companion-animal-surveillance-system-ncasp-a-supercourse-lecture 141. Moe L: Population-based incidence of mammary tumours in some dog breeds. J Reprod Fertility Suppl 2001, 57:439. 117. Glickman LT, Moore GE, Glickman NW, Caldanaro RJ, Aucoin D, Lewis HB: Purdue University-Banfield National Companion Animal Surveillance Pro- gram for emerging and zoonotic diseases. Vector Borne and Zoonotic Diseases 2006, 6:14–23. 142. Støvring M, Moe L, Glattre E: A population-based case–control study of canine mammary tumours and clinical use of medroxyprogesterone acetate. APMIS 1997, 105:590–596. 118. O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 The Canadian Veterinary Journal [La Revue Veterinaire Canadienne] 2006, 47:234–240. 69. Edwards DS, Henley WE, Harding EF, Dobson JM, Wood JLN: Breed incidence of lymphoma in a UK population of insured dogs. Veterinary Comparative Oncology 2003, 1:200–206. 94. Robinson D, Hooker H: The UK veterinary profession in 2006. London: Institute for Employment Studies; 2006. 94. Robinson D, Hooker H: The UK veterinary profession in 2006. London: Institute for Employment Studies; 2006. 70. Egenvall A, Bonnett BN, Öhagen P, Olson P, Hedhammar Å, von Euler H: Incidence of and survival after mammary tumors in a population of over 95. RCVS: RCVS Facts 2012, Part 2. London: Royal College of Veterinary Surgeons; 2012. 28 p. 95. RCVS: RCVS Facts 2012, Part 2. London: Royal College of Veterinary Surgeons; 2012. 28 p. Page 12 of 13 O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 Casey RA, Loftus B, Bolster C, Richards GJ, Blackwell EJ: Inter-dog aggression in a UK owner survey, prevalence, co-occurrence in different contexts and risk factors. Veterinary Record 2013, 172:127. 2004. Veterinary Record 2005, 156:561–563. 181. Dyer F, Diesel G, Cooles S, Tait A: Suspected adverse reactions, 2010. Veterinary Record 2011, 168:610–613. Veterinary Record 2011, 168:610–613. 152. Bland IM, Guthrie-Jones A, Taylor RD, Hill J: Dog obesity, owner attitudes and behaviour. Prev Veterinary Med 2009, 92:333–340. y 182. Dyer F, Diesel G, Cooles S, Tait A: Suspected adverse events y 182. Dyer F, Diesel G, Cooles S, Tait A: Suspected adverse events, 2011. Veterinary Record 2012, 170:640–642. Veterinary Record 2012, 170:640–642. 153. Dufour S, Barkema HW, DesCôteaux L, DeVries TJ, Dohoo IR, Reyher K, Roy, J, Scholl DT: Development and validation of a bilingual questionnaire for measuring udder health related management practices on dairy farms. Prev Veterinary Med 2010, 95:74–85. 183. Dyer F, Diesel G, Cooles S, Tait A: Suspected adverse reactions, 2009. Veterinary Record 2010, 167:118–121. 184. Dyer F, Brown E, Cooles S, Tait A: Suspected adverse reactions, 2008. Veterinary Record 2009, 165:162–164. Veterinary Record 2009, 165:162–164. 154. Dohoo I, Martin W, Stryhn H: Veterinary Epidemiologic Research. Charlottetown, Canada: VER Inc; 2009. 185. Dyer E, Mulugeta R, Evans C, Tait A: Suspected adverse reactions, 2003. Veterinary Record 2004, 154:806–808. 155. Morton LM: Reporting participation in epidemiologic studies, a survey of practice. Am J Epidemiol 2006, 163:197–203. 186. DEFRA: Dog and Cat Travel And Risk Information (DACTARI). Crown; 2011. http://archive.defra.gov.uk/foodfarm/farmanimal/diseases/vetsurveillance/ dactari/summary-report.htm 156. van Gelder MMHJ, Bretveld RW, Roeleveld N: Web-based Questionnaires, The Future in Epidemiology? Am J Epidemiol 2010, 172:1292–1298. 187. Shaw SE, Langton DA, Hillman TJ: Canine leishmaniosis in the UK. Veterinary Record 2008, 163:253–254. 157. Mellersh C: DNA testing, diagnosing and preventing inherited disorders in dogs. Veterinary Record 2013, 172:264–265. 157. Mellersh C: DNA testing, diagnosing and preventing inherited disorders in dogs. Veterinary Record 2013, 172:264–265. 158. Club TK: Assured Breeder Scheme. The Kennel Club: Breed-specific requirements and recommendations including health screening; 2012. 188. Anon: MSD Animal Health Intervet UK Ltd. ; 2013. http://www.msd-animal- health.co.uk/ 158. Club TK: Assured Breeder Scheme. The Kennel Club: Breed-specific requirements and recommendations including health screening; 2012. 189. CICADA: CICADA - The companion animal disease survey. MSD Animal Health; 2012. http://uk.cicadasurvey.com/ 159. O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 172. British Veterinary Association/Kennel Club/International Sheepdog Society: Eye Scheme. British Veterinary Association, 2013. http://www.bva.co.uk/ canine_health_schemes/Eye_Scheme.aspx 145. Vascellari M, Baioni E, Ru G, Carminato A, Mutinelli F: Animal tumour registry of two provinces in northern Italy, incidence of spontaneous tumours in dogs and cats. BMC Veterinary Res 2009, 5:39. 173. Crispin SM, Long SE, Wheeler CA: Incidence and ocular manifestations of multifocal retinal dysplasia in the Golden Retriever in the UK. Veterinary Record 1999, 145:669–672. 146. Merlo DF, Rossi L, Pellegrino C, Ceppi M, Cardellino U, Capurro C, Ratto A, Sambucco PL, Sestito V, Tanara G, Bocchini V: Cancer incidence in pet dogs, findings of the animal tumor registry of Genoa, Italy. J Veterinary In Med 2008, 22:976–984. y 174. Long SE, Crispin SM: Inheritance of multifocal retinal dysplasia in the Golden Retriever in the UK. Veterinary Record 1999, 145:702–704. Golden Retriever in the UK. Veterinary Record 1999, 145:702–704 147. Brønden LB, Lindstrand S, Nielsen SS, Toft N, Kristensen AT: Validation of data collected in the Danish Veterinary Cancer Registry. Veterinary Comparative Oncology 2009, 7:207–211. 175. Curtis R, Barnett KC: A survey of cataracts in Golden and Labrador Retrievers. J Small Animal Pract 1989, 30:277–286. 176. VMD: Veterinary Medicines Directorate: Suspected Adverse Events. Crow 2012. http://www.vmd.defra.gov.uk/vet/adverse.aspx 148. Adams VJ, Evans KM, Sampson J, Wood JLN: Methods and mortality results of a health survey of purebred dogs in the UK. J Small Animal Pract 2010, 51:512–524. 177. Dyer F, Spagnuolo-Weaver M, Tait A: Suspected adverse reactions, 2005. Veterinary Record 2006, 158:464–466. 149. PFMA: The Pet Food Manufacturers’ Association ‘Statistics’. The Pet Food Manufacturers’ Association; 2012. http://www.pfma.org.uk/statistics/ y 178. Dyer F, Spagnuolo-Weaver M, Cooles S, Tait A: Suspected adverse reactions, 2006. Veterinary Record 2007, 160:748–750. 2006. Veterinary Record 2007, 160:748–750. 150. Downes M, Canty MJ, More SJ: Demography of the pet dog and cat population on the island of Ireland and human factors influencing pet ownership. Prev Veterinary Med 2009, 92:140–149. 179. Dyer F, Spagnuolo-Weaver M, Coolees S, Tait A: Suspected adverse reactions, 2007. Veterinary Record 2008, 163:69–72. y 180. Dyer F, Mulugeta R, Spagnuolo-Weaver M, Tait A: Suspected adverse reactions, y 180. Dyer F, Mulugeta R, Spagnuolo-Weaver M, Tait A: Suspected adverse reactions, 2004. Veterinary Record 2005, 156:561–563. y 180. Dyer F, Mulugeta R, Spagnuolo-Weaver M, Tait A: Suspecte 151. O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 Gould D, Pettitt L, McLaughlin B, Holmes N, Forman O, Thomas A, Ahonen S, Lohi H, O’Leary C, Sargan D, Mellersh C: ADAMTS17 mutation associated with primary lens luxation is widespread among breeds. Veterinary Ophthalmol 2011, 14:378–384. 190. Dórea FC, Sanchez J, Revie CW: Veterinary syndromic surveillance, current initiatives and potential for development. Prev Veterinary Med 2011, 101:1–17. 160. Mellersh C: DNA testing and domestic dogs. Mammalian Genome 2012, 23:109–123. 191. Stärk KD, Regula G, Hernandez J, Knopf L, Fuchs K, Morris RS, Davies P: Concepts for risk-based surveillance in the field of veterinary medicine and veterinary public health, review of current approaches. BMC Health Services Res 2006, 6:20. 161. Strain GM: Aetiology, prevalence and diagnosis of deafness in dogs and cats. Br Veterinary J 1996, 152:17–36. 162. Wood JLN, Lakhani KH: Deafness in Dalmatians, does sex matter? Prev Veterinary Med 1998, 36:39–50. 192. Stead WW, Miller RA, Musen MA, Hersh WR: Integration and Beyond, Linking Information from Disparate Sources and into Workflow. J Am Med Inf Assoc 2000, 7:135–145. 163. De Risio L, Lewis T, Freeman J, de Stefani A, Matiasek L, Blott S: Prevalence, heritability and genetic correlations of congenital sensorineural deafness and pigmentation phenotypes in the Border Collie. Veterinary J 2011, 188:286. 193. Egenvall A, Nødtvedt A, Roepstorff L, Bonnett B: Integrating databases for research on health and performance in small animals and horses in the Nordic countries. Acta Veterinaria Scandinavica 2011, 53:1–6. Nordic countries. Acta Veterinaria Scandinavica 2011, 53:1–6. 164. Sommerlad S, Morton J, Haile-Mariam M, Johnstone I, Seddon J, O'Leary C: Prevalence of congenital hereditary sensorineural deafness in Australian Cattle Dogs and associations with coat characteristics and sex. BMC Veterinary Res 2012, 8:202. 194. British Veterinary Association/Kennel Club: Hip Dysplasia Scheme. British Veterinary Association; 2013. http://www.bva.co.uk/canine_health_schemes/ Hip_Scheme.aspx 195. British Veterinary Association/Kennel Club: Elbow Scheme. British Veterinary Association. 2013. http://www.bva.co.uk/canine_health_schemes/ elbow_scheme.aspx 165. Strain GM: Deafness prevalence and pigmentation and gender associations in dog breeds at risk. Veterinary J 2004, 167:23–32. 166. Crispin SM, Turner BM: Changes to the BVA/KC Canine Health Schemes. Veterinary Record 2010, 166:28. doi:10.1186/2052-6687-1-2 Cite this article as: O’Neill et al.: Approaches to canine health surveillance. Canine Genetics and Epidemiology 2014 1:2. 167. Lewis TW: Optimisation of breeding strategies to reduce the prevalence of inherited disease in pedigree dogs. Animal Welfare 2010, 19:93–98. 168. Wilson B, Nicholas FW, Thomson PC: Selection against canine hip dysplasia, success or failure? Veterinary J 2011, 189:160–168. 169. O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 Moore GE, Ward MP, Kulldorff M, Caldanaro RJ, Guptill LF, Lewis HB, Glickman LT: A space–time cluster of adverse events associated with canine rabies vaccine. Vaccine 2005, 23:5557–5562. 143. Brønden LB, Nielsen SS, Toft N, Kristensen AT: Data from the Danish Veterinary Cancer Registry on the occurrence and distribution of neoplasms in dogs in Denmark. Veterinary Record 2010, 166:586–590. 144. Brønden L, Eriksen T, Kristensen AT: Mast cell tumours and other skin neoplasia in Danish dogs - data from the Danish Veterinary Cancer Registry. Acta Veterinaria Scandinavica 2010, 52:1–6. 119. Thompson KA, Parnell NK, Hohenhaus AE, Moore GE, Rondeau MP: Feline exocrine pancreatic insufficiency, 16 cases (1992–2007)⋆. J Feline Med Surg 2009, 11:935–940. Page 13 of 13 O’Neill et al. Canine Genetics and Epidemiology 2014, 1:2 http://www.cgejournal.org/content/1/1/2 Lewis TW: Genetic evaluation of hip score in UK Labrador Retrievers. PLoS One 2010, 5:1–10. 170. Lewis TW, Ilska JJ, Blott SC, Woolliams JA: Genetic evaluation of elbow scores and the relationship with hip scores in UK Labrador Retrievers. Veterinary J 2011, 189:227–233. 171. Dennis R: Interpretation and use of BVA/KC hip scores in dogs. Practice 2012, 34:178–194.
https://openalex.org/W3036085089	http://www.scielo.br/pdf/icse/v24/1807-5762-icse-24-e190740.pdf	Portuguese	null	Atenção Primária à Saúde na perspectiva da formação do profissional médico	Interface	2,020	cc-by	6,865	https://www.interface.org.br eISSN 1807-5762 Artigos (a, c) Curso de Medicina, Universidade de Fortaleza. Avenida Washington Soares, 1321, Edson Queiroz. Fortaleza, CE, Brasil. 60811-905. (b) Programas de Pós-Graduação em Saúde da Família – Renasf e ProfSaude, Escritório-Ceará, Fundação Oswaldo Cruz (Fiocruz). Eusébio, CE, Brasil. (d) Escritório-Ceará, Fundação Oswaldo Cruz (Fiocruz). Eusébio, CE, Brasil. Atenção Primária à Saúde na perspectiva da formação do profissional médico Primary Care in the perspective of medical education (abstract: p. 15) Atención Primaria de la Salud bajo la perspectiva de la formación del profesional médico (resumen: p. 15) Coelho MGM, Machado MFAS, Bessa OAAC, Nuto SAS. Atenção Primária à Saúde na perspectiva da formação do profissional médico. Interface (Botucatu). 2020; 24: e190740 https://doi.org/10.1590/Interface.190740 Introdução O ensino médico no Brasil passa por diversas mudanças nos últimos anos. Ressalta-se o papel do aluno de graduação como protagonista do saber, ressignificando a prática médica e desenvolvendo competências no sentindo de uma formação mais ampla. Nesse contexto e embasada pelas Diretrizes Curriculares Nacionais (DCNs) de 2014, a APS configura-se como campo de prática da graduação em Medicina no qual os discentes entram em contato com as equipes de saúde, com usuários do SUS e com suas comunidades no início da graduação ao longo de toda sua formação, considerando uma oportunidade de adquirir competências necessárias para consolidação do SUS1,2. A articulação entre prática e teoria é um processo importante para a formação médica, pois permite ao discente desenvolver suas competências e sua expertise tendo como foco tanto o perfil de profissional que se pretende formar quanto a consonância com os objetivos educacionais do curso1,2. Para que isso ocorra, é importante que se construa uma relação mais próxima entre os serviços de saúde e as instituições de ensino superior (IES), na qual se preserve a implementação dos princípios e diretrizes do SUS nos serviços de saúde, pressupondo um alinhamento entre o conhecimento teórico e as atividades formativas práticas3. Além disso, nesse novo modelo formativo, agrega-se um elemento-chave para esse processo: o contexto social com as suas limitações, vulnerabilidades e fortalezas, aproximando o aluno das famílias e comunidades. Os problemas nessa realidade se apresentam com toda sua complexidade e indeterminação, longe dos recortes bem delineados da teoria2. A inserção no campo de prática implica, ainda, na permissão para o desenvolvimento de uma maior autonomia e de consciência crítica por meio de uma horizontalidade da relação docente-discente, à luz da Andragogia e fundamentada em uma educação dialógica, na qual o professor estimula o aluno a alcançar o seu potencial, em uma dimensão orientadora do ensino. Para tanto, são necessários os conceitos advindos da aprendizagem significativa, pois, nessa teoria de aprendizagem, o objeto de estudo relaciona-se de forma admissível e não aleatória, ancorando-se em ideias provenientes da bagagem cognitiva do aluno, considerando o contexto e fortalecendo a interação entre as novas informações e conceitos prévios relevantes4,5. O desafio é evitar uma sobrecarga de informações ofertadas ao aluno, muitas vezes com pouca utilidade prática, dispersando seu tempo e sua motivação para aprender6. Primary Care in the perspective of medical education (abstract: p. 15) Primary Care in the perspective of medical education (abstract: p. 15) Atención Primaria de la Salud bajo la perspectiva de la formación del profesional médico (resumen: p. 15) Márcia Gomes Marinheiro Coelho(a) <dramarciagomes@gmail.com> Maria de Fátima Antero Sousa Machado(b) <fatimaantero@uol.com.br>> Olivia Andrea Alencar Costa Bessa(c) <oliviabessa@unifor.br> Sharmênia de Araújo Soares Nuto(d) <shanuto@gmail.com> Márcia Gomes Marinheiro Coelho(a) <dramarciagomes@gmail.com> O presente estudo apresenta como objetivo analisar a Atenção Primária à Saúde (APS) como ambiente de aprendizagem para os discentes do curso de Medicina. Realizou-se um estudo transversal descritivo, com abordagem qualitativa, por meio de quatro grupos focais com internos dos quatro cursos de Medicina em Fortaleza (Ceará, Brasil). Utilizou-se o método de interpretação dos sentidos, tendo sido identificadas duas categorias empíricas. Para os alunos, a Atenção Primária é um cenário de ensino importante para correlação teoria e prática, mas sem dimensionar a importância desta no Sistema Único de Saúde (SUS). Não incluindo, portanto, a defesa do SUS como um principio importante em sua profissão. No entanto, o incentivo à docência na Atenção Primária e o desenvolvimento de aspectos humanísticos durante a formação seriam fatores motivadores para atuação nessa área. Palavras-chave: Atenção Primária à Saúde. Educação médica. Ensino médico. Graduação de Medicina. Coelho MGM, Machado MFAS, Bessa OAAC, Nuto SAS. Atenção Primária à Saúde na perspectiva da formação do profissional médico. Interface (Botucatu). 2020; 24: e190740 https://doi.org/10.1590/Interface.190740 Coelho MGM, Machado MFAS, Bessa OAAC, Nuto SAS. Atenção Primária à Saúde na perspectiva da formação do profissional médico. Interface (Botucatu). 2020; 24: e190740 https://doi.org/10.1590/Interface.190740 Coelho MGM, Machado MFAS, Bessa OAAC, Nuto SAS. Atenção Primária à Saúde na perspectiva da formação do profissional médico. Interface (Botucatu). 2020; 24: e190740 https://doi.org/10.1590/Interface.190740 1/15 Atenção Primária à Saúde na perspectiva da formação do profissional médico ... Coelho MGM, et al. Introdução Por outro lado, sabendo “por que” e “para que” o estudante precisa aprender, espera-se que o aluno tenha uma maior motivação, pois esse cenário favorável permite a contextualização dos problemas abordados, expondo o estudante às limitações e desafios e instigando-o a buscar soluções ao mesmo tempo que o coloca em contato direto com a pessoa que procura o serviço de saúde e os seus cuidados7. Nessa lógica, percebe-se como elementos essenciais para a aprendizagem suas experiências prévias e a aplicação do conhecimento, das atitudes e habilidades em situações reais, trazendo significado e relevância na assimilação de novos saberes, ampliando, dessa forma, a autogestão desse processo8. Interface (Botucatu) https://doi.org/10.1590/Interface.190740 2/15 Atenção Primária à Saúde na perspectiva da formação do profissional médico ... Coelho MGM, et al. Assim, para fortalecer esse entendimento, esta pesquisa possui como objetivo analisar, sob a ótica dos alunos do internato de Medicina, a Atenção Primária à Saúde como ambiente de aprendizagem. Metodologia O estudo é do tipo transversal, descritivo, com abordagem qualitativa e realizado em quatro IES da cidade de Fortaleza (CE) que possuem cursos de Medicina devidamente credenciados pelo Ministério da Educação (MEC). A seleção dos participantes foi intencional, ou seja, baseada no fundamento de que, a partir do pleno conhecimento sobre a seleção amostral e o propósito da pesquisa, os participantes escolhidos seriam representativos desta, fazendo parte do grupo de amostras não probabilísticas9. Em cada IES, a instituição indicou o grupo de alunos que deveriam ser contactados para a pesquisa. Os critérios de inclusão eram: estar regularmente matriculado no curso de Medicina das IES supracitadas e cursando o internato. O critério de exclusão, por sua vez, era: possuir graduação em outro curso de nível superior. A coleta de dados foi realizada por meio de quatro grupo focais, nos meses de agosto de 2018 a janeiro de 2019, um em cada IES, como foco para as experiências do estudante neste cenário de formação. Os grupos focais foram gravados e, posteriormente, transcritos. Utilizou-se para análise dos dados o método de interpretação dos sentidos, proposto por Minayo10, tendo sido constituído dois corpus: alunos de IES pública e alunos de IES privada. Em seguida, compararam-se as categorias empíricas entre os dois corpora. Como não foi encontrada diferença entre os discursos dos dois corpora, decidiu-se tratar todos como estudantes universitários, independentemente da natureza do financiamento da IES, se pública ou privada, e analisar conjuntamente as falas. O estudo foi aprovado sob o parecer nº 2.585.306, em consonância com a Resolução 510/16 do Conselho Nacional de Saúde, e os entrevistados assinaram o Termo de Consentimento Livre e Esclarecido (TCLE). Resultados e discussão Nos quatro grupos focais, participaram 23 internos, sendo 12 do sexo masculino e 11 do sexo feminino, tendo em média entre 24 e 25 anos, com uma idade mínima de 22 e máxima de 32 anos. Em relação à raça/cor, os participantes se identificavam como brancos (três), preto (um) e pardos (19), sendo 17 provenientes da capital e seis, do interior. Em relação à renda familiar, três afirmaram que a família recebe entre três e cinco salários-mínimos, nove recebem de seis a 15 salários-mínimos, três recebem mais de 15 salários-mínimos, seis não souberam e dois não quiseram informar. Esses dados expressam um público de estudantes jovens, a maioria pardos e brancos, provenientes da capital e com renda familiar elevada. Interface (Botucatu) https://doi.org/10.1590/Interface.190740 3/15 Atenção Primária à Saúde na perspectiva da formação do profissional médico ... Coelho MGM, et al. Os temas emergidos das falas foram agrupados em duas categorias empíricas: “Formação na APS: da teoria à prática” e “Perspectivas futuras como profissional na APS: frustração e encantamento”. Formação na APS: da teoria à prática porque a gente vê na teoria, mas quando chega lá na prática é outra coisa. Por exemplo, a gente tava vendo o módulo de saúde da mulher, que é aquela parte de obstetrícia e lá [na unidade de saúde] a gente acompanhou pré-natal, fez puericultura [...]. Foi muito mais enriquecedor do que eu passar horas lendo o Rezende. (IES1) Eu acredito que seja uma experiência valiosa... porque a gente vê na teoria, mas quando chega lá na prática é outra coisa. Por exemplo, a gente tava vendo o módulo de saúde da mulher, que é aquela parte de obstetrícia e lá [na unidade de saúde] a gente acompanhou pré-natal, fez puericultura [...]. Foi muito mais enriquecedor do que eu passar horas lendo o Rezende. (IES1) Ainda sob essa temática, a formação híbrida ganha espaço e reconhecimento na formação médica. Segundo esse conceito, a “teoria emerge da prática”, ou seja, a partir da experiência de situações reais e do enfrentamento de problemas que surgem no cotidiano, o aluno se sente instigado a procurar as informações que precisa para a resolução daqueles, trazendo maior dinamismo para a busca do conhecimento12. Um aspecto importante relatado da inserção no serviço de saúde é o contato com o paciente, momento cercado de diversas sensações. Se por um lado observa-se, por parte dos acadêmicos, a ansiedade advinda da própria imaturidade do ponto de vista técnico, por outro lado, tem-se a oportunidade de vivenciar o enfrentamento na resolução de problemas por parte dos docentes, como expressa uma entrevistada: Vimos a professora fazendo e nos ensinou. Isso deixa a gente mais segura pra no futuro a gente saber, tentar lidar... (IES1) Os internos reforçam também a importância do contato com a APS desde os semestres iniciais, ou seja, do contato com a realidade. Por mais que teoria e prática estejam distantes, existe um reconhecimento da importância da organização curricular com a inserção desde o momento inicial da formação dos estudantes na APS. Um aspecto negativo identificado por um entrevistado foi a grande demanda versus a gestão do tempo. Pela dinâmica mais corrida, não temos tempo de parar e discutir, como às vezes, em ambulatórios especializados, a gente tem. [...]. A gente tem que chegar em casa e lembrar do que viu e ir atrás de atualizar, do que ficou em dúvida. Formação na APS: da teoria à prática Nas falas analisadas encontram-se elementos que apontam para o distanciamento entre a teoria e a prática do SUS/APS, em um tom uníssono do discurso entre internos de todas as IES entrevistadas. O distanciamento entre teoria e prática, no entanto, não é colocado somente como algo negativo para a formação. A vivência da realidade, distante da teoria e longe dos muros das universidades e de suas situações controladas ou simuladas, também pode resultar em grande aprendizado. É possível perceber que o conhecimento sobre a realidade do sistema também tem um lado positivo, pois conhecer os desafios do SUS – e mais especificamente da APS – propicia ao discente a experiência de saber lidar com as dificuldades e procurar soluções para os problemas encontrados: As condições ideais a gente tem no livro. Aqui é o que tem que ser feito, do jeito que tem que ser feito [...]. (IES1) A universidade integra o nosso acesso ao sistema de saúde a fim de que a gente, como estudante, conheça aquela realidade, por mais que não condiga com o que tá no papel [...] e que a gente elabore meios devidos pra contornar aquela situação. (IES1) Vivenciar essa realidade desde os primeiros semestres e observar como os docentes contornavam a situação trouxe uma maior significância para essa integração, na qual as experiências vivenciadas passaram a transmitir maior segurança aos alunos nas futuras decisões em situações adversas ou lidando com as frustrações: Então eu vi ali na prática [...] e me surpreendi com os profissionais, mesmo com aquelas limitações. (IES1) É importante reconhecer que, para uma formação integral, a aprendizagem requer o envolvimento de três domínios11: o cognitivo, o psicomotor e o afetivo, que devem ser trabalhados de forma integrada e podem ser potencializados a partir da análise de situações nas quais vai se demandar do aluno que ele aprenda a conhecer, a ser e a fazer. Não há hierarquia entre as fases do processo de aprendizagem, à medida que se percebe a relação de dinamismo que ocorre no campo prático a partir da perspectiva do “aprender fazendo”, na qual a inserção do aluno no serviço contextualizaria o que vem sendo abordado no campo teórico, facilitando sua assimilação8. Essas considerações podem ser percebidas no relato de um entrevistado: Interface (Botucatu) https://doi.org/10.1590/Interface.190740 4/15 Atenção Primária à Saúde na perspectiva da formação do profissional médico ... Coelho MGM, et al. Eu acredito que seja uma experiência valiosa... Formação na APS: da teoria à prática (IES3) Além do impacto sobre a consulta propriamente dita, eles mencionaram a dificuldade em promover momentos de discussão na unidade de saúde, denotando a necessidade de um melhor planejamento dessas atividades. A inserção dos alunos nos diversos níveis de atenção deve ser acompanhada da conscientização do SUS enquanto política pública e da sua importância para a população brasileira. Problema da nossa inserção no sistema público é o sistema público. A gente tá à mercê dos problemas que já existem e não pode fazer nada. [...] (IES2) Interface (Botucatu) https://doi.org/10.1590/Interface.190740 5/15 Atenção Primária à Saúde na perspectiva da formação do profissional médico ... Coelho MGM, et al. A aproximação com as pessoas acompanhadas pela equipe na Unidade Básica de Saúde (UBS) trouxe também a experiência de se trabalhar a educação em saúde. Os estudantes citaram a atividade de sala de espera como uma prática realizada por todos e relataram que era um momento previamente cercado de dúvidas, tanto relacionadas ao interesse quanto à participação do público-alvo. Os espaços públicos devem ser locais que propiciem o encontro de sujeitos e a valorização das relações interpessoais, em um exercício de alteridade, pautado na ética e no diálogo, no qual os saberes sejam respeitados e percebidos como vivência educativa13. Analogamente, nesse encontro é possível, ainda, ocorrer uma construção do saber compartilhada; promover a autonomia e a consciência coletiva e individual; e dessa forma, ressignificar o papel da pessoa no autocuidado14. A prática da sala de espera foi estimulada na APS pelos docentes. No entanto, a temática muitas vezes vinha do próprio grupo, e não como uma demanda direta da comunidade: Os temas eram, na maioria das vezes, a gente que escolhia. A gente pesquisava e ele [o professor] nos dava a liberdade pra levarmos temas relevantes que a gente achasse naquele período. (IES1) Já meu grupo, a gente abordou a questão do diabetes e pra mim foi uma surpresa, porque logo que os professores passaram esse trabalho eu fiquei: “Meu Deus! Será que essas pessoas vão participar?”. A gente pensou em fazer uma coisa interativa com os pacientes pra que a gente soubesse o que eles tinham de demanda pra gente responder. A gente queria que eles que fizessem as perguntas pra gente. Quando a gente percebeu, tava todo mundo com a mão levantada, todo mundo tirando as dúvidas... Formação na APS: da teoria à prática (IES1) A partir das falas, percebe-se que houve uma surpresa e um impacto positivo ao verem que os usuários tinham uma conduta mais interativa, trocando experiências, questionando e compartilhando suas dúvidas; ou seja, demonstrando interesse pela atividade proposta. A APS constitui-se, ainda, um espaço privilegiado na construção do conhecimento, pois possibilita a aproximação dos discentes com o território e com as famílias que lá vivem. Nesse interim, reforça-se a importância do reconhecimento das necessidades de saúde da população e da organização das unidades de saúde e das equipes para responderem a essa demanda15. Entre as fragilidades, uma das barreiras sociais mais preocupantes da atualidade e que vem impactando no trabalho das equipes da ESF é a violência urbana, refletindo na prática da atenção à saúde principalmente nas ações realizadas na comunidade. Para os profissionais da ESF e discentes em formação, atuar nas comunidades de um grande centro urbano, como na capital cearense, é lidar com um cenário dominado pela baixa escolaridade, pelo desemprego e pelo tráfico de drogas, vivenciando um aumento dos conflitos devido à disputa pelo poder entre facções rivais16,17. Interface (Botucatu) https://doi.org/10.1590/Interface.190740 6/15 Atenção Primária à Saúde na perspectiva da formação do profissional médico ... Coelho MGM, et al. O enfrentamento do fenômeno crescente da violência pelos serviços de saúde e seus profissionais é um desafio, tendo em vista a construção histórica do vínculo com a comunidade trazida com a implantação da ESF no país, processo no qual se lutou para estreitar relações e adentrar o território. A repercussão desse fenômeno pode ser percebida na fala dos alunos: A faculdade, até por motivo de segurança, não deixa os alunos participarem (das visitas domiciliares). (IES2) Foram passadas instruções pra que esse receio que a gente sentisse fosse diminuindo [...] e que a gente fosse preparado, mas o desconforto ainda fica. (IES1) Eu não tava preparado pra ver isso no consultório: o paciente chegar a chorar porque o médico, nosso professor, teve a suspeita de agressão física e dar todo o espaço pra ela [...]. A parte que eu nunca tinha ouvido falar de denúncia de agressão física ou como a gente tem que intervir e se portar como médico foi assim surreal: tudo que eu podia aprender com ele e realmente lidar com a pessoa, e não com o paciente numa doença, estava lá, sendo apresentado pra gente. Formação na APS: da teoria à prática (IES1) Observa-se ainda a prudência das IES ao resguardarem os alunos de situações de maior vulnerabilidade e risco; e a questão do enfrentamento à violência: Não sei se é porque o meu professor parecia estar muito integrado na comunidade. [...] Eu não me senti com medo porque eu tive a sensação de que eles queriam que a gente tivesse ali e que eles tivessem precisando ser ouvidos e serem olhados, entendeu? (IES1) Às vezes até tratavam a gente já como se a gente fosse médico e que a nossa presença ali ia ser positiva pra eles. (IES1) Às vezes até tratavam a gente já como se a gente fosse médico e que a nossa presença ali ia ser positiva pra eles. (IES1) A violência apresenta-se como um dos mais complexos desafios da sociedade atual e interfere diretamente na prática da APS nos territórios de abrangência. É importante saber lidar com as consequências dessa realidade na vida das pessoas e o impacto que gera na saúde destas16-18. A APS constitui a principal porta de entrada do SUS e a demanda muitas vezes é consideravelmente maior do que a oferta de serviços, o que ocasiona uma notória fragilidade na rede de referência e contrarreferência e gera, dessa forma, “um acesso seletivo, focalizado e excludente”19. O acesso ao SUS é confrontado com a legislação vigente que legaliza essa proposta, mas, por outro lado, não assegura a implementação desta na prática. Interface (Botucatu) https://doi.org/10.1590/Interface.190740 7/15 Atenção Primária à Saúde na perspectiva da formação do profissional médico ... Coelho MGM, et al. Eu vi muito a questão de que eles usavam a gente com, assim, o oportunismo de conseguir as coisas [...] e muitas vezes, ela nos abordava querendo vir logo pra cá, pulando essas etapas de entrada no sistema. (IES1) A barreira financeira, a desproporção entre demanda e oferta e a mercantilização da saúde corroboram para uma prática comum nos bastidores da saúde pública: a procura por uma consulta ou exame por intermédio de profissional que atue na rede de saúde e possa viabilizá-las sem os trâmites da fila espera. O contato com o paciente é uma das maiores expectativas do aluno de Medicina durante o início da graduação e é relatado como elemento central em sua aprendizagem. Essa aprendizagem, por sua vez, não envolve somente procedimento clínico ou terapêutico, mas também desenvolvimento da empatia, habilidade de comunicação e manejo de situações. No relato dos alunos, percebe-se a relevância desse momento e como a vivenciaram na prática da APS: Eu acho que isso é o principal porque o resto a gente vê em aula, a gente vê em livro... O paciente tá lá naquele momento, naquela vez ou naquela hora e a gente aprende ou não. (IES4) Uma das coisas mais relevantes é o contato com o paciente. Às vezes até tratavam a gente já como se a gente fosse médico e que a nossa presença ali ia ser positiva pra eles. (IES1) Porque se eu leio alguma coisa no livro de fisiologia, ele me dá uma linguagem X, mas a educação de uma pessoa da comunidade eu tenho que me adequar, eu tenho que me virar com o que ele sabe, com o que eu sei, pra criar aquele diálogo que ele entenda. (IES1) As habilidades podem ser utilizadas tanto para resolver o problema em si quanto para preparar o aluno para seu enfrentamento, inclusive quando este não puder ser alterado, proporcionando medidas adaptativas e amenizando as frustrações que possam advir dessa relação aluno-paciente2. Em estudo sobre as estratégias de enfrentamento no atendimento ao paciente por alunos de Medicina, relatam-se as medidas adaptativas agrupadas em cinco possibilidades: (a) resolução de problemas, na qual o aluno se planeja na busca por estratégias que venham amenizar ou resolver o problema do paciente; (b) aceitação da responsabilidade, na qual ele reconhece suas limitações pessoais e aceita a condição apresentada, tentando minimizar sua carga emocional e lidar melhor com o estresse; (c) fuga-esquiva, na qual o aluno, centrado na sua emoção, protela a busca por ajuda, correndo o risco de perder a capacidade de indignação e a humanização com o sofrimento da pessoa; (d) suporte social, também considerado quando os alunos buscam ajuda profissional para se sentirem emocionalmente amparados ao compartilharem suas emoções; e (e) espiritualidade/religiosidade, que seria utilizada quando o aluno busca explicações para situações difíceis20. Interface (Botucatu) https://doi.org/10.1590/Interface.190740 8/15 Atenção Primária à Saúde na perspectiva da formação do profissional médico ... Coelho MGM, et al. Na fala dos entrevistados, percebe-se que a comunicação é uma preocupação real, na qual a linguagem técnica precisa ser adaptada para a linguagem próxima dos pacientes, viabilizando a troca de informações entre aluno e paciente e a efetividade das orientações a serem transmitidas e pactuadas. Foi frequente ainda, nas falas dos discentes, a empatia e a capacidade de reconhecer diferentes formas de linguagem, inclusive o “não dito”, muitas vezes velado na relação médico-paciente por falta de vínculo e de confiança; e/ou ainda por medo. E é curioso que, foi até comentado, que a gente vê além da necessidade do atendimento, né?! Porque a gente tem a prova aqui [na universidade] que é o paciente simulado e tem algumas horas que já teve provas que a gente tinha que trabalhar essa parte de ver o sentimento do paciente. Às vezes até tratavam a gente já como se a gente fosse médico e que a nossa presença ali ia ser positiva pra eles. (IES1) Só que o nosso paciente simulado, ele deixava bem claro: “Eu estou com medo disso e disso”, sendo que na realidade não é assim que acontece. A gente tem que notar que aquele paciente tá precisando da nossa ajuda. (IES1) A relação médico-paciente é dotada de diversos aspectos que vão desde o contato durante a consulta propriamente dita até a confiança e adesão ao plano terapêutico. Para que essa relação seja de fato eficaz, a empatia, um dos principais aspectos envolvidos, deve ser fortalecida e as escolas médicas, segundo as DCNs1, têm a responsabilidade e o dever de sensibilizar os médicos em formação quanto às habilidades humanísticas. A relação empática, muitas vezes, devido à falta de resolutividade do sistema de saúde, resulta em frustração. Então eu solto o verbo porque eu não gosto de coisa errada. Eu sou leal com os pacientes. Eu acho que a gente vem de uma universidade particular e a maioria tem condição de ter plano de saúde, mas a gente não pode achar que isso é a maioria porque não é. A gente tendo essa consciência, sabe quão injusto é. Porque a gente vê que se fosse nossa mãe, nosso pai, talvez eles teriam conseguido algo que a gente quer pro nosso paciente, mas que a gente não pode fazer e aí a gente fica de mãos atadas. (IES2) A transmissibilidade da empatia está em foco no ensino médico. Esta é dependente da biografia e personalidade do docente, sendo, assim, docente-dependente. Além do conhecimento técnico e científico, o corpo docente deve adentrar nos aspectos humanistas durante a formação20. Interface (Botucatu) https://doi.org/10.1590/Interface.190740 9/15 Atenção Primária à Saúde na perspectiva da formação do profissional médico ... Coelho MGM, et al. Perspectivas futuras como profissional na APS: frustração e encantamento Coelho MGM, et al. É isso que eu ia lhe dizer. A resposta dessa pergunta verbalmente que vem na minha cabeça é “Não, não, Deus me livre!”. Eu sou muito revoltada pra poder baixar a cabeça pra um sistema tão falho como tá agora. Não sei se pela teoria do SUS porque a teoria do SUS é muito linda, né? [...]. (IES1) Perspectivas futuras como profissional na APS: frustração e encantamento Ao serem questionados sobre a possibilidade de se trabalhar no SUS após a sua formação, observaram-se, diferentemente de todos os outros momentos do grupo, um período inicial de silêncio e um certo desconforto, como se houvesse uma necessidade de especificar mais a questão norteadora. Alguns discentes permaneceram com uma postura reflexiva, como se fosse a primeira vez que cogitassem essa possibilidade. O momento foi interrompido com essas falas: Quando você fala de SUS, quer dizer Atenção Primária à Saúde ou SUS como um todo? (IES4) Foi esclarecido que a interpretação deveria partir deles, no sentido de instigar a reflexão sobre a atuação no SUS em um contexto amplo, incluindo, portanto, a APS como uma das possibilidades. O fato de se trabalhar no SUS não quer dizer que se trabalha exclusivamente nesse sistema de saúde, bem como não descarta o trabalho na assistência à saúde ou na docência. Após essa explicação, eles começaram a se manifestar e tivemos os seguintes relatos: Eu acho que a pretensão de um aluno [...] é que a gente quer sempre o SUS, ou algo assim, de modo provisório. (IES2) O meu foco seria a residência, mas posso trabalhar na Atenção Primária. (IES2) O meu foco seria a residência, mas posso trabalhar na Atenção Primária. (IES2) Eu quero trabalhar algum período no SUS, mas não exclusivamente. (IES1) O que motiva é a questão do ensino. Não existe isso em hospital privado: a valorização do ensino e da questão da residência, preceptoria [...] isso é algo que me atrai [no SUS]. (IES4) Quando o discente relatou que o foco da sua escolha seria a residência médica, acredita-se que este quis dizer que escolheria atuar em outra área, mas que não descartava a possibilidade do trabalho na APS. Nesse caso, observamos uma tendência que ocorre com uma certa frequência, historicamente, desde a implantação da ESF no país: a APS como opção de atuação transitória. Porque quando você roda por alguns locais falando da atenção terciária ou falando em questão de saúde primária, você nota quanto o engajamento seu como profissional pode fazer a diferença. Então é nesse sentindo sim, pelo SUS e por ele em si não, porque ele ainda é decepcionante, ele é muito lindo no papel, mas na prática ele destoa muito da situação teórica. É frustrante! (IES2) Interface (Botucatu) https://doi.org/10.1590/Interface.190740 10/15 Atenção Primária à Saúde na perspectiva da formação do profissional médico ... Outros alunos completaram, na mesma linha de pensamento: Com toda sinceridade e falando especificamente SUS, pelo SUS não, mas pelo paciente do SUS, sim! (IES4) Eu não vejo ninguém tendo aquele pensamento: “Meu Deus, eu quero trabalhar no SUS! Eu quero fazer isso da minha vida, eu quero vestir essa camisa pra mim!”. Por quê? Porque a gente sabe que é muito decepcionante mesmo, então isso não faz com que nem que a nova geração queira adentrar nesse mundo dessa forma, entendeu? E é revoltante, gente! (IES2) Acho que a pessoa quando faz Medicina, pelo menos 90%, quer ajudar alguém e eu acho que realmente não é mais pelo dinheiro, pelo tanto de gente que tem [egressos no mercado de trabalho], e não é mais por status, porque nem tem mais. Então eu acho que quem gosta de ajudar alguém vai buscar, vai ter que “pedalar”, como a gente fala. Não gosta de “nãos” e o SUS é um grande “não”, é um “nãozão”. (IES2) A vontade que dá, sinceramente, pra mim hoje, é trabalhar exclusivamente particular e poder fazer as minhas, entre aspas, “caridades”. (IES2) Interface (Botucatu) https://doi.org/10.1590/Interface.190740 A vontade que dá, sinceramente, pra mim hoje, é trabalhar exclusivamente particular e poder fazer as minhas, entre aspas, “caridades”. (IES2) Diante desses relatos, algumas reflexões da formação ética e moral e da concepção hegemônica do ensino médico se fazem necessárias. Do ponto de vista de competência moral, identifica-se três níveis de estágios de raciocínio moral: (a) pré-convencional, (b) convencional e (c) pós-convencional. No primeiro nível, ocorre o julgamento pelo indivíduo do que seria certo e errado, firmado em interesses próprios e incluindo o medo de ser punido. Nesse estágio, considerado do individualismo, ele seguiria as normas por interesse pessoal. No segundo nível, a ação moral do indivíduo é baseada em regras e convenções socialmente aceitas e instituídas por autoridades, correspondendo à necessidade de cumprir com o que se espera do indivíduo para a manutenção da ordem social dentro de uma perspectiva de que os interesses coletivos se sobrepõem aos individuais. Por fim, no terceiro nível – o pós-convencional –, a ação é guiada por princípios universais, pautados pela igualdade e reciprocidade, e não por ordens sociais21. A partir desses conceitos, alguns instrumentos foram propostos para avaliar a competência moral em vários contextos, inclusive entre os universitários. Ao realizar uma pesquisa sobre a competência moral entre os estudantes do curso de Interface (Botucatu) https://doi.org/10.1590/Interface.190740 11/15 Atenção Primária à Saúde na perspectiva da formação do profissional médico ... Coelho MGM, et al. Odontologia21, observou-se que a capacidade de tomada de decisão e julgamento estão intimamente relacionadas à formação escolar e familiar, ou seja, a vivências prévias ao ingresso na universidade. Evidencia-se que a competência moral, caso não seja trabalhada nos processos educacionais seguintes, pode inclusive retroceder. As IES podem e devem dar sua contribuição no desenvolvimento de aspectos cognitivos e morais dos alunos, contribuindo com a sociedade para a formação ética no exercício da profissão e beneficiando os futuros médicos e seus pacientes. A partir desta análise, avalia-se que nenhum dos relatos transmitiram um discurso sobre o SUS no nível pós-convencional, pois nas falas ressalta-se os interesses individuais, às vezes coletivos, mas muito longe de defender a universalidade do SUS como um princípio ético da profissão. Outro aspecto a ser analisado apoia-se na concepção hegemônica do ensino médico. Apesar dos avanços contidos nas DCNs1, a formação médica, seja nos projetos pedagógicos dos cursos ou no currículo oculto22, ainda recebe influência do modelo biologicista, hospitalocêntrico e incentivado para a superespecialização, como preconizado pelo Relatório Flexner23. Considerações finais Os resultados desta pesquisa evidenciaram que a APS permite contextualizar a teoria com situações reais, à medida que aproxima os alunos das necessidades de saúde da população; das fragilidades e limitações do SUS; e das pessoas inseridas em suas famílias e em seu contexto social. Se por um lado as dificuldades nesse percurso contribuíram para um sentimento de frustração e decepção com o SUS e, mais especificamente, com a APS, por outro lado, possibilitou para a maioria dos entrevistados o desenvolvimento de aspectos humanísticos, fundamentais para sua formação. A análise da APS enquanto campo de prática da formação do médico limitou-se por aspectos estruturais, logísticos e de gestão dos serviços de saúde. Não emergiram falas que trouxessem uma compreensão da APS enquanto porta de entrada das redes atenção, sua capacidade resolutiva para a maioria dos problemas de saúde da população e a identificação por parte do estudante de Medicina como futuro profissional de saúde na APS. Reforça-se, portanto, a importância da sensibilização das escolas médicas e dos gestores, a fim de promover uma melhor adequação dos cenários de aprendizagem na APS, mitigando as fragilidades apontadas no estudo. Por fim, sugere-se fortalecer o vínculo ensino-serviço, a fim de repensar e ressignificar o ensino médico; promover a consolidação do SUS; e, consequentemente, valorizar o importante papel da APS na prevenção e promoção da saúde. A vontade que dá, sinceramente, pra mim hoje, é trabalhar exclusivamente particular e poder fazer as minhas, entre aspas, “caridades”. (IES2) Em relação aos motivos que supostamente levariam um médico a permanecer no SUS, algumas hipóteses foram levantadas, como a estabilidade de um emprego público e o retorno financeiro. Não houve, nas falas, a defesa da APS enquanto coordenadora do cuidado; e a valorização e a satisfação do profissional médico em estar inserido nesse sistema. Por que muita gente tá no SUS hoje? Pelo financeiro. Muita gente se submete a isso. Se submete pelo dinheiro, pelo emprego. [...] vai chegar lá de cara feia, faz o mínimo possível e não quer ter o menor trabalho. (IES2) Falta incentivo. [...] não tem reconhecimento. No Brasil sempre teve isso: aquela mentalidade que o médico ganha muito sendo que ele ganha muito trabalhando muito. (IES4) Quando levantaram entre si a questão da interiorização da Medicina, os entraves relatados foram a dificuldade de educação permanente e a infraestrutura. No interior, falta plano de apoio para o aumento de conhecimento profissional porque a gente sabe que médico tem que se atualizar sempre. Sem contar com a falta de estrutura. [...] tudo é mais complicado. (IES2) A gente sabe que, como profissional de saúde, a partir do momento em que a gente se responsabiliza por trabalhar em certo local e com certas condições, a responsabilidade é minha, então posso responder legalmente. (IES4) Interface (Botucatu) https://doi.org/10.1590/Interface.190740 12/15 Atenção Primária à Saúde na perspectiva da formação do profissional médico ... Coelho MGM, et al. Apesar de a maioria das falas reforçar somente aspectos negativos do SUS, a gratidão foi o elemento-chave para afirmação de alguns alunos para inserção profissional nesse setor. Ressaltaram que a criação de vínculos com o paciente nos serviços públicos de saúde é fortalecida pela aproximação do contexto de vida dos indivíduos. Essa realidade supera os problemas relatados, sendo possível uma realização profissional. Contribuições das autoras Márcia Gomes Marinheiro Coelho participou da concepção e delineamento do trabalho, redação do manuscrito e aprovação da versão final do manuscrito. Maria de Fátima Antero de Sousa Machado participou da discussão dos resultados, revisão crítica do conteúdo e aprovação da versão final do manuscrito. Olivia Andrea Alencar Costa Bessa participou da discussão dos resultados, revisão crítica do conteúdo e aprovação da versão final do manuscrito. Sharmênia de Araújo Soares Nuto participou da concepção e delineamento do trabalho, revisão crítica do conteúdo e aprovação da versão final do manuscrito. Referências 1. Brasil. Ministério da Educação. Conselho Nacional de Educação. Resolução CNE/ CES nº 4, de 7 de Novembro de 2001. Institui diretrizes curriculares nacionais do curso de graduação em Medicina. Diário Oficial da União. 9 Nov 2001; Sec. 1, p. 38. 2. Aguiar AC, Ribeiro ECO. Conceito e avaliação de habilidades e competência na educação médica: percepções atuais dos especialistas. Rev Bras Educ Med. 2006; 34(3):371-8. 3. Silva MAM, Mendes AAJ, Souza MCA. Estudo de caso sobre tendências de mudanças nos cenários de prática em escolas médicas. Arq Cienc Saude. 2016; 23(3):80-4. 4. Caldeira ES, Leite MTS, Rodrigues-Neto JF. Estudantes de medicina nos serviços de atenção primária: percepção dos profissionais. Rev Bras Educ Med. 2011; 35(4):477-85. 5. Zompero AF, Sampaio HR, Vieira KM. Investigação da transferência de significados na abordagem da aprendizagem significativa utilizando atividades investigativas. Rev Electron Invest Educ Cienc. 2016; 11(1):40-52. 6. Carvalho MB, Ribeiro MMF, Silva LD, Shimomura FM. A composição do curriculum vitae entre estudantes de medicina e seus condicionantes. Rev Bras Educ Med. 2013; 37(4):483-91. 7. Perim GL, Abdallall IG, Aguilar-da-Silva RH, Lampert JB, Stella RCR, Costa NMSC. Desenvolvimento docente e a formação de médicos. Rev Bras Educ Med. 2009; 33 Supl 1:70-82. 8. Hearn J, Dewji M, Stocker C, Greg Simons. Patient-centered medical education: a proposed definition. Med Teach. 2019; 41(8):934-8. 9. Minayo MC. Amostragem e saturação em pesquisa qualitativa: consensos e controvérsias. Rev Pesqui Qual. 2017; 5(7):1-12. 10. Minayo MC. Pesquisa social: teoria, método e criatividade. Petrópolis: Vozes; 2016. 11. Zeferino AMB, Passeri SMRR. Avaliação da aprendizagem do estudante. Cad ABEM. 2007; 3:39-43. 12. Borges MC, Miranda CH, Santana RC, Bollela VR. Avaliação formativa e feedback como ferramenta de aprendizado na formação de profissionais da saúde. Medicina (Ribeirão Preto). 2014; 47(3):324-31. 13. Caldeira ES, Leite MTS, Rodrigues-Neto JF. Estudantes de medicina nos serviços de atenção primária: percepção dos profissionais. Rev Bras Educ Med. 2011; 35(4):477-85. 14. Reis INC, Silva ILR, Wong JA. Espaço público na atenção básica de saúde: educação popular e promoção da saúde nos centros de saúde-escola do Brasil. Interface (Botucatu). 2014; 18 Supl 2:1161-73. 15. Ruiz DG, Farenzena GJ, Haeffner LSB. Internato regional e formação médica: percepção da primeira turma pós-reforma curricular. Rev Bras Educ Med. 2010; 34(1):21-7. 16. Almeida JF. Exposição à violência comunitária dos agentes da estratégia saúde da família e repercussões sobre suas práticas de trabalho: um estudo qualitativo [dissertação]. Direitos autorais Este artigo está licenciado sob a Licença Internacional Creative Commons 4.0, tipo BY (https://creativecommons.org/licenses/by/4.0/deed.pt_BR).. Este artigo está licenciado sob a Licença Internacional Creative Commons 4.0, tipo BY (https://creativecommons.org/licenses/by/4.0/deed.pt_BR).. 13/15 Interface (Botucatu) https://doi.org/10.1590/Interface.190740 Interface (Botucatu) https://doi.org/10.1590/Interface.190740 Atenção Primária à Saúde na perspectiva da formação do profissional médico ... Coelho MGM, et al. Referências São Paulo: Faculdade de Medicina, Universidade de São Paulo; 2015. 17. Vial EA, Junges JR, Olinto MT, Machado PS, Pattussi MP. Violência urbana e capital social em uma cidade no Sul do Brasil: um estudo quantitativo e qualitativo. Rev Panam Salud Publica. 2010; 28(4):289-97. 18. Machado CB, Daher DV, Teixeira ER, Acioli S. Violência urbana e repercussão nas práticas de cuidado no território da saúde da família. Rev Enferm UERJ. 2016; 24(5):1-6. Interface (Botucatu) https://doi.org/10.1590/Interface.190740 14/15 Atenção Primária à Saúde na perspectiva da formação do profissional médico ... Coelho MGM, et al. 19. Campos RTO, Ferrer AL, Gama CAP, Campos GWS, Trapé TL, Dantas DV. Avaliação da qualidade do acesso na atenção primária de uma grande cidade brasileira na perspectiva dos usuários. Saude Debate. 2014; 38(spe):252-64. 20. Costa FD, Azevedo RCS. Empatia, relação médico-paciente e formação em medicina: um olhar qualitativo. Rev Bras Educ Med. 2010; 34(2):261-9. 21. Landim TP, Silva MSF, Neves HN, Nuto SAS. Competência de juízo moral entre estudantes de odontologia. Rev Bras Educ Med. 2015; 39(1):41-9. 22. Giroux H. Teoria crítica e resistência em educação: para além das teorias de reprodução. Petrópolis: Vozes; 1986. 23. Ribeiro VMB. Discutindo o conceito de inovação curricular na formação dos profissionais de saúde: o longo caminho para as transformações no ensino médico. Trab Educ Saude. 2005; 3(1):91-121. The present study aimed to analyze Primary Care as a learning environment for Medicine students. A cross-sectional descriptive study with a qualitative approach was carried out by means of four focus groups with interns of the four Medicine programs of the city of Fortaleza, State of Ceará, Brazil. Through the method of interpretation of meanings, two empirical categories were identified. Students recognize Primary Care as a space for the correlation between theory and practice in real settings where, many times, individual interests are highlighted and, sometimes, collective interests are emphasized, but the universality of Brazilian National Health System (SUS) is far from being defended as an ethical principle of the profession. However, the incentive to teaching in Primary Care and the development of humanistic aspects during education are motivational aspects for action in this area. Keywords: Primary care. Medical education. Medical teaching. Medicina undergraduate course. El objetivo del presente estudio fue analizar la Atención Primaria de la Salud como ambiente de aprendizaje para los discentes del curso de Medicina. Referências Se realizó un estudio transversal descriptivo, con abordaje cualitativo, por medio de cuatro grupos focales con internos de los cuatro cursos de Medicina en Fortaleza, Ceará, Brasil. Se utilizó el método de interpretación de los sentidos, habiéndose identificado dos categorías empíricas. Para los alumnos, la Atención Primaria se reconoce como espacio para correlación de la teoría con la práctica, en situaciones reales, en las cuales, muchas veces, se subrayan los intereses individuales, otras veces colectivos, pero están muy lejos de defender la universalidad del Sistema Brasileño de Salud (SUS) como un principio ético de la profesión. Sin embargo, el incentivo a la docencia en la Atención Primaria y el desarrollo de aspectos humanísticos durante la formación serían factores motivadores para la actuación en esa área. Palabras clave: Atención primaria de la salud. Educación médica. Enseñanza médica. Graduación en medicina. Submetido em 12/03/20. Aprovado em 30/04/20. Submetido em 12/03/20. Aprovado em 30/04/20. Interface (Botucatu) https://doi.org/10.1590/Interface.190740 15/15
https://openalex.org/W4296926539	https://cienciadigital.org/revistacienciadigital2/index.php/AnatomiaDigital/article/download/2329/5633	es		Eficacia del aceite esencial de Neem sobre la piel y pelaje de gatos con dermatofitosis	Anatomía Digital	2,022	cc-by	4,195	´ ISSN: 2697-3391 Vol. 5 No. 3.3, pp. 46 – 57, septiembre 2022 www.anatomiadigital.org Eficacia del aceite esencial de Neem sobre la piel y pelaje de gatos con dermatofitosis Effectiveness of neem essential oil on the skin and fur of cats with dermatological condition 1 2 3 Pamela Celeste Núñez Sanaguano https://orcid.org/0000-0002-2364-9721 Maestría en Medicina Veterinaria, Universidad Católica de Cuenca, Ecuador. pamela.nunez.70@est.ucacue.edu.ec Pablo Giovanny Rubio Arias https://orcid.org/0000-0002-9185-482 Maestría en Medicina Veterinaria, Universidad Católica de Cuenca, Ecuador. prubioa@ucacue.edu.ec Andrés Renato Ordoñez Palacios https://orcid.org/0000-0002-1916-6778 Facultad de Ciencias Médicas, Universidad de Guayaquil, Ecuador mvz.renato@gmail.con Artículo de Investigación Científica y Tecnológica Enviado: 05/07/2022 Revisado: 07/08/2022 Aceptado: 12/09/2022 Publicado:22/09/2022 DOI: https://doi.org/10.33262/anatomiadigital.v5i3.3.2329 Cítese: Núñez Sanaguano, P. C., Rubio Arias , P. G., & Ordoñez Palacios, A. R. (2022). Eficacia del aceite esencial de Neem sobre la piel y pelaje de gatos con dermatofitosis. Anatomía Digital, 5(3.3), 46-57. https://doi.org/10.33262/anatomiadigital.v5i3.3.2329 ANATOMÍA DIGITAL, es una Revista Electónica, Trimestral, que se publicará en soporte electrónico tiene como misión contribuir a la formación de profesionales competentes con visión humanística y crítica que sean capaces de exponer sus resultados investigativos y científicos en la misma medida que se promueva mediante su intervención cambios positivos en la sociedad. https://anatomiadigital.org La revista es editada por la Editorial Ciencia Digital (Editorial de prestigio registrada en la Cámara Ecuatoriana de Libro con No de Afiliación 663) www.celibro.org.ec Esta revista está protegida bajo una licencia Creative Commons AttributionNonCommercialNoDerivatives 4.0 International. Copia de la licencia: http://creativecommons.org/licenses/by-nc-nd/4.0/ Medicina cooperativa P á g i n a 46 \| 12 ´ ISSN: 2697-3391 Vol. 5 No. 3.3, pp. 46 – 57, septiembre 2022 www.anatomiadigital.org Palabras claves: Azadirachta indica, dermatofito, dermatoscopio, DTM, lámpara de Wood, tricograma. Resumen Objetivo: evaluar el efecto del aceite esencial de Neem (Azadirachta indica) de manera tópica sobre la piel y pelaje de gatos con dermatofitosis. Metodología: la valoración visual fue la metodología de valoración más importante, tomando en cuenta las siguientes variables: piel: (inflamación, descamación, excoriación, hiperpigmentación, alopecia, y pelaje: (seboso, reseco, quebradizo, mal olor), interpretadas con escalas de valoración ( 1: no hay mejoría, 2: mejoría lente, 3: mejoría moderada y 4: mejoría total); la investigación manejó un diseño completamente al azar, conformado por 4 tratamientos: T1 (Testigo), T2 (5 días de aplicación), T3 (10 días de aplicación) y T4 (15 días de aplicación) y T5 (20 días de aplicación), se evaluó en total 30 gatos, 6 animales por cada tratamiento, con el uso de algodón empapado del aceite se cubrió todo la superficie de la piel y pelo, a cada paciente se le realizó una ficha clínica y dermatológica detallados. Resultados: mostraron diferencias significativas en los criterios de valoración de piel y pelo así: no hay mejoría, mejoría leve, mejoría moderada y mejoría total, con respecto a todas las variables existió mejoría leve, moderada y total, muy pocos animales no mostraron mejoría. Conclusiones: el aceite de Neem (Azadirachta indica) funciona en el tratamiento de dermatofitosis y mejora de manera visual la piel como el pelaje de gatos afectados dermatológicamente, todos los tratamientos funcionaron siendo el T4 y T5 los mejores, con aplicaciones de 15 y 20 días consecutivos respectivamente. Keywords: Azadirachta indica; dermatophyte; dermatoscope; DTM; trichogram; Wood's lamp. Abstract Objective: to evaluate the effect of Neem essential oil (Azadirachta indica) topically on the skin and coat of cats with dermatophytosis. Methodology: visual assessment was the most important assessment methodology, taking into account the following variables: skin: (inflammation, flaking, excoriation, hyperpigmentation, alopecia, and fur: (oily, dry, brittle, bad smell), interpreted with scales assessment (1: no improvement, 2: slight improvement, 3: moderate improvement and 4: total improvement); the research used a completely randomized design, made up of 4 treatments: T1 (Control), T2 (5 days of application ), T3 (10 days of application) and T4 (15 days of application) and T5 (20 days of application), a total of 30 cats Medicina cooperativa P á g i n a 47 \| 12 ´ ISSN: 2697-3391 Vol. 5 No. 3.3, pp. 46 – 57, septiembre 2022 www.anatomiadigital.org were evaluated, 6 animals for each treatment, with the use of cotton soaked in the oil, all the surface of the skin and hair, each patient underwent a detailed clinical and dermatological record. Results: they showed significant differences in the evaluation criteria of skin and hair as follows: no improvement, slight improvement, moderate improvement, and total improvement, regarding all the variables there was slight, moderate, and total improvement, very few animals did not show improvement. Conclusions: Neem oil (Azadirachta indica) works in the treatment of dermatophytosis and visually improves the skin as well as the fur of dermatologically affected cats, all treatments worked, being T4 and T5 the best, with applications of 15 and 20 days. consecutive, respectively. Introducción Los estudios manifiestan que en la consulta veterinaria de mascotas especialmente perros y gatos, aproximadamente del 20 al 70% se encuentran relacionadas de manera directa a problemas de piel y anexos y su forma fácil de manifestación visual condiciona a los dueños a que lleven a sus mascotas al especialista (1), las enfermedades dermatológicas han demostrado tener grandes afectaciones a la calidad de vida de los animales, como de sus propietarios (2), sin dejar atrás la posibilidad de transmitir enfermedades zoonóticas e importantes en salud pública, y la capacidad de presentar resistencias a los antimicrobianos, de manera especial en los grupos bacterianos (3). Sobre las enfermedades dermatológicas en los felinos domésticos, existen estudios que demuestran menor prevalencia de dermatofitosis con respecto a otras enfermedades dermatológicas, y los caninos mantienen la mayor prevalencia según varios países europeos como latinoamericanos (4), además las manifestaciones de enfermedades dermatológicas de la piel de los felinos domésticos no se muestran de manera característica como en los caninos (5). Los gatos por sus hábitos de vida, presentan en su piel y pelo agentes fúngicos, los cuales son conocidos como hongos queratinofílicos y queratinolíticos, su presencia en los animales, y cambios en el estado inmunológico, debido a la presencia de otras enfermedades o situaciones de estrés, permitiendo que los hongos se activen y modifiquen el estado de salud de los mismos; con el incremento de riesgos zoonóticos hacia los humanos que conviven con sus mascotas (6). Estudios recientes utilizando aceites esenciales (AE), tales como los presentes en la planta del neem (Azadirachta indica) han logrado una eficacia medicinal de tipo antimicrobiano, anticancerígeno, antiprotozoal, Medicina cooperativa P á g i n a 48 \| 12 ´ ISSN: 2697-3391 Vol. 5 No. 3.3, pp. 46 – 57, septiembre 2022 www.anatomiadigital.org antiinflamatorio, antiviral y principalmente insecticida, convirtiéndolos en una alternativa opcional de tratamiento en la actualidad (7) Dentro de la piel la estructura más importante la constituye la capa córnea ya que es el componente externo de los animales y los humanos, allí se encuentra las proteínas encargadas de la agregación y compactación de estos filamentos de queratina, conocida principalmente como filagrina, el cual proviene del acrónimo filament-aggregating protein (8), esta capa constituye la más externa de la epidermis, actúa como una barrera de protección que no permite el ingreso de agentes patógenos (9) El árbol de Neem (Azadirachta indica) en la india se la llama comúnmente Indian Lila, pertenece a la familia Meliaceae, de la subfamilia Meloideae y tribu Melieae, éste árbol es de los más versátiles del trópico, de elevado potencial al tener varios productos de tipo no madereros muy útiles como su corteza, hojas, frutos, semillas (10); Azadirachta es considerado un árbol de hoja perenne, que va desde los tamaños pequeños a los más grandes de rápido crecimiento , se caracteriza por tener las ramas anchas y extendidas, además que puede tolerar elevadas temperaturas, así como en los suelos pobres de alto degrado (11); el aceite de Neem tiene al menos unos 100 componentes activos biológicamente, de manera principal triterpenos, de los cuales los más importantes los limonoides, de manera especial la azadiractina (12). Los aceites esenciales representan un uso destacado hoy en día gracias a la industria farmacéutica, importantes al convertirse en productos alternativos, de excelente eficiencia y de bajo costo, que inclusive puede sustituir otros fármacos sintéticos tradicionales usados comúnmente (13); por lo tanto la investigación tuvo como objetivo evaluar el efecto del aceite esencial de Neem (Azadirachta indica) de manera tópica sobre la piel y pelaje de gatos con afectaciones dermatológicas y su valoración a través de dermatoscopia y escala de valoración visual. La Investigación tuvo como objetivo evaluar el efecto del aceite esencial de Neem (Azadirachta indica) de manera tópica sobre la piel y pelaje de gatos con dermatofitosis. Metodología Unidades experimentales y diseño del tratamiento. Se evaluaron 30 gatos, que asistieron a consulta al centro de Rescate “Patitas al Rescate”, de la ciudad de Guayaquil en la provincia de Guayas y de la evaluación se le diagnosticó con patologías dermatológicas. A cada paciente, le fue llenada una ficha clínica y dermatológica. Cada felino del Centro de Rescate fue analizado previo el consentimiento de la directiva vigente del lugar, los animales fueron tratados dentro del marco que refleja las cinco libertades, lo cual garantizó que no existiera en el proceso de recolección de muestras, como del tratamiento maltrato animal, se excluyó felinos que no mostraron signos patológicos Medicina cooperativa P á g i n a 49 \| 12 ´ ISSN: 2697-3391 Vol. 5 No. 3.3, pp. 46 – 57, septiembre 2022 www.anatomiadigital.org relacionados a piel o pelo, además de cachorros menores de 6 meses y hembras en estado de gestación. Los pacientes fueron asignados al azar a un experimento en el cual se conformó con 5 tratamientos de 6 gatos cada uno: T1 (Testigo), T2 (5 días de aplicación), T3 (10 días de aplicación), T4 (15 días de aplicación) y T5 (20 días de aplicación). Diagnóstico de la Dermatofitosis. Para el diagnóstico de manera específica de la dermatofitosis se utilizó la metodología de Wood (14), ayudados del dermatoscopio ® Dermlite DL100 se caracterizó las alteraciones cutáneas presentes en la piel, con el raspado de la zona afectada, y mediante citología se diagnosticó si existía la presencia de bacterias con microscopia clínica ®Olympus BX4, además se aplicó como prueba diagnóstica confirmatoria el Test Agar de dermatofitos ®DTM; adicionalmente se realizó el estudio de tricograma para observar características más específicas del folículo piloso como de la estructura capilar superficial. Aplicación del tratamiento. Se aplicó el aceite embebido en algodón a los pacientes cubriendo el lomo, los flancos derecho e izquierdo, extremidades anteriores y posteriores, de manera primaria sobre el pelo, y posteriormente en la piel, realizando un deslizamiento más profundo del algodón sobre las zonas más afectadas; el proceso duró por animal 10 minutos, Luego se dejó reposar a cada gato por 20 minutos más, este proceso se lo realizó por cada día según el tiempo de aplicación, la valoración de las variables en estudio se lo hizo cada cinco días post tratamiento, mediante escalas de valoración visual, con la ayuda del dermatoscopio ®Dermlite DL100, lámpara de Wood y ®DTM. Análisis estadístico. Las variables respuestas estudiadas correspondieron al proceso de evaluación de la piel (inflamación, descamación, escoriación, hiperpigmentación y alopecia) a las cuales se valoró en 4 categorías (1: no hay mejoría; 2: mejoría leve; 3: mejoría moderada y 4: mejoría total). Así mismo se evaluó la condición del pelaje (seboso, quebradizo, reseco, mal olor) a las cuales se valoró de la misma manera (1: no hay mejoría; 2: mejoría leve; 3: mejoría moderada y 4: mejoría total). Todas estas categorías de acuerdo con el tratamiento asignado fueron analizadas de manera estadística mediante ANOVA. Resultados Como se muestra en la Tabla 1, se puede observar el efecto del tratamiento con extracto de Neem sobre las variables de evaluación de piel en estudio (inflamación, descamación, escoriación, hiperpigmentación, alopecia), en las que se observa que la aplicación continua por 20 días se mostró como el mejor tratamiento, en la cual las valoraciones de 4.00, 3.67, 4.00, 3.50, 3.67, interpretado al valor 4 como mejoría total y 3 como mejoría moderada del aceite de Neem, sobre las variables inflamación, descamación, escoriación, Medicina cooperativa P á g i n a 50 \| 12 ISSN: 2697-3391 Vol. 5 No. 3.3, pp. 46 – 57, septiembre 2022 ´ www.anatomiadigital.org hiperpigmentación y alopecia, al presentar diferencias significativas p<0.05 en comparación con los tratamientos T2, T3 y T1 (testigo). Tabla 1. Eficacia del aceite de Neem entre tratamientos sobre las variables de evaluación de la piel de gatos, siguiendo la valoración (1: no hay mejoría, 2: mejoría leve, 3: mejoría moderada y 4: mejoría total). Categorías T1 Valoración de piel Inflamación Descamación Escoriación Hiperpigmentación 1.00b 1.00c 1.00d 1.00d T2 (5 días) 3.00a 2.00a 2.33a 1.83a Tratamientos T3 (10 días) 3.33a 2.50ab 2.83ab 2.33ab Alopecia 1.00c 1.17a 2.83b Letras distintas en la misma fila mostro diferencia significativa p<0.05 T4 (15 días) 3.67a 2.83ab 3.50bc 2.83bc T5 (20 días) 4.00b 3.67bc 4.00d 3.50c 3.33b 3.67b En la Tabla 2 se puede apreciar el efecto del tratamiento con extracto de Neem sobre las variables de evaluación de pelaje en estudio (quebradizo, reseco y mal olor), en las que se observa que la aplicación continua por 20 días se mostró como el mejor tratamiento, en la cual las valoraciones de 3.83, 3.83 y 4.00 interpretado al valor 4 como mejoría total y 3 como mejoría moderada del aceite de Neem, sobre las variables quebradizo, reseco y mal olor, al presentar diferencias significativas p<0.05 en comparación con los tratamientos T2, T3 y T1 (testigo). Tabla 2. Comparación entre tratamientos de la eficacia del aceite de Neem sobre las variables de evaluación del pelaje de los gatos, siguiendo la valoración (1: no hay mejoría; 2: mejoría leve; 3: mejoría moderada y 4: mejoría total) Tratamientos T2 (5 T3 (10 días) días) Quebradizo 1.00c 1.83a 3.17b d a Valoración del Reseco 1.00 2.33 3.00bb c a pelaje Mal Olor 1.00 3.00 3.00a Letras distintas en la misma fila mostro diferencia significativa P<0,05 Categorías T1 (testigo) T4 (15 días) 3.17b 3.67c 3.83b T5 (20 días) 3.83b 3.83c 4.00b Discusión Se puede apreciar el efecto del aceite de Neem sobre el resultado de la mejora de la inflamación independientemente del tratamiento de aplicación del aceite de Neem al que fueron sometidos los felinos, así como lo refiere Singh et al (15), que la aplicación Medicina cooperativa P á g i n a 51 \| 12 ´ ISSN: 2697-3391 Vol. 5 No. 3.3, pp. 46 – 57, septiembre 2022 www.anatomiadigital.org continua del aceite de Neem sobre pacientes tratados con heridas provocadas de diabetes graves provocadas, en la cual más del 50% sanaron en un total de 12 semanas de tratamiento, en el 75% de pacientes. La eficacia del aceite de Neem en la mejora de la descamación de la epidermis de los gatos, se encuentra directamente relacionado con las células existentes en la epidermis, de las cuales los queratinocitos y fibroblastos gracias al efecto del aceite de Neem envuelto en vesículas de argán mediante nanotecnología según lo refiere Manaca et al (16) permitió que disminuya el estrés oxidativo y la migración de estas células para el cierre más rápido de las heridas, lo cual afecta a la integridad de las células epiteliales y la disminución del descame excesivo en enfermedades dermatológicas, demostrado en la mejoría de esta variable en la presente investigación. Con respecto al efecto positivo del aceite de Neem sobre la variable escoriación de la epidermis de los gatos, todos los tratamientos mostraron desde mejoría leve hasta mejora total, siendo el T4 el mejor tratamiento, logrando que el 50% de felinos se recuperen, así lo refiere Giuggioli et al (17), que la aplicación de una crema a base de aceite de Neem conjuntamente con Hypericum perforatum, en úlceras calcinosas de personas con la patología esclerodermia, se recuperaron en un 45% es decir (15/33) de manera total en un tiempo de 40 días, mientras que los otros pacientes 18/33 que representó el 55% se observó mejoría en el eritema. Se puede apreciar el efecto del aceite de Neem sobre el resultado de la mejora de la hiperpigmentación de la piel de los gatos, de 30 animales testeados el 51% mostraron mejoría, lo cual significa que los felinos se recuperaron de manera favorable, tal cual lo refiere Daniel et al (18) en un estudio de 24 perros diagnosticados con Malassezia en la cual valoraron hiperpigmentación como una signología importante de la enfermedad dermatológica, demostraron que la aplicación tópica de aceite de Neem conjuntamente con el consumo vía oral de Itraconazol a los 14 días primarios de tratamiento existió una recuperación de los pacientes del 41,22% y del 91,22% a los 28 días de tratamiento. El efecto del aceite de Neem mostró una notable mejoría de la alopecia de los gatos, de los 30 felinos en estudio, el 53.33% mostraron mejoría, así como lo refiere Daniel et al (18), en la cual los perros con enfermedades fúngicas mejoraron en un 45%, en los primeros 14 días de tratamiento, incluyendo otras sintomatologías adyacentes como alopecia. La eficacia del aceite de Neem sobre el resultado de la mejora de la variable seboso y quebradizo de la evaluación del pelaje de los gatos, de 30 animales testeados 43.33% y 53.33% mejoraron de manera total respectivamente, aunque las investigaciones no detallan de manera específica estos beneficios capilares, los diferentes estudios determinan que el uso solo del Neem como mixto, es decir con otras plantas naturales Medicina cooperativa P á g i n a 52 \| 12 ´ ISSN: 2697-3391 Vol. 5 No. 3.3, pp. 46 – 57, septiembre 2022 www.anatomiadigital.org para el cuidado del cabello generan beneficios en la integridad de la fibra capilar como lo refiere Campos et al (12). El efecto del aceite de Neem sobre la evaluación del pelaje, específicamente la variable reseco de 30 animales testeados el 66.7% mostraron mejoría y aunque no existe información sobre esta valoración en específico, de igual manera las investigaciones del uso de plantas para cosmética y preparaciones de cabello detallan sus múltiples beneficios sobre el brillo, y cura de muchas patologías capilares como caspa, resequedad como lo refiere Tambolli et al (19),y Rathi et al (20). La investigación demostró que el efecto del aceite de Neem fue altamente eficaz sobre el mal olor de la piel de los gatos con dermatofitosis, obteniéndose el 80% de mejora en los 30 animales testeados, lo cual significa que los felinos se recuperaron de manera positiva e inmediata a partir de los primeros cinco días de tratamiento, logrando recuperación absoluta a los 20 días de aplicación del aceite de Neem de la misma manera que lo refiere Daniel et al (18) en un estudio de 24 perros con enfermedad por levaduras, en la cual evaluaron mediante el olfato al mal olor como signo de la enfermedad, la aplicación tópica de aceite de Neem conjuntamente con el consumo vía oral de Itraconazol a los 14 días primarios de tratamiento existió una recuperación de los pacientes del 41.22% y del 91.22% a los 28 días de tratamiento. Conclusiones   El mejor tratamiento de aplicación del aceite de Neem (Azadirachta indica) sobre la piel y pelaje con felinos diagnosticados dermatofitosis, fue los 20 días de aplicación sobre todas las variables de evaluación de piel y pelo. La aplicación del aceite de Neem de 5, 10, 15 y 20 días funciona de manera positiva sobre la piel y pelaje de gatos diagnosticados con dermatofitosis sobre las variables en estudio: inflamación, descamación, escoriación, hiperpigmentación, alopecia, sebosidad, quebradizo, reseco y mal olor; obtuvo mejorías desde leves, moderadas y totales, siendo aún mejor sobre las variables de evaluación de pelo esto es reseco y de mal olor, los cuales obtuvieron mejoras moderadas y totales en todos los felinos analizados desde los 5 días de aplicación. Conflicto de intereses Los autores certifican que no existen conflictos de interés en el presente trabajo. Agradecimientos Cordial agradecimiento al centro de Rescate “Patitas al rescate”, Healthy Pet, RenatoVetDerm, VetSpaLuis. Medicina cooperativa P á g i n a 53 \| 12 ´ ISSN: 2697-3391 Vol. 5 No. 3.3, pp. 46 – 57, septiembre 2022 www.anatomiadigital.org Referencias Bibliográficas 1. Gaspareto N, Trevisan Y, Almeida N, Neves R, Almeida A, Dutra V, et al. Prevalência das doenças de pele não neoplásicas em cães no. Pesq Vet Bras [Internet]. 2013 marzo. (Citado 2022 en. 5); 33(3):359–62. Disponible en: http://www.pvb.com.br/portal/download_artigo/MTM3M3wyMDIyMDYyOTEzMTMz Mw== 2. Madureira R, Sperotto J. Diagnóstico dermatológico em pequenos animais: O que pode influenciar? Arch Vet Sci [Internet]. 2017 nov. (Citado 2022 en. 5);22(4):9–19. Disponible en: http://dx.doi.org/10.5380/avs.v22i4.56944 3. Andrade V, Marques A. Dermatofitose em animais de companhia e sua importância para a Saúde Pública – Revisão de Literatura. Rev Bras Hig e Sanidade Anim Brazilian J Hyg Anim Sanity [Internet]. 2019 marz. (Citado 2022 en. 6); 13(1):142–55. Disponible en: http://www.higieneanimal.ufc.br/seer/index.php/higieneanimal/article/view/502/2648 4. Łagowski, D., Gnat, S., Nowakiewicz, A., Osińska, M., & Zięba, P. The Prevalence of Symptomatic Dermatophytoses in Dogs and Cats and The Pathomechanism of Dermatophyte Infections. Postępy Mikrobiologii - Advancements of Microbiology [Internet]. 2019 ener. (Citato 2022 en. 5); 58(2), 165–176. Disponible en: https://doi.org/10.21307/pm-2019.58.2.165 5. Seals S, Kearney M, Del Piero F, Hammerberg B, Pucheu-Haston C. A study for characterization of IgE-mediated cutaneous immediate and late-phase reactions in nonallergic domestic cats. Vet Immunol Immunopathol [Internet]. 2014 may. (Citato 2022 en. 6); 159(1–2):41–49. Disponible en: http://dx.doi.org/10.1016/j.vetimm.2014.02.007 6. Gallardo J, Piontelli E. Hongos queratinofílicos oportunistas en el pelaje de gatos domésticos (Felix domesticus) en la Ciudad de Valparaíso. Boletín Micológico [Internet]. 2007 dic. (citado 2022 en. 5); 22:9–19. Disponible en: https://doi.org/10.22370/bolmicol.2007.22.0.127 7. Acero-Godoy J, Guzmán-Hernández T, Muñoz-Ruíz C. Revisión documental de uso de los aceites esenciales obtenidos de Lippia alba (Verbenaceae), como alternativa antibacteriana y antifúngica. Rev Tecnol en Marcha [Internet]. 2019 marz. (citado 2022 en. 6); 32:3–11. Disponible en: https://doi.org/10.18845/tm.v32i1.4114 8. Armengot-Carbo M, Hernández-Martín A, Torrelo A. Filagrina: papel en la barrera cutánea y en el desarrollo de patología. Actas Dermosifiliogr [Internet]. 2015 marz. (citado 2022 en. 6); 106(2):86–95. Disponible en: http://dx.doi.org/10.1016/j.ad.2013.10.019 Medicina cooperativa P á g i n a 54 \| 12 ´ ISSN: 2697-3391 Vol. 5 No. 3.3, pp. 46 – 57, septiembre 2022 www.anatomiadigital.org 9. Busi, F. M. Barrera epidérmica en dermatitis atópica. Rev Asoc Col Dermatol [Internet]. 2008 dic. (Citato 2022 en. 6); 16(4), 293–301. Disponible en: https://revista.asocolderma.org.co/index.php/asocolderma/issue/view/11/volumen_16_n umero_4_octubre_diciembre_2008 10. Aneesa N, Gayathri J. Beneficial effects of neem oil-an updated review. J Pharm Sci Res [Internet]. 2016 ener. (Citado 2022 en.6) 8(8):756–758. Disponible en: https://www.jpsr.pharmainfo.in/Documents/Volumes/vol8Issue08/jpsr08081613.pdf 11. Forim M, Fernandes Da Silva M, Fernandes J. Processo de obtencao de nanopartículas biopoliméricas contento óleo e extratos de Azadirachta Indica A. Juss (Neem), nanopartículas biopoliméricas e micropartículas. Organización Mundial de Propiedad Intelectual: Brasil; 2014. Disponible en: https://patentimages.storage.googleapis.com/44/16/25/eeef82ce15e29f/WO2014113860 A1.pdf 12. Campos E, de Oliveira JL, Pascoli M, de Lima R, Fraceto LF. Neem oil and crop protection: From now to the future. Front Plant Sci [Internet]. 2016 oct. (citado 2022 en. 5); 7:1–8. Disponible en: https://doi.org/10.3389/fpls.2016.01494 13. Labrada-Hechavarría, Y., Cordoví-Velázquez, J. M., Ledea-Rodríguez, J. L., Rapado-Paneque, M., & Rosabal-Cordoví, U. M. Caracterización física y química de aceite esencial de Azaridachta indica A Juss expuesto a radiación gamma. Revista Cubana de Química [Internet]. 2018 dic. (citado 2022 en. 6); 30(3), 454–469. Disponible en: https://cubanaquimica.uo.edu.cu/index.php/cq/article/view/4149/3630 14. Moriello K. Dermatophytosis in cats and dogs: A practical guide to diagnosis and treatment. In Pract [Internet]. 2019 feb. (Citado 2022 en. 5); 41(4):138–147. Disponible en: https://doi.org/10.1136/inp.l1539 15. Singh A, Singh A, Narayan G, Singh T, Shukla V. Effect of Neem oil and Haridra on non-healing wounds. An Int Q J Res Ayurveda [Internet]. 2014 dic. (Citado 2022 en. 5); 35(4):398–403. Disponible en: https://doi.org/10.4103/0974-8520.158998 16. Manca M, Manconi M, Meloni M, Marongiu F, Allaw M, Usach I, et al. Nanotechnology for natural medicine: Formulation of neem oil loaded phospholipid vesicles modified with argan oil as a strategy to protect the skin from oxidative stress and promote wound healing. Antioxidants [Internet]. 2021 may. (Citado 2022 en. 6); 10(5):1– 18. Disponible en: https://doi.org/10.3390/antiox10050670 17. Giuggioli D, Lumetti F, Spinella A, Cocchiara E, Sighinolfi G, Citriniti G, et al. Use of Neem oil and Hypericum perforatum for treatment of calcinosis-related skin ulcers Medicina cooperativa P á g i n a 55 \| 12 ´ ISSN: 2697-3391 Vol. 5 No. 3.3, pp. 46 – 57, septiembre 2022 www.anatomiadigital.org in systemic sclerosis. J Int Med Res [Internet]. 2019 dic. (Citado 2022 en. 7); 48(4). Disponible en: https://doi.org/10.1177/0300060519882176 18. Daniel A, David P, Ravishankar C, Sumith K. Evaluation of different therapeutic protocols for canine malasseziosis. Indian J Anim Heal [Internet]. 2022 sept. (Citado 2022 en. 7); 61(1):71–77. Disponible en: https://doi.org/10.36062/ijah.2022.13121 19. Tamboli FA, Kolekar YS, More HN, Mulani SA, Mali NP. Medicinal plants used in cosmetics for skin and hair care. Int J Pharm Chem Anal [Internet]. 2021 jul. (Citado 2022 en. 5); 8(2):36–40. Disponible en: https://doi.org/10.18231/j.ijpca.2021.008 20. Rathi V, Chandra J, Tamizharasi S, Kumar A. Plants used for hair growth promotion: A review. Pharmacogn Rev [Internet]. 2008 ener. (Citado 2022 en. 6) 2(3):185–187. Disponible en: https://www.phcogrev.com/article/2008/2/3-19 Medicina cooperativa P á g i n a 56 \| 12 ´ ISSN: 2697-3391 Vol. 5 No. 3.3, pp. 46 – 57, septiembre 2022 www.anatomiadigital.org El artículo que se publica es de exclusiva responsabilidad de los autores y no necesariamente reflejan el pensamiento de la Revista Anatomía Digital. El artículo queda en propiedad de la revista y, por tanto, su publicación parcial y/o total en otro medio tiene que ser autorizado por el director de la Revista Anatomía Digital. Medicina cooperativa P á g i n a 57 \| 12
https://openalex.org/W4362505830	https://link.springer.com/content/pdf/10.1007/s10143-023-01986-6.pdf	English	null	Survival in patients with surgically treated brain metastases: does infratentorial location matter?	Neurosurgical review	2,023	cc-by	5,880	Abstract Surgical resection is a common treatment modality for brain metastasis (BM). Location of the BM might significantly impact patient survival and therefore might be considered in clinical decision making and patient counseling. In the present study, the authors analyzed infra- and supratentorial BM location for a potential prognostic difference. Between 2013 and 2019, 245 patients with solitary BM received BM resection at the authors’ neuro-oncological center. In order to produce a covari- ate balance for commonly-known prognostic variables (tumor entity, age, preoperative Karnofsky Performance Score, and preoperative Charlson Comorbidity Index), a propensity score matching at a ratio of 1:1 between the cohort of patients with infra- and supratentorial BM location was performed using R. Overall survival (OS) rates were assessed for both matched cohorts of patients with BM. Sixty-one of 245 patients (25%) with solitary BM exhibited an infratentorial tumor location; 184 patients (75%) suffered from supratentorial solitary BM. Patients with infratentorial BM revealed a median OS of 11 months (95% confidence interval (CI) 7.4–14.6 months). Compared with this, median OS for the group of 61 individually matched patients with solitary supratentorial solitary BM was 13 months (95% CI 10.9-15.1 months) (p = 0.32). The present study suggests that the prognostic value of infra- and supratentorial BMs does not significantly differ in patients that undergo surgery for solitary BM. These results might encourage physicians to induce surgical therapy of supra- and infratentorial BM in a similar manner. Keywords Surgery for brain metastasis · Infra- versus supratentorial BM location · Survival RESEARCH RESEARCH RESEARCH Survival in patients with surgically treated brain metastases: does infratentorial location matter? Motaz Hamed1 · Anna‑Laura Potthoff1 · Muriel Heimann1 · Niklas Schäfer2 · Valeri Borger1 · Alexander Radbruch3 · Ulrich Herrlinger2 · Hartmut Vatter1 · Matthias Schneider1 Received: 15 January 2023 / Revised: 9 March 2023 / Accepted: 26 March 2023 / Published online: 30 March 2023 © The Author(s) 2023 Received: 15 January 2023 / Revised: 9 March 2023 / Accepted: 26 March 2023 / Published online: 30 March 2023 © The Author(s) 2023 Received: 15 January 2023 / Revised: 9 March 2023 / Accepted: 26 March 2023 / Published online: 30 March 2023 © The Author(s) 2023 * Motaz Hamed motaz.hamed@ukbonn.de 1 Department of Neurosurgery, University Hospital Bonn, Venusberg‑Campus 1, 53127 Bonn, Germany 2 Division of Clinical Neuro‑Oncology, Department of Neurology, University Hospital Bonn, Venusberg‑Campus 1, 53127 Bonn, Germany 3 Department of Neuroradiology, University Hospital Bonn, Bonn, Germany Neurosurgical Review (2023) 46:80 https://doi.org/10.1007/s10143-023-01986-6 Neurosurgical Review (2023) 46:80 https://doi.org/10.1007/s10143-023-01986-6 * Motaz Hamed motaz.hamed@ukbonn.de Matching procedure All patients who had undergone surgery for solitary BM between 2013 and 2019 at the authors’ neuro-oncological university center were entered into a computerized database. Patients with multiple BM and/or leptomeningeal involve- ment were excluded. The study was conducted in accord- ance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the University Hospi- tal Bonn (No. 250/19). Informed consent was not sought as a retrospective study design was chosen. Matching was used to control for measured pre-treatment variables that are prognostic of the outcome. For the matched-pair analysis, the statistical computing program R (version 4.1.2; The R Foundation for Statistical Computing, https://www.r-project.org/) was used as previously described [11]. A propensity score matching was performed at a ratio of 1:1 between the cohort of 61 patients with infratentorial BM and a cohort of 184 patients with supratentorial BM. To produce a covariate balance in the two groups and therefore increase the robustness of the data, the following known prognostic parameters were selected for matching: age, KPS, and CCI at admission and tumor entity. The balance was measured by the standardized mean differences, variance ratios, and empirical cumulative density function statistics and visualized using a Love plot. Patient characteristics surveyed for further analysis con- sisted of radiological features, preoperative laboratory val- ues, and BM location, as well as the location of the primary malignancy, the preoperative functional status of the affected patient, and the presence of other systemic metastases. In terms of location, two groups were established for further investigation on the basis of the supra- or infratentorial location of the BM. Infratentorial location was defined as within the cerebellum, and brainstem lesions were excluded from analysis. The functional status of patients undergoing surgery was assessed preoperatively according to the Kar- nofsky Performance Score (KPS) [8]. The Charlson Comor- bidity Index (CCI) was used to evaluate the comorbidity burden of patients prior to surgery as previously described [3]. During a weekly tumor board meeting, all the treatment strategies applied and further investigated were determined individually for each individual patient by interdisciplinary consensus and, if necessary, coordinated with the referring physicians and/or taken into account previous oncological therapies [9]. In the present study, only patients with indi- cation for surgical therapy were considered and further analyzed resulting in a consecutive cohort of patients with surgically treated BM. Introduction Despite a myriad of treatment adaptations/optimizations due to ever new findings in the field of different malignan- cies, early surgical/radiotherapy treatment of BM is con- sistently deemed crucial with respect to the prognosis of advanced cancer [4]. Surgical therapy is often considered for solitary/larger BM and requires individualized risk/ben- efit consideration against non-invasive treatment modalities but might nonetheless offer a significant survival benefit [5]. Improved and individualized cancer therapy is leading to a growing number of patients with varying malignancies experiencing metastases to the brain in the course of their disease [1]. The occurrence of brain metastases (BM) rep- resents an important (prognostic) stage in the progression of systemic cancer [2]. In patients with BM, neurosurgi- cal resection often constitutes a key pillar of treatment [3]. fii While BM causing early symptoms can be treated at an early stage, they are often located in eloquent areas [5]. This eloquent location might facilitate early symptom emergence, but it also impairs surgical resectability [5]. Here, BM of the posterior fossa might be a peculiarity as their space-occu- pying effect often leads to early symptoms, and yet they are well-resectable. Even though BM location is not included in commonly used methods of predicting patient outcomes, cli- nicians still consider it important and often make decisions about local treatment based on where the BM is located [6]. According to published studies, some researchers strongly 1 Department of Neurosurgery, University Hospital Bonn, Venusberg‑Campus 1, 53127 Bonn, Germany (0121 3456789) 3 80 Page 2 of 7 Neurosurgical Review (2023) 46:80 80 Page 2 of 7 80 P hemorrhage, while HACs included screening for pneumo- nia, fall injuries, and catheter-associated urinary tract infec- tions. Specific to cranial surgeries, complications such as cerebrospinal fluid leakage and postoperative seizures, were classified as cranial-surgery-related complications (CSCs) as previously described [12]. Any intra- or postoperative adverse events that occurred within 30 days of the initial resection, with or without further surgical interventions, were considered perioperative complications. believe that BMs located in the posterior fossa are associated with worse prognosis and can lead to neurological disability through brainstem damage, hydrocephalus, and herniation of the cerebellum [7]. Therefore, the present study is intended to focus on the prognostic influence of the location in the posterior fossa and to provide clinical data for this important entity. Results To evaluate perioperative complication profiles in patients undergoing BM resection, a list of adverse events known as patient safety indicators (PSIs) and hospital-acquired conditions (HACs) was used as previously described [11]. These events were established by the Agency for Health- care Research and Quality and the Center for Medicare and Medicaid Services. The PSIs included occurrences such as pressure ulcers, transfusion reactions, and postoperative Statistical analysis Data analyses were performed using the SPSS computer software package (version 27, IBM Corp., Armonk, NY). Categorical variables were analyzed in contingency tables using the Fisher’s exact test in case of only two variables. If more than two variables had to be analyzed, the chi-square test was applied. The Mann-Whitney U-test was chosen to compare continuous variables as data were mostly not nor- mally distributed. OS was analyzed by the Kaplan-Meier method. The Gehan-Breslow-Wilcoxon test was used to compare survival rates in case of supratentorial and infraten- torial BMs. Results with p < 0.05 were considered statisti- cally significant. Matching procedure After BM resection, patients are transferred to their transferring hospital in order to conduct postoperative oncological treatment [10].i 1 3 Patient and tumor characteristics Between 01/2013 and 01/2019, 395 patients had undergone resection of BM at the neuro-oncological center of the Uni- versity Hospital Bonn. Thirty-seven patients were excluded from further analysis due to the lack of sufficient follow- up information. One hundred thirteen of the remaining 358 1 3 1 3 Page 3 of 7 80 Neurosurgical Review (2023) 46:80 Table 1 Baseline characteristics (values represent number of patients unless indicated otherwise (%)) BM, brain metastasis; CCI, Charlson Comorbidity Index; CI, con- fidence interval; IQR, interquartile range; KPS, Karnofsky Perfor- mance Score; mo, months; min, minutes; mOS, median overall sur- vival; NSCLC, non-small cell lung carcinoma; OP, operation; SD, standard deviation; yrs, years n = 245 Mean age (yrs, ± SD) 64 ± 12 Female sex 122 (50) Primary site of cancer NSCLC 100 (41) Breast 29 (12) Melanoma 28 (11) Others 88 (36) Preoperative KPS (IQR) 80 (70–90) Preoperative median CCI (IQR) 11 (10–12) Median OP duration (min, IQR) 165 (140–210) Infratentorial BM location 61 (25) Supratentorial BM location 184 (75) 1-Year mortality 126 (51) mOS (mo, 95% CI) 15 (11.4-18.6) patients (32%) revealed multiple BM at admission, and 245 of 358 patients (68%) suffered from solitary BM. Mean age of the patients with solitary BM was 64 years (SD +/- 12 years) with 122 female (50%) and 123 male patients (50%). Most commonly BM originated from lung cancer (n = 100, 41%), followed by breast cancer (n = 29, 12%) and melanoma (n = 28, 11%). Median preoperative KPS for the entire patient cohort was 80 (IQR 70–90). Sixty-one of 245 patients (25%) suffered from infratentorial BM; 184 of 245 patients (75%) exhibited supratentorial BM. One hundred twenty-six patients (51%) died within 1 year after BM resec- tion. mOS for the entire study cohort was 13 months (95% CI 10.4–16.6). Further details are given in Table 1. Table 1 Baseline characteristics (values represent number of patients unless indicated otherwise (%)) Table 2 Comparative matched pair analysis dependent on supra- and infratentorial BM location (values represent number of patients unless indicated otherwise (%)) Comparative matched pair survival analysis for infra‑ and supratentorial BM location Heat map as color-coded illustration of the matching strategy of patients with supratentorial BM to individually matched infratentorial cases by means of age at admission, KPS at admission, CCI at admis- sion, and tumor entity as matching parameters. B Love plot depict- ing the balance of the matching analysis for each matching parameter determined by the standardized mean differences. BM, brain metasta- sis; CCI, Charlson Comorbidity Index; KPS, Karnofsky Performance Score Fig. 1 Illustration of the matching procedure for patients with surgi- cally treated BM dependent on infra- versus supratentorial location of the BM. A Comparative matched pair analysis at a ratio of 1:1 identi- fies 61 out of 184 patients with supratentorial BM that individually correspond to the present series of 61 patients with infratentorial BM. Heat map as color-coded illustration of the matching strategy of patients with supratentorial BM to individually matched infratentorial cases by means of age at admission, KPS at admission, CCI at admis- sion, and tumor entity as matching parameters. B Love plot depict- ing the balance of the matching analysis for each matching parameter determined by the standardized mean differences. BM, brain metasta- sis; CCI, Charlson Comorbidity Index; KPS, Karnofsky Performance Score Fig. 2 Survival rates of patients with supratentorial and infratentorial BMs do not differ. Kaplan-Meier curves for OS of patients with surgi- cally treated BM stratified into supra- versus infratentorial location of the resected BM. BM, brain metastasis; OS, overall survival patients with surgically treated BM dependent on infra- and supratentorial tumor location, a multivariate and propensity score matching with additional balance optimization was per- formed. Patients with infratentorial BM location were indi- vidually matched at a ratio of 1:1 to a cohort of 184 patients with supratentorial BM location that had undergone resection of BM between 2013 and 2018 at our neuro-oncological center (Fig. 1). Patient age, preoperative KPS, preoperative CCI, and primary site of cancer as known prognostic parameters for sur- vival in patients with BM were chosen as matching variables. The matched-pair analysis yielded two individually matched cohorts of 61 patients with infratentorial and 61 patients with supratentorial solitary BM that did not significantly differ with regard to abovementioned prognostic survival param- eters (Table 2). Fifty-two of 61 patients with supratentorial BM (85%) received adjuvant radiation therapy compared to 56 of 61 patients with infratentorial BM (92%) (p = 0.39). Comparative matched pair survival analysis for infra‑ and supratentorial BM location Comparative analysis of age, sex, preoperative KPS, and CCI as well as the presence of extracranial metastasis at the time of BM diagnosis revealed a homogeneous distribution between the groups of synchronous and metachronous BM occur- rence (Table 2, Fig. 1). In order to compare survival rates of BM, brain metastasis; CCI, Charlson Comorbidity Index; CI, con- fidence interval; IQR, interquartile range; KPS, Karnofsky Perfor- mance Score; mo, months; min, minutes; mOS, median overall sur- vival; NSCLC, non-small cell lung carcinoma; OP, operation; SD, standard deviation; yrs, years ASA, American society of anesthesiologists; BM, brain metastasis; CCI, Charlson Comorbidity Index; CI, confidence interval; CSCs, cranial surgery-related complications; HACs, hospital-acquired conditions; IQR, interquartile range; KPS, Karnofsky Performance Score; mo, months; min, minutes; mOS, median overall survival; NSCLC, non-small cell lung carcinoma; OP, operation; PSIs, patient safety indicators; SD, standard deviation; yrs, years Suptratentorial location n = 61 Infratentorial location n = 61 p Value Matching variables Median age (yrs, ± SD) 67 ± 10 66 ± 12 0.66 Primary site of cancer NSCLC 28 (46) 25 (41) 0.59 Breast 5 (8) 11 (18) 0.11 Melanoma 8 (13) 4 (7) 0.22 Others 20 (33) 21 (34) 0.85 Median KPS (IQR) 80 (70–90) 80 (70–90) 0.82 Median CCI (IQR) 11 (10–12) 10 (10–11) 0.51 Outcome variables Female sex 25 (41) 31 (51) 0.37 ASA ≥ 3 40 (66) 38 (62) 0.85 Median OP duration (min, IQR) 177 (143–214) 166 (146–200) 0.3 Perioperative complications 0.95 HACs 1 (2) 1 (2) 1.0 PSIs 1 (2) 1 (2) 1.0 CSCs 1 (2) 2 (3) 0.56 Adjuvant treatment Radiation therapy 52 (85) 56 (92) 0.39 Chemo-/immunotherapy 49 (80) 45 (74) 0.51 1-Year mortality 37 (61) 39 (64) 0.71 mOS (mo, 95% CI) 13 (10.9–15.1) 11 (7.4–14.6) 0.32 Table 2 Comparative matched pair analysis dependent on supra- and infratentorial BM location (values represent number of patients unless indicated otherwise (%)) 1 3 1 3 80 Page 4 of 7 Neurosurgical Review (2023) 46:80 Fig. 1 Illustration of the matching procedure for patients with surgi- cally treated BM dependent on infra- versus supratentorial location of the BM. A Comparative matched pair analysis at a ratio of 1:1 identi- fies 61 out of 184 patients with supratentorial BM that individually correspond to the present series of 61 patients with infratentorial BM. Comparative matched pair survival analysis for infra‑ and supratentorial BM location The respective data for postoperative chemo- and/or immunother- apy were 49 of 61 patients for supratentorial (80%) and 45 of 61 patients with infratentorial BM (74%) (p = 0.51) (Table 2). One-year mortality did not significantly differ between these groups (39 patients infratentorial vs. 37 patients supratentorial, p = 0.71) (Table 2). Similarly, mOS did not significantly dif- fer between the two patient groups of differing BM locations (infratentorial 11 months (95% CI 7.4–14.6) vs. supratentorial 13 months (95% CI 10.9–15.1), p = 0.32) (Table 2, Fig. 2). Fig. 2 Survival rates of patients with supratentorial and infratentorial BMs do not differ. Kaplan-Meier curves for OS of patients with surgi- cally treated BM stratified into supra- versus infratentorial location of the resected BM. BM, brain metastasis; OS, overall survival known prognostic parameters in surgery for BM, a pro- pensity score matching between patients with supra- and infratentorial BM location showed that there were no sig- nificant differences in survival between these two groups. if The mOS for the entire study cohort in the current study was 15 months. For survival comparison with other studies on patients with BM, it is important to note that the patient cohort in the present study only included patients who had undergone resection surgery. This concept is illustrated in a study of 708 patients, where the location of cerebellar brain metastases (BM) was initially associated with poorer sur- vival when all patients were included in the analysis [13]. However, when the analysis was restricted to patients with Discussion These results sug- gest that the location of the tumor—infra- versus supraten- torial (except brainstem lesions)—may not have an impact on patient outcomes, although more research is needed to fully understand the relationship between tumor location and patient outcomes. Further, it has to be mentioned that in addition to OS, local tumor control is an increasingly impor- tant outcome measure following surgery for BM [21]. With In summary, the location of BM (supratentorial or infratentorial) has been a topic of research for decades, but there are conflicting results from various studies. It is impor- tant to note that infratentorial location refers to both the brainstem and cerebellum, which have different outcomes in terms of the accessibility to surgically remove the tumor. Similarly, it is challenging to accurately interpret studies that include patients with differing preoperative patient- and disease-related conditions which in turn do not allow to specifically analyze the impact of tumor location. Here, the authors intend to provide a homogenized cohort of patients based on a comparative matched pair analysis for important patient- and disease-related prognostic factors in BM sur- gery. Further multicenter analyses are needed to fully under- stand the relationship between tumor location and patient outcomes in different populations and settings. Discussion This study included a relatively large and homo- geneous population of patients. The researchers found that overall survival was significantly worse for patients with brainstem tumors when compared to those with supraten- torial or cerebellar tumors [20]. However, in line with the results of the present analysis, where brainstem lesions were excluded from analysis, there was no significant difference in overall survival between patients with cerebellar tumors and those with supratentorial tumors [20]. These results sug- gest that the location of the tumor—infra- versus supraten- torial (except brainstem lesions)—may not have an impact regard to radiation therapy and systemic therapies that have shown significant promise in controlling systemic disease and improving survival rates, local tumor control in the brain after neurosurgcial resection has become a crucial factor in maintaining the patients’ neurological and functional status [22]. Achieving complete resection of tumors and prevent- ing local tumor recurrence can not only maintain or even improve neurological function and quality of life, but can also enhance the efficacy of systemic therapies by reducing the tumor burden in the CNS [22]. Therefore, in addition to OS as a main outcome measure as presented in the present study, follow-up studies might additionally consider local tumor control as an important outcome measure in surgery for BM, particularly in the era of evolving systemic onco- logical therapies. In addition, focal radiation therapy which is the most recommended adjuvant treatment after the sur- gical removal of BM might also contribute in the field of outcome measure dependent on tumor localization. This approach is favored over whole-brain radiation therapy due to its outstanding control of the disease and low side effect profile [9] and available data indicate that radiation therapy provides relief from symptoms in a comparable manner for supra and infratentorial BM localization [23]. resection surgery, there was no significant difference in sur- vival between patients with cerebellar BMs and those with BMs in other locations [13]. Further, as multiple BM are known to be an important negative prognostic predictor of survival following BM surgery [3, 14], the current study focused specifically on solitary BM. Discussion In comparison to sev- eral studies suggesting infratentorial BM location to be asso- ciated with worsened survival outcome parameters [15, 16], the present study made use of a propensity score matching in order to ensure a homogeneous distribution of age [17], tumor entity [14], preoperative KPS [14, 18] and comorbid- ity burden [3] as known important prognostic parameters in patients with BM. One further factor that may contribute to the variability in research findings on the prognostic value of infra- versus supratentorial BM location might be reasoned in undifferentiated analyses of both cerebellar and brainstem lesions. While brainstem tumors are often not able to be sur- gically removed and the dose of radiation used to treat them may be limited due to the risk of side effects, tumors located in the cerebellum are often surgically removable and can be effectively treated with either postoperative radiation or definitive radiosurgery [6]. When studies combine brainstem and cerebellar tumors into a single category (infratentorial location), it can be challenging to accurately interpret the results and understand the specific impact of tumor location on patient outcomes. In a study by Trifiletti et al., the sur- vival outcomes of patients with brainstem tumors who were treated with stereotactic radiosurgery (SRS) were compared to a group of patients with non-brainstem metastases who were also treated with SRS [19]. The authors found that the median survival of patients with brainstem tumors was significantly shorter than that of the comparator group. In a subsequent study, Emery et al. analyzed the outcomes of 817 patients with BMs based on their location in the brain (supratentorial, brainstem, or cerebellum) [20]. All of the patients in this study received SRS treatment, and a small number (9%) had previously undergone surgery to remove the tumor. This study included a relatively large and homo- geneous population of patients. The researchers found that overall survival was significantly worse for patients with brainstem tumors when compared to those with supraten- torial or cerebellar tumors [20]. However, in line with the results of the present analysis, where brainstem lesions were excluded from analysis, there was no significant difference in overall survival between patients with cerebellar tumors and those with supratentorial tumors [20]. Discussion The current study examined survival outcomes in patients who had undergone surgery for BM, either in the supra- or infratentorial region. Applying a covariate balance for 1 3 1 3 Neurosurgical Review (2023) 46:80 Page 5 of 7 80 Page 5 of 7 80 80 resection surgery, there was no significant difference in sur- vival between patients with cerebellar BMs and those with BMs in other locations [13]. Further, as multiple BM are known to be an important negative prognostic predictor of survival following BM surgery [3, 14], the current study focused specifically on solitary BM. In comparison to sev- eral studies suggesting infratentorial BM location to be asso- ciated with worsened survival outcome parameters [15, 16], the present study made use of a propensity score matching in order to ensure a homogeneous distribution of age [17], tumor entity [14], preoperative KPS [14, 18] and comorbid- ity burden [3] as known important prognostic parameters in patients with BM. One further factor that may contribute to the variability in research findings on the prognostic value of infra- versus supratentorial BM location might be reasoned in undifferentiated analyses of both cerebellar and brainstem lesions. While brainstem tumors are often not able to be sur- gically removed and the dose of radiation used to treat them may be limited due to the risk of side effects, tumors located in the cerebellum are often surgically removable and can be effectively treated with either postoperative radiation or definitive radiosurgery [6]. When studies combine brainstem and cerebellar tumors into a single category (infratentorial location), it can be challenging to accurately interpret the results and understand the specific impact of tumor location on patient outcomes. In a study by Trifiletti et al., the sur- vival outcomes of patients with brainstem tumors who were treated with stereotactic radiosurgery (SRS) were compared to a group of patients with non-brainstem metastases who were also treated with SRS [19]. The authors found that the median survival of patients with brainstem tumors was significantly shorter than that of the comparator group. In a subsequent study, Emery et al. analyzed the outcomes of 817 patients with BMs based on their location in the brain (supratentorial, brainstem, or cerebellum) [20]. All of the patients in this study received SRS treatment, and a small number (9%) had previously undergone surgery to remove the tumor. References 1. Boire A, Brastianos PK, Garzia L, Valiente M (2020) Brain metas- tasis. Nature reviews. Cancer 20:4–11. https://doi.org/10.1038/ s41568-019-0220-y 2. Barnholtz-Sloan JS, Sloan AE, Davis FG, Vigneau FD, Lai P, Sawaya RE (2004) Incidence proportions of brain metastases in patients diagnosed (1973 to 2001) in the Metropolitan Detroit Cancer Surveillance System. J Clin Oncol 22:2865–2872. https:// doi.org/10.1200/JCO.2004.12.149f 3. Schneider M, Heimann M, Schaub C, Eichhorn L, Potthoff AL, Giordano FA, Guresir E, Ko YD, Landsberg J, Lehmann F, Radbruch A, Schwab KS, Weinhold L, Weller J, Wispel C, Her- rlinger U, Vatter H, Schafer N, Schuss P (2020) Comorbidity bur- den and presence of multiple intracranial lesions are associated with adverse events after surgical treatment of patients with brain metastases. Cancers 12. https://doi.org/10.3390/cancers12113209fi Conclusions The present study suggests that the prognostic value of infra- and supratentorial BMs does not significantly differ in patients that undergo surgery for solitary BM. These results might encourage physicians to induce surgical therapy of supra- and infratentorial BM in a similar manner. 4. Achrol AS, Rennert RC, Anders C, Soffietti R, Ahluwalia MS, Nayak L, Peters S, Arvold ND, Harsh GR, Steeg PS, Chang SD (2019) Brain metastases. Nat rev Dis prim 5:5. https://doi.org/10. 1038/s41572-018-0055-y 5. Proescholdt MA, Schodel P, Doenitz C, Pukrop T, Hohne J, Schmidt NO, Schebesch KM (2021) The management of brain metastases-systematic review of neurosurgical aspects. Cancers 13. https://doi.org/10.3390/cancers13071616 Authors’ contribution Conceptualization: M.H. and M.S. Data cura- tion: A.-L.P. and M.S. Formal analysis and methodology: M.H., A.- L.P., N.S., and M.S. Writing-original draft preparation: M.H., A.-L.P., and M.S. Writing-review and editing: M.H., A.-L.P., M.H., A.R., N.S., V.B., U.H., H.V., and M.S. Visualization: A-L.P. and M.S. Supervision: M.H., U.H., and M.S. Authors’ contribution Conceptualization: M.H. and M.S. Data cura- tion: A.-L.P. and M.S. Formal analysis and methodology: M.H., A.- L.P., N.S., and M.S. Writing-original draft preparation: M.H., A.-L.P., and M.S. Writing-review and editing: M.H., A.-L.P., M.H., A.R., N.S., V.B., U.H., H.V., and M.S. Visualization: A-L.P. and M.S. Supervision: M.H., U.H., and M.S. 6. Kancharla P, Ivanov A, Chan S, Ashamalla H, Huang RY, Yanagi- hara TK (2019) The effect of brain metastasis location on clinical outcomes: a review of the literature. Neuro-Oncol Adv 1:vdz017. https://doi.org/10.1093/noajnl/vdz017 7. Yoshida S, Takahashi H (2009) Cerebellar metastases in patients with cancer. Surg Neurol 71:184–187; discussion 187. https://doi. org/10.1016/j.surneu.2007.10.010 Funding Open Access funding enabled and organized by Projekt DEAL. Funding Open Access funding enabled and organized by Projekt DEAL. 8. Verger E, Salamero M, Conill C (1992) Can Karnofsky perfor- mance status be transformed to the Eastern Cooperative Oncology Group scoring scale and vice versa? Eur J Cancer 28A:1328– 1330. https://doi.org/10.1016/0959-8049(92)90510-9 Data availability Restrictions apply to the availability of these data due to privacy restrictions. Data availability Restrictions apply to the availability of these data due to privacy restrictions. 9. Mantovani C, Gastino A, Cerrato M, Badellino S, Ricardi U, Levis M (2021) Modern radiation therapy for the management of brain metastases from non-small cell lung cancer: current approaches and future directions. Front Oncol 11:772789. https://doi.org/10. 3389/fonc.2021.772789 Limitations The present study weakens from several other limitations in addition to the design of a retrospective analysis. First, avail- able data reflect the experience of a single neuro-oncological specialized center only. Also, a possible selection bias must be cautioned in regard to the small sample size. Further- more, this analysis was only neurosurgically driven and did not consider subgroup analysis regarding tumor types, course of disease, local irradiation, or systemic treatment. Indication for surgery for this consecutive group of patients surgically treated BM was made within a weekly interdisci- plinary tumor board meeting. It is important to notice that 1 1 3 3 Page 6 of 7 Neurosurgical Review (2023) 46:80 80 need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. within these meetings patients were from various medi- cal centers. After having received surgical BM resection, patients were transferred to the original hospital in order to conduct postoperative oncological treatment. Against this backdrop, outcome assessment in terms of local tumor con- trol rates and “neurological death” instead of death due to systemic disease burden was beyond the scope of this retro- spective analysis. Nevertheless, these data represent inter- disciplinary treatment decisions and might provide a more homogeneous patient population due to the aforementioned propensity score matching for known prognostic predictors in patients with surgically treated BM. Declarations Ethics approval The present study was approved by the local ethics committee at the University of Bonn. Ethics approval The present study was approved by the local ethics committee at the University of Bonn. 10. Schafer N, Bumes E, Eberle F, Fox V, Gessler F, Giordano FA, Konczalla J, Onken J, Ottenhausen M, Scherer M, Schneider M, Vatter H, Herrlinger U, Schuss P (2021) Implementation, relevance, and virtual adaptation of neuro-oncological tumor boards during the COVID-19 pandemic: a nationwide provider survey. J Neuro-Oncol 153:479–485. https://doi.org/10.1007/ s11060-021-03784-wf Consent to participate Informed consent was not sought as a retrospec- tive study design was used. Consent to participate Informed consent was not sought as a retrospec- tive study design was used. Consent for publication All authors agreed to the publication of the manuscript. Consent for publication All authors agreed to the publication of the manuscript. Consent for publication All authors agreed to the publication of the manuscript. Competing interests The authors declare no competing interests. Competing interests The authors declare no competing interests. Competing interests The authors declare no competing interests. Competing interests The authors declare no competing interests. 11. Hamed M, Potthoff AL, Layer JP, Koch D, Borger V, Heimann M, Scafa D, Sarria GR, Holz JA, Schmeel FC, Radbruch A, Guresir E, Schafer N, Schuss P, Garbe S, Giordano FA, Herrlinger U, Vatter H, Schmeel LC, Schneider M (2022) Benchmarking safety indicators of surgical treatment of brain metastases combined with intraoperative radiotherapy: results of prospective observational study with comparative matched-pair analysis. Cancers 14. https:// doi.org/10.3390/cancers14061515f Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will g 12. Schneider M, Ilic I, Potthoff AL, Hamed M, Schafer N, Velten M, Guresir E, Herrlinger U, Borger V, Vatter H, Schuss P (2020) Safety metric profiling in surgery for temporal glioblas- toma: lobectomy as a supra-total resection regime preserves 12. Schneider M, Ilic I, Potthoff AL, Hamed M, Schafer N, Velten M, Guresir E, Herrlinger U, Borger V, Vatter H, Schuss P (2020) Safety metric profiling in surgery for temporal glioblas- toma: lobectomy as a supra-total resection regime preserves 1 3 3 Neurosurgical Review (2023) 46:80 Page 7 of 7 80 perioperative standard quality rates. J Neuro-Oncol 149:455–461. https://doi.org/10.1007/s11060-020-03629-y brain-specific progression-free survival among patients with meta- static breast cancer with brain metastases. Front Oncol 12:867462. https://doi.org/10.3389/fonc.2022.867462i 13. Chaichana KL, Rao K, Gadkaree S, Dangelmajer S, Bettegowda C, Rigamonti D, Weingart J, Olivi A, Gallia GL, Brem H, Lim M, Quinones-Hinojosa A (2014) Factors associated with survival and recurrence for patients undergoing surgery of cerebellar metasta- ses. Neurol Res 36:13–25. https://doi.org/10.1179/1743132813Y. 0000000260 19. p g 19. Competing interests The authors declare no competing interests. Trifiletti DM, Lee CC, Kano H, Cohen J, Janopaul-Naylor J, Alonso-Basanta M, Lee JYK, Simonova G, Liscak R, Wolf A, Kvint S, Grills IS, Johnson M, Liu KD, Lin CJ, Mathieu D, Her- oux F, Silva D, Sharma M et al (2016) Stereotactic radiosurgery for brainstem metastases: an international cooperative study to define response and toxicity. Int J Radiat Oncol Biol Phys 96:280– 288. https://doi.org/10.1016/j.ijrobp.2016.06.009 14. Sperduto PW, Mesko S, Li J, Cagney D, Aizer A, Lin NU, Nesbit E, Kruser TJ, Chan J, Braunstein S, Lee J, Kirkpatrick JP, Breen W, Brown PD, Shi D, Shih HA, Soliman H, Sahgal A, Shanley R et al (2020) Survival in patients with brain metastases: sum- mary report on the updated diagnosis-specific graded prognostic assessment and definition of the eligibility quotient. J Clin Oncol 38:3773–3784. https://doi.org/10.1200/JCO.20.01255 20. Burks C, Hong S, Ho M, Engelman DM (1989) Limitations of the lipid state hypothesis for atherosclerosis are revealed by X-ray diffraction measurements. Atherosclerosis 77:43–51. https://doi. org/10.1016/0021-9150(89)90008-7 g 21. Hughes RT, Black PJ, Page BR, Lucas JT Jr, Qasem SA, Watabe K, Ruiz J, Laxton AW, Tatter SB, Debinski W, Chan MD (2016) Local control of brain metastases after stereotactic radiosurgery: the impact of whole brain radiotherapy and treatment paradigm. Journal Radiosur SBRT 4:89–96 p g 15. Dou Z, Wu J, Wu H, Yu Q, Yan F, Jiang B, Li B, Xu J, Xie Q, Li C, Sun C, Chen G (2021) The infratentorial localization of brain metastases may correlate with specific clinical characteristics and portend worse outcomes based on voxel-wise mapping. Cancers 13. https://doi.org/10.3390/cancers13020324 22. Suh JH, Kotecha R, Chao ST, Ahluwalia MS, Sahgal A, Chang EL (2020) Current approaches to the management of brain metas- tases. Nat Rev Clin Oncol 17:279–299. https://doi.org/10.1038/ s41571-019-0320-3 16. Pietrantonio F, Aprile G, Rimassa L, Franco P, Lonardi S, Cremo- lini C, Biondani P, Sbicego EL, Pasqualetti F, Tomasello G, Niger M, Casagrande M, Ghidini M, Muni R, Montrone S, Bergamo F, Berenato R, Fontanella C, Bozzarelli S et al (2015) A new nomo- gram for estimating survival in patients with brain metastases secondary to colorectal cancer. Radiother Oncol 117:315–321. https://doi.org/10.1016/j.radonc.2015.08.023 23. Cacho-Diaz B, Alvarez-Alvarez A, Salmeron-Moreno K, Rodri- guez-Mayoral O, Santiago-Concha BG, Arrieta Rodriguez OG (2021) Role of whole brain radiotherapy in the management of infratentorial metastases from lung and breast cancer. Rep Pract Oncol Radiother 26:512–517. https://doi.org/10.5603/RPOR. a2021.0060 17. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Competing interests The authors declare no competing interests. Heimann M, Schafer N, Bode C, Borger V, Eichhorn L, Giordano FA, Guresir E, Jacobs AH, Ko YD, Landsberg J, Lehmann F, Radbruch A, Schaub C, Schwab KS, Weller J, Herrlinger U, Vat- ter H, Schuss P, Schneider M (2021) Outcome of elderly patients with surgically treated brain metastases. Front Oncol 11:713965. https://doi.org/10.3389/fonc.2021.713965 Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 18. Freeman M, Ennis M, Jerzak KJ (2022) Karnofsky Perfor- mance Status (KPS) </=60 is strongly associated with shorter 1 3 1 3 1 3
https://openalex.org/W4237386327	https://peerj.com/articles/8314v0.5/submission	English	null	Peer Review #3 of "Relative contribution of ecological and biological attributes in the fine-grain structure of ant-plant networks (v0.2)"	null	2,020	cc-by	22,729	Relative contribution of ecological and biological attributes in the ﬁne-grain structure of ant-plant networks Cecilia Díaz-Castelazo Corresp., 1 , Cristian A. Martínez-Adriano 1, 2 , Wesley Dáttilo 3 , Victor Rico-Gray 4 1 Red de Interacciones Multitróﬁcas, Instituto de Ecología, A.C., Xalapa, Veracruz, México 2 Facultad de Ciencias Forestales, Universidad Autónoma de Nuevo León, Linares, Nuevo León, México 3 Red de Ecoetología, Instituto de Ecología, A.C., Xalapa, Veracruz, México 4 Instituto de Neuroetología, Universidad Veracruzana, Xalapa, Veracruz, México Corresponding Author: Cecilia Díaz-Castelazo Email address: diazcastelazogm@gmail.com 1 Red de Interacciones Multitróﬁcas, Instituto de Ecología, A.C., Xalapa, Veracruz, México 2 Facultad de Ciencias Forestales, Universidad Autónoma de Nuevo León, Linares, Nuevo León, México 3 Red de Ecoetología, Instituto de Ecología, A.C., Xalapa, Veracruz, México 4 Instituto de Neuroetología, Universidad Veracruzana, Xalapa, Veracruz, México Corresponding Author: Cecilia Díaz-Castelazo Email address: diazcastelazogm@gmail.com 1 Red de Interacciones Multitróﬁcas, Instituto de Ecología, A.C., Xalapa, Veracruz, México 2 Facultad de Ciencias Forestales, Universidad Autónoma de Nuevo León, Linares, Nuevo León, México 3 Red de Ecoetología, Instituto de Ecología, A.C., Xalapa, Veracruz, México 4 Instituto de Neuroetología, Universidad Veracruzana, Xalapa, Veracruz, México Corresponding Author: Cecilia Díaz-Castelazo Email address: diazcastelazogm@gmail.com Background. Ecological communities of interacting species analyzed as complex networks have shown that species dependence on their counterparts is more complex than expected at random. As for other potentially mutualistic interactions, ant-plant networks mediated by extraﬂoral nectar show a nested (asymmetric) structure with a core of generalist species dominating the interaction pattern. Proposed factors structuring ecological networks include encounter probability (e.g. species abundances and habitat heterogeneity), behavior, phylogeny, and body size. While the importance of underlying factors that inﬂuence the structure of ant-plant networks have been separately explored, the simultaneous contribution of several biological and ecological attributes inherent to the species, guild or habitat level has not been addressed. Methods. For a tropical seasonal site we recorded (in 48 censuses) the frequency of pairwise ant-plant interactions mediated by extraﬂoral nectaries (EFN) on diﬀerent habitats and studied the resultant network structure. We addressed for the ﬁrst time the role of mechanistic versus neutral determinants at the ‘ﬁne-grain’ structure (pairwise interactions) of ant-plant networks. We explore the simultaneous contribution of several attributes of plant and ant species (i.e. EFN abundance and distribution, ant head length, behavioral dominance and invasive status), and habitat attributes (i.e. vegetation structure) in prevailing interactions as well as in overall network topology (community). Results. Our studied network was highly- nested and non-modular, with core species having high species strengths (higher strength values for ants than plants) and low specialization. Plants had higher dependences on ants than vice versa. Manuscript to be reviewed PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Relative contribution of ecological and biological attributes in the ﬁne-grain structure of ant-plant networks Cecilia Díaz-Castelazo Corresp., 1 , Cristian A. Martínez-Adriano 1, 2 , Wesley Dáttilo 3 , Victor Rico-Gray 4 We found that habitat heterogeneity in vegetation structure (open vs. shaded habitats) was the main factor explaining network and ﬁne-grain structure, with no evidence of neutral (abundance) eﬀects. Discussion. Core ant species are relevant to most plants species at the network showing adaptations to nectar consumption and PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed Manuscript to be reviewed deterrent behavior. Thus larger ants interact with more plant species which, together with higher dependence of plants on ants, suggests potential biotic defense at a community scale. In our study site, heterogeneity in the ant-plant interactions among habitats is so prevalent that it emerges at community-level structural properties. High frequency of morphologically diverse and temporarily-active EFNs in all habitats suggests the relevance and seasonality of plant biotic defense provided by ants. The robust survey of ecological interactions and their biological/ecological correlates that we addressed provides insight of the interplay between adaptive-value traits and neutral eﬀects in ecological networks. deterrent behavior. Thus larger ants interact with more plant species which, together with higher dependence of plants on ants, suggests potential biotic defense at a community scale. In our study site, heterogeneity in the ant-plant interactions among habitats is so prevalent that it emerges at community-level structural properties. High frequency of morphologically diverse and temporarily-active EFNs in all habitats suggests the relevance and seasonality of plant biotic defense provided by ants. The robust survey of ecological interactions and their biological/ecological correlates that we addressed provides insight of the interplay between adaptive-value traits and neutral eﬀects in ecological networks. PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed Manuscript to be reviewed 1 Title: Relative contribution of ecological and biological attributes in the fine-grain structure 2 of ant-plant networks 3 4 Authors: Cecilia Díaz-Castelazo1, Cristian Adrian Martínez-Adriano1,2, Wesley Dáttilo3, and 5 Víctor Rico-Gray4 6 7 8 Affiliations: 1Red de Interacciones Multitróficas, Instituto de Ecología, A.C., Xalapa, Veracruz 9 91073, México. 2Facultad de Ciencias Forestales, Universidad Autónoma de Nuevo León, Linares, 10 Nuevo León, México. 3Red de Ecoetología, Instituto de Ecología, A.C. Xalapa, Veracruz 91073, 11 México. 4Instituto de Neuroetología, Universidad Veracruzana, Xalapa, Veracruz 91190, México. 12 13 Corresponding author: Cecilia Díaz-Castelazo. Carretera Antigua a Coatepec, No. 351, El Haya, 14 Xalapa, 91073, Veracruz, México. diazcastelazogm@gmail.com, cecilia.diaz@inecol.mx 15 Manuscript to be reviewed 16 Abstract 17 Background. Ecological communities of interacting species analyzed as complex networks have 18 shown that species dependence on their counterparts is more complex than expected at random. 19 As for other potentially mutualistic interactions, ant-plant networks mediated by extrafloral 20 nectar show a nested (asymmetric) structure with a core of generalist species dominating the 21 interaction pattern. Proposed factors structuring ecological networks include encounter 22 probability (e.g. species abundances and habitat heterogeneity), behavior, phylogeny, and body 23 size. While the importance of underlying factors that influence the structure of ant-plant 24 networks have been separately explored, the simultaneous contribution of several biological and 25 ecological attributes inherent to the species, guild or habitat level has not been addressed. 26 Methods. For a tropical seasonal site we recorded (in 48 censuses) the frequency of pairwise ant- 27 plant interactions mediated by extrafloral nectaries (EFN) on different habitats and studied the 28 resultant network structure. We addressed for the first time the role of mechanistic versus neutral 29 determinants at the ‘fine-grain’ structure (pairwise interactions) of ant-plant networks. We explore 30 the simultaneous contribution of several attributes of plant and ant species (i.e. EFN abundance 31 and distribution, ant head length, behavioral dominance and invasive status), and habitat attributes 32 (i.e. vegetation structure) in prevailing interactions as well as in overall network topology 33 (community). 16 Abstract 17 Background. Ecological communities of interacting species analyzed as complex networks have 18 shown that species dependence on their counterparts is more complex than expected at random. 19 As for other potentially mutualistic interactions, ant-plant networks mediated by extrafloral 20 nectar show a nested (asymmetric) structure with a core of generalist species dominating the 21 interaction pattern. Proposed factors structuring ecological networks include encounter 22 probability (e.g. species abundances and habitat heterogeneity), behavior, phylogeny, and body 23 size. While the importance of underlying factors that influence the structure of ant-plant 24 networks have been separately explored, the simultaneous contribution of several biological and 25 ecological attributes inherent to the species, guild or habitat level has not been addressed. 34 Results. Our studied network was highly-nested and non-modular, with core species having high 35 species strengths (higher strength values for ants than plants) and low specialization. Plants had 36 higher dependences on ants than vice versa. We found that habitat heterogeneity in vegetation 37 structure (open vs. 1 Title: Relative contribution of ecological and biological attributes in the fine-grain structure 2 of ant-plant networks 13 Corresponding author: Cecilia Díaz-Castelazo. Carretera Antigua a Coatepec, No. 351, El Haya, 14 Xalapa, 91073, Veracruz, México. diazcastelazogm@gmail.com, cecilia.diaz@inecol.mx 15 15 15 PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed shaded habitats) was the main factor explaining network and fine-grain 38 structure, with no evidence of neutral (abundance) effects. 49 Introduction 50 The interactions among species occurring at a community have been studied recently with 51 a complex network perspective, where interacting species (i.e. plants and animals) are 52 graphically represented as nodes and their trophic interactions as links (Bascompte et al., 2003). 53 Such studies have paid important attention to network structure and its underlying factors, both 54 for mutualistic or antagonistic interactions (Bascompte & Jordano, 2007; Vázquez, Chacoff & 55 Cagnolo, 2009; Díaz-Castelazo et al., 2013; López-Carretero et al., 2014). Unraveling how 56 interactions among species are structured in communities or ecosystems is crucial for 57 understanding the ecological and evolutionary processes that support ecosystem function and 58 diversity (Herrera & Pellmyr, 2002). Furthermore, understanding the architecture of species 59 relationships may help predict how ecosystems respond either to abiotic or human-derived 60 changes (Bascompte, 2010). 50 The interactions among species occurring at a community have been studied recently with 51 a complex network perspective, where interacting species (i.e. plants and animals) are 52 graphically represented as nodes and their trophic interactions as links (Bascompte et al., 2003). 53 Such studies have paid important attention to network structure and its underlying factors, both 54 for mutualistic or antagonistic interactions (Bascompte & Jordano, 2007; Vázquez, Chacoff & 55 Cagnolo, 2009; Díaz-Castelazo et al., 2013; López-Carretero et al., 2014). Unraveling how 56 interactions among species are structured in communities or ecosystems is crucial for 57 understanding the ecological and evolutionary processes that support ecosystem function and 58 diversity (Herrera & Pellmyr, 2002). Furthermore, understanding the architecture of species 59 relationships may help predict how ecosystems respond either to abiotic or human-derived 60 changes (Bascompte, 2010). 61 Ecological network studies have shown that interactions among species are frequently 62 asymmetric and species dependence on their counterparts is more complex than expected at 63 random (Bascompte, Jordano & Olesen, 2006; Guimarães et al., 2007). For networks of 64 mutualistic interactions a nonrandom “nested” structure is frequently observed, where more 65 specialist species tend to interact with specific subsets of those species interacting with the more 66 generalist species (Almeida-Neto et al., 2008; Bascompte, 2010). Thus, nested structure implies 67 that interactions occur asymmetrically in a group of generalist species that comprise most 68 interactions in the network (network core) (Dáttilo et al., 2013b) and a group of specialist species 69 that maintain few interactions mostly or exclusively with generalist species. Manuscript to be reviewed 49 Introduction 50 The interactions among species occurring at a community have been studied recently with 51 a complex network perspective, where interacting species (i.e. plants and animals) are 52 graphically represented as nodes and their trophic interactions as links (Bascompte et al., 2003) 53 Such studies have paid important attention to network structure and its underlying factors, both 54 for mutualistic or antagonistic interactions (Bascompte & Jordano, 2007; Vázquez, Chacoff & 55 Cagnolo, 2009; Díaz-Castelazo et al., 2013; López-Carretero et al., 2014). Unraveling how 56 interactions among species are structured in communities or ecosystems is crucial for 57 understanding the ecological and evolutionary processes that support ecosystem function and 58 diversity (Herrera & Pellmyr, 2002). Furthermore, understanding the architecture of species 59 relationships may help predict how ecosystems respond either to abiotic or human-derived 60 changes (Bascompte, 2010). 61 Ecological network studies have shown that interactions among species are frequently 62 asymmetric and species dependence on their counterparts is more complex than expected at 63 random (Bascompte, Jordano & Olesen, 2006; Guimarães et al., 2007). For networks of 64 mutualistic interactions a nonrandom “nested” structure is frequently observed, where more 65 specialist species tend to interact with specific subsets of those species interacting with the mor 66 generalist species (Almeida-Neto et al., 2008; Bascompte, 2010). Thus, nested structure implie 67 that interactions occur asymmetrically in a group of generalist species that comprise most 68 interactions in the network (network core) (Dáttilo et al., 2013b) and a group of specialist speci 69 that maintain few interactions mostly or exclusively with generalist species. Such as a nested 70 architecture has been found to increase network robustness against loss of species (Memmott et 71 al., 2004; Bascompte et al., 2006) and to maximize the number of coexisting species supported Manuscript to be reviewed 39 Discussion. Core ant species are relevant to most plants species at the network showing 40 adaptations to nectar consumption and deterrent behavior. Thus larger ants interact with more plant 41 species which, together with higher dependence of plants on ants, suggests potential biotic defense 42 at a community scale. In our study site, heterogeneity in the ant-plant interactions among habitats 43 is so prevalent that it emerges at community-level structural properties. High frequency of 44 morphologically diverse and temporarily-active EFNs in all habitats suggests the relevance and 45 seasonality of plant biotic defense provided by ants. The robust survey of ecological interactions 46 and their biological/ecological correlates that we addressed provides insight of the interplay 47 between adaptive-value traits and neutral effects in ecological networks. 39 Discussion. Core ant species are relevant to most plants species at the network showing 40 adaptations to nectar consumption and deterrent behavior. Thus larger ants interact with more plant 41 species which, together with higher dependence of plants on ants, suggests potential biotic defense 42 at a community scale. In our study site, heterogeneity in the ant-plant interactions among habitats 43 is so prevalent that it emerges at community-level structural properties. High frequency of 44 morphologically diverse and temporarily-active EFNs in all habitats suggests the relevance and 45 seasonality of plant biotic defense provided by ants. The robust survey of ecological interactions 46 and their biological/ecological correlates that we addressed provides insight of the interplay 47 between adaptive-value traits and neutral effects in ecological networks. 48 PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) 49 Introduction Such as a nested 70 architecture has been found to increase network robustness against loss of species (Memmott et 71 al., 2004; Bascompte et al., 2006) and to maximize the number of coexisting species supported PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 72 by these networks (Bastolla et al., 2009; Thébault & Fontaine, 2010). A nested pattern of links in 73 mutualistic interaction networks could result from several ecological and evolutionary processes: 74 for instance, the complementarity and convergence of phenotypic traits between both sets of 75 interacting species (Thompson, 2005; Stang et al., 2006; Stang, 2007; Rezende, Bascompte & 76 Jordano, 2007). 72 by these networks (Bastolla et al., 2009; Thébault & Fontaine, 2010). A nested pattern of links in 73 mutualistic interaction networks could result from several ecological and evolutionary processes: 74 for instance, the complementarity and convergence of phenotypic traits between both sets of 75 interacting species (Thompson, 2005; Stang et al., 2006; Stang, 2007; Rezende, Bascompte & 76 Jordano, 2007). 77 Another nonrandom structural pattern in ecological interaction networks is the 78 ‘modularity’ or ‘compartmentalization’, which is characterized by a group of species interacting 79 more strongly among themselves than with other species or subsets in the network (Bascompte, 80 2010). The modular pattern is more frequently observed for networks of antagonistic interactions 81 (Cagnolo, Salvo & Valladares, 2011). Like nestedness in mutualistic networks, modularity is 82 thought to increase the persistence of species in antagonistic networks (Thébault & Fontaine, 83 2010). 84 Proposed mechanisms affecting overall network structure are diverse, including habitat 85 heterogeneity constraints (Pimm & Lawton, 1980; López-Carretero et al., 2014), phylogeny 86 (Rezende et al., 2007; Cagnolo, Salvo & Valladares, 2011), body size (Cohen et al., 2005; 87 Rezende, Bascompte & Jordano, 2007; Chamberlain & Holland, 2009;), encounter probability 88 based on natural abundance of species (Vázquez, Chacoff & Cagnolo, 2009; Blüthgen, 2010; 89 Dáttilo et al., 2014a), and variation in spatiotemporal co-occurrence (Rico-Gray et al., 2012; 90 Sánchez-Galván et al., 2012; Díaz-Castelazo et al., 2013; Junker et al., 2013; López-Carretero et 91 al., 2014). Environmental changes may alter network structure and thus, favor evolutionary 92 responses in opposing directions for different species (Guimarães, Jordano & Thompson, 2011). 93 The reorganization of network structure due to the indirect effects of coevolution, may explain PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 94 why and how mutualisms persist amid the turnover of species and interactions through space and 95 time (Guimarães et al., 2017). 96 Several aggregate network properties such as nestedness, connectance (the proportion of 97 realized interactions from all the ones possible given the number of species), and interaction 98 asymmetry (i.e. asymmetry of dependence of plants on animals and vice versa) may also emerge 99 due to properties inherent to communities (Jordano, Bascompte & Olesen, 2006). These causes 100 include the different abundances of species, community sampling biases (that affect the 101 detectability of some interactions), and the spatio-temporal overlap of species (i.e. co- 102 occurrence) (Vázquez et al., 2007, 2009; Blüthgen et al., 2008). 94 why and how mutualisms persist amid the turnover of species and interactions through space and 95 time (Guimarães et al., 2017). 94 why and how mutualisms persist amid the turnover of species and interactions through space and 95 time (Guimarães et al., 2017). 95 time (Guimarães et al., 2017). 96 Several aggregate network properties such as nestedness, connectance (the proportion of 97 realized interactions from all the ones possible given the number of species), and interaction 98 asymmetry (i.e. asymmetry of dependence of plants on animals and vice versa) may also emerge 99 due to properties inherent to communities (Jordano, Bascompte & Olesen, 2006). These causes 100 include the different abundances of species, community sampling biases (that affect the 101 detectability of some interactions), and the spatio-temporal overlap of species (i.e. co- 102 occurrence) (Vázquez et al., 2007, 2009; Blüthgen et al., 2008). 103 Although relative species abundance (Vázquez et al., 2009; Dáttilo et al., 2014a) and 104 spatio-temporal overlap –considered as ‘neutral’ causes of network structure– could explain 105 overall network structure, they fall short of predicting the frequency of pairwise interactions 106 (Vázquez et al., 2009; Poisot, Stouffer & Gravel, 2015). Indeed, the frequency of interactions 107 occurring for any given pair of species within the network can vary significantly even if the 108 overall network topology remains the same (Vázquez et al., 2005, 2007, 2009; Díaz-Castelazo e 109 al., 2010; Dáttilo et al., 2014d; Sánchez-Galván et al., 2012). Manuscript to be reviewed 103 Although relative species abundance (Vázquez et al., 2009; Dáttilo et al., 2014a) and 104 spatio-temporal overlap –considered as ‘neutral’ causes of network structure– could explain 105 overall network structure, they fall short of predicting the frequency of pairwise interactions 106 (Vázquez et al., 2009; Poisot, Stouffer & Gravel, 2015). Indeed, the frequency of interactions 107 occurring for any given pair of species within the network can vary significantly even if the 108 overall network topology remains the same (Vázquez et al., 2005, 2007, 2009; Díaz-Castelazo et 109 al., 2010; Dáttilo et al., 2014d; Sánchez-Galván et al., 2012). 110 The pattern and frequency of pairwise interactions is what we refer to here as, the ‘fine- 111 grain’ structure of the network, and is relevant since it could potentially demonstrate 112 convergence or complementarity between species (Thomson, 2005; Guimarães, Jordano & 113 Thompson, 2011). Thus, a current challenge in ecological network studies is to infer which 114 processes are involved in the structuring the fine-scale patterns of interaction networks and how 115 these may change over time (Ramos-Robles et al., 2016). Temporal changes in network structure 116 and species composition may occur because of seasonal variability in weather (Rico-Gray et al., 110 The pattern and frequency of pairwise interactions is what we refer to here as, the ‘fine- 111 grain’ structure of the network, and is relevant since it could potentially demonstrate 112 convergence or complementarity between species (Thomson, 2005; Guimarães, Jordano & 113 Thompson, 2011). Thus, a current challenge in ecological network studies is to infer which 114 processes are involved in the structuring the fine-scale patterns of interaction networks and how 115 these may change over time (Ramos-Robles et al., 2016). Temporal changes in network structure 116 and species composition may occur because of seasonal variability in weather (Rico-Gray et al., 110 The pattern and frequency of pairwise interactions is what we refer to here as, the ‘fine- 111 grain’ structure of the network, and is relevant since it could potentially demonstrate 112 convergence or complementarity between species (Thomson, 2005; Guimarães, Jordano & 113 Thompson, 2011). Thus, a current challenge in ecological network studies is to infer which 114 processes are involved in the structuring the fine-scale patterns of interaction networks and how 115 these may change over time (Ramos-Robles et al., 2016). Manuscript to be reviewed Temporal changes in network structure 116 and species composition may occur because of seasonal variability in weather (Rico-Gray et al., PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 117 2012), food abundance (Carnicer, Jordano & Melián, 2009; López-Carretero et al., 2014; Ramos- 118 Robles, Andresen & Díaz-Castelazo et al., 2016), or plant traits (López-Carretero et al., 2016). 119 Progress in understanding the determinants of network patterns requires datasets with detailed 120 information of natural history such as spatial or temporal variation, morphological, behavioral, or 121 life-history traits, which explain interspecific differences observed between species in the 122 number and strength of interactions (Stang et al., 2006; Carnicer, Jordano & Melian, 2009; 123 Junker et al., 2013; López-Carretero et al., 2016). 117 2012), food abundance (Carnicer, Jordano & Melián, 2009; López-Carretero et al., 2014; Ramos- 118 Robles, Andresen & Díaz-Castelazo et al., 2016), or plant traits (López-Carretero et al., 2016). 119 Progress in understanding the determinants of network patterns requires datasets with detailed 120 information of natural history such as spatial or temporal variation, morphological, behavioral, or 121 life-history traits, which explain interspecific differences observed between species in the 122 number and strength of interactions (Stang et al., 2006; Carnicer, Jordano & Melian, 2009; 123 Junker et al., 2013; López-Carretero et al., 2016). 124 The study of ant-plant interactions at a community level has also been approached with the 125 theoretical/analytical framework of ecological networks (Chamberlain & Holland, 2009; Díaz- 126 Castelazo et al., 2010; Dáttilo et al., 2013a; Fagundes et al., 2017). These ant-plant interactions 127 are mediated by several plant rewards for ants, such as extrafloral nectar, food bodies, fleshy 128 diaspores, or plant domatia (Rico-Gray & Oliveira, 2007). At a community-level, plants 129 providing good-quality extrafloral nectar are highly attractive to ants and accumulate more 130 interactions with aggressive and territorial ant species (Blüthgen et al., 2004; Dáttilo et al., 131 2014c), but more importantly, ant visits may result in a reduced herbivory damage (Oliveira et 132 al., 1999; Cuautle & Rico-Gray, 2003; Fagundes et al., 2017). Plenty of variation in extrafloral 133 nectaries (EFN thereafter) attributes exist, including nectar volume, the amount of secreted 134 sugar, variable gland size and morphology, position of EFNs within plant organs, and differential 135 attractiveness to ant foragers (Koptur, 1992; Wäckers & Bonifay, 2004, Díaz-Castelazo, 136 Chavarro-Rodríguez & Rico-Gray, 2017). Many of these attributes show phenotypic plasticity or 137 context-dependency (Koptur, 1992; Rudgers, 2004; Wäckers & Bonifay, 2004). Manuscript to be reviewed In this context, 138 plant investment in nectar production and quality is a very important factor modifying the benefit 124 The study of ant-plant interactions at a community level has also been approached with the 125 theoretical/analytical framework of ecological networks (Chamberlain & Holland, 2009; Díaz- 126 Castelazo et al., 2010; Dáttilo et al., 2013a; Fagundes et al., 2017). These ant-plant interactions 127 are mediated by several plant rewards for ants, such as extrafloral nectar, food bodies, fleshy 128 diaspores, or plant domatia (Rico-Gray & Oliveira, 2007). At a community-level, plants 129 providing good-quality extrafloral nectar are highly attractive to ants and accumulate more 130 interactions with aggressive and territorial ant species (Blüthgen et al., 2004; Dáttilo et al., 124 The study of ant-plant interactions at a community level has also been approached with the 125 theoretical/analytical framework of ecological networks (Chamberlain & Holland, 2009; Díaz- 126 Castelazo et al., 2010; Dáttilo et al., 2013a; Fagundes et al., 2017). These ant-plant interactions 127 are mediated by several plant rewards for ants, such as extrafloral nectar, food bodies, fleshy 128 diaspores, or plant domatia (Rico-Gray & Oliveira, 2007). At a community-level, plants 129 providing good-quality extrafloral nectar are highly attractive to ants and accumulate more 130 interactions with aggressive and territorial ant species (Blüthgen et al., 2004; Dáttilo et al., 131 2014c), but more importantly, ant visits may result in a reduced herbivory damage (Oliveira et 132 al., 1999; Cuautle & Rico-Gray, 2003; Fagundes et al., 2017). Plenty of variation in extrafloral 133 nectaries (EFN thereafter) attributes exist, including nectar volume, the amount of secreted 134 sugar, variable gland size and morphology, position of EFNs within plant organs, and differential 135 attractiveness to ant foragers (Koptur, 1992; Wäckers & Bonifay, 2004, Díaz-Castelazo, 136 Chavarro-Rodríguez & Rico-Gray, 2017). Many of these attributes show phenotypic plasticity or 137 context-dependency (Koptur, 1992; Rudgers, 2004; Wäckers & Bonifay, 2004). In this context, PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 139 received by plants through biotic defense, and fitness-related outcomes of the interaction 140 (Rudgers & Gardner, 2004; Holland, Chamberlain & Horn, 2009). 139 received by plants through biotic defense, and fitness-related outcomes of the interaction 140 (Rudgers & Gardner, 2004; Holland, Chamberlain & Horn, 2009). 140 (Rudgers & Gardner, 2004; Holland, Chamberlain & Horn, 2009). 141 Ant-plant networks, including potentially mutualistic interactions (‘potentially’, because 142 benefits were assessed only for few interactions, see: Horvitz & Schemske, 1984; Rico-Gray et 143 al., 1989; Cuautle, Rico-Gray & Díaz-Castelazo, 2005; Rico-Gray & Oliveira, 2007), have been 144 recently addressed focusing on their spatio-temporal variation (Díaz-Castelazo et al., 2010; 145 Sánchez-Galván et al., 2012; Díaz-Castelazo et al., 2013; Dáttilo et al., 2013b, 2014b) and/or 146 determining biotic/abiotic factors; among the latter, temperature and precipitation (Rico-Gray et 147 al., 2012), soil pH (Dáttilo et al., 2013a), and the temporal variation in the percentage of plants 148 with active extrafloral nectaries that mediate these interactions (Lange, Dáttilo & Del-Claro, 149 2013), have important effects on the structure (i.e. nestedness, specialization) of ant–plant 150 networks mediated by extrafloral nectaries (EFNs). 141 Ant-plant networks, including potentially mutualistic interactions (‘potentially’, because 142 benefits were assessed only for few interactions, see: Horvitz & Schemske, 1984; Rico-Gray et 143 al., 1989; Cuautle, Rico-Gray & Díaz-Castelazo, 2005; Rico-Gray & Oliveira, 2007), have been 144 recently addressed focusing on their spatio-temporal variation (Díaz-Castelazo et al., 2010; 145 Sánchez-Galván et al., 2012; Díaz-Castelazo et al., 2013; Dáttilo et al., 2013b, 2014b) and/or 146 determining biotic/abiotic factors; among the latter, temperature and precipitation (Rico-Gray et 147 al., 2012), soil pH (Dáttilo et al., 2013a), and the temporal variation in the percentage of plants 148 with active extrafloral nectaries that mediate these interactions (Lange, Dáttilo & Del-Claro, 149 2013), have important effects on the structure (i.e. nestedness, specialization) of ant–plant 150 networks mediated by extrafloral nectaries (EFNs). 151 Some studies have shown that variation in abundance of ants among different types of 152 vegetation, partially explains the network structure of mutualistic interactions, where abundant 153 ant species usually interact with more plant species (Dáttilo et al., 2014b). Similarly, the 154 abundance of plants-bearing extrafloral nectaries and plant size (Lange, Dáttilo & Del-Claro, 155 2013) are important predictors of asymmetric (i.e. nested) interactions between plants and ants in 156 ant-plant networks. PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 162 variable depending on ant species/genus or ecological context (Ness & Bronstein, 2004; Lach 163 &Hooper-Bui, 2010). Often, aggressive behavior of ants and numerical dominance are attributes 164 that influence the recruitment and competition abilities (Parr & Gibb, 2010). Ant dominance 165 hierarchy determined by ant behavior also influences network structure since ant species found 166 in the central core of the network are frequently competitively superior (i.e. showing massive 167 recruitment and resource domination), compared with peripheral species with fewer interactions 168 (Dáttilo et al., 2014c). Furthermore, invasive ant species, given their opportunism, recruitment 169 behavior, and numeric dominance (Ness & Bronstein, 2004; Lach & Hooper-Bui, 2010), could 170 rapidly become important components of the core of ant-plant networks even if they do not 171 displace other ant species (Díaz-Castelazo et al., 2010; Falcão et al., 2017). 162 variable depending on ant species/genus or ecological context (Ness & Bronstein, 2004; Lach 163 &Hooper-Bui, 2010). Often, aggressive behavior of ants and numerical dominance are attributes 164 that influence the recruitment and competition abilities (Parr & Gibb, 2010). Ant dominance 165 hierarchy determined by ant behavior also influences network structure since ant species found 166 in the central core of the network are frequently competitively superior (i.e. showing massive 167 recruitment and resource domination), compared with peripheral species with fewer interactions 168 (Dáttilo et al., 2014c). Furthermore, invasive ant species, given their opportunism, recruitment 169 behavior, and numeric dominance (Ness & Bronstein, 2004; Lach & Hooper-Bui, 2010), could 170 rapidly become important components of the core of ant-plant networks even if they do not 171 displace other ant species (Díaz-Castelazo et al., 2010; Falcão et al., 2017). 172 While the importance of abiotic/biotic factors have been separately explored for ant-plant 173 networks, the simultaneous relative contribution of biological attributes of species and ecological 174 and habitat level attributes (i.e. ecological correlates) in a facultative mutualistic ant-plant 175 network, is addressed here for the first time. Attributes of the species sets considered here are in 176 accordance with the foraging theory perspective required for a mechanistic understanding of 177 ecological networks (Ings et al., 2009). Our study system provides the opportunity to test 178 simultaneously the effect of several ecological and biological attributes of interacting species, 179 including morphology, behavior, and abundance as well as their inter-habitat (spatial) variation, 180 on the overall and ‘fine-grain’ structure of a quantitative mutualistic network. Manuscript to be reviewed Ant species attributes may influence the structure in ant-plant networks, 157 include the social recruitment behavior of ants (Dáttilo et al. 2014b), as well as its invasive 158 potential (Ness & Bronstein, 2004). Once a worker ant forager finds a profitable food source (i.e. 159 extrafloral nectar) it will (or not if it is a solitary forager) recruit nestmates using variable 160 strategies (Dornhaus & Powell, 2010). These strategies includes group recruitment, tandem 161 running, mass recruitment by pheromone trail, trunk trails, team transport, etc. which are highly 151 Some studies have shown that variation in abundance of ants among different types of 152 vegetation, partially explains the network structure of mutualistic interactions, where abundant 153 ant species usually interact with more plant species (Dáttilo et al., 2014b). Similarly, the 154 abundance of plants-bearing extrafloral nectaries and plant size (Lange, Dáttilo & Del-Claro, 155 2013) are important predictors of asymmetric (i.e. nested) interactions between plants and ants in 156 ant-plant networks. Ant species attributes may influence the structure in ant-plant networks, 157 include the social recruitment behavior of ants (Dáttilo et al. 2014b), as well as its invasive 158 potential (Ness & Bronstein, 2004). Once a worker ant forager finds a profitable food source (i.e. 159 extrafloral nectar) it will (or not if it is a solitary forager) recruit nestmates using variable 160 strategies (Dornhaus & Powell, 2010). These strategies includes group recruitment, tandem 161 running, mass recruitment by pheromone trail, trunk trails, team transport, etc. which are highly PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed In particular we 181 addressed the following questions: 1) What is the network structure of this intensively-sampled 182 ant-plant community-mediated by extrafloral nectaries?; 2) Which is the “fine-grain” structure 183 emerging from the frequency (strength) of pairwise interactions?; 3) Which is the position of 184 species in the core/periphery structure of the network?; and 4) Which is the relative contribution 162 variable depending on ant species/genus or ecological context (Ness & Bronstein, 2004; Lach 163 &Hooper-Bui, 2010). Often, aggressive behavior of ants and numerical dominance are attributes 164 that influence the recruitment and competition abilities (Parr & Gibb, 2010). Ant dominance 165 hierarchy determined by ant behavior also influences network structure since ant species found 166 in the central core of the network are frequently competitively superior (i.e. showing massive 167 recruitment and resource domination), compared with peripheral species with fewer interactions 168 (Dáttilo et al., 2014c). Furthermore, invasive ant species, given their opportunism, recruitment 169 behavior, and numeric dominance (Ness & Bronstein, 2004; Lach & Hooper-Bui, 2010), could 170 rapidly become important components of the core of ant-plant networks even if they do not 171 displace other ant species (Díaz-Castelazo et al., 2010; Falcão et al., 2017). 172 While the importance of abiotic/biotic factors have been separately explored for ant-plant 173 networks, the simultaneous relative contribution of biological attributes of species and ecological 174 and habitat level attributes (i.e. ecological correlates) in a facultative mutualistic ant-plant 175 network, is addressed here for the first time. Attributes of the species sets considered here are in 176 accordance with the foraging theory perspective required for a mechanistic understanding of 177 ecological networks (Ings et al., 2009). Our study system provides the opportunity to test 178 simultaneously the effect of several ecological and biological attributes of interacting species, 179 including morphology, behavior, and abundance as well as their inter-habitat (spatial) variation, 180 on the overall and ‘fine-grain’ structure of a quantitative mutualistic network. In particular we 181 addressed the following questions: 1) What is the network structure of this intensively-sampled 182 ant-plant community-mediated by extrafloral nectaries?; 2) Which is the “fine-grain” structure 183 emerging from the frequency (strength) of pairwise interactions?; 3) Which is the position of 184 species in the core/periphery structure of the network?; and 4) Which is the relative contribution PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 208 deciduous flood forest with wetland (TDF-W), and 6) mangrove forest ecotone (MFE) 209 (nomenclature as in Martínez-Adriano et al., 2016; derived from Castillo-Campos and Travieso- 210 Bello, 2006, following methods from Rico-Gray, 1993 and Díaz-Castelazo et al., 2004). 211 Vegetation associations differ in their structural complexity provided partly by arboreal plant 212 cover and contrasting physiognomies occur between “open” and “shaded” habitats. In 213 CICOLMA the first three habitats (PDV, CDS, and (TSF-Y) being included in the former 214 physiognomy and the other three habitats (TSF-O, TDF-W, and MFE) included in the latter 215 (Díaz-Castelazo et al., 2004; López-Carretero et al., 2014). Habitats 1, 2, and 3 are also different 216 from 4, 5, and 6 in their floristic similarity of flowering plants (Chao-Jaccard Similarity Index, 217 see Martínez-Adriano et al., 2016) and in the mean density of ants observed in honey baits 218 placed in (Díaz-Castelazo et al., 2004). In these six vegetation types we recorded all occurrences 219 of ants collecting liquids directly from all plants within each transect (ant-plant interactions). We 220 considered all plant life forms but only from those below 4 m in height, since no canopy 221 censuses were performed. 208 deciduous flood forest with wetland (TDF-W), and 6) mangrove forest ecotone (MFE) 209 (nomenclature as in Martínez-Adriano et al., 2016; derived from Castillo-Campos and Travieso- 210 Bello, 2006, following methods from Rico-Gray, 1993 and Díaz-Castelazo et al., 2004). 211 Vegetation associations differ in their structural complexity provided partly by arboreal plant 212 cover and contrasting physiognomies occur between “open” and “shaded” habitats. In 213 CICOLMA the first three habitats (PDV, CDS, and (TSF-Y) being included in the former 214 physiognomy and the other three habitats (TSF-O, TDF-W, and MFE) included in the latter 215 (Díaz-Castelazo et al., 2004; López-Carretero et al., 2014). Habitats 1, 2, and 3 are also different 216 from 4, 5, and 6 in their floristic similarity of flowering plants (Chao-Jaccard Similarity Index, 217 see Martínez-Adriano et al., 2016) and in the mean density of ants observed in honey baits 218 placed in (Díaz-Castelazo et al., 2004). In these six vegetation types we recorded all occurrences 219 of ants collecting liquids directly from all plants within each transect (ant-plant interactions). We 220 considered all plant life forms but only from those below 4 m in height, since no canopy 221 censuses were performed. Manuscript to be reviewed 185 of biological or ecological correlates (ant, plant, or habitat attributes) in rendering the “fine- 186 grain” and overall network structure? 187 188 Materials & Methods 189 Study site and data collection 190 Field work was carried out in the Centro de Investigaciones Costeras La Mancha (CICOLMA), 191 located on the coast of the state of Veracruz, Mexico (19o 36' N, 96 o 22' W; elevation <100 m). 192 The climate is warm and sub-humid with rainy season between June and September, a total 193 annual precipitation is ca. 1500 mm, and mean annual temperature is 22°-26°C (Rico-Gray, 194 1993). The major vegetation types in the study area are tropical sub-deciduous forest, tropical 195 deciduous forest, coastal dune scrub, mangrove forest, freshwater marsh, and deciduous flood 196 forest (Castillo-Campos & Travieso-Bello, 2006). Changes in the abundance of associations 197 between ants and plants bearing EFNs suggest that ant–plant interactions are strongly influenced 198 by climatic conditions as a result of marked seasonality (Díaz-Castelazo et al., 2004; Rico-Gray 199 & Oliveira, 2007). Marked seasonality at the study site (rainy, dry, and cold-front seasons) 200 influences primary productivity and have a strong effect in animal-plant interactions (Rico-Gray 201 1993; Díaz-Castelazo et al., 2004; Sánchez-Galván et al., 2012; López-Carretero et al., 2014; 202 Ramos-Robles, Andresen & Díaz-Castelazo, 2016; Martínez-Adriano, 2017). 203 Biweekly observations were conducted between October 1998 and September 2000 (Rico- 204 Gray, 1993; Díaz-Castelazo et al., 2004), rendering an intensive sampling of 48 censuses along 185 of biological or ecological correlates (ant, plant, or habitat attributes) in rendering the “fine- 186 grain” and overall network structure? 203 Biweekly observations were conducted between October 1998 and September 2000 (Rico- 204 Gray, 1993; Díaz-Castelazo et al., 2004), rendering an intensive sampling of 48 censuses along 205 six 1km trails that sampled vegetation types representative of the plant communities in the study 206 area: 1) pioneer dune vegetation (PDV), 2) coastal dune scrub (CDS), 3) tropical sub-deciduous 207 forest in young soil (TSF-Y), 4) tropical sub-deciduous forest in old soil (TSF-O), 5) tropical PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 231 when they were immobile, with mouthparts in contact with nectar secreting tissues for periods of 232 up to five minutes (Rico-Gray, 1993), although for some species, particularly when recruitment 233 of nestmates to the nectar source occurred, ant feeding was very evident and thus, recorded in 234 shorter time periods. Further information on the ant-plant interaction censuses showed at the 235 present study (including seasonal variations of species and attributes) is detailed in Díaz- 236 Castelazo et al. (2004) (Appendix 1, Figure 1). 231 when they were immobile, with mouthparts in contact with nectar secreting tissues for periods of 232 up to five minutes (Rico-Gray, 1993), although for some species, particularly when recruitment 233 of nestmates to the nectar source occurred, ant feeding was very evident and thus, recorded in 234 shorter time periods. Further information on the ant-plant interaction censuses showed at the 235 present study (including seasonal variations of species and attributes) is detailed in Díaz- 236 Castelazo et al. (2004) (Appendix 1, Figure 1). Manuscript to be reviewed 222 We also estimated the abundance of EFN-bearing plants through their line cover within 223 each transect (please see Díaz-Castelazo et al., 2004 or Sánchez-Galván et al., 2012 for details). 224 On each visit at each transect we recorded: ant species, plant species, the plant organ where the 225 extrafloral nectaries were located, and its distribution. Once an individual plant was marked as 226 visited by ants, it was subsequently re-checked throughout the study. When doubt existed on the 227 nectar source, EFN-secretion, we corroborated this with glucose reagent strips (Clinistix, Bayer). 228 We considered extrafloral nectar either produced by the surface of reproductive structures such 229 as the spike, pedicel, bud, calyx, or fruit, or secreted by special structures on vegetative parts 230 such as leaves, shoots, petioles, bracts, or stems. Ants were considered to be feeding on nectar PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 254 the following: (1) behavioral dominance based in the classification of ant functional groups 255 proposed by Andersen (1995; 2000) in relation to plant life-forms, stress, and disturbance, (2) 256 head length, a robust estimator of body mass in ant species (Kaspari & Weiser, 1999), and (3) 257 species status as invasive. The invasive status that we used was based in Holway et al. (2002), 258 with adjustments to include ‘tramp’ species status as well; invasive ants are those non-native ant 259 species which establish long-term populations and expand their range upon introduction to new 260 areas; while tramp ants are non-native transferred populations of ants closely tied with urban 261 areas and human activities (considered thus as “human commensals”) (McGlynn, 1999; Lach & 262 Hooper-Bui, 2010; Falcão et al., 2017); (Table 2). 263 We provide further detail on species attribute selection at the present study as follow. 263 We provide further detail on species attribute selection at the present study as follow. 264 Cover and distribution of EFN-bearing plant species (among habitats with different vegetation 265 structures) is an important factor influencing the richness and abundance of ant-plant interactions 266 (Díaz-Castelazo et al., 2004), interactions with other insects (López-Carretero et al., 2014), and 267 the spatio-temporal variations due to seasonality (Rico-Gray, 1993; Rico-Gray & Oliveira, 2007; 268 Díaz-Castelazo et al., 2010). Similarly, the differential distribution of EFNs among plant organs 269 could favor different ant assemblages (Majer, 1993; Blüthgen & Fiedler, 2004). This is essential 270 for the optimal defense of valuable reproductive plant organs compared to vegetative ones (Rico- 271 Gray, 1993; Wäckers & Bonifay, 2004; Holland, Chamberlain & Horn, 2009). Related to ant 272 attributes, behavioral dominance is a relevant feature in mutualistic ant-plant interactions given 273 that competitive species may exclude submissive ones (Andersen, 2000; Ness & Bronstein, 274 2004; Lach & Hooper-Bui, 2010; Dáttilo et al., 2014c). For example, head length has been 275 shown to be positively correlated with the number of plant species that each ant species interact 276 in ant-plant networks (knows as “degree”; Chamberlain & Holland, 2009). Other relevant 263 We provide further detail on species attribute selection at the present study as follow. 238 Plant and ant attributes 239 Regarding the distribution of EFNs among plant organs, we used a general characterization 240 (see Díaz-Castelazo et al., 2005) differentiating the EFNs which are glands circumscribed to 241 particular plant organs or whorls (at specific or modular locations) from the ones dispersed 242 among plant organs (i.e. secretory trichomes on leaves or surfaces of vegetative tissues). Díaz- 243 Castelazo et al. (2005) results raised the idea that gland distribution on plant organs could follow 244 an aggregate (i.e. circumscribed) location against a widely dispersed location and this could 245 result in distinct ant visitor arrays (Díaz-Castelazo et al., 2004). In a similar way that extrafloral 246 nectar sources may differ from honeydew sources in their associated ant assemblages (Blüthgen 247 & Fiedler, 2004; Blüthgen et al., 2000). Circumscribed EFNs include: elevated glands, hollow 248 glands (vascularized), transformed glands (vascularized), capitated trichomes (non-vascularized), 249 and unicellular trichomes (non-vascularized). Disperse EFNs include: flattened glands, peltate 250 trichomes, and scale-like trichomes (Díaz-Castelazo et al., 2005). 251 Attributes for plants included: (1) the abundance of plants with EFNs, (2) species 252 distribution in vegetation associations with distinct habitat structure (open or shaded habitats), 253 and (3) the distribution of the EFNs among plant organs (Table 1). Attributes for ants included PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 264 Cover and distribution of EFN-bearing plant species (among habitats with different vegetation 265 structures) is an important factor influencing the richness and abundance of ant-plant interactions 266 (Díaz-Castelazo et al., 2004), interactions with other insects (López-Carretero et al., 2014), and 267 the spatio-temporal variations due to seasonality (Rico-Gray, 1993; Rico-Gray & Oliveira, 2007; 268 Díaz-Castelazo et al., 2010). Similarly, the differential distribution of EFNs among plant organs 269 could favor different ant assemblages (Majer, 1993; Blüthgen & Fiedler, 2004). This is essential 270 for the optimal defense of valuable reproductive plant organs compared to vegetative ones (Rico- 271 Gray, 1993; Wäckers & Bonifay, 2004; Holland, Chamberlain & Horn, 2009). Related to ant 272 attributes, behavioral dominance is a relevant feature in mutualistic ant-plant interactions given 273 that competitive species may exclude submissive ones (Andersen, 2000; Ness & Bronstein, 274 2004; Lach & Hooper-Bui, 2010; Dáttilo et al., 2014c). For example, head length has been 275 shown to be positively correlated with the number of plant species that each ant species interact 276 in ant-plant networks (knows as “degree”; Chamberlain & Holland, 2009). Other relevant PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 277 attribute that could affect the ant-plant interactions is the presence of invasive ant species since 278 many invasive species have behavior or foraging strategies that overcome their native 279 counterparts (Díaz-Castelazo et al., 2010) or disrupt the mutualistic interactions (Schultz & 280 McGlynn 2000; Holway et al., 2002). 281 282 Data analysis 283 The ant-plant network analyzed here consists of a quantitative species-species matrix given by 284 the frequency of occurrence of each pairwise ant-plant interaction. Ecological and biological 285 attributes of the species were of different kinds and considered as highly important in modulating 286 the mutualistic interaction (Díaz-Castelazo et al., 2004; 2005). 287 The pairwise interaction matrix here considered is a highly informative sub-web taken 288 from Díaz-Castelazo et al. (2010), where we excluded those interactions that occurred at 289 considerably low frequencies (interactions recorded on less than three occasions from the whole 290 48 censuses), in order to perform better multivariate analysis (NMDS), interpretation of biplot 291 ordinations, and adjustment of explanatory variables. This also reduced the probability of 292 considering a species with a single or very few interactions as a “specialist”, when it was just a 293 very rare species and helped to avoid the overestimation of specialization, nestedness, and 294 strength asymmetry (Blüthgen et al., 2008). 295 For this informative network we analyzed nestedness (NODF) (Nestedness based on 295 For this informative network we analyzed nestedness (NODF) (Nestedness based on 296 Overlap and Decreasing Fill) (Almeida-Neto et al., 2008) using ANINHADO (Guimarães & 297 Guimarães, 2006). This metric is robust to detect a nested pattern since it is less sensitive to 298 matrix size and shape than other measures such as nestedness derived from matrix Temperature 299 Almeida-Neto et al., 2008). Significance of the NODF value for our network was obtained with 295 For this informative network we analyzed nestedness (NODF) (Nestedness based on 296 Overlap and Decreasing Fill) (Almeida-Neto et al., 2008) using ANINHADO (Guimarães & 297 Guimarães, 2006). This metric is robust to detect a nested pattern since it is less sensitive to 298 matrix size and shape than other measures such as nestedness derived from matrix Temperature 299 Almeida-Neto et al., 2008). Significance of the NODF value for our network was obtained with PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 300 ANINHADO after comparing it with 1000 simulations using null model Ce (Guimarães & 301 Guimarães, 2006), which corresponds to Null Model II of Bascompte et al. (2003). It assumes 302 that the probability of occurrence of an interaction is proportional to the observed number of 303 interactions of both plant and ant species (Bascompte et al., 2003; Dáttilo et al., 2013b). We then 304 estimated network topology or structural metrics (connectance, dependence asymmetry, 305 weighted nestedness, and niche overlap) using different indexes included in the function 306 “network-level” of the “bipartite” package (Dormann & Gruber, 2009) in ‘R’ software ver. 3.5.1 307 (R Core Team, 2014). 300 ANINHADO after comparing it with 1000 simulations using null model Ce (Guimarães & 301 Guimarães, 2006), which corresponds to Null Model II of Bascompte et al. (2003). It assumes 302 that the probability of occurrence of an interaction is proportional to the observed number of 303 interactions of both plant and ant species (Bascompte et al., 2003; Dáttilo et al., 2013b). We then 304 estimated network topology or structural metrics (connectance, dependence asymmetry, 305 weighted nestedness, and niche overlap) using different indexes included in the function 306 “network-level” of the “bipartite” package (Dormann & Gruber, 2009) in ‘R’ software ver. 3.5.1 307 (R Core Team, 2014). 308 Additionally, with the software MODULAR (Marquitti et al., 2013) we tested the existence 309 of a modular structure in the network with the modularity index (M). This index ranges 0-1 and 310 was calculated with simulated annealing optimization approach (SA) (Guimerà & Amaral, 308 Additionally, with the software MODULAR (Marquitti et al., 2013) we tested the existence 309 of a modular structure in the network with the modularity index (M). This index ranges 0-1 and 310 was calculated with simulated annealing optimization approach (SA) (Guimerà & Amaral, 311 2005). This metric was based on Barbers modularity metric which are recommended for bipartite 312 networks (QB) (Barber, 2007). The statistical significance of modularity (M) was calculated using 313 Monte Carlo tests with 1000 randomizations (Guimerà, Sales-Pardo & Amaral, 2004). High 314 values of M indicate the occurrence of ants and plants in cohesive subgroups that generate 315 compartments or modules in which these species interact more closely than with the other 316 species in the network (Olesen et al., 2007). 311 2005). Manuscript to be reviewed This metric was based on Barbers modularity metric which are recommended for bipartite 312 networks (QB) (Barber, 2007). The statistical significance of modularity (M) was calculated using 313 Monte Carlo tests with 1000 randomizations (Guimerà, Sales-Pardo & Amaral, 2004). High 314 values of M indicate the occurrence of ants and plants in cohesive subgroups that generate 315 compartments or modules in which these species interact more closely than with the other 316 species in the network (Olesen et al., 2007). 311 2005). This metric was based on Barbers modularity metric which are recommended for bipartite 312 networks (QB) (Barber, 2007). The statistical significance of modularity (M) was calculated using 313 Monte Carlo tests with 1000 randomizations (Guimerà, Sales-Pardo & Amaral, 2004). High 314 values of M indicate the occurrence of ants and plants in cohesive subgroups that generate 315 compartments or modules in which these species interact more closely than with the other 316 species in the network (Olesen et al., 2007). 317 For calculation of the “fine-grain” structure of the network we used the “species-level” 318 function (Dorman, 2011) in the “bipartite” package. The metrics calculated for this objective 319 were “species strength” and d’. The first is defined as the sum of dependences of the plants 320 visited by this animal (or vice versa). Thus, species strength is a quantitative extension of the 321 metric “species degree” and provides information about the relevance of a species for their 322 interacting counterpart, being thus a meaningful measure of network complexity (Bascompte, 317 For calculation of the “fine-grain” structure of the network we used the “species-level” 318 function (Dorman, 2011) in the “bipartite” package. The metrics calculated for this objective 319 were “species strength” and d’. The first is defined as the sum of dependences of the plants 320 visited by this animal (or vice versa). Thus, species strength is a quantitative extension of the 321 metric “species degree” and provides information about the relevance of a species for their 322 interacting counterpart, being thus a meaningful measure of network complexity (Bascompte, PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 346 smallest stress value using the metaMDS function in “Vegan” package (Dixon, 2009) for R 347 software (R Core Team, 2014). 348 In order to explore the simultaneous relative contribution of several biological and 349 ecological species attributes and habitat level attributes on the interaction pattern (NMDS 350 ordination), we fitted those ecological/biological factors and vectors using the envfit function 351 from the “Vegan” package (Dixon, 2009) on R software (R Core Team, 2014). This function fit 352 the vectors (continuous variables) and factors (categorical variables) from the environmental 353 variables to the NMDS ordination, providing statistical significance by comparing our real model 354 of pairwise interactions with 1000 permutations of a given null model. The envfit function 355 provides a measure of correlation (r) and a significance value (p) based on the probability that 356 1000 random permutations of simulated (environmental) variables would have a better fit than 357 the real variables (Oksanen, 2009). 346 smallest stress value using the metaMDS function in “Vegan” package (Dixon, 2009) for R 347 software (R Core Team, 2014). 348 In order to explore the simultaneous relative contribution of several biological and 349 ecological species attributes and habitat level attributes on the interaction pattern (NMDS 350 ordination), we fitted those ecological/biological factors and vectors using the envfit function 351 from the “Vegan” package (Dixon, 2009) on R software (R Core Team, 2014). This function fit 352 the vectors (continuous variables) and factors (categorical variables) from the environmental 353 variables to the NMDS ordination, providing statistical significance by comparing our real model 354 of pairwise interactions with 1000 permutations of a given null model. The envfit function 355 provides a measure of correlation (r) and a significance value (p) based on the probability that 356 1000 random permutations of simulated (environmental) variables would have a better fit than 357 the real variables (Oksanen, 2009). 346 smallest stress value using the metaMDS function in “Vegan” package (Dixon, 2009) for R 347 software (R Core Team, 2014). 358 To test if the frequency of ants was different when foraging in the different EFN 359 morphological types, we performed a χ2 test. To test if between-group floristic similarity 360 (Sorensen´s floristic similarity index, Češka, 1966) was higher than within group floristic 361 similarity we performed one-way ANOVA contrasting open and shaded habitats. Manuscript to be reviewed 323 Jordano & Olesen, 2006). While, d’ is the specialization of each species based on its 324 discrimination from random selection of partners (Blüthgen et al., 2008). Finally, we calculated 325 core-periphery structure of the network and its component species (i.e. which species constitute 326 the cohesive core are generalists, and which the low-degree species constitute the peripheral). 327 This metric was calculated with a function developed by Martínez-Adriano (2017) in R software 328 based on the formula proposed by Dáttilo, Guimarães & Izzo (2013), where the species with 329 values equal or larger than 1 are considered as core components and species < 1 are considered 330 peripheral. 331 In order to explore the among-species dissimilarities resulting in the interaction pattern of 332 the network, we generated the ordination of interaction frequencies with “Non-metric 333 Multidimensional Scaling (NMDS)” multivariate technique (Quinn & Keough, 2002). This 334 method is specifically designed to graphically represent relationship between objects (i.e. 335 species/sites) in a multidimensional space provided by non-metric dissimilarities among objects 336 (Quinn & Keough, 2002). NMDS is one of the most effective methods for the ordination of 337 ecological data and the identification of underlying gradients because it does not assume a linear 338 relationship among all variables (Quinn & Keough, 2002). NMDS reduces the dimensionality of 339 a matrix among sample similarity coefficients, based on particular number of dimensions (Borg 340 & Groenen, 1997). We chose the Bray-Curtis dissimilarity coefficient to construct the similarity 341 matrices because joint absences do not influence among sample similarity, and then we chose a 342 two-dimension configuration. The fit of an NMDS ordination, known as “stress”, is determined 343 by how well the ordination preserved the actual sample dissimilarities, where values range from 344 zero to one (values of 0.2 and below are valid configurations to be interpreted). Because NMDS 345 analysis offers more than one solution, we carried out an iterative process to find the model with PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed With this 362 analysis we further confirm that open and shaded habitats differ in their vegetation structure and 363 in turn, provide differential biotic and abiotic conditions for inhabitant species, presumably 364 affecting the resultant network structure of ant-plant interactions. To explore if there was a 365 relation between ant head length and species degree in the network (the number of plant species 366 interacting with ants), we performed a Spearman rank correlation (Quinn & Keough, 2002). 367 358 To test if the frequency of ants was different when foraging in the different EFN 359 morphological types, we performed a χ2 test. To test if between-group floristic similarity 360 (Sorensen´s floristic similarity index, Češka, 1966) was higher than within group floristic 361 similarity we performed one-way ANOVA contrasting open and shaded habitats. With this 362 analysis we further confirm that open and shaded habitats differ in their vegetation structure and 363 in turn, provide differential biotic and abiotic conditions for inhabitant species, presumably 364 affecting the resultant network structure of ant-plant interactions. To explore if there was a 365 relation between ant head length and species degree in the network (the number of plant species 366 interacting with ants), we performed a Spearman rank correlation (Quinn & Keough, 2002). 367 368 Results PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) 369 Network-level and fine-grain structure 370 Our ant-plant network involved 31 EFN-bearing plant species and 19 ant-forager species linked 371 by 1302 quantitative interactions (overall frequency of interactions) derived from 157 species 372 associations (links among species). The general topology shows a highly and significantly nested 373 network (NODFObs = 49.13, NODF (Ce) = 34.93, P (Ce) < 0.001). Although five modules were 374 detected in the modularity analysis (Barber´s QB) the network was not significantly modular (M= 375 0.288, P = 0.55), thus no true compartments exist in the network (Fig 1). Network-level indexes 376 were: connectance = 0.267, dependence asymmetry = 0.669 (implying that plants depend more 377 on ants than the opposite), niche overlap among ant species = 0.223, niche overlap among plant 378 species = 0.425, and weighted nestedness = 0.554 (implying that the network is still nested when 379 considering the frequency of pair-wise interactions). Four plant species and three ant species 380 constituted the central core of this network, the remaining species were peripheral. Plant core 381 species were: Cordia spinescens, Cedrela odorata, Callicarpa acuminata, and Crotalaria 382 incana, while ant core species were: Camponotus planatus, Camponotus mucronatus, and 383 Camponotus atriceps. 384 In terms of ‘species strength’, most plant species exhibited low strength values (below 1), 385 thus having a modest relevance for the ant community. However, some plant species stand out 386 with higher strength values (around 2) which are Cordia spinescens, Cedrela odorata, 387 Callicarpa acuminata, and Turnera ulmifolia. These plant species are the most important EF 388 nectar sources for ant foragers at a community level. Species-level specialization values (d’, 389 considered as a measure of selectiveness) for plant species were also generally low (around 0.1), 390 and only those plant species with few associated ant species (ant species with interaction patterns 391 atypical or different from the rest) showed values above 0.3, such as Ficus obtusifolia (d´= 0.43) 384 In terms of ‘species strength’, most plant species exhibited low strength values (below 1), 385 thus having a modest relevance for the ant community. However, some plant species stand out 386 with higher strength values (around 2) which are Cordia spinescens, Cedrela odorata, 384 In terms of ‘species strength’, most plant species exhibited low strength values (below 1), 385 thus having a modest relevance for the ant community. Manuscript to be reviewed The frequency of ants foraging on the different EFN morphological types ( 407 Castelazo et al. 2005; Díaz-Castelazo, Chavarro-Rodríguez & Rico-Gray, 2017) wer 408 (χ28= 1091.7, P <0.01). Moreover, the range of total associated ant species visiting p 409 considered to have each type of nectary is different among EFN distribution types. T 401 Relative contribution of attributes to the assemblage of pair-wise interactions 369 Network-level and fine-grain structure However, some plant species stand out 386 with higher strength values (around 2) which are Cordia spinescens, Cedrela odorata, PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 392 and Senna occidentalis (d´= 0.37). In contrast with plants, some ant species had higher strength 393 values. Seven ant species had values above 1, and two core ant species, Camponotus planatus 394 and C. mucronatus, have strength values over 6, being thus very important visitors of EFN- 395 bearing plants; these ant species with high-strength values are relevant at a community level. On 396 the other hand, specialization (selectiveness) for ants was generally low (around 0.3, thus, higher 397 than plants), but few ant species had intermediate d´values such as Tetramorium spinosum (d´ = 398 0.53) or Wasmannia auropunctata (d´= 0.43) not only because they interact with few plant 399 species but with plants visited by few ant species. 392 and Senna occidentalis (d´= 0.37). In contrast with plants, some ant species had higher strength 393 values. Seven ant species had values above 1, and two core ant species, Camponotus planatus 394 and C. mucronatus, have strength values over 6, being thus very important visitors of EFN- 395 bearing plants; these ant species with high-strength values are relevant at a community level. On 396 the other hand, specialization (selectiveness) for ants was generally low (around 0.3, thus, higher 397 than plants), but few ant species had intermediate d´values such as Tetramorium spinosum (d´ = 398 0.53) or Wasmannia auropunctata (d´= 0.43) not only because they interact with few plant 399 species but with plants visited by few ant species. 394 and C. mucronatus, have strength values over 6, being thus very important visitors o 395 bearing plants; these ant species with high-strength values are relevant at a communi 396 the other hand, specialization (selectiveness) for ants was generally low (around 0.3, 397 than plants), but few ant species had intermediate d´values such as Tetramorium spin 398 0.53) or Wasmannia auropunctata (d´= 0.43) not only because they interact with few 399 species but with plants visited by few ant species. 400 401 Relative contribution of attributes to the assemblage of pair-wise interactions 402 Attributes of species are summarized as follows: plant species with circumscrib 403 (Table 1) produced larger mean nectar volumes (2.06 µl), than those plants with disp 404 nectaries (0.53 µl). However, the number of active glands in a plant individual may b 405 dispersed nectaries, since these glands are structurally simpler than those of circumsc 406 nectaries. 401 Relative contribution of attributes to the assemblage of pair-wise interactions 402 Attributes of species are summarized as follows: plant species with circumscribed nectaries 403 (Table 1) produced larger mean nectar volumes (2.06 µl), than those plants with dispersed 404 nectaries (0.53 µl). However, the number of active glands in a plant individual may be higher for 405 dispersed nectaries, since these glands are structurally simpler than those of circumscribed 406 nectaries. The frequency of ants foraging on the different EFN morphological types (Díaz- 407 Castelazo et al. 2005; Díaz-Castelazo, Chavarro-Rodríguez & Rico-Gray, 2017) were different 408 (χ28= 1091.7, P <0.01). Moreover, the range of total associated ant species visiting plants 409 considered to have each type of nectary is different among EFN distribution types. The range of 410 visits to circumscribed nectaries was between nine and 17 ant species, while it was between 20 411 and 23 ant species for disperse nectaries. 412 We considered the two main vegetation structural associations (“open” vs. “shaded 413 habitats”) to be natural groups (Table 1), because floristic similarity between them is 414 considerably lower and significantly different (F1,13 = 15.79, P <0.01) to that occurring within PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 415 each group (36.06 and 41.28 for open and shaded habitats, respectively). See Methods for 416 information on the vegetation associations, either of “open or shaded habitats”. 417 The stress value of the multivariate Non-Metric Multidimensional Scaling (NMDS) 418 obtained at the fourth run of the iterative process was the lowest (0.17), and suggests that the 419 NMDS two-dimensional solution of the ordination suitably represented ant-plant assemblage 420 dissimilarity. This configuration also had very low residuals (max res = 0.0004) indicating a 421 good concordance between the calculated dissimilarities and the distances among objects. In Fig. 422 2, axis NMDS1 is related to the contribution or importance of plant species to the ant forager 423 community. Those plant species that are ordered at the extremes of axis NMDS1, either with low 424 (negative) or higher (positive) values for component NMDS1, have low species strength values; 425 thus, these plant species have ‘atypical’ interaction patterns (and occupy in Fig. 1, the lowest or 426 basal position within the network). In contrast, at axis 1 of NMDS, those plant species 427 aggregated near zero are those plant species with the highest species strength or relevance for the 428 associated ant community. For ants, no generic or grouping trends are apparent. Axis NMDS2 429 divides plant species according to the main habitats where they occur. Starting from the upper 430 part of the ordination and ending at the lower part, we see an arrangement of the plant species 431 according to a ‘humidity’ gradient of habitats; first we see plants of shaded habitats with modest 432 light incidence and higher humidity and at the lower part of the biplot along axis 2 we see the 433 plants of the drier habitats. The higher values for NMDS2 show (in decreasing order) plants (and 434 associated ants) from the MFE, followed TSF-W, and TSF-O. At the bottom of the bi-plot, the 435 plants and ants occurring mostly in open vegetation types with high light incidence: from zero to 436 the lowest values of NMDS2, the interacting species are arranged through TSF-Y, CDS, and 437 PDV. 415 each group (36.06 and 41.28 for open and shaded habitats, respectively). See Methods for 416 information on the vegetation associations, either of “open or shaded habitats”. 415 each group (36.06 and 41.28 for open and shaded habitats, respectively). Manuscript to be reviewed 438 The results of fitting among the biological/ecological variables and the NMDS ordination 439 showed that vegetation associations with differential structure (open vs. shaded habitats) were 440 the variables that determined the variation in the frequency of ant-plant pairwise interactions- 441 mediated by EFNs (r2 = 0.24, P = 0.005). Two contrasting groups were formed along NMDS2, 442 which were plant species (and their associated ant forager species) located either in open or 443 shaded habitats (Fig. 2). Neither the distribution of EFNs on plant organs, nor the abundance of 444 extrafloral-nectary bearing plants at each vegetation type, had a significant contribution to the 445 variation in the observed ant-plant association patterns. 446 None of the variables of ant species (behavioral dominance, invasive status or head length) 447 showed in Table 2, explain the network´s fine-grained structure. However, we found a significant 448 positive correlation between ant head length and species degree (the number of plant species 449 interacting with ants) (Spearman rank correlation, rs = 0.565, P < 0.05). A trend at the NMDS 450 ordination is that the invasive (tramp) ant species in our study (Solenopsis geminata, Wasmannia 451 auropunctata, Tetramorium spinosum, and Paratrechina longicornis) separate from the rest of 452 ant species at the interaction display. However, when all ant attributes are simultaneously 446 None of the variables of ant species (behavioral dominance, invasive status or head length) 447 showed in Table 2, explain the network´s fine-grained structure. However, we found a significant 448 positive correlation between ant head length and species degree (the number of plant species 449 interacting with ants) (Spearman rank correlation, rs = 0.565, P < 0.05). A trend at the NMDS 450 ordination is that the invasive (tramp) ant species in our study (Solenopsis geminata, Wasmannia 451 auropunctata, Tetramorium spinosum, and Paratrechina longicornis) separate from the rest of 452 ant species at the interaction display. However, when all ant attributes are simultaneously 453 considered, they do not provide significant contribution to the variation in the observed ant-plant 454 association pattern. PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed See Methods for 416 information on the vegetation associations, either of “open or shaded habitats”. 415 each group (36.06 and 41.28 for open and shaded habitats, respectively). See Methods for 416 information on the vegetation associations, either of “open or shaded habitats”. PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 457 Network-level and fine-grain network structure 458 Our studied network, comprising 31 plant and 19 ant species attached by 157 interaction 459 links, has a general nested structure and is thus asymmetric in its specialization patterns (see also 460 Díaz-Castelazo et al., 2010; Díaz-Castelazo et al., 2013). The network shows no modular PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 461 structure, as the non-modular structure that occurs commonly in theoretical mutualistic networks, 462 especially for facultative non-symbiotic interactions (Guimarães et al., 2007). Few species with 463 very high interaction frequencies exists within our network (eight plant and four ant species), 464 referred as ‘super-generalists’. Super-generalist species are fundamental components for the 465 maintenance of convergence at the community level within highly diversified mutualistic 466 assemblages (Guimarães, Jordano & Thompson, 2011). In our study, super-generalist species 467 may favor trait convergence. That is, core ant species belong to the same functional group 468 (Subordinate Camponotini) and share adaptations for foraging on plant-derived liquids resources 469 such as extrafloral nectar (Davidson, Cook & Snelling, 2004). Similarly, core plants species 470 show mostly “disperse” EFNs, a gland distribution that may favor a more diverse array of 471 associated ant visitors. 461 structure, as the non-modular structure that occurs commonly in theoretical mutualistic networks, 462 especially for facultative non-symbiotic interactions (Guimarães et al., 2007). Few species with 463 very high interaction frequencies exists within our network (eight plant and four ant species), 464 referred as ‘super-generalists’. Super-generalist species are fundamental components for the 465 maintenance of convergence at the community level within highly diversified mutualistic 466 assemblages (Guimarães, Jordano & Thompson, 2011). In our study, super-generalist species 467 may favor trait convergence. That is, core ant species belong to the same functional group 468 (Subordinate Camponotini) and share adaptations for foraging on plant-derived liquids resources 469 such as extrafloral nectar (Davidson, Cook & Snelling, 2004). Similarly, core plants species 470 show mostly “disperse” EFNs, a gland distribution that may favor a more diverse array of 471 associated ant visitors. 472 In our study system, the fact that the plant ‘guild’ shows higher dependence asymmetry 473 values than ants, implying that the studied community plants ‘depend’ more on ants as a guild 474 than the opposite. It is also reinforced by the higher species strength values of ants than those 475 given for plants. This asymmetry could reflect a higher temporal turnover of plants at the 476 network –perhaps caused by seasonality or disturbance versus higher ant resilience– probably 477 derived from facultative foraging of ants. Manuscript to be reviewed Three of four plant species constituting the core of this 478 network had high strength values (Cordia spinescens, Cedrela odorata, and Callicarpa 479 acuminata); these results suggest that the most connected plant species are important resources 480 for the ants at the community level. However, the relative importance of specific plant species 481 for this ant community do not seem related to specific biological attributes or neutral effects 482 since neither mean nectar volumes secreted by each plant species (Díaz-Castelazo et al., 2005; 483 Díaz-Castelazo, Chavarro-Rodríguez & Rico-Gray, 2017), nor gland distribution or plant 472 In our study system, the fact that the plant ‘guild’ shows higher dependence asymmetry 473 values than ants, implying that the studied community plants ‘depend’ more on ants as a guild 474 than the opposite. It is also reinforced by the higher species strength values of ants than those 475 given for plants. This asymmetry could reflect a higher temporal turnover of plants at the 476 network –perhaps caused by seasonality or disturbance versus higher ant resilience– probably 477 derived from facultative foraging of ants. Three of four plant species constituting the core of this 478 network had high strength values (Cordia spinescens, Cedrela odorata, and Callicarpa 479 acuminata); these results suggest that the most connected plant species are important resources 480 for the ants at the community level. However, the relative importance of specific plant species 481 for this ant community do not seem related to specific biological attributes or neutral effects 482 since neither mean nectar volumes secreted by each plant species (Díaz-Castelazo et al., 2005; 483 Díaz-Castelazo, Chavarro-Rodríguez & Rico-Gray, 2017), nor gland distribution or plant PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 484 abundance explained core composition and species strength of plant species. Instead, this patter 485 seems to emerge from degree and interaction frequencies, possibly driven by other higher-scale 486 factors (i.e. habitat structure, species co-occurrence, abiotic variables, etc.). 487 The rest of plant species showed very low species strength values, having thus a modest 488 relevance for the ant community. Species-level specialization values (d’) for plant species were 489 also generally low (around 0.1) and only those plant species with few associated ant species 490 (with an atypical interaction pattern) exhibited values above 0.3. These findings are in 491 accordance with the generalized, highly nested structure of this network. For potentially 492 mutualistic networks (such as this) and for facultative ant-plant interactions (such as those 493 mediated by extrafloral nectar), low specialization or selectiveness for each species (and the 494 whole network) is the general trend (Bascompte et al., 2003; Vázquez & Aizen, 2004; Díaz- 495 Castelazo et al., 2010). 496 In contrast with plants (which have lowest species strength values), the ant species that 497 constitute the core of this network (Camponotus planatus, Camponotus mucronatus, and 498 Camponotus atriceps) had species strength values above 1. Camponotus planatus and C. 499 mucronatus have strength values above 6, being thus remarkably important visitors of EFN- 500 bearing plants. Species belonging to this genus are frequent visitors of EFNs (Díaz-Castelazo et 501 al., 2004; Díaz-Castelazo et al., 2013) and solitary leaf foragers that cover high foliar areas. 502 Camponotus species have high ability to rapidly take up nectar given their proventricular 503 adaptations that allow passive damming of sugary liquids, large crop capacities, and seeping 504 canals to nourish the midgut (Davidson, Cook & Snelling, 2004). Thus, this group of ants is 505 highly adapted to forage on nectar and sugary liquids. It is understandable that at the present 506 study Camponotus species have high degree, high strength values, and low levels of 484 abundance explained core composition and species strength of plant species. Instead, this pattern 485 seems to emerge from degree and interaction frequencies, possibly driven by other higher-scale 486 factors (i.e. habitat structure, species co-occurrence, abiotic variables, etc.). 484 abundance explained core composition and species strength of plant species. Instead, this pattern 485 seems to emerge from degree and interaction frequencies, possibly driven by other higher-scale 486 factors (i.e. habitat structure, species co-occurrence, abiotic variables, etc.). Manuscript to be reviewed 507 specialization or selectiveness (d’). Given that these ants are physiologically adapted to forage in 508 all available extrafloral nectar sources and not having any trophic restriction, they tend to be 509 generalist visitors of EFN-bearing plants. Although some other ecological aspects –such as 510 competition ability of other ant species and resource attractiveness (Dáttilo et al., 2014c)– may 511 differentiate visitation pattern of these core ant species. In our studied community core ant 512 species are relevant to most plants species at the network and the plant species depend more of 513 ant species than the opposite. Adaptations to nectar consumption and deterrent behavior of core 514 ants (as well as their high interaction frequency), suggest that these species may provide 515 potential biotic defense at a community scale; this do not exclude the possibility that many other 516 ant species provide biotic defense at smaller scales or for particular plant species. 517 507 specialization or selectiveness (d’). Given that these ants are physiologically adapted to forage in 508 all available extrafloral nectar sources and not having any trophic restriction, they tend to be 509 generalist visitors of EFN-bearing plants. Although some other ecological aspects –such as 510 competition ability of other ant species and resource attractiveness (Dáttilo et al., 2014c)– may 511 differentiate visitation pattern of these core ant species. In our studied community core ant 512 species are relevant to most plants species at the network and the plant species depend more of 513 ant species than the opposite. Adaptations to nectar consumption and deterrent behavior of core 514 ants (as well as their high interaction frequency), suggest that these species may provide 515 potential biotic defense at a community scale; this do not exclude the possibility that many other 516 ant species provide biotic defense at smaller scales or for particular plant species. 517 Manuscript to be reviewed 487 The rest of plant species showed very low species strength values, having thus a modest 488 relevance for the ant community. Species-level specialization values (d’) for plant species were 489 also generally low (around 0.1) and only those plant species with few associated ant species 490 (with an atypical interaction pattern) exhibited values above 0.3. These findings are in 491 accordance with the generalized, highly nested structure of this network. For potentially 492 mutualistic networks (such as this) and for facultative ant-plant interactions (such as those 493 mediated by extrafloral nectar), low specialization or selectiveness for each species (and the 494 whole network) is the general trend (Bascompte et al., 2003; Vázquez & Aizen, 2004; Díaz- 495 Castelazo et al., 2010). PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) 518 Relative contributions of the attributes to the assemblage of pairwise interactions 518 Relative contributions of the attributes to the assemblage of pairwise interactions 519 Major vegetation associations grouped according to habitat structure were the only factors that 520 explained variations in pair-wise interactions or fine-grain structure of the network. Open and 521 shaded habitats at the study site seem to differ structurally in vegetation and on their abiotic 522 conditions, which may in turn be important determinants for insect-plant interactions (López- 523 Carretero et al., 2014). Although, some studies have discussed the possible effects of abiotic 524 variables on ant-plant networks (Díaz-Castelazo et al., 2010; Rico-Gray & Oliveira, 2007 525 (references therein); Rico-Gray et al., 2012, Sánchez-Galván et al., 2012), our study is the only 526 one addressing habitat abiotic effects jointly with species-level biological attributes and neutral 527 explanations (i.e. abundance) in a quantitative ant-plant network. 519 Major vegetation associations grouped according to habitat structure were the only factors that 520 explained variations in pair-wise interactions or fine-grain structure of the network. Open and 521 shaded habitats at the study site seem to differ structurally in vegetation and on their abiotic 522 conditions, which may in turn be important determinants for insect-plant interactions (López- 523 Carretero et al., 2014). Although, some studies have discussed the possible effects of abiotic 524 variables on ant-plant networks (Díaz-Castelazo et al., 2010; Rico-Gray & Oliveira, 2007 525 (references therein); Rico-Gray et al., 2012, Sánchez-Galván et al., 2012), our study is the only 526 one addressing habitat abiotic effects jointly with species-level biological attributes and neutral 527 explanations (i.e. abundance) in a quantitative ant-plant network. 528 A mechanistic explanation for the differential ant-plant association pattern between open 529 and shaded habitats (suggesting habitat complexity effects; Dáttilo et al., 2013b), could include 528 A mechanistic explanation for the differential ant-plant association pattern between open 529 and shaded habitats (suggesting habitat complexity effects; Dáttilo et al., 2013b), could include PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 530 light incidence (under light conditions, jasmonic acid-induced EFN secretion is higher than in 531 dark conditions), ‘attractiveness’ or nutritional value of extrafloral nectar secreted by ‘light 532 demanding’ plant species compared to ‘shade tolerant’ ones, and the physiological tolerance of 533 ants to high temperatures (Radhika et al., 2010). Increased photosynthetic activity of plants in 534 open light-rich habitats could result in higher carbohydrate availability in extrafloral nectar and 535 thus increased attractiveness to ants (Radhika et al., 2010), or a higher density of EFN-bearing 536 plant life forms (such as vines). EFN-bearing plants growing in sunlight obtain a measurable 537 benefit from ant visitation, whereas the same plant species growing under shaded conditions has 538 no such a benefit (e.g., Bentley, 1976; Frank & Fonseca, 2005). For some plant species size of 539 EFNs and nectar secretion are higher under intense light conditions compared to low light 540 conditions (Yamawo & Hada, 2010) and a similar trend is found for the ant abundance foraging 541 on these glands (Rudgers & Gardener 2004, Yamawo & Hada, 2010). This effect of site 542 conditions on EFN abundance and secretory activity could also exist in our study system since 543 vegetation types with canopy cover (shaded) versus open habitats do sustain different species 544 abundances, floristic similarities (Díaz-Castelazo et al., 2004), and patterns of specific insect- 545 plant interactions (López-Carretero et al., 2014). 546 Among-habitat heterogeneity in vegetation structure (as well as seasonality) in our study 547 site is so prevalent (having a strong effect in animal-plant interactions as seen in Rico-Gray, 548 1993; Díaz-Castelazo et al., 2004; Sánchez-Galván et al., 2012; López-Carretero et al., 2014; and 549 Ramos-Robles, Andresen & Díaz-Castelazo, 2016). This is clearly detected in the ant-plant 550 interaction pattern, in contrast to other studies where vegetation structure differences are not so 551 outstanding as have an effect in other ant-plant networks (Dáttilo et al., 2013b). Further evidence 552 of among-habitat heterogeneity translating to ant-plant network structure is provided in the 546 Among-habitat heterogeneity in vegetation structure (as well as seasonality) in our study 547 site is so prevalent (having a strong effect in animal-plant interactions as seen in Rico-Gray, 548 1993; Díaz-Castelazo et al., 2004; Sánchez-Galván et al., 2012; López-Carretero et al., 2014; and 549 Ramos-Robles, Andresen & Díaz-Castelazo, 2016). PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed This is clearly detected in the ant-plant 550 interaction pattern, in contrast to other studies where vegetation structure differences are not so 551 outstanding as have an effect in other ant-plant networks (Dáttilo et al., 2013b). Further evidence 552 of among-habitat heterogeneity translating to ant-plant network structure is provided in the PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 553 present study by the multivariate analysis. In this analysis one of the components explaining the 554 variance in the lack of independence among ant and plant species (NMDS2) displays habitats 555 following a decreasing humidity gradient, from MFE, followed by TSF-W and TSF-O, TSF-Y, 556 CDS and PDV. Indeed, open habitats at the study site, such as coastal dune scrub (CDS) and 557 pioneer dune vegetation (PDV) have the most extreme temperatures, solar radiation (Moreno- 558 Casasola, 1982; Moreno-Casasola & Travieso-Bello, 2006), and are exposed to continual 559 disturbance (López-Carretero et al., 2014) like sand movement, strong winds, and abrasion 560 (Pérez-Maqueo, 1995). 561 Our results showed that no neutral effects derived from variation in species abundances 562 are structuring the studied ant-plant network. Abundance of EFN-bearing plant species was 563 considered in our analysis but rendered no significant contribution to explain the frequency of 564 pairwise ant-plant interactions. Similar results were found for another ecological network at the 565 same study site such as a plant-herbivore network (López-Carretero et al. 2016), where network 566 parameters were not influenced by plant cover (abundance) but by biological and seasonality 567 aspects. In our study, although ant abundance was not included, we know from robust estimates 568 of ant density (honey baits) at the same periods of time and vegetation types that average ant 569 density is higher in open habitats than in shaded ones (Díaz-Castelazo et al., 2004). In other 570 studies of ant-plant interactions, the abundance of interacting species partially explain some 571 features of network structure (Vázquez et al. 2007; Dáttilo et al., 2014a). However, Dáttilo et al. 572 (2014a) show that although more abundant ant species interact with more plant species with 573 EFNs, information on the difference in abundance among interacting species was insufficient to 574 explain ant-plant network organization. That is, nestedness was higher in networks of ants and 575 plants with EFNs than that observed in networks of ants and plants without EFNs. Thus, the 561 Our results showed that no neutral effects derived from variation in species abundances 562 are structuring the studied ant-plant network. Abundance of EFN-bearing plant species was 563 considered in our analysis but rendered no significant contribution to explain the frequency of 564 pairwise ant-plant interactions. Similar results were found for another ecological network at the 565 same study site such as a plant-herbivore network (López-Carretero et al. Manuscript to be reviewed 576 differences in nestedness, connectance, and heterogeneity of interactions remained after 577 controlling for the effects of species richness structure. 578 Other potentially mutualistic networks have shown that species abundance or temporal 579 overlap is far from accurately predicting the frequency of pair-wise interactions (Jordano, 580 Bascompte & Olesen, 2006; Vázquez et al., 2009). Poisot, Stouffer & Gravel (2015) outline 581 several direct (abundance-based and trait-based) and indirect (biotic modifiers and indirect 582 effects of co-occurrence) effects to account for variation in interactions occurrence. Given that 583 perspective, at the sampling intensity and duration of our ant-plant interaction survey (this reflect 584 temporal and spatial variation; see Díaz-Castelazo et al. 2004), neither the abundance-based nor 585 the trait-based modifiers seems to be enough relevant to explain the variation in pairwise ant- 586 plant interactions, even if at other scales ant abundance could partially explain an overall 587 network pattern (Dáttilo et al., 2014a). In contrast, an indirect effect given by habitat structure 588 (biotic modifiers through co-occurrence, sensu Poisot, Stouffer & Gravel, 2015) more thoroughly 589 explains the quantitative interaction pattern at the present study. 576 differences in nestedness, connectance, and heterogeneity of interactions remained after 577 controlling for the effects of species richness structure. 576 differences in nestedness, connectance, and heterogeneity of interactions remained after 577 controlling for the effects of species richness structure. 576 differences in nestedness, connectance, and heterogeneity of interactions remained after 577 controlling for the effects of species richness structure. 590 The fact that neither the distribution of EFNs on plant organs nor the abundance of 591 extrafloral-nectary bearing plants at each vegetation type had a significant contribution to the 592 variation in the observed ant-plant association patterns, does not rule out its potential effect on 593 ant foraging patterns in other ecosystems or spatial scales (Dáttilo et al. 2013a, 2014b). At our 594 study site, besides the overwhelming evidence of seasonality and habitat heterogeneity (we did 595 find an effect of habitat structure in ant-plant interactions within the network), the high 596 occurrence frequency and seasonal activity of morphologically diverse EFNs at vegetation 597 associations (Díaz-Castelazo et al., 2004; Díaz-Castelazo et al., 2005) suggests a temporal 598 variation in benefits provided by ant visitors to EFN-bearing plants. Manuscript to be reviewed 2016), where network 566 parameters were not influenced by plant cover (abundance) but by biological and seasonality 567 aspects. In our study, although ant abundance was not included, we know from robust estimates 568 of ant density (honey baits) at the same periods of time and vegetation types that average ant 569 density is higher in open habitats than in shaded ones (Díaz-Castelazo et al., 2004). In other 570 studies of ant-plant interactions, the abundance of interacting species partially explain some 571 features of network structure (Vázquez et al. 2007; Dáttilo et al., 2014a). However, Dáttilo et al. 572 (2014a) show that although more abundant ant species interact with more plant species with 573 EFNs, information on the difference in abundance among interacting species was insufficient to 574 explain ant-plant network organization. That is, nestedness was higher in networks of ants and 575 plants with EFNs than that observed in networks of ants and plants without EFNs. Thus, the PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed Indeed, at the studied 590 The fact that neither the distribution of EFNs on plant organs nor the abundance of PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 599 habitats, several plant species receive anti-herbivory defense from ants foraging on EFNs 600 (Oliveira et al. 1999; Cuautle and Rico-Gray 2003; Chavarro-Rodríguez et al. 2013). There is 601 also evidence that the frequency of ants foraging on different EFN morphologies and 602 distributions among plant organs differs (Díaz-Castelazo, Chavarro-Rodríguez & Rico-Gray, 603 2017). Other anti-herbivory plant defenses of plant species at the study site have shown spatial 604 and temporal variation (López-Carretero et al., 2016; López-Carretero et al., 2018). Thus, 605 information on the contribution to plant fitness of the EFN occurring among plant organs (and 606 their temporal activity patterns) could shed light on the optimal defense-value of EFN resources 607 as an indirect defense (Holland, Chamberlain & Horn, 2009) mediated by ants, an issue not yet 608 explored at a community-level. 599 habitats, several plant species receive anti-herbivory defense from ants foraging on EFNs 600 (Oliveira et al. 1999; Cuautle and Rico-Gray 2003; Chavarro-Rodríguez et al. 2013). There is 601 also evidence that the frequency of ants foraging on different EFN morphologies and 602 distributions among plant organs differs (Díaz-Castelazo, Chavarro-Rodríguez & Rico-Gray, 603 2017). Other anti-herbivory plant defenses of plant species at the study site have shown spatial 604 and temporal variation (López-Carretero et al., 2016; López-Carretero et al., 2018). Thus, 605 information on the contribution to plant fitness of the EFN occurring among plant organs (and 606 their temporal activity patterns) could shed light on the optimal defense-value of EFN resources 607 as an indirect defense (Holland, Chamberlain & Horn, 2009) mediated by ants, an issue not yet 608 explored at a community-level. 609 For ant variables, although ant size (head length) was not a significant factor explaining 610 frequency of pairwise interactions, it was important in explaining other attributes such as species 611 degree within our mutualistic network. This may occur because competition among ant species 612 foraging at EFNs could vary with ant body size, size of ants contributing thus to the species 613 degree values (Chamberlain & Holland 2009). Larger ant species can forage over a greater area 614 than small species, and thus interact with more plant species; in addition, it has been shown that 615 recruitment of ant foragers to a resource is negatively correlated with ant body size (LeBrun, 616 2005). PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 622 and circumscribed EFNs that could provide resources for both dominant and non-competitive ant 623 species. However, ant invasive/tramp species in the study site (Solenopsis geminata, Wasmannia 624 auropunctata, Tetramorium spinosum, and Paratrechina longicornis) seem to have a slightly 625 different pattern of interaction from the other species (separate from the rest of ant species in the 626 interaction display), probably due to their ability to access new habitats or food resources (Ness 627 & Bronstein, 2004; Lach & Hooper-Bui, 2010). This makes sense in such a human-altered 628 ecosystem as La Mancha, that seems to rapidly reflect ant invasions. At smaller time-scales, in 629 the same study site ant invasiveness does not alter the core structure of the network (Falcão et al., 630 2017), despite of other possible functional effects in the community that are just about to be 631 explored. 632 633 Conclusions 634 Our extrafloral-nectary mediated ant-plant network was highly nested, non-modular, 635 showed high species strength for core species, low specialization or selectiveness, and higher 636 dependence of plants on ants. These results in accordance to a facultative mutualism scenario, 637 mainly considering that the core ant species in this interaction network are known as good plant- 638 defenders in general (Oliveira et al., 1999; Cuautle & Rico-Gray, 2003; Dáttilo et al., 2014c). 639 When simultaneously exploring plants, ants, and habitat attributes on a network-level and 640 fine-grain structure, the only factor that significantly affects the pair-wise interactions is habitat 641 heterogeneity in the vegetation structure (and distribution of EFN-bearing plant species). At our 642 study site this heterogeneity is so strong that is clearly detected in the ant-plant interaction 643 network patterns, both in network topology and in the fine-grain network structure provided by 622 and circumscribed EFNs that could provide resources for both dominant and non-competitive ant 623 species. However, ant invasive/tramp species in the study site (Solenopsis geminata, Wasmannia 624 auropunctata, Tetramorium spinosum, and Paratrechina longicornis) seem to have a slightly 625 different pattern of interaction from the other species (separate from the rest of ant species in the 626 interaction display), probably due to their ability to access new habitats or food resources (Ness 627 & Bronstein, 2004; Lach & Hooper-Bui, 2010). This makes sense in such a human-altered 628 ecosystem as La Mancha, that seems to rapidly reflect ant invasions. Manuscript to be reviewed That is, while ant body size increases, the number of recruiting foragers decreases, which 617 can lead to a body size-driven competition hierarchy in which larger ant species visit more plant 618 species. 619 Overall, behavioral dominance as a factor was not significant to explain variations in the 620 frequency of pair-wise interactions, possibly due to the spectrum of factors considered PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed In our study the 648 plant species in shaded habitats have disperse EFNs more frequently , while plants at open 649 habitats have circumscribed EFNs with most frequency. The latter EFNs are more structurally 650 complex glands (i.e. elevated or pit nectaries) and are more effectively protected against nectar 651 evaporation (Koptur, 1992; Nepi, 2007), which is important at these open, insolated, high- 652 temperature sites. 653 Non-neutral effects were detected in the ant-plant interacting community since EFN- 654 bearing plant abundance per se had no effects in the ant-plant interaction pattern. As we showed 655 before, more ecological/biological factors, such as habitat/vegetation structure, could affect 656 network structure. Thus, possible convergence effects of interacting species in open vs. shaded 657 habitats could be occurring presided by supergeneralist species and consequently, the possibility 658 of cascading coevolutionary events taking place. This may deserve further study considering 659 ecological/abiotic and coevolutionary contexts for mutualistic interaction networks (Guimarães 660 et al. 2017). 653 Non-neutral effects were detected in the ant-plant interacting community since EFN- 654 bearing plant abundance per se had no effects in the ant-plant interaction pattern. As we showed 655 before, more ecological/biological factors, such as habitat/vegetation structure, could affect 656 network structure. Thus, possible convergence effects of interacting species in open vs. shaded 657 habitats could be occurring presided by supergeneralist species and consequently, the possibility 658 of cascading coevolutionary events taking place. This may deserve further study considering 659 ecological/abiotic and coevolutionary contexts for mutualistic interaction networks (Guimarães 660 et al. 2017). Manuscript to be reviewed At smaller time-scales, in 629 the same study site ant invasiveness does not alter the core structure of the network (Falcão et al., 630 2017), despite of other possible functional effects in the community that are just about to be 631 explored. PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 644 the frequency of pair-wise interactions. This provides further evidence of abiotic factor influence 645 on facultative mutualism and biotic plant defense. 646 Habitat heterogeneity in vegetation structure and distribution of EFN-bearing plant 647 species suggest variability in plant strategies for biotic anti-herbivory defense. In our study the 648 plant species in shaded habitats have disperse EFNs more frequently , while plants at open 649 habitats have circumscribed EFNs with most frequency. The latter EFNs are more structurally 650 complex glands (i.e. elevated or pit nectaries) and are more effectively protected against nectar 651 evaporation (Koptur, 1992; Nepi, 2007), which is important at these open, insolated, high- 652 temperature sites. 653 Non-neutral effects were detected in the ant-plant interacting community since EFN- 654 bearing plant abundance per se had no effects in the ant-plant interaction pattern. As we showed 655 before, more ecological/biological factors, such as habitat/vegetation structure, could affect 656 network structure. Thus, possible convergence effects of interacting species in open vs. shaded 657 habitats could be occurring presided by supergeneralist species and consequently, the possibility 658 of cascading coevolutionary events taking place. This may deserve further study considering 659 ecological/abiotic and coevolutionary contexts for mutualistic interaction networks (Guimarães 660 et al. 2017). 661 662 Acknowledgements 663 We wish to acknowledge Mariana Cuautle, Rosa Linda Robles, and Gloria Castelazo for their 664 assistance during fieldwork Fernando Ortega helped with the characterization of extrafloral 644 the frequency of pair-wise interactions. This provides further evidence of abiotic factor influence 645 on facultative mutualism and biotic plant defense. 644 the frequency of pair-wise interactions. This provides further evidence of abiotic factor influence 645 on facultative mutualism and biotic plant defense. 646 Habitat heterogeneity in vegetation structure and distribution of EFN-bearing plant 647 species suggest variability in plant strategies for biotic anti-herbivory defense. In our study the 648 plant species in shaded habitats have disperse EFNs more frequently , while plants at open 649 habitats have circumscribed EFNs with most frequency. The latter EFNs are more structurally 650 complex glands (i.e. elevated or pit nectaries) and are more effectively protected against nectar 651 evaporation (Koptur, 1992; Nepi, 2007), which is important at these open, insolated, high- 652 temperature sites. 646 Habitat heterogeneity in vegetation structure and distribution of EFN-bearing plant 647 species suggest variability in plant strategies for biotic anti-herbivory defense. PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 666 manuscript at early stages. Exclusively CDC thanks for the achievement of this study to God 667 Almighty for the gifts of life, of Christ and his unchanging love and grace. 668 669 References 670 Almeida-Neto M, Guimarães P, Guimarães PRJr, Loyola RD, Ulrich W. 2008. A consistent 671 metric for nestedness analysis in ecological systems: reconciling concept and measurement. 672 Oikos 117:1227–1239. DOI:10.1111/j.2008.0030-1299.16644.x 673 Andersen AN. 1995. A classification of Australian ant communities, based on functional groups 674 which parallel plant life-forms in relation to stress and disturbance. Journal of Biogeography 675 22:15–29. 676 Andersen AN. 2000. A global ecology of rainforest ants: functional groups in relation to 677 environmental stress and disturbance. In: Agosti D, Majer JD, Alonso LE, Schultz TR, eds. 678 Ants: standard methods for measuring and monitoring biodiversity. . Washington: 679 Smithsonian Institution Press, 25–34. 680 Barber MJ. 2007. Modularity and community detection in bipartite networks. Physical Review E. 681 76:066102. 682 Bascompte J, Jordano P, Melián CJ, Olesen JM. 2003. The nested assembly of plant–animal 683 mutualistic networks. Proceedings of the National Academy of Sciences of the United States 684 of America 100:9383–9387. DOI:10.1073/pnas.1633576100. 685 Bascompte J, Jordano P, Olesen JM. 2006. Asymmetric coevolutionary networks facilitate 686 biodiversity maintenance. Science 312:431-433. 666 manuscript at early stages. Exclusively CDC thanks for the achievement of this study to God 667 Almighty for the gifts of life, of Christ and his unchanging love and grace. 669 References 670 Almeida-Neto M, Guimarães P, Guimarães PRJr, Loyola RD, Ulrich W. 2008. A consistent 671 metric for nestedness analysis in ecological systems: reconciling concept and measurement. 672 Oikos 117:1227–1239. DOI:10.1111/j.2008.0030-1299.16644.x 673 Andersen AN. 1995. A classification of Australian ant communities, based on functional groups 674 which parallel plant life-forms in relation to stress and disturbance. Journal of Biogeography 675 22:15–29. 673 Andersen AN. 1995. A classification of Australian ant communities, based on functional groups 674 which parallel plant life-forms in relation to stress and disturbance. Journal of Biogeography 675 22:15–29. 676 Andersen AN. 2000. A global ecology of rainforest ants: functional groups in relation to 677 environmental stress and disturbance. In: Agosti D, Majer JD, Alonso LE, Schultz TR, eds. 678 Ants: standard methods for measuring and monitoring biodiversity. . Washington: 679 Smithsonian Institution Press, 25–34. 680 Barber MJ. 2007. Modularity and community detection in bipartite networks. Physical Review E. 681 76:066102. 662 Acknowledgements 663 We wish to acknowledge Mariana Cuautle, Rosa Linda Robles, and Gloria Castelazo for their 664 assistance during fieldwork. Fernando Ortega helped with the characterization of extrafloral 665 nectaries. Paulo R. Guimarães and Pedro Jordano made suggestions that improved this PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 682 Bascompte J, Jordano P, Melián CJ, Olesen JM. 2003. The nested assembly of plant–animal 683 mutualistic networks. Proceedings of the National Academy of Sciences of the United States 684 of America 100:9383–9387. DOI:10.1073/pnas.1633576100. 684 of America 100:9383–9387. DOI:10.1073/pnas.1633576100. 685 Bascompte J, Jordano P, Olesen JM. 2006. Asymmetric coevolutionary networks facilitate 686 biodiversity maintenance. Science 312:431-433. 687 Bascompte J. 2009. Disentangling the web of life. Science 325:416-419. mpte J. 2009. Disentangling the web of life. Science 325:416-419. 688 Bascompte J. 2010. Structure and dynamics of ecological networks. Science 329:765–766. PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 689 Bastolla U, Fortuna MA, Pascual-García A, Ferrera A, Luque B, Bascompte J. 2009. The 690 architecture of mutualistic networks minimizes competition and increases biodiversity. 691 Nature 458(7241):1018. 693 Interhabitat differences in the reduction of herbivore damage. Ecology 57:815–820. 694 Blüthgen N, Verhaagh M, Goitía W, Jaffé K, Morawetz W, Barthlott W. 2000. How plants shape 695 the ant community in the Amazonian rainforest canopy: the key role of extrafloral nectaries 696 and homopteran honeydew. Oecologia 125:229-240. 697 Blüthgen N, Fiedler K. 2004. Competition for composition: lessons from nectar-feeding ant 698 communities. Ecology 85:1479–1485. 699 Blüthgen N, Fründ J, Vázquez DP, Menzel F. 2008. What do interaction network metrics tell us 700 about specialization and biological traits? Ecology 89:3387–3399. 700 about specialization and biological traits? Ecology 89:3387–3399. 700 about specialization and biological traits? Ecology 89:3387–3399. 701 Blüthgen N. 2010. Why network analysis is often disconnected from community ecology: A 702 critique and an ecologist's guide. Basic and Applied Ecology 11:85-195. 703 Borg I, Groenen P. 1997. Modern Multidimensional Scaling. Springer, New York. 704 Cagnolo L, Salvo A, Valladares G. 2011. Network topology: patterns and mechanisms in 705 plant‐herbivore and host‐parasitoid food webs. Journal of Animal Ecology 80(2): 342-351. 705 plant‐herbivore and host‐parasitoid food webs. Journal of Animal Ecology 80(2): 342-351. 706 Carnicer J, Jordano P, Melián CJ. 2009. The temporal dynamics of resource use by frugivorous 707 birds: a network approach. Ecology 90:1958-1970. 707 birds: a network approach. Ecology 90:1958-1970. 08 Castillo-Campos G, Travieso-Bello AC. 2006. La flora. In: Moreno-Casasola 709 Veracruzanos: la costa de La Mancha. Instituto de Ecología, A.C., México. Instituto de 710 Ecología, A.C., México. 711 Češka, A. (1966). Estimation of the mean floristic similarity between and within sets of PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed Manuscript to be reviewed 712 vegetational relevés. Folia Geobotanica et Phytotaxonomica, 1(2), 93-100. 12 vegetational relevés. Folia Geobotanica et Phytotaxonomica, 1(2), 93-100 713 Chamberlain SA, Holland JN 2009. Body size predicts degree in ant-plant mutualistic networks. 714 Functional Ecology 23: 196-202. 715 Chavarro-Rodríguez N, Díaz-Castelazo C, Rico-Gray V. 2013. Characterization and functional 716 ecology of the extrafloral nectar of Cedrela odorata in contrasting growth environmets in 717 central Veracruz, Mexico. Botany 91, 695-701. 716 ecology of the extrafloral nectar of Cedrela odorata in contrasting growth environmets in 717 central Veracruz, Mexico. Botany 91, 695-701. 718 Cohen JE, Jonsson T, Müller CB, Godfray HCJ, Savage VM. 2005. Body sizes of hosts and 719 parasitoids in individual feeding relationships. Proceedings of the National Academy of 720 Sciences of the United States of America 102:684–689. 718 Cohen JE, Jonsson T, Müller CB, Godfray HCJ, Savage VM. 2005. Body sizes of hosts and 719 parasitoids in individual feeding relationships. Proceedings of the National Academy of 721 Cuautle M, Rico-Gray V. 2003. The effect of wasps and ants on the reproductive success of the 722 extrafloral nectaried plant Turnera ulmifolia L. Turneraceae. Functional Ecology 17:417- 723 423. 724 Cuautle M, Rico-Gray V, Díaz-Castelazo C. 2005. Effects of ant behaviour and presence of 725 extrafloral nectaries on seed dispersal of the Neotropical myrmecochore Turnera ulmifolia 726 L. Turneraceae. Biological Journal of the Linnean Society 86:67-77. 727 Dáttilo W, Guimarães PR, Izzo TJ. 2013b. Spatial structure of ant–plant mutualistic networks. 728 Oikos 122:1643-1648. 729 Dáttilo W, Rico-Gray V, Rodrigues DSJ, Izzo T. 2013a. Soil and vegetation features determine 730 the nested pattern of ant–plant networks in a tropical rainforest. Ecological Entomology 731 38:374-380. 732 Dáttilo W, Sánchez-Galván I, Lange D, Del-Claro K, Rico-Gray V. 2014d. Importance of 733 interaction frequency in analysis of ant-plant networks in tropical environments. Journal of 734 Tropical Ecology 30:165-168. 732 Dáttilo W, Sánchez-Galván I, Lange D, Del-Claro K, Rico-Gray V. 2014d. Importance of 733 interaction frequency in analysis of ant-plant networks in tropical environments. Journal of 734 Tropical Ecology 30:165-168. 732 Dáttilo W, Sánchez-Galván I, Lange D, Del-Claro K, Rico-Gray V. 2014d. Importance of 733 interaction frequency in analysis of ant-plant networks in tropical environments. Journal of 734 Tropical Ecology 30:165-168. PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 735 Dáttilo W, Marquitti FMD, Guimarães PR, Izzo TJ. 2014a. The structure of ant–plant ecologica 736 networks: is abundance enough? Ecology 95:475-485. 737 Dáttilo W, Fagundes R, Gurka CAQ, Silva MSA, Vieira MCL, Izzo TJ, Díaz-Castelazo C, Del- 738 Claro K, Rico-Gray V. 2014b. Individual-based ant-plant networks: diurnal-nocturnal 739 structure and species-area relationship. PLoS ONE 9:e99838. 740 Dáttilo W, Díaz-Castelazo C, Rico-Gray V. 2014c. Ant dominance hierarchy determines the 741 nested pattern in ant–plant networks. Biological Journal of the Linnean Society 113:405- 742 414. 743 Davidson DW, Cook SC, Snelling RR. 2004. Liquid-feeding performances of ants (Formicidae 744 ecological and evolutionary implications. Oecologia 139:255–266. 745 Díaz–Castelazo C, Rico–Gray V, Oliveira PS, Cuautle M. 2004. Extrafloral nectary-mediated 746 ant-plant interactions in the coastal vegetation of Veracruz, Mexico: richness, occurrence, 747 seasonality and ant foraging patterns. Ecoscience 11:472–481. 748 Díaz-Castelazo C, Rico-Gray V, Ortega F, Ángeles G. 2005. Morphological and secretory 749 characterization of extrafloral nectaries in plants of coastal Veracruz, Mexico. Annals of 750 Botany 96:1175-1189. 751 Díaz-Castelazo C, Guimarães PR, Jordano P, Thompson JN, Marquis RJ, Rico-Gray V. 2010. 752 Changes of a mutualistic network over time: reanalysis over a 10-year period. Ecology 753 91:793–801. 754 Díaz-Castelazo C, Sánchez-Galván IR, Guimarães PR, Raimundo RLG, Rico-Gray V. 2013. 755 Long term temporal variation in the organization of an ant plant network Annals of Botan 735 Dáttilo W, Marquitti FMD, Guimarães PR, Izzo TJ. 2014a. The structure of ant–plant ecological 736 networks: is abundance enough? Ecology 95:475-485. 735 Dáttilo W, Marquitti FMD, Guimarães PR, Izzo TJ. 2014a. The structure of ant–plant ecological 736 networks: is abundance enough? Ecology 95:475-485. 737 Dáttilo W, Fagundes R, Gurka CAQ, Silva MSA, Vieira MCL, Izzo TJ, Díaz-Castelazo C, Del- 738 Claro K, Rico-Gray V. 2014b. Individual-based ant-plant networks: diurnal-nocturnal 739 structure and species-area relationship. PLoS ONE 9:e99838. 737 Dáttilo W, Fagundes R, Gurka CAQ, Silva MSA, Vieira MCL, Izzo TJ, Díaz-Castelazo C, Del- 738 Claro K, Rico-Gray V. 2014b. Individual-based ant-plant networks: diurnal-nocturnal 739 structure and species-area relationship. PLoS ONE 9:e99838. 740 Dáttilo W, Díaz-Castelazo C, Rico-Gray V. 2014c. Ant dominance hierarchy determines the 741 nested pattern in ant–plant networks. Biological Journal of the Linnean Society 113:405- 742 414. 741 nested pattern in ant–plant networks. Biological Journal of the Linnean Society 113:405- 742 414. 743 Davidson DW, Cook SC, Snelling RR. 2004. Liquid-feeding performances of ants (Formicidae): 744 ecological and evolutionary implications. Oecologia 139:255–266. Manuscript to be reviewed 758 ecology of extrafloral nectar production and associated ant assemblages in Mexican 759 landscapes. In: Oliveira PS, Koptur S. Ant-Plant Interactions: Impacts of Humans on 760 Terrestrial Ecosystems. Cambridge University Press. Pp. 179-199. 758 ecology of extrafloral nectar production and associated ant assemblages in Mexican 759 landscapes. In: Oliveira PS, Koptur S. Ant-Plant Interactions: Impacts of Humans on 760 Terrestrial Ecosystems. Cambridge University Press. Pp. 179-199. 762 Vegetation Science 14:927–930. DOI:10.1111/j.1654-1103.2003.tb02228.x. 762 Vegetation Science 14:927–930. DOI:10.1111/j.1654-1103.2003.tb02228.x. 763 Dormann CF, Fruend J, Blüthgen N, Gruber B. 2009. Indices, graphs and null models: analyzing 763 Dormann CF, Fruend J, Blüthgen N, Gruber B. 2009. Indices, graphs and null models: analyzing 764 bipartite ecological networks. The Open Ecology Journal 2:7–24. 765 Dormann CF, Gruber B. 2009. Package ‘Bipartite’: visualizing bipartite networks and calculating 765 Dormann CF, Gruber B. 2009. Package ‘Bipartite’: visualizing bipartite networks and calculating 766 some ecological indices R statistical soft are ‘R gro p’ 765 Dormann CF, Gruber B. 2009. Package Bipartite : visualizing bipartite networks and calculating 766 some ecological indices. R statistical software. ‘R group’. 766 some ecological indices. R statistical software. ‘R group’. 767 Dormann CF. 2011. How to be a specialist? quantifying specialization in pollination networks. 768 Network Biology 1:1–20. URL:http://www.iaees.org/publications/journals/nb/articles/2011- 769 1(1)/How-to-be-a-specialist.pdf 769 1(1)/How-to-be-a-specialist.pdf 770 Dornhaus A, Powell S. 2010. Foraging and defence strategies in ants. In: Lach L, Parr CL, 771 Abbott KL eds. Ant ecology, Oxford, UK: Oxford University Press, 210–230. 772 Falcão JC, Dáttilo W, Díaz-Castelazo C, Rico-Gray V. 2017. Assessing the impacts of tramp and 773 invasive species on the structure and dynamic of ant-plant interaction networks. Biological 774 Conservation 209:517–523. 772 Falcão JC, Dáttilo W, Díaz-Castelazo C, Rico-Gray V. 2017. Assessing the impacts of tramp and 773 invasive species on the structure and dynamic of ant-plant interaction networks. Biological 772 Falcão JC, Dáttilo W, Díaz-Castelazo C, Rico-Gray V. 2017. Assessing the impacts of tramp and 773 invasive species on the structure and dynamic of ant-plant interaction networks. Biological 774 Conservation 209:517–523. 775 Frank MK, Fonseca CR. 2005. Abiotic factors and the conditional outcome of an ant-plant 776 mutualism. Ecology 86:2117–2126. 775 Frank MK, Fonseca CR. 2005. Abiotic factors and the conditional outcome of an ant-plant 776 mutualism. Ecology 86:2117–2126. 777 Guimarães PR, Guimarães P. 2006. Improving the analyses of nestedness for large sets of 778 matrices. Environmental Modelling and Software 21:1512-1513. 778 matrices. Environmental Modelling and Software 21:1512-1513. Manuscript to be reviewed 745 Díaz–Castelazo C, Rico–Gray V, Oliveira PS, Cuautle M. 2004. Extrafloral nectary-mediated 746 ant-plant interactions in the coastal vegetation of Veracruz, Mexico: richness, occurrence, 747 seasonality and ant foraging patterns Ecoscience 11:472 481 745 Díaz–Castelazo C, Rico–Gray V, Oliveira PS, Cuautle M. 2004. Extrafloral nectary-mediated 746 ant plant interactions in the coastal egetation of Veracr Me ico: richness occ rrence 746 ant-plant interactions in the coastal vegetation of Veracruz, Mexico: richness, occurrence, 747 seasonality and ant foraging patterns. Ecoscience 11:472–481. 748 Díaz-Castelazo C, Rico-Gray V, Ortega F, Ángeles G. 2005. Morphological and secretory 749 characterization of extrafloral nectaries in plants of coastal Veracruz, Mexico. Annals of 750 Botany 96:1175-1189. 751 Díaz-Castelazo C, Guimarães PR, Jordano P, Thompson JN, Marquis RJ, Rico-Gray V. 2010. 752 Changes of a mutualistic network over time: reanalysis over a 10-year period. Ecology 751 Díaz-Castelazo C, Guimarães PR, Jordano P, Thompson JN, Marquis RJ, Rico-Gray V. 2010. 751 Díaz-Castelazo C, Guimarães PR, Jordano P, Thompson JN, Marquis RJ, Rico-Gray V. 2010. 752 Changes of a mutualistic network over time: reanalysis over a 10-year period. Ecology 753 91:793–801. 754 Díaz-Castelazo C, Sánchez-Galván IR, Guimarães PR, Raimundo RLG, Rico-Gray V. 2013. 755 Long–term temporal variation in the organization of an ant-plant network. Annals of Botany 756 111:1285-1293. 757 Díaz-Castelazo C, Chavarro-Rodríguez N, Rico-Gray V. 2017. Interhabitat variation in the 757 Díaz-Castelazo C, Chavarro-Rodríguez N, Rico-Gray V. 2017. Interhabitat variation in the PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed Manuscript to be reviewed 779 Guimarães PR, Rico-Gray V, Oliveira PS, Izzo TJ, dos Reis SF, Thompson JN. 2007. Interaction 780 intimacy affects structure and coevolutionary dynamics in mutualistic networks. Current PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 782 Guimarães PR, Jordano P, Thompson JN. 2011. Evolution and coevolution in mutualistic 783 networks. Ecology Letters 14: 877–885. 784 Guimarães PR, Pires MM, Jordano P, Bascompte J, Thompson JN. 2017. Indirect effects drive 785 coevolution in mutualistic networks. Nature 550: 511–514. 785 coevolution in mutualistic networks. Nature 550: 511–514. 786 Guimerà R, Sales‐Pardo M, Amaral LAN. 2004. Modularity from fluctuations in random graphs 786 Guimerà R, Sales‐Pardo M, Amaral LAN. 2004. Modularity from fluctuations in random graphs 787 and complex networks. Physical Review E 70:025101. 787 and complex networks. Physical Review E 70:025101. 788 Guimerà R, Amaral LAN. 2005. Functional cartography of complex metabolic networks. Nature 789 433:895–900. 790 Herrera CM, Pellmyr O. 2009. Plant animal interactions: an evolutionary approach. John Wiley 791 & Sons. 792 Holland JN, Chamberlain SA, Horn KC. 2009. Optimal defence theory predicts investment in 793 extrafloral nectar resources in an ant-plant mutualism. Journal of Ecology 97: 89–96. 792 Holland JN, Chamberlain SA, Horn KC. 2009. Optimal defence theory predicts investment in 793 t fl l t i t l t t li J l f E l 97 89 96 792 Holland JN, Chamberlain SA, Horn KC. 2009. Optimal defence theory predicts investment in 793 extrafloral nectar resources in an ant-plant mutualism. Journal of Ecology 97: 89–96. 794 Holway DA, Lach L, Suarez AV, Tsutsui ND, Case1 TJ. 2002. The causes and consequences of 795 ant invasions. Annual Review of Ecology and Systematics 33:181–233. 796 Horvitz CC, Schemske DW. 1984. Effects of ants and an ant tended herbivore on seed 797 production of a Neotropical herb. Ecology 65:1369-1378. 798 Ings TC, Montoya JM, Bascompte J, Blüthgen N, Brown L, Dormann CF, Edwards F, Figueroa 799 D Jacob U Jones JI Lauridsen RB Ledger ME Lewis HM Olesen JM van Veen FJF 798 Ings TC, Montoya JM, Bascompte J, Blüthgen N, Brown L, Dormann CF, Edwards F, Figueroa 799 D, Jacob U, Jones JI, Lauridsen RB, Ledger ME, Lewis HM, Olesen JM, van Veen FJF, 800 Warren PH, Woodward G. 2009. Ecological networks–beyond food webs. Journal of 801 Animal Ecology 78:253–269. 798 Ings TC, Montoya JM, Bascompte J, Blüthgen N, Brown L, Dormann CF, Edwards F, Figueroa 799 D, Jacob U, Jones JI, Lauridsen RB, Ledger ME, Lewis HM, Olesen JM, van Veen FJF, 800 Warren PH, Woodward G. 2009. Ecological networks–beyond food webs. Journal of 801 Animal Ecology 78:253–269. Manuscript to be reviewed 03 plant–animal interactions. Ecology Letters 6:69-81. 803 plant–animal interactions. Ecology Letters 6:69-81. 804 Jordano P, Bascompte J, Olesen JM. 2006. Ecological consequences of complex topology and 805 nested structure in pollination webs. In: Waser N.W. & Ollerton J. Plant-Pollinator 806 Interactions: From Specialization to Generalization. The University of Chicago Press. 807 Junker RR, Blüthgen N, Brehm T, Binkenstein J, Paulus J, Martin Schaefer H, Stang M. 2013. 808 Specialization on traits as basis for the niche‐breadth of flower visitors and as structuring 809 mechanism of ecological networks. Functional Ecology 27(2):329-341. 810 Kaspari M, Weiser MD. 1999. The size-grain hypothesis and interspecific scaling in ants. 811 Functional Ecology 13:530–538. p g yp p g 811 Functional Ecology 13:530–538. 812 Koptur, S. 1992. Interactions between insects and plants mediated by extrafloral nectaries. In: 813 Bernays E. Insect-Plant Interactions (4), p. 85-132. Insect/Plant Interactions. CRC Press. 814 Lach L, & Hooper-Bui, LM. 2010. Consequences of ant invasions. In: Lach L, Parr CL, Abbott 815 KL eds. Ant ecology, Oxford, UK: Oxford University Press, 261-286. 812 Koptur, S. 1992. Interactions between insects and plants mediated by extrafloral nectaries. In: 813 Bernays E Insect-Plant Interactions (4) p 85-132 Insect/Plant Interactions CRC Press 812 Koptur, S. 1992. Interactions between insects and plants mediated by extrafloral nectaries. In: 813 Bernays E. Insect-Plant Interactions (4), p. 85-132. Insect/Plant Interactions. CRC Press. 814 Lach L, & Hooper-Bui, LM. 2010. Consequences of ant invasions. In: Lach L, Parr CL, Abbott 812 Koptur, S. 1992. Interactions between insects and plants mediated by extrafloral nectaries. In: 813 Bernays E. Insect-Plant Interactions (4), p. 85-132. Insect/Plant Interactions. CRC Press. 814 Lach L, & Hooper-Bui, LM. 2010. Consequences of ant invasions. In: Lach L, Parr CL, Abbott 813 Bernays E. Insect-Plant Interactions (4), p. 85-132. Insect/Plant Interactions. CRC Press. 814 Lach L, & Hooper-Bui, LM. 2010. Consequences of ant invasions. In: Lach L, Parr CL, Abbott 815 KL eds. Ant ecology, Oxford, UK: Oxford University Press, 261-286. 814 Lach L, & Hooper Bui, LM. 2010. Consequences of ant invasions. In: Lach L, Parr CL, Abbott 815 KL eds. Ant ecology, Oxford, UK: Oxford University Press, 261-286. 815 KL eds. Ant ecology, Oxford, UK: Oxford University Press, 261-286. 816 Lange D, Dáttilo W, Del-Claro K. 2013. Influence of extrafloral nectary phenology on ant–plant 817 mutualistic networks in a neotropical savanna. Ecological Entomology 38:463-469. 816 Lange D, Dáttilo W, Del-Claro K. 2013. Manuscript to be reviewed 802 Jordano P, Bascompte J, Olesen JM. 2003. Invariant properties in coevolutionary networks of 802 Jordano P, Bascompte J, Olesen JM. 2003. Invariant properties in coevolutionary networks of 802 Jordano P, Bascompte J, Olesen JM. 2003. Invariant properties in coevolutionary networks of PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewe PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed Influence of extrafloral nectary phenology on ant–plant li i k i i l E l i l E l 38 463 469 816 Lange D, Dáttilo W, Del-Claro K. 2013. Influence of extrafloral nectary phenology on ant–plant 817 mutualistic networks in a neotropical savanna. Ecological Entomology 38:463-469. 818 LeBrun EG. 2005. Who is the top dog in ant communities? Resources, parasitoids, and multiple 819 competitive hierarchies. Oecologia 142:643–652. 820 López-Carretero A, Díaz-Castelazo C, Boege K, Rico-Gray V. 2014. Evaluating the Spatio- 821 Temporal Factors that Structure Network Parameters of Plant-Herbivore Interactions. PLoS 822 ONE 9: e110430. 823 Majer JD. 1993. Comparison of the arboreal ant mosaic in Ghana, Brazil, Papua New Guinea 824 and Australia – its structure and influence on arthropod diversity. In: LaSalle J, Gauld ID 825 eds. Hymenoptera and biodiversity, Wallingford, UK: CAB International, 115-141. PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 826 Marquitti FMD, Guimaraes PR, Pires MM, Bittencourt LF. 2014. MODULAR: Software for the 827 autonomous computation of modularity in large network sets. Ecography 37:221–224. 828 Martínez-Adriano CA, Aguirre-Jaimes A, Díaz-Castelazo C. 2016. Floristic survey of flowering 829 plants in a tropical coastal ecosystem in Veracruz, Mexico. Botanical Sciences 94:185–197. 830 Martínez-Adriano CA. 2017. Fenología reproductiva de angiospermas, producción de flores y 831 visitantes florales en un ecosistema costero en Veracruz, México. Ph.D. Thesis, Instituto de 832 Ecología, A. C. 826 Marquitti FMD, Guimaraes PR, Pires MM, Bittencourt LF. 2014. MODULAR: Software for the 827 autonomous computation of modularity in large network sets. Ecography 37:221–224. 826 Marquitti FMD, Guimaraes PR, Pires MM, Bittencourt LF. 2014. MODULAR: Software for the 827 autonomous computation of modularity in large network sets. Ecography 37:221–224. 828 Martínez-Adriano CA, Aguirre-Jaimes A, Díaz-Castelazo C. 2016. Floristic survey of flowering 829 plants in a tropical coastal ecosystem in Veracruz, Mexico. Botanical Sciences 94:185–197. 830 Martínez-Adriano CA. 2017. Fenología reproductiva de angiospermas, producción de flores y 831 visitantes florales en un ecosistema costero en Veracruz, México. Ph.D. Thesis, Instituto de 832 Ecología, A. C. 831 visitantes florales en un ecosistema costero en Veracruz, México. Ph.D. Thesis, Instituto de 832 Ecología, A. C. 833 McGlynn TP. 1999. The worldwide transfer of ants: geographical distribution and ecological 834 invasions. Journal of Biogeography 26:535–548. 835 Memmott J, Waser NM, Price MV. 2004. Tolerance of pollination networks to species 836 extinctions. Proceedings of the Royal Society of London B: Biological Sciences, 271(1557): 837 2605-2611. 835 Memmott J, Waser NM, Price MV. 2004. Tolerance of pollination networks to species 836 extinctions. Proceedings of the Royal Society of London B: Biological Sciences, 271(1557): 835 Memmott J, Waser NM, Price MV. 2004. Tolerance of pollination networks to species 836 extinctions. Proceedings of the Royal Society of London B: Biological Sciences, 271(1557): 837 2605-2611. 838 Moreno-Casasola P. 1982. Ecología de la vegetación de dunas costeras: factores 839 físicos Biotica 7:577 602 840 Moreno-Casasola P, Travieso-Bello AC. 2006. Las playas y dunas. In: Moreno-Casasola ed. 841 Entornos Veracruzanos: la costa de La Mancha Instituto de Ecología A C México 840 Moreno-Casasola P, Travieso-Bello AC. 2006. Las playas y dunas. In: Moreno-Casasola ed. 840 Moreno-Casasola P, Travieso-Bello AC. 2006. Las playas y dunas. In: Moreno-Casasola ed. 841 Entornos Veracruzanos: la costa de La Mancha. Instituto de Ecología, A.C., México. 842 Nepi, M. 2007. Nectary structure and ultrastructure. Manuscript to be reviewed In Nectaries and nectar. pp. 129-166. 843 Springer, Dordrecht. 844 Ness JH, Bronstein JL. 2004. The effects of invasive ants on prospective ant mutualists. 845 Biological Invasions 6:445–461. 846 Oksanen J. 2009. Vegan: an introduction to ordination. R statistical software. ‘R group’. 846 Oksanen J. 2009. Vegan: an introduction to ordination. R statistical software. ‘R group’. 847 Olesen JM, Bascompte J, Dupont YL, Jordano P. 2007. The modularity of pollination networks. 848 Proceedings of the National Academy of Sciences of the United States of America PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 850 Oliveira PS, Rico-Gray V, Díaz-Castelazo C, Moreno-Casasola -Guevara C. 1999. Interaction 851 between ants, extrafloral nectaries and insect herbivores in Neotropical coastal sand dunes: 852 herbivore deterrence by visiting ants increases fruit set in Opuntia stricta Cactaceae. 853 Functional Ecology 13:623-631. 854 Parr CL, Gibb H. 2010. Competition and the role of dominant ants. In: Lach L, Parr CL, Abbott 854 Parr CL, Gibb H. 2010. Competition and the role of dominant ants. In: Lach L, Parr CL, Abbott 855 KL eds. Ant ecology, Oxford, UK: Oxford University Press, 77–96. 855 KL eds. Ant ecology, Oxford, UK: Oxford University Press, 77–96. 855 KL eds. Ant ecology, Oxford, UK: Oxford University Press, 77–96. 856 Pérez-Maqueo O. 1995. Análisis del efecto de los disturbios en la dinámica de la playa de El 857 Morro de La Mancha Veracruz México DF: UNAM 856 Pérez-Maqueo O. 1995. Análisis del efecto de los disturbios en la dinámica de la playa de El 856 Pérez-Maqueo O. 1995. Análisis del efecto de los disturbios en la dinámica de la playa de El 857 Morro de La Mancha, Veracruz. México DF: UNAM. 857 Morro de La Mancha, Veracruz. México DF: UNAM. 858 Pimm SL, Lawton JH. 1980. Are food webs divided into compartments?. The Journal of Animal 859 Ecology 49:879–898. 860 Poisot T, Stouffer DB, Gravel D. 2015. Beyond species: why ecological interaction networks 861 vary through space and time. Oikos 124(3): 243-251. 862 Quinn GP, Keough MJ. 2002. Experimental design and data analysis for biologists. Cambridge 863 University Press. 864 R Core Team. 2014. R: A language and environment for statistical computing. R Foundation for 865 Statistical Computing, Vienna, Austria. URL: http://www.R-project.org/. 866 Radhika V, Kost C, Mithöfer A, Boland W. 2010. Regulation of extrafloral nectar secretion by 867 jasmonates in lima bean is light dependent. Proceedings of the National Academy of S f h U d S f A 107(4) 17228 17233 j g p g f y f 868 Sciences of the United States of America 107(4):17228–17233. 868 Sciences of the United States of America 107(4):17228–17233. 869 Ramos-Robles M, Andresen E, Díaz-Castelazo C. 2016. Temporal changes in the structure of a 870 plant-frugivore network are influenced by bird migration and fruit availability. PeerJ 871 4:e2048. doi:10.7717/peerj.2048. PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 872 Rezende, EL, Jordano P, Bascompte J. 2007. Effects of phenotypic complementarity and 873 phylogeny on the nested structure of mutualistic networks. Oikos 116(11): 1919-1929. 874 Rezende EL, Lavabre JE, Guimarães PR, Jordano P, Bascompte J. 2007. Non-random 875 coextinctions in phylogenetically structured mutualistic networks Nature 448:9257–9259. 876 Rico-GrayV, Barber JT, Thien LB, Ellgaard EG, Toney JJ. 1989. An Unusual Animal-Plant 877 Interaction: Feeding of Schomburgkia tibicinis (Orchidaceae) by ants. American Journal of 878 Botany 76:603-608. 872 Rezende, EL, Jordano P, Bascompte J. 2007. Effects of phenotypic complementarity and 873 phylogeny on the nested structure of mutualistic networks. Oikos 116(11): 1919-1929. 874 Rezende EL, Lavabre JE, Guimarães PR, Jordano P, Bascompte J. 2007. Non-random 875 coextinctions in phylogenetically structured mutualistic networks Nature 448:9257–9259. 876 Rico-GrayV, Barber JT, Thien LB, Ellgaard EG, Toney JJ. 1989. An Unusual Animal-Plant 877 Interaction: Feeding of Schomburgkia tibicinis (Orchidaceae) by ants. American Journal of 878 Botany 76:603-608. 879 Rico-Gray V. 1993. Use of plant-derived food resources by ants in the dry tropical lowlands of 880 coastal Veracruz, Mexico. Biotropica 25:301-315. 881 Rico-Gray V, Oliveira PS. 2007. The ecology and evolution of ant-plant interactions. University 882 of Chicago Press. 883 Rico-Gray V, Díaz-Castelazo C, Ramírez-Hernández A, Guimarães PR, Holland JN. 2012. 884 Abiotic factors shape temporal variation in the structure of an ant–plant network. 885 Arthropod–Plant Interactions 6:289-295. 886 Rudgers JA, 2004. Enemies of herbivores can shape plant traits: selection in a facultative ant– 887 plant mutualism. Ecology 85:192–201. 886 Rudgers JA, 2004. Enemies of herbivores can shape plant traits: selection in a facultative ant– 887 plant mutualism Ecology 85:192 201 887 plant mutualism. Ecology 85:192–201. 888 Rudgers JA, Gardener MC. 2004. Extrafloral nectar as a resource mediating multispecies 889 interactions. Ecology 85:1495–1502. 888 Rudgers JA, Gardener MC. 2004. Extrafloral nectar as a resource mediating multispecies 889 interactions. Ecology 85:1495–1502. 890 Sánchez-Galván IR, Díaz-Castelazo C, Rico-Gray V. 2012. Effect of Hurricane Karl on a plant– 891 ant network occurring in coastal Veracruz, Mexico. Journal of Tropical Ecology 28:603- 892 609. 893 Schultz TR, McGlynn TP. 2000. The interactions of ants with other organisms. In: Agosti D, 894 Majer J.D. Alonso LE, Schultz TR. Ants: standard methods for measuring and monitoring PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 895 biodiversity. Washington: Smithsonian Institution Press,35–44. 896 Stang M, Klinkhamer PGL, van der Meijden E, Memmott J. 2006. Size constraints and flower 897 abundance determine the number of interactions in a plant-flower visitor web. Oikos 898 112:111-121. 897 abundance determine the number of interactions in a plant-flower visitor web. Oikos 898 112:111-121. 898 112:111-121. 899 Stang M. 2007. Asymmetric specialization and extinction risk in plant-flower visitor webs: a 899 Stang M. 2007. Asymmetric specialization and extinction risk in plant-flower visitor webs: a 900 matter of morphology or abundance? Oecologia 151:442-453. 900 matter of morphology or abundance? Oecologia 151:442-453. 901 Thébault E, Fontaine C. 2010. Stability of ecological communities and the architecture of 902 mutualistic and trophic networks. Science 329:853–856. 903 Thompson JN 2005. The geographic mosaic of coevolution. University of Chicago Press, 904 Chicago. 905 Vázquez DP, Aizen MA. 2004. Asymmetric specialization: a pervasive feature of plant– 906 pollinator interactions. Ecology 85:1251–1257. 907 Vázquez DP, Morris WF, Jordano P. 2005. Interaction frequency as a surrogate for the total 908 effect of animal mutualists on plants. Ecology Letters 8:1088–1094. 907 Vázquez DP, Morris WF, Jordano P. 2005. Interaction frequency as a surrogate for the total 908 effect of animal mutualists on plants. Ecology Letters 8:1088–1094. 908 effect of animal mutualists on plants. Ecology Letters 8:1088–1094. 909 Vázquez DP, Melián CJ, Williams NM, Blüthgen N, Krasnov BR, Poulin R. 2007. Species 909 Vázquez DP, Melián CJ, Williams NM, Blüthgen N, Krasnov BR, Poulin R. 2007. Species 910 abundance and asymmetric interaction strength in ecological networks. Oikos 116:1120– 909 Vázquez DP, Melián CJ, Williams NM, Blüthgen N, Krasnov BR, Poulin R. 2007. Species 910 abundance and asymmetric interaction strength in ecological networks. Oikos 116:1120– 911 1127. 910 abundance and asymmetric interaction strength in ecological networks. Oikos 116:1120– 911 1127. 911 1127. 912 Vázquez DP, Blüthgen N, Cagnolo L, Chacoff NP. 2009. The determinants of patterns in 913 t li ti t k i A l f B t 103 1445 1457 912 Vázquez DP, Blüthgen N, Cagnolo L, Chacoff NP. 2009. The determinants of patterns in 912 Vázquez DP, Blüthgen N, Cagnolo L, Chacoff NP. 2009. The determinants of patterns in 913 mutualistic networks: a review. Annals of Botany 103:1445-1457. 913 mutualistic networks: a review. Annals of Botany 103:1445-1457. 914 Vázquez DP, Chacoff NP, Cagnolo L. 2009. Evaluating multiple determinants of the structure of 915 plant–animal mutualistic networks. Ecology 90:2039–2046. Manuscript to be reviewed 914 Vázquez DP, Chacoff NP, Cagnolo L. 2009. Evaluating multiple determinants of the structure of 915 plant–animal mutualistic networks. Ecology 90:2039–2046. 916 Wäckers FL, Bonifay C. 2004. How to be sweet? Extrafloral nectar allocation by Gossypium 917 hirsutum fits optimal defense theory predictions. Ecology 85:1512–1518. 17 hirsutum fits optimal defense theory predictions. Ecology 85:1512–1518. PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) 918 Yamawo A, Hada Y. 2010. Effects of light on direct and indirect defences against herbivores of 919 young plants of Mallotus japonicus demonstrate a trade-off between two indirect defence 920 traits. Annals of Botany 106:143–148. Manuscript to be reviewed Manuscript to be reviewed 918 Yamawo A, Hada Y. 2010. Effects of light on direct and indirect defences against herbivores of 919 young plants of Mallotus japonicus demonstrate a trade-off between two indirect defence 920 traits. Annals of Botany 106:143–148. 921 922 PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 923 Table 1. EFN-bearing plant species within the network and its attributes. Plant attributes considered also 924 in Figure 2 are: EFN= Distribution of extrafloral nectaries within a plant species (‘C’ are circumscribed 925 glands and ‘D’ are disperse glands). Habitat= distribution of plant species between habitats with 926 contrasting vegetation structure (‘S’ is shaded vegetation and ‘O’ is open vegetation). Abundance = 927 percentage cover of EFN-bearing plant species. 923 Table 1. EFN-bearing plant species within the network and its attributes. Plant attributes considered also 924 in Figure 2 are: EFN= Distribution of extrafloral nectaries within a plant species (‘C’ are circumscribed 925 glands and ‘D’ are disperse glands). Habitat= distribution of plant species between habitats with 926 contrasting vegetation structure (‘S’ is shaded vegetation and ‘O’ is open vegetation). Abundance = 927 percentage cover of EFN-bearing plant species. Plant species Plant species code Distribution of EFNs Habitat structure Abundance (%cover) Cordia spinescens CorSpi D S 38.833 Turnera ulmifolia TurUlm C O 6.66 Crotalaria indica CrotIn C O 12.38 Cedrela odorata CedOdo D S 36.143 Callicarpa acuminata CallAc D B 68.797 Caesalpinia crista CaeCri C O 27.15 Bidens pilosa BidPil C S 27.95 Canavalia rosea CanRos C O 76.057 Calopogonium caeruleum CalCae C O 16.85 Terminalia catappa TerCat C S 0.35 Senna occidentalis SenOcc C S 3.717 Opuntia stricta OpuStri D O 64.35 Hibiscus tiliaceus HibTill C O 2.4 Amphilophium paniculatum AmphPa D O 17.55 Ipomoea pescaprae IpoPes C O 49.1 Conocarpus erectus ConEre C S 16.383 Ficus obtusifolia FicObt C S 8.15 Cornutia grandiflora CorGra D O 2.5 Manuscript to be reviewed PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed Macroptilium atropurpureum MacAtr C O 16.3 Cissus rhombifolia CisRho C O 3.55 Ipomoea sp. IpoSp. C S 12.167 Mansoa hymenaea ManHym C S 16.3 Tabebuia rosea TabRos D S 6.66 Acacia macracantha AcaMac C B 2.75 Trichilia havanensis TriHav C S 28.33 Arundo donax AruDon C O 151.66 Petrea volubilis PetVol D O 74.1 Chamaecrista chamaecristoides ChaCha C O 32.4 Iresine celosia IreCel C O 16.55 Cordia dentata CorDen D S 3.615 Bunchosia lindeliana BunLin C S 1.7 929 930 931 932 933 934 935 936 937 938 939 PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be review Macroptilium atropurpureum MacAtr C O 16.3 Cissus rhombifolia CisRho C O 3.55 Ipomoea sp. IpoSp. C S 12.167 Mansoa hymenaea ManHym C S 16.3 Tabebuia rosea TabRos D S 6.66 Acacia macracantha AcaMac C B 2.75 Trichilia havanensis TriHav C S 28.33 Arundo donax AruDon C O 151.66 Petrea volubilis PetVol D O 74.1 Chamaecrista chamaecristoides ChaCha C O 32.4 Iresine celosia IreCel C O 16.55 Cordia dentata CorDen D S 3.615 Bunchosia lindeliana BunLin C S 1.7 PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 940 Table 2. Ant species within the network and its attributes. Ant attributes considered also in Figure 2 are: 941 invasive status = status as invasive/tramp ant species (INV or NO), Dominance = hierarchies of 942 behavioral dominance (from the most dominant to the least) are: A= Dominant Dolichoderine, B= 943 Generalized Myrmicine, C= Subordinate Camponotini, D= Tropical Climate Specialists, E= 944 Opportunistic, F= Specialist Predators; Head length= length (mm) from head apex to anterior clypeal 945 margin of species (minor worker). 940 Table 2. Ant species within the network and its attributes. Ant attributes considered also in Figure 2 are: 941 invasive status = status as invasive/tramp ant species (INV or NO), Dominance = hierarchies of 942 behavioral dominance (from the most dominant to the least) are: A= Dominant Dolichoderine, B= 943 Generalized Myrmicine, C= Subordinate Camponotini, D= Tropical Climate Specialists, E= 944 Opportunistic, F= Specialist Predators; Head length= length (mm) from head apex to anterior clypeal 945 margin of species (minor worker). 946 Ant species Ant species code Invasive status Dominance hierarchy Head length Camponotus planatus CamPla NO C 1.198 Camponotus mucronatus CamMu NO C 1.418 Camponotus atriceps CamAt NO C 1.946 Azteca sp. 1 AztSp NO A 1.471 Paratrechina longicornis ParLo INV E 0.638 Tetramorium spinosum TetSpi INV E 0.968 Cephalotes minutus CepMin NO D 1.155 Dorymyrmex bicolor DorBi NO A 0.973 Pseudomyrmex gracilis PseGra NO F 1.738 Monomorium cyaneum MonCy NO B 0.482 Camponotus mucronatus hirsutinasus CamHi NO C 1.076 Pachycondyla villosa PachVi NO F 2.880 Forelius analis ForAna NO A 0.631 Crematogaster brevispinosa CreBre NO B 1.031 Pheidole sp. PheSp NO B 0.553 Solenopsis geminata SolGe INV D 0.684 Wassmannia auropunctata WasAu INV D 0.479 Pseudomyrmex PseEje NO F 0.800 PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed ejectus Pseudomyrmex brunneus PseBru NO F 0.768 947 948 949 FIGURE LEGENDS 950 951 Figure 1. Quantitative mutualistic networks between EFN-bearing plants (lower trophic level, 952 green nodes) and ant visitor species (higher trophic level, red nodes). Blue-colored nodes depict 953 species constituting the core of the network. Species codes as in Tables 1 and 2. 954 955 Figure 2. Ordination of NMDS representing the assemblage of interactions given the ant-plant 956 distances (Bray-Curtis) at the network. Plant species in black; ant species in red. Species names 957 appear as in Tables 1 and 2: at the ordination, first plant species (P1), second plant species (P2) 958 and so on, correspond to the first plant species, and the second plant species in Table 1 and so on. 959 First ant species (A1), second ant species (A2) and so on, correspond to the first ant species, and 960 the second ant species in Table 2 and so on. NMDS Stress = 0.17 (fourth iteration) indicates a 961 good two-dimensional solution of the ordination suitably representing ant-plant assemblage 962 dissimilarity; this configuration also has very low residuals (max res = 0.0004) showing a good 963 concordance between the calculated dissimilarities and the distances among objects. Non- 964 overlapping ellipses (i.e. orange and green) circle the attribute (factor) that significantly 965 explained (r2 =0.24, P = 0.005) the pairwise interaction pattern (habitat types). 966 0.768 F NO PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed Manuscript to be reviewed 970 971 Figure 1. 970 972 PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) Manuscript to be reviewed 975 976 977 978 979 980 PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019) 5 6 7 976 PeerJ reviewing PDF \| (2015:11:7572:4:1:NEW 21 Nov 2019)
https://openalex.org/W2607624821	http://centreofexcellence.net/index.php/JSS/article/view/jss.2017.6.2.319.326/2017.6.2.319.326	English	null	Instructional leadership in enhancing pedagogic teachers’ competence at three universities guided schools in Malang City	Journal of social sciences	2,017	cc-by	3,738	Journal of Social Sciences (COES&RJ-JSS) ISSN (E): 2305-9249 ISSN (P): 2305-9494 Publisher: Centre of Excellence for Scientific & Research Journalism, COES&RJ LLC Online Publication Date: 1st April 2017 Online Issue: Volume 6, Number 2, April 2017 http://centreofexcellence.net/J/JSS/JSS%20Mainpage.htm Journal of Social Sciences (COES&RJ-JSS) ISSN (E): 2305-9249 ISSN (P): 2305-9494 Publisher: Centre of Excellence for Scientific & Research Journalism, COES&RJ LLC Online Publication Date: 1st April 2017 Online Issue: Volume 6, Number 2, April 2017 http://centreofexcellence.net/J/JSS/JSS%20Mainpage.htm Instructional leadership in enhancing pedagogic teachers’ competence at three universities guided schools in Malang City Abdul Tedy Candidate of Doctor, EducationManagement, Post Graduate School, State University of Malang, Malang, East Java, Indonesia Ahmad Sonhadji K.H. Professor, Education Management, Post Graduate School, State University of Malang, Malang, East Java, Indonesia Imron Arifin Associate Professor, Education Management, Post Graduate School, State University of Malang, Malang, East Java, Indonesia Ali Imron Professor, Education Management, Post Graduate School, State University of Malang, Malang, East Java, Indonesia Ab t t Abstact: This research was aimed at finding out the following focuses as universities guided models at thee high schools: (1) instructional leadership, (2) Teachers’ pedagogic competences, (3) instructional leadership behavior in the teachers’ pedagogic enhancement, (4) roles of the guiding universities in enhancing the instructional leadership and teachers’ peagogic competences. Data were collected by using three techniques including (1) indepht interview, (2) participative observation, and (3) documentation. Data were analized in two steps of individual case analysis and cross case analysis. The individual case analysis was done by (1) data reduction, (2) data display, and (3) conclusion, and the cross case analysis was done through constant comparative analysis. The validity and reliability was measured by examining the credibility, transferability, and confirmability. This research revealed that (1) the instructional leadership covered vission, mission, learning or instructional services, motivation, community relation, governmental relation, and the guiding universities relation, (2) the teachers’ competences covered ability to understand learners, instructional planning and implementation, instructional evaluation, and students’ potential development, (3) the instructional leadership behaviors covered management engineer, comunicator, clinical practitioner, role model, and high priest, (4) the roles of guiding universities covered guiding and developing: teachers, educational staffs, learners, parents, and schools’ committees Keywords: This work is licensed under a Creative Commons Attribution 4.0 International License. Journal of Social Sciences (COES&RJ-JSS), 6(2), pp. 319-326 Journal of Social Sciences (COES&RJ-JSS), 6(2), pp. 319-326 Introduction Education plays primary roles in developing educated and characterized Indonesian human resources. Qualified human resources can only be resulted from quality education with professional educators. The Law no 20, 2003 of the Indonesia Republic stated that educators are professional work forces having the key main duties to plan and execute learning processes, evaluate learning outcomes, guide and train learners, research, and work for community services. Teachers with pedagogic, professional, personal, and social competences have been expected to be the primary driving force of instructional and learning processes and to be open for changes. Educators must not only be smart, but also adaptive to changes. University guided schools are those immersing in the format of partnership education conducted by schools, universities, and government. Such partnership program is aimed at enahncing thr education quality and school effectiveness as expected by the community. Several prominent universities in Indonesia have involved in such program. In Malang City, East Java, there have been three high schools experiencing this program, which are Brawijaya Smart School Senior High School, State University of Malang Laboratory Senior High School, and Widyagama Senior High School. This research was aimed at (1) analyzing and describing the instructional leadership at those three schools, (2) analyzing and describing the teachers’ pedagogic competences at those three schools, (3) finding out and describing effective instructional leadership towards the teachers’ competences inhancement at those three schools. (4) finding and describing the roles of guiding universities in enhancing instuctional leadership and teachers’ pedgogic competences at those three schools as universited guided school models. p g Keywords: Instructional leadership, Principal, Teachers’ pedagogic competence Citation: Tedy, Abdul; Sonhadji, Ahmad K.H.; Arifin, Imron; Imron, Ali (2017) ; Instructional leadership in enhancing pedagogic teachers’ competence at three universities guided schools in Malang City; Journal of Social Sciences (COES&RJ-JSS), Vol.6, No.2, pp:319- 326. This work is licensed under a Creative Commons Attribution 4.0 International License. This work is licensed under a Creative Commons Attribution 4.0 International License. Instructional leadership in enhancing pedagogic teahcers’ competence…. The cross case analysis procedures was done by following Yin (1984:107) with expalanation building tehnique. This was guided towards explaining meaningful causal fenomena which was presented naratively. The procedures of this cross case analysis were: (1) Comparing conceptual findings of each individual case by using conceptual inductive approach, (2) Results of these analysis were used to develop propositions of the overal cases which were in line with the empirical data, (3) mengevaluasi kesesuaian proposisi dengan fakta yang diacu; (4) Reconstructing the relevant propositions based on the empirical data found in each individual case, and (5) repeating the process as far as these were needed. Holistically and integratively with the consideration of relevant data focusing on the research objectives, the data collection techniques were conducted by doing: (1) indepth interview; (2) participant observation; and (3) study of documents. Data were analized qualitatively within individual case and cross cases. The validity and reliability was measured in terms of: (1) the credibility by consistantly comparing facts among resources and methods, (2) tranferability, meliputi; (1) kredibilitas, yaitu pengecekan kredibilitas atau derajat kepercayaan by making detail explanation on the research focus, (3) dependabilitas, by auditing, checking mistakes and inconsistances, and (4) confirmability by auditing done at the same time with the dependability. Method This research used descriptive qualitative multicase studies. Multicase studies designed was chosen due to the nature of this research to comprehend two or more subjects and data storage places. Such design made effort in examing several identified subjects and comparing those similarities and differences. This research had the characteristics of (1) progressing in the scientific background, (2) having the researcher as the main instrument, and (3) employing inductive data analysis analisis (Moleong, 2007). Locations of this research were: (1) Brawijaya Smart School Senior High School, Malang; (2) State University of Malang Laboratory Senior High School, Malang, and (3) Widyagama Senior High School, Malang. Data analysis of this research comprised (1) individual case analysis, and (2) cross cases analysis. The individual case analysis was analysis within the individual schools of each location. The analysis was done constantly at the same time using data colleting technique as advised by Bogdan dan Biklen (1982) and practiceds by Mantja (1989) covering the following procedures: (1) limiting the scope of analysis, (2) desiding the type of assessments, (3) developing analysis questions, (4) planning data collection steps by considering the previous observation; (5) writing comments of the observants on the appearing ideas, (6)writing self managed memo on the issues being assessed, and (7) exploring relevant resources while having the research. 320 Journal of Social Sciences (COES&RJ-JSS), 6(2), pp. 319-326 Journal of Social Sciences (COES&RJ-JSS), 6(2), pp. 319-326 At the fourth focus, it was found out that the roles of guiding univeristies had relationship with stakeholders which empowered the universities in guiding schools for the instuctional leadership of principals and enhancing teachers’ pedagogic competence. It was found out that principals in those three schools conducted formal relationship among teachers, educational staffs, and school communities by having formal meeting and informal meeting in social activities. Towards the students potential development, it was found out the the principals provided facilities needed by the students both for learning and for healthy activities, provided developmental programs in terms of the academic and non-academic activities, and provided guidance and counselling. Principals ensured the parents’ involvement and provided regulation involving parents. Principals turned out to involve alumni to improve school facilities. Such involvement evidenced to improve the schools. Principals turned out to involve school supervisors for monitoring, supervision, evaluation, reports, and continuous professional development. There evidenced the productive relationship among goovernment, schools, and the guiding universities characterized by intensive communication and community involvement. This geared to the fact that the community also provided positive contribution in the forms of fund and non materials contribution. There was also found out that the guided universities model also proved of having the involvement of other educational organization for operational educational improvement. From the findings, there have been obvious that the instructional leadership involved the (1) community expectation values, (2) unique instructional leadership characteristics, (3) unique guided school model; and (4) school organization structures. These have been important factors in the instructional leadership. These can be productive parts of fostering leadership responsibility in enhancing the teachers’ profesionalism and instructional processes at schools (Gorton & Schneider, 1991; Hallinger & Leithwood, 2002). Such instructional leadership practices conforms the notion of instructional leadership mentioned byHeck & Rossow (1990), which is, all principals’ actions influence the students’ learning outcomes improvement directly and indirectly. Furthermore, Gorton & Schneider (1991) said that Instructional leadership is basically operational actions to attain better working environment, satisfactory performance of teachers, and good outcomes of learning. The instructional leadership actions, of course, also deal with learners. Newman, King & Young (2000) says that the instructinal leadership involve principals to form professional learning community to develop productive environment, teachers’ satisfaction, and students’ enhancement in their learning outcomes (Eggen & Kauchak 2004). Such situation also touches classroom situation. Results and discussion Research on the cross individual case for the first focus, which was the instructional leadership at the three Senior High Schools were: (1) charismatic leaders influencing the followers based on the followers perception on the supernatural ability, but not on the formal authority, (2) collaboration, which were the ability to determine vision, providing energies, having sense of togetherness, optimizing potentials, and making the follwers aware of becoming one team, and productive performance, (3) innovative, which resulted new paradigm and products as ell as new practical ideas, (4) creative having ideas, opinions, and innovation for the improvement of the schools, (5) visioner having future perspective which enabled people to productively contribute efforts for the success of the schools, (6) inspirative givinng opportunities to the followers to actively participate in works and thinking as well as to innovate and produce new ideas for refference of the schooldevelopment, and (7) collaboration which was the ability to determine vision, providing energy, fostering sense of collaboration and togethernes, optimaizing potentials, and making every followers aware of mking contribution in the good performance. At the second focus, the teachers’ competences turned out to cover: (1) pedagogic competence, which indicated the ability to manage instructional learning including understanding learners, planning and executing instruction, developing learners, and evaluating the instruction (2) personal competence, which indicated the charactesitics of teachers as being firm, stable, mature, wise, commanding, faithful, and exemplary, (3) professional competence, which was teachers mastery in knowledge of teaching, knowledge of subject studies, and skills of leading students to learn, and (4) social competence which indicated the ability to communicate and interact among educational entities. At the third focus, it was found out that the instructional behavior in three schools could be summarized as having:(1) technical behavior; (2) human behavior; (3) educational behavior; (4) symbolic behavior, dan; (5) cultural behavior. 321 Instructional leadership in enhancing pedagogic teahcers’ competence…. The instructional leadership practiced at the three univerity guided schools also conforms the instructional leadership elements stated by the Learning Centered Leadership Policy (2008), which include curriculum, learning process, assessment, teachers appraishals, and customer success provision. The universities guided schools had excellences in being effective schools. The universities targetted to guide the schools in 7 national schools standard including content standard, competence standard, process standard, teachers and educational staff standard, educational facilities standard, and funding standard. In complying these standards, the three schools employed goor instructional leadership covering (1) understanding the principle duties of the principals as leaders; (2) developing teachers’ potential to effectively work for instructional activities, (3) developing the learners’ poential, (4) being fully respobsible for at school activities, and (5) collaborating with stakeholders to implement education. In working for these, apparently the principals were in line with the notion of principals as instructional leaders as stated by Kemendiknas (2010) covering (1) understanding the roles of principals, (2) actuating the principals responsibility accountably, (3) doing work professionally, (4) managing sustainable quality of work and performance, (5) improving teachers’ pedagogic competence, and (6) improving learninf process and outcomes. It has also evidenced that teachers’ pedagogic competence improved. The pedagogic competence is the main competence element which has to be paid attention. This supports the attainment of learning outcomes (Hasanuddin & Nurmaliah, 2012). Among others, this is the most important providing as the driving force of other competencies, which include pedagogic competence, personal competence, sosial competence, and professional competence (Peraturan Pemerintah Nomor 74 Tahun 2008). Results of the third focus concerning instructional aspects showed that the three Senior High Schools implemented effective and efficient instructional management. The principals used technical behavior, human behavior, educational behavior, symbolic behavior, and cultural behavior. The technical behaviors were evidenced with the implementation of the education management principles to manage the effective and efficient instructional and learning processes. Such behaviors were shown with the discipline monitoring and supervision in all aspects of teachers functions including praparation, instruction, evaluation, feedback, and folow up. The rincipals could successfully provide easy access and assistance to the teachers and made teachers aware of how to use the school time effectively. The educational behaviors appeared to be having the principals provided clinical superision and assistance which can be called as clinical practitioner (Ubben & Hughes, 1992, Sergiovanni, 1991). Journal of Social Sciences (COES&RJ-JSS), 6(2), pp. 319-326 Sergiovani (2009) says that: “to extent to which principal focus directly on teaching and learning, the importance of increasing student achievement, curriculum and assessment, and the development of improved instructional program”. The instructional leadership of the three schools turned out to significantly contribute to the improvement of the students’ achievements. Such results showed that the instructional leadership complied the 5 aspects of leadership which are instructional and learning, collaboration, students’ achievvement analysis, teachers development, and curriculum renewal, instruction, and evaluation (Lunenburg & Irby, 2006). These are conducted to attain the vision, mision, and goals of the schools and to develop the so-called learning schools (Kemendiknas, 2010). 322 Instructional leadership in enhancing pedagogic teahcers’ competence…. The principals had the capability of diagnosing learning problems and developing learning program fostering students to learn productively with better outcomes. These schools were guided by the different universities with different members of faculties and background, education based and non-education based. The human relation behaviors of the three Senior High Schools Principals turned out to be evidenced in their democratic ways of leading the school community to solve problems. They often walked through the teachers’ rooms and classrooms and communicate collegiately. They also discussed ideas on the spot. It was obvious that most of the Brawijaya Smart School Senior High School had principals developed from their teachers. Such practices conforms the notion of participatory management (Ubben, 323 Journal of Social Sciences (COES&RJ-JSS), 6(2), pp. 319-326 Huges, and Sergiovanni, 1992). All principals of these trhee schools employed human enginner leadership approach. Being effective leaders wirth positive behaviors can foster individual motivation and team work collaboration to achieve the school goals (Muliati, 2010). Principals who are able to create conducive atmosfir for learners and teachers to develop their personal potential and professionalism can fulfill the community expectation (Dubin,1991). Such practices can produce succesful-school. Such principals, according to Bernard, in McPherson (1986), are characterized by having no ulterior motives, eager to coaborate in team work eager to communicate, having been authorized,having been smart in taking decition, having balancing dynamics, and being responsible. The evidence of the guiding universities’ roles in succeeding to mentor and develop all school community members can be prooved as having successful indicators in making principals form effective school community and develop successful network with stakeholders (Gurr, et all, 2005). Conclusion This research concluded that (1) community expetation valuesand instructional leadership were the principals actions to create productive and satisfactory work environement and to create learning effectiveness for students to have high achievements. One of the factors, the community chose one of the three schools were the fact that the schools delivered educational services as they expected, that the schools were good schools, and the schools made their graduates become accepted in higher institutions; (2) they have different unique characteristics that enabled them to succeed their schools as leaders. The three schools were guided by different universities with different main majors, education based and non-education based. However, they had similar outputs of the teachers’ competence improvement and effective instructional leadership practices. As they were guided by different universities, such as the Brawijaya Smart School Senior High School guided by the University of Brawijaya, the State University of Malang Laboratory Senior High School was guided by State University of Malang, and Widyagama Senior High School was guided by the University of Widyagaman. They had diferencs in their organizational structures. The similarities turned out to focus on the instructional leadership practices, teachers’competencies enhancement, leadership behavior, and roles of the guiding universities. The leadership behavior turned out to be technical behavior, human relation, educational behavior, symbolc behavior, and cultural behavior. There were stakehoders empowered the universities and the schools including teachers and educational staffs, learners, parents, alumni, supervisors, government, public community, and other educational organizations. It is suggested that the principals were to maintain the quality of the schools withh sustainable strategies, to make effective supervision, to enlarge teachers’ knowlege and to enhance teachers’ competence, to conduct benchmarking program for teachers, and to empower alumni. For the universities, it is suggested to consider the schools’ excellences, teachers commitment, students’ potential, and success factors of the schools in managing the partnership programs. To the government, it is suggested to pay attention to the 324 Mulyasa, E. 2007. Standar Kompetensi dan Sertifikasi Guru. Bandung: Remaja Rosdakarya. References References Arifin, I. 2008. Evaluasi Pembelajaran. Bandung: Remaja Rosdakarya. Arifin, I. 2010. Kepemimpinan Kepala PAUD dalam Inovasi Pembelajaran: Studi \TK Anak Saleh. Yogyakarta: Aditya Media. Cotton, K. 2003. Principals and Student Achievement. Virginia USA: ASCD. Depdiknas. 2004. Pedoman Sekolah Standar Nasional. Jakarta: Direktorat PLP, \Ditjen Dikdasmen, Depdiknas. Depdiknas. 2008. Kurikulum Tingkat Satuan Pendidikan Jakarta: Dikmenum. Arifin, I. 2008. Evaluasi Pembelajaran. Bandung: Remaja Rosdakarya. Arifin, I. 2010. Kepemimpinan Kepala PAUD dalam Inovasi Pembelajaran: Studi \TK A k S l h Y k t Adit M di gy y Cotton, K. 2003. Principals and Student Achievement. Virginia USA: ASCD. Depdiknas. 2004. Pedoman Sekolah Standar Nasional. Jakarta: Direktorat PLP, \Ditjen Dikdasmen, Depdiknas. Depdiknas. 2008. Kurikulum Tingkat Satuan Pendidikan Jakarta: Dikmenum. Depdiknas. Direktorat Jenderal Peningkatan Mutu Pendidik dan Tenaga Kependidikan 2010. Pedoman Pelaksanaan Penilaian Kinerja Guru (PK Guru). Jakarta: Kementerian Pendidikan Nasional. Eggen, P & Kauchak, D. 2004. Educational Psychology Windows on Classroom, Sixth Edition. New Jersey. Pearson Prentice-Hall, inc. Fattah, N. 2004. Konsep Manajemen Berbasis Sekolah (MBS) dan Dewan S k l h B d P k B i Q i Fattah, N. 2004. Konsep Manajemen Berbasis Sekolah (MBS) dan Dewan Sekolah. Bandung: Pustaka Bani Quraisy. Ghufron, A. 2008. Kompetensi Guru Sekolah Dasar. Yogyakarta: Fakultas Ilmu Sekolah. Bandung: Pustaka Bani Quraisy. Ghufron, A. 2008. Kompetensi Guru Sekolah Dasar. Yogyakarta: Fakultas Ilmu Pendidikan UNY. Glickman, C. D. 2002. Leadership for Learning. Virginia USA. ASCD. Gomes, S. 2013. Quality Management Philosophies. (Online),(http://xisspm. mfiles.wordpress.com/2011/07/chap2-quality-management-philosophy.pdf), retrieved on 29 Agutus 2013. Gurr, C.D., Gordon P.S., & Gordor, R. 2010. Supervision and Instructional Leadership. Boston USA: Pearson. Hasanuddin & Nurmaliah, C. 2013. Kompetensi Pedagogik Guru Biologi yang Telah Lulus Sertifikasi di SMA Negeri Kota Banda Aceh. Darussalam Banda Aceh: Program Studi Pendidikan Biologi FKIP, Universitas Syiah Kuala. g g y Kumars. D. 2010. Sistem Pendidikan Dasar dan Menengah dan Pendidikan Tinggi Suatu Perbandingan di Beberapa Negara. Jakarta: Depdikbud, Dikti, P2LPTK Lie A. 2012. Cooperative Learning: Mempraktekkan Cooperative Learning di Ruang-Ruang Kelas. Jakarta: Grasindo. Lipham J M et al 2008 The Principalship: Concepts Competences and Cases Kumars. D. 2010. Sistem Pendidikan Dasar dan Menengah dan Pendidikan Tinggi Suatu Perbandingan di Beberapa Negara. Jakarta: Depdikbud, Dikti, P2LPTK Kumars. D. 2010. Sistem Pendidikan Dasar dan Menengah dan Pendidikan Kumars. D. 2010. Sistem Pendidikan Dasar dan Menengah dan Pendidikan Tinggi Suatu Perbandingan di Beberapa Negara. Jakarta: Depdikbud, Dikti, P2LPTK Lie A. 2012. Cooperative Learning: Mempraktekkan Cooperative Learning di g g Lipham, J.M.et al. Instructional leadership in enhancing pedagogic teahcers’ competence…. educational administration, educational management, and specifically the recruitment and man power development. To researchers it is suggested that this research can be a reference to conduct further or other researchers. References 2008. The Principalship: Concepts, Competences, and Cases. New York: Logman, Inc Lipham, J.M.et al. 2008. The Principalship: Concepts, Competences, and Cases. New York: Logman, Inc Lunenburg, F.C., & Irby, B.J. 2006. The Principalship: Vision to Action. Wadsworth: Cengange Learning. Lunenburg, F.C., & Irby, B.J. 2006. The Principalship: Vision to Action. Wadsworth: Cengange Learning. Mantja, W. 2003. Etnografi: Desain Penelitian Kualitatif dan Manajemen Pendidikan. Malang: Wineka Media. Mantja, W. 2005. Kerangka Acuan Penulisan Modul Strategi Pengelolaan Pendidikan Universitas Terbuka. Jakarta: PAU-UT. Mantja, W. 2008. Profesionalisasi Tenaga Kependidikan: Manajemen dan Moleong, L.J. 1989. Metodologi Penelitian Kualitatif. Bandung: Remaja Rosda Karya. Mulyasa, E. 2007. Standar Kompetensi dan Sertifikasi Guru. Bandung: Remaja Rosdakarya. 325 Journal of Social Sciences (COES&RJ-JSS), 6(2), pp. 319-326 Nawawi, H. 2008. Administrasi Pendidikan. Jakarta: Gunung Agung. Sergiovanni, T. J., McCarthy, M. M., & Fowler, F. C. 2009. Educational \Governance and Adiminstration. USA: Pearson Education,Inc. Sonhadji, A. 2003. Alternatif Penyempurnaan Pembaharuan Penyelenggaraan Pendidikan di Sekolah Menengah Kejuruan Makalah. Jakarta: Depdiknas. Sulistiyorini. 2009. Peranan kepala Sekolah Sebagai Pemimpin Pembelajaran. Jakarta: Taa’lum. Suriansyah, A. & Aslamiah. 2007. Strategi Pembelajaran Anak Usia Dini. Banjarmasin: Comdes. Uji Kompetensi Guru (UKG) Tahun 2012. Kementerian Pendidikan dan Kebudayaan. (Online), (sergur.kemendiknas.go.id), diakses 11 Oktober 2013. Wahyudi. 2010. Kepemimpinan Kepala Sekolah dalam Organisasi Pembelajar (Learning Organization). Bandung: Alfabeta. Yin, R.K. 2002. Case Study Research: Design and Methods. Washington DC: Crounus Corporations. Nawawi, H. 2008. Administrasi Pendidikan. Jakarta: Gunung Agung. Sergiovanni, T. J., McCarthy, M. M., & Fowler, F. C. 2009. Educational \Governance and Adiminstration. USA: Pearson Education,Inc. Sonhadji, A. 2003. Alternatif Penyempurnaan Pembaharuan Penyelenggaraan Pendidikan di Sekolah Menengah Kejuruan Makalah. Jakarta: Depdiknas. Sulistiyorini. 2009. Peranan kepala Sekolah Sebagai Pemimpin Pembelajaran. Jakarta: Taa’lum. Suriansyah, A. & Aslamiah. 2007. Strategi Pembelajaran Anak Usia Dini. Banjarmasin: Comdes. Uji Kompetensi Guru (UKG) Tahun 2012. Kementerian Pendidikan dan Kebudayaan. (Online), (sergur.kemendiknas.go.id), diakses 11 Oktober 2013. Wahyudi. 2010. Kepemimpinan Kepala Sekolah dalam Organisasi Pembelajar (Learning Organization). Bandung: Alfabeta. Yin, R.K. 2002. Case Study Research: Design and Methods. Washington DC: Crounus Corporations. 326
https://openalex.org/W2898436590	http://juser.fz-juelich.de/record/858692/files/acp-18-15345-2018.pdf	English	null	Peroxy acetyl nitrate (PAN) measurements at northern midlatitude mountain sites in April: a constraint on continental source–receptor relationships	Atmospheric chemistry and physics	2,018	cc-by	17,620	Peroxy acetyl nitrate (PAN) measurements at northern midlatitude mountain sites in April: a constraint on continental source–receptor relationships Fiore (amﬁore@ldeo.columbia.edu) Received: 27 January 2018 – Discussion started: 26 February 2018 Revised: 28 August 2018 – Accepted: 1 October 2018 – Published: 25 October 2018 Received: 27 January 2018 – Discussion started: 26 February 2018 Revised: 28 August 2018 – Accepted: 1 October 2018 – Published: 25 October 2018 Atmos. Chem. Phys., 18, 15345–15361, 2018 https://doi.org/10.5194/acp-18-15345-2018 © Author(s) 2018. This work is distributed under the Creative Commons Attribution 4.0 License. Peroxy acetyl nitrate (PAN) measurements at northern midlatitude mountain sites in April: a constraint on continental source–receptor relationships Stevenson11, Sophie Szopa24, Christoph Zellweger23, and Guang Zeng25 1Department of Earth and Environmental Science, Columbia University, Palisades, NY 10964, USA 2Lamont-Doherty Earth Observatory of Columbia University, Palisades, NY 10964, USA 3Department of Atmospheric Science, Colorado State University, Fort Collins, CO 80521, USA y y y, , , 3Department of Atmospheric Science, Colorado State University, Fort Collins, CO 80521, USA 4Institute for Atmospheric and Climate Science, ETH Zürich, Switzerland 5Lancaster Environment Centre, Lancaster University, Lancaster, LA1 4YQ, UK 6School of STEM, University of Washington, Bothell, WA 98011, USA 6School of STEM, University of Washington, Bothell, WA 98011, USA Department of Atmospheric Science, University of Washington, Seattle, WA 98195, USA 7Department of Atmospheric Science, University of Washington, Seattle, WA 98195, Lawrence Livermore National Laboratory, Livermore, CA 94550, USA 8Lawrence Livermore National Laboratory, Livermore, CA 94550, USA Department of Meteorology, University of Reading, Reading, RG6 6BB, UK 9Department of Meteorology, University of Reading, Reading, RG6 6BB, UK 10European Commission, Joint Research Centre, Ispra, 21027, Italy 10European Commission, Joint Research Centre, Ispra, 21027, Italy 11School of GeoSciences, The University of Edinburgh, Edinburgh, EH9 3FF, UK 11School of GeoSciences, The University of Edinburgh, Edinburgh, EH9 3FF, UK 12Atmospheric Chemistry and Dynamics Laboratory, NASA GSFC, Greenbelt, MD 20720, USA 12Atmospheric Chemistry and Dynamics Laboratory, NASA GSFC, Greenbelt, MD 2072 Federal Environment Agency (UBA), Schauinsland, 79254, Oberried, Germany g y y 14Meteorological Observatory Hohenpeissenberg, German Meteorological Service (D 14Meteorological Observatory Hohenpeissenberg, German Meteorological Service (DWD), Hohenpeissenberg, Germany g y p g g p Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853, U p g g g y 16Geophysical Fluid Dynamics Laboratory, National Oceanic and Atmospheric Administration, Princeton, NJ 08540, USA 17 16Geophysical Fluid Dynamics Laboratory, National Oceanic and Atmospheric Administration 16Geophysical Fluid Dynamics Laboratory, National Oceanic and Atmospheric Administration, Princeton, NJ 08540, USA 1 Centre for Research in Earth and Space Science, York University, Toronto, M3J 1P3, Canada p , y, , , 18School of Earth and Environmental Sciences, Seoul National University, Seoul, 08826, Republic of Korea 19II4 5 7 German Environment Agency (UBA) Zugspitze 82475 Germany p y 18School of Earth and Environmental Sciences, Seoul National University, Seoul, 08826, Republic of Korea 19II4.5.7, German Environment Agency (UBA), Zugspitze, 82475, Germany 20 School of Earth and Environmental Sciences, Seoul National University, Seoul, 08826, Republic 18School of Earth and Environmental Sciences, Seoul National University, Seoul 19II4.5.7, German Environment Agency (UBA), Zugspitze, 82475, Germany 20Met Ofﬁce, Exeter, EX1 3PB, UK School of Earth and Environmental Sciences, Seoul National University, Seo II4.5.7, German Environment Agency (UBA), Zugspitze, 82475, Germany 21Jülich Supercomputing Centre, Forschungszentrum Jülich, 52425 Jülich, Germany 22 22Nicholas School of the Environment, Duke University, Durham, NC 27708, USA 23 Laboratory for Air Pollution/Environmental Technology, Empa – Swiss Federal Laboratories for aterials Science and Technology Dübendorf 8600 Switzerland 23Laboratory for Air Pollution/Environmental Technology, Empa – Swiss Federal Laboratorie Materials Science and Technology, Dübendorf, 8600, Switzerland 23Laboratory for Air Pollution/Environmental Technology, Empa – Swiss Federal Laboratories Materials Science and Technology, Dübendorf, 8600, Switzerland 24Laboratoire des Sciences du Climat et de l’Environnement, Institut Pierre Simon Laplace, CEA/CNRS/UVSQ, Gif-sur-Yvette, France CEA/CNRS/UVSQ, Gif-sur-Yvette, France 25National Institute of Water and Atmospheric Research, Wellington, 6021, New Zealand anow at: Advanced Study Program, National Center for Atmospheric Research, Boulder, CO, USA 25National Institute of Water and Atmospheric Research, Wellington, 6021, New Zealand a Ad d S d P N i l C f A h i R h B ld CO USA y g p bnow at: DWD, Research Center Human Biometeorology, Freiburg, Germany bnow at: DWD, Research Center Human Biometeorology, Freiburg, Germany cnow at: Air Quality Research Division, Environment and Climate Change Canada, Toronto, M3H Correspondence: Arlene M. Peroxy acetyl nitrate (PAN) measurements at northern midlatitude mountain sites in April: a constraint on continental source–receptor relationships Arlene M. Fiore1,2, Emily V. Fischer3, George P. Milly2, Shubha Pandey Deolal4, Oliver Wild5, Daniel A. Jaffe6,7, Johannes Staehelin4, Olivia E. Clifton1,2,a, Dan Bergmann8, William Collins9, Frank Dentener10, Ruth M. Doherty11, Bryan N. Duncan12, Bernd Fischer13, Stefan Gilge14,b, Peter G. Hess15, Larry W. Horowitz16, Alexandru Lupu17,c, Ian A. MacKenzie11, Rokjin Park18, Ludwig Ries19, Michael G. Sanderson20, Martin G. Schultz21, Drew T. Shindell22, Martin Steinbacher23, David S. A. M. Fiore et al.: A constraint on continental source–receptor relationships 15346 Abstract. Abundance-based model evaluations with obser- vations provide critical tests for the simulated mean state in models of intercontinental pollution transport, and under cer- tain conditions may also offer constraints on model responses to emission changes. We compile multiyear measurements of peroxy acetyl nitrate (PAN) available from ﬁve mountain- top sites and apply them in a proof-of-concept approach that exploits an ensemble of global chemical transport models (HTAP1) to identify an observational “emergent constraint”. In April, when the signal from anthropogenic emissions on PAN is strongest, simulated PAN at northern midlatitude mountaintops correlates strongly with PAN source–receptor relationships (the response to 20 % reductions in precursor emissions within northern midlatitude continents; hereafter, SRRs). This ﬁnding implies that PAN measurements can pro- vide constraints on PAN SRRs by limiting the SRR range to that spanned by the subset of models simulating PAN within the observed range. In some cases, regional anthropogenic volatile organic compound (AVOC) emissions, tracers of transport from different source regions, and SRRs for ozone also correlate with PAN SRRs. Given the large observed in- terannual variability in the limited available datasets, estab- lishing strong constraints will require matching meteorology in the models to the PAN measurements. Application of this evaluation approach to the chemistry–climate models used to project changes in atmospheric composition will require routine, long-term mountaintop PAN measurements to dis- cern both the climatological SRR signal and its interannual variability. tinents, and (2) O3 can be produced in transit from the export and subsequent chemical evolution of PAN and other pre- cursors. Below, we examine the extent to which springtime PAN observations at northern midlatitude mountaintop sites can be used to constrain the spread in multi-model estimates of source–receptor relationships (SRRs), where the sources are continental-scale regions and the receptors are the moun- taintop sites, for both PAN and O3. p Observations during several aircraft ﬁeld campaigns in the eastern Paciﬁc and at mountaintop sites in the western US and North Atlantic document efﬁcient O3 production in the lower troposphere following subsidence of PAN-containing air masses (Fischer et al., 2010; Heald et al., 2003; Hudman et al., 2004; Kotchenruther et al., 2001a, b; Val Martin et al., 2008; Zhang et al., 2008). A. M. Fiore et al.: A constraint on continental source–receptor relationships When PAN decomposes in low- NOx regions of the atmosphere, the NOx released can pro- duce O3 up to 8 times more efﬁciently than in polluted (high- NOx) regions (Liang et al., 1998; Liu et al., 1987) and thus increase global O3 abundances (Moxim et al., 1996; Wang and Jacob, 1998), as O3 formation is NOx limited in most of the free troposphere (Chameides et al., 1992). The life- time of PAN against thermal decomposition is about 1 h at 20 ◦C, and it approximately doubles for every 4 ◦C decrease in temperature, leading to a lifetime of at least a month in the mid-troposphere during spring. This strong temperature de- pendence implies that a warmer climate will decrease PAN export from polluted continental boundary layers, although a rise in temperature-sensitive biogenic precursor emissions may temper this response (e.g., Doherty et al., 2013). Future projections of atmospheric composition under global change scenarios will thus beneﬁt from a thorough understanding of the role PAN plays in transporting oxidized reactive nitrogen and thereby altering ozone production throughout the tropo- sphere. A. M. Fiore et al.: A constraint on continental source–receptor relationships 15347 free-tropospheric O3 abundance over western North Amer- ica. Over East Asia, Lin et al. (2010) found that the export of PAN produced from European anthropogenic emission changes and subsequent downwind O3 formation contributed 20 % of the spatially averaged response of surface O3 levels, and up to 50 % of the O3 response at mountain sites. 2014), we hypothesize that PAN measurements may offer much-needed constraints for discriminating across model es- timates of intercontinental transport of PAN, and possibly O3. The number of models contributing to the HTAP1 study, which was designed to maximize comparability across in- dividual model estimates of ozone responses to changes in precursor emissions within northern midlatitude continental- scale source regions, offers an opportunity to evaluate this hypothesis. p 3 p In addition to the direct inﬂuence of PAN on interconti- nental O3 transport, PAN may serve as a sensitive diagnos- tic of model uncertainties in O3 production chemistry and transport (Emmerson and Evans, 2009; Kuhn et al., 1998). Prior analysis of measurements and global model simulations suggests that PAN abundances at high-altitude sites may be more sensitive than O3 itself to changes in precursor emis- sions (Fiore et al., 2011; Fischer et al., 2011; Jaffe et al., 2007). We interpret this stronger sensitivity of PAN than O3 to changes in precursor emissions as reﬂecting buffering of O3 by changes to O3 losses that compensate for changes in production, whereas PAN loss pathways are far less sensi- tive to changes in precursor emissions. PAN loss pathways include thermal decomposition (which dominates below ap- proximately 7 km), photolysis in the upper troposphere, and dry deposition within the boundary layer (Kirchner et al., 1999; Roberts, 2007; Turnipseed et al., 2006). All of the HTAP1 models include PAN formation, but the chemical mechanisms and kinetic rate coefﬁcients differ, with likely implications for long-range transport (Emmerson and Evans, 2009; Knote et al., 2015). A prior multi-model study found that even with the same emissions, PAN differs widely across models, reﬂecting differences in simulated photochemistry (Emmons et al., 2015). While the absence of direct emis- sions and its low background make PAN a useful tracer of photochemistry, we note that O3 typically responds more strongly to changes in NOx emissions, while PAN responds more strongly to changes in VOC emissions in many regions (Fischer et al., 2014; see their Fig. 4). A. M. Fiore et al.: A constraint on continental source–receptor relationships We describe the HTAP1 model simulations, mountaintop measurements, and our strategy to sample the models at these sites (Sect. 2) before illustrating our rationale for selecting the month of April for our analysis (Sect. 3). We then borrow from the emergent constraint approach in climate science to show that correlations between simulated total PAN and SRRs for PAN are sufﬁciently strong to permit PAN mea- surements at mountaintop sites (one in each of the three ma- jor northern midlatitude source regions) to narrow the wide inter-model spread in estimates of PAN origin (Sect. 4). We further examine inter-model relationships among the simu- lated PAN SRRs at these three mountaintop sites and re- gional precursor emissions, and with a proxy for model trans- port (Sect. 5). Finally, we assess the relationship between PAN and O3 SRRs (Sect. 6) and conclude with a summary and recommendations for future work based on our proof- of-concept analysis (Sect. 7). 1 Introduction Peroxy acetyl nitrate (PAN) is produced alongside ozone (O3) from photochemical reactions involving precursor emis- sions of nitrogen oxides (NOx) and non-methane volatile or- ganic compounds (VOCs). Once ventilated from a source re- gion to the free troposphere where it is more stable at colder temperatures, PAN can be efﬁciently transported throughout the hemisphere (Singh, 1987; Singh and Hanst, 1981). When a PAN-containing free-tropospheric air mass subsides, PAN thermally decomposes to release NOx and can thus facili- tate O3 formation far downwind (Wild et al., 1996; Schultz et al., 1999; Jaeglé et al., 2003; Kotchenruther et al., 2001a; Hudman et al., 2004). Both PAN and O3 distributions over any northern midlatitude region reﬂect the combined inﬂu- ence of production from sources within the region and trans- port from outside that region. At northern midlatitudes, the intercontinental inﬂuence from anthropogenic emissions on surface O3 levels is largest during spring (e.g., HTAP 2010) and occurs via at least two pathways: (1) O3 can be produced within a polluted continental boundary layer, ventilated to the free troposphere and efﬁciently transported to other con- To better distinguish among disparate estimates for inter- continental O3 transport in the published literature, the Task Force on Hemispheric Transport of Air Pollution (HTAP) or- ganized an international global modeling study, referred to here as HTAP1. The HTAP1 study identiﬁed a range of a factor of 2 across individual model estimates of surface O3 response to changes in anthropogenic precursor emissions from continental-scale, northern midlatitude source regions (HTAP, 2007, 2010; Fiore et al., 2009; Wild et al., 2012). The HTAP1 models do not distinguish between intercontinental O3 transport occurring due to O3 produced from PAN chem- istry and direct transport of O3 formed in a remote boundary layer, but other work indicates that both pathways contribute. Jaegle et al. (2003) ﬁnd that 28 % of the O3 in the Paciﬁc Northwest free troposphere between 0 and 6 km is associ- ated with PAN-to-NOx conversion, consistent with Jiang et al. (2016), who found that PAN produced from East Asian emissions and exported to the free troposphere contributes 35 % and 25 % in spring and summer, respectively, to the www.atmos-chem-phys.net/18/15345/2018/ www.atmos-chem-phys.net/18/15345/2018/ Atmos. Chem. Phys., 18, 15345–15361, 2018 2.1 HTAP1 model simulations Fiore et al.: A constraint on continental source–receptor relationships 15348 15348 A. M. Fiore et al.: A constraint on continental source–receptor relationships Table 1. Models contributing to the HTAP1 simulations (SR1, SR6xx, and COfromXX) used in this study. Model Resolution (lat × long × layer) Institute Model contact SR1 SR6xx COfrom xx Plotting symbol CAMCHEM-3311m13 2.5◦× 2◦× 30 NCAR, USA Peter Hess X X X Filled circle FRSGCUCI-v01 2.81◦× 2.81◦× 37 Lancaster Univ., UK Oliver Wild X X X Filled upward triangle GEMAQ-v1p0 2◦× 2◦× 28 York Univ., Canada Alex Lupu X X X Filled downward triangle GEOSChem-v07 2.5◦× 2◦× 30 Harvard Univ., USA Rokjin Park X X X Filled diamond GISS-PUCCINI- modelE 5◦× 4◦× 23 NASA GISS, USA Drew Shindell X X X Filled square GMI-v02f 2.5◦× 2◦× 42 NASA GSFC, USA Bryan Duncan X X X Open circle LMDZ3-INCA1 3.75◦× 2◦× 19 CEA, France Sophie Szopa X X Open upward triangle LLNL-IMPACT-T5a 2.5◦× 2◦× 48 LLNL, USA Dan Bergmann X Open downward triangle MOZARTGFDL-v2 1.88◦× 1.88◦× 28 NOAA GFDL, USA Arlene Fiore X X X Open diamond MOZECH-v16 1.88◦× 1.88◦× 28 FZ Jülich, Germany Martin Schultz X X X Open square STOC-HadAM3-v01 5◦× 5◦× 19 University of Edinburgh, UK Ruth Doherty, David Stevenson X X X Plus sign STOCHEM-v02 3.75 × 2.5◦× 20 Met Ofﬁce, Hadley Center, UK Bill Collins, Michael Sanderson X X TM5-JRC-cy2-ipcc-v1 1◦× 1◦× 25 JRC, Italy Frank Dentener X X X Filled right-facing triangle UM-CAM-v01 3.75◦× 2.5◦× 19 University of Cambridge, UK, and NIWA, New Zealand Guang Zeng X X X Filled left-facing triangle ble 1. Models contributing to the HTAP1 simulations (SR1, SR6xx, and COfromXX) used in this study. denote the tracers emitted from EA, NA, and EU, respec- tively (Table 2; see also Doherty et al., 2013, and Shindell et al., 2008). For HTAP1, each model used its own emissions invento- ries (see Table A1 of Fiore et al., 2009); Fiore et al. (2009) provide emission totals within each HTAP1 source region for all (their Table A2) and anthropogenic (their Table A3) emissions of NOx, non-methane volatile organic compounds (NMVOCs), and CO. The relative inter-model spread in re- gional anthropogenic emissions is smallest for NOx emis- sions in EU and NA (< 10 %) and largest for VOCs from EU (58 %) (Fiore et al., 2009). 2.1 HTAP1 model simulations We use monthly mean PAN mixing ratios for the year 2001 simulated by 14 global chemistry transport models (Table 1); the temporal resolution for three-dimensional chemical ﬁelds archived from the HTAP1 models is limited to monthly. We use four HTAP1 source–receptor (SR) simulations (Ta- ble 2): a base case (SR1) and three perturbation simula- tions in which anthropogenic O3 precursor emissions (NOx, VOCs, carbon monoxide, and aerosols) are reduced simul- taneously by 20 % within East Asia (EA, SR6EA), Europe and northern Africa (EU, SR6EU), and North America (NA, SR6NA). We calculate PAN SRRs by differencing the per- turbation and base simulations (SR1-SR6XX), for which XX refers to the region in which emissions of PAN precursors were decreased by 20 %. A challenge in discriminating among model estimates of O3 produced from different source regions is the lack of di- rect observational constraints on SRRs. For example, Fiore et al. (2009) did not ﬁnd any relationship across models be- tween their biases against surface O3 observations and the strength of their response to emission changes. In the absence of an observable quantity to constrain these relationships, one approach is to identify an “emergent constraint” (Borod- ina et al., 2017), whereby an unobservable quantity corre- lates strongly across a multi-model ensemble with an ob- served variable. The inter-model range of the non-observable quantity is then narrowed by limiting it to the range encom- passed by the models closest to the observed variable. This approach has gained traction for narrowing the spread across future climate projections (e.g., Hall and Qu, 2006; Cox et al., 2018). Given that PAN both directly facilitates interconti- nental ozone transport and can serve as a proxy for ozone for- mation chemistry, and that prior work indicates a large sig- nature of PAN originating from the European boundary layer during spring at Jungfraujoch (Pandey Deolal et al., 2013, Of the models in Table 1, 11 used 2001 meteorological ﬁelds. Two models are chemistry-transport models coupled directly to a general circulation model forced by observed sea surface temperatures (STOC-HadAM3 and STOCHEM) and one model incorporates chemistry directly into a general cir- culation model (UM-CAM). We include these models as our evaluation compiles PAN measurements across several years (Sect. 2.2). The individual model speciﬁcations and emis- sions are described in Tables 1 and 2 of Fiore et al. (2009). www.atmos-chem-phys.net/18/15345/2018/ Atmos. Chem. Phys., 18, 15345–15361, 2018 A. M. 2.2 Multiyear PAN measurements at mountaintop sites and model sampling (2010, 2011) 121.687◦W 30 Aug–7 Oct 2008, https://digital.lib.washington.edu/researchworks/ 26 Mar–20 May 2009, (last access: 22 October 2018) 23 Mar–25 May 2010 Hohenpeissenberg 47.80◦N, 985 m Jan 2003–Dec 2008 http://ds.data.jma.go.jp/gmd/wdcgg/cgi-bin/wdcgg 11.02◦E (last access: 22 October 2018) Gilge et al. (2010) Jungfraujoch 46.55◦N, 3580 m Apr 1997–May 1998, Balzani Lööv et al. (2008); 7.98◦E Aug 30 2005–16 Sep 2005, Carpenter et al. (2000); Throughout (2005); Zellweger et al. (2000, 2003) but not continuous Zugspitze 47.42◦N, 2960 m May 2004–Dec 2008 https://gaw.kishou.go.jp 10.98◦E (last access: 16 October 2018) Schauinsland 47.92◦N, 1205m Jan 1995–Dec 2010 https://www.umweltbundesamt.de 7.92◦E (last access: 22 October 2018) A. M. Fiore et al.: A constraint on continental source–receptor relationships A. M. Fiore et al.: A constraint on continental source–receptor relationships Table 2. Simulations from HTAP1 used in this study. Simulation Description SR1 Base case (see Sect. 2.1 for details). SR6EA SR1 but with anthropogenic emissions of all O3 precursors (NOx + CO + NMVOC) and aerosols within EA decreased by 20 %. SR6EU SR1 but with 20 % emissions reductions within the EU region SR6NA SR1 but with 20 % emissions reductions within the NA region. COfromEA Idealized tracer simulation in which all models use identical CO emissions, emitted within the EA region, with a 50-day e-folding lifetime. COfromEU Same as COfromEA but for the EU region. COfromNA Same as COfromEA but for the NA region. 15349 Table 2. Simulations from HTAP1 used in this study. Table 3. Mountaintop sites with multiple years of PAN observations used in this study. Site Location Elevation Measurement period (s) Reference (s) Mount Bachelor 43.979◦N, 2763 m 3 Apr–18 Jun 2008, Fischer et al. (2010, 2011) 121.687◦W 30 Aug–7 Oct 2008, https://digital.lib.washington.edu/researchworks/ 26 Mar–20 May 2009, (last access: 22 October 2018) 23 Mar–25 May 2010 Hohenpeissenberg 47.80◦N, 985 m Jan 2003–Dec 2008 http://ds.data.jma.go.jp/gmd/wdcgg/cgi-bin/wdcgg 11.02◦E (last access: 22 October 2018) Gilge et al. (2010) Jungfraujoch 46.55◦N, 3580 m Apr 1997–May 1998, Balzani Lööv et al. (2008); 7.98◦E Aug 30 2005–16 Sep 2005, Carpenter et al. (2000); Throughout (2005); Zellweger et al. (2000, 2003) but not continuous Zugspitze 47.42◦N, 2960 m May 2004–Dec 2008 https://gaw.kishou.go.jp 10.98◦E (last access: 16 October 2018) Schauinsland 47.92◦N, 1205m Jan 1995–Dec 2010 https://www.umweltbundesamt.de 7.92◦E (last access: 22 October 2018) Table 3. Mountaintop sites with multiple years of PAN observations used in this study. at Mount Bachelor (Fischer et al., 2010). 2.2 Multiyear PAN measurements at mountaintop sites and model sampling To evaluate the HTAP1 models, we compiled April mean climatologies of lower-tropospheric PAN measurements from northern midlatitude mountain observatories (Table 3). Given the large interannual variability in PAN abundances, we require at least 2 years of observations in April. PAN observations from Mount Bachelor (USA), Jungfraujoch (Switzerland), and Zugspitze (Schneefernerhaus), Hohen- peissenberg, and Schauinsland (all in Germany) meet these criteria. Taken together, these mountaintop measurements span 15 years, from 1995 to 2010 (Table 3), although only one site (Schauinsland) overlaps with the HTAP1 simulation year of 2001. To separate the role of inter-model differences in trans- port from the combined impacts of inter-model differences in emissions and chemistry on simulated PAN at the mountain- top sites, we analyze an additional set of idealized tracer sim- ulations available from 11 models (COfromXX in Table 1, for which XX is the source region). In these simulations, a set of tagged carbon-monoxide-like tracers are emitted, each from a single HTAP1 source region with a 50-day lifetime, and with identical emissions across models. Biomass burning emissions for the CO tracers are from GFED (van der Werf et al., 2006, 2010) and other emissions are from the RETRO project (Schultz et al., 2007, 2008). We refer to these tracers as “COfromEA”, “COfromNA”, and “COfromEU”, which PAN was measured at all ﬁve mountain sites using gas chromatography with electron capture detection (ECD). A custom system using a Shimadzu Mini-2 ECD was employed www.atmos-chem-phys.net/18/15345/2018/ Atmos. Chem. Phys., 18, 15345–15361, 2018 A. M. Fiore et al.: A constraint on continental source–receptor relationships 15349 Table 2. Simulations from HTAP1 used in this study. Simulation Description SR1 Base case (see Sect. 2.1 for details). SR6EA SR1 but with anthropogenic emissions of all O3 precursors (NOx + CO + NMVOC) and aerosols within EA decreased by 20 %. SR6EU SR1 but with 20 % emissions reductions within the EU region SR6NA SR1 but with 20 % emissions reductions within the NA region. COfromEA Idealized tracer simulation in which all models use identical CO emissions, emitted within the EA region, with a 50-day e-folding lifetime. COfromEU Same as COfromEA but for the EU region. COfromNA Same as COfromEA but for the NA region. Table 3. Mountaintop sites with multiple years of PAN observations used in this study. Site Location Elevation Measurement period (s) Reference (s) Mount Bachelor 43.979◦N, 2763 m 3 Apr–18 Jun 2008, Fischer et al. 2.2 Multiyear PAN measurements at mountaintop sites and model sampling The commercially available Meteorologie Consult (GmbH) system was used at the European sites (Zellweger et al., 2000). Calibrations generate PAN from the photolysis of excess acetone and NO in air (Warneck and Zerbach, 1992; Volz-Thomas et al., 2002). Reported detection limits are ∼20 ppt for PAN mea- surements at Mount Bachelor and ∼50 ppt for the European sites, with total uncertainties of < 10 % (Fischer et al., 2010; Zellweger et al., 2003). For comparison with the observations, we sample each model on its native grid (Table 1) at the horizontal grid cell containing the latitude and longitude of each mountain site. Orography at these mountain sites is poorly resolved at the relatively coarse HTAP1 model horizontal resolutions. This mismatch requires us to apply some approximations for ver- tical sampling. We convert the station altitude to an approxi- mate pressure level by assuming a mean tropospheric temper- ature of 260 K and a corresponding atmospheric scale height of 7.6 km. We then use monthly mean pressure ﬁelds from each model to linearly interpolate PAN based on the pres- sures of the two model grid cells that vertically bound the station pressure. While different sampling strategies may al- ter the exact value of simulated PAN and its comparison to observations, our primary interest is in the inter-model differ- ences. Although the Zugspitze and Hohenpeissenberg sites fall within the same horizontal grid cell in the HTAP1 mod- els, the station altitudes differ, so we consider the two sites separately. We include all available data at these sites without ﬁlter- ing for upslope winds or any other criteria. At Mount Bach- elor, the cleanest of the ﬁve mountaintop sites (Supplement Fig. S1), Fischer et al. (2010) have shown that PAN mix- ing ratios are not primarily controlled by diurnal wind pat- terns, which lead to variations an order of magnitude smaller than the total observed range in measured PAN. When mea- surements fall below the detection limit, we include half of the detection limit. This assumption should not affect our conclusions as mountaintop sites generally sample free- tropospheric air at night (e.g., Weiss-Penzias et al., 2004), but PAN values below the detection limit typically occur due to deposition in a shallow nocturnal boundary layer. Given that we seek constraints on intercontinental trans- port from the three major midlatitude source regions, we con- duct a more in-depth analysis at the highest-altitude Euro- Atmos. Chem. Phys., 18, 15345–15361, 2018 A. M. Fiore et al.: A constraint on continental source–receptor relationships 15350 pean site (Jungfraujoch), the most likely of the available sites to measure PAN transported among continents in the free tro- posphere, as well as at Mount Bachelor in North America. At Jungfraujoch, we also evaluate SRRs in the models with an estimate of PAN originating in the European boundary layer based on an analysis of 20-day back trajectories (Pandey De- olal et al., 2013). We conduct a proof-of-concept analysis at Mount Waliguan in Asia (36.28◦N, 100.90◦E, 3816 m) to assess the potential for future PAN measurements at this site to narrow the inter-model range in SRRs. Short-term mea- surements have previously been collected at this site (Xue et al., 2011). While aircraft and satellite observations have ad- vanced the understanding of the chemistry and dynamics of individual PAN plumes using models that archived chemi- cal ﬁelds at high temporal frequencies (e.g., Alvarado et al., 2010; Payne et al., 2014; Emmons et al., 2015), their limited temporal coverage is not well suited for comparison with the HTAP1 monthly mean PAN mixing ratios. across the models. The multi-model mean falls in the range of the measurements at four of the sites but is higher than ob- served in any year at Mount Bachelor. The model rankings show some consistency across the different sites, suggest- ing systematic model differences that can be narrowed with a limited set of observational constraints, especially for mod- els that rank similarly across the sites on all three continents (Fig. 3). For example, CAMCHEM and GEMAQ are consis- tently at the higher end of the range while GISS-PUCCINI and LLNL-IMPACT are at the low end. The two mod- els falling closest to the observed 2001 value at Schauins- land (MOZECH and MOZARTGFDL) fall into the observed range at either Mount Bachelor or Jungfraujoch; we analyze these two sites further in the following sections. The longest observational dataset at Schauinsland varies by over a factor of 3 across years, consistent with large in- terannual variability found in prior analyses at mountaintop sites (Zellweger et al., 2003; Fischer et al., 2011; Pandey De- olal et al., 2013, 2014). All but one of the models (LLNL- IMPACT) fall within the wide range of observed interan- nual variability at Schauinsland, underscoring the tenuous nature of conclusions regarding model performance drawn from short observational records unless the modeled and ob- served meteorological years match. A. M. Fiore et al.: A constraint on continental source–receptor relationships Future work to coor- dinate consistent time periods between measurements and models would provide tighter constraints than are possible with our proof-of-concept analysis described in the follow- ing sections. 3 Modeled and measured lower tropospheric PAN at northern midlatitudes in April Our goal is to assess the potential for mountaintop PAN mea- surements to discriminate among model estimates of PAN and O3 produced by regional anthropogenic emissions and transported to the mountaintop sites. We thus focus our anal- ysis on April when measured PAN reaches its seasonal max- imum (Penkett and Brice, 1986; Singh and Salas, 1989; Bottenheim et al., 1994; Schmitt and Volz-Thomas, 1997; Fig. S1) and when the HTAP1 models indicate that the pro- duction of PAN from the EA, EU, and NA source regions dominates total simulated PAN (Fig. 1). April thus offers the strongest possible signal of the inﬂuence of anthropogenic emissions from these three northern midlatitude source re- gions in the mountaintop measurements. 4 Exploring emergent constraints on model SRRs from measured total PAN The range of the PAN SRRs across the HTAP1 models at Jungfraujoch, Mount Bachelor, and Mount Waliguan is wide for all three source regions, spanning a factor of 5 or more in several cases (Fig. 4). The key to a successful emer- gent constraint analysis is for this range in inter-model PAN SRRs, our unobservable quantity, to correlate with the total PAN simulated at the mountaintop site, our observable vari- able. The strongest correlations emerge for PAN originating in the region where the mountain is located, but some in- tercontinental SRR pairs also show signiﬁcant correlations (p ≤0.05) with total simulated PAN (Fig. 4). Figure 2 shows the spatial distribution of the HTAP1 model ensemble mean PAN mixing ratios at 650 hPa (∼3 km), the level sampled by the highest-altitude sites on which we focus the majority of our analysis. PAN mixing ratios in April generally increase with latitude, as expected from the strong thermal dependence of the PAN lifetime, al- though some of the highest mixing ratios are simulated over the Asian source region. The multi-model spread in lower- tropospheric PAN, represented by the coefﬁcient of variation (standard deviation over the 14 models divided by the model ensemble mean) is within ±45 % across much of the North- ern Hemisphere (Fig. 2). The large inter-model spread over much of Europe in Fig. 2b implies that observational con- straints in this region would be particularly valuable. We illustrate here how PAN measurements can be used to narrow the inter-model range in the SRR pairs. For the sites with signiﬁcant correlations, the range across years (i.e., red vertical lines in Fig. 4) bound the April mean values observed at Jungfraujoch and Mount Bachelor. The models falling in this range are highlighted in red. We select these models to narrow the range in SRRs, indicated by the red horizontal dashed lines extending from the bounding models (red sym- bols) to the ordinate axis. Figure 4 shows that the constraint from total measured PAN narrows the inter-model range in SRRs for PAN by at least half, revealing some models as out- Observed and modeled PAN mixing ratios at the north- ern midlatitude mountain sites are compared in Fig. 3 (see Fig. S1 for a comparison extended throughout the year). We consider the measured range across years to bound the “plausible” portion of the wide range in simulated total PAN Atmos. Chem. www.atmos-chem-phys.net/18/15345/2018/ Atmos. Chem. Phys., 18, 15345–15361, 2018 A. M. Fiore et al.: A constraint on continental source–receptor relationships Multi-model ensemble (n = 14; Table 1) average PAN mixing ratios (ppt; a) and relative standard deviation (the absolute standard deviation across the models divided by the ensemble mean; b) at 650 hPa in April; relative standard deviations are masked out (white) for regions where multi-model mean PAN falls below 100 ppt. The models were sampled at 650 hPa by vertically interpolating among the bounding grid cells and then re-gridded horizontally to a common 1◦×1◦grid. White lines denote the HTAP1 source regions: North America (NA), Europe and North Africa (EU), and East Asia (EA) from left to right. White circles indicate the ﬁve mountain sites with multiyear PAN observations used in our analysis (note that Zugspitze and Hohenpeissenberg are too close to differentiate on the map; see Table 3). Mount Waliguan in Asia, where we lack multiyear measurements but conduct model analysis, is denoted by the white square. 10o N 20o N 30o N 40o N 50o N 60o N 70o N 80o N 180o 120o W 60o W 0o 60o E 120o E 180o 25 100 175 250 325 400 10o N 20o N 30o N 40o N 50o N 60o N 70o N 80o N 180o 120o W 60o W 0o 60o E 120o E 180o 0.30 0.40 0.50 0.60 0.70 (a) Multi-model mean PAN (ppt) (b) Multi-model relative standard deviation April 650 hPa (b) Multi-model relative standard deviation Figure 2. Multi-model ensemble (n = 14; Table 1) average PAN mixing ratios (ppt; a) and relative standard deviation (the absolute standard deviation across the models divided by the ensemble mean; b) at 650 hPa in April; relative standard deviations are masked out (white) for regions where multi-model mean PAN falls below 100 ppt. The models were sampled at 650 hPa by vertically interpolating among the bounding grid cells and then re-gridded horizontally to a common 1◦×1◦grid. White lines denote the HTAP1 source regions: North America (NA), Europe and North Africa (EU), and East Asia (EA) from left to right. White circles indicate the ﬁve mountain sites with multiyear PAN observations used in our analysis (note that Zugspitze and Hohenpeissenberg are too close to differentiate on the map; see Table 3). Mount Waliguan in Asia, where we lack multiyear measurements but conduct model analysis, is denoted by the white square. liers. A. M. Fiore et al.: A constraint on continental source–receptor relationships 15351 0 2 4 6 8 10 12 Month 0 50 100 150 200 250 PAN (ppt) 0 2 4 6 8 10 12 Month 0 100 200 300 400 PAN (ppt) (a) Mount Bachelor (b) Jungfraujoch Figure 1. Multi-model monthly mean total PAN mixing ratios (black circles and solid lines) at Mount Bachelor (a) and Jungfraujoch (b). We take the difference between the base simulation (SR1) and one in which emissions are decreased by 20 % and then multiply the difference by 5 to estimate a 100 % contribution associated with anthropogenic precursor emissions from Europe (green), North America (red), and East Asia (blue). The sum of the anthropogenic contribution from these three regions is shown (dashed black) for comparison with total simulated PAN. 0 2 4 6 8 10 12 Month 0 50 100 150 200 250 PAN (ppt) 0 2 4 6 8 10 12 Month 0 100 200 300 400 PAN (ppt) (a) Mount Bachelor (b) Jungfraujoch 12 0 2 4 6 8 10 12 Month 0 100 200 300 400 PAN (ppt) (b) Jungfraujoch (b) Jungfraujoch Figure 1. Multi-model monthly mean total PAN mixing ratios (black circles and solid lines) at Mount Bachelor (a) and Jungfraujoch (b). We take the difference between the base simulation (SR1) and one in which emissions are decreased by 20 % and then multiply the difference by 5 to estimate a 100 % contribution associated with anthropogenic precursor emissions from Europe (green), North America (red), and East Asia (blue). The sum of the anthropogenic contribution from these three regions is shown (dashed black) for comparison with total simulated PAN. 10o N 20o N 30o N 40o N 50o N 60o N 70o N 80o N 180o 120o W 60o W 0o 60o E 120o E 180o 25 100 175 250 325 400 10o N 20o N 30o N 40o N 50o N 60o N 70o N 80o N 180o 120o W 60o W 0o 60o E 120o E 180o 0.30 0.40 0.50 0.60 0.70 (a) Multi-model mean PAN (ppt) (b) Multi-model relative standard deviation April 650 hPa Figure 2. A. M. Fiore et al.: A constraint on continental source–receptor relationships Other models simulate SRRs within the observationally constrained range (between the dashed red horizontal lines) despite falling outside the observed range for total PAN, pos- sibly indicating a role for inter-model differences in non- anthropogenic sources of PAN or in the relative contributions from the individual midlatitude source regions, which we in- vestigate further in the next section. Given the year-to-year variability in total PAN, stronger constraints could be placed in future work in which the model meteorology corresponds to the same year as the measurements. region. The horizontal blue dashed lines indicate the bounds obtained from this trajectory-based approach to estimating PAN from EU. The models falling in these bounds overlap with those constrained by the total PAN measurements, lend- ing some conﬁdence that these two independent approaches (one using total PAN and the correlated inter-model spread in SRRs; the other using back trajectories to estimate SRRs) yield useful constraints on the inﬂuence of the EU source re- gion on PAN measured at Jungfraujoch. We note that for consistency with the model SRRs in Fig. 4, which are the responses to 20 % emission reduc- tions in the source region, we divide the Pandey Deolal et al. (2013) EU SRRs by 5 to scale back from their estimated “full contribution” (100 %). This linear scaling of the PAN response between 20 % and 100 % may incur errors due to nonlinear chemistry. With an additional simulation in which the FRSGCUCI model sets European anthropogenic emis- sions of NOx, CO, and VOCs to zero (a 100 % perturbation), we estimate this error to be ∼10 %. For intercontinental re- gions, this error reduces to < 3 %. Earlier work shows that At Jungfraujoch, we additionally consider PAN SRRs for the EU source region with those estimated previously using back-trajectory analysis (Pandey Deolal et al., 2013). While Pandey Deolal et al. (2013) also attribute trajectories to NA and EA, fewer than 15 % and 4 % of trajectories are attributed to those regions compared to 25 %–50 % from EU (range across years; see Fig. 1 of Pandey Deolal et al., 2013). Com- bining these low frequencies with the inevitable growth in uncertainty as trajectories lengthen, we have the most conﬁ- dence in the Pandey Deolal et al. (2013) estimates for the EU 4 Exploring emergent constraints on model SRRs from measured total PAN Phys., 18, 15345–15361, 2018 www.atmos-chem-phys.net/18/15345/2018/ A. M. Fiore et al.: A constraint on continental source–receptor relationships ore et al.: A constraint on continental source–receptor relationships A. M. Fiore et al.: A constraint on continental source–receptor relationships 15352 Total PAN MBO ZUG JFJ HOH SCH MTW 0 500 1000 1500 2000 ppt Figure 3. April mean PAN abundances (ppt) simulated (black sym- bols, one per model as deﬁned in Table 1; blue circles offset to the left show multi-model mean values) and measured (red circles offset to the right of the model values) at northern midlatitude mountain- top sites: Mount Bachelor (MBO), Zugspitze (ZUG), Jungfraujoch (JFJ), Hohenpeissenberg (HOH), Schauinsland (SCH), and Mount Waliguan (MTW). The observed year 2001 April mean, which cor- responds to the meteorological year used by most of the models at Schauinsland, is shown in blue to the right of the models. Total PAN MBO ZUG JFJ HOH SCH MTW 0 500 1000 1500 2000 ppt differences in regional AVOC emissions in contributing to the inter-model range in PAN SRRs. Differences in model transport (e.g., Arnold et al., 2015; Orbe et al., 2017) may also contribute to the inter-model dif- ferences in PAN SRRs. Our analysis of the HTAP1 ideal- ized CO tracers, however, reveals little correlation between inter-model differences in these idealized tracers (which have identical regional emissions and lifetimes applied in all of the models) and in the PAN SRRs sampled at these sites. Although we do not ﬁnd any clear overall correlation, differ- ences in the idealized CO tracers explain some of the scatter in Fig. 5. For example, at Jungfraujoch for EU AVOC emis- sions of 22 Tg C a−1, the lowest model (GISS-PUCCINI) has one of the smallest values for the COfromEU tracer, whereas the highest model (STOC-HadAM3) has the largest value of COfromEU. Figure 3. April mean PAN abundances (ppt) simulated (black sym- bols, one per model as deﬁned in Table 1; blue circles offset to the left show multi-model mean values) and measured (red circles offset to the right of the model values) at northern midlatitude mountain- top sites: Mount Bachelor (MBO), Zugspitze (ZUG), Jungfraujoch (JFJ), Hohenpeissenberg (HOH), Schauinsland (SCH), and Mount Waliguan (MTW). The observed year 2001 April mean, which cor- responds to the meteorological year used by most of the models at Schauinsland, is shown in blue to the right of the models. In light of the dependence of inter-model differences in PAN attributed to EU and NA during April and the corre- sponding regional AVOC emissions, we illustrate how one could extend our emergent constraints in Fig. A. M. Fiore et al.: A constraint on continental source–receptor relationships 4 (horizontal dashed red lines) to the regional AVOC emission estimates shown in Fig. 5. A major caveat underlying this analysis is the mismatch between meteorological years for the models and measurements as discussed above, and the underlying assumption that the relationships in Fig. 5 can exclusively be attributed to differences in the AVOC emissions (as opposed to chemistry or transport). The observationally constrained SRRs between PAN from NA and total PAN measured at Jungfraujoch and Mount Bachelor can be used to narrow the range of NA AVOC emissions to 12–18 Tg C a−1 (the low end is ruled out by the constraint imposed by PAN from NA at Jungfraujoch; the high end is ruled out by PAN from NA at Mount Bachelor). Similarly, the range for EU AVOC emis- sions would narrow to 16–25 Tg C a−1. the smaller nonlinearity in PAN for intercontinental versus regional source–receptor pairs also holds for ozone (Fiore et al., 2009; Wu et al., 2009; Wild et al., 2012) and demon- strates approximate linearity between the simulated tropo- spheric ozone burden and ±50 % of present-day global NOx emissions (Stevenson et al., 2006). 5 Factors contributing to the inter-model range in PAN SRRs We consider next the importance that various models as- cribe to a given source region relative to another source re- gion. We ﬁrst correlate the ratios of PAN from two differ- ent source regions with the total PAN simulated by the indi- vidual models in April. We ﬁnd little relationship, with the exception of Mount Bachelor, where the observational con- straint implies that more PAN originating from EA should be present at Mount Bachelor than PAN originating from NA (Fig. 6a). We interpret this as indicating that models with higher total PAN at Mount Bachelor are overestimating North American inﬂuence at this mountain site (which sam- ples free-tropospheric air). This interpretation is supported by the idealized CO tracer simulations (with identical re- gional emissions and the same lifetime applied in all the models), which suggest that some of the variance in the ratio of PAN from NA to EA at Mount Bachelor is due to differ- ences in transport from the two regions (Fig. 6b). We empha- size that these transport differences do not simply reﬂect the use of different meteorology in the models (Fig. 6b). We investigate the role of inter-model differences in re- gional emissions of PAN precursors versus transport in con- tributing to inter-model differences in the PAN response to continental-scale emission changes at the three mountaintop sites shown in Fig. 4. At each site, we examine the corre- lation across models between simulated PAN SRRs and re- gional anthropogenic emissions of VOCs (AVOCs; Fig. 5) or NOx (ANOx). The relationships for the EA SRRs are not sig- niﬁcant, even at Mount Waliguan. We ﬁnd, however, that the inter-model range in regional AVOC emissions explains as much as 64 % of the variation in PAN attributed to EU emis- sions, and at least 25 % of the variance in PAN attributed to the NA region (Fig. 5). In contrast to AVOCs, we ﬁnd lit- tle relationship between the range in simulated PAN SRRs at the mountain sites and the model spread in regional ANOx emissions. Fischer et al. (2014) have previously shown that PAN abundances respond more strongly to changes in emis- sions of VOCs than of NOx. www.atmos-chem-phys.net/18/15345/2018/ Atmos. Chem. Phys., 18, 15345–15361, 2018 A. M. Fiore et al.: A constraint on continental source–receptor relationships A. M. Fiore et al.: A constraint on continental source–receptor relationships At Jungfraujoch, the HTAP1 multi-model mean attributes much of the PAN to emissions from the EU and NA source regions during April (Fig. 1). The ratio of PAN attributed to EU to PAN attributed to NA at Jungfraujoch, however, varies from approximately 0.5 to 2 across the individual HTAP1 models (Fig. S2). In contrast to our ﬁndings at Mount Bachelor, this ratio at Jungfraujoch depends most strongly on the ratio of ANOx emissions in the EU to NA regions (r = 0.6) and more the relative importance of emissions within the source region, where the measurement site is located, versus the upwind intercontinental source region of PAN (Fig. S2). At Mount Bachelor, the HTAP1 multi-model mean SRRs from NA, EA, and EU are roughly equal in April (Fig. 1). The differences across the HTAP models in the relative importance of the NA : EA source regions of PAN (which range from about 0.5 to 2.5) correlate roughly equally with the ratio of the NA : EA CO transport tracers and with the ratio of the NA : EA AVOC emissions (Spearman’s rank correlation coefﬁcient (r) = 0.6 for both cases); we ﬁnd no relationship with the ratio of the NA : EA ANOx emissions (Figs. 6b and S2, upper left). At Jungfraujoch, the HTAP1 multi-model mean attributes much of the PAN to emissions from the EU and NA source regions during April (Fig. 1). The ratio of PAN attributed to EU to PAN attributed to NA at Jungfraujoch, however, varies from approximately 0.5 to 2 across the individual HTAP1 models (Fig. S2). In contrast to our ﬁndings at Mount Bachelor, this ratio at Jungfraujoch depends most strongly on the ratio of ANOx emissions in the EU to NA regions (r = 0.6) and more We repeat this correlation analysis of inter-model differ- ences in ratios of ANOx emissions, AVOC emissions, or the idealized CO tracers of transport from a region, but for the ratio of PAN SRRs from two intercontinental regions. At Mount Bachelor, the EU and EA source regions con- tribute similar amounts to multi-model mean PAN during April (Fig. 1). Across the individual models, however, the ratio of the EU to EA source regions on PAN at Mount Bach- elor varies from less than half to a factor of 2 (Fig. S3). A. M. Fiore et al.: A constraint on continental source–receptor relationships We ﬁnd that the ratio of PAN attributed to the EU versus EA source regions at Mount Bachelor correlates strongly across the models with the ratio of the AVOC emissions in the re- spective source regions (r = 0.8; Fig. S3). In contrast, the 5 Factors contributing to the inter-model range in PAN SRRs Our analysis supports that ear- lier ﬁnding and furthermore highlights a key role for model By comparing NA : EA at Mount Bachelor, EU : NA at Jungfraujoch, and EA : EU at Mount Waliguan, we examine www.atmos-chem-phys.net/18/15345/2018/ www.atmos-chem-phys.net/18/15345/2018/ Atmos. Chem. Phys., 18, 15345–15361, 2018 A. M. Fiore et al.: A constraint on continental source–receptor relationships For source–receptor pairs with signiﬁcant correlations (p ≤0.05), models falling within the observed range (across years) are colored red, and horizontal red dashed lines extend to the ordinate, representing the emergent constraint (narrower range resulting from selecting only those models falling in the observed range of total PAN). At Jungfraujoch, the range (across years) in PAN attributed to the EU source region by back-trajectory analysis (Pandey Deolal et al., 2013) is indicated by horizontal dashed blue lines. Individual models are denoted by the symbols deﬁned in Table 1. p p g j Mount Bachelor, vertical red lines bound the observed range in total PAN. For source–receptor pairs with signiﬁcant correlations (p ≤0.05), models falling within the observed range (across years) are colored red, and horizontal red dashed lines extend to the ordinate, representing the emergent constraint (narrower range resulting from selecting only those models falling in the observed range of total PAN). At Jungfraujoch, the range (across years) in PAN attributed to the EU source region by back-trajectory analysis (Pandey Deolal et al., 2013) is indicated by horizontal dashed blue lines. Individual models are denoted by the symbols deﬁned in Table 1. weakly on the ratio of EU : NA AVOC emissions (r = 0.5; Fig. S2). The correlation is even weaker between the ratio of PAN SRRs for these two regions with inter-model dif- ferences in transport as diagnosed with the CO tracers from EU to NA (r = 0.4). At Mount Waliguan, the strongest rela- tionship is found for the ratio of AVOC emissions (r = 0.5; Fig. S2). the relative importance of emissions within the source region, where the measurement site is located, versus the upwind intercontinental source region of PAN (Fig. S2). At Mount Bachelor, the HTAP1 multi-model mean SRRs from NA, EA, and EU are roughly equal in April (Fig. 1). The differences across the HTAP models in the relative importance of the NA : EA source regions of PAN (which range from about 0.5 to 2.5) correlate roughly equally with the ratio of the NA : EA CO transport tracers and with the ratio of the NA : EA AVOC emissions (Spearman’s rank correlation coefﬁcient (r) = 0.6 for both cases); we ﬁnd no relationship with the ratio of the NA : EA ANOx emissions (Figs. 6b and S2, upper left). A. M. Fiore et al.: A constraint on continental source–receptor relationships ore et al.: A constraint on continental source–receptor relationships 15353 100 200 300 400 500 600 700 Total PAN (ppt) 0 5 10 15 20 25 30 PAN from NA (ppt) r=0.58 p=0.05 100 200 300 400 500 600 700 Total PAN (ppt) 0 20 40 60 80 100 120 PAN from EU (ppt) r=0.85 p=0.01 100 200 300 400 500 600 700 Total PAN (ppt) 0 2 4 6 8 10 12 PAN from EA (ppt) r=0.77 p=0.01 100 200 300 400 500 Total PAN (ppt) 0 10 20 30 PAN from NA (ppt) r=0.97 p=0.00 100 200 300 400 500 Total PAN (ppt) 0 10 20 30 PAN from EU (ppt) r=0.76 p=0.01 100 200 300 400 500 Total PAN (ppt) 0 5 10 15 20 PAN from EA (ppt) r=0.22 p=0.46 100 200 300 400 500 600 700 Total PAN (ppt) 0 5 10 15 20 PAN from NA (ppt) r=0.45 p=0.14 100 200 300 400 500 600 700 Total PAN (ppt) 0 10 20 30 40 PAN from EU (ppt) r=0.22 p=0.46 100 200 300 400 500 600 700 Total PAN (ppt) 0 20 40 60 PAN from EA (ppt) r=0.59 p=0.05 Jungfraujoch Mount Bachelor Mount Waliguan Figure 4. Simulated total PAN versus source–receptor relationships (SRRs) at each of three northern midlatitude sites. For Jungfraujoch and Mount Bachelor, vertical red lines bound the observed range in total PAN. For source–receptor pairs with signiﬁcant correlations (p ≤0.05), models falling within the observed range (across years) are colored red, and horizontal red dashed lines extend to the ordinate, representing the emergent constraint (narrower range resulting from selecting only those models falling in the observed range of total PAN). At Jungfraujoch, the range (across years) in PAN attributed to the EU source region by back-trajectory analysis (Pandey Deolal et al., 2013) is indicated by horizontal dashed blue lines. Individual models are denoted by the symbols deﬁned in Table 1. A. M. Fiore et al.: A constraint on continental source–receptor relationships 100 200 300 400 500 600 700 Total PAN (ppt) 0 20 40 60 80 100 120 PAN from EU (ppt) r=0.85 p=0.01 Jungfraujoch 100 200 300 400 500 600 700 Total PAN (ppt) 0 5 10 15 20 25 30 PAN from NA (ppt) r=0.58 p=0.05 100 200 300 400 500 600 700 Total PAN (ppt) 0 20 40 60 80 100 120 PAN from EU (ppt) r=0.85 p=0.01 PAN from EA (ppt) Jungfraujoch 100 200 300 400 500 600 700 Total PAN (ppt) 0 5 10 15 20 25 30 PAN from NA (ppt) r=0.58 p=0.05 100 200 300 400 500 600 700 Total PAN (ppt) 0 20 40 60 80 100 120 PAN from EU (ppt) r=0.85 p=0.01 100 200 300 400 500 60 Total PAN (ppt) 0 2 4 6 8 10 12 PAN from EA (ppt) r=0.77 p=0.01 Jungfraujoch 100 200 300 400 500 600 700 Total PAN (ppt) 0 2 4 6 8 10 12 PAN from EA (ppt) r=0.77 p=0.01 PAN from EA (ppt) 300 400 500 Total PAN (ppt) Total PAN (ppt) Total PAN (ppt) 0 100 200 300 400 500 Total PAN (ppt) 0 10 20 30 PAN from EU (ppt) r=0.76 p=0.01 100 200 300 400 500 Total PAN (ppt) 0 5 10 15 20 PAN from EA (ppt) r=0.22 p=0.46 Mount Bachelor Mount Bachelor 100 200 300 400 500 Total PAN (ppt) 0 10 20 30 PAN from NA (ppt) r=0.97 p=0.00 Mount Waliguan 100 200 300 400 500 600 700 Total PAN (ppt) 0 10 20 30 40 PAN from EU (ppt) r=0.22 p=0.46 Mount Waliguan 100 200 300 400 500 600 700 Total PAN (ppt) 0 5 10 15 20 PAN from NA (ppt) r=0.45 p=0.14 100 200 300 400 500 600 700 Total PAN (ppt) 0 20 40 60 PAN from EA (ppt) r=0.59 p=0.05 total PAN versus source–receptor relationships (SRRs) at each of three northern midlatitude sites. For Jungfra or relationships (SRRs) at each of three northern midlati Figure 4. Simulated total PAN versus source receptor relationships (SRRs) at each of three northern midlatitude sites. For Jungfraujoch and Mount Bachelor, vertical red lines bound the observed range in total PAN. A. M. Fiore et al.: A constraint on continental source–receptor relationships SRRs diagnosed as the difference between the SR1 and SR6xx simulations in Table 1 for PAN (ppt) at Jungfraujoch (a, b, c), Mount Bachelor (d, e, f), and Mount Waliguan (g, h, i) in each HTAP1 model (see Table 1 for the symbol assigned to each model) versus the annual emission of anthropogenic VOCs (AVOCs; Tg C a−1) within the NA (a, d, g), EU (b, e, h), and EA (c, f, i) source regions. The Spearman’s rank correlation coefﬁcient (more robust to outliers than the traditional Pearson coefﬁcient) and associated p value are shown in each panel. The horizontal red lines correspond to the values identiﬁed with the red symbols in Fig. 4. he difference between the SR1 and SR6xx simulations in Table 1 for PAN (ppt) at Jungfraujoch (a, b, c), Figure 5. SRRs diagnosed as the difference between the SR1 and SR6xx simulations in Table 1 for PAN (ppt) at Jungfraujoch (a, b, c), Mount Bachelor (d, e, f), and Mount Waliguan (g, h, i) in each HTAP1 model (see Table 1 for the symbol assigned to each model) versus the annual emission of anthropogenic VOCs (AVOCs; Tg C a−1) within the NA (a, d, g), EU (b, e, h), and EA (c, f, i) source regions. The Spearman’s rank correlation coefﬁcient (more robust to outliers than the traditional Pearson coefﬁcient) and associated p value are shown in each panel. The horizontal red lines correspond to the values identiﬁed with the red symbols in Fig. 4. A. M. Fiore et al.: A constraint on continental source–receptor relationships 15354 5 10 15 20 25 NA AVOC (Tg C a -1) 0 5 10 15 20 25 30 PAN from NA (ppt) r=0.49 p=0.10 0 10 20 30 40 EU AVOC (Tg C a -1) 0 20 40 60 80 100 120 PAN from EU (ppt) r=0.80 p=0.01 5 10 15 20 25 EA AVOC (Tg C a -1) 0 2 4 6 8 10 12 PAN from EA (ppt) r=0.21 p=0.48 5 10 15 20 25 NA AVOC (Tg C a -1) 0 10 20 30 PAN from NA (ppt) r=0.52 p=0.08 0 10 20 30 40 EU AVOC (Tg C a -1) 0 10 20 30 PAN from EU (ppt) r=0.56 p=0.06 5 10 15 20 25 EA AVOC (Tg C a -1) 0 5 10 15 20 PAN from EA (ppt) r=0.33 p=0.27 5 10 15 20 25 NA AVOC (Tg C a -1) 0 5 10 15 20 PAN from NA (ppt) r=0.58 p=0.05 0 10 20 30 40 EU AVOC (Tg C a -1) 0 10 20 30 40 PAN from EU (ppt) r=0.60 p=0.05 5 10 15 20 25 EA AVOC (Tg C a -1) 0 20 40 60 PAN from EA (ppt) r=-0.20 p=0.50 (a) (d) (g) (b) (e) (h) (c) (f) (i) Jungfraujoch Mount Bachelor Mount Waliguan Figure 5. SRRs diagnosed as the difference between the SR1 and SR6xx simulations in Table 1 for PAN (ppt) at Jungfraujoch (a, b, c), Mount Bachelor (d, e, f), and Mount Waliguan (g, h, i) in each HTAP1 model (see Table 1 for the symbol assigned to each model) versus the annual emission of anthropogenic VOCs (AVOCs; Tg C a−1) within the NA (a, d, g), EU (b, e, h), and EA (c, f, i) source regions. The Spearman’s rank correlation coefﬁcient (more robust to outliers than the traditional Pearson coefﬁcient) and associated p value are shown in each panel. The horizontal red lines correspond to the values identiﬁed with the red symbols in Fig. 4. www.atmos-chem-phys.net/18/15345/2018/ Atmos. Chem. Phys., 18, 15345–15361, 2018 A. M. Fiore et al.: A constraint on continental source–receptor relationships 20 25 g C a -1) 0 10 20 30 40 EU AVOC (Tg C a -1) 0 20 40 60 80 100 120 PAN from EU (ppt) r=0.80 p=0.01 5 10 15 20 25 EA AVOC (Tg C a -1) 0 2 4 6 8 10 12 PAN from EA (ppt) r=0.21 p=0.48 (b) (c) Jungfraujoch 5 10 15 20 25 NA AVOC (Tg C a -1) 0 5 10 15 20 25 30 PAN from NA (ppt) r=0.49 p=0.10 0 10 20 30 40 EU AVOC (Tg C a -1) 0 20 40 60 80 100 120 PAN from EU (ppt) r=0.80 p=0.01 5 10 15 20 25 EA AVOC (Tg C a -1) 0 2 4 6 8 10 12 PAN from EA (ppt) r=0.21 p=0.48 (a) (b) (c) Jungfraujoch 5 10 15 20 25 NA AVOC (Tg C a -1) 0 10 20 30 PAN from NA (ppt) r=0.52 p=0.08 0 10 20 30 40 EU AVOC (Tg C a -1) 0 10 20 30 PAN from EU (ppt) r=0.56 p=0.06 5 10 15 20 EA AVOC (Tg C a -1 0 5 10 15 20 PAN from EA (ppt) r=0.33 p=0.27 (d) (e) (f) Mount Bachelor 5 20 25 (Tg C a -1) 0 10 20 30 40 EU AVOC (Tg C a -1) 0 10 20 30 PAN from EU (ppt) r=0.56 p=0.06 5 10 15 20 25 EA AVOC (Tg C a -1) 0 5 10 15 20 PAN from EA (ppt) r=0.33 p=0.27 (e) (f) Mount Bachelor Mount Bachelor 5 10 15 20 25 NA AVOC (Tg C a -1) 0 5 10 15 20 PAN from NA (ppt) r=0.58 p=0.05 0 10 20 30 40 EU AVOC (Tg C a -1) 0 10 20 30 40 PAN from EU (ppt) r=0.60 p=0.05 5 10 EA A 0 20 40 60 PAN from EA (ppt) r=-0.20 p=0.50 (g) (h) (i) Mount Waliguan 15 20 25 VOC (Tg C a -1) 0 10 20 30 40 EU AVOC (Tg C a -1) 0 10 20 30 40 PAN from EU (ppt) r=0.60 p=0.05 5 10 15 20 25 EA AVOC (Tg C a -1) 0 20 40 60 PAN from EA (ppt) r=-0.20 p=0.50 (h) (i) Mount Waliguan Figure 5. A. M. Fiore et al.: A constraint on continental source–receptor relationships 15355 (a) 100 200 300 400 500 Total PAN (ppt) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Ratio of NA/EA PAN r=0.66 p=0.03 (b) 0.2 0.4 0.6 0.8 1.0 COFROMNA/COFROMEA 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Ratio of NA/EA PAN r=0.62 p=0.05 Figure 6. Ratio of the PAN response to 20 % emission reductions within NA to that from EA plotted against (a) total PAN and (b) the ratio of idealized tracers of model transport emitted from NA to that from EA (COfromNA/COfromEA; see Table 2) at Mount Bachelor as simulated by the HTAP1 models. Each symbol in (a) represents a model as deﬁned in Table 1; the range of observed total PAN at Mount Bachelor is indicated by the black vertical lines. The col- ored symbols in (b) represent the meteorological ﬁelds used in the simulation: blue triangles for GEOS winds, red circles for NCEP, black diamonds for ECMWF, cyan upside-down triangles for the Canadian Meteorological Centre, and green squares for general cir- culation models forced by observed sea surface temperatures and sea ice. Both panels show Spearman’s rank correlation coefﬁcients and p values, as well as a black dashed horizontal line at 1 to sep- arate the models suggesting a higher NA inﬂuence (above) versus higher EA inﬂuence (below) on PAN SRRs. (a) 100 200 300 400 500 Total PAN (ppt) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Ratio of NA/EA PAN r=0.66 p=0.03 (b) 0.2 0.4 0.6 0.8 1.0 COFROMNA/COFROMEA 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Ratio of NA/EA PAN r=0.62 p=0.05 (a) 100 200 300 400 500 Total PAN (ppt) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Ratio of NA/EA PAN r=0.66 p=0.03 sions. Model differences in transport as diagnosed by the idealized regional CO tracers correlate more with O3 SRRs than for PAN for all source–receptor pairs, though the cor- relations remain weak except for COfromEU with O3 SRRs at Jungfraujoch. Overall, this analysis supports earlier ﬁnd- ings that PAN is more sensitive to changes in emissions (and subsequent chemistry), particularly for VOC precursors, than O3. The correlations between SRRs for PAN and O3 could re- ﬂect a role for PAN transport in contributing to O3 production over the receptor region, or may instead reﬂect coproduction of PAN and O3 from oxidation of regional precursor emis- sions followed by transport in the same air mass. A. M. Fiore et al.: A constraint on continental source–receptor relationships In the lat- ter case, PAN serves as a proxy for O3 transport, whereas in the former case PAN serves as the actual pathway by which O3 is transported. We do not have model diagnostics that al- low us to distinguish between these two roles for PAN. The correlations between PAN and O3 SRRs, however, suggest that long-term PAN measurements contain signals relevant for constraining the relative importance of regional vs. in- tercontinental emissions on both PAN and O3. We examine the strength of these signals by correlating the O3 SRRs at each site with total PAN as simulated at each site. Relation- ships are far weaker than for the PAN SRRs and total PAN shown in Fig. 4, but correlations are signiﬁcant between total PAN at Jungfraujoch for O3 from EU (r = 0.67; p = 0.03) and at Mount Bachelor for O3 from NA (r = 0.61; p = 0.04; Fig. S4). Figure 6. Ratio of the PAN response to 20 % emission reductions Figure 6. Ratio of the PAN response to 20 % emission reductions within NA to that from EA plotted against (a) total PAN and (b) the ratio of idealized tracers of model transport emitted from NA to that from EA (COfromNA/COfromEA; see Table 2) at Mount Bachelor as simulated by the HTAP1 models. Each symbol in (a) represents a model as deﬁned in Table 1; the range of observed total PAN at Mount Bachelor is indicated by the black vertical lines. The col- ored symbols in (b) represent the meteorological ﬁelds used in the simulation: blue triangles for GEOS winds, red circles for NCEP, black diamonds for ECMWF, cyan upside-down triangles for the Canadian Meteorological Centre, and green squares for general cir- culation models forced by observed sea surface temperatures and sea ice. Both panels show Spearman’s rank correlation coefﬁcients and p values, as well as a black dashed horizontal line at 1 to sep- arate the models suggesting a higher NA inﬂuence (above) versus higher EA inﬂuence (below) on PAN SRRs. is relevant for interpreting O3 SRRs by correlating PAN and O3 SRRs at the three mountaintop sites (Fig. 7). Rela- tionships vary across the individual source–receptor pairs, with the inter-model variability in PAN explaining 16 %– 60 % of the inter-model differences in O3 at the mountain sites. A. M. Fiore et al.: A constraint on continental source–receptor relationships The strongest relationships occur for the inﬂuence of regional sources at Mount Bachelor (from NA) and Jungfrau- joch (from EU). At Mount Waliguan, the EU and EA source– receptor relationships for PAN and O3 are of similar strength (r = 0.7). Intercontinental source–receptor pairs for O3 and PAN at Mount Bachelor and Mount Waliguan are also sig- niﬁcant to within 90 %, with variability in the PAN attributed to intercontinental source regions explaining 25 %–35 % and 30 %–45 %, respectively, of the variability in the correspond- ing O3 SRRs. 6 Linking PAN and O3 SRRs ratio of EU : EA anthropogenic emission inﬂuence on PAN at Mount Bachelor shows little correlation with the respec- tive regional NOx emissions used in the models, or with the differences in the simulated transport tracers (r = 0.3 for both cases). As at Mount Bachelor, the model spread in the contribution to total simulated PAN from the EA versus NA source regions at both Jungfraujoch and Mount Waliguan de- pends most on the regional AVOC ratios (r = 0.8 and 0.6, re- spectively; Fig. S3), with little correlation with inter-model differences in NA : EA ANOx emissions. Some correlation also emerges between the NA : EA SRRs for PAN and the NA : EA transport tracers (r = 0.6 at both sites; Fig. S3). Fi- nally, we do not ﬁnd any obvious link between PAN SRRs and the choice of meteorological ﬁelds (the individual sym- bols in Figs. S2 and S3). We address here the extent to which observational constraints on PAN SRRs might also serve to narrow the range of un- certainty in the inter-model spread in intercontinental SRRs for O3 (e.g., Fiore et al., 2009). We expect some common- ality between the sensitivity of PAN and O3 to changes in precursor emissions because (1) both species are pro- duced from chemical reactions involving NOx and VOCs and (2) PAN serves as a NOx reservoir, which upon decomposi- tion releases NOx that can then produce O3 far downwind of the region where the PAN (and O3) precursors were origi- nally emitted. Furthermore, earlier analysis of HTAP1 ozone continental-scale SRRs also identiﬁed a correlation with the model AVOC emissions, particularly over EU (Fiore et al., 2009). We assess the extent to which the inter-model range in source region inﬂuence on mountaintop PAN levels in April Atmos. Chem. Phys., 18, 15345–15361, 2018 www.atmos-chem-phys.net/18/15345/2018/ 7 Conclusions and recommendations Our proof-of-concept approach applies the HTAP1 multi- model ensemble to identify a strong inter-model correlation between PAN source–receptor relationships (SRRs; deﬁned as the difference in simulations with 20 % emission reduc- tions separately within each of the northern midlatitude con- tinents) and simulated total PAN at mountaintop sites during April. Our ﬁndings imply promise for developing emergent constraints (e.g., Hall and Qu, 2006; Borodina et al., 2017; Cox et al., 2018) from more routine PAN measurements to narrow uncertainty in wide-ranging model estimates of PAN SRRs, quantities that are not directly observable yet relevant to air quality policy (e.g., HTAP, 2010). Inter-model correla- tions of the responses of PAN versus O3 to perturbations in regional anthropogenic emissions (Figs. 7 and 8) imply that constraints on PAN SRRs are relevant for lowering uncer- tainty in O3 SRR estimates. This connection between PAN and O3 likely reﬂects the dual role of PAN as both a pathway for O3 transport (by producing O3 upon its decomposition following transport), and as a proxy for O3 transport (as it is produced alongside O3 in the polluted continental boundary layer). We expand the correlation analysis of ozone and PAN SRRs from the free troposphere sampled at the mountain- top sites to large-scale SRRs in surface air over the HTAP1 continental regions. Of the signiﬁcant relationships in Fig. 7 (p < 0.10), six out of seven also emerge as signiﬁcant in Fig. 8. We infer that conclusions drawn from a limited num- ber of mountaintop sites regarding PAN SRRs and their rela- tionship to ozone SRRs are relevant, at least according to the models, on much broader scales. We repeat the analysis in Fig. 5 but for O3 to consider the inﬂuence of the three source regions on the three mountain- top sites (nine total source–receptor pairs) but ﬁnd little rela- tionship between the model spread in the simulated O3 SRRs and in the magnitude of the regional AVOCs or ANOx emis- Establishing the strongest constraints possible on simu- lated SRRs for PAN and O3 will require (1) measurements www.atmos-chem-phys.net/18/15345/2018/ A. M. Fiore et al.: A constraint on continental source–receptor relationships 30 0 20 40 60 80 100 120 PAN from EU (ppt) 0.0 0.5 1.0 1.5 2.0 2.5 O3 from EU (ppb) r=0.77 p=0.01 2 4 6 8 10 12 PAN from EA (ppt) 0.0 0.2 0.4 0.6 0.8 O3 from EA (ppb) r=0.40 p=0.18 (b) (c) Jungfraujoch 5 10 15 20 25 30 PAN from NA (ppt) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 O3 from NA (ppb) r=0.46 p=0.13 0 20 40 60 80 100 120 PAN from EU (ppt) 0.0 0.5 1.0 1.5 2.0 2.5 O3 from EU (ppb) r=0.77 p=0.01 2 4 6 8 10 12 PAN from EA (ppt) 0.0 0.2 0.4 0.6 0.8 O3 from EA (ppb) r=0.40 p=0.18 (a) (b) (c) Jungfraujoch 5 20 25 30 35 om NA (ppt) 5 10 15 20 25 30 PAN from EU (ppt) 0.0 0.2 0.4 0.6 0.8 1.0 O3 from EU (ppb) r=0.59 p=0.05 0 5 10 15 20 PAN from EA (ppt) 0.0 0.2 0.4 0.6 0.8 1.0 O3 from EA (ppb) r=0.51 p=0.09 (e) (f) Mount Bachelor 20 0 5 10 15 20 25 30 35 PAN from EU (ppt) 0.0 0.2 0.4 0.6 0.8 1.0 O3 from EU (ppb) r=0.72 p=0.02 0 10 20 30 40 50 60 PAN from EA (ppt) 0.0 0.5 1.0 1.5 2.0 O3 from EA (ppb) r=0.67 p=0.03 (h) (i) Mount Waliguan Figure 7. SRRs for O3 versus PAN at Jungfraujoch (a, b, c), Mount Bachelor (d, e, f), and Mount Waliguan (g, h, i), obtained by subtracting the SR6XX from the SR1 simulations (Table 2) available from 12 models, for which XX denotes the NA (a, d, g), EU (b, e, h), or EA (c, f, i) source region. Each model thus contributes one point (symbols deﬁned in Table 1) in each panel. Spearman’s (rank) correlation coefﬁcient and p values are also shown. Waliguan would provide constraints on PAN SRRs, partic- ularly for PAN originating in EA (Fig. 4). Additional work could systematically examine over 60 stations at altitudes above 2500 m in the Tropospheric Ozone Assessment Report (TOAR) database (Schultz et al., 2017). and simulations with chemical transport models that coincide and (2) a sufﬁciently long measurement record to build a cli- matology suitable for evaluating chemistry–climate models that generate their own meteorology. Repeated sampling for the month of April may be sufﬁcient to provide constraints on model responses to changes in anthropogenic emissions. A. M. Fiore et al.: A constraint on continental source–receptor relationships PAN measurements over multiple seasons are necessary to evaluate model responses of PAN to climate change (e.g., by changing temperature and weather-sensitive precursor emis- sions) and the resulting inﬂuence on atmospheric O3 and ox- idizing capacity (e.g., Doherty et al., 2013). For example, changes in meteorology and biomass burning (Fischer et al., 2011; Zhu et al., 2015) such as those driven by El Niño– Southern Oscillation (Koumoutsaris et al., 2008), as well as biogenic and lightning sources (Payne et al., 2017) vary from year to year and are expected to change as climate warms. and simulations with chemical transport models that coincide and (2) a sufﬁciently long measurement record to build a cli- matology suitable for evaluating chemistry–climate models that generate their own meteorology. Repeated sampling for the month of April may be sufﬁcient to provide constraints on model responses to changes in anthropogenic emissions. PAN measurements over multiple seasons are necessary to evaluate model responses of PAN to climate change (e.g., by changing temperature and weather-sensitive precursor emis- sions) and the resulting inﬂuence on atmospheric O3 and ox- idizing capacity (e.g., Doherty et al., 2013). For example, changes in meteorology and biomass burning (Fischer et al., 2011; Zhu et al., 2015) such as those driven by El Niño– Southern Oscillation (Koumoutsaris et al., 2008), as well as biogenic and lightning sources (Payne et al., 2017) vary from year to year and are expected to change as climate warms. We recommend archiving daily model ﬁelds for future ap- plications of this multi-model emergent constraint approach to SRRs. Access to daily model ﬁelds permits (1) a more rigorous process-oriented evaluation of speciﬁc events (e.g., Fischer et al., 2010; Alvarado et al., 2010; Arnold et al., 2015) and (2) comparison with satellite-derived tropospheric PAN columns, which show promise for documenting PAN distributions, particularly in the upper troposphere, and their temporal variability and spatial patterns across the globe (e.g., Fadnavis et al., 2014; Jiang et al., 2016; Payne et al., 2014, 2017; Zhu et al., 2015, 2017). We also suggest archiv- ing daily tracers tagged by emission region to isolate the role of model differences in transport during individual events. In addition, Lin et al. A. M. Fiore et al.: A constraint on continental source–receptor relationships A. M. Fiore et al.: A constraint on continental source–receptor relationships 5 10 15 20 25 30 PAN from NA (ppt) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 O3 from NA (ppb) r=0.46 p=0.13 0 20 40 60 80 100 120 PAN from EU (ppt) 0.0 0.5 1.0 1.5 2.0 2.5 O3 from EU (ppb) r=0.77 p=0.01 2 4 6 8 10 12 PAN from EA (ppt) 0.0 0.2 0.4 0.6 0.8 O3 from EA (ppb) r=0.40 p=0.18 0 5 10 15 20 25 30 35 PAN from NA (ppt) 0.0 0.2 0.4 0.6 0.8 1.0 O3 from NA (ppb) r=0.67 p=0.03 5 10 15 20 25 30 PAN from EU (ppt) 0.0 0.2 0.4 0.6 0.8 1.0 O3 from EU (ppb) r=0.59 p=0.05 0 5 10 15 20 PAN from EA (ppt) 0.0 0.2 0.4 0.6 0.8 1.0 O3 from EA (ppb) r=0.51 p=0.09 0 5 10 15 20 PAN from NA (ppt) 0.0 0.2 0.4 0.6 0.8 O3 from NA (ppb) r=0.56 p=0.06 0 5 10 15 20 25 30 35 PAN from EU (ppt) 0.0 0.2 0.4 0.6 0.8 1.0 O3 from EU (ppb) r=0.72 p=0.02 0 10 20 30 40 50 60 PAN from EA (ppt) 0.0 0.5 1.0 1.5 2.0 O3 from EA (ppb) r=0.67 p=0.03 (a) (d) (g) (b) (e) (h) (c) (f) (i) Jungfraujoch Mount Bachelor Mount Waliguan Figure 7. SRRs for O3 versus PAN at Jungfraujoch (a, b, c), Mount Bachelor (d, e, f), and Mount Waliguan (g, h, i), obtained by subtracting the SR6XX from the SR1 simulations (Table 2) available from 12 models, for which XX denotes the NA (a, d, g), EU (b, e, h), or EA (c, f, i) source region. Each model thus contributes one point (symbols deﬁned in Table 1) in each panel. Spearman’s (rank) correlation coefﬁcient and p values are also shown. www.atmos-chem-phys.net/18/15345/2018/ Atmos. Chem. Phys., 18, 15345–15361, 2018 15356 A. M. Fiore et al.: A constraint on continental source–receptor relationships EU mean 0 5 10 15 PAN (SR1-SR6NA) 0.0 0.2 0.4 0.6 0.8 O3 (SR1-SR6NA) r=0.28 p=0.33 EU mean 0 20 40 60 80 100 120 PAN (SR1-SR6EU) 0.0 0.5 1.0 1.5 2.0 O3 (SR1-SR6EU) r=0.62 p=0.03 O3 (SR1 SR6EA) (a) (b) EU mean 0 2 4 6 8 10 PAN (SR1-SR6EA) 0.0 0.1 0.2 0.3 0.4 O3 (SR1-SR6EA) r=0.61 p=0.04 (c) PAN (SR1 SR6NA) PAN (SR1 SR6EU) NA mean 0 20 40 60 PAN (SR1-SR6NA) 0.0 0.5 1.0 1.5 2.0 O3 (SR1-SR6NA) r=0.49 p=0.09 NA mean 0 5 10 15 20 PAN (SR1-SR6EU) 0.0 0.1 0.2 0.3 0.4 0.5 O3 (SR1-SR6EU) r=0.62 p=0.03 0 0.0 0.1 0.2 0.3 0.4 0.5 O3 (SR1-SR6EA) (d) (e) (f) ( ) ( ) 60 NA mean 0 5 10 15 20 PAN (SR1-SR6EU) 0.0 0.1 0.2 0.3 0.4 0.5 O3 (SR1-SR6EU) r=0.62 p=0.03 NA mean 0 2 4 6 8 PAN (SR1-SR6EA) 0.0 0.1 0.2 0.3 0.4 0.5 O3 (SR1-SR6EA) r=0.56 p=0.05 (e) (f) EA mean 0 2 4 6 8 10 PAN (SR1-SR6NA) 0.0 0.1 0.2 0.3 0.4 O3 (SR1-SR6NA) r=0.56 p=0.05 EA mean 0 5 10 15 20 25 30 PAN (SR1-SR6EU) 0.0 0.2 0.4 0.6 O3 (SR1-SR6EU) r=0.70 p=0.02 EA mean 0 10 20 30 40 PAN (SR1-SR6EA 0.0 0.5 1.0 1.5 O3 (SR1-SR6EA) r=0 p= (g) (h) (i) 8 10 A) 0.56 0.05 EA mean 0 5 10 15 20 25 30 PAN (SR1-SR6EU) 0.0 0.2 0.4 0.6 O3 (SR1-SR6EU) r=0.70 p=0.02 EA mean 0 10 20 30 40 50 60 PAN (SR1-SR6EA) 0.0 0.5 1.0 1.5 O3 (SR1-SR6EA) r=0.31 p=0.29 (h) (i) Figure 8. SRRs for O3 versus PAN in surface air over each of the HTAP1 northern midlatitude continental regions: EU (a, b, c), NA (d, e, f), and EA (g, h, i), obtained by subtracting the SR6XX from the SR1 simulations, for which XX denotes the NA (a, d, g), EU (b, e, h), or EA (c, f, i) source region. Spearman’s (rank) correlation coefﬁcient and p values are also shown. Symbols denote individual models as deﬁned in Table 1. STOC-HadAM3-v01 is excluded here as an outlier that artiﬁcially raised the correlation signiﬁcance. ture model simulations (and multi-model ensembles) can be assessed. tracers can better isolate free-tropospheric air from surface air masses when comparing coarse-resolution models with high-altitude measurements. A. M. Fiore et al.: A constraint on continental source–receptor relationships By focusing on April, our analysis largely minimizes com- plexities introduced by inter-model differences in biogenic, ﬁre, and lightning sources that further complicate disentan- gling summertime discrepancies in simulated PAN and O3 (e.g., Arnold et al., 2015; Emmons et al., 2015) and re- stricts inter-model differences to those associated with an- thropogenic emissions and the subsequent chemistry and transport. Nevertheless, we ﬁnd a wide range in inter-model SRR relationships that reﬂects uncertainties in emissions and different model representations of VOC chemistry, including PAN yields from VOCs (Fig. 5; Emmerson and Evans, 2009; Fischer et al., 2014; Arnold et al., 2015; Emmons et al., 2015; Knote et al., 2015). Future multi-model efforts could seek to separately parse the inﬂuence of differences in total anthro- pogenic VOC emissions, the mix of emitted VOC species and their reactivity, and the chemical production of PAN and O3. Documenting these aspects of model conﬁguration would help to establish benchmarks for inter-model differences in simulated total PAN, O3, and their SRRs, against which fu- Data availability. Upon publication, the data used to generate the ﬁgures can be found in a CSU digital repository that we established for this paper (https://hdl.handle.net/10217/185610). A. M. Fiore et al.: A constraint on continental source–receptor relationships (2017) have demonstrated that applying a ﬁltering technique based on daily idealized CO regional We identiﬁed only ﬁve multiyear datasets at mountain sites, four of which are located near each other in Europe, and only one of which continues at present (Schauinsland). Our analysis suggests that future measurements at Mount www.atmos-chem-phys.net/18/15345/2018/ Atmos. Chem. Phys., 18, 15345–15361, 2018 The Supplement related to this article is available online at: https://doi.org/10.5194/acp-18-15345-2018- supplement Author contributions. AMF and EVF conceived the analysis with guidance from SPD, OW, DAJ, and JS. EVF compiled monthly mean PAN measurements from EVF, DAJ, SPD, JS, BF, SG, LR, MGSa, MS, and CZ. AMF led the model evaluation, emergent con- straint analysis, and writing. AMF, GPM, and OEC prepared the ﬁgures. SPD and JS contributed trajectory analysis at Jungfrau- joch. OW contributed to linearity analysis. AMF, OW, DB, WC, FD, RMD, BND, PGH, LWH, AL, IAM, RP, MGSa, MGSc, DTS, DSS, SS, and GZ contributed HTAP Phase 1 model simulations. All A. M. Fiore et al.: A constraint on continental source–receptor relationships 15357 EU mean 0 5 10 15 PAN (SR1-SR6NA) 0.0 0.2 0.4 0.6 0.8 O3 (SR1-SR6NA) r=0.28 p=0.33 EU mean 0 20 40 60 80 100 120 PAN (SR1-SR6EU) 0.0 0.5 1.0 1.5 2.0 O3 (SR1-SR6EU) r=0.62 p=0.03 EU mean 0 2 4 6 8 10 PAN (SR1-SR6EA) 0.0 0.1 0.2 0.3 0.4 O3 (SR1-SR6EA) r=0.61 p=0.04 NA mean 0 20 40 60 PAN (SR1-SR6NA) 0.0 0.5 1.0 1.5 2.0 O3 (SR1-SR6NA) r=0.49 p=0.09 NA mean 0 5 10 15 20 PAN (SR1-SR6EU) 0.0 0.1 0.2 0.3 0.4 0.5 O3 (SR1-SR6EU) r=0.62 p=0.03 NA mean 0 2 4 6 8 PAN (SR1-SR6EA) 0.0 0.1 0.2 0.3 0.4 0.5 O3 (SR1-SR6EA) r=0.56 p=0.05 EA mean 0 2 4 6 8 10 PAN (SR1-SR6NA) 0.0 0.1 0.2 0.3 0.4 O3 (SR1-SR6NA) r=0.56 p=0.05 EA mean 0 5 10 15 20 25 30 PAN (SR1-SR6EU) 0.0 0.2 0.4 0.6 O3 (SR1-SR6EU) r=0.70 p=0.02 EA mean 0 10 20 30 40 50 60 PAN (SR1-SR6EA) 0.0 0.5 1.0 1.5 O3 (SR1-SR6EA) r=0.31 p=0.29 (a) (d) (g) (b) (e) (h) (c) (f) (i) Figure 8. SRRs for O3 versus PAN in surface air over each of the HTAP1 northern midlatitude continental regions: EU (a, b, c), NA (d, e, f), and EA (g, h, i), obtained by subtracting the SR6XX from the SR1 simulations, for which XX denotes the NA (a, d, g), EU (b, e, h), or EA (c, f, i) source region. Spearman’s (rank) correlation coefﬁcient and p values are also shown. Symbols denote individual models as deﬁned in Table 1. STOC-HadAM3-v01 is excluded here as an outlier that artiﬁcially raised the correlation signiﬁcance. References Emmons, L. K., Arnold, S. R., Monks, S. A., Huijnen, V., Tilmes, S., Law, K. S., Thomas, J. L., Raut, J.-C., Bouarar, I., Tur- quety, S., Long, Y., Duncan, B., Steenrod, S., Strode, S., Flem- ming, J., Mao, J., Langner, J., Thompson, A. M., Tarasick, D., Apel, E. C., Blake, D. R., Cohen, R. C., Dibb, J., Diskin, G. S., Fried, A., Hall, S. R., Huey, L. G., Weinheimer, A. J., Wisthaler, A., Mikoviny, T., Nowak, J., Peischl, J., Roberts, J. M., Ryerson, T., Warneke, C., and Helmig, D.: The POLARCAT Model Intercomparison Project (POLMIP): overview and eval- uation with observations, Atmos. Chem. Phys., 15, 6721–6744, https://doi.org/10.5194/acp-15-6721-2015, 2015. Alvarado, M. J., Logan, J. A., Mao, J., Apel, E., Riemer, D., Blake, D., Cohen, R. C., Min, K.-E., Perring, A. E., Browne, E. C., Wooldridge, P. J., Diskin, G. S., Sachse, G. W., Fuel- berg, H., Sessions, W. R., Harrigan, D. L., Huey, G., Liao, J., Case-Hanks, A., Jimenez, J. L., Cubison, M. J., Vay, S. A., Wein- heimer, A. J., Knapp, D. J., Montzka, D. D., Flocke, F. M., Pol- lack, I. B., Wennberg, P. O., Kurten, A., Crounse, J., Clair, J. M. St., Wisthaler, A., Mikoviny, T., Yantosca, R. M., Carouge, C. C., and Le Sager, P.: Nitrogen oxides and PAN in plumes from boreal ﬁres during ARCTAS-B and their impact on ozone: an integrated analysis of aircraft and satellite observations, At- mos. Chem. Phys., 10, 9739–9760, https://doi.org/10.5194/acp- 10-9739-2010, 2010. Fadnavis, S., Schultz, M. G., Semeniuk, K., Mahajan, A. S., Poz- zoli, L., Sonbawne, S., Ghude, S. D., Kiefer, M., and Eck- ert, E.: Trends in peroxyacetyl nitrate (PAN) in the upper tro- posphere and lower stratosphere over southern Asia during the summer monsoon season: regional impacts, Atmos. Chem. Phys., 14, 12725–12743, https://doi.org/10.5194/acp-14-12725- 2014, 2014. Arnold, S. R., Emmons, L. K., Monks, S. A., Law, K. S., Rid- ley, D. A., Turquety, S., Tilmes, S., Thomas, J. L., Bouarar, I., Flemming, J., Huijnen, V., Mao, J., Duncan, B. N., Steen- rod, S., Yoshida, Y., Langner, J., and Long, Y.: Biomass burn- ing inﬂuence on high-latitude tropospheric ozone and reac- tive nitrogen in summer 2008: a multi-model analysis based on POLMIP simulations, Atmos. Chem. Phys., 15, 6047–6068, https://doi.org/10.5194/acp-15-6047-2015, 2015. Fiore, A. M., Dentener, F. J., Wild, O., Cuvelier, C., Schultz, M. G., Hess, P., Textor, C., Schulz, M., Doherty, R. M., Horowitz, L. W., MacKenzie, I. A., Sanderson, M. Competing interests. The authors declare that they have no conﬂict of interest. Competing interests. The authors declare that they have no conﬂict of interest. Bottenheim, J. W., Sirois, A., Brice, K. A., and Gallant, A. J.: Five years of continuous observations of PAN and ozone at a rural location in eastern Canada, J. Geophys. Res.-Atmos., 99, 5333– 5352, 10.1029/93JD02716, 1994. Special issue statement. This article is part of the special issue “Global and regional assessment of intercontinental transport of air pollution: results from HTAP, AQMEII and MICS”. It is not associ- ated with a conference. Carpenter, L. J., Green, T. J., Mills, G. P., Bauguitte, S., Penkett, S. A., Zanis, P., Schuepbach, E., Schmidbauer, N., Monks, P. S., and Zellweger, C: Oxidized nitrogen and ozone production efﬁcien- cies in the springtime free troposphere over the Alps, J. Geophys. Res., 105, 14547–14559, 2000. Acknowledgements. We thank Mathew Evans (York University, UK) and Terry Keating (U.S. EPA) for useful discussions and the two anonymous referees for their constructive comments. Arlene M. Fiore and Bryan N. Duncan acknowledge NASA MAP (NNX14AM38G). David S. Stevenson acknowledges NERC (grants NE/K001329/1 and NE/N003411/1) and the ARCHER UK National Supercomputing Service (http://www.archer.ac.uk, last access: 17 October 2018). Drew T. Shindell acknowledges the NASA High-End Computing Program through the NASA Center for Climate Simulation at GSFC for computational resources. Michael G. Sanderson was supported by the Met Ofﬁce Hadley Centre Climate Programme funded by BEIS and Defra. This is Lamont contribution number 8251. Chameides, W. L., Fehsenfeld, F. C., Rodgers, M. O., Cardelino, C., Martinez, J., Parrish, D., Lonnerman, W., Lawson, D. R., Ras- mussen, R. A., Zimmerman, P., Greenberg, J., Middleton, P., and Wang, T.: Ozone precursor relationships in the ambient atmo- sphere, J. Geophys. Res., 97, 6037–6055, 1992. Cox, P. M., Huntingford, C., and Williamson, M. S., Emer- gent constraint on equilibrium climate sensitivity from global temperature variability: Nature, 553, 319–323, https://doi.org/10.1038/nature25450, 2018. Doherty, R. M., Wild, O., Shindell, D. T., Zeng, G., MacKenzie, I. A., Collins, W. J., Fiore, A. M., Stevenson, D. S., Dentener, F. J., Schultz, M. G., Hess, P., Derwent, R. G., and Keating, T. J.: Impacts of climate change on surface ozone and intercontinental ozone pollution: A multi-model study, J. Geophys. Res.-Atmos., 118, 3744–3763, https://doi.org/10.1002/jgrd.50266, 2013. Edited by: Kathy Law Reviewed by: two anonymous referees Edited by: Kathy Law Reviewed by: two anonymous referees Edited by: Kathy Law Reviewed by: two anonymous referees Emmerson, K. M. and Evans, M. Competing interests. The authors declare that they have no conﬂict of interest. J.: Comparison of tropospheric gas-phase chemistry schemes for use within global models, At- mos. Chem. Phys., 9, 1831–1845, https://doi.org/10.5194/acp-9- 1831-2009, 2009. www.atmos-chem-phys.net/18/15345/2018/ www.atmos-chem-phys.net/18/15345/2018/ Atmos. Chem. Phys., 18, 15345–15361, 2018 A. M. Fiore et al.: A constraint on continental source–receptor relationships 15358 Alpine site Jungfraujoch (3580 m asl), J. Geophys. Res., 113, D22305, https://doi.org/10.1029/2007JD009751, 2008. Alpine site Jungfraujoch (3580 m asl), J. Geophys. Res., 113, D22305, https://doi.org/10.1029/2007JD009751, 2008. authors helped to shape the paper content by editing prior versions of the paper. Borodina, A., Fischer, E. M., and Knutti, R.: Emergent Constraints in Climate Projections: A Case Study of Changes in High- Latitude Temperature Variability, J. Climate, 30, 3655–3670, https://doi.org/10.1175/JCLI-D-16-0662.1, 2017. A. M. Fiore et al.: A constraint on continental source–receptor relationships 15359 Jonson, J. E., Kaminski, J. W., Keating, T. J., Lupu, A., Marmer, E., Montanaro, V., Park, R. J., Pitari, G., Pringle, K. J., Pyle, J. A., Schroeder, S., Vivanco, M. G., Wind, P., Wojcik, G., Wu, S., and Zuber, A.: Multimodel estimates of intercontinental source- receptor relationships for ozone pollution, J. Geophys. Res., 114, D04301, https://doi.org/10.1029/2008jd010816, 2009. Jonson, J. E., Kaminski, J. W., Keating, T. J., Lupu, A., Marmer, E., Montanaro, V., Park, R. J., Pitari, G., Pringle, K. J., Pyle, J. Jaffe, D., Thornton, J., Wolfe, G., Reidmiller, D., Fischer, E. V., Ja- cob, D. J., Cohen, R., Singh, H., Weinheimer, A., and Flocke, F.: Can we Detect an Inﬂuence over North America From Increas- ing Asian NOx Emissions?, EOS Trans. AGU, 88(52), Fall Meet. Suppl., Abstract A51E-04, 2007. Jiang, Z., Worden, J. R., Payne, V. H., Zhu, L., Fischer, E., Walker, T., and Jones, D. B. A.: Ozone export from East Asia: The role of PAN, J. Geophys. Res.-Atmos., 121, 6555–6563, https://doi.org/10.1002/2016JD024952, 2016. Fiore, A. M., Levy II, H., and Jaffe, D. A.: North American isoprene inﬂuence on intercontinental ozone pollution, Atmos. Chem. Phys., 11, 1697–1710, https://doi.org/10.5194/acp-11- 1697-2011, 2011. Kirchner, F., Mayer-Figge, A., Zabel, F., and Becker, K. H.: Ther- mal stability of peroxynitrates, Int. J. Chem. Kinet., 31, 127–144, 1999. Fischer, E. V., Jaffe, D. A., Reidmiller, D. R., and Jaegle, L.: Me- teorological controls on observed peroxyacetyl nitrate (PAN) at Mount Bachelor during the spring of 2008, J. Geophys. Res., 115, D03302, https://doi.org/10.1029/2009JD012776, 2010. Knote, C., Tuccella, P., Curci, G., Emmons, L., Orlando, J. J., Madronich, S., Baro, R., Jimenez-Guerrero, P., Luecken, D., Hogrefe, C., Forkel, R. , Werhahn, J., Hirtl, M., Perez, J., San Jose, R., Giordano, L., Brunner, D., Yahya, K., and Zhang, Y.: Inﬂuence of the choice of gas-phase mechanism on predictions of key gaseous pollutants during the AQMEII phase-2 intercom- parison, Atmos. Environ., 115, 553–568, 2015. Fischer, E. V., Jaffe, D. A., and Weatherhead, E. C.: Free tro- pospheric peroxyacetyl nitrate (PAN) and ozone at Mount Bachelor: potential causes of variability and timescale for trend detection, Atmos. Chem. Phys., 11, 5641–5654, https://doi.org/10.5194/acp-11-5641-2011, 2011. Kotchenruther, R. A., Jaffe, D. A., and Jaeglé, L.: Ozone potochem- istry and the role of PAN in the springtime northeastern Paciﬁc Troposphere: Results from the PHOBEA Campaign, J. Geophys. Res., 106, 28731–28741, 2001a. Fischer, E. V., Jacob, D. J., Yantosca, R. M., Sulprizio, M. P., Millet, D. A. M. Fiore et al.: A constraint on continental source–receptor relationships B., Mao, J., Paulot, F., Singh, H. B., Roiger, A., Ries, L., Talbot, R. W., Dzepina, K., and Pandey Deolal, S.: Atmospheric peroxyacetyl nitrate (PAN): a global budget and source attribution, Atmos. Chem. Phys., 14, 2679–2698, https://doi.org/10.5194/acp-14-2679-2014, 2014. Kotchenruther, R. A., Jaffe, D. A., Beine, H. J., Anderson,T., Bot- tenheim, J. W., Harris, J. M., Blake, D., and Schmitt, R.: Obser- vations of ozone and related species in the Northeast Paciﬁc dur- ing the PHOBEA Campaigns: 2. Airborne observations, J. Geo- phys. Res., 106, 7463–7483, 2001b. Gilge, S., Plass-Duelmer, C., Fricke, W., Kaiser, A., Ries, L., Buch- mann, B., and Steinbacher, M.: Ozone, carbon monoxide and nitrogen oxides time series at four alpine GAW mountain sta- tions in central Europe, Atmos. Chem. Phys., 10, 12295–12316, https://doi.org/10.5194/acp-10-12295-2010, 2010. Koumoutsaris, S., Bey, I., Generoso, S., and Thouret, V.: Inﬂuence of El Niño-Southern Oscillation on the interannual variability of tropospheric ozone in the northern midlatitudes, J. Geophys. Res., 113, D19301, https://doi.org/10.1029/2007jd009753, 2008. Hall, A. and Qu, X., Using the current seasonal cycle to constrain snow albedo feedback in future climate change, Geophys. Res. Lett., 33, L03502, https://doi.org/10.1029/2005GL025127, 2006. Kuhn, M., Builtjes, P. J. H., Poppe, D., Simpson, D., Stockwell, W. R., Andersson-Sköld, Y., Baart, A., Das, M., Fiedler, F., Hov, Ø., Kirchner, F., Makar, P. A., Milford, J. B., Roemer, M. G. M., Ruhnke, R., Strand, A., Vogel, B., and Vogel, H.: Intercompari- son of the gas-phase chemistry in several chemistry and transport models, Atmos. Environ., 32, 693–709, 1998. p g Heald, C. L., Jacob, D. J., Fiore, A. M., Emmons, L. K., Gille, J. C., Deeter, M. N., Warner, J., Edwards, D. P., Crawford, J. H., Hamlin, A. J., Sachse, G. W., Browell, E. V., Avery, M. A., Vay, S. A., Westberg, D. J., Blake, D. R., Singh, H. B., Sandholm, S. T., Talbot, R. W., and Fuelberg, H. E.: Asian outﬂow and trans- Paciﬁc transport of carbon monoxide and ozone pollution: An integrated satellite, aircraft, and model perspective, J. Geophys. Res., 108, 4804, https://doi.org/10.1029/2003jd003507, 2003. Liang, J., Horowitz, L. W., Jacob, D. J., Wang, Y., Fiore, A. M., Logan, J. A., Gardner, G. M., and Munger, J. W.: Seasonal vari- ations of reactive nitrogen species and ozone over the United States and export ﬂuxes to the global atmosphere, J. Geophys. Res., 103, 13435–413450, 1998. References G., Shindell, D. T., Stevenson, D. S., Szopa, S., Van Dingenen, R., Zeng, G., Ather- ton, C., Bergmann, D., Bey, I., Carmichael, G., Collins, W. J., Duncan, B. N., Faluvegi, G., Folberth, G., Gauss, M., Gong, S., Hauglustaine, D., Holloway, T., Isaksen, I. S. A., Jacob, D. J., Balzani Lööv, J. M., Henne, S., Legreid, G., Staehelin, J., Reimann, S., Prévôt, A. S. H., Steinbacher, M., and Vollmer, M. K.: Esti- mation of background concentrations of trace gases at the Swiss www.atmos-chem-phys.net/18/15345/2018/ Atmos. Chem. Phys., 18, 15345–15361, 2018 A. M. Fiore et al.: A constraint on continental source–receptor relationships 15360 Res., 101, 12621–12638, https://doi.org/10.1029/96jd00338, 1996. Surface Ozone Observations, Elem. Sci. Anth., 5, 58, https://doi.org/10.1525/elementa.244, 2017. Orbe, C., Waugh, D. W., Yang, H., Lamarque, J.-F., Tilmes, S., and Kinnison, D. E.: Tropospheric transport differ- ences between models using the same large-scale me- teorological ﬁelds, Geophys. Res. Lett., 44, 1068–1078, https://doi.org/10.1002/2016GL071339, 2007. Shindell, D. T., Chin, M., Dentener, F., Doherty, R. M., Faluvegi, G., Fiore, A. M., Hess, P., Koch, D. M., MacKenzie, I. A., Sanderson, M. G., Schultz, M. G., Schulz, M., Stevenson, D. S., Teich, H., Textor, C., Wild, O., Bergmann, D. J., Bey, I., Bian, H., Cuvelier, C., Duncan, B. N., Folberth, G., Horowitz, L. W., Jonson, J., Kaminski, J. W., Marmer, E., Park, R., Pringle, K. J., Schroeder, S., Szopa, S., Takemura, T., Zeng, G., Keat- ing, T. J., and Zuber, A.: A multi-model assessment of pollu- tion transport to the Arctic, Atmos. Chem. Phys., 8, 5353–5372, https://doi.org/10.5194/acp-8-5353-2008, 2008. g y Bian, H., Cuvelier, C., Duncan, B. N., Folberth, G., Horowitz, L. W., Jonson, J., Kaminski, J. W., Marmer, E., Park, R., Pringle, Pandey Deolal, S., Staehelin, J., Brunner, D., Cui, J., Steinbacher, M., Zellweger, C., Henne, S., and Vollmer, M. K.: Transport of PAN and NOy from different source regions to the Swiss high alpine site Jungfraujoch, Atmos. Environ., 64, 103–115, https://doi.org/10.1016/j.atmosenv.2012.08.021, 2013. Singh, H. B.: Reactive nitrogen in the troposphere, Environ. Sci. Technol., 21, 320–327, 1987. Pandey Deolal, S., Henne, S., Ries, L., Gilge, S., Weers, U., Stein- bacher, M., Staehelin, J., and Peter, T.: Analysis of elevated springtime levels of Peroxyacetyl nitrate (PAN) at the high Alpine research sites Jungfraujoch and Zugspitze, Atmos. Chem. Phys., 14, 12553–12571, https://doi.org/10.5194/acp-14-12553- 2014, 2014. Singh, H. B. and Hanst, P. L.: Peroxyacetyl nitrate (PAN) in the un- polluted atmosphere: An important reservoir for nitrogen oxides, Geophys. Res. Lett., 8, 941–944, 1981. Singh, H. B. and Salas, L. J.: Measurements of peroxyacetyl ni- trate (pan) and peroxypropionyl nitrate (ppn) at selected ur- ban, rural and remote sites, Atmos. Environ., 23, 231–238, https://doi.org/10.1016/0004-6981(89)90115-7, 1989. Payne, V. H., Alvarado, M. J., Cady-Pereira, K. E., Worden, J. R., Kulawik, S. S., and Fischer, E. V.: Satellite obser- vations of peroxyacetyl nitrate from the Aura Tropospheric Emission Spectrometer, Atmos. Meas. Tech., 7, 3737–3749, https://doi.org/10.5194/amt-7-3737-2014, 2014. Stevenson, D. S., Dentener, F. J., Schultz, M. G., Ellingsen, K., van Noije, T. P. C., Wild, O., Zeng, G., Amann, M., Ather- ton, C. A. M. Fiore et al.: A constraint on continental source–receptor relationships S., Bell, N., Bergmann, D. J., Bey, I., Butler, T., Co- fala, J., Collins, W. J., Derwent, R. G., Doherty, R. M., Drevet, J., Eskes, H. J., Fiore, A. M, Gauss, M., Hauglustaine, D. A., Horowitz, L. W., Isaksen, I. S. A, Krol, M. C., Lamarque, J.-F., Lawrence, M. G., Montanaro, V., Müller, J.-F., Pitari, G., Prather, M. J., Pyle, J. A., Rast, S., Rodriguez, J. M., Sanderson, M. G., Savage, N. H., Shindell, D. T., Strahan, S. E., Sudo, K., and Szopa, S.: Multimodel ensemble simulations of present-day and near-future tropospheric ozone, J. Geophys. Res., 111, D08301, https://doi.org/10.1029/2005JD006338, 2006. Payne, V. H., Fischer, E. V., Worden, J. R., Jiang, Z., Zhu, L., Kurosu, T. P., and Kulawik, S. S.: Spatial variability in tropo- spheric peroxyacetyl nitrate in the tropics from infrared satellite observations in 2005 and 2006, Atmos. Chem. Phys., 17, 6341– 6351, https://doi.org/10.5194/acp-17-6341-2017, 2017. J., Eskes, H. J., Fiore, A. M, Gauss, M., Hauglustaine, D. A., Penkett, S. A. and Brice, K. A.: The spring maximum in photo- oxidants in the Northern Hemisphere troposphere, Nature, 319, 655–657, 1986. M. J., Pyle, J. A., Rast, S., Rodriguez, J. M., Sanderson, M. G., Savage, N. H., Shindell, D. T., Strahan, S. E., Sudo, K., and Szopa, S.: Multimodel ensemble simulations of present-day and near-future tropospheric ozone, J. Geophys. Res., 111, D08301, https://doi.org/10.1029/2005JD006338, 2006. Roberts, J. M.: PAN and Related Compounds, in: Volatile Organic Compounds in the Atmosphere, edited by: Koppmann, R., Blackwell Publishing, 221–268, https://doi.org/10.1002/9780470988657.ch6, 2007. Turnipseed, A. A., Huey, L. G., Nemitz, E., Stickel, R., Higgs, J., Tanner, D. J., Slusher, D. L., Sparks, J. P., Flocke, F., and Guen- ther, A.: Eddy covariance ﬂuxes of peroxyacetyl nitrates (PANs) and NOy to a coniferous forest, J. Geophys. Res.-Atmos., 111, D09304, https://doi.org/10.1029/2005jd006631, 2006. Schmitt, R. and Volz-Thomas, A.: Climatology of Ozone, PAN, CO, and NMHC in the Free Troposphere Over the Southern North Atlantic, J. Atmos. Chem., 28, 245–262, https://doi.org/10.1023/A:1005801515531, 1997. Val Martin, M., Honrath, R. E., Owen, R. C., and Lapina, K.: Large-scale impacts of anthropogenic pollution and bo- real wildﬁres on the nitrogen oxide levels over the cen- tral North Atlantic region, J. Geophys. Res., 113, D17308, https://doi.org/10.1029/2007JD009689, 2008. A. M. Fiore et al.: A constraint on continental source–receptor relationships HTAP: Task Force on Hemispheric Transport of Air Pollution 2007 Interim Report, United Nations Economic Commission for Eu- rope, New York and Geneva, 2007. Lin, M., Holloway, T., Carmichael, G. R., and Fiore, A. M.: Quan- tifying pollution inﬂow and outﬂow over East Asia in spring with regional and global models, Atmos. Chem. Phys., 10, 4221– 4239, https://doi.org/10.5194/acp-10-4221-2010, 2010. HTAP: Hemispheric transport of air pollution 2010 part a: ozone and particulate matter, Air Pollution Studies No. 17, UNITED NATIONS, New York, 2010. Lin, M., Horowitz, L. W., Payton, R., Fiore, A. M., and Tonnesen, G.: US surface ozone trends and extremes from 1980 to 2014: quantifying the roles of rising Asian emissions, domestic con- trols, wildﬁres, and climate, Atmos. Chem. Phys., 17, 2943– 2970, https://doi.org/10.5194/acp-17-2943-2017, 2017. Hudman, R. C., Jacob, D. J., Cooper, O. R., Evans, M. J., Heald, C. L., Park, R. J., Fehsenfeld, F., Flocke, F., Holloway, J., Hübler, G., Kita, K., Koike, M., Kondo, Y., Neuman, A., Nowak, J., Olt- mans, S., Parrish, D., Roberts, J. M., and Ryerson, T.: Ozone production in transpaciﬁc Asian pollution plumes and implica- tions for ozone air quality in California, J. Geophys. Res., 109, D23S10, https://doi.org/10.1029/2004jd004974, 2004. Liu, S. C., Trainer, M., Fehsenfeld, F. C., Parrish, D. D., Williams, E. J., Fahey, D. W., Hubler, G., and Murphy, P. C.: Ozone pro- duction in the Rural Troposphere and the Implications for Re- gional and Global Ozone Distributions, J. Geophys. Res., 92, 4191–4207, 1987. Jaeglé, L., Jaffe, D. A., Price, H. U., Weiss-Penzias, P., Palmer, P. I., Evans, M. J., Jacob, D. J., and Bey, I.: Sources and budgets for CO and O3 in the northeastern Paciﬁc during the spring of 2001: Results from the PHOBEA-II Experiment, J. Geophys. Res., 108, D20, 8802, https://doi.org/10.1029/2002JD003121, 2003. Moxim, W. J., Levy II, H., and Kasibhatla, P. S.: Simulated global tropospheric PAN: Its transport and impact on NOx, J. Geophys. www.atmos-chem-phys.net/18/15345/2018/ Atmos. Chem. Phys., 18, 15345–15361, 2018 A. M. Fiore et al.: A constraint on continental source–receptor relationships 15361 Zellweger, C., Ammann, M., Buchmann, B., Hofer, P., Lu- gauer, M., Rüttimann, R., Streit, N., Weingartner, E., and Bal- tensperger, U.: Summertime NOy speciation at the Jungfrau- joch, 3580 m asl, Switzerland, J. Geophys. Res., 105, 6655– 6667, https://doi.org/10.1029/1999JD901126, 2000. Wang, Y. and Jacob, D. J.: Anthropogenic forcing on tropospheric ozone and OH since preindustrial times, J. Geophys. Res., 103, 31123–131135, 1998. Warneck, P. and Zerbach, T.: Synthesis of peroxyacetyl nitrate by acetone photolysis, Environ. Sci. Technol., 26, 74–79, 1992. Zellweger, C., Forrer, J., Hofer, P., Nyeki, S., Schwarzenbach, B., Weingartner, E., Ammann, M., and Baltensperger, U.: Par- titioning of reactive nitrogen (NOy) and dependence on me- teorological conditions in the lower free troposphere, Atmos. Chem. Phys., 3, 779–796, https://doi.org/10.5194/acp-3-779- 2003, 2003. Weiss-Penzias, P., Jaffe, D. A., Jaeglé, L., and Liang, Q.: In- ﬂuence of long-range-transported pollution on the annual and diurnal cycles of carbon monoxide and ozone at Cheeka Peak Observatory, J. Geophys. Res.-Atmos., 109, D23S14, https://doi.org/10.1029/2004JD004505, 2004. Wild, O., Law, K., McKenna, D., Bandy, B., Penkett, S., and Pyle, J.: Photochemical trajectory modeling studies of the North At- lantic region during August 1993, J. Geophys. Res.-Atmos., 101, 29269–29288, 1996. Zhang, L., Jacob, D. J., Boersma, K. F., Jaffe, D. A., Olson, J. R., Bowman, K. W., Worden, J. R., Thompson, A. M., Avery, M. A., Cohen, R. C., Dibb, J. E., Flock, F. M., Fuelberg, H. E., Huey, L. G., McMillan, W. W., Singh, H. B., and Weinheimer, A. J.: Transpaciﬁc transport of ozone pollution and the effect of recent Asian emission increases on air quality in North Amer- ica: an integrated analysis using satellite, aircraft, ozonesonde, and surface observations, Atmos. Chem. Phys., 8, 6117–6136, https://doi.org/10.5194/acp-8-6117-2008, 2008. Wild, O., Fiore, A. M., Shindell, D. T., Doherty, R. M., Collins, W. J., Dentener, F. J., Schultz, M. G., Gong, S., MacKenzie, I. A., Zeng, G., Hess, P., Duncan, B. N., Bergmann, D. J., Szopa, S., Jonson, J. E., Keating, T. J., and Zuber, A.: Modelling fu- ture changes in surface ozone: a parameterized approach, At- mos. Chem. Phys., 12, 2037–2054, https://doi.org/10.5194/acp- 12-2037-2012, 2012. Zhu, L., Fischer, E. V., Payne, V. H., Worden, J. R., and Jiang, Z.: TES observations of the interannual variabil- ity of PAN over Northern Eurasia and the relationship to springtime ﬁres, Geophys. Res. Lett., 42, 7230–7237, https://doi.org/10.1002/2015GL065328, 2015. Wu, S., Duncan, B. N., Jacob, D. J., Fiore, A. A. M. Fiore et al.: A constraint on continental source–receptor relationships Schultz, M., Rast, S., van het Bolscher, M., Pulles, T., Brand, R., Pereira, J., Mota, B., Spessa, A., Dalsoren, S., van Noije, T., and Szopa, S.: Emission data sets and methodologies for estimat- ing emissions, Hamburg, Max Planck Institute for Meteorology, 2007. van der Werf, G. R., Randerson, J. T., Giglio, L., Collatz, G. J., Kasibhatla, P. S., and Arellano Jr., A. F.: Interannual variability in global biomass burning emissions from 1997 to 2004, Atmos. Chem. Phys., 6, 3423–3441, https://doi.org/10.5194/acp-6-3423- 2006, 2006. Schultz, M. G., Jacob, D. J., Wang, Y., Logan, J. A., Atlas, E. L., Blake, D. R., Blake, N. J., Bradshaw, J. D., Browell, E. G., Fenn, M. A., Flocke, F., Gregory, G. L., Heikes, B. G., Sachse, G. W., Sandholm, G. W., Shetter, R. E., Singh, H. B., and Talbot, R. W.: On the origin of tropospheric ozone and NOx over the tropical South Paciﬁc, J. Geophys. Res., 104, 5829–5843, 1999. van der Werf, G. R., Randerson, J. T., Giglio, L., Collatz, G. J., Mu, M., Kasibhatla, P. S., Morton, D. C., DeFries, R. S., Jin, Y., and van Leeuwen, T. T.: Global ﬁre emissions and the contribution of deforestation, savanna, forest, agricultural, and peat ﬁres (1997–2009), Atmos. Chem. Phys., 10, 11707–11735, https://doi.org/10.5194/acp-10-11707-2010, 2010. Schultz, M. G., Heil, A., Hoelzemann, J. J., Spessa, A., Thon- icke, K., Goldammer, J. G., Held, A. C., Pereira, J. M. C., and van het Bolscher, M.: Global wildland ﬁre emissions from 1960 to 2000, Global Biogeochem. Cy., 22, GB2002, https://doi.org/10.1029/2007GB003031, 2008. Volz-Thomas, A., Xueref, I., and Schmitt, R.: An automatic gas chromatograph and calibration system for ambient measure- ments of PAN and PPN, Environ. Sci. Pollut. R., 4, 72–76, 2002. p g Schultz, M. G., Schröder, S., Lyapina, O., Cooper, O., Gal- bally, I., Petropavlovskikh, I., et al.: Tropospheric Ozone Assessment Report: Database and Metrics Data of Global Atmos. Chem. Phys., 18, 15345–15361, 2018 www.atmos-chem-phys.net/18/15345/2018/ A. M. Fiore et al.: A constraint on continental source–receptor relationships M., and Wild, O.: Chemical nonlinearities in relating intercontinental ozone pollution to anthropogenic emissions, Geophys. Res. Lett., 36, L05806, https://doi.org/10.1029/2008gl036607, 2009. Zhu, L., Payne, V. H., Walker, T. W., Worden, J. R., Jiang, Z., Kulawik, S. S., and Fischer, E. V.: PAN in the eastern Paciﬁc free troposphere: A satellite view of the sources, seasonality, interannual variability, and timeline for trend detection, J. Geophys. Res.-Atmos., 122, 3614–3629, https://doi.org/10.1002/2016JD025868, 2017. Xue, L. K., Wang, T., Zhang, J. M., Zhang, X. C., Deliger, Poon, C. N., Ding, A. J., Zhou, X. H., Wu, W. S., Tang, J., Zhang, Q. Z., and Wang, W. X.: Source of surface ozone and reactive nitrogen speciation at Mount Waliguan in western China: New insights from the 2006 summer study, J. Geophys. Res., 116, D07306, https://doi.org/10.1029/2010JD014735, 2011. Atmos. Chem. Phys., 18, 15345–15361, 2018 www.atmos-chem-phys.net/18/15345/2018/ Atmos. Chem. Phys., 18, 15345–15361, 2018
https://openalex.org/W4393148182	https://www.qeios.com/read/V7VX4M/pdf	English	null	Review of: "Assessment of Quality, Bacterial Population and Diversity of Irrigation Water in Selected Areas of Minna, Niger State, Nigeria"	null	2,024	cc-by	287	Qeios, CC-BY 4.0 · Review, March 25, 2024 Qeios ID: V7VX4M · https://doi.org/10.32388/V7VX4M Potential competing interests: No potential competing interests to declare. Potential competing interests: No potential competing interests to declare. It is a valuable paper, but I have some suggestions: Ionela Hotea1 1 University of Agriculture and Veterinary Medicine Timisoara Review of: "Assessment of Quality, Bacterial Population and Diversity of Irrigation Water in Selected Areas of Minna, Niger State, Nigeria" Ionela Hotea1 It is a valuable paper, but I have some suggestions: Please add information in the Abstract and Materials and Methods about the months when the samples were collected. Mention if you collected water samples every month of the cropping season of 2016, or just once. Please add information in the Abstract and Materials and Methods about the months when the samples were collected. Mention if you collected water samples every month of the cropping season of 2016, or just once. If you collected water samples every month of the cropping season of 2016, please add the results separately and do the correlations. The results can be influenced by the month of collection. If you collected water samples every month of the cropping season of 2016, please add the results separately and do the correlations. The results can be influenced by the month of collection. Please add some information regarding the meteorological conditions from that period. It could be of great relevance to do some correlations between your results and meteorological conditions. Please add some information regarding the meteorological conditions from that period. It could be of great relevance to do some correlations between your results and meteorological conditions. Please update your references. The newest one is from 2014….. Qeios ID: V7VX4M · https://doi.org/10.32388/V7VX4M 1/1
https://openalex.org/W3211277111	https://bovine-ojs-tamu.tdl.org/AABP/article/download/7460/6211	English	null	Economic Aspects of Nutritional Monitoring on Dairy Herds	American Association of Bovine Practitioners Conference	1,987	cc-by	2,362	(Q) n 0 "'O '-< '"i ....... (JQ g > 8 (D '"i ....... (") § > 00 00 0 (") ....... a ....... 0 ~ 0 1-i; to 0 < 5· (D ~ ~ (") ,-+- ....... ,-+- ....... 0 ~ (D '"i 00 0 "'O (D ~ f:; (") (D 00 00 0.. ....... 00 ,-+- '"i ~ ~ ....... 0 p David T. Galligan, V.M.D., M.B.A. Section of Animal Health Economics University of Pennsylvania TABLE 1. Nutrient Analysis. Veterinary nutritional services are an integral part of the herd health services offered to dairy clients (1). Nutrition plays a vital role influencing the production efficiency of the herd both on a disease and input cost level. Feeds form approximately 40-60% of the total input cost of milk production. More importantly the feeding program is a cost center that can be effectively controlled at minimal addition- al cost. The veterinarian can play the vital role in providing unbiased nutritional services at relatively low cost. The purpose of this talk is to go over the nature of this service. A nutritional service can be broken down into three main areas of routine activity in which the veterinarian can play a vital role: I) Feed Selection, 2) Feed distribution, and 3) Monitoring production efficiency. Each of these areas will be discussed in detail. Veterinary nutritional services are an integral part of the herd health services offered to dairy clients (1). Nutrition plays a vital role influencing the production efficiency of the herd both on a disease and input cost level. Feeds form approximately 40-60% of the total input cost of milk production. More importantly the feeding program is a cost center that can be effectively controlled at minimal addition- al cost. The veterinarian can play the vital role in providing unbiased nutritional services at relatively low cost. The purpose of this talk is to go over the nature of this service. TABLE 1. Nutrient Analysis. Nutrient Dry Matter % Crude Protein % Acid Det. Fiber % Calcium % Phosphorus % Magnesium% Potassium % Copper PPM Zinc PPM Iron PPM Manganese PPM Mean 31.9 17.8 39.1 1.06 .32 .40 3.3 9.9 30.2 489 57 * 6 Replicate Samples Sent to 5 Forage Labs. Coefficient of Variation 5.3 8.8 6.5 12.1 11.7 18 21 22 12 29 11 A nutritional service can be broken down into three main areas of routine activity in which the veterinarian can play a vital role: I) Feed Selection, 2) Feed distribution, and 3) Monitoring production efficiency. Each of these areas will be discussed in detail. * 6 Replicate Samples Sent to 5 Forage Labs. Feed Selection Feed selection is an important part of the nutritional service and forms the backbone of the feeding program. Producers are faced with the problem of selecting from a variety of sources the combination of feeds that will help maximize his long term profits. Selected feeds should fit in with the feeding program and be economically efficient. Veterinarians can help in this selection process by giving unbiased advice. The first part of the feed selection process involves knowing the nutrient content of the feed. For grains that are commonly traded, NRC (National Research Council) (2) tables can provide their approximate nutrient analysis. Forages and unusual feeds require nutrient analysis to be performed by laboratory methods. It is important to realize the limitations of laboratory analysis. A recent laboratory survey project conducted by the Nutrition Section of New Bolton Center, found that dry matter, acid detergent fiber, crude protein, calcium and phosphorus had coefficients of variation (standard deviation/ means of samples) of ranging 5-12% while magnesium, potassium, copper, zinc, iron and manganese had coefficients of variation ranging from 11- 29% (Table I). Hence our ability to accurately access nutrient quality and therefore economic value is somewhat limited. This valuation format allows us to estimate the economic value associated with forage nutrient quality. Economic substitution values are calculated for a forage and then compared to the economic substitution value for a "goal" forage (usually the NRC nutrient analysis for the forage). The difference represents the potential loss or gain associ- TABLE 2. Base feeds SMB 48% and Shelled Corn GP% NE (MCALS/LB) SBM $200/ton = x 48% + Y .75 Corn $100/ton = x 8.9% + Y .82 Solution: Y = 92.3 x = 271.6 Corn Silage Hay GP% NE 271 * .028 + 92.3* .25 271 * .082 + 92.3* .53 Substitution $ $30.66/ton $71.14/ton Base feeds SMB 48% and Shelled Corn GP% NE (MCALS/LB) SBM $200/ton = x 48% + Y .75 Corn $100/ton = x 8.9% + Y .82 Solution: Y = 92.3 x = 271.6 Corn Silage Hay GP% NE 271 * .028 + 92.3* .25 271 * .082 + 92.3* .53 Substitution $ $30.66/ton $71.14/ton Forages and grains are evaluated according to nutrient composition and market prices by an algebraic procedure similar to a formula described over 50 years ago (3). (Q) n 0 "'O '-< '"i ....... (JQ g > 8 (D '"i ....... (") § > 00 00 0 (") ....... a ....... 0 ~ 0 1-i; to 0 < 5· (D ~ ~ (") ,-+- ....... ,-+- ....... 0 ~ (D '"i 00 0 "'O (D ~ f:; (") (D 00 00 0.. ....... 00 ,-+- '"i ~ ~ ....... 0 p Nutrient price parameters (X, Y) are calculated by using two reference feeds of known market price, normally in our area soybean meal (48%) and shelled corn (Table 2). A replace- ment cost or implicit value for each feed is calculated using these price parameters and the feed's energy and protein composition. The economic value of a feed is related to its nutrient composition. Nutrients most commonly used to economically value feeds are crude protein and net energy. These nutrients account for approximately 90% of the variation in price seen in feeds. We will commonly use soy bean meal and shelled corn as base feeds to estimate the economic nutrient value($ value of CP%, $ value of energy), since these feeds have well established market prices. These nutrient values can also be used to estimate the substitution value for forages. Feed Delivery Feed delivery is an important part of the feeding program. In general, feed delivery systems which have a method of weighing feed realized higher production levels (Keown, 1987). For any herd, one extreme feeding system would be to feed each cow individually according to her maintenance and production requirements. This feed delivery system would have the cheapest feed cost but the highest labor cost. Grouping cows allows the labor cost to be reduced while the feed cost will increase according to the degree of overfeeding within any group. TABLE 4. Sample Calculations. Output Period One Period Two Change Ratio Milk Lb. (Q) Value $ (V) Price $ (P) Input Gain Lb. (Q) Value $ (V) Cost $ (P) IOFC (1) = $1,517 ICFC (2) = $1,544 19500 2,048 10.50 5900 531 190 % Change in Profit = 2.18% TABLE 5. Performance Ratios. Output Milk (Lb.) (Q) Value $ (V) Price $ (V) Input Gram (Lb.) (Q) Value$ (V) Price $ (P) Prof its Increased Output% 1.01 1.01 1.00 Input% 1.017 .989 .972 (Value Performance Ratio - 1) Productivity Decreased (Quantity Performance Ratio - 1) Price Recovery Increased (Price Performance Ratio - 1) 19700 2,069 10.50 6000 525 175 Q2/Q1 = 1.01 V2/V1 = 1.01 P2/P1 = 1.00 02/01 = 1.017 V2/V1 = .989 P2/P1 = .972 Performance Ratios Output% /Input% .993 1.0218 1.0286 = 2.18% = -.66% = 2.86% 0 "'O (D ~ ~ (") (D 00 00 0...... 00 ,-+- '"i ~ ~ ..... 0 p The feed delivery system is also important in ensuring a balanced ration is delivered to the appropriate cows. Thus nutrient delivery, and economic efficiency must be consider- ed when selecting a delivery system. New applications of linear programming (multi-period linear programming) are being applied to the ration formulation problem. Current linear programming tech- niques balance rations using current feed prices. The proposed multi-period linear programs will additionally use future prices of feeds, physical and economic inventory constraints to base the selection offeed ingredients for ration formulation (5). This will enable the producer to better plan his feed expenditures and storage and minimize feed price swings. duced, price, economic value and input used to produce this grain (it's quantity, cost, and value). From these values one can calculate the CHANGE RA TIO which is simply the ratio of period 2/ period l's parameters. These represent the % change in quantity value or price for both inputs and outputs. PERFORMANCE RATIOS (change ratio out- put/ change ratio input) can be calculated for the quantities, values and prices of the outputs and inputs. In the simple example, profits between the two periods have increased by 2.18%. Productivity ( conversion of grain to milk) actually decreased profits by -.66% while price changes allowed profits to increase by 2.86- the total effect being a change in profit by 2.86%-.66% = 2.18%. TABLE 4. Sample Calculations. Output Period One Period Two Change Ratio Milk Lb. (Q) Value $ (V) Price $ (P) Input Gain Lb. (Q) Value $ (V) Cost $ (P) IOFC (1) = $1,517 ICFC (2) = $1,544 19500 2,048 10.50 5900 531 190 % Change in Profit = 2.18% TABLE 5. Performance Ratios. Output Milk (Lb.) (Q) Value $ (V) Price $ (V) Input Gram (Lb.) (Q) Value$ (V) Price $ (P) Prof its Increased Output% 1.01 1.01 1.00 Input% 1.017 .989 .972 (Value Performance Ratio - 1) Productivity Decreased (Quantity Performance Ratio - 1) Price Recovery Increased (Price Performance Ratio - 1) 19700 2,069 10.50 6000 525 175 Q2/Q1 = 1.01 V2/V1 = 1.01 P2/P1 = 1.00 02/01 = 1.017 V2/V1 = .989 P2/P1 = .972 Performance Ratios Output% /Input% .993 1.0218 1.0286 = 2.18% = -.66% = 2.86% 0 "'O (D ~ ~ (") (D 00 00 0...... 00 ,-+- '"i ~ ~ ..... 0 p Feed Selection The algorithm uses Net Energy Lactation and crude protein as the nutrients to determine the economic value of a feed (4). APRIL, 1988 85 that are due to actual changes in production efficiency versus changes associated with price changes. Performance ratios allow one to monitor these effects on profitability. Table 4 shows two periods of production information: milk pro- duced, price, economic value and input used to produce this grain (it's quantity, cost, and value). From these values one can calculate the CHANGE RA TIO which is simply the ratio of period 2/ period l's parameters. These represent the % change in quantity value or price for both inputs and outputs. PERFORMANCE RATIOS (change ratio out- put/ change ratio input) can be calculated for the quantities, values and prices of the outputs and inputs. In the simple example, profits between the two periods have increased by 2.18%. Productivity ( conversion of grain to milk) actually decreased profits by -.66% while price changes allowed profits to increase by 2.86- the total effect being a change in profit by 2.86%-.66% = 2.18%. ated by having a protein and energy composition different from the goal. This method of economically monitoring forage quality forms a powerful incentive for producers to improve nutrient quality. Nutritional Monitoring The last important aspect of a nutritional service is monitoring the production and profit impact of the feeding program. Monitoring production has received a lot of emphasis and a number of indices have been developed. These indices usually measure a physical flow of input to output, such as grain to milk ratios or the measure ratio of a value of input to output such as feed cost/ cwt. The producer is to use these indices as a guide to improving profit. To test how these indices correlate with profit/ cow (income over feed cost IOFC) two farms were followed for five years and the correlations between indices and OIFC were calculated. TABLE 3. Correlation of Common Production Measures with Income Over Feed Cost. p Milk/Grain -.18 0.5 Feed Cost/ CWT -.317 <.001 Grain $/CWT -.154 .10 TABLE 3. Correlation of Common Production Measures with Income Over Feed Cost. The poor correlations show that these production indices are poor monitors of profitability. The proposed methodology can be expanded to incor- porate several inputs (forage, grain, mineral etc) and several outputs. We feel that the above method will allow the producer to evaluate economic efficiency as a function of both productivity changes as well as prices changes. A feed monitoring system is needed that is sensitive to changes in feed values and amounts as well as milk values and amounts. The system needs to reflect changes in profit THE BOVINE PROCEEDINGS-No. 20 86 3. Peterson, WE ( 1932). A formula for evaluating feeds on the Basis of digestible nutrients. J. Dairy Sci. 15:293. 4. Galligan OT, JD Fer!?uson. CF Ramberg, and W Chalupa ( 1986). Dairy Ration Formulation and Evalua- tion Program for Microcomputers. J. Dairy Sci. 69: 1656-1664. 5. Galligan OT. Multi-period ration formulation. (1987) In preparation for J . Dairy Sci. References 3. Peterson, WE ( 1932). A formula for evaluating feeds on the Basis of digestible nutrients. J. Dairy Sci. 15:293. 4. Galligan OT, JD Fer!?uson. CF Ramberg, and W Chalupa ( 1986). Dairy Ration Formulation and Evalua- tion Program for Microcomputers. J. Dairy Sci. 69: 1656-1664. 5. Galligan OT. Multi-period ration formulation. (1987) In preparation for J . Dairy Sci. I. Ferguson, JD, OT Galligan, CF Ramberg, OS Kronfeld (1987). Veterinary Nutritional Advisory Services to Dairy Farms. Conpendium Food Animal, Vol 9 No. 5. 2. National Research Council. 1978. Nutrient requirements for dairy cattle. 5th rev. ed. Natl. Aca. Sci., Washington DC. I. Ferguson, JD, OT Galligan, CF Ramberg, OS Kronfeld (1987). Veterinary Nutritional Advisory Services to Dairy Farms. Conpendium Food Animal, Vol 9 No. 5. 2. National Research Council. 1978. Nutrient requirements for dairy cattle. 5th rev. ed. Natl. Aca. Sci., Washington DC. 0 "'O (D ~ ~ (") (D 00 00 0.. ...... 00 ,-+- '"i ~ ~ ...... 0 p 0 "'O (D 0 "'O (D ~ ~ (") (D 00 00 0.. ...... 00 ,-+- '"i ~ ~ ...... 0 p APRIL, 1988 87
https://openalex.org/W2399197571	https://www.rrh.org.au/journal/download/pdf/2883/	English	null	Supporting rural remote physicians to conduct a study and write a paper: Experience of Clinical Research Support Team (CRST)-Jichi	Rural and remote health	2,014	cc-by	4,542	Supporting rural remote physicians to conduct a study and write a paper: Experience of Clinical Research Support Team (CRST)-Jichi Matsubara1, A Ohkuchi1, T Kamesaki1, S Ishikawa1, Y Nakamura1, M Matsumoto2 1Jichi Medical University, Shimotsuke, Tochigi, Japan 2Department of Community-Based Medical System, Faculty of Medicine, Hiroshima University, Hiroshima, Japan Supporting rural remote physicians to conduct a study and write a paper: Experience of Clinical Research Support Team (CRST)-Jichi Rural and Remote Health 14: 2883. (Online) 2014 © S Matsubara, A Ohkuchi, T Kamesaki, S Ishikawa, Y Nakamura, M Matsumoto, 2014. A licence to publish this material has been given to James Cook University, http://www.rrh.org.au 1 Why was CRST-Jichi founded? A rural remote area is a 'frontline' not only of medical practice but also of medical research. A new technology, although perhaps first introduced in an urban center, can only have its real-world efficacy confirmed after widespread use: its use in rural areas will provide important data. Observations by rural doctors may lead to 'discovery' of a new clinical entity: Huntington’s disease was first reported by a rural doctor1. In this context, conducting research and writing a paper in a rural remote area is quite important. But is this working well? One graduate’s bad experience and a faculty member’s sympathy for it triggered the establishment of CRST-Jichi. At a conference for JMU graduates, a faculty member of JMU, who later became the director of the CRST, was moved by a young graduate’s experience, 'I wrote a paper based on my experience in rural practice and submitted it to a journal without success; I have no experience to publishing an English paper and have no support, and thus the paper will surely be buried in my desk'. The faculty member, also a graduate of JMU, had a similar experience 30 years ago, when he was a rural doctor. Therefore, he and a few other JMU-graduated faculty members decided to make a 'system' to support such rural doctors. It is hard for any young doctor to continually produce academic publications2. In rural and remote areas, it is even harder3. Lack of mentorship is one of the largest barriers for young practitioners to conduct research2. Some government- and university-based research support programs for rural practitioners in which mentorship and other services are provided have been reported, mainly from Australia and Canada, but not from Asian countries4-8. A B S T R A C T Context: Jichi Medical University (JMU) is the only medical school in Japan that is devoted solely to producing rural and remote doctors. To support research activities of its graduates, mainly young graduates under obligatory rural service, JMU established a voluntary team, Clinical Research Support Team (CRST)-Jichi. Issues: CRST-Jichi consists of current and past JMU faculty members; all of them are specialists of certain medical fields and many are also graduates of JMU who have completed rural service. A client who asks the CRST for advice on study design or editing a paper emails the CRST to ask for support in conducting a study. Then, core members of the CRST assign the job to a registered specialist of the corresponding topic, who becomes a ‘responsible supporter’ and continues to support the client until a paper has been published. During the 3 years from July 2010, 12 English papers have been published in international peer-review journals, two Japanese papers in domestic journals, and 13 studies are in progress. Ninety-one percent of clients were satisfied with the service, and eighty-two percent considered their papers would not have been published if they had not used the service. Sense of commitment, existence of JMU-graduated specialists, and quick response were reported by clients as major strengths of CRST-Jichi. Lessons learned: The experience of CRST-Jichi can potentially be transferred to not only other Japanese medical schools with rural doctor production programs, which are now rapidly increasing as part of a national policy, but also rural medical education systems in other countries. Key words: clinical research support team, designing study, emailing, Japan, research support, rural medicine, writing a paper. © S Matsubara, A Ohkuchi, T Kamesaki, S Ishikawa, Y Nakamura, M Matsumoto, 2014. A licence to publish this material has been given to James Cook University, http://www.rrh.org.au 1 © S Matsubara, A Ohkuchi, T Kamesaki, S Ishikawa, Y Nakamura, M Matsumoto, 2014. A licence to publish this material has been given to James Cook University, http://www.rrh.org.au 2 © S Matsubara, A Ohkuchi, T Kamesaki, S Ishikawa, Y Nakamura, M Matsumoto, 2014. A licence to publish this material has been given to James Cook University, http://www.rrh.org.au 2 we asked all JMU academic staff members to join this support team on a voluntary basis. Approximately 60 members accepted this proposal within a couple of days after emailing and then the CRST started on 1 July 2010. The CRST members currently number 88, including 43 JMU graduates. means a 'happy engagement' of the supporter and the client. As mentioned, on an open mailing list forum among CRST members, the issue in progress is sometimes discussed, and the theme is shared by all CRST members. CRST members advise whenever they want to do so, and are regarded as 'cooperating witnesses'. How does the CRST system work? 7. If the paper has been accepted, the responsible supporter is listed as a co-author. Depending on their contribution, the responsible supporter can become the corresponding author. The second or the third supporter, if they have contributed significantly, can also become a co-author of the paper. Who becomes a co-author is not strictly defined by the CRST. Simply, those who significantly contributed to the study/paper become co-authors. Therefore, those only giving advice via email (cooperating witnesses) do not become co-authors. The director can decide the authorship in the event of disagreement, but this has never happened. Figure 1 shows how CRST-Jichi supports the client, a JMU graduate who asks CRST for advice on study design or about editing a paper. 1. The client e-mails the CRST that he/she wishes the CRST to support designing or modifying a study, or editing a paper. 2. Usually, the director reviews the study design or reads the draft of the paper, and appoints an appropriate staff member ('responsible supporter'), based on the topic dealt with there. The core members of the CRST also review the request on the day it arrives and help the director to find a candidate. If the theme happens to be a core member’s specialty, then, the core member takes on the role of a responsible supporter who is responsible for guiding the client through the authorship process. © S Matsubara, A Ohkuchi, T Kamesaki, S Ishikawa, Y Nakamura, M Matsumoto, 2014. A licence to publish this material has been given to James Cook University, http://www.rrh.org.au 3 How did CRST-Jichi start? The founding director and vice-director are both obstetrics/gynecology physicians. Although obstetrics/ gynecology is one of the main themes of rural and remote medicine, many studies and papers from rural areas are naturally not confined to obstetrics/gynecology, and thus as a research support team, CRST-Jichi requires a number of experienced researcher-clinicians from various medical fields. For example, if a rural doctor encounters some insect bite disorders and wishes to write about it, then specialists in parasitology, pharmacology, and infection medicine are required. Specialists for medical statistics are needed for studies requiring statistics. Jichi Medical University (JMU) in Shimotsuke, Tochigi, established in 1972, is a unique medical university that aims to produce doctors who engage in rural remote practice in Japan. In most cases, JMU graduates work as general practitioners in rural areas for about 6 years after their residency training. The rural practice is conducted as an obligatory service in exchange for exemption of medical school tuition9,10. During this rural service, there is no or little opportunity to receive advice on research or writing a paper. JMU, thus, established the Clinical Research Support Team (CRST)-Jichi, in which JMU faculty members support JMU graduates in designing a study or writing a paper. During a 3-year period since its beginning in July 2010, support requests to CRST-Jichi have increased, resulting in the publishing of 12 English papers in peer-reviewed international journals and two papers in Japanese journals. Here, we briefly describe the system and its significance. CRST-Jichi started with the director asking some experienced JMU staff researchers to join the 'support team' (later becoming CRST) in a person-by-person manner. Twenty-one researchers, all well experienced both in study design and writing papers, joined, forming the core of the present CRST. Among the 21 members, 19 were JMU graduates. Later, via email and periodical JMU newsletters, 2 © S Matsubara, A Ohkuchi, T Kamesaki, S Ishikawa, Y Nakamura, M Matsumoto, 2014. A licence to publish this material has been given to James Cook University http://www rrh org au 3 © S Matsubara, A Ohkuchi, T Kamesaki, S Ishikawa, Y Nakamura, M Matsumoto, 2014. A licence to publish this material has been given to James Cook University, http://www.rrh.org.au 4 † In order of request. Papers 2, 3, 7, 9, 10, 11, and 12 were published in a rural medicine journal. ¶ From request to paper acceptance What are the CRST outcomes? Table 1 shows the achievements of CRST-Jichi at the time of writing. So far, we received a total of 27 requests; all have already been published or are in progress. Of them, 12 English papers1,11-21 and two Japanese papers have been accepted for publication in peer-reviewed journals. Of the 12 English papers, seven were accepted in rural medicine journals. The median time from client email request to paper acceptance was 7 months (range 1– 32 months). Table 2 shows the research (13 themes) currently under the guidance of CRST-Jichi. 3. After the candidate specialist accepts the role of the supporter, the client then emails the details of the study to him/her, yielding a 'happy engagement'. 4. If a candidate chosen by the director does not accept the offer, or the director is not sure who best knows the corresponding issue, then all CRST members will be emailed, and asked if they wish to become a responsible supporter of the client. To date, steps 2–4 have always resulted in a 'happy engagement'. In September 2013, CRST sent a questionnaire to all the 11 clients (one client wrote two English papers) whose papers were accepted. All have responded; 91% were satisfied with CRST service, and 82% considered their papers would not have been published if they had not used the service (Table 3). A sense of commitment, access to JMU-graduated specialists, and quick response were reported by clients as the major strengths of CRST (Table 3). The variety of specialists and their ability to lead research consultation were also cited as strengths. 5. If a responsible supporter has some difficulty in performing their job (designing a study or writing a paper), then they raise an 'open question' via the CRST mailing list to all 88 CRST members. If the theme is multidisciplinary, a second or third supporter in the relevant specialties provides assistance. 6. The responsible supporter and the client discuss the issue by email and complete the final paper, which 3 Satisfied 10 90.9 Moderately satisfied 0 Borderline 1 9.1 Moderately dissatisfied 0 0 Dissatisfied 0 0 If you had not used Clinical Research Support Team services, your paper would have been … unpublished 9 81.8 published in a lower-rank journal 0 0 published in the same journal with delay 2 18.2 published in the same journal without delay 0 0 What are the strengths of the Clinical Research Support Team? (multiple answers per client) Sense of commitment 7 63.6 JMU-graduated supporters 7 63.6 Quick response 6 54.5 Variety of supporters 5 45.5 Supporter’s capability to lead 4 36.4 JMU, Jichi Medical University Table 3: Summary of questionnaire responses from the 11 clients whose papers were accepted by peer-r journals Question n % Are you satisfied with Clinical Research Support Team services? Satisfied 10 90.9 Moderately satisfied 0 Borderline 1 9.1 Moderately dissatisfied 0 0 Dissatisfied 0 0 If you had not used Clinical Research Support Team services, your paper would have been … unpublished 9 81.8 published in a lower-rank journal 0 0 published in the same journal with delay 2 18.2 published in the same journal without delay 0 0 What are the strengths of the Clinical Research Support Team? (multiple answers per client) Sense of commitment 7 63.6 JMU-graduated supporters 7 63.6 Quick response 6 54.5 Variety of supporters 5 45.5 Supporter’s capability to lead 4 36.4 JMU, Jichi Medical University re responses from the 11 clients whose papers were accepted by peer-reviewed journals Table 3: Summary of questionnaire responses from the 11 clients whose papers were accepted by peer-reviewed journals Table 2: Research themes under the guidance of the Clinical Research Support Team, as of March 2014 No. Theme 1 Prospective study on osteoporosis in rural areas 2 Circulatory effect caused by gastric endoscopy: its difference among the routes 3 Education of critical care obstetrics with special emphasis on advanced life support in obstetrics 4 Injury of the hand muscle caused by Mycobacterium marium infection 5 Effectiveness of tissue plasminogen activator administration for acute myocardial infarction: analysis in a rural area 6 Central vein port placement via subclavian vein: practical considerations in a rural setting 7 Possible cause for the high occurrence of myelodysplastic syndrome in an island of Japan 8 Systemic septicemia due to Clostridium welchi: liver abscess in a rural area 9 Trauma-induced myelopathy in patients with retro-odontoid pseudotumor combined with ossification of the anterior longitudinal ligament 10 Home blood pressure monitoring in a rural setting 11 Oxyuriasis in a remote area 12 Primary hepatic choriocarcinoma in a rural area 13 Pneumococcus vaccine: its effectiveness to outpatients . Table 2: Research themes under the guidance of the Clinical Research Support Team, as of March 2014 No. Theme 1 Prospective study on osteoporosis in rural areas 2 Circulatory effect caused by gastric endoscopy: its difference among the routes 3 Education of critical care obstetrics with special emphasis on advanced life support in obstetrics 4 Injury of the hand muscle caused by Mycobacterium marium infection 5 Effectiveness of tissue plasminogen activator administration for acute myocardial infarction: analysis in a rural area 6 Central vein port placement via subclavian vein: practical considerations in a rural setting 7 Possible cause for the high occurrence of myelodysplastic syndrome in an island of Japan 8 Systemic septicemia due to Clostridium welchi: liver abscess in a rural area 9 Trauma-induced myelopathy in patients with retro-odontoid pseudotumor combined with ossification of the anterior longitudinal ligament 10 Home blood pressure monitoring in a rural setting 11 Oxyuriasis in a remote area 12 Primary hepatic choriocarcinoma in a rural area 13 Pneumococcus vaccine: its effectiveness to outpatients . earch themes under the guidance of the Clinical Research Support Team, as of March 2014 Table 3: Summary of questionnaire responses from the 11 clients whose papers were accepted by peer-reviewed journals Question n % Are you satisfied with Clinical Research Support Team services? Lessons learned First, rapid response on the CRST side is important. For example, within a day after a manuscript’s arrival, the director usually reads the manuscript, retrieves the references via the PubMed database, reads UpToDate (an evidence-based clinical support resource) for the corresponding theme, and then roughly estimates the significance of the study. It is usually within 1 week that a Figure 1: How the Clinical Research Support Team supports a client. Figure 1: How the Clinical Research Support Team supports a client. able 1: Clinical Research Support Team-supported English papers published in peer-reviewed journa † In order of request. Papers 2, 3, 7, 9, 10, 11, and 12 were published in a rural medicine journal. Paper no.† Reference Main category Topic Interval (months)¶ 1 11 Urology Bladder cancer 1 2 12 Rural medicine Internet at rural medicine 3 3 13 Rural medicine Decompression sickness 3 4 14 Infectious disease Dose of vancomycin administration 13 5 1 Neurology Huntington’s disease 1 6 15 Neurology Head injury 16 7 16 Rural medicine Home palliative care 9 8 17 Obstetrics Hyperlipidemia during pregnancy 5 9 18 Surgery Colonic rupture 1 10 19 Rural medicine Cancer screening 21 11 20 Gastroenterology Dysphagia 32 12 21 Gastroenterology Screening for gastric cancer 9 © S Matsubara, A Ohkuchi, T Kamesaki, S Ishikawa, Y Nakamura, M Matsumoto, 2014. A licence to publish this material has been given to James Cook University http://www rrh org au 5 © S Matsubara, A Ohkuchi, T Kamesaki, S Ishikawa, Y Nakamura, M Matsumoto, 2014. A licence to publish this material has been given to James Cook University, http://www.rrh.org.au 5 CRST wish them to ask for support. Being involved in the current issues in rural health makes a refreshing change for CRST members, many of whom used to be rural doctors but now are in academic posts. Thus, supporting and being supported will become a real win–win relationship, making this system more sustainable. responsible supporter is selected. The client continues to be informed at each step. Designing a study and writing a paper is better performed while both a client and CRST members have a keen interest. Delays may weaken the motivation of the client. Thus, a rapid response at this stage is paramount to success. After a responsible supporter has been selected, progress depends on the client and the supporter. The 3-year trial of CRST has a potential impact on schemes that produce rural doctors in other medical schools in Japan. Since 2008 the government has implemented JMU-like rural medical education programs at most of the 80 medical schools in Japan. The number of entrants to these programs has now reached 15% of medical school entrants in Japan22. Although these programs are currently only for undergraduate students, their graduates will need systemic academic support in the future. The experience of CRST may be helpful for these new programs to create such a support system. Second, a sense of commitment of CRST members is essential. Even without the CRST system, the university staff may advise a client if asked. However, merely receiving advice is by no means sufficient for eventual acceptance of the paper. The responsible supporter must be responsible and commit to the paper. This becomes in some ways a burden to the supporter. This is unavoidable, and so, in a sense, may be a necessary evil for the paper acceptance. However, being an author is important to one’s academic career, especially for university posts. Thus, a win–win relationship develops between the client and the supporter. This is important for the system to continue. Some medical schools and public sectors support research conducted by rural health professionals4-8. The support includes providing mentors, short courses such as research design and statistics, research funds, conferences, and arrangement of research groups4-8. Flinders University in South Australia, for example, utilizing government funding scheme, provided funds and mentoring to rural health professionals and achieved a substantial effect7. Because CRST-Jichi is a voluntary group, it neither uses nor provides funds. However, the experience of the CRST showed that providing responsible and rural-minded mentors in a timely fashion is of crucial importance. Third, many CRST members are JMU graduates (note that it is a custom of Japanese medical schools that graduates of a school return to the school to work as faculty). The JMU-graduated specialists not only provide knowledge and experience but also bring a rural perspective to the paper, which adds value to the paper, particularly for readers of rural health journals. Also a sense of community and strong volunteerism are easily fostered among CRST members and clients. This may be partly because JMU is a unique school in which all the students live in the same dormitory and all the graduates serve in rural areas, often in the same hospitals. This enables the members to voluntarily devote themselves to the time- and energy-consuming job. Thus, JMU may be in a better position in this sense. We believe that a strong sense of confidence becomes fostered both between a client and the supporter, and among CRST members during the effort required to study or to write a paper. CRST has created a strong bond among the participants. The CRST system is applicable to other institutions under some conditions. It needs a substantial number and variety of rural-experienced clinical/academic specialists. This does not necessarily apply to all research support institutions. However, many medical schools or hospitals in the world may have a role to academically support rural practitioners. At the same time, many of them may have limited funding resources and thus have to provide the service on a voluntary basis. However, in such institutions, there may be specialists who had once worked in rural areas and are now working as © S Matsubara, A Ohkuchi, T Kamesaki, S Ishikawa, Y Nakamura, M Matsumoto, 2014. A licence to publish this material has been given to James Cook University, http://www.rrh.org.au 6 © S Matsubara, A Ohkuchi, T Kamesaki, S Ishikawa, Y Nakamura, M Matsumoto, 2014. A licence to publish this material has been given to James Cook University http://www rrh org au 6 Three possible reasons for success CRST-Jichi was successful in supporting rural doctors. As shown in the results of the client survey (Table 3), the following are possible reasons why CRST worked well. 5 References 9. Inoue K, Hirayama Y, Igarashi M. A medical school for rural areas. Medical Education 1997; 31(6): 430-434. 1. Nakamura K, Ota M, Kawata A, Isozaki E, Muramatsu S, Matsubara S. Careful clinical observation is essential for diagnosis of Huntington’s disease. Arquivos de Neuro-psiquiatria 2012; 70(8): 646. 1. Nakamura K, Ota M, Kawata A, Isozaki E, Muramatsu S, Matsubara S. Careful clinical observation is essential for diagnosis of Huntington’s disease. Arquivos de Neuro-psiquiatria 2012; 70(8): 646. 10. Matsumoto M, Inoue K, Kajii E. Contract-based training system for rural physicians: follow-up of Jichi Medical University graduates (1978–2006). Journal of Rural Health 2008; 24(4): 360- 368. 2. Reid MB, Misky GJ, Harrison RA, Sharpe B, Auerbach A, Glasheen JJ. Mentorship, productivity, and promotion among academic hospitalists. Journal of General Internal Medicine 2012; 27(1): 23-27. 11. Ohzawa H, Arai W, Kondo Y, Endo S, Morita T, Matsubara S. Urinary bladder rupture associated with squamous cell carcinoma of the bladder; a cause of acute peritonitis. Internet Journal of Urology 7: 2. (Online) 2010. Available: http://ispub.com/IJU/7/2/13354 (Accessed 24 March 2014). 3. Cusick A, Lannin N. On becoming a practitioner-researcher in remote northern Australia: personal commitment and resources compensate for structural deterrents to research. Disability and Rehabilitation 2008; 30(26): 1984-1998. 12. Tateno Y, Sato T, Matsubara S: Sharing area-specific information via an internet forum benefits rural clinicians: a lesson learned from a case with decompression sickness on a remote island. Rural and Remote Health 11(3): 1856. (Online) 2011. Available: www.rrh.org.au (Accessed 31 March 2014). 4. Macleod ML, Dosman JA, Kulig JC, Medves JM. The development of the Canadian Rural Health Research Society: creating capacity through connection. Rural and Remote Health 7(1): 622. (Online) 2007. Available: www.rrh.org.au (Accessed 31 March 2014). 13. Sato T, Iga T, Nagashima K, Matsubara S. Is centralization in emergency rural medicine always right? Lessons learned from two cases of decompression sickness. Rural Remote Health 11(2): 1711. (Online) 2011. Available: www.rrh.org.au (Accessed 31 March 2014). 5. Birden HH. The researcher development program: how to extend the involvement of Australian general practitioners in research? Rural and Remote Health 7(3): 776. (Online) 2007. Available: www.rrh.org.au (Accessed 31 March 2014). 6. Miller J, Bryant Maclean L, Coward P, Broemeling AM. Developing strategies to enhance health services research capacity in a predominantly rural Canadian health authority. Rural and Remote Health 9(4): 1266. (Online) 2009. Available: www.rrh. org.au (Accessed 31 March 2014). 14. academic or specialist clinicians. This situation is quite similar to that of JMU. JMU has some features favoring the success of this CRST trial, and our experience can be applied to this type of institution both in Japan and other countries. 8. Taylor J, Hughes C, Petkov J, Williams M. Unique issues in research and evaluation in rural and remote locations: is there a place for specific research training? Rural and Remote Health 5(2): 351. (Online) 2005. Available: www.rrh.org.au (Accessed 31 March 2014). Potential impact and future plan CRST- Jichi has made a good start; however, there may be many more JMU graduates who seek help. Members of 6 © S Matsubara, A Ohkuchi, T Kamesaki, S Ishikawa, Y Nakamura, M Matsumoto, 2014. A licence to publish this material has been given to James Cook University, http://www.rrh.org.au 7 References Maki N, Ohkuchi A, Tashiro Y, Kim MR, Le M, Sakamoto T, et al. Initial dose of vancomycin based on body weight and creatinine clearance to minimize inadequate trough levels in Japanese adults. European Journal of Clinical Microbiology and Infectious Diseases 2012; 31(10): 2537-2543. 7. Ried K, Farmer EA, Weston KM. Bursaries, writing grants and fellowships: a strategy to develop research capacity in primary health care. BMC Family Practice 8: 19. (Online) 2007. Available: http://www.biomedcentral.com/1471-2296/8/19 (Accessed 24 March 2014). 15. Tokushige J, Matsubara S, Tanaka Y, Kato S. Trephination for acute epidural hematoma using stainless wire on a remote island. Journal of Emergency Medicine 2012; 43(6): e489-490. 15. Tokushige J, Matsubara S, Tanaka Y, Kato S. Trephination for acute epidural hematoma using stainless wire on a remote island. Journal of Emergency Medicine 2012; 43(6): e489-490. © S Matsubara, A Ohkuchi, T Kamesaki, S Ishikawa, Y Nakamura, M Matsumoto, 2014. A licence to publish this material has been given to James Cook University, http://www.rrh.org.au 7 © S Matsubara, A Ohkuchi, T Kamesaki, S Ishikawa, Y Nakamura, M Matsumoto, 2014. A licence to publish this material has been given to James Cook University, http://www.rrh.org.au 8 uchi, T Kamesaki, S Ishikawa, Y Nakamura, M Matsumoto, 2014. A licence to publish this material has been given to James C .rrh.org.au 8 19. Tateno Y, Miyazaki Y, Tsuboi S. Can screening invitations from primary care physicians increase participation in cancer screenings on remote islands? General Medicine 2013; 14(1): 40-47. 19. Tateno Y, Miyazaki Y, Tsuboi S. Can screening invitations from primary care physicians increase participation in cancer screenings on remote islands? General Medicine 2013; 14(1): 40-47. 16. Tateno Y, Ishikawa S. Clinical pathways can improve the quality of pain management in home palliative care in remote locations: retrospective study on Kozu Island, Japan. Rural Remote Health 12: 1992. (Online) 2012. Available: www.rrh.org.au (Accessed 31 March 2014). 20. Murata K, Ishikawa S, Sugioka T. Investigation of dysphagia symptoms and their association with subjective symptoms in inhabitants of an island. General Medicine 2013; 14(1): 32-39. 17. Sato S, Ohkuchi A, Kawano M, Iwanaga N, Furukawa Y, Matsumoto H. Effect of eicosapentaenoic acid agent on aggravated hypertriglyceridemia during pregnancy. Journal of Obstetrics and Gynaecology Research 2013; 39(11): 1541-1544. 21. Matsumoto S, Ishikawa S, Yoshida Y. Reduction of gastric cancer mortality by endoscopic and radiographic screening in an isolated island: a retrospective cohort study. Australian Journal of Rural Health 2013; 21(6): 319-324. 18. Okada Y, Okamoto D, Matsubara S. Successful drainage for life-threatening colonic perforation by a young physician on a remote island. Rural and Remote Health 13(2): 2638. (Online) 2013. Available: www.rrh.org.au (Accessed 31 March 2014). 22. Ministry of Education, Culture, Sports, Science and Technology. Survey on community health. (Japanese) (Online) 2014. Available: http://www.mext.go.jp/component/a_menu/ education/detail/__icsFiles/afieldfile/2013/07/11/1324090_6.p df (Accessed 24 March 2014). 8
https://openalex.org/W2158220148	https://hal-amu.archives-ouvertes.fr/hal-03274814/file/PIIS0021925820376626.pdf	English	null	HxcQ Liposecretin Is Self-piloted to the Outer Membrane by Its N-terminal Lipid Anchor	Journal of biological chemistry/The Journal of biological chemistry	2,009	cc-by	9,436	HxcQ Liposecretin Is Self-piloted to the Outer Membrane by Its N-terminal Lipid Anchor Véronique Viarre, E. Cascales, Geneviève Ball, Gérard P.F. Michel, Alain Filloux, Romé Voulhoux HxcQ Liposecretin Is Self-piloted to the Outer Membrane by Its N-terminal Lipid Anchor Véronique Viarre, E. Cascales, Geneviève Ball, Gérard P.F. Michel, Alain Filloux, Romé Voulhoux To cite this version: Véronique Viarre, E. Cascales, Geneviève Ball, Gérard P.F. Michel, Alain Filloux, et al.. HxcQ Liposecretin Is Self-piloted to the Outer Membrane by Its N-terminal Lipid Anchor. Journal of Biological Chemistry, 2009, 284 (49), pp.33815-33823. 10.1074/jbc.M109.065938. hal-03274814 Distributed under a Creative Commons Attribution 4.0 International License HxcQ Liposecretin Is Self-piloted to the Outer Membrane by Its N-terminal Lipid Anchor□ S Received for publication,September 14, 2009, and in revised form, October 6, 2009 Published, JBC Papers in Press,October 8, 2009, DOI 10.1074/jbc.M109.065938 Ve´ronique Viarre‡1, Eric Cascales‡, Genevie`ve Ball‡, Ge´rard P. F. Michel‡, Alain Filloux‡§2, and Rome´ Voulhoux‡3 From the ‡Laboratoire d’Inge´nierie des Syste`mes Macromole´culaires (LISM-UPR 9027), CNRS, Institut de Microbiologie de la Me´diterrane´e (IMM), 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France and the §Centre for Molecular Microbiology and Infection, Division of Cell and Molecular Biology, Imperial College London, London SW7 2AZ, United Kingdom the Sec or Tat translocons. Following removal of the SP, they are released into the periplasm (3–4). The periplasmic inter- mediates are specifically recognized by the type II secretion system (T2SS), also called secreton, for their transport across the OM. This pathway, therefore, promotes the specific trans- port of exoproteins requiring intracellular folding, like periplas- mic disulfide bridge formation, and, in some cases, assembly into multimeric complexes prior to their secretion. Such a requirement implies that the secretion process uses a large and tightly controlled secretion channel in the OM. The T2SS is a highly complex nanomachine embedded in the bacterial enve- lope consisting of 12–16 different proteins, depending on the organism (1, 5). Interestingly, there is only one integral OM protein in this system, which therefore constitutes the only can- didate for the OM translocation channel. This OM component belongs to a family of proteins generically designated as secre- tins (6). This family also includes members that are involved in type III protein secretion (T3SS), type IV pilus assembly, type IV bundle-forming pili, toxin co-regulated pili, and assembly and export of filamentous phage (7–12). Therefore, secretins constitute an important group of transporters specialized in the translocation of bulky macromolecules or macromolecular complexes across the OM. Secretins are an unusual and important class of bacterial outer membrane (OM) proteins. They are involved in the trans- port of single proteins or macromolecular structures such as pili, needle complexes, and bacteriophages across the OM. Secretins are multimeric ring-shaped structures that form large pores in the OM. The targeting of such macromolecular struc- tures to the OM often requires special assistance, conferred by specific pilotins or pilot proteins. Here, we investigated HxcQ, the OM component of the second Pseudomonas aeruginosa type II secretion system. We found that HxcQ forms high molecular mass structures resistant to heat and SDS, revealing its secretin nature. HAL Id: hal-03274814 https://amu.hal.science/hal-03274814v1 Submitted on 14 Sep 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 49, pp. 33815–33823, December 4, 2009 © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Research in the Rome´ Voulhoux laboratory was supported by Agence NationaledelaRechercheprograms,“Youngresearcher”(GrantANR-JC07- 183230) and “ERA-NET Pathogenomics” (Grant ANR-08-PATH-004-01). □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental “Experimental Procedures,” additional references, and Figs. S1 and S2. 1 Supported by a grant from the French Ministry of Research and Technology. 2 Supported by the Royal Society. 3 To whom correspondence should be addressed: Laboratoire d’Inge´nierie des Syste`mes Macromole´culaires UPR 9027, CNRS, IMM, 31 Chemin Joseph Aiguier, 13402 Marseille cedex 20, France. Tel.: 33-4-91-16-41-26; Fax: 33-4- 91-71-21-24; E-mail: voulhoux@ifr88.cnrs-mrs.fr. 4 The abbreviations used are: OM, outer membrane; T2SS, type II secretion system; IM, inner membrane; MES, 2-(N-morpholino)ethanesulfonic acid; HMM, high molecular mass; SP, signal peptide. DECEMBER 4, 2009•VOLUME 284•NUMBER 49 This is an Open Access article under the CC BY license. * Research in the Rome´ Voulhoux laboratory was supported by Agence NationaledelaRechercheprograms,“Youngresearcher”(GrantANR-JC07- 183230) and “ERA-NET Pathogenomics” (Grant ANR-08-PATH-004-01). □ □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental “Experimental Procedures,” additional references, and Figs. S1 and S2. 1 Supported by a grant from the French Ministry of Research and Technology. 2 This is an Open Access article under the CC BY license. 2 Supported by the Royal Society. 3 To whom correspondence should be addressed: Laboratoire d’Inge´nierie des Syste`mes Macromole´culaires UPR 9027, CNRS, IMM, 31 Chemin Joseph Aiguier, 13402 Marseille cedex 20, France. Tel.: 33-4-91-16-41-26; Fax: 33-4- 91-71-21-24; E-mail: voulhoux@ifr88.cnrs-mrs.fr. HxcQ Liposecretin Is Self-piloted to the Outer Membrane by Its N-terminal Lipid Anchor□ S Interestingly, we showed that HxcQ is a lipoprotein. Construction of a recombinant nonlipidated HxcQ (HxcQnl) revealed that lipidation is essential for HxcQ function. Further phenotypic analysis indicated that HxcQnl accumulates as mul- timers in the inner membrane of P. aeruginosa, a typical pheno- type observed for secretins in the absence of their cognate pilo- tin. Our observations led us to the conclusion that the lipid anchor of HxcQ plays a pilotin role. The self-piloting of HxcQ to the OM was further confirmed by its correct multimeric OM localization when expressed in the heterologous host Esche- richia coli. Altogether, our results reveal an original and unprec- edented pathway for secretin transport to the OM. Several secretins have been purified and analyzed by electron microscopy, revealing that 12–14 identical secretin monomers form ring-like complexes with a central channel large enough to accommodate their substrates (7, 13–14). The homology between the members of the secretin family is contained within the C-terminal half of the protein (see Fig. 2) (15). Therefore, this domain has been proposed to form the secretion channel, whereas the much less conserved N-terminal domain that largely protrudes into the periplasm probably undertakes more specific functions, such as substrate recognition and/or inter- action with the other components of the corresponding machineries (13, 16). The presence of an outer membrane (OM)4 in Gram-nega- tive bacteria constitutes a second barrier for the secretion of exoproteins into the extracellular medium. At least six different secretion pathways have evolved in these bacteria for the secre- tion of a very diverse pool of extracellular proteins (1–2). Among them, the type II secretion pathway is a two-step proc- ess in which exoproteins with an N-terminal signal peptide (SP) are first exported through the cytoplasmic membrane by either Among the large diversity of identified secretins, most of them depend on a small pilot protein for their correct final insertion into the outer membrane. In most cases, pilot proteins are outer membrane-linked lipoproteins called pilotins. To date, characterized secretin/pilotin couples are: PulD/PulS of Klebsiella (17–18), OutD/OutS of Erwinia (19) for T2SS; YscC/ YscW of Yersinia (8), InvG/InvH of Salmonella (12), MxiD/ MxiM of Shigella (20) for T3SS, and PilQ/Tgl of Myxococcus (21) for Type IV pilus systems. For T2SS secretin/pilotin cou- ples, the specific pilotin binding domain is localized at the extreme C terminus of the secretin (19, 22). 4 The abbreviations used are: OM, outer membrane; T2SS, type II secretion system; IM, inner membrane; MES, 2-(N-morpholino)ethanesulfonic acid; HMM, high molecular mass; SP, signal peptide. DECEMBER 4, 2009•VOLUME 284•NUMBER 49 EXPERIMENTAL PROCEDURES Bacterial Strains, Plasmids, and Growth Conditions—The bacterial strains, vectors, and plasmids used in this study are listed in Table 1. Recombinant DNA methods were performed essentially as described previously (30). Oligonucleotides used for PCR are listed Table 2. P. aeruginosa and E. coli strains were grown at 37 °C in Luria-Bertani medium. To induce LapA pro- duction and secretion via the P. aeruginosa Hxc T2SS, cells were grown at 30 °C under phosphate-limiting conditions using proteose peptone medium (Difco Laboratories) containing 0.4% glucose, with horizontal shaking (29). When required, media were supplemented with the following antibiotics used at the indicated concentrations: 50 gml1 kanamycin, 20 gml1 gentamicin; and 50 gml1 ampicillin; 50 gml1 for E. coli and 250 gml1 carbenicillin; 50 gml1 gentamycin; and 2,000 gml1 streptomycin for P. aeruginosa. Bacterial growth was measured by optical density at 600 nm (A600). 1 A600 unit corresponds to 109 cells/ml. The E. coli CC118pir strain was used to propagate pKNG101 and derivative plasmids, while TG1 and the DH5 strains were used for other plasmid manip- ulations. Plasmids were transferred to P. aeruginosa using the conjugative properties of the helper plasmid pRK2013 in tripa- rental matings (31). Transconjugants were selected on Pseudo- monas isolation agar (Difco) containing 2.5% glycerol (v/v) sup- plemented with corresponding antibiotic(s). For classical arabinose induction, bacterial cultures were induced with 0.2% filtered L-arabinose (Sigma) at A600 0.8 for 2.5 h. In Gram-negative bacteria, most lipoproteins are periplas- mic proteins anchored to the inner or outer membrane through a lipid moiety attached to their invariable N-terminal cysteine residue (3). Lipidation and maturation of lipoproteins take place after their translocation through the inner membrane via Sec machinery (3). Lipoprotein-specific signal peptides (SPs) are characterized by a specific consensus motif (V/L)XXC called the Lipobox (26). The Lipobox is both the lipidation site and the maturation site recognized by the lipoprotein signal peptidase II, which cleaves the SP upstream of the cysteine (27–28). P. aeruginosa strain PAO1 possesses two complete and non- redundant T2SS, referred to as the Xcp and Hxc systems (1). While more than a dozen exoproteins utilize the Xcp T2SS for their secretion, the Hxc T2SS, which is induced under phos- phate starvation, is dedicated to the secretion of one single low molecular mass protein, the alkaline phosphatase LapA (29). In the present work, we reveal that the atypical HxcQ secre- tin of the Hxc T2SS of P. Strains and plasmids used in this study Source or Ref. Relevant characteristics OM via its N-terminal lipid anchor, therefore revealing a new pathway for secretin biogenesis. genes encoding pilotins are found in the same cluster as the genes encoding the corresponding secretion systems. However, in several secretin-containing systems, a pilotin gene has yet to be identified, suggesting the existence of possible alternatives to the pilotin biogenesis pathway. Recently, a soluble nonlipidated periplasmic protein has been shown to be important for the OM localization of XcpQ secretin in P. aeruginosa (23). Inter- estingly, three secretins are themselves lipoproteins, but no function has so far been attributed to their atypical N-terminal lipid anchor. One, XpsD of Xanthomonas campestris pv. campestris, belongs to a T2SS (24), and two others, BfpB of enteropathogenic E. coli (25) and TcpC of Vibrio cholerae (11) are members of type IV pili systems. HxcQ Liposecretin Is Self-piloted to the Outer Membrane by Its N-terminal Lipid Anchor□ S The majority of the 4 The abbreviations used are: OM, outer membrane; T2SS, type II secretion system; IM, inner membrane; MES, 2-(N-morpholino)ethanesulfonic acid; HMM, high molecular mass; SP, signal peptide. 4 The abbreviations used are: OM, outer membrane; T2SS, type II secretion system; IM, inner membrane; MES, 2-(N-morpholino)ethanesulfonic acid; HMM, high molecular mass; SP, signal peptide. This is an Open Access article under the CC BY license. 33815 JOURNAL OF BIOLOGICAL CHEMISTRY 33815 DECEMBER 4, 2009•VOLUME 284•NUMBER 49 DECEMBER 4, 2009•VOLUME 284•NUMBER 49 DECEMBER 4, 2009•VOLUME 284•NUMBER 49 TABLE 1 Strains and plasmids used in this study Ap, ampicillin; Gm, gentamicin; Km, kanamycin; Sm, streptomycin. Strains, vectors, and plasmids Relevant characteristics Source or Ref. Strains E. coli TG1 SupE (lac-proAB) thi hsd R5 (F’:traD36 rpo AB lacIq lacZM15) Lab collection DH5 supE44 hsdR17 recA1 endA1 gyrA96 thi-1 relA1 Lab collection CC118pir (ara-leu) araD lacX74 galE galK phoA20 thi-1 rpsE rpoB argE(Am) recA1 RfR(pir) Ref. 46 P. aeruginosa PAO1 Prototrophe, chl-2 B. Holloway PAO1xcpQ Non polar deletion of the xcpQ gene in PAO1 Ref. 47 PAO1hxcQ Non polar deletion of the hxcQ gene in PAO1 This study PAO1hxcQxcpQ Non polar deletion of the hxcQ gene in PAO1xcpQ This study PAO1hxcQxcpQpAqA Non polar deletion of the xphA, and xqhA genes in PAO1hxcQxcpQ This study Vectors and Plasmids pKNG101 SmR, mobRK2, sacBR (suicide vector) Ref. 48 pRK2013 Km R, ColE1, Tra Mob (RK2) Ref. 30 pCR2.1 ApR Km R, ColE1, f1 ori Invitrogen pJN105 GmR, araC-pBAD, (broad-host-range vector) Ref. 49 pMMB67HE ApR, IncQ tac promoter; lacIq, (broad-host-range vector) Ref. 50 pMMB67HEhxcQV5 hxcQV5 cloned into pMMB67HE (XbaI/SmaI) This study pMB4 xcpQ cloned in pMMB67HE (PstI-SmaI) Ref. 47 pET-DEST42 ApR CmR, Gateway destination vector Invitrogen pET-DEST42xcpQV5 xcpQV5 cloned in the Gateway vector pET-DEST42 This study pET-DEST42hxcQV5 hxcQV5 cloned in the Gateway vector pET-DEST42 This study pKNGhxcQ Mutator plasmid for hxcQ deletion This study pKNGpAqA Mutator plasmid for xphA-xqhA deletion Ref. 31 pJNhxcQV5 hxcQV5 cloned into pJN105, pBAD This study pJNhxcQnlV5 hxcQnlV5 cloned into pJN105, pBAD This study pJNhxcQnl hxcQnl cloned into pJN105, pBAD This study Self-targeted HxcQ Liposecretin Self-targeted HxcQ Liposecretin Self-targeted HxcQ Liposecretin TABLE 2 Oligonucleotides used in this study TABLE 2 Oligonucleotides used in this study Name Nucleotide sequence (53 3) hxcQ-500 CAGGCCTACTGGCGGCAACTGGCGCCG hxcQ for CCCATGAGGCGTCGGCGACATGCAGGC hxcQ rev TCGCCGACGCCTCATGGGGAATCCTTG hxcQ 500 AATGGGTCTCGAAGGGCTCGATGTGGA petDEST42 for TCTAGAAATAATTTTGTTTAACTTTAA xcpQ114 rev GACCTTCGGCGTCTCGCTGCCGCTGTTTTCGGCGTGCG hxcQ85 for CACGCCGAAAACAGCGGCAGCGAGACGCCGAAGGTCCC petDEST42 rev TGTTAGCAGCCGGATCAAACTCAATGG hQnl_1 for GTCGACGAGCGCGCCAGCACCGCCGCCGGG hQnl_4 rev ATGGATATCTGCGCAGAATTCGCCTCATAGCGGCGCCGCCT CGCCC Oligonucleotides used in this study Name Nucleotide sequence (53 3) hxcQ-500 CAGGCCTACTGGCGGCAACTGGCGCCG hxcQ for CCCATGAGGCGTCGGCGACATGCAGGC hxcQ rev TCGCCGACGCCTCATGGGGAATCCTTG hxcQ 500 AATGGGTCTCGAAGGGCTCGATGTGGA petDEST42 for TCTAGAAATAATTTTGTTTAACTTTAA xcpQ114 rev GACCTTCGGCGTCTCGCTGCCGCTGTTTTCGGCGTGCG hxcQ85 for CACGCCGAAAACAGCGGCAGCGAGACGCCGAAGGTCCC petDEST42 rev TGTTAGCAGCCGGATCAAACTCAATGG hQnl_1 for GTCGACGAGCGCGCCAGCACCGCCGCCGGG hQnl_4 rev ATGGATATCTGCGCAGAATTCGCCTCATAGCGGCGCCGCCT CGCCC Q Isolation and Separation of P. aeruginosa Membranes by Density Sucrose Gradient Centrifugation—250 A600 units equivalent of bacterial cells were harvested by centrifugation at 2,000 g. The pellet was resuspended in 1.5 ml buffer A (10 mM Tris, pH 7.4, 1 mM p-toluenesulfonyl fluoride (Sigma-Aldrich); 10 gml1 DNase and RNase (Roche); and sucrose 20% (w/w)). The cells were passed twice through a French press cell dis- rupter (Thermo) at 15,000 pressure units using a 3/8-inch diameter piston (20K French pressure cell, AMINCO). Unbro- ken cells were removed by centrifugation at 4 °C for 15 min at 1,600 g. The supernatant was centrifuged at 4 °C for 30 min at 125,000 g. The crude membrane pellet was resuspended in 0.5 ml buffer M (10 mM Tris, pH 7.4, “Complete EDTA-free” proteases inhibitor mixture (Roche) and 5 mM EDTA) contain- ing 20% (w/w) sucrose and then loaded on top of a discontinu- ous sucrose gradient consisting of 1.5 ml layers of buffer M solution containing 60 (bottom), 55, 50, 45, 40, 35, and 30% (w/w) sucrose. The membrane separation was performed by centrifugation at 4 °C for 65 h at 39,000 rpm in a Beckman SW41 rotor. The gradients were visually checked, and pre- dicted inner membrane (IM) (upper disc) and OM (lower disc) fractions were collected for experiments as presented (Fig. 5A). Fractions were electrophoresed in (i) 11% denaturing SDS- polyacrylamide gel followed by Coomassie Blue staining and visual identification of the OM protein OprF; (ii) 11% denatur- ing SDS-polyacrylamide gel followed by Western blotting for XcpY detection; (iii) 3.5–9% gradient seminative polyacryl- amide gel followed by Western blotting with antibody against HxcQ peptide 28 for detection of multimers of secretins; and (iv) 9% denaturing SDS-polyacrylamide gel followed by West- ern blotting with antibody against HxcQ peptide 29 for detec- tion of monomers of secretins. TABLE 2 Oligonucleotides used in this study SDS-PAGE and Immunoblotting—Protein samples were ana- lyzed under denaturing or semi-native conditions as described previously (32). Protein samples were solubilized in SDS-PAGE sample buffer (33) containing 2% SDS and mercaptoethanol (denaturing) or 0.2% SDS without mercaptoethanol (semi-na- tive). Samples were heated for 10 min at 95 °C (denaturing) or stored at 4 °C (semi-native). Electrophoresis was performed using 11% SDS-polyacrylamide gel at room temperature and 25 mA/gel (denaturing) or 3.5–9% gradient SDS-polyacrylamide- free gel at 4 °C and 100 V (semi-native conditions) or a different percentage of polyacrylamide when indicated. For Western blotting, proteins were transferred from gels onto nitrocellu- lose membranes. The membranes were blocked overnight in Tris-buffered saline (pH 7.6), 5% milk, and 0.05% Tween 20 and incubated with primary antibodies directed against the V5 epitope (Bethyl/Interchim), LapA (laboratory stock), TolR (lab- oratory stock), XcpY and XcpR (laboratory stock), HxcQ (EUROGENTEC peptides based polyclonal antibody protocol (AS-DOUB-LXP), peptide 28 (H2N-GGEGNEGDQQRARLS- G-CONH2) for specific multimer detection, and peptide 29 (H2N-SSVDERASTAAGVC-CONH2) for specific monomer detection in blocking buffer, followed by a second incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies (A6154, Sigma) in blocking buffer. Membranes were developed using the enhanced chemiluminescence protocol (Pierce). Isolation and Separation of E. coli Membranes by Density Sucrose Gradient Centrifugation—500 A600 units equivalent of bacterial cells were harvested by centrifugation at 2,000 g. The pellet was resuspended in 5 ml of buffer B (10 mM Tris, pH 7.4; 1 mM p-toluenesulfonyl fluoride; 10 gml1 DNase and RNase (Roche); sucrose 20% (w/w); and 400 gml1 lysozyme (Euromedex)). The cells were passed twice through a French press cell disrupter (Thermo) at 15,000 pressure units using a 3/8-inch diameter piston (20K French pressure cell, AMINCO). Unbroken cells were removed by centrifugation at 4 °C for 15 min at 1,600 g. The supernatant was centrifuged at 4 °C for 30 min at 125,000 g. The crude membrane pellet was resuspended in 0.5 ml buffer M (10 mM Tris, pH 7.4; “Complete EDTA-free” proteases inhibitor mixture (Roche), and 5 mM EDTA), containing 20% (w/w) sucrose and then loaded on top of a discontinuous sucrose gradient consisting of 1.5 ml layers of buffer M solution containing 60 (bottom), 55, 50, 45, 40, 35, and 30% (w/w) sucrose. The membrane separation was per- formed by centrifugation at 4 °C for 18 h at 39,000 rpm in a Beckman SW41 rotor. EXPERIMENTAL PROCEDURES aeruginosa is a lipoprotein. Moreover, we demonstrate that the HxcQ liposecretin is self-piloted to the Construction of P. aeruginosa Mutants and Plasmids— Details are available under supplemental “Experimental Procedures.” VOLUME 284•NUMBER 49•DECEMBER 4, 2009 33816 JOURNAL OF BIOLOGICAL CHEMISTRY 33816 VOLUME 284•NUMBER 49•DECEMBER 4, 2009 DECEMBER 4, 2009•VOLUME 284•NUMBER 49 Self-targeted HxcQ Liposecretin Gene expression was induced with 0.2% L-arabinose. At the same time, 50 Ci of [3H]-palmitic acid was added to 1 ml of the culture. Cells were grown for 150 min and collected, and total proteins were analyzed by SDS-PAGE. Gels were then either dried on filter paper and subjected to autoradiography for 5 months at 80 °C and revealed or blotted for HxcQ detection. TABLE 2 Oligonucleotides used in this study Self-targeted HxcQ Liposecretin Self-targeted HxcQ Liposecretin FIGURE 1. HxcQ forms SDS and heat-resistant HMM complexes. Immu- nodetection of HxcQ secretin with anti-HxcQ multimers and anti-HxcQ monomer antibodies. The PAO1 hxcQ mutant complemented with pJNhxcQV5 was grown under standard conditions to induce HxcQV5 produc- tion. Whole cell extracts were collected, resuspended in SDS-PAGE sample buffer containing 2% SDS and incubated at the indicated temperature for 10 min. The proteins were separated using 3.5% acrylamide stacking and 9% running gel. The positions of HxcQ multimers and monomers are indicated on the left. Self-targeted HxcQ Liposecretin fractions of about 550 l each. Fractions were electrophoresed in 3.5–11% denaturing SDS-polyacrylamide gel followed by (i) Coomassie Blue staining and visual identification of the OM porins; and (ii) Western blotting for TolR, HxcQ monomers, and multimers detection. NADH oxidase activity measurement was carried out essentially as described by Osborn et al. (51). Briefly, incubation mixtures containing 50 mM Tris HCl, pH 7.4, 0,12 mM -nicotinamide adenine dinucleotide, reduced form (NADH) disodium salt hydrate (Sigma, N-8129), and 0.2 mM dithiothreitol (Sigma, D-0632), and the membrane frac- tions (20 l) in a final volume of 200 l were prepared. The rate of decrease in absorbance at 340 nm was measured in micro- plates at 23 °C in a Multiskan Ascent recording spectropho- tometer (Thermo Labsystems). Differential Detergent Solubilization of Membrane-associ- ated Secretins—The equivalent of 10 A600 units of a crude mem- brane pellet (obtained following the density sucrose gradient protocol) was resuspended in 500 l of either 2% Triton X-100 (w/v) (T-9284, Sigma) solution, 100 mM sodium carbonate pH 11 (S-6139 Sigma) solution, or 4 M urea (161-0731, Bio-Rad) in 20 mM MES, 99% (M-3023, Sigma) solution. Samples were incu- bated for 30 min at 4 °C with gentle shaking. Soluble and insol- uble membrane proteins were separated by centrifugation at 4 °C for 30 min at 125,000 g. Insoluble membrane proteins were recovered in the pellet fraction, whereas solubilized mem- brane proteins present in the soluble fraction were precipitated by adding tRNA (100 gml1 final) and trichloroacetic acid FIGURE 1. HxcQ forms SDS and heat-resistant HMM complexes. Immu- nodetection of HxcQ secretin with anti-HxcQ multimers and anti-HxcQ monomer antibodies. The PAO1 hxcQ mutant complemented with pJNhxcQV5 was grown under standard conditions to induce HxcQV5 produc- tion. Whole cell extracts were collected, resuspended in SDS-PAGE sample buffer containing 2% SDS and incubated at the indicated temperature for 10 min. Self-targeted HxcQ Liposecretin The proteins were separated using 3.5% acrylamide stacking and 9% running gel. The positions of HxcQ multimers and monomers are indicated on the left. FIGURE 2. Primary sequence comparison of HxcQ and other secretins listed in this study. Secretin primary sequence are presented as followed from their N terminus to their C terminus: the signal sequence (SS), an L1 domain (when present), followed by the variable N-terminal domain, the L2 domain (when present) and finally, the C-terminal domain. Linker amino acids are indicated on the top of each schematic representation. Type I signal sequences are indicated in light gray whereas type II lipoprotein signal sequences are indicated in black. The L1 domain, present only in HxcQ and XpsD primary sequences is represented with black dotted lines. The typical N-terminal domain found in T2SS secretins (43) is indicated in gray, whereas N-terminal domains from other types of secretins are indicated in white. The L2 domain, bridging the N and C terminus domains of OutD and the four lipidated secretins (HxcQ, XpsD, BfpB, and TcpC) is indicated with gray dotted lines. The highly conserved C-terminal domain among all secretins is striped. Homologous domains between HxcQ and XcpQ are connected with small dotted lines. The transport systems to which each secretin belongs are indicated on the left. FIGURE 2. Primary sequence comparison of HxcQ and other secretins listed in this study. Secretin primary sequence are presented as followed from their N terminus to their C terminus: the signal sequence (SS), an L1 domain (when present), followed by the variable N-terminal domain, the L2 domain (when present) and finally, the C-terminal domain. Linker amino acids are indicated on the top of each schematic representation. Type I signal sequences are indicated in light gray whereas type II lipoprotein signal sequences are indicated in black. The L1 domain, present only in HxcQ and XpsD primary sequences is represented with black dotted lines. The typical N-terminal domain found in T2SS secretins (43) is indicated in gray, whereas N-terminal domains from other types of secretins are indicated in white. The L2 domain, bridging the N and C terminus domains of OutD and the four lipidated secretins (HxcQ, XpsD, BfpB, and TcpC) is indicated with gray dotted lines. The highly conserved C-terminal domain among all secretins is striped. Homologous domains between HxcQ and XcpQ are connected with small dotted lines. Self-targeted HxcQ Liposecretin The transport systems to which each secretin belongs are indicated on the left. VOLUME 284•NUMBER 49•DECEMBER 4, 2009 TABLE 2 Oligonucleotides used in this study The gradient was further collected in 16 Preparation of Culture Supernatants—P. aeruginosa strains were grown under phosphate-limiting conditions to an A600 of 1.5. Cells and extracellular medium were separated by centrifugation; proteins contained in the supernatants were precipitated by adding trichloroacetic acid (10% (w/v) final concentration) and incubated overnight at 4 °C. Samples were subsequently centrifuged (30 min at 15,000 g), the pellets were washed with 90% (v/v) acetone, resuspended in SDS-PAGE sample buffer, and analyzed under denaturing conditions. Inhibition of Lipoprotein Signal Peptidase with Globomycin— Bacteria were grown in Luria-Bertani medium to an A600 of 0.8, and arabinose at 0.2% final and globomycin at 100 gml1 final were added to the culture, and incubation was continued for 30 min at 37 °C. Bacteria were harvested by centrifugation and resuspended in SDS sample buffer, and solubilized proteins were examined by SDS-PAGE and immunoblotting. [3H]-Palmitic Acid Labeling—Bacteria were grown at 37 °C in a liquid minimal medium, proteose peptone 1 (supple- mented with 0.4% glucose as carbon source) to an A600 of 0.3. DECEMBER 4, 2009•VOLUME 284•NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 33817 Self-targeted HxcQ Liposecretin RESULTS HxcQ Is a Member of the Secretin Family—Among the different T2SS Hxc proteins encoded by the hxc cluster, HxcQ is predicted to be the secretin component of the system (29). As presented in Fig. 1, HxcQ forms SDS-resistant high molecular mass (HMM) complexes, which has been shown to be a general charac- teristic of secretins. We observed such HMM complexes for HxcQ secretin when total cell fractions of P. aeruginosa hxcQ producing a C-terminal V5-hexahistidine tagged HxcQ (HxcQV5) were loaded on a standard SDS-polyacrylamide gel (Fig. 1). However, secretin com- plexes can show different behaviors in response to heat treatment. For example, HMM complexes formed by PulD or pIV secretins are fully resistant to heat (18, 35), whereas HMM complexes formed by XcpQ, BfpB, TcpC, or OutD secretins are totally dissociated after boiling (7, 10–11, 36). We found that HxcQ multimers are partially heat-resist- e incubated at up to 95 °C for 10 min Family—Among the different T2SS Hxc proteins encoded by the hxc cluster, HxcQ is predicted to be the secretin component of the system (29). As presented in Fig. 1, HxcQ forms SDS-resistant high molecular mass (HMM) complexes, which has been shown to be a general charac- teristic of secretins. We observed such HMM complexes for HxcQ secretin when total cell fractions of P. aeruginosa hxcQ producing a C-terminal V5-hexahistidine tagged HxcQ (HxcQV5) were loaded on a standard SDS-polyacrylamide gel (Fig. 1). However, secretin com- plexes can show different behaviors in response to heat treatment. For example, HMM complexes formed by PulD or pIV secretins are fully resistant to heat (18, 35), whereas HMM complexes formed by XcpQ, BfpB, TcpC, or OutD secretins are totally dissociated after boiling (7, 10–11, 36). We found that HxcQ multimers are partially heat-resist- en samples are incubated at up to 95 °C for 10 min 4). ition of HxcQV5 bed with either tection. In the ed, whereas in multimers and withorwithout re labeled with acrylamide gel molecular mass V5 protein and abeled protein FIGURE 3. HxcQV5 globomycin sensitivity and [3H]-palmitic acid labeling. Globomycin inhibition of HxcQV5 maturation. Immunoblotting of total cell proteins from strain PAO1hxcQ/pJN105hxcQV5 probed with either V5 antibody (top panel) for HxcQV5 detection or XcpQ antibody (bottom panel) for XcpQ detection. RESULTS In the absence of globomycin (), mature HxcQV5 monomers as well as HxcQV5 multimers are observed, whereas in the presence of globomycin (), the maturation of HxcQV5 is inhibited leading to the loss of multimers and accumulationoftheprecursorformofHxcQV5monomers.Incontrast,matureXcpQisdetectedwithorwithout globomycin treatment. A, PAO1hxcQ cells producing HxcQV5 or HxcQnlV5 from plasmids were labeled with [3H]-palmitic acid. Cell samples were electrophoresed on an 8% stacking/9% running SDS-polyacrylamide gel and radiolabeled lipoproteins were detected by autoradiography. Similar amounts of low molecular mass radiolabeled proteins are detected at the migration front in both samples. Full-length HxcQV5 protein and molecular mass markers (in kDa) are indicated on the left. The asterisk indicates a specific radiolabeled protein that might correspond to an HxcQV5 degradation product (B). (10% (w/v) final) and incubated overnight at 4 °C. Samples were subsequently centrifuged (30 min at 15,000 g), and the pellets washed with 90% acetone. Pellets containing insoluble or pre- cipitated soluble membrane proteins were resuspended in denaturing or seminative SDS-PAGE sample buffer for SDS- PAGE and immunoblotting. HxcQ secretin encodes an 803-amino acid protein that is 30% identical and 49% similar to XcpQ, a well characterized T2SS secretin of P. aeruginosa. HxcQ primary sequence analysis revealed the typical two subdomains found in XcpQ, the highly conserved C-terminal domain (residues 424–803) involved in pore formation, and the dissimilar N-terminal domain (residues 81–362), predicted to be periplasmic (Fig. 2). Primary sequence comparison between HxcQ and XcpQ also revealed the presence of two supplemental linker regions on both sides of HxcQ N-terminal domain that are absent in XcpQ (Fig. 2). The region located between the signal peptide and the N-terminal domain is called L1. L1 is 71-amino acids long and is mostly composed of small amino acids such as alanine, serine, and glycine. A comparable linker region is also present in the Xanthomonas campestris XpsD T2SS secretin (Fig. 2). The second linker region, L2, located between the N- and the C-terminal domains, is 62- amino acids long and has a composition of 58% serine and glycine. A similar polyserine/glycine region has already been described for OutD and BfpB (16, 37) and is also present in XpsD and TcpC secretins (Fig. 2). Transmembrane Potential Measurements—P. aeruginosa strains were grown exponentially for 2 h in the presence of 0.2% L-arabinose and harvested by centrifugation at room tempera- ture. The transmembrane potential () was measured essen- tially as described previously (34). 33818 JOURNAL OF BIOLOGICAL CHEMISTRY 33818 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284•NUMBER 49•DECEMBER 4, 2009 Self-targeted HxcQ Liposecretin FIGURE 3. HxcQV5 globomycin sensitivity and [3H]-palmitic acid labeling. Globomycin inhibition of HxcQV5 maturation. Immunoblotting of total cell proteins from strain PAO1hxcQ/pJN105hxcQV5 probed with either V5 antibody (top panel) for HxcQV5 detection or XcpQ antibody (bottom panel) for XcpQ detection. In the absence of globomycin (), mature HxcQV5 monomers as well as HxcQV5 multimers are observed, whereas in the presence of globomycin (), the maturation of HxcQV5 is inhibited leading to the loss of multimers and accumulationoftheprecursorformofHxcQV5monomers.Incontrast,matureXcpQisdetectedwithorwithout globomycin treatment. A, PAO1hxcQ cells producing HxcQV5 or HxcQnlV5 from plasmids were labeled with [3H]-palmitic acid. Cell samples were electrophoresed on an 8% stacking/9% running SDS-polyacrylamide gel and radiolabeled lipoproteins were detected by autoradiography. Similar amounts of low molecular mass radiolabeled proteins are detected at the migration front in both samples. Full-length HxcQV5 protein and molecular mass markers (in kDa) are indicated on the left. The asterisk indicates a specific radiolabeled protein that might correspond to an HxcQV5 degradation product (B). quots were first incubated with 10 M of carbonyl cyanide m-chloro- phenylhydrazone for 15 min at room temperature before addition of the Br-TPP solution, incubation, and filtration. quots were first incubated with 10 M of carbonyl cyanide m-chloro- phenylhydrazone for 15 min at room temperature before addition of the Br-TPP solution, incubation, and filtration. Self-targeted HxcQ Liposecretin Cellular (C) and extracellular (E) proteins separated on a 12% (w/v) SDS-polyacrylamide gels were stained with Coomassie Blue (upper panel) or immunoblotted for LapA detection (lower panel). The dots indicate Xcp-de- pendent exoproteins, whereas LapA is indicated by a triangle. The asterisk indicates a nonspecific band reacting with the LapA antiserum. This band is also present in a lapA mutant (not shown). The HxcQ Lipid Anchor Has a Pilotin Function—In order to understand why HxcQnlV5 was not functional, we examined its cellular localization. These studies were carried out in P. aeruginosa PAO1hxcQ producing wild type or nonlipi- dated HxcQ from plasmids and under arabinose-inducing con- ditions. Bacterial cells were disrupted and both HxcQ secretins localized in the total membrane fraction (data not shown). In order to investigate the presence of the secretin multimers in the inner membrane (IM) or the OM, total membrane fractions were then separated by centrifugation on a sucrose density gra- dient. Regions corresponding to the IM and OM (Fig. 5A) were directly sampled from the tube and analyzed. The quality of the fractionation procedure was verified by the presence in the cor- responding fractions of the integral IM protein XcpY and the major P. aeruginosa outer membrane protein OprF (Fig. 5B). Interestingly, we clearly detected both wild type and nonlipi- dated HxcQ multimers. However, whereas HxcQV5 multimers were correctly localized in the OM (Fig. 5B, lane 2), HxcQnl multimers were mislocalized and accumulated in the IM frac- tion (Fig. 5B, lane 3). In contrast to wild type HxcQ multimers, multimers of HxcQnl could only be detected under seminative conditions (see experimental procedure). Multimers of HxcQnl indeed appeared to be more sensitive to heat than wild type HxcQ multimers since they could not be detected under classi- cal denaturing conditions (supplemental Fig. S2). We therefore used semi-native conditions for all HxcQnl multimers detec- tion described in this study. p Lipidation of HxcQ Is Essential for Its Function—Given that most secretins, including the P. aeruginosa type II secretin XcpQ, are not lipoproteins (6), we wanted to determine if the N-terminal lipid anchor of HxcQ is required for its function. For this purpose, we constructed a nonlipidated HxcQV5 vari- ant (HxcQnlV5). This construction was made by substituting the type II SP of the HxcQ wild type for the type I SP of XcpQ. Self-targeted HxcQ Liposecretin FIGURE 4. N-terminal lipid anchor of HxcQ is required for function. Anal- ysis of LapA secretion in supernatant fractions from P. aeruginosa PAO1 wild type and hxcQxcpQxphAxqhA mutant strains producing HxcQV5 and HxcQnlV5. Cellular (C) and extracellular (E) proteins separated on a 12% (w/v) SDS-polyacrylamide gels were stained with Coomassie Blue (upper panel) or immunoblotted for LapA detection (lower panel). The dots indicate Xcp-de- pendent exoproteins, whereas LapA is indicated by a triangle. The asterisk indicates a nonspecific band reacting with the LapA antiserum. This band is also present in a lapA mutant (not shown). HxcQ Secretin Is a Lipoprotein—The comparison of Xcp and Hxc SPs revealed that, in contrast to XcpQ, which has a classical type I SP, HxcQ presents a characteristic type II or lipoprotein SP ending with a typical lipobox (supplemental Fig. S1). This observation was also supported by the lipoprotein prediction program DOLOP (38). To experimentally demonstrate the lipoprotein nature of HxcQ, we treated P. aeruginosa cells with globomycin, a specific lipoprotein signal peptidase II inhibitor (39). As shown in Fig. 3A, the maturation of HxcQV5 was sig- nificantly affected by the globomycin treatment, leading to the accumulation of the precursor form and loss of HxcQ multim- ers. As a control, we found that XcpQ remained unaffected in agreement with the resistance of signal peptidase I to globomy- cin. The lipidation of HxcQ was furthermore confirmed by the recovery of radiolabeled HxcQV5 when the bacteria were grown in the presence of [3H]palmitic acid (Fig. 3B). As a negative control, no radiolabeling was observed for a nonlipidated form of HxcQV5 called HxcQnlV5 (see below, and for description, see supplemental Fig. S1). A control experiment where proteins from palmitic acid-treated cells were blotted following SDS- PAGE and probed with antibody against the V5 epitope indi- cated that both HxcQV5 and HxcQnlV5 were equally produced (data not shown) and that HxcQV5 did migrate at the position corresponding to the band designated as HxcQV5 in Fig. 3B. In conclusion, both globomycin treatment and [3H]palmitic acid- labeling assays clearly demonstrated that, in contrast to XcpQ, HxcQ is a lipoprotein. From now on, we will refer to this variant of secretin as liposecretin. FIGURE 4. N-terminal lipid anchor of HxcQ is required for function. Anal- ysis of LapA secretion in supernatant fractions from P. aeruginosa PAO1 wild type and hxcQxcpQxphAxqhA mutant strains producing HxcQV5 and HxcQnlV5. RESULTS Briefly, the cell pellet from two A600 units was resuspended in 100 l of 100 mM Tris-HCl, pH 7.8, and 1 mM EDTA for outer membrane permeabilization and incubated for 3 min at 37 °C. The cell suspension was then diluted 20-fold in the transport buffer (100 mM phosphate buffer, pH 7.8, 1 mM KCl, and 0.4% glycerol). A tritiated tri- phenylphosphonium bromide (Br-TPP) solution ([3H]Br-TPP; Amersham Biosciences; diluted 40-fold in 2 mM cold Br-TPP) was added at the final concentration of 10 M in 200 l of the cell suspension and further incubated at 37 °C for 10 min. Cells were recovered by filtration (Whatman) and washed twice with transport buffer and once with transport buffer without glycerol. As a control for nonspecific TPP binding, cell ali- DECEMBER 4, 2009•VOLUME 284•NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 33 33819 DECEMBER 4, 2009•VOLUME 284•NUMBER 49 Self-targeted HxcQ Liposecretin To maintain a compatible environment for type I signal pepti- dase recognition, we also included the four amino acids down- stream of the XcpQ SP cleavage site (supplemental Fig. S1). As both the wild type and non-lipidated HxcQV5 possess a C-terminal V5-hexahistidine tag, we first tested the influ- ence of the tag on HxcQV5 function in the quadruple PAO1hxcQxcpQpAqA mutant that is deficient in Hxc, Xcp, and hybrid Xcp T2SSs (40, 5) (Fig. 4, lane 4 versus lane 2). The expression of hxcQV5 from pJNhxcQV5 in this mutant spe- cifically restored secretion of the unique Hxc substrate LapA in the extracellular medium (Fig. 4, lane 6), indicating a functional complementation and no influence of the V5 tag on HxcQ func- tion. We then tested the functionality of the nonlipidated recombinant HxcQ. Although the amount of HxcQnlV5 pro- duced by P. aeruginosa was similar to that of the lipidated form (data not shown), HxcQnlV5 was unable to restore secretion of LapA (Fig. 4, lane 8). Instead, LapA accumulated within the cells (Fig. 4, lower panel, lane 7), which indicates that the HxcQ N-terminal lipid anchor fulfills an essential secretion function. We constructed a tag-free HxcQnl to definitely exclude a pos- sible effect of the V5 tag in HxcQnl nonfunctionality. We did not observe any phenotypic differences between tagged and untagged HxcQnl variants (data not shown). To determine whether IM-recovered HxcQnl multimers were integrated or peripherically associated with the IM, total membrane fractions containing HxcQV5 or HxcQnlV5 were treated with various solubilizing agents. As shown in figure 6, 33820 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284•NUMBER 49•DECEMBER 4, 2009 33820 VOLUME 284•NUMBER 49•DECEMBER 4, 2009 Self-targeted HxcQ Liposecretin FIGURE 5. Membrane localization of HxcQV5 and HxcQnl recombinant secretin in P. aeruginosa. Inner and outer membrane fractions were visually detectable after sucrose gradient sedimentation (A). IM and OM frac- tions were collected, and their proteins content analyzed by semi-native PAGE for immunodetection of secretin monomers and multimers with the HxcQ antibodies or denaturing SDS-PAGE for OprF and XcpY detection (B). In contrast, treatment with the non- ionic detergent Triton X-100, which typically solubilizes proteins in- serted into the IM (XcpY), specifi- cally affected the non-lipidated secretin (Fig. 6, lane 10), indicating its IM insertion. Since no HxcQnlV5 was recovered in the soluble frac- tion, its solubilisation probably led to its degradation or at least the deg- radation of the V5 epitope used for HxcQ detection. As a control, the wild type HxcQV5 was not found to be solubilized by Triton X-100 (Fig. 6, lane 9), which is congruent with its OM localization. The IM localization of secretin multimers in the absence of a func- tional pilotin has already been reported for the PulD and YscC secretins, respectively involved in type II and type III secretion (41, 8). As shown for PulD, the absence of the pilotin led to partial dissipation of the proton-motive force (pmf) indicative for IM perturba- tion. This increase in IM permeability was attributed to IM insertion of the mislocalized secretin multimers. Interestingly, we found similar and significant pmf dissipation when Hxc- QnlV5 was produced in P. aeruginosa hxcQ (Table 3) and we attribute this effect to the integral IM insertion of HxcQnlV5 multimers. Together, our results show that lipidation of HxcQV5 is essential for correct localization of the protein in the OM. Moreover, the recovery of HxcQnlV5 multimers inserted in the IM suggests that the N-terminal lipid anchor of HxcQ plays a pilotin role, since such behavior was earlier reported for secretins produced in the absence of their cognate pilotin. tin in P. aeruginosa. Inner and mentation (A). IM and OM frac- or immunodetection of secretin OprF and XcpY detection (B). FIGURE 5. Membrane localization of HxcQV5 and HxcQnl recombinant secretin in P. aeruginosa. Inner and outer membrane fractions were visually detectable after sucrose gradient sedimentation (A). IM and OM frac- tions were collected, and their proteins content analyzed by semi-native PAGE for immunodetection of secretin monomers and multimers with the HxcQ antibodies or denaturing SDS-PAGE for OprF and XcpY detection (B). FIGURE 6. Measurement of in cells producing HxcQnlV5 or HxcQV5 Measurement of in cells producing HxcQnlV5 or HxcQV5 Accumulation of 3H-TPP was measured by the ratio of radioactivity inside and outside the cells (second column) and used to calculate the (third column), as described under “Experimental Procedures.” Strains 3H-TPP in/out mV Wild type/pJN105 1,000 180 4 hxcQ/pJN105 1,113.4 183 2 hxcQ/pJN105hxcQV5 857.7 176 8 hxcQ/pJN105hxcQnlV5 256 145 11 Self-targeted HxcQ Liposecretin HxcQV5 and HxcQnlV5 were probed with anti-V5 antibody. Molecular masses (in kDa) are indicated on the left. Self-targeted HxcQ Liposecretin Differential solubilization of membrane-associated HxcQV5 and HxcQnlV5. Membrane fractions containing HxcQV5 or HxcQnlV5 were treated with 2% (v/v) Triton X-100, 100 mM sodium carbonate, pH 11, or 4 M urea at pH 6.5 for differential solubilization. Soluble and insoluble fractions were analyzed under semi-native conditions for HxcQV5 and HxcQnlV5 mul- timer detection and under denaturing conditions on a 12% (w/v) SDS-poly- acrylamide gel for XcpR and XcpY. HxcQV5 and HxcQnlV5 were probed with anti-V5 antibody. Molecular masses (in kDa) are indicated on the left. p g p HxcQ Correctly Localized in the OM of the Heterologous Host E. coli—The biogenesis of secretins is impaired in the absence of their cognate pilotins or pilot proteins, and they either remain monomeric or mislocalize to the IM. We tested whether HxcQ liposecretin also requires the assistance of another P. aeruginosa protein for its biogenesis i.e. correct insertion of multimers into the OM. To this end, HxcQV5 was expressed in the heterologous host E. coli. Membrane samples were fraction- ated by sucrose density gradient and analyzed by SDS-PAGE. Data presented in Fig. 7 clearly indicates that in such a heterol- ogous environment, HxcQ remains correctly localized in the OM as a multimer. This result contrasts with those observed for other secretins such as PulD which requires co-expression of a pilotin for proper localization in E. coli. This important finding demonstrates for the first time the pilotin-independent biogen- esis of a member of the secretin family. FIGURE 6. Differential solubilization of membrane-associated HxcQV5 and HxcQnlV5. Membrane fractions containing HxcQV5 or HxcQnlV5 were treated with 2% (v/v) Triton X-100, 100 mM sodium carbonate, pH 11, or 4 M urea at pH 6.5 for differential solubilization. Soluble and insoluble fractions were analyzed under semi-native conditions for HxcQV5 and HxcQnlV5 mul- timer detection and under denaturing conditions on a 12% (w/v) SDS-poly- acrylamide gel for XcpR and XcpY. HxcQV5 and HxcQnlV5 were probed with anti-V5 antibody. Molecular masses (in kDa) are indicated on the left. FIGURE 6. Differential solubilization of membrane-associated HxcQV5 and HxcQnlV5. Membrane fractions containing HxcQV5 or HxcQnlV5 were treated with 2% (v/v) Triton X-100, 100 mM sodium carbonate, pH 11, or 4 M urea at pH 6.5 for differential solubilization. Soluble and insoluble fractions were analyzed under semi-native conditions for HxcQV5 and HxcQnlV5 mul- timer detection and under denaturing conditions on a 12% (w/v) SDS-poly- acrylamide gel for XcpR and XcpY. DISCUSSION Previ- ously, BfpB, TcpC and XpsD were y p p p experimentally demonstrated to be lipoproteins (10–11, 24). The involvement of lipidation in secretin biogenesis was only tested for XpsD where this post translational fatty acylation turned out to be dispensable for secretin function (24). For BfpB and TcpC two small nonlipidated periplasmic proteins have been shown to be required for their stabilization and multim- erization respectively (25, 11). N-terminal lipidation plays a key role for HxcQ transport and no additional specific partner is required. We therefore suggest that among the liposecretins, HxcQ defines a distinctive subclass whose biogenesis is guided by a new and unprecedented transport pathway. multimeric pore-forming structures that display relatively low -strand content (13) and high resistance to dissociation in SDS (17). Such specialized and complex OM proteins require custom-made biogenesis pathways involving addi- tional partners. Depending on the secretin, different routes and partners have been described (42), but so far no secretin has been shown to be self-transported to its final destination. In the present work we report on HxcQ liposecretin, the first example of a self-piloted secretin. Interestingly, we showed that the N-terminal lipid anchor of HxcQ which plays a crit- ical role in its biogenesis might compensate for the lack of specific partner and directly participate in the proper target- ing of HxcQ to the OM. Altogether our data reveal a new pathway for secretin transport. experimentally demonstrated to be lipoproteins (10–11, 24). The involvement of lipidation in secretin biogenesis was only tested for XpsD where this post translational fatty acylation turned out to be dispensable for secretin function (24). For BfpB and TcpC two small nonlipidated periplasmic proteins have been shown to be required for their stabilization and multim- erization respectively (25, 11). N-terminal lipidation plays a key role for HxcQ transport and no additional specific partner is required. We therefore suggest that among the liposecretins, HxcQ defines a distinctive subclass whose biogenesis is guided by a new and unprecedented transport pathway. Although the presence of a lipoprotein is often associated with secretin transport, the involvement of the Lol lipoprotein sorting pathway (44) in this process is still an open question. The discovery here that HxcQ is itself a lipoprotein might sug- gest that the Lol pathway is directly involved; however the Lol- dependent transport of HxcQ remains to be demonstrated. DISCUSSION HxcQnlV5 behaves as an integral IM protein since it remained insoluble upon treatment of the membranes with 100 mM sodium carbonate or 4 M urea (Fig. 6, lane 2 and 6), both known to solubilize only peripheral membrane proteins, such as XcpR. Secretins are an unusual and important class of bacterial OM protein involved in various membrane transport pathways such as T2SS and T3SS, type IV pili assembly, and export and assem- bly of filamentous phage. They form, in the OM, about 1 MDa DECEMBER 4, 2009•VOLUME 284•NUMBER 49 DECEMBER 4, 2009•VOLUME 284•NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 33821 Self-targeted HxcQ Liposecretin FIGURE 7. Membrane localization of HxcQV5 secretin in E. coli. Membrane fractions were collected after centrifugation on a density sucrose gradient and their protein content analyzed under denaturing SDS-PAGE conditions. HxcQV5 multimers and monomers were detected with HxcQ antibodies. IM fractions were defined by NADH activity and TolR detection. OM fractions were defined by the visual identification of E. coli OM porins on Coomassie Blue-stained gel. we propose that HxcQ liposecretin carries an intramolecular pilotin. In type II secretion, the fatty-acy- lated pilotin binds the C-terminal domain of the secretin (22) whereas in HxcQ liposecretin, the secretin is fatty-acylated at its extreme N ter- minus. The C terminus of a secretin is embedded in the OM and is there- fore well situated for interacting with a pilotin which is also anchored in the OM. The situation seems more conflicting for the N-terminal domain which needs some flexibil- ity to interact with other periplas- mic or inner membrane compo- nents (43). The extra glycine/ alanine/serine rich domain between the lipid anchor and the N-terminal domain that we identified in HxcQ (Fig. 2) could give to the N-terminal extremity the flexibility necessary for its function. It is interesting to note that this domain, absent in nonlipidated PulD and XcpQ T2SS secretins, is also present in XpsD (Fig. 2), another T2SS-lipidated secretin. FIGURE 7. Membrane localization of HxcQV5 secretin in E. coli. Membrane fractions were collected after centrifugation on a density sucrose gradient and their protein content analyzed under denaturing SDS-PAGE conditions. HxcQV5 multimers and monomers were detected with HxcQ antibodies. IM fractions were defined by NADH activity and TolR detection. OM fractions were defined by the visual identification of E. coli OM porins on Coomassie Blue-stained gel. HxcQ is the fourth secretin described to be a lipoprotein. REFERENCES J., Sch Donnenberg, M. S. (2006) Microbiology 152, 2405–2420 Donnenberg, M. S. (2006) Microbiology 152, 2405–2420 41. Guilvout, I., Chami, M., Engel, A., Pugsley, A. P., and Bayan, N. (2006) EMBO J. 25, 5241–5249 11. Bose, N., and Taylor, R. K. (2005) J. Bacteriol. 187, 2225–2232 12. Crago, A. M., and Koronakis, V. (1998) Mol. Microbiol. 30, 47–56 42. Bayan, N., Guilvout, I., and Pugsley, A. P. (2006) Mol. Microbiol. 60, 1–4 13. Chami, M., Guilvout, I., Gregorini, M., Re´migy, H. W., Mu¨ller, S. A., Vale- rio, M., Engel, A., Pugsley, A. P., and Bayan, N. (2005) J. Biol. Chem. 280, 37732–37741 43. Korotkov, K. V., Pardon, E., Steyaert, J., and Hol, W. G. (2009) Structure 17, 255–265 rio, M., Engel, A., Pugsley, A. P., and Bayan, N. (2005) J. Biol. Chem. 280, 37732–37741 44. Tokuda, H., and Matsuyama, S. (2004) Biochim. Biophys. Acta. 1693, 5–13 14. Koster, M., Bitter, W., de Cock, H., Allaoui, A., Cornelis, G. R., and Tom- massen, J. (1997) Mol. Microbiol. 26, 789–797 45. Collin, S., Guilvout, I., Chami, M., and Pugsley, A. P. (2007) Mol. Micro- biol. 64, 1350–1357 15. Genin, S., and Boucher, C. A. (1994) Mol. Gen. Genet. 243, 112–118 46. Herrero, M., de Lorenzo, V., and Timmis, K. N. (1990) J. Bacteriol. 172, 6557–6567 16. Bouley, J., Condemine, G., and Shevchik, V. E. (2001) J. Mol. Biol. 308, 205–219 47. de Groot, A., Koster, M., Ge´rard-Vincent, M., Gerritse, G., Lazdunski, A., 47. de Groot, A., Koster, M., Ge´rard-Vincent, M., Gerritse, G., Lazduns Tommassen, J., and Filloux, A. (2001) J. Bacteriol. 183, 959–967 17. Hardie, K. R., Seydel, A., Guilvout, I., and Pugsley, A. P. (1996) Mol. Mi- crobiol. 22, 967–976 Tommassen, J., and Filloux, A. (2001) J. Bacteriol. 183, 959–967 48. Kaniga, K., Delor, I., and Cornelis, G. R. (1991) Gene 109, 137–141 18. Hardie, K. R., Lory, S., and Pugsley, A. P. (1996) EMBO J. 15, 978–988 49. Newman, J. R., and Fuqua, C. (1999) Gene 227, 197–203 19. Shevchik, V. E., and Condemine, G. (1998) Microbiology 144, 3219–3228 50. Fu¨rste, J. P., Pansegrau, W., Frank, R., Blo¨cker, H., Scholz, P., Bagdasarian, M., and Lanka, E. (1986) Gene 48, 119–131 20. Schuch, R., and Maurelli, A. T. (2001) J. Bacteriol. 183, 6991–6998 21. Nudleman, E., Wall, D., and Kaiser, D. (2006) Mol. Microbiol. 60, 16–29 51. Osborn, H. J., Gander, J. E., Parisi, E., and Carson, J. (1972) J. Biol. DISCUSSION On the other hand and based on the broad diversity of secretin transport pathways it is also possible that certain secretins might follow an alternative Lol-independent pathway. This is particularly true when looking at XcpQ, another P. aeruginosa secretin. XcpQ is not a lipoprotein and so far, no cognate lipi- dated pilotin has yet been identified. The situation is also puz- zling regarding the implication of the Bam general OM protein assembly machinery in secretin transport (32). Bam depend- ence was demonstrated for PilQ secretin (32) but invalidated for PulD (45). It would therefore be interesting to experimen- As proposed earlier, the biogenesis of secretins sometimes requires special assistance conferred by pilotin lipoproteins (17–18, 19). In the case of the T2SS PulD/PulS secretin/pilotin pair, the pilotin binds to the secretin emerging from the IM translocon and either keeps it in a competent state, or prevents its non-productive aggregation, before its insertion into the OM. The pilotin may first maintain the secretin in its mono- meric form and, second, assist its transport through the periplasm (41). Here, we demonstrate that the HxcQ N-termi- nal lipid moiety functions as a pilotin since a nonlipidated ver- sion of HxcQ behaves like a secretin in the absence of its cog- nate pilotin i.e. multimers accumulation in the IM. Given that HxcQ does not need any additional partner for its biogenesis, 33822 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284•NUMBER 49•DECEMBER 4, 2009 33822 Self-targeted HxcQ Liposecretin tally test the Bam- and Lol-dependence of HxcQ in order to reveal the contribution of these systems to the biogenesis of this liposecretin. Lol dependence should also be tested for other secretins, although it will be difficult to discriminate between the requirement of secretin and pilotin for Lol. 23. Seo, J., Brencic, A., and Darwin, A. J. (2009) J. Bacteriol. 191, 898–908 24. Hu, N. T., Hung, M. N., Liao, C. T., and Lin, M. H. (1995) Microbiology 141, 1395–1406 25. Schmidt, S. A., Bieber, D., Ramer, S. W., Hwang, J., Wu, C. Y., and Schoolnik, G. (2001) J. Bacteriol. 183, 4848–4859 26. Sankaran, K., and Wu, H. C. (1994) J. Biol. Chem. 269, 19701–19706 26. Sankaran, K., and Wu, H. C. (1994) J. Biol. Chem. 269 27. Yamaguchi, K., Yu, F., and Inouye, M. (1988) Cell 53, 423–432 Acknowledgments—We thank Berenge`re Ize and Ben Field for careful reading of the manuscript; Jan Tommassen, Margot Koster, Steve Garvis, and Marc Despoints for helpful discussions; Elise Termine for Gateway cloning; Margot Koster for anti-XcpR antibody; Vincent Ore´al for assistance with sucrose density gradients and discussions; and Shunichi Miyakoshi (Sankyo, Japan) for the generous gift of globomycin. Acknowledgments—We thank Berenge`re Ize and Ben Field for careful reading of the manuscript; Jan Tommassen, Margot Koster, Steve Garvis, and Marc Despoints for helpful discussions; Elise Termine for Gateway cloning; Margot Koster for anti-XcpR antibody; Vincent Ore´al for assistance with sucrose density gradients and discussions; and Shunichi Miyakoshi (Sankyo, Japan) for the generous gift of globomycin. 28. Seydel, A., Gounon, P., and Pugsley, A. P. (1999) Mol. Microbiol. 34, 810–821 29. Ball, G., Durand, E., Lazdunski, A., and Filloux, A. (2002) Mol. Microbiol. 43, 475–485 30. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) in Molecular Cloning: A Laboratory Manual (Nolan, C., ed) 2nd Ed., pp. 1.2–1.110, C. S. H. L. Press, New York 31. Figurski, D. H., and Helinski, D. R. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 1648–1652 REFERENCES 32. Voulhoux, R., Bos, M. P., Geurtsen, J., Mols, M., and Tommassen, J. (2003) Science 299, 262–265 1. Filloux, A. (2004) Biochim. Biophys. Acta. 1694, 163–179 33. Lugtenberg, B., Meijers, J., Peters, R., van der Hoek, P., and van Alphen, L. (1975) FEBS Lett. 58, 254–258 2. Remaut, H., and Waksman, G. (2004) Curr. Opin. Struct. Biol. 14, 161–170 3 Pugsley A P (1993) Microbiol Rev 57 50 108 2. Remaut, H., and Waksman, G. (2004) Curr. Opin. Struct. Biol. 14, 161–170 3. Pugsley, A. P. (1993) Microbiol. Rev. 57, 50–108 34. Cascales, E., and Christie, P. J. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 17228–17233 4. Voulhoux, R., Ball, G., Ize, B., Vasil, M. L., Lazdunski, A., Wu, L. F., and Filloux, A. (2001) EMBO J. 20, 6735–6741 35. Linderoth, N. A., Model, P., and Russel, M. (1996) J. Bacteriol. 178, 1962–1970 5. Michel, G. P., and Voulhoux, R. (2009) Bacterial Secreted Proteins in Se- cretory Mechanisms and Role in Pathogenesis (Wooldridge, K., ed). pp. 67–92, Caister Academic Press, Nottingham 36. Shevchik, V. E., Robert-Baudouy, J., and Condemine, G. (1997) EMBO J. 16, 3007–3016 6. Bitter, W. (2003) Arch. Microbiol. 179, 307–314 7. Bitter, W., Koster, M., Latijnhouwers, M., de Cock, H., and Tommassen, J. (1998) Mol. Microbiol. 27, 209–219 37. Sohel, I., Puente, J. L., Ramer, S. W., Bieber, D., Wu, C. Y., and Schoolnik, G. K. (1996) J. Bacteriol. 178, 2613–2628 8. Burghout, P., Beckers, F., de Wit, E., van Boxtel, R., Cornelis, G. R., Tom- 8. Burghout, P., Beckers, F., de Wit, E., van Boxtel, R., Cornelis, G. R., Tom- massen, J., and Koster, M. (2004) J. Bacteriol. 186, 5366–5375 38. Madan Babu, M., and Sankaran, K. (2002) Bioinformatics 18, 641–643 massen, J., and Koster, M. (2004) J. Bacteriol. 186, 5366–5375 39. Hussain, M., Ichihara, S., and Mizushima, S. (1980) J. Biol. Chem. 255, 3707–3712 9. Collins, R. F., Frye, S. A., Kitmitto, A., Ford, R. C., Tønjum, T., and Derrick, J. P. (2004) J. Biol. Chem. 279, 39750–39756 Collins, R. F., Frye, S. A., Kitmitto, A., Ford, R. C., Tønjum, T. J. P. (2004) J. Biol. Chem. 279, 39750–39756 40. Michel, G. P., Durand, E., and Filloux, A. (2007) J. Bacteriol. 189, 3776–3783 10. Daniel, A., Singh, A., Crowther, L. J., Fernandes, P. J., Schr Donnenberg, M. S. (2006) Microbiology 152, 2405–2420 10. Daniel, A., Singh, A., Crowther, L. J., Fernandes, P. REFERENCES Chem. 247, 3962–3972 22. Daefler, S., Guilvout, I., Hardie, K. R., Pugsley, A. P., and Russel, M. (1997) Mol. Microbiol. 24, 465–475 DECEMBER 4, 2009•VOLUME 284•NUMBER 49 DECEMBER 4, 2009•VOLUME 284•NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 33823
https://openalex.org/W3035856320	https://europepmc.org/articles/pmc7302433?pdf=render	English	null	Intermittent pain of the pelvis in a Syrian woman	PLoS neglected tropical diseases	2,020	cc-by	1,812	Benjamin T. SchleenvoigtID1, Bernhard Theis2, Michaela Wu¨st3, Christina Forstner1,4, Mathias W. Pletz1, Stefan HagelID1 1 Institute for Infectious Diseases and Infection Control, Jena University Hospital, Jena, Germany, 2 Section Pathology of the Institute of Forensic Medicine, Jena University Hospital, Jena, Germany, 3 Department of Internal Medicine IV (Gastroenterology, Hepatology, and Infectious Diseases), Jena University Hospital, Jena, Germany, 4 Department of Medicine I, Division of Infectious Diseases and Tropical Medicine, Medical University of Vienna, Vienna, Austria benjamin.schleenvoigt@med.uni-jena.de OPEN ACCESS Citation: Schleenvoigt BT, Theis B, Wu¨st M, Forstner C, Pletz MW, Hagel S (2020) Intermittent pain of the pelvis in a Syrian woman. PLoS Negl Trop Dis 14(6): e0008183. https://doi.org/10.1371/ journal.pntd.0008183 Citation: Schleenvoigt BT, Theis B, Wu¨st M, Forstner C, Pletz MW, Hagel S (2020) Intermittent pain of the pelvis in a Syrian woman. PLoS Negl Trop Dis 14(6): e0008183. https://doi.org/10.1371/ journal.pntd.0008183 PLOS NEGLECTED TROPICAL DISEASES PLOS NEGLECTED TROPICAL DISEASES a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Case discussion Echinococcosis is a neglected tropical disease recognized by the World Health Organization (WHO). Cystic Echinococcosis (CE) is caused by metacestodes—the larval stage of the species E. granulosus. Adult parasites live in the intestine of the definitive host. These are dogs and other carnivores. The intermediate hosts—usually sheep and other herbivores—get infected by oral intake of the eggs. The human is an intermediate incidental host who can harbor the vesic- ular metacestode in any anatomical site. The liver is the most frequent location of the fluid filled cystic lesions (70%), followed by the lungs (20%). The central nervous system is affected in 3%. Osseous echinococcosis is a rare disease (1% to 4%). It can involve any bone, but has a predilection for the vertebral column (45%), pelvis (14%), and femur (10%) [1,2]. Multiosseous infestation and concomitant soft tissue or organ involvement are frequent, particularly the combination of proximal femur involvement with the pelvic girdle. In a small study from Kazakhstan including 8 patients with CE of the bone, the pelvis was the predominant localiza- tion. In all cases, malignancy was initially suspected [3]. Editor: Zvi Bentwich, Ben-Gurion University of the Negev, ISRAEL Presentation of case A 66-year-old immunocompetent woman from Syria presented with a two-months history of intermittent pain of the right pelvis to the Jena University Hospital (Germany). Fever, night sweats and weight loss were denied. An MRI scan of the pelvis showed marked destruction of os ilium and os sacrum and a sinus tract draining in multiple subcutaneous cystic lesions (Fig 1 and Fig 2). Erythrocyte sedimentation rate was slightly elevated (30 mm/h), and C-reactive protein and white blood cell count were normal. There was no eosinophilia or Immunoglobu- lin E (IgE) elevation. To establish diagnosis, a bone biopsy was performed. Histopathology showed loose edematous connective tissue, a mixed inflammatory infiltrate, and regressive cal- cifications. PAS-positive cuticular components with typical lamellar layering, consistent with echinococcosis were detectable. Furthermore, wall fragments of cystic lesions were visible (Fig 3 and Fig 4). Immunohistochemistry directed against specific antigens of Echinococcus granu- losus and E. multilocularis confirmed the former as causative agent. Due to the localization and extensive bone destruction, no surgical excision was possible. Consequently, empirical treat- ment for tuberculosis was stopped, and therapy with albendazole (400 mg twice daily) was ini- tiated for an indefinite period. SYMPOSIUM Intermittent pain of the pelvis in a Syrian woman Benjamin T. SchleenvoigtID1*, Bernhard Theis2, Michaela Wu¨st3, Christina Forstner1,4, Mathias W. Pletz1, Stefan HagelID1 Published: June 18, 2020 Copyright: © 2020 Schleenvoigt et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors received no specific funding for this work. Competing interests: The authors have no conflicts of interest to declare. Competing interests: The authors have no conflicts of interest to declare. 1 / 5 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0008183 June 18, 2020 PLOS NEGLECTED TROPICAL DISEASES Fig 1. Cystic formation with intra- and extrapelvic parts as well as migration of the right Ala ossis ilii and sacroiliac joint (T1 post-contrast agent). Image credit: S. Gießler. T1, T1-weighted imaging https://doi org/10 1371/journal pntd 0008183 g001 Fig 1. Cystic formation with intra- and extrapelvic parts as well as migration of the right Ala ossis ilii and sacroiliac joint (T1 post-contrast agent). Image credit: S. Gießler. T1, T1-weighted imaging https://doi.org/10.1371/journal.pntd.0008183.g001 Fig 2. Cystic formation with intra- and extrapelvic parts as well as migration of the right Ala ossis ilii and sacroiliac joint (T2). Image credit: S. Gießler. T2, T2-weighted imaging https://doi.org/10.1371/journal.pntd.0008183.g002 Fig 2. Cystic formation with intra- and extrapelvic parts as well as migration of the right Ala ossis ilii and sacroiliac joint (T2). Image credit: S. Gießler. T2, T2-weighted imaging Fig 2. Cystic formation with intra- and extrapelvic parts as well as migration of the right Ala ossis ilii and sacroiliac joint (T2). Image credit: S. Gießler. T2, T2-weighted imaging https://doi.org/10.1371/journal.pntd.0008183.g002 https://doi.org/10.1371/journal.pntd.0008183.g002 2 / 5 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0008183 June 18, 2020 PLOS NEGLECTED TROPICAL DISEASES Fig 3. Numerous fragments of the hydatid cyst wall at low magnification, 25x (HE stain). Image credit: B. Theis. HE, hematoxylin–eosin Fig 3. Numerous fragments of the hydatid cyst wall at low magnification, 25x (HE stain). Image credit: B. Theis. HE, hematoxylin–eosin https://doi.org/10.1371/journal.pntd.0008183.g003 The prevalence of CE in the population of migrants from Middle East countries is not known. Diagnoses relies on imaging, in addition to serology, and occasionally microscopic Fig 4. Characteristic lamellar membranous structure of the hydatid cyst wall, 400x (PAS stain). Neither a well- defined germinal epithelium, nor protoscolices were observed, which may indicate an infertile state of the hydatid cyst. Image credit: B. Theis. PAS, Periodic acid–Schiff. https://doi.org/10.1371/journal.pntd.0008183.g004 Fig 4. Characteristic lamellar membranous structure of the hydatid cyst wall, 400x (PAS stain). Published: June 18, 2020 Neither a well- defined germinal epithelium, nor protoscolices were observed, which may indicate an infertile state of the hydatid cyst. Image credit: B. Theis. PAS, Periodic acid–Schiff. https://doi.org/10.1371/journal.pntd.0008183.g004 https://doi.org/10.1371/journal.pntd.0008183.g004 3 / 5 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0008183 June 18, 2020 PLOS NEGLECTED TROPICAL DISEASES identification of tissue samples. Ultrasound is the cornerstone for diagnosis, staging, and fol- low-up. It can be supplemented by computed tomography (CT) and MRI. When ultrasound is unavailable, MRI should be the preferred cut imaging [4]. Diagnosis is often protracted as CE of the bone lacks a typical clinical appearance and image characteristics on X-ray or CT scan are similar to those of tuberculosis, metastases, and giant cell tumors or bone cysts. Complete surgical excision of the lesions combined with albendazole chemotherapy are the standard of care with a 17% risk of recurrence [5]. The dual therapeutic combination of albendazole with praziquantel seems to be more effective than albendazole alone and is occasionally used for therapy of CE [6]. In most cases, complete surgical intervention is not possible and thus the prognosis is very poor. Frequent sequels of the highly destructive parasitic process are chronic pain, pseudarthrosis, fractures, and death [5, 7, 8]. Physicians should be aware of this rare differential diagnosis in cystic bone lesions, espe- cially in migrating populations. Key learning points • CE is an important differential diagnosis of cystic bone lesions, especially in migrating populations. • CE can involve any bone, but has a predilection for the vertebral column (45%), pelvis (14%), and femur (10%). • In histopathology, PAS-positive cuticular components with typical lamellar layering of the hydatid cyst can be observed in CE. • If possible, surgical excision of the lesions combined with albendazole chemotherapy are standard of care for CE of the bone. References References 1. Agudelo Higuita NI, Brunetti E, McCloskey C. Cystic Echinococcosis. J Clin Microbiol. 2016; 54(3):518– 23. https://doi.org/10.1128/JCM.02420-15 PMID: 26677245; PubMed Central PMCID: PMC4767951. 2. Neumayr A, Tamarozzi F, Goblirsch S, Blum J, Brunetti E. Spinal cystic echinococcosis—a systematic analysis and review of the literature: part 2. Treatment, follow-up and outcome. PLoS Negl Trop Dis. 2013; 7(9):e2458. https://doi.org/10.1371/journal.pntd.0002458 PMID: 24069501; PubMed Central PMCID: PMC3777903. 3. Manciulli T, Mustapayeva A, Juszkiewicz K, Sokolenko E, Maulenov Z, Vola A, et al. Cystic Echinococ- cosis of the Bone in Kazakhstan. Case Rep Infect Dis. 2018; 2018:9682508. https://doi.org/10.1155/ 2018/9682508 PMID: 30319824; PubMed Central PMCID: PMC6167588. 4. Stojkovic M, Rosenberger K, Kauczor HU, Junghanss T, Hosch W. Diagnosing and staging of cystic echinococcosis: how do CT and MRI perform in comparison to ultrasound? PLoS Negl Trop Dis. 2012; 6(10):e1880. https://doi.org/10.1371/journal.pntd.0001880 PMID: 23145199; PubMed Central PMCID: PMC3493391. 5. Steinmetz S, Racloz G, Stern R, Dominguez D, Al-Mayahi M, Schibler M, et al. Treatment challenges associated with bone echinococcosis. J Antimicrob Chemother. 2014; 69(3):821–6. https://doi.org/10. 1093/jac/dkt429 PMID: 24222611. 6. Velasco-Tirado V, Alonso-Sardon M, Lopez-Bernus A, Romero-Alegria A, Burguillo FJ, Muro A, et al. Medical treatment of cystic echinococcosis: systematic review and meta-analysis. BMC Infect Dis. 1. Agudelo Higuita NI, Brunetti E, McCloskey C. Cystic Echinococcosis. J Clin Microbiol. 2016; 54(3):518– 23. https://doi.org/10.1128/JCM.02420-15 PMID: 26677245; PubMed Central PMCID: PMC4767951. 2. Neumayr A, Tamarozzi F, Goblirsch S, Blum J, Brunetti E. Spinal cystic echinococcosis—a systematic analysis and review of the literature: part 2. Treatment, follow-up and outcome. PLoS Negl Trop Dis. 2013; 7(9):e2458. https://doi.org/10.1371/journal.pntd.0002458 PMID: 24069501; PubMed Central PMCID: PMC3777903. 3. Manciulli T, Mustapayeva A, Juszkiewicz K, Sokolenko E, Maulenov Z, Vola A, et al. Cystic Echinococ- cosis of the Bone in Kazakhstan. Case Rep Infect Dis. 2018; 2018:9682508. https://doi.org/10.1155/ 2018/9682508 PMID: 30319824; PubMed Central PMCID: PMC6167588. 4. Stojkovic M, Rosenberger K, Kauczor HU, Junghanss T, Hosch W. Diagnosing and staging of cystic echinococcosis: how do CT and MRI perform in comparison to ultrasound? PLoS Negl Trop Dis. 2012; 6(10):e1880. https://doi.org/10.1371/journal.pntd.0001880 PMID: 23145199; PubMed Central PMCID: PMC3493391. 5. Steinmetz S, Racloz G, Stern R, Dominguez D, Al-Mayahi M, Schibler M, et al. Treatment challenges associated with bone echinococcosis. J Antimicrob Chemother. 2014; 69(3):821–6. https://doi.org/10. 1093/jac/dkt429 PMID: 24222611. 6. Velasco-Tirado V, Alonso-Sardon M, Lopez-Bernus A, Romero-Alegria A, Burguillo FJ, Muro A, et al. Medical treatment of cystic echinococcosis: systematic review and meta-analysis. BMC Infect Dis. PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0008183 June 18, 2020 2018; 18(1):306. https://doi.org/10.1186/s12879-018-3201-y PMID: 29976137; PubMed Central PMCID: PMC6034244. 7. Cattaneo L, Manciulli T, Cretu CM, Giordani MT, Angheben A, Bartoloni A, et al. Cystic Echinococcosis of the Bone: A European Multicenter Study. Am J Trop Med Hyg. 2019; 100(3):617–21. https://doi.org/ 10.4269/ajtmh.18-0758 PMID: 30693857; PubMed Central PMCID: PMC6402919. 8. Monge-Maillo B, Chamorro Tojeiro S, Lopez-Velez R. Management of osseous cystic echinococcosis. Expert Rev Anti Infect Ther. 2017; 15(12):1075–82. https://doi.org/10.1080/14787210.2017.1401466 PMID: 29110551. 8. Monge-Maillo B, Chamorro Tojeiro S, Lopez-Velez R. Management of osseous cystic echinococcosis. Expert Rev Anti Infect Ther. 2017; 15(12):1075–82. https://doi.org/10.1080/14787210.2017.1401466 PMID: 29110551. References 4 / 5 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0008183 June 18, 2020 PLOS NEGLECTED TROPICAL DISEASES 2018; 18(1):306. https://doi.org/10.1186/s12879-018-3201-y PMID: 29976137; PubMed Central PMCID: PMC6034244. 8. Monge-Maillo B, Chamorro Tojeiro S, Lopez-Velez R. Management of osseous cystic echinococcosis. Expert Rev Anti Infect Ther. 2017; 15(12):1075–82. https://doi.org/10.1080/14787210.2017.1401466 PMID: 29110551. 5 / 5
https://openalex.org/W2789236759	https://europepmc.org/articles/pmc5852478?pdf=render	English	null	Microbial Degradation of Amino Acid-Containing Compounds Using the Microcystin-Degrading Bacterial Strain B-9	Marine drugs	2,018	cc-by	7,399	Microbial Degradation of Amino Acid-Containing Compounds Using the Microcystin-Degrading Bacterial Strain B 9 Haiyan Jin 1,, Yoshiko Hiraoka 2, Yurie Okuma 2, Elisabete Hiromi Hashimoto 2, Miki Kurita 2, Andrea Roxanne J. Anas 2, Hitoshi Uemura 3, Kiyomi Tsuji 3 and Ken-Ichi Harada 1,2, yan Jin 1,, Yoshiko Hiraoka 2, Yurie Okuma 2, Elisabete Hiromi Hashimoto 2, Miki Kurita 2, drea Roxanne J. Anas 2, Hitoshi Uemura 3, Kiyomi Tsuji 3 and Ken-Ichi Harada 1,2, j y p g y p 2 Faculty of Pharmacy, Meijo University, Tempaku, Nagoya 468-8503, Japan; y715o.c5@gmail.com (Y.H.); g0773315@ccalumni.meijo-u.ac.jp (Y.O.); elisabete.utfpr@gmail.com (E.H.H.); g0673319@ccalumni.meijo-u.ac.jp (M.K.); anasaroj@meijo-u.ac.jp (A.R.J.A.) 3 g j jp ( ); j j jp ( J ) 3 Kanagawa Prefectural Institute of Public Health, Shimomachiya, Chigasaki, Kanagawa 253-0087, Ja uemura.aklt@pref.kanagawa.jp (H.U.); tsuji.df7@pref.kanagawa.jp (K.T.) p g jp j p g jp * Correspondence: 163891501@ccalumni.meijo-u.ac.jp (H.J.); kiharada@meijo-u.ac.jp (K.-I.H.); Tel./Fax: +81-52-839-2720 (H.J.); +81-52-834-8780 (K.-I.H.) Received: 25 November 2017; Accepted: 30 January 2018; Published: 6 February 2018 Received: 25 November 2017; Accepted: 30 January 2018; Published: 6 February 2018 Abstract: Strain B-9, which has a 99% similarity to Sphingosinicella microcystinivorans strain Y2, is a Gram-negative bacterium with potential for use in the degradation of microcystin-related compounds and nodularin. We attempted to extend the application area of strain B-9 and applied it to mycotoxins produced by fungi. Among the tested mycotoxins, only ochratoxin A was completely hydrolyzed to provide the constituents ochratoxin α and L-phenylalanine, and levels of fumonisin B1 gradually decreased after 96 h. However, although drugs including antibiotics released into the aquatic environment were applied for microbial degradation using strain B-9, no degradation occurred. These results suggest that strain B-9 can only degrade amino acid-containing compounds. As expected, the tested compounds with amide and ester bonds, such as 3,4-dimethyl hippuric acid and 4-benzyl aspartate, were readily hydrolyzed by strain B-9, although the sulfonamides remained unchanged. The ester compounds were characteristically and rapidly hydrolyzed as soon as they came into contact with strain B-9. Furthermore, the degradation of amide and ester compounds with amino acids was not inhibited by the addition of ethylenediaminetetraacetic acid (EDTA), indicating that the responsible enzyme was not MlrC. These results suggest that strain B-9 possesses an additional hydrolytic enzyme that should be designated as MlrE, as well as an esterase. Keywords: microcystin-degrading bacteria; mycotoxin; protease; esterase; inhibitor marine drugs marine drugs Article 1. Introduction Microcystins (MCs) are typical compounds produced by cyanobacteria, such as Microcystis, Anabaena, and Planktothrix [1]. They are cyclic heptapeptides showing potent hepatotoxicity and tumor-promoting activity [1]. In the environment, there are many bacteria which work to degrade such hazardous and harmful compounds. The ﬁrst MC-degrading bacterium was isolated and identiﬁed as a Sphingomonas strain (ACM-3962) in 1994 [2]. Similar bacteria capable of degrading MC were reported by Dziga et al. [3]. As per their review [3], many MC-degrading microorganisms have been found and identiﬁed, and the corresponding genetic aspects with respect to MC degradation have been studied. However, in related published papers, no substrates other than MCs have been applied [4]. The purpose of the present study is to elucidate the inherent function and role of MC-degrading microorganisms in the aquatic environment. Mar. Drugs 2018, 16, 50; doi:10.3390/md16020050 www.mdpi.com/journal/marinedrugs www.mdpi.com/journal/marinedrugs 2 of 11 Mar. Drugs 2018, 16, 50 Mar. Drugs 2018, 16, 50 Strain B-9, isolated from Lake Tsukui, Japan, exhibits degradation activity against MCs [1]. This strain belongs to the genus Sphingosinecella sp., and, based on the 16S rDNA sequence (GenBank accession no. AB084247), has 99% similarity to Sphingosinecella microcystinivorans strain Y2, a type of MC-degrading bacteria [5]. The Sphingomonas sp. strain ACM-3962 [2] was the ﬁrst strain reported to degrade MCs. The cloning and molecular characterization of four genes from strain ACM-3962 revealed the presence of three hydrolytic enzymes (MlrA, MlrB, and MlrC), together with a putative oligopeptide transporter (MlrD) [6,7]. The three hydrolytic enzymes were putatively characterized as metalloproteases (MlrA and MlrC) or serine proteases (MlrB). The microcystinase MlrA catalyzes the initial ring opening of microcystin-LR (MC-LR) at the (2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4(E),6(E)-dienoic acid (Adda)-Arg peptide bond to give linearized MC-LR (Adda-Glu-Mdha-Ala-Leu-β-MeAsp-Arg). This further degrades to Adda-Glu-Mdha-Ala by MlrB, and the third enzyme, MlrC, hydrolyzes the tetrapeptide into smaller peptides and amino acids [6–8]. In terms of advancements, recombinant MlrA and MlrC have been prepared, and the degradation scheme has been almost completely veriﬁed [9,10]. The use of typical protease inhibitors, such as ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline, results in the accumulation of linear MC-LR and the tetrapeptide, which allows for the classiﬁcation of the enzymes MlrA and MlrC as metalloproteases, [6,7]. Meanwhile, phenylmethylsulfonyl ﬂuoride (PMSF) characterizes MlrB as a possible serine protease [6,7]. 1. Introduction We extended the area of application of strain B-9 for bioremediation and applied it to the secondary fungal metabolites of mycotoxins that may have mutagenic, carcinogenic, cytotoxic, and endocrine-disrupting effects. These substances frequently contaminate agricultural commodities despite efforts for prevention, so successful detoxiﬁcation methods are needed. The application of microorganisms to degrade mycotoxins is a possible strategy that shows potential for example in food and feed processing [11]; in antibiotics used in human and veterinary medicine (which can enter the environment via wastewater treatment plant efﬂuents, hospitals, and processing plant efﬂuents); in the application of agricultural waste and biosolids to ﬁelds; and in the case of leakage from waste-storage containers and landﬁlls [12,13]. Such antibiotic pollution may facilitate the development and spread of antibiotic resistance [4]. Strain B-9 degrades MC-LR, the most toxic of the MCs, within 24 h [14,15]. After the discovery of strain B-9, we advanced our research on the degradation of the following compounds: non-toxic cyanobacterial cyclic peptides that are structurally different from MCs [16]; representative cyclic peptides (antibiotics) produced by bacteria [17]; physiologically-active cardiovascular and neuropeptides [18]; and the glucagon/vasoactive intestinal polypeptide (VIP) family of peptides [19]. The aforementioned experiments [17–19] confirmed that strain B-9 could degrade the tested peptides completely. During the application of strain B-9 to remove mycotoxins and drugs released in the aquatic environment, we obtained interesting results concerning the function of this strain. The purpose of this study was to demonstrate the additional hydrolytic enzymes (such as protease and/or esterase) of strain B-9. 2.1. Mycotoxin and Drugs with Amide, Ester, and Sulfonamide Bonds 2.1. Mycotoxin and Drugs with Amide, Ester, and Sulfonamide Bonds As already mentioned [14,15], strain B-9 can degrade the phycotoxins microcystin and nodularin, as well as non-toxic cyclic peptides and linear peptides. In this study, we applied strain B-9 to mycotoxins. We monitored the degradation behavior of the ﬁve mycotoxins using HPLC and LC/MS. No degradation was observed in zearalenone, deoxynivalenol, or patulin, while the peak of ochratoxin A completely disappeared (Figure 1). The peak of fumonisin B1 reduced to a certain extent after 96 h (Figure S1). Figure 1 shows (a) HPLC chromatograms; (b) total ion chromatograms, and selected ion monitoring (SIM) (c) at m/z 166.1 and (d) at m/z 257.0 of the reaction products of ochratoxin A by microbial degradation using strain B-9 at 0 h. Although ochratoxin A appeared at 18.7 min in the HPLC chromatogram, such a peak was not observed in the data at 0 h (Figure 1A). It was found that the peak 3 of 11 Mar. Drugs 2018, 16, 50 at 20.28 min was derived from strain B-9 itself by comparison of the chromatograms of ochratoxin A and a mixture of the ochratoxin A and strain B-9 broth. Consequently, the peaks at 3.66 and 13.78 min were derived from ochratoxin A, which corresponded to phenylalanine and ochratoxin α, respectively (Figure 2). After 96 h, the latter was still observed, whereas the presence of the former was signiﬁcantly reduced (Figure 1B). Mar. Drugs 2018, 16, x FOR PEER REVIEW 3 of 11 chromatograms of ochratoxin A and a mixture of the ochratoxin A and strain B-9 broth. Consequently, the peaks at 3.66 and 13.78 min were derived from ochratoxin A, which corresponded to phenylalanine and ochratoxin α, respectively (Figure 2). After 96 h, the latter was still observed, whereas the presence of the former was significantly reduced (Figure 1B). Mar. Drugs 2018, 16, x FOR PEER REVIEW 3 of 11 chromatograms of ochratoxin A and a mixture of the ochratoxin A and strain B-9 broth. Consequently, the peaks at 3.66 and 13.78 min were derived from ochratoxin A, which corresponded to phenylalanine and ochratoxin α, respectively (Figure 2). After 96 h, the latter was still observed, whereas the presence of the former was significantly reduced (Figure 1B). Figure 1. Degradation behavior of ochratoxin A at (A) 0 h and (B) 96 h after microbial degradation by strain B-9. 2.1. Mycotoxin and Drugs with Amide, Ester, and Sulfonamide Bonds (a) HPLC chromatograms; (b) total ion chromatograms, and selected ion monitoring (SIM) (c) at m/z 166.1 and (d) m/z 257.0 of the reaction products of ochratoxin A. Figure 1. Degradation behavior of ochratoxin A at (A) 0 h and (B) 96 h after microbial degradation by strain B-9. (a) HPLC chromatograms; (b) total ion chromatograms, and selected ion monitoring (SIM) (c) at m/z 166.1 and (d) m/z 257.0 of the reaction products of ochratoxin A. Figure 1. Degradation behavior of ochratoxin A at (A) 0 h and (B) 96 h after microbial degradation by strain B-9. (a) HPLC chromatograms; (b) total ion chromatograms, and selected ion monitoring (SIM) (c) at m/z 166.1 and (d) m/z 257.0 of the reaction products of ochratoxin A. Figure 1. Degradation behavior of ochratoxin A at (A) 0 h and (B) 96 h after microbial degradation by strain B-9. (a) HPLC chromatograms; (b) total ion chromatograms, and selected ion monitoring (SIM) (c) at m/z 166.1 and (d) m/z 257.0 of the reaction products of ochratoxin A. Figure 1. Degradation behavior of ochratoxin A at (A) 0 h and (B) 96 h after microbial degradation by strain B-9. (a) HPLC chromatograms; (b) total ion chromatograms, and selected ion monitoring (SIM) (c) at m/z 166.1 and (d) m/z 257.0 of the reaction products of ochratoxin A. Figure 1. Degradation behavior of ochratoxin A at (A) 0 h and (B) 96 h after microbial degradation by strain B-9. (a) HPLC chromatograms; (b) total ion chromatograms, and selected ion monitoring (SIM) (c) at m/z 166.1 and (d) m/z 257.0 of the reaction products of ochratoxin A. Figure 2. Microbial degradation of ochratoxin A (molecular weight (MW): 403.0) using strain B-9 to provide phenylalanine (MW: 165.1) and ochratoxin α (MW: 256.0). Figure 2. Microbial degradation of ochratoxin A (molecular weight (MW): 403.0) using strain B-9 to provide phenylalanine (MW: 165.1) and ochratoxin α (MW: 256.0). Figure 2. Microbial degradation of ochratoxin A (molecular weight (MW): 403.0) using strain B-9 to provide phenylalanine (MW: 165.1) and ochratoxin α (MW: 256.0). Figure 2. Microbial degradation of ochratoxin A (molecular weight (MW): 403.0) using strain B-9 to provide phenylalanine (MW: 165.1) and ochratoxin α (MW: 256.0). Figure 2. Microbial degradation of ochratoxin A (molecular weight (MW): 403.0) using strain B-9 to provide phenylalanine (MW: 165.1) and ochratoxin α (MW: 256.0). Figure 2. 2.2. Amino Acid-Containing Compounds (Amides, Esters, and Sulfonamides) amino acid containing compounds. The following commercially available amino acid-containing compounds with different bonds were selected. Amides: 3,4-dimethylhippuric acid, D- and L-N-acetylphenylalanine, N-carboben zoxy-L-phenylalanine-L-phenylalanine, and L-leucine-2-naphthylamide; esters: L-serine benzyl ester, and 4-benzyl L-aspartate; and sulfonamides: N-(p-toluenesulfonyl)-L-phenylalanine, and N-(1-naphthalenesulfonyl)-L-phenylalanine (Figure 3). L-Leucine-2-naphthylamide was treated in the same manner as the mycotoxins. The degradation proceeded smoothly and the starting material peak disappeared within 3 h (Figure 4a). A new peak was formed in the HPLC chromatogram. LC/MS showed that the starting material peak appeared after 18.13 min and the new peaks at 2.63 and 9.41 min were detected at 3 h (Figure 5). These peaks were determined to be 2-naphthylamine and leucine by selected ion chromatograms (SIM) at m/z 144.1 and m/z 132.03, respectively. The results indicated that L-leucine-2-naphthylamide was subjected to microbial degradation using strain B-9 to provide 2-naphthyl amine and leucine. As shown in Figure 4, the remaining amide compounds were also degraded and characteristic degradation behavior was observed. In addition, 3,4-dimethylhippuric acid was similarly degraded in the case of L-leucine-2-naphthylamide (Figure 4b). While N-acetyl-L-phenylalanine disappeared within 24 h, the D-amino acid derivative continued to appear at 96 h (Figure 4c,d). In the case of N-carbobenzoxy-L-phenylalanine-L-phenylalanine, the starting material peak disappeared as soon as it came into contact with strain B-9 (Figure 4e). 2.2. Amino Acid-Containing Compounds (Amides, Esters, and Sulfonamides) The following commercially available amino acid-containing compounds with different bonds were selected. Amides: 3,4-dimethylhippuric acid, D- and L-N-acetylphenylalanine, N-carboben zoxy-L-phenylalanine-L-phenylalanine, and L-leucine-2-naphthylamide; esters: L-serine benzyl ester, and 4-benzyl L-aspartate; and sulfonamides: N-(p-toluenesulfonyl)-L-phenylalanine, and N-(1-naphthalenesulfonyl)-L-phenylalanine (Figure 3). L-Leucine-2-naphthylamide was treated in the same manner as the mycotoxins. The degradation proceeded smoothly and the starting material peak disappeared within 3 h (Figure 4a). A new peak was formed in the HPLC chromatogram. LC/MS showed that the starting material peak appeared after 18.13 min and the new peaks at 2.63 and 9.41 min were detected at 3 h (Figure 5). These peaks were determined to be 2-naphthylamine and leucine by selected ion chromatograms (SIM) at m/z 144.1 and m/z 132.03, respectively. The results indicated that L-leucine-2-naphthylamide was subjected to microbial degradation using strain B-9 to provide 2-naphthyl amine and leucine. As shown in Figure 4, the remaining amide compounds were also degraded and characteristic degradation behavior was observed. In addition, 3,4-dimethylhippuric acid was similarly degraded in the case of L-leucine-2-naphthylamide (Figure 4b). 2.1. Mycotoxin and Drugs with Amide, Ester, and Sulfonamide Bonds Microbial degradation of ochratoxin A (molecular weight (MW): 403.0) using strain B-9 to provide phenylalanine (MW: 165.1) and ochratoxin α (MW: 256.0). We tried to degrade drugs including antibiotics released in the aquatic environment using strain B-9 and selected the following drugs with amide, ester, and sulfonamide bonds: oxytetracycline (OTC), sulphaminomethoxime, sulfadimethoxime, oseltamivir, crotamiton, N,N-diethyl-m-toluamide, We tried to degrade drugs including antibiotics released in the aquatic environment using strain B-9 and selected the following drugs with amide, ester, and sulfonamide bonds: oxytetracycline (OTC), sulphaminomethoxime, sulfadimethoxime, oseltamivir, crotamiton, N,N-diethyl-m-toluamide, We tried to degrade drugs including antibiotics released in the aquatic environment using strain B-9 and selected the following drugs with amide, ester, and sulfonamide bonds: oxytetracycline (OTC), 4 of 11 Mar. Drugs 2018, 16, 50 sulphaminomethoxime, sulfadimethoxime, oseltamivir, crotamiton, N,N-diethyl-m-toluamide, and acetaminophen. Although these were treated in the same manner as the mycotoxins, no degradation was observed (data not shown). These results suggested that strain B-9 can degrade only amino acid-containing compounds. Mar. Drugs 2018, 16, x FOR PEER REVIEW 4 of 11 degradation was observed (data not shown). These results suggested that strain B-9 can degrade only 2.2. Amino Acid-Containing Compounds (Amides, Esters, and Sulfonamides) amino acid containing compounds. While N-acetyl-L-phenylalanine disappeared within 24 h, the D-amino acid derivative continued to appear at 96 h (Figure 4c,d). In the case of N-carbobenzoxy-L-phenylalanine-L-phenylalanine, the starting material peak disappeared as soon as it came into contact with strain B-9 (Figure 4e). 3,4-Dimethylhippuric acid N-Acetyl-D-phenylalanine N-Acetyl-L-phenylalanine N-Carbobenzoxy-L-phenylalanyl- L-phenylalanine L-Leucine-2-naphthylamide N-(p-Toluenesulfonyl)-L-phenylalanine L-Serine benzyl ester 4-Benzyl L-aspartate N-(1-Naphthalenesulfonyl)-L-phenylalanine Figure 3. Structures of the tested amino acid-containing compounds. Amides: 3,4-dimethylhip puric acid, D- and L-N-acetylphenylalanines, N-carbobenzoxy-L-phenylalanine-L-phenylalanine, and L-leucine-2-naphthylamide; esters: serine benzyl ester, and 4-benzyl aspartate; and sulfonamides: N-(1-naphthalenesulfonyl)-phenylalanine, and N-(p-toluenesulfonyl)-L-phenylalanine. H N HO O O OH HN O O OH HN O O O N H NH HO O O O H N O NH2 HCl HO H N S O O O O OH O NH2 O OH O O NH2 HO H N S O O O Figure 3. Structures of the tested amino acid-containing compounds. Amides: 3,4-dimethylhip puric acid, D- and L-N-acetylphenylalanines, N-carbobenzoxy-L-phenylalanine-L-phenylalanine, and L-leucine-2-naphthylamide; esters: serine benzyl ester, and 4-benzyl aspartate; and sulfonamides: N-(1-naphthalenesulfonyl)-phenylalanine, and N-(p-toluenesulfonyl)-L-phenylalanine. N-Acetyl-L-phenylalanine OH HN O O N-Acetyl-D-phenylalanine OH HN O O 3,4-Dimethylhippuric acid H N HO O O 3,4-Dimethylhippuric acid N-Acetyl-L-phenylalanine N-Acetyl-D-phenylalanine N-Carbobenzoxy-L-phenylalanyl- L-phenylalanine O N H NH HO O O O L-Leucine-2-naphthylamide H N O NH2 HCl HO H N S O O O HCl N-Carbobenzoxy-L-phenylalanyl- L-phenylalanine N-(p-Toluenesulfonyl)-L-phenylalanine L-Leucine-2-naphthylamide y p y L-phenylalanine N-(1-Naphthalenesulfonyl)-L-phenylalanine HO H N S O O O 4-Benzyl L-aspartate O OH O O NH2 L-Serine benzyl ester O OH O NH2 4-Benzyl L-aspartate L-Serine benzyl ester N-(1-Naphthalenesulfonyl)-L-phenylalanine O O Figure 3. Structures of the tested amino acid-containing compounds. Amides: 3,4-dimethylhip puric acid, D- and L-N-acetylphenylalanines, N-carbobenzoxy-L-phenylalanine-L-phenylalanine, and L-leucine-2-naphthylamide; esters: serine benzyl ester, and 4-benzyl aspartate; and sulfonamides: N-(1-naphthalenesulfonyl)-phenylalanine, and N-(p-toluenesulfonyl)-L-phenylalanine. Figure 3. Structures of the tested amino acid-containing compounds. Amides: 3,4-dimethylhip puric acid, D- and L-N-acetylphenylalanines, N-carbobenzoxy-L-phenylalanine-L-phenylalanine, and L-leucine-2-naphthylamide; esters: serine benzyl ester, and 4-benzyl aspartate; and sulfonamides: N-(1-naphthalenesulfonyl)-phenylalanine, and N-(p-toluenesulfonyl)-L-phenylalanine. 5 of 11 Mar. Drugs 2018, 16, 50 4,000,000 (a) Figure 4. Time courses for the degradation of the tested compounds by strain B-9. (a) L-leucine-2-naph thylamide; (b) 3,4-dimethylhippuric acid; (c) N-acetyl-L-phenylalanine; (d) N-acetyl-D-phenyl alanine; and (e) N-carbobenzoxy-L-phenyl-L-phenylalanine. 2.2. Amino Acid-Containing Compounds (Amides, Esters, and Sulfonamides) amino acid containing compounds. 0 1,000,000 2,000,000 0 3 6 12 24 Value of peak area Time（h） B-9 反応液コントロール B-9 control 0 500,000 1,000,000 , , 0 3 6 12 24 Value of peak area Time（h） B-9 反応液コントロール B-9 control 0 500,000 036 12 24 48 72 96 Value of peak area Time (h） (c) B-9 反応液コントロール B-9 control 0 200,000 400,000 036 12 24 48 72 96 Value of peak area Time（h） (d) B-9 反応液コントロール B-9 control 0 200,000 400,000 0 3 6 12 24 Value of peak area Time（h） (e) B-9 反応液コントロール B-9 control 0 500,000 1,000,000 1,500,000 2,000,000 0 3 6 12 24 Value of peak area Time（h） (b) B-9 反応液コントロール B-9 control 0 200,000 400,000 036 12 24 48 72 96 Value of peak area Time（h） (d) B-9 反応液コントロール B-9 control 0 500,000 1,000,000 0 3 6 12 Value of peak are Time（h） B-9 反応液コントロール B-9 control 0 200,000 400,000 036 12 24 48 72 Value of peak area Time（h） (d) B-9 反応液コントロール B-9 control 0 1,000,000 2,000,000 3,000,000 4,000,000 0 3 6 12 24 Value of peak area Time（h） (a) B-9 反応液コントロール B-9 control 0 500,000 036 12 24 48 72 96 Value of peak area Time (h） (c) B-9 反応液コントロール B-9 control 0 ,000,000 ,000,000 0 3 6 12 24 Time（h） B-9 反応液コントロール B-9 control 0 500,000 036 12 24 48 72 96 Time (h） B-9 反応液コントロール B-9 control Figure 4. Time courses for the degradation of the tested compounds by strain B-9. (a) L-leucine-2-naph thylamide; (b) 3,4-dimethylhippuric acid; (c) N-acetyl-L-phenylalanine; (d) N-acetyl-D-phenyl alanine; and (e) N-carbobenzoxy-L-phenyl-L-phenylalanine. Figure 4. Time courses for the degradation of the tested compounds by strain B-9. (a) L-leucine-2-naph thylamide; (b) 3,4-dimethylhippuric acid; (c) N-acetyl-L-phenylalanine; (d) N-acetyl-D-phenyl alanine; and (e) N-carbobenzoxy-L-phenyl-L-phenylalanine. Figure 4. Time courses for the degradation of the tested compounds by strain B-9. (a) L-leucine-2-naph thylamide; (b) 3,4-dimethylhippuric acid; (c) N-acetyl-L-phenylalanine; (d) N-acetyl-D-phenyl alanine; and (e) N-carbobenzoxy-L-phenyl-L-phenylalanine. Figure 4. Time courses for the degradation of the tested compounds by strain B-9. (a) L-leucine-2-naph thylamide; (b) 3,4-dimethylhippuric acid; (c) N-acetyl-L-phenylalanine; (d) N-acetyl-D-phenyl alanine; and (e) N-carbobenzoxy-L-phenyl-L-phenylalanine. Figure 4. Time courses for the degradation of the tested compounds by strain B-9. (a) L-leucine-2-naph thylamide; (b) 3,4-dimethylhippuric acid; (c) N-acetyl-L-phenylalanine; (d) N-acetyl-D-phenyl alanine; and (e) N-carbobenzoxy-L-phenyl-L-phenylalanine. Figure 4. Time courses for the degradation of the tested compounds by strain B-9. 2.2. Amino Acid-Containing Compounds (Amides, Esters, and Sulfonamides) amino acid containing compounds. 0 1,000,000 2,000,000 3,000,000 4,000,000 0 3 6 12 24 Value of peak area Time（h） (a) B-9 反応液コントロール B-9 control 0 500,000 1,000,000 1,500,000 2,000,000 0 3 6 12 24 Value of peak area Time（h） (b) B-9 反応液コントロール B-9 control 0 500,000 036 12 24 48 72 96 Value of peak area Time (h） (c) B-9 反応液コントロール B-9 control 0 200,000 400,000 036 12 24 48 72 96 Value of peak area Time（h） (d) B-9 反応液コントロール B-9 control 0 200,000 400,000 0 3 6 12 24 Value of peak area Time（h） (e) B-9 反応液コントロール B-9 control Figure 4. Time courses for the degradation of the tested compounds by strain B-9. (a) L-leucine-2-naph thylamide; (b) 3,4-dimethylhippuric acid; (c) N-acetyl-L-phenylalanine; (d) N-acetyl-D-phenyl alanine; and (e) N-carbobenzoxy-L-phenyl-L-phenylalanine. Figure 4. Time courses for the degradation of the tested compounds by strain B-9. (a) L-leucine-2-naph thylamide; (b) 3,4-dimethylhippuric acid; (c) N-acetyl-L-phenylalanine; (d) N-acetyl-D-phenyl alanine; and (e) N-carbobenzoxy-L-phenyl-L-phenylalanine. 2.2. Amino Acid-Containing Compounds (Amides, Esters, and Sulfonamides) amino acid containing compounds. (a) L-leucine-2-naph thylamide; (b) 3,4-dimethylhippuric acid; (c) N-acetyl-L-phenylalanine; (d) N-acetyl-D-phenyl alanine; and (e) N-carbobenzoxy-L-phenyl-L-phenylalanine. Figure 5. Selected ion chromatograms (SIMs) (a) at m/z 257.1 at 0 h; (b) at m/z 144.1 at 0 h; and (c) at m/z 132.03 at 3 h for reaction products of L-leucine-2-naphthylamide on microbial degradation using strain B-9. Figure 5. Selected ion chromatograms (SIMs) (a) at m/z 257.1 at 0 h; (b) at m/z 144.1 at 0 h; and (c) at Figure 5. Selected ion chromatograms (SIMs) (a) at m/z 257.1 at 0 h; (b) at m/z 144.1 at 0 h; and (c) at m/z 132.03 at 3 h for reaction products of L-leucine-2-naphthylamide on microbial degradation using strain B-9. 6 of 11 ng Mar. Drugs 2018, 16, 50 g m/z 132.03 at 3 h There was a common degradation behavior of the tested ester compounds, in which the starting material peaks disappeared as soon as strain B-9 came into contact with the compounds, as shown in Figure 6a,b. The resulting benzyl alcohol continued to be detected by LC/MS during the experiment (data not shown). Figure 6c,d show the degradation behavior of the sulfonamide compounds. These compounds could not be degraded by strain B-9 during the experimental period. There was a common degradation behavior of the tested ester compounds, in which the starting material peaks disappeared as soon as strain B-9 came into contact with the compounds, as shown in Figure 6a,b. The resulting benzyl alcohol continued to be detected by LC/MS during the experiment (data not shown). Figure 6c,d show the degradation behavior of the sulfonamide compounds. These compounds could not be degraded by strain B-9 during the experimental period. Figure 6. Time courses for the degradation of the tested compounds by strain B-9. (a) L-serine benzyl ester; (b) 4-benzyl L-aspartate; (c) N-(1-naphthalenesulfonyl)-L-phenylalanine; and (d) N-(p-toluenesulfonyl)-L-phenylalanine. 0 100,000 200,000 300,000 0 24 48 72 96 Value of peak area Time（h） (a) B-9 反応液コントロール B-9 control 0 200,000 400,000 600,000 0 3 6 12 24 Value of peak area Time（h） (b) B-9 反応液コントロール B-9 control 0 200,000 400,000 600,000 0 24 48 72 96 Value of peak area Time（h） (c) B-9 反応液コントロール B-9 control 0 100,000 200,000 0 24 48 72 96 Value of peak area Time（h） (d) B-9 反応液コントロール B-9 control Figure 6. Time courses for the degradation of the tested compounds by strain B-9. 2.2. Amino Acid-Containing Compounds (Amides, Esters, and Sulfonamides) amino acid containing compounds. (a) L-serine benzyl ester; (b) 4-benzyl L-aspartate; (c) N-(1-naphthalenesulfonyl)-L-phenylalanine; and (d) N-(p-toluenesulfonyl)-L-phenylalanine. 0 100,000 200,000 300,000 0 24 48 72 96 Value of peak area Time（h） (a) B-9 反応液コントロール B-9 control 0 200,000 400,000 600,000 0 24 48 72 96 Value of peak area Time（h） (c) B-9 反応液コントロール B-9 control 0 200,000 400,000 600,000 0 3 6 12 24 Value of peak area Time（h） (b) B-9 反応液コントロール B-9 control 0 100,000 200,000 0 24 48 72 96 Value of peak area Time（h） (d) B-9 反応液コントロール B-9 control Figure 6. Time courses for the degradation of the tested compounds by strain B-9. (a) L-serine benzyl ester; (b) 4-benzyl L-aspartate; (c) N-(1-naphthalenesulfonyl)-L-phenylalanine; and (d) N-(p-toluenesulfonyl)-L-phenylalanine Figure 6. Time courses for the degradation of the tested compounds by strain B-9. (a) L-serine benzyl ester; (b) 4-benzyl L-aspartate; (c) N-(1-naphthalenesulfonyl)-L-phenylalanine; and (d) N-(p-toluenesulfonyl)-L-phenylalanine. 2.3. Inhibition of Hydrolysis of Amino Acid-Containing Compounds Using EDTA and PMSF 2.3. Inhibition of Hydrolysis of Amino Acid-Containing Compounds Using EDTA and PMSF When inhibitors such as EDTA or PMSF were used at 10-mM concentrations to inhibit the degradation of MCs, the microbial degradation was effectively inhibited. Consequently, the concentration was set at 10 mM in this study. To check the inhibitory activity of the prepared solution, MC-LR was subjected to the microbial degradation in the presence or absence of the inhibitor. While MC-LR (22.7 min) and the resulting tetrapeptide (20.9 min) disappeared within 24 h in the HPLC chromatogram without the inhibitor (Figure S2A), the MC-LR disappeared and the tetrapeptide continued to appear even after 72 h in the HPLC chromatogram with the inhibitor (Figure S2B). These results were consistent with our previous findings that EDTA inhibits MlrC. Figure 7 shows the time course of the degradation using EDTA and the tested compounds. These were: (a) 3,4-dimethylhippuric acid; (b) N-carbobenzoxy-L-phenyl-L-phenylalanine; (c) L-leucine-2-naphthylamide; (d) 4-benzyl L-aspartate; and (e) L-serine benzyl ester. They showed a common degradation behavior in that the microbial degradation of the amide and ester compounds using strain B-9 was not inhibited by EDTA. These results indicated that the degradation mentioned above was not related to MlrC. 2.2. Amino Acid-Containing Compounds (Amides, Esters, and Sulfonamides) amino acid containing compounds. In the case of PMSF, the following compounds tested positive: 3,4-dimethylhippuric acid; L-leucine-2-naphthylamide; and 4-benzyl-L-aspartate; while other compounds tested negative: N-carbobenzoxy-L-phenylalanine-L-phenylalanine and a-serine benzyl ester No definitive conclusive information was obtained When inhibitors such as EDTA or PMSF were used at 10-mM concentrations to inhibit the degradation of MCs, the microbial degradation was effectively inhibited. Consequently, the concentration was set at 10 mM in this study. To check the inhibitory activity of the prepared solution, MC-LR was subjected to the microbial degradation in the presence or absence of the inhibitor. While MC-LR (22.7 min) and the resulting tetrapeptide (20.9 min) disappeared within 24 h in the HPLC chromatogram without the inhibitor (Figure S2A), the MC-LR disappeared and the tetrapeptide continued to appear even after 72 h in the HPLC chromatogram with the inhibitor (Figure S2B). These results were consistent with our previous findings that EDTA inhibits MlrC. Figure 7 shows the time course of the degradation using EDTA and the tested compounds. These were: (a) 3,4-dimethylhippuric acid; (b) N-carbobenzoxy-L-phenyl-L-phenylalanine; (c) L-leucine-2-naphthylamide; (d) 4-benzyl L-aspartate; and (e) L-serine benzyl ester. They showed a common degradation behavior in that the microbial degradation of the amide and ester compounds using strain B-9 was not inhibited by EDTA. These results indicated that the degradation mentioned above was not related to MlrC. In the case of PMSF, the following compounds tested positive: 3,4-dimethylhippuric acid; L-leucine-2-naphthylamide; and 4-benzyl-L-aspartate; while other compounds tested negative: N-carbobenzoxy-L-phenylalanine-L-phenylalanine and a-serine benzyl ester. No definitive conclusive information was obtained. 7 of 11 Mar. Drugs 2018, 16, 50 8, 16, x FOR PEER REVIEW 7. Time courses for the degradation of the tested compounds by B-9 in the pre ediaminetetraacetic acid (EDTA): (a) 3,4-dimethylhippuric acid; (b) N-carbob yl-L-phenylalanine; (c) L-leucine-2naphthyl amide; (d) 4-benzyl L-aspartate; and (e) ester. 3. Discussion Strain B-9, which has 99% similarity to Sphingosinicella microcystinivorans strain Y2 [5], is Gram-negative and has, in several studies, shown promise for the degradation of MC-related compounds and nodularin [14,15]. Subsequently, we applied strain B-9 to other types of substrates, such as cyanobacterial peptides including depsipeptides [16], and bacterial cyclic peptides including depsipeptides [16], which are structurally different from the MCs and nodularin. Based on these results, the hydrolytic behavior using this strain is suggested as follows: (1) the reaction essentially occurs at a peptide bond in a cyclic peptide moiety to give a linearized peptide, which is more quickly hydrolyzed compared to their original ones; (2) strain B-9 primarily hydrolyzes an ester bond in a depsipeptide, in which the resulting peptides are further hydrolyzed; (3) a cyclic peptide is hydrolyzed at the acyclic part, and no further reaction occurs; and (4) the resulting linearized peptide is more quickly hydrolyzed compared to the cyclic one. In some cases, it is hard to detect the degraded peptides or amino acids due to rapid hydrolysis [16]. To conﬁrm these observations and to further investigate the hydrolytic activities of the strain, we extended our study to physiologically active peptides such as neuropeptides and cardiovascular peptides [18]. The tested peptides were classiﬁed into two groups: (1) linear peptides, and (2) cyclic peptides with a loop formed by disulﬁde bond formation. The linearized peptides degraded faster than the loop-containing peptides because the loop formed by the disulﬁde bond was regarded as one of the degradation-resistant factors. Hydrolysis of the peptides occurred through the cleavage of various peptide bonds, and strain B-9 may bear similarities to the mammalian neutral endopeptidase (NEP) 24.11, a 94-kDa zinc metalloendoprotease widely distributed in humans and involved in the processing of peptide hormones due to its broad selectivity [20]. In a separate study, we observed the degradation behavior of the linear peptides—the glucagon/VIP family peptides (3200–5000 Da)—by strain B-9 in the absence or presence of two protease inhibitors, EDTA and PMSF. Consequently, we conﬁrmed that one of the B-9 proteases (presumed to be MlrB), which is not inhibited by EDTA, cleaved bioactive peptides in the manner of an endopeptidase similar to NEP. Another protease, which is not inhibited by PMSF, corresponded to MlrC and cleaved the resulting middle-sized peptides to smaller peptides or amino acids [19]. 2.2. Amino Acid-Containing Compounds (Amides, Esters, and Sulfonamides) amino acid containing compounds. 0 500,000 1,000,000 1,500,000 2,000,000 0 3 6 12 24 Value of peak area Time（h） (a) B-9 反応液コントロール EDTA B-9 control EDTA 0 100,000 200,000 300,000 400,000 500,000 0 3 6 12 24 Value of peak area Time（h） (b) B-9 反応液コントロール EDTA B-9 control EDTA 0 1,000,000 2,000,000 3,000,000 4,000,000 5,000,000 0 3 6 12 24 Value of peak area Time（h） (c) B-9 反応液コントロール EDTA B-9 control EDTA 0 100,000 200,000 300,000 400,000 500,000 600,000 0 3 6 12 24 Value of peak area Time（h） (d) B-9 反応液コントロール EDTA B-9 control EDTA 0 50,000 100,000 150,000 200,000 250,000 0 3 6 12 24 Value of peak area Time (h) (e) B-9 反応液コントロール EDTA B-9 control EDTA Time courses for the degradation of the tested compounds by B-9 in the p iaminetetraacetic acid (EDTA): (a) 3,4-dimethylhippuric acid; (b) N-carbobenzoxy- nine; (c) L-leucine-2naphthyl amide; (d) 4-benzyl L-aspartate; and (e) L-serine be 0 500,000 1,000,000 1,500,000 2,000,000 0 3 6 12 24 Value of peak area Time（h） (a) B-9 反応液コントロール EDTA B-9 control EDTA 0 100,000 200,000 300,000 400,000 500,000 0 3 6 12 24 Value of peak area Time（h） (b) B-9 反応液コントロール EDTA B-9 control EDTA 0 1,000,000 2,000,000 3,000,000 4,000,000 5,000,000 0 3 6 12 24 Value of peak area Time（h） (c) B-9 反応液コントロール EDTA B-9 control EDTA 0 100,000 200,000 300,000 400,000 500,000 600,000 0 3 6 12 24 Value of peak area Time（h） (d) B-9 反応液コントロール EDTA B-9 control EDTA 0 50,000 100,000 150,000 200,000 250,000 0 3 6 12 24 Value of peak area Time (h) (e) B-9 反応液コントロール EDTA B-9 control EDTA Figure 7. Time courses for the degradation of the tested compounds by B-9 in the presence of ethylenediaminetetraacetic acid (EDTA): (a) 3,4-dimethylhippuric acid; (b) N-carbobenzoxy- L-phenyl-L-phenylalanine; (c) L-leucine-2naphthyl amide; (d) 4-benzyl L-aspartate; and (e) L-serine benzyl ester. Figure 7. Time courses for the degradation of the tested compounds by B-9 in the presence of ethylenediaminetetraacetic acid (EDTA): (a) 3,4-dimethylhippuric acid; (b) N-carbobenzoxy-L-phenyl-L- phenylalanine; (c) L-leucine-2naphthyl amide; (d) 4-benzyl L-aspartate; and (e) L-serine benzyl ester. Mar. Drugs 2018, 16, 50 8 of 11 3. Discussion In the present study, we attempted to extend the applications of strain B-9, applying it to mycotoxins produced by fungi. Among the tested mycotoxins, only ochratoxin A was completely hydrolyzed to provide the constituents ochratoxin α and L-phenylalanine (Figure 2), and fumonisin B1 levels gradually decreased to a certain extent after 96 h due to the formation of a new peak at 12.7 min (Figure S1). However, although drugs including antibiotics released into the aquatic environment were applied for microbial degradation using strain B-9, no degradation occurred. These results suggest that strain B-9 can only degrade amino acid-containing compounds. As expected, the tested compounds with amide and ester bonds were readily hydrolyzed by strain B-9, although the sulfonamides were not degraded (Figures 4 and 6). In particular, the ester compounds were characteristically and rapidly hydrolyzed as soon as they came into contact with strain B-9 (Figure 6). Furthermore, the degradations of the amide and ester compounds containing amino acids were not inhibited by the addition of EDTA, suggesting that the responsible enzyme is not MlrC. It is understood that MlrC is found in the ﬁnal stage in the microbial degradation of MC, catalyzing the degradation from linearized MC and tetrapeptide to smaller peptides and amino acids. Indeed, Dziga et al. reported the role of MlrC in MC degradation, in which linearized MC and tetrapeptides could be degraded to provide Adda by the cleavage of a peptide bond between Adda and Glu by a recombinant MlrC [14]. These results suggest that strain B-9 possesses an additional hydrolytic enzyme that should be designated as MlrE. Furthermore, the results of the present study suggest that strain B-9 possesses an esterase. As mentioned above, strain B-9 degraded depsipeptides such as aeruginosins and mikamycin A, in which the cleavage at the ester bond was predominant over that of other peptide bonds [16]. However, there may be a possibility that known proteases are responsible for the ester bond cleavage. 9 of 11 Mar. Drugs 2018, 16, 50 Mar. Drugs 2018, 16, 50 Since their discovery in 1994, it has been believed that MC-degrading microorganisms are only responsible for MC degradation [2]. Although many reports on MC-degrading microorganisms have appeared since then [3], few papers have described their substantial function and role in the aquatic environment. 4.4. Enzyme Inhibition Enzyme inhibitors were prepared as follows: EDTA was prepared as a 200-mM stock solution in water and was used at an assay concentration of 10 mM. PMSF was prepared as a 200-mM stock solution in EtOH and was used at an assay concentration of 10 mM. The cell broth and required inhibitor were preincubated at 27 ◦C for 30 min. 4.2. MC-Degrading Bacterium Bacterial strain B-9, isolated from the surface water of Lake Tsukui, Kanagawa, Japan, was previously reported to degrade various MCs and nodularin [15]. This bacterium was inoculated into a ﬂask containing 100 mL of Sakurai medium composed of peptone, yeast extract, and glucose, and incubated at 27 ◦C at 200 revolutions per minute (rpm) for 3 days. 3. Discussion As reported by our group, one of the MC-degrading microorganisms, strain B-9, is applicable to structurally diverse peptide compounds, suggesting a different function. We should investigate the detailed function of each hydrolytic and transporter enzyme, as well as a system composed of these enzymes to understand their inherent roles under aquatic conditions. 4.1. Chemicals As protease inhibitors, EDTA-2Na (purity: >99.5%) and PMSF (purity: >98.5%), were purchased from Dojindo Laboratories (Kumamoto, Japan) and Sigma-Aldrich Japan (Tokyo, Japan), respectively. Acetonitrile (ACN, LC/MS grade, purity: 99.8%), methanol (MeOH, LC/MS grade, purity: 99.7%), ethanol (EtOH, special grade, purity: 99.5%), formic acid (FA, LC/MS grade, purity: 99.5%), acetic acid (AcOH, LC/MS grade, purity: 99.5%), triﬂuoroacetic acid (TFA, special grade, purity: 98.0%), ammonium carbonate (special grade), and 28% ammonia solution (NH4OH, special grade) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Water used for the preparation of all the solutions was puriﬁed using a Milli-Q apparatus (Millipore, Billerica, MA, USA); LC/MS analysis used ultrapure water from Wako. The mycotoxins (ochratoxin A, fumonisin B1, zearalenone, deoxynivalenol, patulin) were purchased from Sigma (St. Louis, MO, USA). Drugs with amide, ester, and sulfonamide bonds and amino acid-containing compounds were obtained from the following companies: Aldrich and Sigma Japan (Tokyo, Japan), Nacalai Tesque (Kyoto, Japan), Tokyo Chemical Industry (Tokyo, Japan), and Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 4.3. Degradation of Tested Compounds Two milligrams of the tested compounds was dissolved in 1 mL of EtOH and 50 µL of the solution was evaporated to dryness. One milliliter of the preincubated cell broth of strain B-9 (containing approximately 3 × 106 colony forming units (CFU) mL−1) was added to the residue. The resulting solution was mixed, and then incubated at 27 ◦C for 5, 15, 30, 60, and 120 min. After incubation, 50 µL of each of these mixtures was added to 50 µL of MeOH containing 0.2% FA and ﬁltered using an Ultrafree-MC membrane centrifuge-ﬁltration unit (hydrophilic polytetraﬂuoroethylene (PTFA), 0.20 µm, Millipore, Bedford, MA, USA) to stop the degradation and to eliminate proteins. Each supernatant was then analyzed by HPLC and LC/MS. 4.6. Liquid Chromatography/Ion Trap Mass Spectrometry The sample, column, mobile phase, and gradient conditions were the same as those used for the HPLC analysis (12). The LC separation was performed using the Agilent 1100 HPLC system (Agilent Technologies, Palo Alto, CA, USA). Five microliters of the sample was ﬁltrated using an Ultrafree-MC membrane centrifuge ﬁltration unit (hydrophilic PTFE, 0.20 µm, Millipore, Bedford, MA, USA) and loaded onto a TSK-gel Super ODS column (2.0 µm, 2.0 × 100 mm, TOSOH, Tokyo, Japan) at 40 ◦C. The mobile phase was 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The ﬂow rate was 200 µL/min with UV detection at 254 nm. The gradient conditions were initially 10–90% B for 40 min. The entire eluate was directed into the mass spectrometer, where it was diverted to waste 2.5 min after injection to avoid any introduction of salts into the ion source. The MS analysis was accomplished using a Finnigan LCQ Deca XP plus ITMS (Thermo Fischer Scientiﬁc, San Jose, CA, USA) equipped with an electrospray ionization (ESI) interface. The ESI conditions in the positive ion mode were as follows: capillary temperature 300 ◦C, sheath gas ﬂow rate 35 (arbitrary unit), ESI source voltage 5000 V, capillary voltage 43 V, and tube lens offset 15 V. Various scan ranges were used according to the molecular weights of the tested compounds. Supplementary Materials: The following are available online at http://www.mdpi.com/1660-3397/16/2/50/s1, Figure S1: Total ion chromatograms (a) and (b) and selected ion monitoring (SIM) at m/z 722.4 (c) and m/z 564.3 (d) of fumonisin B1 and a reaction product by microbial degradation using B-9 at 0 h (A) and 96 h (B), respectively, Figure S2: (A) HPLC chromatograms of MC-LR by B-9 without EDTA after 0, 6 and 24 h. (B) HPLC chromatograms of MC-LR by B-9 with EDTA after 0, 6 and 72 h. Acknowledgments: K.-I.H. and H.J. gratefully acknowledge Tatsuko Sakai at the Analytical Services Ce Faculty of Pharmacy, Meijo University, for assistance and support. Author Contributions: K.-I.H. and H.J. conceptualized the research. K.T., H.U. and Y.O. performed the degradation experiments of drugs including antibiotics with the supervision of K.-I.H.; E.H.H. and M.K. performed the experiments of mycotoxin degradation with the guidance of K.-I.H.; Y.H. and H.J. performed degradation of amide and esters with the guidance of K.-I.H.; M.K., Y.H. and H.J. ran the LC/MS and analyzed the LC/MS data under the supervision of A.R.J.A.; H.J. 4.5. High-Performance Liquid Chromatography The degradation process was monitored by HPLC-photo diode array (PDA) at 220 or 254 nm. The system consisted of a pair of LC 10AD VP pumps, a DGU 12A degasser, a CTO 6A column oven, 10 of 11 Mar. Drugs 2018, 16, 50 an SPD 10A VP photodiode array detector, and an SCL 10A VP system controller (Shimadzu, Kyoto, Japan). Five microliters of the ﬁltered sample were loaded onto a TSK-gel Super ODS column (2.0 µm, 2.0 × 100 mm, TOSOH, Tokyo, Japan) at 40 ◦C. The mobile phase was 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B). The gradient conditions were initially 40–90% B for 20 min, and the ﬂow rate was 200 µL/min. 4.6. Liquid Chromatography/Ion Trap Mass Spectrometry and K.-I.H. wrote the manuscript. All co-authors agreed to the contents of the paper. Conﬂicts of Interest: The authors declare no conﬂict of interest. References Tsuji, K.; Asakawa, M.; Anzai, Y.; Sumino, T.; Harada, K.-I. Degradation of microcystins using immobilized microorganism isolated in an eutrophic lake. Chemosphere 2006, 65, 117–124. [CrossRef] [PubMed] j g y g microorganism isolated in an eutrophic lake. Chemosphere 2006, 65, 117–124. [CrossRef] [PubMed] 2. Jones, G.J.; Bourne, D.G.; Blakeley, R.L.; Doelle, H. Degradation of the cyanobacterial hepatotoxin microcystin by aquatic bacteria Nat Toxins 1994 2 228–235 [CrossRef] [PubMed] microorganism isolated in an eutrophic lake. Chemosphere 2006, 65, 117–124. [CrossRef] [PubMed] 2. Jones, G.J.; Bourne, D.G.; Blakeley, R.L.; Doelle, H. Degradation of the cyanobacterial hepatotoxin microcystin by aquatic bacteria. Nat. Toxins 1994, 2, 228–235. [CrossRef] [PubMed] Jones, G.J.; Bourne, D.G.; Blakeley, R.L.; Doelle, H. Degradation of the cyanobacterial hepatotoxin microcystin by aquatic bacteria. Nat. Toxins 1994, 2, 228–235. [CrossRef] [PubMed] 3. Dziga, D.; Wasylewski, M.; Wladyka, B.; Nybom, S.; Meriluoto, J. Microbial degradation of microcystins. Chem. Res. Toxicol. 2013, 26, 841–852. [CrossRef] [PubMed] 3. Dziga, D.; Wasylewski, M.; Wladyka, B.; Nybom, S.; Meriluoto, J. Microbial degradation of microcystins. Chem. Res. Toxicol. 2013, 26, 841–852. [CrossRef] [PubMed] 4. Martinez, J.L. Antibiotics and antibiotic resistance genes in natural environments. Science 2008, 321, 365–367. [CrossRef] [PubMed] 4. Martinez, J.L. Antibiotics and antibiotic resistance genes in natural environments. Science 2008, 321, 365–367. [CrossRef] [PubMed] 5. Maruyama, T.; Park, H.D.; Ozawa, K.; Tanaka, Y.; Sumino, T.; Hamana, K.; Hiraishi, A.; Kato, K. Sphingosinicella microcystinivorans gen. nov., sp. nov., a microcystin-degrading bacterium. Int. J. Syst. Evol. Microbiol. 2006, 56, 85–89. [CrossRef] [PubMed] 6. Bourne, D.G.; Jones, G.J.; Blakeley, R.L.; Jones, A.; Negri, A.P.; Riddles, P. Enzymatic pathway for the bacterial degradation of the cyanobacterial cyclic peptide toxin microcystin, LR. Appl. Environ. Microbiol. 1996, 62, 4086–4094. [PubMed] 6. Bourne, D.G.; Jones, G.J.; Blakeley, R.L.; Jones, A.; Negri, A.P.; Riddles, P. Enzymatic pathway for the bacterial degradation of the cyanobacterial cyclic peptide toxin microcystin, LR. Appl. Environ. Microbiol. 1996, 62, 4086–4094. [PubMed] 11 of 11 Mar. Drugs 2018, 16, 50 7. Bourne, D.G.; Riddles, P.; Jones, G.J.; Smith, W.; Blakeley, R.L. Characterisation of a gene cluster involved in bacterial degradation of the cyanobacterial toxin microcystin, LR. Environ. Toxicol. 2001, 16, 523–534. [CrossRef] [PubMed] 8. Hashimoto, E.H.; Kato, H.; Kawasaki, Y.; Nozawa, Y.; Tsuji, K.; Hirooka, E.Y.; Harada, K.-I. Further investigation of microbial degradation of microcystin using advanced Marfey’s method. Chem. Res. Toxicol. 2009, 22, 391–398. [CrossRef] [PubMed] 9. Dziga, D.; Wasylewski, M.; Zielinska, G.; Meriluoto, J.; Wasylewski, M. References Heterologous expression and characterization of microcystinase. Toxicon 2012, 59, 578–586. [CrossRef] [PubMed] 10. Dziga, D.; Wasylewski, M.; Szetela, A.; Bochenska, O.; Wladyka, B. Veriﬁcation of the role of MlrC in microcystin biodegradation by studies using a heterologously expressed enzyme. Chem. Res. Toxicol. 2012, 25, 1192–1194. [CrossRef] [PubMed] 11. Cserháti, M.; Kriszt, B.; Krifaton, C.; Szoboszlay, S.; Háhn, J.; Tóth, S.; Nagy, I.; Kukolya, J. Mycotoxin-degradation proﬁle of Rhodococcus strains. Int. J. Food Microbiol. 2013, 166, 176–185. [CrossRef] [PubMed] 12. Kümmerer, K. The presence of pharmaceuticals in the environment due to human use—Present knowledge and future challenges. J. Environ. Manag. 2009, 90, 2354–2366. [CrossRef] [PubMed] 13. Sarmah, A.K.; Meyer, M.T.; Boxall, A.B.A. A global perspective on the use, sales, exposure pathways occurrence, fate and effects of veterinary antibiotics (VAs) in the environment. Chemosphere 2006, 65, 725–759. [CrossRef] [PubMed] 14. Harada, K.-I.; Imanishi, S.; Kato, H.; Mizuno, M.; Ito, E.; Tsuji, K. Isolation of Adda from microcystin-LR by microbial degradation. Toxicon 2004, 44, 107–109. [CrossRef] [PubMed] 15. Imanishi, S.; Kato, H.; Mizuno, M.; Tsuji, K.; Harada, K.-I. Bacterial degradation of microcystins and nodularin. Chem. Res. Toxicol. 2005, 18, 591–598. [CrossRef] [PubMed] 16. Kato, H.; Imanishi, S.Y.; Tsuji, K.; Harada, K.-I. Microbial degradation of cyanobacterial cyclic peptides. Water Res. 2007, 41, 1754–1762. [CrossRef] [PubMed] 17. Kato, H.; Tsuji, K.; Harada, K.-I. Microbial degradation of cyclic peptides produced by bacteria. J. Antibiot. 2009, 62, 181–190. [CrossRef] [PubMed] 8. Kondo, F.; Okada, S.; Miyachi, A.; Kurita, M.; Tsuji, K.; Harada, K.-I. Microbial degradation of physiologic active peptides by strain B-9. Anal. Bioanal. Chem. 2012, 403, 1783–1791. [CrossRef] [PubMed] 19. Miyachi, A.; Kondo, F.; Kurita, M.; Tsuji, K.; Harada, K.-I. Microbial Degradation of linear peptides by strain B-9 of Sphingosinicella and its application in peptide quantiﬁcation using liquid chromatography-mass spectrometry. J. Biosci. Bioeng. 2015, 119, 724–728. [CrossRef] [PubMed] p y g 20. Stephenson, S.L.; Kenny, A.J. The hydrolysis of α-human atrial natriuretic peptide by pig kidney microvillar membranes is initiated by endopeptidase-24.11. Biochem. J. 1987, 243, 183–187. [CrossRef] [PubMed] © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W4293317182	https://dspace.lib.cranfield.ac.uk/bitstream/1826/18370/1/Hydrogen-fuelled_combined-cycle_gas_turbine-2022.pdf	English	null	Techno-environmental assessment of a hydrogen-fuelled combined-cycle gas turbine for a liquid hydrogen tanker	Energy reports	2,022	cc-by	8,119	∗Corresponding author at: Thermal Power and Propulsion Engineering, Cranfield University, College Road, Cranfield, Bedford MK43 0AL, United Kingdom. Techno-environmental assessment of a hydrogen-fuelled combined-cycle gas turbine for a liquid hydrogen tanker Abdullah NFNR Alkhaledi a,b,∗, Amit Batra a, Suresh Sampath a, Pericles Pilidis a a Thermal Power and Propulsion Engineering, Cranfield University, College Road, Cranfield, Bedford MK43 0AL, United Kingdom b Department of Automotive and Marine Engineering, College of Technological Studies, PAAET, P.O. Box 42325 Shuwaikh, Kuwait Keywords: Keywords: Liquid hydrogen tanker Engine thermal efficiency Combined-cycle gas turbine NOx emissions Hydrogen-fuelled engine LH2 tanker mission p p © 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Energy Reports 8 (2022) 10561–10569 Energy Reports 8 (2022) 10561–10569 p // g/ /j gy 2352-4847/© 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). E-mail address: a.alkhaledi@cranfield.ac.uk (A.N. Alkhaledi). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1016/j.egyr.2022.08.202 2352-4847/© 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). a r t i c l e i n f o Article history: Received 4 May 2022 Received in revised form 31 July 2022 Accepted 14 August 2022 Available online xxxx Keywords: Liquid hydrogen tanker Engine thermal efficiency Combined-cycle gas turbine NOx emissions Hydrogen-fuelled engine LH2 tanker mission Article history: Received 4 May 2022 Received in revised form 31 July 2022 Accepted 14 August 2022 Available online xxxx Article history: Received 4 May 2022 Received in revised form 31 July 2022 Accepted 14 August 2022 Available online xxxx The purpose of this study was a techno-environmental assessment analysis for a 280,000 m3 liquid hydrogen (LH2) tanker powered by a hydrogen-fuelled combined-cycle gas turbine (COGAS) as the prime mover. This study utilised established and reliable methods to simulate the LH2 tanker in different journeys and conditions for analysing the advantages of the hydrogen-fuelled COGAS in terms of performance and emission reduction. The assessment was based on engine behaviour on different journeys under cargo loaded and unloaded conditions, with a normal and a 6% degraded engine, and under various ambient conditions. According to the results, the single-pressure hydrogen-fuelled COGAS reached a maximum overall thermal efficiency of 55.5% in winter under unloaded and normal engine conditions at a ship speed of 18 knots during the journey from Marseille to Algeria. From an environmental perspective, the maximum NOx emission of the COGAS was 3.5 kg/h during the journey from Tangier to Southampton in summer with a 6% degraded engine under the loaded condition at a ship speed of 18 knots. The results of the techno-environmental assessment indicated that the LH2 tanker powered by the hydrogen-fuelled COGAS could successfully achieve high efficiency and low emission targets in off-design conditions. The outcome of this study will lead to a future investigation of the LH2 tankers operation economic aspects. https://doi.org/10.1016/j.egyr.2022.08.202 2352-4847/© 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http: 1. Introduction Batra, S. Sampath et al. Abbreviations COGAS Combined-cycle gas turbine LH2 Liquid hydrogen IMO International Maritime Organisation 3D Three-dimensional NOx Nitrogen oxide CO2 Carbon Dioxide CO Carbon monoxide SOx Sulphur oxide Units Tonnes Total displacement m Lightweight, Deadweight, LOA, Length between perpendiculars, Length on the waterline, Extreme breadth, Depth, Draught, Propeller diameter m2 Wetted surface area knots Vessel speed MW Output power of combined-cycle gas and steam, Output power, Steam tur- bine output power, Pump work K Ambient temperature, Exhaust gas tem- perature, Turbine inlet temperature; kPa Ambient pressure kg/kWh Specific fuel consumption kg/s Exhaust gas mass flow rate, Fuel flow rate, Steam mass flow rate % Thermal efficiency, Open-water effi- ciency ◦C Superheated steam temperature, Pinch point temperature difference, Approach points temperature difference, Feed water temperature bar Steam pressure, Condenser pressure rpm Propeller rotational speed g/kWh Emission factor ppm Concentration of gaseous species m3/h Engine exhaust flow rate J/(mol K) Ideal gas constant kW Engine power motors, renewable energy, and hybrid propulsion as future ma- rine propulsion technology with low fuel consumption and emis- sion reduction solutions (Parker, 2013). Benvenuto et al. provided vital fundamental information for evaluating the per- formance of marine COGAS propulsion systems using a single- and dual-pressure heat recovery steam generator without using LH2 fuel (Benvenuto et al., 2012, 2014). McKinlay et al. evaluated several viable options to achieve ships zero-emission target by 2025, using three promising alternative fuels on board large-scale international vessels; hydrogens, ammonia and methanol. This is essentially owing to fuel’s possibility to generate emission- free electricity using fuel cells (McKinlay et al., 2021). Yan et al. studied the design optimisation of a cruise ship energy system using a multi-objective mathematical programming model that considered the technical, environmental, and economic aspects of ship function (Yan et al., 2019). Ahn et al. evaluated a four propulsion system options for a hydrogen tanker in terms of their economic, technological, and environmental feasibilities by con- sidering factors such as lifecycle cost analysis (Ahn et al., 2017). Doulgeris et al. developed a Techno-economic, Environmental and Risk Analysis (TERA) computational method for marine propul- sion systems. The method includes several numerical models to simulate the life cycle operation of marine gas turbines as a propulsion system for marine vessels in different journeys and conditions (Doulgeris et al., 2012). Talluri et al. 1. Introduction applied a Techno- economic, Environmental and Risk Assessment (TERA) method to evaluate the performance and economic benefits of a 4.5 MW simple cycle, twin-shaft marine gas turbine fuelled by natural gas and twin-diesel engines, each of 3 MW rating, operating on marine diesel oil as ship power plants in conjunction with three Flettner towers as assistance propulsion system (Talluri et al., 2018). To the best of the authors’ knowledge, the techno- environmental evaluation of hydrogen-fuelled combined cycle gas turbines in different journeys and conditions for marine appli- cations to support the acceleration of manufacturing and utilising the liquified hydrogen tankers has not yet been investigated. Therefore, this study makes a novel contribution by employing and examining a combined cycle gas turbine fuelled by hydrogen as an alternative efficient prime mover for a new design of hydrogen carrier ships in different journeys and ambient condi- tions to achieve an almost zero-emission target for the maritime transportation sector. This study aimed to assess the missions of a novel improved hydrogen tanker powered by hydrogen-fuelled COGASs using the techno-environmental analysis method in off-design conditions in terms of the performance and environmental impact; for this purpose, the authors used state-of-the-art design and analysis methods as a feasible and sustainable approach to the large- scale investigation. This assessment is a very important stage and one of the research and development stages for implementing liquefied hydrogen tankers as a new design in the future. many regions. Hydrogen can also be used with a thermal engine for waste heat recovery, such as a combined-cycle gas turbine (COGAS), to achieve a higher thermal efficiency and lower emis- sions compared to those achieved using conventional internal combustion engines (Alkhaledi et al., 2022). The combined cycle fuelled by hydrogen could be a successful alternative for ship propulsion systems to achieve almost zero- emission targets in the maritime sector. In this regard, the key issues that necessitate a techno- environmental examination of the combined cycle fuelled by hydrogen as a prime mover of liquefied hydrogen tankers (Alkhaledi et al., 2022) through dif- ferent mission assessments in terms of the performance and environmental impact represent an essential stage in achieving the reliability of the combined cycle gas turbine fuelled by hydro- gen as a propulsion system of liquified hydrogen carrier ships in the design implementation stages of these tankers in the future. 1. Introduction constructed ships. The IMO also increased the strictness of the regulations on the NOx and SOx emissions from vessels and designated emission control areas, such as the Baltic and North seas (Van Hoecke et al., 2021). In 2019, the United Kingdom was one of the countries that took action in this regard by publishing the UK Clean Maritime Plan to achieve zero-emission shipping by 2025. The plan indicated that the use of hydrogen fuel in vessel propulsion systems can play a significant role in the global transition to zero-emission shipping and transportation in the maritime sector (UK Department for Transport, 2019). Global sustainability requires urgent action to tackle climate change and global warming. Accordingly, world leaders at the UN climate change conference (COP26) in Glasgow took action to apply the 2015 Paris climate change agreement for overcoming the negative impact of climate change and limiting the global temperature increase to approximately 1.5 ◦C (COP26, 2021). In the same direction, the International Maritime Organisation (IMO) aims to reduce 50% of greenhouse gas emissions from global shipping by 2050 compared to 2008 (IMO, 2018). Re- garding the environment, maritime transportation and shipping sectors are responsible for approximately 3% of the CO2 emissions globally (Tillig and Ringsberg, 2020). Moreover, several studies have shown that the continually growing CO2, CO, and NOX emissions from shipping are expected to play a crucial role in IMO regulations in future (Skjolsvik et al., 2000; Buhaug et al., 2009). ( p p ) Consequently, employing clean hydrogen can be a promising solution for global decarbonisation and climate change mitiga- tion (Alkhaledi et al., 2021). In actuality, the expectation indicates that hydrogen fuel has considerable potential to decarbonise the maritime sector if used in thermal engines by utilising the green hydrogen produced from renewable energy sources through elec- trolysis. Furthermore, hydrogen removes unburnt hydrocarbons, aromatic compounds, sulphur oxides, soot, and smoke as harmful emissions (Alkhaledi et al., 2022). The use of hydrogen as an energy storage mechanism is considered particularly useful in the context of electricity generated from sources where a sig- nificant mismatch between supply and demand is present; these include wind, solar, and nuclear sources, which could be used to electrolyse seawater rather than fresh water, which is scarce in Additionally, the IMO applied the energy efficiency design index, a design instrument for reducing the emissions from newly Energy Reports 8 (2022) 10561–10569 A.N. Alkhaledi, A. 2.2.1. Ship module p An improved design of the LH2 tanker named JAMILA was simulated in this study, as described in Section 2.1. The LH2 tanker evaluated in this research was 370 m long, 75 m wide, and had draughts of 9.263 and 10.012 m under the unloaded and fully loaded conditions, respectively. The full-load displacement was 230,000 tonnes for the transportation of 20,000 tonnes of LH2. Furthermore, the Lightweight of the ship was 208,000 tonnes which represents; the LH2 ship cargo tanks, fuel tank, engine, hull structure, Deck gear, anchor, cables, pipes and LH2 tanks accessories and equipment weights, and the ship’s Deadweight was 22,000 which represents; the cargo, accommodation, crew, and fuel weights (Alkhaledi et al., 2021). The specifications of the LH2 tanker are highly unusual compared with other traditional tankers designs because the Deadweight is 22000 tonnes which is very light compared with the ship’s immense size. This is the result of the very low density and a substantial volume of hydrogen as a cargo. However, increasing the ship’s Lightweight in the ship design stage to 208,000 tonnes directly supported the ship stability requirements based on the 2008 IMO Intact Stability Code (ISC) (Alkhaledi et al., 2021; IMO, 2008). steam turbine was modelled and simulated using MATLAB soft- ware (Mathworks, 2020). Additionally, the gas turbine cycle was inspired by an existing advanced LM2500+ gas turbine engine model, which is widely used in marine propulsion systems (Luck, 1998; Oosterveld and van Oossanen, 1975), and a single-pressure non-reheated steam cycle. The general specifications of the gas turbine and steam cycles are presented in Tables 2 and 3, respec- tively (Alkhaledi et al., 2022). Consequently, in the design and off-design assessment, am- bient temperature variations and a component degradation of 6% were considered. Moreover, two fixed-pitch propellers were assumed to be installed in the pods of the LH2 tanker. The vessel speed guided this selection and power requirement con- sidering the size of JAMILA in accordance with the maximum allowable unloaded draught, which was 9.263 m, as mentioned in Table 1 (Alkhaledi et al., 2021). The propeller design was based on the open-water characteristics of the Wageningen B- series propellers (Oosterveld and van Oossanen, 1975). The main propeller design characteristics are listed in Table 4 (Alkhaledi et al., 2022). Moreover, the vessel used a single-pressure COGAS as the prime mover with an output power of approximately 50 MW (Alkhaledi et al., 2021). 2. Methods In addition, the assessment considered the influence of the cargo weight, which was approximately 20,000 tonnes (Alkhaledi et al., 2021), and an engine degradation of 6% (Alkhaledi et al., 2022). Further, the analysis provided a techno-environmental method for evaluating the ability of a proposed LH2 tanker to satisfy the zero-emission target with an efficient power system. was performed using the in-house TURBOMATCH gas turbine per- formance code (MacMillan, 1974), MATLAB (Mathworks, 2020), and SOLIDWORKS software to design and simulate an LH2 tanker powered by a COGAS within POSEIDON, a ship-wide in-house simulation software of Cranfield University. This approach evalu- ates proposed systems using the COGAS engine thermodynamic and environmental performance models. This study employed the hydrogen-fuelled COGAS as a prime mover to achieve the off-design requirements from geographic location and ambient conditions for LH2 tankers in different journeys. In addition, the assessment considered the influence of the cargo weight, which was approximately 20,000 tonnes (Alkhaledi et al., 2021), and an engine degradation of 6% (Alkhaledi et al., 2022). Further, the analysis provided a techno-environmental method for evaluating the ability of a proposed LH2 tanker to satisfy the zero-emission target with an efficient power system. tics of the simulated liquid hydrogen (LH2) tanker Alkhaledi 2. Methods An established methodology was developed to improve the novel design of the LH2 tanker with a total capacity of 280,000 m3 of LH2, which was designed in a previous study (Alkhaledi et al., 2021). In addition, the methodology was analytically extended to assess the performance and emission for the improved design of the LH2 tanker using a techno-environmental assessment frame- work by applying a computational method for marine propulsion systems (Doulgeris et al., 2012). Developing the utilised method in this research could facilitate the design of a COGAS power plant fuelled by hydrogen as a prime mover to optimally reduce the fuel consumption and emissions of LH2 tankers. This improved design However, interesting, exciting studies presented organic Rank- ine cycle, fuel cells, nuclear energy, batteries, superconducting 10562 Energy Reports 8 (2022) 10561–10569 A.N. Alkhaledi, A. Batra, S. Sampath et al. Table 1 Main characteristics of the simulated liquid hydrogen (LH2) tanker Alkhaledi et al. (2021). LH2 tanker parameters Values Units Class JAMILA – Vessel type LH2 Tanker – Total displacement (∆total) 230,000 tonnes Lightweight 208,000 tonnes Deadweight 22,000 tonnes Length overall (LOA) 370 m Length between perpendiculars 367.5 m Length on the waterline 367.9 m Extreme breadth (B) 75 m Depth (D) 35 m Full-loaded condition Draught (T) 10.012 m Block coefficient (CB) 0.819 – Prismatic coefficient (Cp) 0.824 – Midship coefficient (Cm) 0.995 – Water plane coefficient (Cwp) 0.900 – Wetted surface area 29,550.906 m2 Unloaded condition Draught (T) 9.263 m Block coefficient (CB) 0.813 – Prismatic coefficient (Cp) 0.818 – Midship coefficient (Cm) 0.994 – Water plane coefficient (Cwp) 0.894 – Wetted surface area 28,916.458 m2 Vessel speed 18 knots Minimum propulsion power required for the full-load condition 27.3 MW Output power of combined-cycle gas and steam 50 MW was performed using the in-house TURBOMATCH gas turbine per- formance code (MacMillan, 1974), MATLAB (Mathworks, 2020), and SOLIDWORKS software to design and simulate an LH2 tanker powered by a COGAS within POSEIDON, a ship-wide in-house simulation software of Cranfield University. This approach evalu- ates proposed systems using the COGAS engine thermodynamic and environmental performance models. This study employed the hydrogen-fuelled COGAS as a prime mover to achieve the off-design requirements from geographic location and ambient conditions for LH2 tankers in different journeys. 2.2.3. Vessel resistance prediction h hi i d l h 2.2.3. Vessel resistance prediction 2.2. Description of techno-environmental modelling modules 2.2.1. Ship module 2.2.1. Ship module The specifications of the LH2 tanker are listed in Table 1. To achieve adequate simulation accuracy, the authors assumed that the ship maintained a maximum operating speed of 18 knots during all journeys. 2.1. Overall improved design philosophy The overall design philosophy of the LH2 tanker has been presented in a previous study (Alkhaledi et al., 2021). In the present study, only a few modifications were implemented to improve the previous design of the LH2 tanker; these modifi- cations concerned adding new design elements represented by the LH2 tank pumps, ship anchors, storage facilities, and crew service tunnel. The updated design of the LH2 tanker is expected to improve applicability in the manufacturing sector. 2.2. Description of techno-environmental modelling modules 2.2.4. Weather and journey management module In practice, the weather module input variables are the ambi- ent temperatures of air and sea between the hydrogen exporting and importing ports (Doulgeris et al., 2012). In this study, the operation period of the LH2 tanker was assumed to be 330 days per year; this period was divided into three seasons (winter, summer, and middle) to achieve the actual evaluation results of the ship performance and emission. The winter months were December, January, and February; summer months were June, July, and August; and middle-season months were March, April, May, September, October, and November. The average ambient temperature of each season was applied in the LH2 tanker sim- ulation for different journeys. The journey management model includes the trip distance, ship speed, and time periods with changing weather conditions and estimates the total trip time and the total fuel consumption of the engine (Doulgeris et al., 2012). This study selected the trips based on the expectation of the hydrogen exporting hubs, which are sunny countries with ports. Therefore, two journeys were selected: from Marseille to Algeria (22 h) and from Tangier to Southampton (81 h). Each season, the journeys were simulated to realise adequate accuracy in the engine performance and emission analysis. In this stage of hydrogen storage technologies, the journeys are assumed to be short (not more than 4 d), owing to the relatively high boil-off rate. wave and wind resistance in addition to the calm water resis- tance. These additional resistance modules, which are presented in the following sections, were expected to increase the accu- racy of resistance estimation under the loaded and unloaded conditions for the LH2 tanker in different weather conditions. 2.2.3.1. Added wave resistance module. The added resistance due to the wave motion resulting from the interaction of the vessel with normal waves was considered in this study, in contrast to the calm water resistance model presented in a previous study (Alkhaledi et al., 2021). The combination of wave and ship motions produces a 20% to 40% increase in water resistance; this percentage cannot be neglected in the total resistance cal- culations (Liu et al., 2011). According to Alexandersson (Alexan- dersson, 2009), the added wave resistance can be estimated by considering the incident wave angles. This method is one of the most common procedures for accurately estimating the added resistance due to wave motion. 2.2.2. COGAS and propeller module 2.2.2. COGAS and propeller module The ship resistance model that affects the ship and power requirements in calm water has been comprehensively presented in a previous study on propulsion LH2 tankers (Alkhaledi et al., 2021). The total resistance and power requirements of JAMILA were estimated using the Holtrop and Mennen method based on the ship shape and size (Holtrop and Mennen, 1982; Holtrop, 1984). In the present study, the authors considered the added The primary propulsion system of the ship was designed to be a 50-MW combined-cycle gas and steam turbine (Alkhaledi et al., 2022). The performance of the power plant was analysed using an in-house gas turbine performance software (MacMil- lan, 1974) with hydrogen as an alternative fuel based on the gas turbine performance evaluation code (Nikolaidis, 2017). The 10563 A.N. Alkhaledi, A. Batra, S. Sampath et al. Energy Reports 8 (2022) 10561–10569 Table 2 General specifications of a gas turbine cycle Alkhaledi et al. (2022). Table 2 General specifications of a gas turbine cycle Alkhaledi et al. (2022). Gas turbine cycle Values Units Ambient temperature 288.15 K Ambient pressure 101.3 kPa Pressure ratio 23.1 – Specific fuel consumption 0.07416 kg/kWh Exhaust gas mass flow rate 84.948 kg/s Exhaust gas temperature 840.3 K Turbine inlet temperature 1,550 K Fuel flow 0.7485 kg/s Output power 36.2 MW Thermal efficiency 40.3 % vessels, especially when radiation-induced resistance is applied. The added wave resistance module used in the present study is based on a method described by Talluri et al. (2016). 2.2.3.2. Wind resistance module. The method used here to esti- mate the aerodynamic resistance due to headwind was intro- duced by Jensen (Schneekluth and Bertram, 1998), expressed as follows: RAA = CAA ρa 2 · (V + VW)2 · AFP, (1) (1) where RAA is the wind resistance, CAA is a coefficient in the range of 0.8–1.0 for cargo ships, ρa is the air density, V is the speed of the ship, VW is the absolute value of wind speed, and AFP is the frontal project area of the ship above sea level. For all wind directions, the Blendermann model (Blender- mann, 1993, 1996) was used by applying the following equation: Table 3 General specifications of a steam cycle [9]. Table 4 2.2.4. Weather and journey management module 2.2.2. COGAS and propeller module Steam plant parameters Values Units Superheated steam temperature (Ta) 559.8 ◦C Steam pressure (PS) 60 bar Pinch point temperature difference ( ∆Tp ) 7.5 ◦C Approach point temperature difference (∆Ta) 20 ◦C Steam mass flow rate (ms) 10.51 kg/s Condenser pressure (Pcond) 0.06 bar Feed water temperature (Te) 36.2 ◦C Steam turbine output power (WST) 14.35 MW Steam turbine isentropic efficiency (ηST ) 89.9 % Pump work (Wpump) 4.1388 MW RAA = ρa 2 u2 · AL · CDl cos ε 1 −δ 2 ( 1 −CDl CDt sin22ε ), (2) (2) where u is the apparent wind velocity; AL is the lateral plane area; ε is the apparent wind angle, which is zero in the case of headwind; δ is the cross-force parameter; CDl is the non- dimensional drag under the headwind condition; and CDt is the non-dimensional drag under the beam wind condition. 2.2.3.3. Engine performance. The engine performance model EN- GINE was one of the main modules in the numerical software PO- SEIDON, which was used to implement the combined-cycle model and LH2 tanker design for simulating the performance of the LH2 tanker powered by the hydrogen-fuelled COGAS in different jour- neys and conditions (Doulgeris et al., 2012). ENGINE estimated the engine performance using a three-dimensional (3D) interpo- lation function that obtained the engine power at each operating point. The input values were obtained by varying the turbine inlet temperature and ambient conditions through the combined-cycle performance and stored as a 3D table (Talluri et al., 2016). In this study, ENGINE was used to simulate the COGAS model with the turbine inlet temperature ranging from 1025 to 1700 K in increments of 25 K and the ambient temperature ranging from −25 to 45 ◦C in increments of 10 ◦C; these values were selected to cover a large scale of the turbine inlet temperature variation in the winter, summer, and middle seasons. Table 4 Propeller design parameters and performance [9]. Propeller characteristic Values Units Propeller type Fixed pitch – Number of blades (Z) 4 – Number of propellers Twin-screw fixed on the pods – Propeller diameter (Dpr) 6.4 m Pitch ratio (P/Dpr) 1.0 – Blade area ratio (AE/AO) 0.9 – Thickness-to-chord ratio (t/c) 0.029 – Propeller rotational speed (n) 124.8 rpm Open-water efficiency (ηO) 56 % 2.2.4. Weather and journey management module The method of Gerritsma and Beukelman (Gerritsma and Beukelman, 1972) is also used to estimate the added wave resistance. In this method, the linearised formulation is acceptable only for the head waves, for even keel 10564 10564 Energy Reports 8 (2022) 10561–10569 A.N. Alkhaledi, A. Batra, S. Sampath et al. Fig. 1. JAMILA three-dimensional design; (a) side elevation; (b) plan view; (c) rear elevation; and (d) front elevation. Fig. 1. JAMILA three-dimensional design; (a) side elevation; (b) plan view; (c) rear elevation; and (d) front elevation. 2.2.5. Emission module 2.2.5. Emission module Southampton). The simulation of ship NOx emission was studied under the impact of a normal and a 6% degraded engine under loaded and unloaded conditions. Southampton). The simulation of ship NOx emission was studied under the impact of a normal and a 6% degraded engine under loaded and unloaded conditions. The LH2 tanker in this study was assumed to be transporting and fuelled by zero-carbon green hydrogen, producing only low NOx gaseous emissions (Enable H2, 2022). From an environmen- tal perspective, the model for assessing the NOx output emissions from a loaded and unloaded green LH2 tanker powered by the hydrogen-fuelled COGAS in different off-design operation condi- tions and journeys was predicted using emission factor estima- tion. The emission factor of the 50-MW hydrogen-fuelled COGAS as a prime mover can be calculated using Eq. (3) (Garaniya, 2016): 3. Results and discussion The performance and emission results for the improved de- sign of JAMILA are presented in this section. In this study, a techno-environmental assessment based on engine behaviour for different journeys under loaded and unloaded conditions, a nor- mal and a 6% degraded engine, and varying ambient conditions was conducted to assess the prime mover as an environmentally friendly propulsion system. EFi,k = ( Ei,k × 10−6) ( P.V R.T × MWk Pp ) , (3) (3) where EFi,k is the emission factor (g/kWh); i refers to the engine type; k refers to the emission type; E is the concentration of gaseous species (ppm); P is the pressure (N/m2); V is the engine exhaust flow rate (m3/h); R is the ideal gas constant, which equals 8.3145 J/(mol K); T is the exhaust gas temperature (K); MW is the molecular weight; and Pp is the engine power (kW). The LM2500+ gas turbine, which was used in this study, was not designed to be exclusively fuelled by hydrogen, and there is much scope for future research in this field. Therefore, the authors adopted the maximum value of the concentration of gaseous species for this type of gas turbine, which is 25 ppm (General Electric 2019, 2022). Consequently, the NOx emission for each journey hour using fuel flow can be calculated by Eq. (4) (Trozzi, 2010): where EFi,k is the emission factor (g/kWh); i refers to the engine type; k refers to the emission type; E is the concentration of gaseous species (ppm); P is the pressure (N/m2); V is the engine exhaust flow rate (m3/h); R is the ideal gas constant, which equals 8.3145 J/(mol K); T is the exhaust gas temperature (K); MW is the molecular weight; and Pp is the engine power (kW). The LM2500+ gas turbine, which was used in this study, was not designed to be exclusively fuelled by hydrogen, and there is much scope for future research in this field. Therefore, the authors adopted the maximum value of the concentration of gaseous species for this type of gas turbine, which is 25 ppm (General Electric 2019, 2022). Consequently, the NOx emission for each journey hour using fuel flow can be calculated by Eq. (4) (Trozzi, 2010): 3.1. Results of design assessment Fig. 1 shows the design of the improved LH2 tanker after adding new design elements, such as hydrogen tank pumps, ship anchors, storage facilities, and the crew service tunnel (Alkhaledi et al., 2021). The improved design that includes a hydrogen- fuelled COGAS as the prime mover without any additional propul- sion technologies can be used in future applications. 3.1.1. Results of weather and journey routes In this study, the weather estimation showed a variety of ambient temperatures between different journeys for the win- ter, summer, and middle seasons. For this reason, two jour- neys were analysed in terms of the ambient conditions. The Marseille–Algeria journey showed that the maximum tempera- ture in summer was more than 30 ◦C, compared to 25 ◦C for the Tangier–Southampton journey. The increase in the ambient temperature may negatively influence the ship engine perfor- mance and emissions. The middle season assumed in this study ENOx = F.F. × EFi,k, (4) (4) ENOx = F.F. × EFi,k, where ENOx is the NOx emission and F.F. is the gas turbine fuel flow per hour during the journey. The LH2 tanker was simulated for three seasons (winter, summer, and middle) using COGAS NOx emission estimation considering the ship operation hours during two journeys (from Marseille to Algeria and from Tangier to 10565 Energy Reports 8 (2022) 10561–10569 A.N. Alkhaledi, A. Batra, S. Sampath et al. p gy p ( ) Fig. 2. Weather conditions in different seasons during the (a) Marseille–Algeria and (b) Tangier–Southampton journeys. Credits: timeanddate.com. . 3. Overall thermal efficiency of the LH2 tanker with a combined-cycle gas turbine (COGAS) as a function of journey time under different conditions for th rseille–Algeria journey. N: normal engine, D: degraded engine, W: winter, S: summer, M: middle season, L: loaded condition, and UN: unloaded condition. presented approximately six months of the year, and the max- mum temperature during the Marseille–Algeria journey in this i t l 22 ◦C d t 24 ◦C f th Fig. 3 shows the overall thermal efficiency of the hydrogen fuelled COGAS during the one-way Marseille–Algeria journey fo three seasons (winter summer and middle) The result show Fig. 2. Weather conditions in different seasons during the (a) Marseille–Algeria and (b) Tangier–Southampton journeys. Credits: timeanddate.com. Fig. 2. Weather conditions in different seasons during the (a) Marseille–Algeria and (b) Tangier–Southampton journeys. Credits: timeanddate.com. he LH2 tanker with a combined-cycle gas turbine (COGAS) as a function of journey time under different conditions for the engine, D: degraded engine, W: winter, S: summer, M: middle season, L: loaded condition, and UN: unloaded condition. Fig. 3. Overall thermal efficiency of the LH2 tanker with a combined-cycle gas turbine (COGAS) as a function of journey time under different conditions for the Marseille–Algeria journey. 3.1.1. Results of weather and journey routes N: normal engine, D: degraded engine, W: winter, S: summer, M: middle season, L: loaded condition, and UN: unloaded condition. Fig. 3 shows the overall thermal efficiency of the hydrogen- fuelled COGAS during the one-way Marseille–Algeria journey for three seasons (winter, summer, and middle). The result shows that the COGAS as a prime mover could achieve the required power with a ship speed of 18 knots in all conditions except in summer with a degraded engine; in summer, the vessel speed re- quired reducing to 17 knots because the required ship power was more than the COGAS power output due to the high ambient tem- perature and the influence of 6% degradation. The results showed that the overall thermal efficiency of the normal COGAS under the unloaded condition in winter could reach 55.5%, representing the maximum overall thermal efficiency of all conditions. The minimum overall thermal efficiency was 49.7%, which occurred in summer under the loaded condition with a 6% degraded engine. Fig. 3 shows the overall thermal efficiency of the hydrogen- fuelled COGAS during the one-way Marseille–Algeria journey for three seasons (winter, summer, and middle). The result shows that the COGAS as a prime mover could achieve the required power with a ship speed of 18 knots in all conditions except in summer with a degraded engine; in summer, the vessel speed re- quired reducing to 17 knots because the required ship power was more than the COGAS power output due to the high ambient tem- perature and the influence of 6% degradation. The results showed that the overall thermal efficiency of the normal COGAS under the unloaded condition in winter could reach 55.5%, representing the maximum overall thermal efficiency of all conditions. The minimum overall thermal efficiency was 49.7%, which occurred in summer under the loaded condition with a 6% degraded engine. i il l h i h j i l d represented approximately six months of the year, and the max- imum temperature during the Marseille–Algeria journey in this season was approximately 22 ◦C, compared to 24 ◦C for the Tangier–Southampton journey. In winter, the maximum temper- ature during the Marseille–Algeria journey was less than 15 ◦C, compared to 21 ◦C for the Tangier–Southampton journey. Fig. 2 shows the weather conditions in the winter, summer, and middle seasons during the Marseille–Algeria and Tangier–Southampton journeys. 3.1.2. Ship performance analysis Two journeys were selected to assess the performance of the COGAS as a prime mover. The ship off-design performance was analysed in terms of the overall thermal efficiency of the COGAS as a prime mover. The prime mover performance was simulated under the unloaded condition and a loaded of 20,000 tonnes of LH2 for a one-way journey in each case. In addition, the perfor- mance of the ship engine was assessed under different weather conditions to investigate the single-pressure COGAS behaviour. Similarly, the Tangier–Southampton journey was simulated for three seasons under different conditions, as shown in Fig. 4. The results showed that the maximum overall thermal efficiency of the normal COGAS under the unloaded condition was 55%. The minimum overall thermal efficiency was 49.2%, which occurred under the full-loaded condition with a 6% degraded engine. 10566 A.N. Alkhaledi, A. Batra, S. Sampath et al. Energy Reports 8 (2022) 10561–10569 A.N. Alkhaledi, A. Batra, S. Sampath et al. Energy Reports 8 (2022) 10561 10569 Fig. 4. Overall thermal efficiency of the LH2 tanker with a COGAS as a function of journey time under different conditions for the Tangier–Southampton journey. Fig. 4. Overall thermal efficiency of the LH2 tanker with a COGAS as a function of journey time under different conditions for the Tangier–Southampton journey. Fig. 4. Overall thermal efficiency of the LH2 tanker with a COGAS as a function of journey time under different conditions for the Tangier–Southampton journey. he LH2 tanker with a COGAS as a function of journey time under different conditions for the Tangier–Southampton journey. of the LH2 tanker with a COGAS as a function of journey time under different conditions for the Tangier–Southampton jour 4. Overall thermal efficiency of the LH2 tanker with a COGAS as a function of journey time under different conditions for th Fig. 4. Overall thermal efficiency of the LH2 tanker with a COGAS as a function of journey time under different conditio The difference of the maximum and minimum overall thermal efficiencies of the LH2 tanker propulsion system between the Marseille–Algeria and Tangier–Southampton journeys was ap- proximately 0.5%. This difference resulted from the ambient tem- perature variation between the different seasons. In particular, during the Marseille–Algeria journey in summer, the maximum ambient temperature was higher than 30 ◦C, while the corre- sponding maximum for the Tangier–Southampton journey was 27 ◦C, as shown in Fig. 2. 3.1.3. Environmental assessment results 3.1.3. Environmental assessment results This section describes the emission analysis of the LH2 tanker propelled by the COGAS during two different journeys (Marseille– Algeria and Tangier–Southampton). The LH2 tanker was simu- lated for three seasons (winter, summer, and middle) in terms of the COGAS NOx emissions with respect to the ship operation hours for each journey. The ship NOx emissions were examined in the case of a normal and a 6% degraded engine under the loaded and unloaded conditions. Because the fuel consumed in the CO- GAS engine is zero-carbon green hydrogen, which produces NOx emissions, only NOx emissions were considered in this study. Fig. 5 shows the NOx emissions from the LH2 tanker propelled by the hydrogen-fuelled COGAS during the one-way Marseille– Algeria journey, which lasted for 22 h, under different ambient conditions. The maximum amount of NOx emissions from the LH2 tanker engine was 3.5 kg/h in the case of degraded COGAS in the middle season at a ship speed of 18 knots. The minimum amount of NOx emissions was 2.6 kg/h in the case of degraded COGAS in summer at a ship speed of 17 knots. From Figs. 3 and 5, the results indicated that the thermal efficiency and NOx emission are higher in the winter and middle seasons compared with the summer season; this represents a particular case for the journey from Mar- seille to Algeria because usually, increasing the thermal efficiency 3.1.2. Ship performance analysis Hence, the minimum overall thermal efficiency of the ship engine in the Marseille–Algeria journey should be lower than that in the Tangier–Southampton journey; however, the results showed were the opposite. This happened because the vessel speed decrease from 18 to 17 knots increased the minimum overall thermal efficiency of the LH2 tanker engine in the Marseille–Algeria journey. will decrease the NOx emissions this is achieved provided that the speed of the ship constant and unchanged for all scenarios during the journey. However, the results clarify that the NOx emissions may decrease with decreasing ship speed regardless of the season and engine condition. Fig. 6 presents the estimation of the NOx emissions during the one-way Tangier–Southampton journey, which lasted for 81 h, under different ambient conditions for three seasons. The results show that the maximum amount of NOx emissions was 3.5 kg/h in the case of a degraded COGAS under the full-loaded condition in summer at a ship speed of 18 knots. The minimum amount of NOx emissions was 2.95 kg/h at a ship speed of 18 knots. In the case of a constant ship speed of 18 knots, the journey season and cargo weight played a key role in controlling the NOx emissions from the LH2 tankers. Between the Marseille–Algeria and Tangier–Southampton jour- neys, the maximum amount of NOx emissions from the LH2 tanker did not vary. The reason for these unusual results is that the maximum amount of NOx emissions in the Marseille–Algeria journey was generated in the middle season, whereas that in the Tangier–Southampton journey was produced in summer. The minimum amount of NOx emissions in the Tangier–Southampton journey was approximately 10% higher than that in the Marseille– Algeria journey. The increased NOx emissions resulted from the increased speed of the LH2 tanker from 17 to 18 knots. 4. Conclusions An established techno-environmental assessment method was applied to a 280,000 m3 LH2 tanker propulsion system with a hydrogen-fuelled COGAS as the prime mover. The performance and emissions were analysed based on the engine behaviour for different journeys (Marseille–Algeria and Tangier–Southampton) under loaded and unloaded conditions, a normal and a 6% de- graded engine, and varying ambient conditions for the LH2 tanker prime mover in different three seasons (winter, middle, and sum- mer). The results were promising in terms of the propulsion system performance and environmental benefits; the key results can be summarised as follows. 10567 A.N. Alkhaledi, A. Batra, S. Sampath et al. Energy Reports 8 (2022) 10561–10569 Fig. 5. NOx emissions from the LH2 tanker with a COGAS as a function of journey time under different conditions for the Marseille–Algeria journey. Fig. 5. NOx emissions from the LH2 tanker with a COGAS as a function of journey time under different conditions for the Marseille–Algeria journey. Fig. 5. NOx emissions from the LH2 tanker with a COGAS as a function of journey time under different conditions for Fig. 6. NOx emissions from the LH2 tanker with a COGAS as a function of journey time under different conditions for the Tangier–Southampton journey. the LH2 tanker with a COGAS as a function of journey time under different conditions for the Tangier–Southampton journey. Fig. 6. NOx emissions from the LH2 tanker with a COGAS as a function of journey time under different conditions for th • An LH2 tanker design was improved to enhance its applica- bility by adding new design elements, such as hydrogen tank pumps, ship anchors, storage facilities, and a crew service tunnel. middle season at a ship speed of 18 knots. On the other hand, the minimum amount of NOx emissions was 2.6 kg/h for the same journey in the case of degraded COGAS in sum- mer at a ship speed of 17 knots. Moreover, the minimum amount of NOx emissions during the Marseille–Algeria jour- ney was 10% lower than that in the Tangier–Southampton journey. The decreased NOx emissions resulted from the decreased speed of the LH2 tanker from 18 to 17 knots in the Marseille–Algeria journey. Hence, the amount of NOx emission was substantially influenced by the ship’s speed. middle season at a ship speed of 18 knots. 4. Conclusions On the other hand, the minimum amount of NOx emissions was 2.6 kg/h for the same journey in the case of degraded COGAS in sum- mer at a ship speed of 17 knots. Moreover, the minimum amount of NOx emissions during the Marseille–Algeria jour- ney was 10% lower than that in the Tangier–Southampton journey. The decreased NOx emissions resulted from the decreased speed of the LH2 tanker from 18 to 17 knots in the Marseille–Algeria journey. Hence, the amount of NOx emission was substantially influenced by the ship’s speed. • Technically, the assessment results for the LH2 tanker propul- sion system performance indicated that the hydrogen-fuelled COGAS could achieve a maximum overall thermal efficiency of 55.5% in the Marseille–Algeria journey in winter with a normal engine under unloaded conditions; the correspond- ing maximum for the Tangier–Southampton journey was 55%. The variation in the overall thermal efficiencies de- pended on ambient temperature, cargo weight, and engine condition. To conclude, the assessment results signify that the employment of the hydrogen-fuelled COGAS for a large-scale LH2 tanker has significant benefits, compared to the conventional propulsion sys- tems, in terms of achieving the required overall thermal efficiency and reducing the NOx emissions of the prime mover. • Environmentally, the maximum amount of NOx emissions from the 50-MW hydrogen-fuelled COGAS was 3.5 kg/h in the Marseille–Algeria journey with a degraded engine in the 10568 10568 A.N. Alkhaledi, A. Batra, S. Sampath et al. Energy Reports 8 (2022) 10561–10569 Acknowledgements MacMillan, W.L., 1974. Development of a Modular-Type Computer Program for Thecalculation of Gas Turbine Off-Design Performance (Doctoral disserta- tion). Schoolof Mechanical Engineering, Cranfield Institute of Technology, Cranfield, UK. The authors are grateful to the Government of the State of Kuwait and the Public Authority for Applied Education and Train- ing (PAAET), State of Kuwait for the provisions and financial support. Mathworks, 2020. Matlab documentations. [Online] https://www.mathworks. com/help/matlab/. (Accessed 16 March 2022). McKinlay, C.J., Turnock, S.R., Hudson, D.A., 2021. Route to zero emission shipping: hydrogen, ammonia or methanol? Int. J. Hydrog. Energy 46 (55), 28282e97, Aug 10. CRediT authorship contribution statement Gerritsma, J., Beukelman, W., 1972. Analysis of the resistanceincrease in waves of a fast cargo ship. Int. Shipbuild Prog. 19, 285–293. http://dx.doi.org/10. 3233/ISP-1972-1921701. Abdullah NFNR Alkhaledi: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software. Amit Batra: POSEI- DON Software. Suresh Sampath: Supervision. Pericles Pilidis: Supervision. Holtrop, J., 1984. A statistical re-analysis of resistance and propulsion data. Int. Shipbuild Prog. 31, 272–276. Holtrop, J., Mennen, G.G.J., 1982. An approximate power prediction method. Int. Shipbuild Prog. 29, 166–170. IMO, 2008. Intact Stability Code. International MaritimeOrganization, London, UK. Declaration of competing interest IMO, 2018. Report of the Marine environment protection committee (MEPC) on its 72nd session. 9–13. https://www.imo.org/en/MediaCentre/ MeetingSummaries/Pages/MEPC-72nd-session.aspx. (Accessed 16 March 2022). The authors declare that they have no known competing finan- cial interests or personal relationships that could have appeared to influence the work reported in this paper. Liu, S., Papanikolaou, A., Zaraphonitis, G., 2011. Prediction of added resistance of shipsin waves. Ocean Eng. 38, 641–650. http://dx.doi.org/10.1016/j.oceaneng. 2010.12.007. References Nikolaidis, T., 2017. TURBOMATCH – Gas Turbine Performance Simulation – User Manual, Unpublished. Cranfield University. Ahn, J., You, H., Ryu, J., Chang, D., 2017. Strategy for selecting an optimal propulsion system of a liquefied hydrogen tanker. Int. J. Hydrog. Energy 42 (8), 5366–5380, Feb 23. Oosterveld, M.W.C., van Oossanen, P., 1975. Further computer-analyzed data of the wageningen B-screw series. Int. Shipbuild Prog. 22, 251–262. http: //dx.doi.org/10.3233/ISP-1975-2225102. Alexandersson, M., 2009. A Study of Methods to Predict Added Resistance in Waves (MSc Thesis). KTH Centre for Naval Architecture, Stockholm. Parker, J., 2013. Future Ship Powering Options: Exploring Alternative Methods of Ship Propulsion. Royal Academy of Engineering, London. Alkhaledi, Anfnr, Sampath, S., Pilidis, P., 2021. A hydrogen fuelled LH2 tanker ship design. Ships Offshore Struct. http://dx.doi.org/10.1080/17445302.2021. 1935626. Schneekluth, H., Bertram, V., 1998. Ship Design for Efficiency and Economy, second ed. Butterworth-Heinemann, Oxford, UK. Alkhaledi, Anfnr, Sampath, S., Pilidis, P., 2022. Propulsion of a hydrogen-fuelled LH2 tanker ship. Int. J. Hydrog. Energy 47 (39), 17407–17422. Skjolsvik, K.O., Andersen, A.B., Corbett, J.J., Skjelvik, J.M., 2000. Study of greenhouse gas emissions from ships. Int. Marit. Organ. https: //citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.112.1805%20&%20rep= rep1%20&%20type=pdf. Benvenuto, G., Bertetta, D., Carollo, F., Campora, U., 2012. COGAS plant as possi- ble future alternative to the diesel engine for the propulsion of large ships. In: Rizzuto, E., C., Guedes Soares. (Eds.), Sustainable Maritime Transportation and Exploitation of Sea Resources Taylor & Francis Group London Talluri, L., Nalianda, D.K., Giuliani, E., 2018. Techno economic and environmental assessment of flettner rotors for marine propulsion. Ocean Eng. 154, 1–15. and Exploitation of Sea Resources. Taylor & Francis Group, London. Benvenuto, G., Laviola, M., Campora, U., 2014. Assessment of steam cycle layouts for COGAS ship propulsion systems. In: Guedes Soares, C., Santos, T.A. (Eds.), p y p, Benvenuto, G., Laviola, M., Campora, U., 2014. Assessment of steam cycle layouts f COGAS hi l i I G d S C S T A (Ed ) Benvenuto, G., Laviola, M., Campora, U., 2014. Assessment of steam cycle layouts for COGAS ship propulsion systems. In: Guedes Soares, C., Santos, T.A. (Eds.), Maritime Technology and Engineering. CRC Press, London. Talluri, L., Nalianda, D.K., Kyprianidis, K.G., Nikolaidis, T., Pilidis, P., 2016. Techno economic and environmental assessment of wind assisted marine propulsion systems. Ocean Eng. 121, 301–311. http://dx.doi.org/10.1016/j. oceaneng.2016.05.047. for COGAS ship propulsion systems. In: Guedes Soares, C., Santos, T.A. (Eds.), Maritime Technology and Engineering. CRC Press, London. Blendermann, W., 1993. Data availability Luck, D.L., 1998. Extending use of marine gas turbines through application of the LM2500+. In: Proceedings of the ASME 1998 International Gas Turbine and Aeroengine Congress and Exhibition. Volume 2: Aircraft Engine; Marine; Microturbines and Small Turbomachinery. Stockholm, Sweden, pp. 2–5. http: //dx.doi.org/10.1115/98-GT-041, June. The data that has been used is confidential. References Parameter identification of wind loads on ships. J. Wind Eng. Ind. Aerodyn. 5, 339–351. l d i d di f hi ll d f i d Blendermann, W., 1996. Wind Loading of Ships Collected Data from Wind Tests Tunnel in Uniform Flow. Inst Schiffbau Univ Hambg, p. 574. Tillig, F., Ringsberg, J.W., 2020. Design, operation and analysis of wind-assisted cargo ships. Ocean Eng. 211, 107603. http://dx.doi.org/10.1016/j.oceaneng. 2020.107603. Buhaug, Ø., Corbett, J.J., Endresen, Ø., Eyring, V., Faber, J., Hanayama, S., Lee, D.S., Lee, D., Lindstad, H., Markowska, A.Z., Mjelde, A., Nelissen, D., Nilsen, J., Pålsson, C., Winebrake, J.J., Wu, W-Q., Yoshida, K., 2009. Second IMO GHG study 2009. Int. Marit. Organ.. Trozzi, C., 2010. Emission estimate methodology for maritime navigation. https: //gaftp.epa.gov/air/nei/ei_conference/EI19/session10/trozzi.pdf. (Accessed 16 March 2022). COP26, 2021. COP26 goals. https://ukcop26.org/cop26-goals/. (Accessed 16 March 2022). UK Department for Transport, 2019. Clean maritime plan. UK, London. https://www.gov.uk/government/publications/clean-maritime-plan- maritime-2050-environment-route-ma. (Accessed 18 May 2021). Doulgeris, G., Korakianitis, T., Pilidis, P., Tsoudis, E., 2012. Techno-economic and environmental risk analysis for advanced marine propulsion systems. Appl. Energy 99, 1–12. http://dx.doi.org/10.1016/j.apenergy.2012.04.026. Van Hoecke, L., Laffineur, L., Campe, R., Perreault, P., Verbruggen, S.W., Lenaerts, S., 2021. Challenges in the use of hydrogen for maritime ap- plications. Energy Environ. Sci. 14, 815–843. http://dx.doi.org/10.1039/ D0EE01545H. ble H2, https://www.enableh2.eu/. (Accessed 16 March 2022). Garaniya, V., 2016. Development of a Methodology to Measure and Assess Ship Emissions. Theme 2 - Marine Environmental Issues. International Association of Maritime Universities (IAMU) Secretariat, Tokyo, https://www.iamu-edu. org. 2016 (Accessed 16 March 2022). Yan, Y., Zhang, H., Long, Y., Wang, Y., Liang, Y., Song, X., Yu, J.J.Q., 2019. Multi-objective design optimization of combined cooling, heating and power system for cruise ship application. J. Clean Prod. 233, 264–279. http://dx.doi. org/10.1016/j.jclepro.2019.06.047. General Electric 2019, 2022. LM2500 power plants. https://www.ge.com/gas- power/products/gas-turbines/lm2500. (Accessed 20 March 2022). 10569 Cranfield University CERES Research Repository https://dspace.lib.cranfield.ac.uk/ School of Aerospace, Transport and Manufacturing (SATM) Staff publications (SATM) School of Aerospace, Transport and Manufacturing (SATM) Staff publications (SATM) Alkhaledi ANFNR, Batra A, Sampath S, Pilidis P. (2022) Techno-environmental assessment of a hydrogen-fuelled combined-cycle gas turbine for a liquid hydrogen tanker, Energy Reports, Volume 8, November 2022, pp. 10561-10569 https://doi.org/10.1016/j.egyr.2022.08.202 Downloaded from CERES Research Repository, Cranfield University Techno-environmental assessment of a hydrogen-fuelled combined-cycle gas turbine for a liquid hydrogen tanker Alkhaledi, Abdullah N. F. N. R. 2022-08-27 Attribution 4.0 International Alkhaledi, Abdullah N. F. N. R. 2022-08-27 Attribution 4.0 International
https://openalex.org/W2052538238	https://europepmc.org/articles/pmc2386802?pdf=render	English	null	Differential protein expression following low temperature culture of suspension CHO-K1 cells	BMC biotechnology	2,008	cc-by	9,869	BioMed Central BioMed Central Received: 11 September 2007 Accepted: 22 April 2008 Received: 11 September 2007 Accepted: 22 April 2008 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Open Acc Research article Differential protein expression following low temperature culture of suspension CHO-K1 cells Niraj Kumar, Patrick Gammell, Paula Meleady, Michael Henry and Martin Clynes Open Access Differential protein expression following low temperature culture of suspension CHO-K1 cells umar, Patrick Gammell, Paula Meleady, Michael Henry and Address: National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland Email: Niraj Kumar* - niraj.kumar2@mail.dcu.ie; Patrick Gammell - patrick.gammell@dcu.ie; Paula Meleady - paula.meleady@dcu.ie; Michael Henry - michael.henry@dcu.ie; Martin Clynes - martin.clynes@dcu.ie * Corresponding author * Corresponding author Background up regulated during low temperature culture [12,13,17,18]. Of these proteins, CRIP and RBM3 have been studied extensively [15,17-20]. Both CIRP and RBM3 are involved in modulation of transcription and translation by functioning as RNA chaperones [18-20]. CRIP improves recombinant protein productivity and can arrest cell growth in a cell line-specific manner whereas RBM3 facilitates cap-independent protein synthesis at low temperature [15,17,20]. RBM3 is also reported to interact with and partially block the activity of miRNAs at low temperature [19]. Altered miRNA expression following temperature shift has recently been reported and may be an important component of regulating protein expression and cell behavior at low temperature [13]. g Since the introduction of human tissue plasminogen acti- vator (tPA) as the first recombinant therapeutic protein product from mammalian cells to get approval in 1986, approximately 60–70% of all recombinant proteins are being produced in mammalian cell lines [1], predomi- nantly in Chinese Hamster Ovary (CHO) cells. The increasing demands for biologicals necessitates improved productivity from CHO cells and the methods currently applied include optimization of culture conditions (tem- perature, pH, aeration rate, etc.), improved reactor designs, medium formulation and cell engineering. Despite this, however, many aspects of CHO cell biology remain poorly understood. Many culture processes operate a biphasic culture whereby cells are grown at 37°C to maximise biomass and then the culture is shifted to a lower temperature (28– 33°C) to encourage protein production while maintain- ing a longer and more viable stationary/production phase [2-6]. Low temperature cultivation of mammalian cells is an established method to maintain cell viability while at the same time inhibiting growth; this inhibition is associ- ated with G1-phase cell cycle arrest which has been posi- tively correlated with increased productivity [2-9]. However, the effect of low temperature on recombinant protein productivity can be product, clone or cell line spe- cific [10]. Increased levels of mRNA coding for the recom- binant protein, either due to enhanced transcription or increased mRNA stability of the recombinant gene of interest, have also been observed during low temperature culture and also correlate with improved recombinant protein productivity [3,8]. Post-translational modifica- tions, particularly glycosylation, which are important for biological activity of recombinant proteins, are main- tained or even improved at low temperature [3,4]. Background In addi- tion to affecting productivity, low temperature culture has been shown to reduce cellular metabolism (glucose/ medium consumption and oxygen uptake), waste accu- mulation (lactate and ammonia production) and apopto- sis [7,8,11]. In this study, we have used two-dimensional difference gel electrophoresis (2D-DIGE) to investigate the pro- teomic changes associated with the shift to low tempera- ture in a biphasic batch culture of suspension-adapted CHO-K1 cells. In this manner we wished to gain insights into the effects of low temperature culture on mammalian cells, with potential relevance to mammalian cell-based recombinant protein production. http://www.biomedcentral.com/1472-6750/8/42 BMC Biotechnology 2008, 8:42 Results Cell Culture Suspension-adapted CHO-K1 cells were seeded at 1 × 105 cells/mL in spinner flasks and maintained at 37°C for 144 hrs or for 72 hrs at 37°C followed by a temperature shift to 31°C for a further 72 hrs. 72 hrs was chosen for this temperature shift because it represents the mid-exponen- tial phase of cell growth. 31°C was selected for the tem- perature shift as it represents the average temperature employed by others performing similar studies, i.e. 28– 33°C [2,4,6,9,10,13]. As can be seen in Figure 1 the cells that were temperature shifted immediately ceased loga- rithmic growth and did not exceed a peak viable cell den- sity of 1.45 × 106 ± 0.013 cells/mL, whereas the cells maintained in standard culture at 37°C continued in log- arithmic growth for a further 48 hrs and achieved a peak viable density of 2.02 × 106 ± 0.11 cells/mL. The cells that were temperature shifted displayed a steady if slightly increasing viable cell density throughout, whereas the cells cultured at 37°C had entered the late stationary/ death phase of the growth cycle by 144 hrs (Figure 1A&B). Cultures were sampled at 72 hrs and 144 hrs for protein extraction from 3 independent experiments. The molecular mechanisms regulating sub-physiological temperature response of cells in culture are still poorly understood, despite a number of recent transcriptomic and proteomic profiling studies [2,12-14]. It is generally accepted that global cap-dependent protein translation decreases at sub-physiological temperatures, however IRES mediated cap-independent protein translation is active [15,16]. More than 20 proteins including cold- inducible RNA binding protein (CIRP), RNA binding motif protein 3 (RBM3), protein disulphide isomerase (PDI), nuclear localization sequence binding protein (NSR1), vimentin, phosphoglycerate kinase and heat shock cognate 71 kDa protein have been reported as being Abstract Background: To ensure maximal productivity of recombinant proteins (rP) during production culture it is typical to encourage an initial phase of rapid cell proliferation to achieve high biomass followed by a stationary phase where cellular energies are directed towards production of rP. During many such biphasic cultures, the initial phase of rapid cell growth at 37°C is followed by a growth arrest phase induced through reduction of the culture temperature. Low temperature induced growth arrest is associated with many positive phenotypes including increased productivity, sustained viability and an extended production phase, although the mechanisms regulating these phenotypes during mild hypothermia are poorly understood. Results: In this study differential protein expression in suspension CHO-K1 cells was investigated following a reduction of the culture temperature from 37°C to 31°C in comparison to standard batch culture maintained at 37°C using 2D-DIGE (Fluorescence 2-D Difference Gel Electrophoresis) and mass spectrometry (MS). There is only limited proteomic analysis of suspension-grown CHO cells describing a direct comparison of temperature shifted versus non- temperature shifted cultures using 2D-DIGE. This investigation has enabled the identification of temperature-dependent as well as temperature-independent proteomic changes. 201 proteins were observed as differentially expressed following temperature shift, of which 118 were up regulated. Of the 53 proteins identified by MALDI-ToF MS, 23 were specifically differentially expressed upon reduction of the culture temperature and were found related to a variety of cellular functions such as regulation of growth (HNRPC), cap-independent translation (EIF4A), apoptosis (importin-α), the cytoskeleton (vimentin) and glycoprotein quality control (alpha glucosidase 2). Conclusion: These results indicate the extent of the temperature response in CHO-K1 cells and suggest a number of key regulatory proteins and pathways that are involved in modulating the response of cells to mild hypothermia. Regulation of these identified proteins and pathways could be useful for future approaches to engineer CHO cells for improved recombinant protein production. Page 1 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1472-6750/8/42 Compariso ature shift Figure 1 Comparison of the viable cell number (A) and percentage viability (B) of CHO K1 cells cultured using either a biphasic temper ature shifted culture ( ) or using a standard culture at 37 C ( ▲). Figure 1 Comparison of the viable cell number (A) and percentage viability (B) of CHO-K1 cells cultured using either a biphasic temperature shifted culture (----) or using a standard culture at 37°C (--▲--). Error bars represent the standard deviation calculated from three biological replicate cultures. '' indicates statistically significant (p-value ≤ 0.02) differ- ences in cell growth and viability. care) using the Profound database search engine for pep- tide mass fingerprints. An expectation value of 0.01 or better means that there is ≤ 1% chance that the identifica- tion is random. Although many spots resulted in high quality spectra, they were not identified which may be due to insufficient amounts of protein, modification and/or variation of the amino acid sequences in CHO proteins or the paucity of CHO proteins in the protein databases. Despite this 53 proteins were identified (Additional file 1) and the representative spectra of 3 of these proteins (vimentin, HNRPC and GAPDH) are presented in Figure 3. Following identification of the 53 spots, all identified proteins were compared for differential expression in both temperature shifted and standard culture experi- ments to identify temperature-dependent and tempera- ture-independent protein changes (Additional file 1). For example, GANAB, a glycan-processing enzyme that is involved in glycoprotein quality control in the endoplas- mic reticulum [21], was 2.19 fold up regulated at 144 hrs compared to 72 hrs following temperature shift while it was unchanged when cells were maintained at 37°C. Acti- vator of 90 kDa heat shock protein ATPase homolog 1 (AHSA1) however, was differentially expressed in both experiments (1.69 and 1.73 fold down regulated at 144 hrs following temperature shift and standard culture respectively), hence could be considered a temperature- independent protein change. A number of the identified proteins including vimentin and GAPDH have been pre- viously identified as differentially expressed following use of a pooled internal standard which facilitated spot matching and relative spot quantitation while minimiz- ing the gel-to-gel variations. Spots originating from dust particles and gel impurities in each gel image were checked and removed manually to improve the quantita- tion and matching process. Compariso ature shift Figure 1 Landmarks, which aid the gel- to-gel spot matching process, were also defined in the gel to increase the accuracy of the matching algorithm. Man- ual checks were carried out to allow for cases where spots were detected more than once (due to spot splits by the spot detection algorithms), these spots were then merged back to improve spot matching. After spot matching and filtering, 2852 and 2885 spots were detected for the tem- perature shifted and standard culture experiments respec- tively. Spots that were 1.5 fold up/down regulated at 144 hrs compared to 72 hrs with a t-test score of ≤ 0.05 and present on all gels were considered differentially expressed (DE). A total of 201 DE spots (118 up regulated and 83 down regulated) were observed in the temperature shift experiment and 404 DE spots (210 up regulated and 194 down regulated) were observed in the standard cul- ture experiment (Figure 2). Differential analysis of proteins 2D-DIGE protein expression maps (PEMs) for the biolog- ical triplicate samples were generated for both tempera- ture shifted and standard cultures for CHO-K1 cells at 72 hrs and 144 hrs. The 72 hrs PEMs were then compared to that at 144 hrs using the Biological Variance Analysis (BVA) module of DeCyder software. This was aided by the Page 2 of 13 (page number not for citation purposes) BMC Biotechnology 2008, 8:42 http://www.biomedcentral.com/1472-6750/8/42 Comparison of the viable cell number (A) and percentage viability (B) of CHO-K1 cells cultured using either a biphasic temper- ature shifted culture (----) or using a standard culture at 37°C (--▲--). Figure 1 Comparison of the viable cell number (A) and percentage viability (B) of CHO-K1 cells cultured using either a biphasic temperature shifted culture (----) or using a standard culture at 37°C (--▲--). Error bars represent the standard deviation calculated from three biological replicate cultures. '' indicates statistically significant (p-value ≤ 0.02) differ- ences in cell growth and viability. Differential analysis of proteins 0 5 10 15 20 25 0 24 48 72 96 120 144 95 96 97 98 99 100 101 0 24 48 72 96 120 144 A B Viable Cells/mL (x105) Time (Hours) Viability % Time (Hours) 0 5 10 15 20 25 0 24 48 72 96 120 144 95 96 97 98 99 100 101 0 24 48 72 96 120 144 A B Viable Cells/mL (x105) Time (Hours) Viability % Time (Hours) 0 5 10 15 20 25 0 24 48 72 96 120 144 95 96 97 98 99 100 101 0 24 48 72 96 120 144 A B Viable Cells/mL (x105) Time (Hours) Viability % Time (Hours) 0 5 10 15 20 25 0 24 48 72 96 120 144 95 96 97 98 99 100 101 0 24 48 72 96 120 144 A B Viable Cells/mL (x105) Time (Hours) Viability % Time (Hours) 95 96 97 98 99 100 101 0 24 48 72 96 120 144 B Viability % Time (Hours) 95 96 97 98 99 100 101 0 24 48 72 96 120 144 B Viability % Time (Hours) 95 96 97 98 99 100 101 0 24 48 72 96 120 144 B Viability % Time (Hours) 95 96 97 98 99 100 101 0 24 48 72 96 120 144 B Viability % Time (Hours) 0 5 10 15 20 25 0 24 48 72 96 120 144 A Viable Cells/mL (x105) Time (Hours) 0 5 10 15 20 25 0 24 48 72 96 120 144 A Viable Cells/mL (x105) Time (Hours) 0 5 10 15 20 25 0 24 48 72 96 120 144 A Viable Cells/mL (x105) Time (Hours) 0 5 10 15 20 25 0 24 48 72 96 120 144 A Viable Cells/mL (x105) Time (Hours) B Page 3 of 13 (page number not for citation purposes) Spot Identification Following spot picking and tryptic digestion, protein identification was carried out using MALDI-ToF MS. All identified proteins had an expectation value of 0.01 or better. Expectation value for proteins was determined by Ettan MALDI-ToF Pro evaluation software (GE Health- Page 3 of 13 (page number not for citation purposes) Page 3 of 13 (page number not for citation purposes) BMC Biotechnology 2008, 8:42 http://www.biomedcentral.com/1472-6750/8/42 Representative overlapping 2D-DIGE expression map of CHO-K1 proteins labelled with fluorescent dyes (Cy2, Cy3 and Cy Figure 2 Representative overlapping 2D-DIGE expression map of CHO-K1 proteins labelled with fluorescent dyes (Cy2 Cy3 and Cy5). The location of differentially expressed proteins identified using MALDI-ToF MS are indicated by arrows. Representative overlapping 2D-DIGE expression map of CHO-K1 proteins labelled with fluorescent dyes (Cy2, Cy3 and Cy5) Figure 2 Representative overlapping 2D-DIGE expression map of CHO-K1 proteins labelled with fluorescent dyes (Cy2, Cy3 and Cy5). The location of differentially expressed proteins identified using MALDI-ToF MS are indicated by arrows. p pp g p p p y ( y , y y ) g Representative overlapping 2D-DIGE expression map of CHO-K1 proteins labelled with fluorescent dyes (Cy2, Cy3 and Cy5). The location of differentially expressed proteins identified using MALDI-ToF MS are indicated by arrows. temperature shift (Table 1). The identified differentially expressed proteins were also compared between both temperature shifted and standard cultures at 144 hrs (Table 2 &3). Three proteins, ACTB, ALDH and DPYSL2 were identified at more than one location on the gel pos- sibly indicating some form of post-translational modifica- tion (PTM), of which PTMs in ALDH and DPYSL2 were specific to the temperature shift to 31°C. The identified proteins could be linked to a broad range of biological functions including structural, metabolic, differentiation, secretory pathways, translation, transcription, protein binding, signal transduction, cell adhesion and apoptosis. Validation of results by Western Blotting Analysis Two proteins, vimentin and HNRPC, were selected from the list of differentially expressed identified proteins for validation by Western blotting. RBM3 is already known to be induced following shift to low temperature and was included as a positive control. α-Tubulin was used as a loading control. Western blot analysis reflected a similar Page 4 of 13 (page number not for citation purposes) Page 4 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1472-6750/8/42 BMC Biotechnology 2008, 8:42 oF generated spectra of Heterogeneous nuclear ribonucleoprotein C (HNRPC) (A), Vimentin (VIM) (B) and 3-phosphate dehydrogenase (GAPDH) (C) ToF generated spectra of Heterogeneous nuclear ribonucleoprotein C (HNRPC) (A), Vimentin Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (C). Trypsin digested peptides were used for h MALDI-ToF. Trypsin gives peaks at specific molecular weight (842.51, 1045.5 and 2211.1 m/z) and can be u in the MS spectra. Peaks other than trypsin are from target proteins. Trypsin calibration peaks are represente belonging to identified proteins by ▲. A B C B C B C MALDI ToF generated spectra of Heterogeneous nuclear ribonucleoprotein C (HNRPC) (A), Vimentin (VIM) (B) and Glycer aldehyde 3 phosphate dehydrogenase (GAPDH) (C) Figure 3 MALDI-ToF generated spectra of Heterogeneous nuclear ribonucleoprotein C (HNRPC) (A), Vimentin (VIM) (B) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (C). Trypsin digested peptides were used for identifi- cation with MALDI-ToF. Trypsin gives peaks at specific molecular weight (842.51, 1045.5 and 2211.1 m/z) and can be used as calibrants in the MS spectra. Peaks other than trypsin are from target proteins. Trypsin calibration peaks are represented by and peaks belonging to identified proteins by ▲. Page 5 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1472-6750/8/42 BMC Biotechnology 2008, 8:42 Table 1: Comparison of differentially expressed proteins identified in this study with previous studies on the effect of temperature. Protein This Study Previous Study Function Author Protein/Transcript Effect Vimentin Up regulated Baik et al, 2006 Vimentin Up regulated Cell size, proliferation, cell growth rate and apoptosis. Validation of results by Western Blotting Analysis GAPDH Up regulated Baik et al, 2006 GAPDH Up regulated Apoptosis, nuclear tRNA export, DNA replication, DNA repair and transcription LGALS1 Up regulated Baik et al, 2006 Galectin 1 No change Cell adhesion and proliferation ACTB No Change Baik et al, 2006 B-actin (Identified at multiple spots) Up regulated at 2 spots and down regulated at 1 spot Cell size, shape and proliferation, transcription and apoptosis PHB No Change Baik et al, 2006 Prohibitin Up regulated Proliferation, differentiation and apoptosis TPI1 No Change Baik et al, 2006 Triosephosphate isomerise No change Stress conditions and cell death mparison of differentially expressed proteins identified in this study with previous studies on the effect of te pattern of expression to those observed using 2D-DIGE (Figure 4). Vimentin was 2.05 fold up regulated following temperature shift and 1.08 up regulated in standard cul- ture from 2D-DIGE analysis. Densitometry analysis of Western blot data revealed that vimentin was 1.9 fold up regulated following temperature shift and 1.11 fold down regulated in standard culture. HNRPC was 1.52 fold down regulated following temperature shift and 1.26 fold down regulated in standard culture from 2D-DIGE analysis, while from densitometry analysis of western blot data, it was 1.33 fold down regulated at low temperature and 1.07 fold down regulated in standard culture. as a means of simultaneously inducing growth arrest and extending long term culture viability thus increasing recombinant protein productivity and yield [2-9]. To investigate the mechanisms regulating the effects of low temperature in CHO-K1 cells we employed 2D-DIGE fol- lowed by MALDI-ToF mass spectrometry. When suspension-adapted CHO-K1 cells were cultured at 37°C for 72 hrs and then shifted to low temperature (31°C), cell growth was immediately arrested compared to cells maintained in standard cultures (37°C) (Figure 1A). Following the temperature shift, the cells were observed to maintain a steady viable cell density whereas the cells at 37°C began entering the death phase by 120 hrs of culture. While the viabilities of both cultures were comparable throughout the 144 hrs of culture this changed towards the end of the experiment for the cells Discussion h The incorporation of a temperature reduction step is com- monly employed during production cell culture in the biopharmaceutical industry. This temperature shift is used Table 2: Differential expression of process-dependent proteins at 144 hrs between temperature shifted and standard culture conditions. Number Protein Description Fold Change at temperature shift (72 hrs vs. 144 hrs) Fold Change at standard culture (72 hrs vs. 144 hrs) Fold Change at 144 hrs (144 hrs at 31°C vs. 144 hrs at 37°C) 1 Calponin 3, acidic -1.96 -3.37 1.72 2 Chain A, Structure Of Bovine Beta-Actin- Profilin Complex (1HLUA) -1.59 -1.53 -1.04 3 PREDICTED: similar to NADH dehydrogenase (ubiquinone) Fe-S protein 1, 75 kDa precursor 2 1.59 1.26 4 Succinate dehydrogenase flavoprotein subunit 1.7 1.87 -1.1 5 PREDICTED: similar to Dihydropyrimidinase related protein-2 2.58 1.97 1.31 6 Eukaryotic translation initiation factor 3, subunit I -1.72 -2 1.16 7 Eukaryotic translation initiation factor 5A -1.81 -2.07 1.14 8 PREDICTED: similar to Activator of 90 kDa heat shock protein ATPase homolog 1 -1.69 -1.73 1.02 9 Thioredoxin-like 2 -1.8 -1.8 1 10 Menkes disease gene product 1.79 1.6 1.12 11 FK506 binding protein 4 -2.5 -2.43 -1.02 12 PREDICTED: similar to heat shock protein 8 isoform 3 -1.9 -2.09 1.1 l expression of process-dependent proteins at 144 hrs between temperature shifted and standard culture Table 2: Differential expression of process-dependent proteins at 144 hrs between temperature shifted a conditions. http://www.biomedcentral.com/1472-6750/8/42 BMC Biotechnology 2008, 8:42 Table 3: Differential expression of proteins at 144 hrs between temperature shifted and standard culture conditions. Number Protein Description Fold Change at 144 hrs (144 hrs at 31°C vs. Discussion h 144 hrs at 37°C) 1 Vimentin 1.9 2 Calponin 3, acidic 1.72 3 Beta actin 2.2 4 Capping protein (actin filament) muscle Z-line, beta 1.88 5 Tubulin T beta15 1.76 6 Aldehyde dehydrogenase family 1, subfamily A1 1.76 7 Glyceraldehyde-3-phosphate dehydrogenase 1.64 8 PREDICTED: similar to UDP-N-acetylhexosamine pyrophosphorylase (Antigen X) -2.90 9 Acetyl-Coenzyme A dehydrogenase, long-chain 1.71 10 PREDICTED: similar to alpha enolase -1.51 11 PREDICTED: similar to ATP synthase beta chain, mitochondrial precursor isoform 1 1.6 12 Annexin A4 1.9 13 PREDICTED: similar to eukaryotic translation initiation factor 4E isoform 1 -2.42 14 PREDICTED: similar to prohibitin 2.32 15 Lectin, galactose binding, soluble 1 1.56 16 Chain A, Importin Alpha, Mouse -2.09 17 PREDICTED: similar to von Hippel-Lindau binding protein 1 isoform 2 1.51 18 PREDICTED: similar to chaperonin containing TCP1, subunit 2 isoform 2 1.74 19 Hypothetical protein LOC499689 1.53 20 MHC class II antigen beta chain 2.08 21 Chain A, Crystal Structure Of The Human Sh3 Binding Glutamic-Rich Protein Like 1.56 mediated cap-independent protein translation [23]. EIF4A is 1.81 fold up regulated following temperature shift. This indicates that EIF4A may play an important role in facilitating translation in this system. EIF4A has been shown to be up regulated in G2 arrested CHO cells [24], which may also indicate a role for this protein in the observed growth arrest. mediated cap-independent protein translation [23]. EIF4A is 1.81 fold up regulated following temperature shift. This indicates that EIF4A may play an important role in facilitating translation in this system. EIF4A has been shown to be up regulated in G2 arrested CHO cells [24], which may also indicate a role for this protein in the observed growth arrest. maintained at 37°C (Figure 1). It is possible that the high percentage of serum (10%) in the culture medium pro- tected both cultures from apoptotic cell death, therefore maintaining the high viabilities recorded. Serum is known to protect cells against apoptosis in suspension culture [22]. Following 2D-DIGE analysis to compare the differences in global protein expression at 72 hrs and 144 hrs of culture, 201 DE spots were observed in the temperature shifted cells whereas 404 DE spots were observed for the cells maintained in standard culture. It is apparent from this data that cells at the end of a temperature shifted batch culture are more similar to healthy exponentially growing cells than cells which have achieved late stationary/death phase at 37°C. Discussion h Reduced cellular metabolism at low tem- perature could be responsible for the smaller number of differentially expressed proteins observed after tempera- ture shift in comparison to the cells grown at 37°C. It has been observed that protein synthesis rates are reduced at low temperature due to a general inhibition of cap- dependent protein synthesis. However some proteins, e.g. RBM3, are translated under cold stress via an IRES medi- ated cap-independent mechanism [15,16]. This type of protein translation initiation requires both eukaryotic ini- tiation factors (EIF2, EIF3, EIF4A, EIF4B, and EIF4F) and message-specific cellular IRES trans-acting factors (ITAFs) [23]. Eukaryotic translation initiation factor 4A, isoform 1 (EIF4A), an RNA helicase enzyme, is one of the critical translational initiation factors that are required in IRES Of the 247 spots picked (127 up regulated and 120 down regulated at 144hrs), a total of 53 proteins were success- fully identified (Additional file 1). Of these, 23 proteins (18 up regulated and 5 down regulated at 144 hrs) were specifically altered following temperature shift (Addi- tional file 1). Two of these proteins (ALDH and DPYSL2) were present at more than one location in the gel indicat- ing a low-temperature-specific protein modification. Con- versely, 18 proteins were differentially regulated specifically in standard culture, of which one protein, ACTB, was present at two different positions in the gel. Grouping of the identified proteins by biological function indicated that proteins involved in metabolism were the dominant group affected in both temperature shifted and standard cultures (31 and 28% respectively), followed by structural proteins (14 and 21%). The indication that low temperature affects metabolism and structure is in keep- ing with other studies [12,14]. However, the number of proteins identified here is insufficient for a comprehen- sive analysis of the main biological functions affected in this system, as we may be observing a bias towards the identification of the more highly expressed metabolic and Page 7 of 13 (page number not for citation purposes) Page 7 of 13 (page number not for citation purposes) BMC Biotechnology 2008, 8:42 http://www.biomedcentral.com/1472-6750/8/42 Comparative Western blot analysis for HNRPC, vimentin and RBM3 proteins between 72 hrs and 144 hrs of culture in tem- perature shifted and standard culture Figure 4 Comparative Western blot analysis for HNRPC, vimentin and RBM3 proteins between 72 hrs and 144 hrs of culture in temperature shifted and standard culture. α-tubulin is used as a loading control. Discussion h The numbers below each band represents the intensities of bands after normalization to α-tubulin. The graph indicates the fold change, derived from densitometry analysis, of vimentin and HNRPC between 72 hrs and 144 hrs of culture. Error bars represent the standard devi- ation calculated from normalized intensities of bands from three biological replicate samples for vimentin and HNRPC. Comparat perature s Figure 4 Comparative Western blot analysis for HNRPC, vimentin and RBM3 proteins between 72 hrs and 144 hrs of culture in tem perature shifted and standard culture Figure 4 Comparative Western blot analysis for HNRPC, vimentin and RBM3 proteins between 72 hrs and 144 hrs of culture in temperature shifted and standard culture. α-tubulin is used as a loading control. The numbers below each band represents the intensities of bands after normalization to α-tubulin. The graph indicates the fold change, derived from densitometry analysis, of vimentin and HNRPC between 72 hrs and 144 hrs of culture. Error bars represent the standard devi- ation calculated from normalized intensities of bands from three biological replicate samples for vimentin and HNRPC. structural proteins within the cell such as GAPDH and vimentin. This is important because during mitosis, similar to low temperature culture inductions, protein synthesis is rap- idly and severely repressed possibly due to inhibition of global cap-dependent translation [27]. The fact that HNRPC and EIF4A are both differentially expressed in this experiment clearly indicates the importance of IRES-based regulation of the translational machinery. This is important because during mitosis, similar to low temperature culture inductions, protein synthesis is rap- idly and severely repressed possibly due to inhibition of global cap-dependent translation [27]. The fact that HNRPC and EIF4A are both differentially expressed in this experiment clearly indicates the importance of IRES-based regulation of the translational machinery. The most obvious effect following reduction of culture temperature is the immediate decrease in growth observed. A number of growth-related proteins were iden- tified as being differentially expressed in a temperature- specific manner, including HNRPC, RRN3 and LGALS1. HNRPC is a nuclear pre-mRNA binding protein that enhances the translation of c-myc mRNA by interacting with an IRES in the 5' UTR and thus impacting prolifera- tion [25]. Silencing of HNRP C1/C2 protein expression by siRNA has also been shown to result in inhibition of cell proliferation [26]. http://www.biomedcentral.com/1472-6750/8/42 BMC Biotechnology 2008, 8:42 Another common feature of cells that are cultured at reduced temperatures is that they display delayed apopto- sis compared to cells cultured at 37°C [7,16]. It has been proposed that the reduction in apoptosis is as a result of the reduced cellular metabolism at low temperatures [7]. However, others have reported the induction of anti- apoptotic proteins (e.g. BCL-2) following culture at reduced temperature [30]. BCL-2 was not identified as a DE protein in this study but a number of apoptosis-asso- ciated proteins were identified including Importin-α, NADH dehydrogenase (ubiquinone) Fe-S protein 3 (NDUFS3) and Annexin A4. Importin-α, a nuclear import receptor, facilitates trafficking of the growth promoting Ras effector, RASSF5, into the nucleus [31]. Over expres- sion of importin-α also results in significant increases in p21(waf1/cip1) transcript levels and apoptosis, implicat- ing it in the nuclear import of p53 [32]. The temperature- specific 1.8 fold down regulation of importin-α levels therefore may have an impact on both proliferation and protection against apoptosis. Unlike importin-α, the other two apoptosis-related proteins Annexin A4 and NDUSF3 were both up regulated (1.95 and 1.51 fold respectively), and are positive regulators of apoptosis. Annexin A4 is selectively translocated from the nucleus to the cytosol during apoptosis and is recognized as an early marker of apoptotic cell death [33]. NDUFS3 is part of the mitochondrial complex I. Inhibition of NDUFS3 expres- sion by siRNA has been shown to provide resistance to IFN-β/Retinoic Acid-induced apoptosis [34]. The data pre- sented indicates that by the end of the temperature shifted culture the cells were beginning to initiate apoptosis. This is in agreement with other findings that despite the delay in apoptosis, once initiated, the rate and manner of cell death is similar to that observed in standard culture at 37°C [7]. inhibits cell growth and induce apoptosis in neuroblast- oma cells [37]. PHB and ALDH1A1 were 1.66 and 1.64 fold down regulated respectively, while ENO1 was 1.51 fold up regulated at 144 hrs at 37°C in comparison to cul- ture at 72 hrs. These observations are in keeping with the reduced viability observed at 144 hrs in standard culture in comparison to temperature shifted cultures. A number of cytoskeletal proteins including vimentin, profilin, beta actin (ACTB) and capping protein (actin fil- ament) muscle Z-line beta were differentially expressed following temperature shift and/or in standard culture (Additional file 1). http://www.biomedcentral.com/1472-6750/8/42 Vimentin, profilin and beta-actin were also observed to be differentially expressed by Baik et. al. [12]. Vimentin is one of the major cytoskeletal proteins and its increased expression has previously been shown in the heat-resistant phenotypes of CHO cells [38]. Vimentin was 2.05 fold up regulated following temperature shift to 31°C and was unchanged at standard culture. Profilin is a small ubiquitous actin monomer sequestering protein that is required for normal actin polymerization in response to thermal stress [39], and was 1.69 fold up reg- ulated following temperature shift. However profilin was unchanged at standard culture. ACTB is involved in many biological functions in cell such as regulating cell shape and growth. Increased expression of ACTB has been shown to provide resistance against apoptosis [40]. ACTB was 1.97 fold down regulated at 144 hrs at 37°C and was not significantly altered following temperature shift. CAPZB binds to one end of actin filaments, regulates actin polymerization including the number and length of actin filaments, and strengthens the actin cytoskeleton in the cytoplasm [41]. CAPZB was 1.5 fold down regulated at 144 hrs at 37°C, whereas no change was observed follow- ing temperature shift. Although the role of vimentin and profilin in regulating low temperature specific phenotypes is unclear at this point, the reduced expression of ACTB and CAPZB could possibly be associated with increased apoptosis in standard culture [40,41]. By comparison, cells at 144 hrs in standard culture were already in early death phase (Figure 1). This is likely due to a variety of reasons including nutrient limitation, oxy- gen depletion and waste accumulation at 144 hrs in stand- ard culture. A number of apoptosis related proteins, including prohibitin (PHB), Aldehyde dehydrogenase family 1, subfamily A1 (ALDH1A1) and alpha-enolase (ENO1) were found to be differentially regulated in this system (Additional file 1). Over expression of PHB inhib- its cell proliferation by arresting cells in G0/G1 phase and has also been shown to increase cell survival [35]. ALDH1A1 is a member of aldehyde dehydrogenase family that can oxidize a wide variety of aldehydes (retinalde- hyde, acetaldehyde, etc.) to the corresponding carboxylic acids (retinoic acid, acetic acid, etc.). Down regulation of ALDH1A1 has been correlated with the increased suscep- tibility of cells towards apoptosis [36]. ENO1 catalyses the formation of phosphoenolpyruvate from 2-phosphoglyc- erate. Discussion h These results are in keeping with our finding that HNRPC is 1.52 fold down regulated follow- ing reduction of the culture temperature. HNRPC also interacts with IRESs in the 5'UTR of 'upstream of N-Ras' (Unr) and affects cell proliferation through IRESs, includ- ing the PITSLRE IRES, which is activated at mitosis [26]. RRN3 protein levels were found to be 1.53 fold down reg- ulated at the end of the temperature shifted culture which is unsurprising as its activity is known to be reduced in sta- tionary cells [28]. LGALS1 (lectin, galactoside-binding, soluble, 1 or galectin 1) has been shown to act as a nega- tive autocrine growth factor that regulates cell prolifera- tion [29] which correlates with the 1.62 fold up regulation of LGALS1 observed in the temperature shifted samples and growth data. Page 8 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1472-6750/8/42 Methods Cell Culture Suspension-adapted CHO-K1 cells were used in this study. The culture medium consisted of ATCC medium (DMEM/F-12 Ham containing glutamine and sodium pyruvate; Sigma) supplemented with 10% fetal bovine serum (Sigma). Clearly serum concentration or use of serum-free medium could impact on protein expression profiles. Cells were maintained in 250 mL spinner vessels (Techne) at 60 rpm on spinner platforms in 37°C or 31°C incubators as appropriate. For batch culture experiments, exponentially growing cells were inoculated at 1 × 105 cells/mL into spinner vessels at a final volume of 100 mL. All cultures were gassed with compressed air (Air Prod- ucts) each day for 1 min. Cell counts were taken every 24 hrs using a hemacytometer and the trypan blue exclusion method. For both temperature shifted and standard cul- tures, triplicate spinner vessels were taken for sampling following 72 and 144 hrs of culture. Three proteins, ACTB, ALDH and DPYSL2, were identified from multiple spots. ACTB was present at two different pIs and molecular weights in the gel containing samples from standard culture. ALDH and DPYSL2 were identified at two different pIs on gels from the temperature shifted cul- ture suggesting potential low temperature-induced post translational modifications. ALDH is a NAD(P)+-depend- ent enzyme that can protect against hyperoxia-induced cell death through reduction of ROS, activation of ERK/ MAPK, and PI3K-Akt cell survival signalling pathways [42]. Analysis of the ALDH spots revealed 1.63 and 1.98 fold up regulation following temperature shift and no change in the cultures maintained at 37°C. The role of this protein in low temperature response is unknown. DPYSL2 promotes microtubule formation by binding to tubulin heterodimers [43]. Different variants, possibly phosphorylated, of DPYSL2 have been reported previ- ously [44]. In this study, DPYSL2 was 2.58 fold up regu- lated following temperature shift whereas it was 1.97 fold up regulated in the cultures maintained at 37°C. How- ever, the second spot identified as DPYSL2 was specifically 2.51 fold down regulated at 31°C. The functional associ- ation of DPYSL2 with low temperature response is unclear. In-silico phosphoproteome analysis predicted that both ALDH and DPYSL2 contain phosphorylation sites and hence the multiple spots may indicate that they are phosphorylated. A previous study also identified two proteins as being phosphorylated at tyrosine residues in CHO cells cultured at low temperature [2] suggesting that active cell signalling occurs in response to temperature shifts. http://www.biomedcentral.com/1472-6750/8/42 The over expression of ENO1 has been shown to Cellular metabolism is generally reduced in low culture temperature [7] but this does not apply to every enzyme and pathway. GAPDH, a multi-functional glycolytic enzyme, was almost 2-fold up regulated following tem- perature shift. Similar results have been reported in other published data [12] (Table 1). In contrast metabolic enzymes involved in energy generation (PYP and ATP5B) were down regulated in dying cells (Additional file 1) which indicates reduced energy generation in standard culture at 37°C. This may be due to depletion of nutrients in the media at 144 hrs of culture. Out of 53 identified proteins, 12 proteins (4 upregulated and 8 down regulated at 144 hrs compared to 72 hrs) were differentially expressed in both the temperature shifted Page 9 of 13 (page number not for citation purposes) Page 9 of 13 (page number not for citation purposes) BMC Biotechnology 2008, 8:42 http://www.biomedcentral.com/1472-6750/8/42 http://www.biomedcentral.com/1472-6750/8/42 http://www.biomedcentral.com/1472-6750/8/42 cells and those maintained at 37°C (Table 2). In most cases the regulation between 72 hrs and 144 hrs was sim- ilar in the temperature shifted and standard cultures indi- cating that these are culture-dependent changes. As with the DE proteins identified from the temperature shift, these proteins cover a range of functions including stress response e.g. AHSA1. Of the 53 identified proteins, 21 were found to be differentially expressed between temper- ature shifted and non-shifted cultures at 144 hrs (Table 3). Methods Cell Culture Sample Preparation and Protein Labelling for 2D-DIGE Frozen cell pellets containing 2 × 107 cells were thawed and re-suspended in 400 µL of lysis buffer (7 M urea, 2 M thiourea, 30 mM Tris, 4% CHAPS, 5 mM magnesium ace- tate, pH 8.5), and then homogenised by carefully passing the samples through a 20 gauge needle 5 times. Samples were left on a shaker for 1 hr at room temp to allow extrac- tion to take place, and then centrifuged at 14,000 rpm (or equivalent g) for 15 min at room temperature to remove insoluble material. The supernatant was removed and stored at -80°C until required for use. Protein concentra- tion was determined using the thiourea-compatible Brad- ford protein assay (Bio-Rad). For 2D-DIGE experiments, control and test cell lysates were labelled with 200 pmol of either Cy3 or Cy5 fluores- cent dyes (GE Healthcare) for comparison on the same gel. Labelling reactions were performed on ice in the dark for 30 min and then quenched with a 50-fold molar excess of free lysine to dye for 10 min on ice. An internal stand- ard containing a pool of all samples (both control and test) was labelled with Cy2 fluorescent dye, and this was used as a standard on all gels to aid image matching and cross-gel statistical analysis. The Cy3 and Cy5 labelling reactions (50 µg of each) from each lysate were mixed and run on the same gels with an equal amount (i.e. 50 µg per gel) of Cy2-labelled standard. Technical duplicates of 3 independent biological replicates resulted in 36 fluores- cent dye gel images being analysed in this study. Conclusion l In conclusion, the present study has identified a number of temperature-specific as well as temperature-independ- ent changes in protein expression that occur during batch suspension culture. A number of the protein changes can be linked to the growth inhibition observed subsequent to temperature shift. Further studies targeting the proteins identified in this study using CHO-K1 cells producing a recombinant protein product will improve the under- standing of the cold shock response and may be useful in identifying candidate targets to re-engineer CHO cells to produce recombinant therapeutic proteins more effi- ciently. Page 10 of 13 (page number not for citation purposes) MALDI-ToF analysis of peptides using the IPGphor (GE Healthcare) for a total of 50 kV/hrs at 20°C with the resistance set at 50 mA per strip. After IEF the strips were equilibrated for 15 min in 50 mM Tris-HCl pH 8.8, 6 M urea, 30% (v/v) glycerol, 1% (w/v) SDS sup- plemented with 65 mM DTT, followed by a second 15 min equilibration in the same buffer containing 240 mM iodoacetamide instead of DTT. The equilibrated strips were then transferred onto large format 12.5% acrylamide gels poured between low fluorescence glass plates, and then overlaid with 0.5% agarose solution containing bromophenol blue. The gels were placed in the Ettan Dalt 12 electrophoresis unit (GE Healthcare) and run at 1.5 W/ gel at 10°C overnight until the dye-front had reached ~1 cm from the bottom of the gel. using the IPGphor (GE Healthcare) for a total of 50 kV/hrs at 20°C with the resistance set at 50 mA per strip. After IEF the strips were equilibrated for 15 min in 50 mM Tris-HCl pH 8.8, 6 M urea, 30% (v/v) glycerol, 1% (w/v) SDS sup- plemented with 65 mM DTT, followed by a second 15 min equilibration in the same buffer containing 240 mM iodoacetamide instead of DTT. The equilibrated strips were then transferred onto large format 12.5% acrylamide gels poured between low fluorescence glass plates, and then overlaid with 0.5% agarose solution containing bromophenol blue. The gels were placed in the Ettan Dalt 12 electrophoresis unit (GE Healthcare) and run at 1.5 W/ gel at 10°C overnight until the dye-front had reached ~1 cm from the bottom of the gel. For MALDI-ToF analysis, the peptide samples were desalted and concentrated using C-18 Zip-Tips (Milli- pore). Each tip was wetted firstly using 100% ACN, then with 50% ACN solution with 0.1% trifluoroacetic acid (TFA), and then equilibrated with 0.1% TFA in LC-MS grade water (Sigma). The bound peptides were eluted directly onto MALDI-ToF MS target slides with 5 mg/mL of α-cyano-4-hydroxycinnamic acid (Laser Biolabs) in 50% acetonitrile/0.1% TFA (v/v) solution. An internal sample mix, Pep4 (Laser Biolabs) was also spotted onto target slides and used as an internal calibrant. Mass spectra were recorded in the positive ion, reflectron mode using the Ettan MALDI-ToF Pro mass spectrometer (GE Healthcare) equipped with delayed extraction and a standard nitrogen laser (337 nm). MALDI-ToF analysis of peptides The spectra were inter- nally calibrated with trypsin enzyme autolysis peptide peaks at m/z 842.51 and m/z 2211.10, and Pep4 mix when possible. Spectra were acquired by selective accu- mulation of 250 individual laser shots and processed using Ettan MALDI evaluation software. Known contami- nant peaks were removed from the resulting mass spectra and remaining sample-related peaks were used for data- base searching. The artificial modifications of peptides (carbamidomethylation of cysteines and partial oxidation of methionines) were also considered. Protein identifica- tion was achieved using Ettan MALDI-ToF Pro evaluation software (GE Healthcare) incorporating Profound data- base search engine for peptide mass fingerprints. The sequence database searched was NCBI-nr database (2007/ 12/02) using subset species, Mammalian, Rodentia and Homo sapiens. Western Blot Analysis 10–40 µg protein were separated on 10% SDS polyacryla- mide gels and transferred onto Hybond ECL nitrocellu- lose membranes (GE) using the semi-dry transfer method. Membranes were blocked with 1–5% Marvel-PBS for 1 hr at room temperature and probed overnight at 4°C with mouse anti-hnRNP C (Sigma), goat anti-vimentin (Sigma), rabbit anti-RBM3 (kindly supplied by Peter W. Vanderklish, Scripps Research Institute) and mouse anti- tubulin (Sigma) antibodies. Blots were then washed and incubated with the appropriate peroxidase-labelled sec- ondary antibodies (anti-mouse secondary (DakoCytoma- tion), anti-goat secondary (Sigma), and anti-rabbit secondary (DakoCytomation)). After washing, bands were visualized by the enhanced chemiluminescence (ECL) method (GE Healthcare). α-Tubulin was used as a loading control. Image Acquisition and Data Analysis All of the gels were scanned using the Typhoon 9400 Var- iable Mode Imager (GE Heathcare) to generate gel images at the appropriate excitation and emission wavelengths from the Cy2, Cy3 and Cy5 labelled samples. The result- ant gel images were cropped using the ImageQuant soft- ware tool and imported into Decyder 6.5 software. The Biological Variation Analysis (BVA) module of Decyder 6.5 was used to compare the control versus test samples to generate lists of differentially expressed proteins. For sta- tistical purposes, a cut-off of 1.5 fold up/down regulated with a t-test score < 0.05 was used. Spot Digestion p g Preparative gels containing 400 µg of protein were fixed and then post-stained with colloidal coomassie blue stain (Sigma). The gels were scanned and images imported into ImageMaster Platinum software (GE Healthcare) and matched to the Master gel image generated from the DIGE experiment. Spots of interest were selected and a pick list was generated and imported into the software of the Ettan Spot Picker robot (GE Healthcare). Gel plugs were placed into presiliconised microtitre plates and stored at 4°C until digestion. Tryptic digestions were performed using the Ettan Digestor robot (GE Healthcare). Excess liquid was removed from each plug, and washed for 3 cycles of 20 min using 50 mM NH4HCO3 in 50% methanol solu- tion. The plugs were then washed for 2 cycles of 15 min using 70% ACN and left to air dry for 1 hour. Lyophilised sequencing grade tryspin (Promega) was reconstituted with 50 mM acetic acid as a stock solution and then diluted to a working solution with 40 mM NH4HCO3 in 10% ACN solution, to a concentration of 12.5 ng trypsin per µL. Samples were digested at 37°C overnight and were then extracted twice with 50% ACN and 0.1% TFA solu- tion for 20 min. each. All extracts were pooled and con- centrated by SpeedVac (Thermo Scientific) for 40 min. Protein Separation by 2D-DIGE Protein Separation by 2D-DIGE 18 cm IPG 4–7 strips (GE Healthcare) were rehydrated overnight in buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.5% IPG buffer, 50 mM DTT) according to manufac- turer's guidelines. Isoelectric focussing was performed Page 10 of 13 (page number not for citation purposes) Page 10 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1472-6750/8/42 http://www.biomedcentral.com/1472-6750/8/42 http://www.biomedcentral.com/1472-6750/8/42 http://www.biomedcentral.com/1472-6750/8/42 BMC Biotechnology 2008, 8:42 References 1. Wurm FM: Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 2004, 22(11):1393-1398. 1. Wurm FM: Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 2004, 22(11):1393-1398. ( ) 21. Totani K, Ihara Y, Matsuo I, Ito Y: Substrate specificity analysis of endoplasmic reticulum glucosidase II using synthetic high mannose-type glycans. J Biol Chem 2006, 281(42):31502-31508. ( ) 2. Kaufmann H, Mazur X, Fussenegger M, Bailey JE: Influence of low temperature on productivity, proteome and protein phos- phorylation of CHO cells. Biotechnol Bioeng 1999, 63(5):573-582. 22. Zanghi JA, Fussenegger M, Bailey JE: Serum protects protein-free competent Chinese hamster ovary cells against apoptosis induced by nutrient deprivation in batch culture. Biotechnol Bioeng 1999, 64(1):108-119. p g ( ) 3. Yoon SK, Song JY, Lee GM: Effect of low culture temperature on specific productivity, transcription level, and heterogeneity of erythropoietin in Chinese hamster ovary cells. Biotechnol Bioeng 2003, 82(3):289-298. g ( ) 23. Pilipenko EV, Pestova TV, Kolupaeva VG, Khitrina EV, Poperechnaya AN, Agol VI, Hellen CU: A cell cycle-dependent protein serves as a template-specific translation initiation factor. Genes Dev 2000, 14(16):2028-2045. g ( ) 4. Bollati-Fogolin M, Forno G, Nimtz M, Conradt HS, Etcheverrigaray M, Kratje R: Temperature reduction in cultures of hGM-CSF- expressing CHO cells: effect on productivity and product quality. Biotechnol Prog 2005, 21(1):17-21. 24. Sasaki Y, Itoh F, Suzuki H, Kobayashi T, Kakiuchi H, Hareyama M, Imai K: Identification of genes highly expressed in G2-arrested Chinese hamster ovary cells by differential display analysis. J Clin Lab Anal 2000, 14(6):314-319. q y g ( ) 5. Butler M: Animal cell cultures: recent achievements and per- spectives in the production of biopharmaceuticals. Appl Micro- biol Biotechnol 2005, 68(3):283-291. 25. Kim JH, Paek KY, Choi K, Kim TD, Hahm B, Kim KT, Jang SK: Het- erogeneous nuclear ribonucleoprotein C modulates transla- tion of c-myc mRNA in a cell cycle phase-dependent manner. Mol Cell Biol 2003, 23(2):708-720. ( ) 6. Trummer E, Fauland K, Seidinger S, Schriebl K, Lattenmayer C, Kun- ert R, Vorauer-Uhl K, Weik R, Borth N, Katinger H, Muller D: Proc- ess parameter shifting: Part II. Biphasic cultivation-A tool for enhancing the volumetric productivity of batch processes using Epo-Fc expressing CHO cells. Biotechnol Bioeng 2006, 94(6):1045-1052. ( ) 26. Additional file 1 MALDI-ToF MS identification of proteins that were differentially expressed at 144 hrs compared to 72 hrs using either temperature shifted or standard culture. Click here for file MALDI-ToF MS identification of proteins that were differentially expressed at 144 hrs compared to 72 hrs using either temperature shifted or standard culture. Click here for file 16. Al-Fageeh MB, Smales CM: Control and regulation of the cellu- lar responses to cold shock: the responses in yeast and mam- malian systems. Biochem J 2006, 397(2):247-259. 17. Nishiyama H, Itoh K, Kaneko Y, Kishishita M, Yoshida O, Fujita J: A glycine-rich RNA-binding protein mediating cold-inducible suppression of mammalian cell growth. J Cell Biol 1997, 137(4):899-908. ( ) 18. Danno S, Itoh K, Matsuda T, Fujita J: Decreased expression of mouse Rbm3, a cold-shock protein, in Sertoli cells of cryp- torchid testis. Am J Pathol 2000, 156(5):1685-1692. J ( ) 19. Dresios J, Aschrafi A, Owens GC, Vanderklish PW, Edelman GM, Mauro VP: Cold stress-induced protein Rbm3 binds 60S ribos- omal subunits, alters microRNA levels, and enhances global protein synthesis. Proc Natl Acad Sci USA 2005, 102(6):1865-1870. http://www.biomedcentral.com/1472-6750/8/42 http://www.biomedcentral.com/1472-6750/8/42 BMC Biotechnology 2008, 8:42 11. Chuppa S, Tsai YS, Yoon S, Shackleford S, Rozales C, Bhat R, Tsay G, Matanguihan C, Konstantinov K, Naveh D: Fermentor tempera- ture as a tool for control of high density perfusion cultures of mammalian cells. Biotechnol Bioeng 1997, 55(2):328-338. PG participated in the experimental design of the study, cell culture, interpretation of results and manuscript prep- aration. PM participated in the experimental design of the proteomics part of the study, statistical analysis of the 2D- DIGE gels and manuscript preparation. MH participated in the identification of differentially expressed proteins by mass spectrometry. MC conceived the study, participated in its design and coordination. All authors read and approved the final manuscript. PG participated in the experimental design of the study, cell culture, interpretation of results and manuscript prep- aration. PM participated in the experimental design of the proteomics part of the study, statistical analysis of the 2D- DIGE gels and manuscript preparation. MH participated in the identification of differentially expressed proteins by mass spectrometry. MC conceived the study, participated in its design and coordination. All authors read and approved the final manuscript. 12. Baik JY, Lee MS, An SR, Yoon SK, Joo EJ, Kim YH, Park HW, Lee GM: Initial transcriptome and proteome analyses of low culture temperature-induced expression in CHO cells producing erythropoietin. Biotechnol Bioeng 2006, 93(2):361-371. y p g ( ) 13. Gammell P, Barron N, Kumar N, Clynes M: Initial identification of low temperature and culture stage induction of miRNA expression in suspension CHO-K1 cells. J Biotechnol 2007, 130(3):213-8. ( ) 14. Underhill MF, Smales CM: The cold-shock response in mamma- lian cells: investigating the HeLa cell cold-shock proteome. Cytotechnology 2007, 53(1–3):47-53. Additional material 15. Chappell SA, Owens GC, Mauro VP: A 5' Leader of Rbm3, a Cold Stress-induced mRNA, Mediates Internal Initiation of Trans- lation with Increased Efficiency under Conditions of Mild Hypothermia. J Biol Chem 2001, 276(40):36917-36922. Additional file 1 MALDI-ToF MS identification of proteins that were differentially expressed at 144 hrs compared to 72 hrs using either temperature shifted or standard culture. Click here for file [http://www.biomedcentral.com/content/supplementary/1472- 6750-8-42-S1.doc] Additional file 1 MALDI-ToF MS identification of proteins that were differentially expressed at 144 hrs compared to 72 hrs using either temperature shifted or standard culture. Click here for file [http://www.biomedcentral.com/content/supplementary/1472- 6750-8-42-S1.doc] Authors' contributions NK carried out the cell culture, proteomics work, interpre- tation of results and manuscript preparation of this study. Page 11 of 13 (page number not for citation purposes) Page 11 of 13 (page number not for citation purposes) Acknowledgements This work was supported by funding from Science Foundation Ireland (SFI) grant 03/IN3/B395. p y ( ) 20. Tan HK, Lee MM, Yap MG, Wang DI: Overexpression of cold- inducible RNA-binding protein increases interferon-gamma production in Chinese-hamster ovary cells. Biotechnol Appl Bio- chem 2008, 49(Pt 4):247-257. References Kim IS, Kim DH, Han SM, Chin MU, Nam HJ, Cho HP, Choi SY, Song BJ, Kim ER, Bae YS, Moon YH: Truncated form of importin alpha identified in breast cancer cell inhibits nuclear import of p53. J Biol Chem 2000, 275(30):23139-23145. 33. Gerner C, Frohwein U, Gotzmann J, Bayer E, Gelbmann D, Bursch W, Schulte-Hermann R: The Fas-induced apoptosis analyzed by high throughput proteome analysis. J Biol Chem 2000, 275(50):39018-39026. ( ) 34. Huang G, Chen Y, Lu H, Cao X: Coupling mitochondrial respira- tory chain to cell death: an essential role of mitochondrial complex I in the interferon-beta and retinoic acid-induced cancer cell death. Cell Death Differ 2007, 14(2):327-337. ( ) 35. Wang S, Fusaro G, Padmanabhan J, Chellappan SP: Prohibitin co- localizes with Rb in the nucleus and recruits N-CoR and HDAC1 for transcriptional repression. Oncogene 2002, 21(55):8388-8396. ( ) 36. Choudhary S, Xiao T, Vergara LA, Srivastava S, Nees D, Piatigorsky J, Ansari NH: Role of aldehyde dehydrogenase isozymes in the defense of rat lens and human lens epithelial cells against oxi- dative stress. Invest Ophthalmol Vis Sci 2005, 46(1):259-267. p ( ) 37. Ejeskär K, Krona C, Carén H, Zaibak F, Li L, Martinsson T, Ioannou PA: Introduction of in vitro transcribed ENO1 mRNA into neuroblastoma cells induces cell death. BMC Cancer 2005, 5:161-175. 38. Lee YJ, Hou ZZ, Curetty L, Armour EP, al-Saadi A, Bernstein J, Corry PM: Heat-resistant variants of the Chinese hamster ovary cell: alteration of cellular structure and expression of vimen- tin. J Cell Physiol 1992, 151(1):138-146. J y ( ) 39. Yeh J, Haarer BK: Profilin is required for the normal timing of actin polymerization in response to thermal stress. FEBS Lett 1996, 398(2–3):303-307. ( ) 40. Li J, Li Q, Xie C, Zhou H, Wang Y, Zhang N, Shao H, Chan SC, Peng X, Lin SC, Han J: β-Actin is required for mitochondria cluster- ing and ROS generation in TNF-induced, caspase-independ- ent cell death. J Cell Sci 2004, 117:4673-4680. J 41. Casella JF, Craig SW, Maack DJ, Brown AE: Cap Z(36/32), a barbed end actin-capping protein, is a component of the Z-line of skeletal muscle. J Cell Biol 1987, 105(1):371-379. J ( ) 42. Xu D, Guthrie JR, Mabry S, Sack TM, Truog WE: Mitochondrial aldehyde dehydrogenase attenuates hyperoxia-induced cell death through activation of ERK/MAPK and PI3K-Akt path- ways in lung epithelial cells. References Schepens B, Tinton SA, Bruynooghe Y, Parthoens E, Haegman M, Bey- aert R, Cornelis S: A role for hnRNP C1/C2 and Unr in internal initiation of translation during mitosis. EMBO J 2007, 26(1):158-169. ( ) 7. Moore A, Mercer J, Dutina G, Donahue CJ, Bauer KD, Mather JP, Etcheverry T, Ryll T: Effects of temperature shift on cell cycle, apoptosis and nucleotide pools in CHO cell batch cultures. Cytotechnology 1997, 23(1–3):47-54. 27. Fan H, Penman S: Regulation of protein synthesis in mamma- lian cells. II. Inhibition of protein synthesis at the level of ini- tiation during mitosis. J Mol Biol 1970, 50(3):655-670. 28. Milkereit P, Tschochner H: A specialized form of RNA polymer- ase I, essential for initiation and growth-dependent regula- tion of rRNA synthesis, is disrupted during transcription. EMBO J 1998, 17(13):3692-3703. y gy ( ) 8. Furukawa K, Ohsuye K: Effect of culture temperature on a recombinant CHO cell line producing a C-terminal a-ami- dating enzyme. Cytotechnology 1998, 26(2):153-164. g y y gy ( ) 9. Kumar N, Gammell P, Clynes M: Proliferation control strategies to improve productivity and survival during CHO based pro- duction culture: A summary of recent methods employed and the effects of proliferation control in product secreting CHO cell lines. Cytotechnology 2007, 53(1–3):33-46. J ( ) 29. Wells V, Mallucci L: dentification of an autocrine negative growth factor: mouse beta-galactoside-binding protein is a cytostatic factor and cell growth regulator. Cell 1991, 64(1):91-97. ( ) 30. Zhang Z, Sobel RA, Cheng D, Steinberg GK, Yenari MA: Mild hypo- thermia increases Bcl-2 protein expression following global cerebral ischemia. Brain Res Mol Brain Res 2001, 95(1–2):75-85. y gy ( ) 10. Yoon SK, Hwang SO, Lee GM: Enhancing effect of low culture temperature on specific antibody productivity of recom- binant Chinese hamster ovary cells: clonal variation. Biotech- nol Prog 2004, 20(6):1683-1688. ( ) 31. Kumari G, Singhal PK, Rao MR, Mahalingam S: Nuclear transport of Ras-associated tumor suppressor proteins: different trans- 31. Kumari G, Singhal PK, Rao MR, Mahalingam S: Nuclear transport of Ras-associated tumor suppressor proteins: different trans- Page 12 of 13 (page number not for citation purposes) Page 12 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1472-6750/8/42 http://www.biomedcentral.com/1472-6750/8/42 BMC Biotechnology 2008, 8:42 port receptor binding specificities for arginine-rich nuclear targeting signals. J Mol Biol 2007, 367(5):1294-1311. g g g J ( ) 32. References Am J Physiol Lung Cell Mol Physiol 2006, 291(5):L966-L975. ( ) 43. Fukata Y, Itoh TJ, Kimura T, Menager C, Nishimura T, Shiromizu T, Watanabe H, Inagaki N, Iwamatsu A, Hotani H, Kaibuchi K: CRMP- 2 binds to tubulin heterodimers to promote microtubule assembly. Nat Cell Biol 2002, 4(8):583-591. y ( ) 44. Franzen B, Yang Y, Sunnemark D, Wickman M, Ottervald J, Opper- mann M, Sandberg K: Dihydropyrimidinase related protein-2 as a biomarker for temperature and time dependent post mor- tem changes in the mouse brain proteome. Proteomics 2003, 3(10):1920-1929. y ( ) 44. Franzen B, Yang Y, Sunnemark D, Wickman M, Ottervald J, Opper- mann M, Sandberg K: Dihydropyrimidinase related protein-2 as a biomarker for temperature and time dependent post mor- tem changes in the mouse brain proteome. Proteomics 2003, 3(10):1920-1929. References Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 13 of 13 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge Page 13 of 13 (page number not for citation purposes)
https://openalex.org/W2096780782	https://bmcpublichealth.biomedcentral.com/counter/pdf/10.1186/1471-2458-7-145	English	null	Response rates for providing a blood specimen for HIV testing in a population-based survey of young adults in Zimbabwe	BMC public health	2,007	cc-by	4,426	BioMed Central BioMed Central Received: 22 January 2007 Accepted: 5 July 2007 This article is available from: http://www.biomedcentral.com/1471-2458/7/145 This article is available from: http://www.biomedcentral.co This article is available from: http://www.biomedcentral.co © 2007 McNaghten et al; licensee BioMed Central Ltd. © 2007 McNaghten et al; licensee BioMed Central Ltd. © 2007 McNaghten et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creative which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cit Abstract Background: To determine differences among persons who provided blood specimens for HIV testing compared with those who did not among those interviewed for the population-based Zimbabwe Young Adult Survey (YAS). Methods: Chi-square analysis of weighted data to compare demographic and behavioral data of persons interviewed who provided specimens for anonymous testing with those who did not. Prevalence estimation to determine the impact if persons not providing specimens had higher prevalence rates than those who did. Results: Comparing those who provided specimens with those who did not, there was no significant difference by age, residence, education, marital status, perceived risk, sexual experience or number of sex partners for women. A significant difference by sexual experience was found for men. Prevalence estimates did not change substantially when prevalence was assumed to be two times higher for persons not providing specimens. Conclusion: When comparing persons who provided specimens for HIV testing with those who did not, few significant differences were found. If those who did not provide specimens had prevalence rates twice that of those who did, overall prevalence would not be substantially affected. Refusal to provide blood specimens does not appear to have contributed to an underestimation of HIV prevalence. BMC Public Health Open Access p Research article Response rates for providing a blood specimen for HIV testing in a population-based survey of young adults in Zimbabwe AD McNaghten1, Joan M Herold2,3, Hazel M Dube4 and Michael E St Louis1 Address: 1Division of HIV/AIDS Prevention, National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention, Centers for Disease Control and Prevention, Atlanta, USA, 2Division of Reproductive Health, National Center for Chronic Disease Prevention and Health Promotion, Centers for Disease Control and Prevention, Atlanta, USA, 3Department of Behavioral Sciences and Health Education, Emory University, Atlanta, USA and 4Operations Research, Family Health International, Harare, Zimbabwe Email: AD McNaghten - aom5@cdc.gov; Joan M Herold - JHerold@cdc.gov; Hazel M Dube - hdube@zol.co.zw; Michael E St Louis mes2@cdc gov Email: AD McNaghten* - aom5@cdc.gov; Joan M Herold - JHerold@cdc.gov; Hazel M Dube - hdube@zol.co.zw; Michael E St Louis - mes2@cdc.gov * Corresponding author Published: 5 July 2007 BMC Public Health 2007, 7:145 doi:10.1186/1471-2458-7-145 Received: 22 January 2007 Accepted: 5 July 2007 This article is available from: http://www.biomedcentral.com/1471-2458/7/145 © 2007 McNaghten et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Received: 22 January 2007 Accepted: 5 July 2007 Page 1 of 6 (page number not for citation purposes) Methods h i b The Zimbabwe YAS was a nationally representative survey of men and women aged 15–29 years designed to esti- mate the prevalence of behaviors that may place young adults at risk for HIV, to evaluate the coverage and quality of services for HIV prevention and care, and to assess HIV prevalence among young adults in Zimbabwe. The design was a multi-stage household probability sample: the pri- mary sampling units were 187 census enumeration areas in four geographic strata; the secondary sampling units were households in the selected census enumeration areas; and the tertiary sampling units were all eligible respondents in the selected households. In the second stage, households were randomly selected to belong to either the female sample or the male sample. Female interview teams went to households in the female sample and male interview teams went to households in the male sample to administer the household questionnaire. A ros- ter in the household questionnaire identified eligible respondents living in the household; an eligible respond- ent in a female sample household would be a female aged 15–29 and in a male sample household a male aged 15– 29. Household and individual interviews were conducted between September 2001 and February 2002. Filter paper was used to collect finger prick blood specimens, which were unlinked and tested anonymously for HIV antibod- ies. All persons interviewed were given a voucher for free HIV testing at a voluntary counseling and testing center and a transport subsidy. The Medical Research Council of Zimbabwe and the U.S. Centers for Disease Control and Prevention reviewed the protocol for data and specimen collection methods, including ethical considerations, and approved the YAS. Written informed consent was obtained from all participants who agreed to provide a blood specimen; additional parental consent was obtained for participants less than 16 years of age. To determine how prevalence estimates would be affected if persons who were interviewed but elected not to pro- vide a specimen for testing had higher HIV infection rates, prevalence estimates were computed assuming persons who were interviewed but did not provide a specimen for testing had prevalence rates 2 times higher than those who provided a specimen. "Positives" were randomly assigned among those who did not provide a specimen. ferent from those who consent to both the interview and HIV testing [1]. Because the sample design resulted in the oversampling of urban areas, weights were constructed to adjust for dispro- portionate stratification. Non-response weights were con- structed using demographic data available from the household rosters collected during the household inter- views. The sample design weights and the non-response weights were combined to produce a total weight for the individual respondents. With the exception of the response rates, all percentages and numbers presented were weighted by the total weights [2]. Among those who agreed to be interviewed in the Zimba- bwe Young Adult Survey (YAS), we compared demo- graphic and behavioral data from those who consented to and provided a blood specimen for anonymous HIV test- ing with those who declined to provide a specimen. We also estimated the impact on HIV prevalence if persons who did not provide a specimen had higher prevalence rates than those who provided specimens for testing to determine how this would impact overall prevalence rates. Chi-square analyses were performed using SAS version 8.2 (SAS Institute, Cary, NC) to compare demographic and behavioral data of persons interviewed who provided specimens for HIV testing with those who did not by age group, area of residence, education level, marital status, perceived risk of HIV infection, sexual experience and for sexually experienced respondents, number of lifetime sex partners. Background exclusion of individuals who were not at home during the survey period or who refuse to participate can bias the results, possibly leading to an underestimation or overes- timation of national HIV prevalence. The risk of HIV infection among persons who do not participate in the survey because they refuse or are not at home, or who agree to be interviewed but refuse to be tested may be dif- g Many countries in sub-Saharan African have conducted national population-based surveys that include HIV-1 testing. Although such surveys represent a major technical advance over convenience samples such as surveys in antenatal care settings, they introduce other technical issues. The representativeness of such surveys is an issue as Page 1 of 6 (page number not for citation purposes) Page 1 of 6 (page number not for citation purposes) http://www.biomedcentral.com/1471-2458/7/145 http://www.biomedcentral.com/1471-2458/7/145 BMC Public Health 2007, 7:145 ferent from those who consent to both the interview and HIV testing [1]. ferent from those who consent to both the interview and HIV testing [1]. Results Female Household Sample 6,671 Household Interview 6,369 (97%) Households with No Women Eligible for Individual Interview 1,893 Households with Women Eligible for Individual Interview 4,476 Total Women Eligible for Individual Interview 5,469 No Individual Interview 661 (12%) Individual Interview 4,808 (88%) Refused Interview or Respondent Absent 204 (3%) Dwelling Vacant or not a Household 98 No Blood Specimen 545 (11%) Blood Specimen 4,263 (89%) Household and individual response rates among males Figure 2 Household and individual response rates among males. Unweighted data. Male Household Sample 7,662 Household Interview 7,295 (96%) Households with No Men Eligible for Individual Interview 3,209 Households with Men Eligible for Individual Interview 4,086 Total Men Eligible for Individual Interview 5,082 No Individual Interview 878 (17%) Individual Interview 4,204 (83%) Refused Interview or Respondent Absent 275 (4%) Dwelling Vacant or not a Household 92 No Blood Specimen 371 (9%) Blood Specimen 3,833 (91%) Househol Figure 2 Household and individual response rates among females Figure 1 Household and individual response rates among females. Unweighted data. Household and individual response rates among males Figure 2 Household and individual response rates among males. Unweighted data. Household and individual response rates among females Figure 1 Household and individual response rates among females. Unweighted data. Household and individual response rates among males Figure 2 Household and individual response rates among males. Unweighted data. Among women who were selected for individual inter- view, the nonresponse comprised 4.6% refusals, 4.4% not at home, 1.2% incapacitated and 2.0% incomplete inter- views. Among men selected for individual interview, non- response comprised 4.5% refusals, 9.7% not at home, 1.1% incapacitated and 2.3% incomplete interviews. ital status, perceived risk of HIV infection or number of lifetime sex partners. Overall estimates of HIV prevalence assuming those who were interviewed but did not provide a specimen for HIV testing had a prevalence rate 2 times higher than those who did provide a specimen for testing were not substan- tially higher than the overall prevalence based on women and men aged 15–29 years who provided specimens (Table 3). Estimates of prevalence would increase from 21.8% to 24.2% among women and from 10.3% to 11.1% among men. This effect is also shown by age group for women and men in Table 3. Results The number of female and male household and individ- ual questionnaires completed and acceptance or refusal of testing is outlined in Figures 1 and 2. Of 6,671 female and 7,662 male households visited, 97% of female and 96% of male households were interviewed. Among female households interviewed, 70% had at least one female eli- gible for individual interview and 30% did not have an eligible female. Among male households interviewed, 56% had at least one male eligible for individual inter- view and 44% did not have an eligible male. Of 5,469 eli- gible young women identified in the 4,476 female households, 88% were interviewed. Of 5,082 eligible young men identified in the 4,086 male households, 83% were interviewed. Eighty-nine percent of women and 91% of men interviewed also agreed to provide a specimen for HIV testing for an overall response rate (household inter- view rate multiplied by individual interview rate multi- plied by blood specimen rate) of 76% among women and 72% among men. Of female households selected, 1.6% had residents who were not at home, 1.5% was vacant, destroyed or not found and 1.0% refused. Of male households selected, 1.3% had residents who were not at home, 1.2% was vacant, destroyed or not found and 1.6% refused. Interview data were weighted to adjust for the sample design and for non-response. The original sample was stratified by four geographic areas: the two major cities, Harare and Bulawayo, other urban areas, and rural areas. Page 2 of 6 (page number not for citation purposes) Page 2 of 6 (page number not for citation purposes) BMC Public Health 2007, 7:145 http://www.biomedcentral.com/1471-2458/7/145 Household and individual response rates among males Figure 2 Household and individual response rates among males. Unweighted data. Male Household Sample 7,662 Household Interview 7,295 (96%) Households with No Men Eligible for Individual Interview 3,209 Households with Men Eligible for Individual Interview 4,086 Total Men Eligible for Individual Interview 5,082 No Individual Interview 878 (17%) Individual Interview 4,204 (83%) Refused Interview or Respondent Absent 275 (4%) Dwelling Vacant or not a Household 92 No Blood Specimen 371 (9%) Blood Specimen 3,833 (91%) Household and individual response rates among females Figure 1 Household and individual response rates among females. Unweighted data. Page 3 of 6 (page number not for citation purposes) Results % Provided Specimen (No.) weighted n = 4293 % Did Not Provide Specimen (No.) weighted n = 517 p-value WOMEN Age Group 0.4137 15–19 42.1 (1807) 39.3 (203) 20–24 32.5 (1395) 35.0 (181) 25–29 25.4 (1091) 25.7 (133) Area of Residence 0.0733 Urban 38.0 (1629) 42.0 (217) Rural 62.0 (2664) 58.0 (300) Education Level 0.0835 Primary or less 33.3 (1428) 34.2 (177) 1–3 years secondary 32.0 (1374) 27.4 (142) ≥ 4 years secondary 34.7 (1491) 38.4 (199) Marital Status 0.8701 Previously Married 8.2 (352) 8.9 (46) Currently Married 46.2 (1985) 45.8 (237) Never Married 45.6 (1956) 45.3 (235) Perceived Risk of HIV Infection 0.0574 None 56.8 (2288) 62.6 (303) Low 12.6 (507) 13.0 (63) Medium 13.4 (539) 10.4 (50) High 7.6 (304) 7.2 (35) Don't Know 9.5 (382) 6.8 (33) Sexual Experience 0.4795 Ever had sex 66.1 (2834) 64.6 (334) Never had sex 33.9 (1455) 35.4 (183) Number of Lifetime Sex Partners 0.8990 1 71.0 (2014) 71.8 (239) 2 19.8 (563) 18.8 (62) 3 or more 9.2 (260) 9.4 (31) Table 1: Provision of blood specimen for anonymous HIV testing among women who were interviewed. WOMEN Age Group 15–19 20–24 25–29 Area of Residence Urban Rural Education Level Primary or less 1–3 years secondary ≥ 4 years secondary Marital Status Previously Married Currently Married Never Married Perceived Risk of HIV Infection None Low Medium High Don't Know Sexual Experience Ever had sex Never had sex Number of Lifetime Sex Partners 1 2 3 or more Results It is important to note that if respondents who did not provide a specimen had a lower prevalence, say, 2 times lower than those who did provide a specimen, the decrease in total prevalence would produce the same magnitude of difference as the increase but in the opposite direction (data not shown). When comparing women who provided a specimen for HIV testing and those who did not, we found no signifi- cant difference by age group, residence, education level, marital status, perceived risk of HIV infection, sexual expe- rience or number of lifetime sex partners (Table 1). Com- paring men who provided a specimen for HIV testing with men who did not, we found a significant difference by sex- ual experience (Table 2). A larger percentage of men who provided a specimen for testing reported they had ever had sex. There was no significant difference between men who provided a specimen for testing and men who did not by age group, area of residence, education level, mar- Page 3 of 6 (page number not for citation purposes) Page 3 of 6 (page number not for citation purposes) http://www.biomedcentral.com/1471-2458/7/145 BMC Public Health 2007, 7:145 Table 1: Provision of blood specimen for anonymous HIV testing among women who were interviewed. Table 1: Provision of blood specimen for anonymous HIV testing among women who were interviewed. Page 4 of 6 (page number not for citation purposes) Discussion f We examined these data to determine if there were any potential sources of bias that could have resulted in the underestimation or overestimation of HIV prevalence in the YAS. We found no significant demographic or behav- ioral differences among women who provided a specimen for testing and women who did not. The only significant finding among men was by sexual experience. Among men who provided a specimen, a larger percentage reported ever having sex. In the YAS, HIV prevalence among sexually experienced men was over 5 times higher than men who reported never having sex [2]. The overrep- resentation of sexually experienced males in the group that provided specimens therefore could have contributed to an overestimation of HIV prevalence. Both female and male household interview response rates were high (97% and 96%, respectively) compared with other national population-based surveys in sub-Saharan Africa (75.4%–99.7%) [3][4]. Individual interview response rates were relatively high as well. Similar to other population-based surveys conducted in African countries during this same time period, overall response rates declined when HIV testing was included [3]. Gregson et al. found that HIV prevalence was higher in working men in rural Zimbabwe [5], and it is possible that some of the men who were not interviewed may have been working away from home. Lydie et al. found that among men in Cameroon, as time away from town increased, so did HIV prevalence [6]. However, individu- als who were not interviewed because they were not at home during the survey period or refused to participate should not have biased the sample because these data were weighted to adjust for such non-response. This weighting to adjust for non-response should have ensured the representativeness of the survey. If those interviewed but not providing a specimen for HIV testing had prevalence rates 2 times higher or 2 times lower than those who provided specimens, YAS preva- lence estimates would not be greatly affected. The magni- tude of difference in prevalence for those providing specimens versus those electing not to was greatest in the 25–29 year age group for both women and men. Page 4 of 6 (page number not for citation purposes) Page 4 of 6 (page number not for citation purposes) http://www.biomedcentral.com/1471-2458/7/145 BMC Public Health 2007, 7:145 Table 2: Provision of blood specimen for anonymous HIV testing among men who were interviewed. Discussion f Table 2: Provision of blood specimen for anonymous HIV testing among men who were interviewed. Table 2: Provision of blood specimen for anonymous HIV testing among men who were interviewed. % Provided Specimen (No.) weighted n = 3844 % Did Not Provide Specimen (No.) weighted n = 356 p-value MEN Age Group 0.2277 15–19 43.0 (1653) 47.8 (170) 20–24 29.3 (1125) 27.0 (96) 25–29 27.7 (1065) 25.3 (90) Area of Residence 0.2759 Urban 41.4 (1591) 44.4 (158) Rural 58.6 (2253) 55.6 (198) Education Level 0.0830 Primary or less 21.9 (842) 27.1 (96) 1–3 years secondary 30.3 (1164) 28.6 (102) ≥ 4 years secondary 47.8 (1838) 44.3 (158) Marital Status 0.1210 Previously Married 2.2 (86) 0.6 (2) Currently Married 21.1 (812) 21.3 (76) Never Married 76.7 (2946) 78.1 (278) Perceived Risk of HIV Infection 0.1849 None 73.2 (2729) 72.3 (243) Low 11.8 (439) 12.4 (42) Medium 8.4 (312) 7.0 (24) High 4.8 (178) 4.4 (15) Don't Know 1.9 (72) 3.8 (13) Sexual Experience 0.0026* Ever had sex 62.5 (2401) 54.4 (194) Never had sex 37.5 (1443) 45.6 (163) Number of Lifetime Sex Partners 0.1655 1 30.6 (733) 36.7 (71) 2 20.8 (498) 17.1 (33) 3 or more 48.6 (1165) 46.2 (90) *p < 0.05 surveillance, including a lack of HIV prevalence data on men, non-pregnant women, women receiving care from private providers and non-sexually active women. Studies in sub-Saharan Africa have shown that HIV prevalence in High rates of infection among women aged 15–29 years attending antenatal clinics (ANC) (24.6%) have been doc- umented by Zimbabwe's ANC sentinel surveillance pro- gram [7]. However, there are limitations of ANC sentinel Table 3: Effect of higher prevalence among interviewees who did not provide a blood specimen for HIV testing on YAS prevalence estimates by sex. Prevalence (%) If persons not providing a specimen were 2x higher (%) WOMEN Age Group 15–19 10.6 11.7 20–24 26.1 29.1 25–29 34.7 38.5 Total (15–29) 21.8 24.2 MEN Age Group 15–19 2.1 2.3 20–24 9.2 9.9 25–29 24.4 26.3 Total (15–29) 10.3 11.1 Table 3: Effect of higher prevalence among interviewees who did not provide a blood specimen for HIV testing on YAS prevalence estimates by sex. References 1. Buvé A, Lagarde E, Carael M, Rutenberg N, Ferry B, Glynn JR, et al.: Interpreting Sexual Behaviour Data: validity issues in the multicentre study on factors determining the differential spread of HIV in four African cities. AIDS 2001, 15(Suppl 4):S117-126. ) 2. Ministry of Health and Child Welfare: The Zimbabwe Young Adult Sur- vey 2001–2002. Zimbabwe 2004. y 3. Boerma JT, Ghys PD, Walker N: Estimates of HIV-1 prevalence from national population-based surveys as a new gold stand- ard. Lancet 2003, 362:1929-31. 4. Garcia-Calleja JM, Gous E, Ghys PD: National population based HIV prevalence surveys in sub-Saharan Africa: results and implications for HIV and AIDS estimates. Sex Transm Infect 2006, 82(Suppl 3):iii64-70. ( pp ) 5. Gregson S, Mason PR, Garnett GP, Zhuwau T, Nyamukapa CA, Aner- son RM, et al.: A rural HIV epidemic in Zimbabwe? Findings from a population-based survey. Int J STD AIDS 2001, 12(3):189-196. ( ) 6. Lydie N, Robinson NJ, Ferry B, Akam E, De Loenzien M, Abega S, Study Group on Heterogeneity of HIV Epidemics in African Cities: Mobility, sexual behavior, and HIV infection in an urban pop- ulation in Cameroon. J Acquir Immune Defic Syndr 2004, 35(1):67-74. Although there are many advantages of using a popula- tion-based survey to estimate HIV prevalence among young Zimbabwean adults, there were some limitations in the YAS and this analysis. Reasons for not providing a specimen for HIV testing were not collected, but likely rea- sons include fear of the procedure and fear that confiden- tiality may not be upheld. Because the sample was drawn from households, police, military and incarcerated popu- lations were not included in the YAS. The prevalence of HIV in young adults in these populations may be very dif- ferent than that of the general population. ( ) 7. Ministry of Health and Child Welfare: National Survey of HIV and Syph- ilis Prevalence Among Women Attending Antenatal Clinics in Zimbabwe, 2002. Zimbabwe 2004. 8. Zaba B, Gregson S: Measuring the impact of HIV on fertility in Africa. AIDS 1998, 12(Suppl 1):S41-50. , ( pp ) 9. UNAIDS: Report on the UNAIDS Epidemiology Reference Group Meeting, Rome, October 8–10, 2000. Oxford 2000. 10. Gregson S, Garnett GP: Contrasting gender differentials in HIV-1 prevalence and associated mortality increase in east- ern and southern Africa: artifact of data or natural course of epidemics? AIDS 2000, 14(Suppl 3):S85-99. p ( pp ) 11. References Central Statistical Office [Zimbabwe] and Macro International Inc: Zimbabwe Demographic and Health Survey 1999. Calverton, Maryland 2000. When comparing demographic and behavioral character- istics of persons who provided specimens for HIV testing with those who did not, few significant differences were found that could have potentially contributed to overesti- mating or underestimating HIV prevalence in our study population. If those who did not provide specimens had prevalence rates even twice (or one-half) that of those who did, prevalence would not be substantially affected. Therefore, the estimates obtained from the YAS appear representative of the population aged 15–29 years in Zim- babwe. Competing interests p g The author(s) declare that they have no competing inter- ests. Pre-publication history p y The pre-publication history for this paper can be accessed here: The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2458/7/145/pre pub http://www.biomedcentral.com/1471-2458/7/145 BMC Public Health 2007, 7:145 pregnant women frequently underestimates prevalence in women of reproductive age in the general population [8], are generally higher than the prevalence in men [9] but fairly accurately represents prevalence of both men and women in the general population [10]. Periodic popula- tion-based surveys can serve not only to provide reasona- bly accurate estimates of HIV prevalence to supplement sentinel surveillance data, but can help validate estimates obtained from sentinel sites or produced using mathemat- ical models. Similarities in the Zimbabwe ANC and YAS prevalence data are probably due to the high ANC attend- ance in the country [11]. Of the women in the YAS who reported being pregnant in the five years prior to being interviewed, 95.0% reported receiving antenatal care [2]. pregnant women frequently underestimates prevalence in women of reproductive age in the general population [8], are generally higher than the prevalence in men [9] but fairly accurately represents prevalence of both men and women in the general population [10]. Periodic popula- tion-based surveys can serve not only to provide reasona- bly accurate estimates of HIV prevalence to supplement sentinel surveillance data, but can help validate estimates obtained from sentinel sites or produced using mathemat- ical models. Similarities in the Zimbabwe ANC and YAS prevalence data are probably due to the high ANC attend- ance in the country [11]. Of the women in the YAS who reported being pregnant in the five years prior to being interviewed, 95.0% reported receiving antenatal care [2]. Authors' contributions Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge AM conceived the study, its design, and drafted the man- uscript. JH performed analyses and contributed to the writing of the manuscript. HD and MS contributed to the content and writing. All authors read and approved the final manuscript. Discussion f Prevalence (%) If persons not providing a specimen were 2x higher (%) ce among interviewees who did not provide a blood specimen for HIV testing on YAS prevalence Table 3: Effect of higher prevalence among interviewees who did not provide a blood specimen for HIV t estimates by sex. Page 5 of 6 (page number not for citation purposes) http://www.biomedcentral.com/1471-2458/7/145 http://www.biomedcentral.com/1471-2458/7/145 Acknowledgements g Sources of support for the Zimbabwe Young Adult Survey included the Zimbabwe Ministry of Health and Child Welfare, the Zimbabwe National Family Planning Council, the University of Zimbabwe School of Medicine, and the U.S. Centers for Disease Control and Prevention, Global AIDS Pro- gram.
https://openalex.org/W4361936295	https://aacr.figshare.com/articles/journal_contribution/Figure_S1_from_Identification_of_Recurrent_Activating_i_HER2_i_Mutations_in_Primary_Canine_Pulmonary_Adenocarcinoma/22474644/1/files/39926139.pdf	Aragonese	null	Figure S1 from Identification of Recurrent Activating <i>HER2</i> Mutations in Primary Canine Pulmonary Adenocarcinoma	null	2,023	cc-by	1,562	Primary Tumor location Total=76 Left cranial lobe (10.53%) Left caudal lobe (28.95%) Right cranial lobe (9.21%) Right middle lobe(5.26%) Right caudal lobe (17.11%) Unspecified (28.95%) Treatment Figure S1 Lung lobectomy (71.05%) Vinorelbine only (1.32%) Lobectomy and radiation(1.32%) Lobectomy, cytotoxic chemo or toceranib (14.47%) None(2.63%) Unknown (7.89%) Other (1.32) Total=76 7 T o ta l= 7 6 T re a tm e n t L u n g L o b e c to m y T o ta l= 7 6 P rim a ry T u m o r L o c a tio n L e ft c a u d a l r r yr yr 0 yr yr 2 yr 3 yr yr yr kn o w n Primary Tumor location Total=76 Left cranial lobe (10.53%) Left caudal lobe (28.95%) Right cranial lobe (9.21%) Right middle lobe(5.26%) Right caudal lobe (17.11%) Unspecified (28.95%) Treatment Figure S1 Lung lobectomy (71.05%) Vinorelbine only (1.32%) Lobectomy and radiation(1.32%) Lobectomy, cytotoxic chemo or toceranib (14.47%) None(2.63%) Unknown (7.89%) Other (1.32) Total=76 7 T o ta l= 7 6 T re a tm e n t L u n g L o b e c to m y T o ta l= 7 6 P rim a ry T u m o r L o c a tio n L e ft c a u d a l r r yr yr 0 yr yr 2 yr 3 yr yr yr kn o w n Affected Breeds Total=76 Mixed (25.00%) Labrador Retriever (21.05%) Bichon Frise (3.95%) Cocker Spaniel (3.95%) Miniature Schnautzer (3.95%) Shih Tzu (2.63%) Basset Hound(2.63%) Boston terrier(2.63%) Golden Retriever (2.63%) Other Purbreeds of n=1 (31.58%) Age at Diagnosis Total=76 6 yr (1.32%) 7 yr(3.95%) 8 yr (11.84%) 9 yr (11.84%) 10 yr (14.47%) 11 yr (21.05%) 12 yr(17.11%) 13 yr (13.16%) 14 yr (2.53%) 15 yr (1.32%) Unknown (1.32%) Sex Distribution Adenocarcinoma Subtype Spayed Female (48.68%) Unknown Female (1.32%) Castrated Male (42.11%) Intact Male (6.58%) Unknown (1.32%) Papillary (61.84%) Tubulopapillary (11.84%) Mucinous Papillary( 6.58%) Acinar(10.53%) Solid (3.95%) Unespecified Subtype (5.26) Total=76 Total=76 T o ta l= 7 6 A g e a t D ia g n o s is 1 1 y r 4 8 .6 8 % S p a y e d F e m a le 1 .3 2 % U n k n o w n F e m a le 4 2 .1 1 % C a s tra te d M a le 6 .5 8 % In ta c t M a le T o ta l= 7 6 S e x D is trib u tio n 1 .3 2 % U n k n o w n S p a y e d F e m a le 2 5 .0 0 % M ix e d 2 1 .0 5 % L a b ra d o r R e trie v e r 3 .9 5 % B ic h o n F rise 3 .9 5 % C o c k e r S p a n ie l 3 .9 5 % M in ia tu re S ch n a u ze r 2 .6 3 % S h ih T zu 2 .6 3 % B a ss e t H o u n d 2 .6 3 % B o sto n T e rrie r 2 .6 3 % G o ld e n R e trie v e r 3 1 .5 8 % O th e r P u re b re e d s o f n = 1 T o ta l= 7 6 A ffe c te d B re e d s M ix e d B re e d L a b ra d o r R e trie v e r Primary Tumor location P rim a ry T u m o r L o c a tio n Affected Breeds Total=76 Mixed (25.00%) Labrador Retriever (21.05%) Bichon Frise (3.95%) Cocker Spaniel (3.95%) Miniature Schnautzer (3.95%) Shih Tzu (2.63%) Basset Hound(2.63%) Boston terrier(2.63%) Golden Retriever (2.63%) Other Purbreeds of n=1 (31.58%) Age at Diagnosis Total=76 6 yr (1.32%) 7 yr(3.95%) 8 yr (11.84%) 9 yr (11.84%) 10 yr (14.47%) 11 yr (21.05%) 12 yr(17.11%) 13 yr (13.16%) 14 yr (2.53%) 15 yr (1.32%) Unknown (1.32%) T o ta l= 7 6 A g e a t D ia g n o s is 1 1 y r 2 5 .0 0 % M ix e d 2 1 .0 5 % L a b ra d o r R e trie v e r 3 .9 5 % B ic h o n F rise 3 .9 5 % C o c k e r S p a n ie l 3 .9 5 % M in ia tu re S ch n a u ze r 2 .6 3 % S h ih T zu 2 .6 3 % B a ss e t H o u n d 2 .6 3 % B o sto n T e rrie r 2 .6 3 % G o ld e n R e trie v e r 3 1 .5 8 % O th e r P u re b re e d s o f n = 1 T o ta l= 7 6 A ffe c te d B re e d s M ix e d B re e d L a b ra d o r R e trie v e r Affected Breeds A ffe c te d B re e d s Age at Diagnosis A g e a t D ia g n o s is Age at Diagnosis Total=76 6 yr (1.32%) 7 yr(3.95%) 8 yr (11.84%) 9 yr (11.84%) 10 yr (14.47%) 11 yr (21.05%) 12 yr(17.11%) 13 yr (13.16%) 14 yr (2.53%) 15 yr (1.32%) Unknown (1.32%) T o ta l= 7 6 A g e a t D ia g n o s is 1 1 y r Primary Tumor location Total=76 Left cranial lobe (10.53%) Left caudal lobe (28.95%) Right cranial lobe (9.21%) Right middle lobe(5.26%) Right caudal lobe (17.11%) Unspecified (28.95%) T o ta l= 7 6 P rim a ry T u m o r L o c a tio n L e ft c a u d a l r r r r o w n Affected Breeds Total=76 Mixed (25.00%) Labrador Retriever (21.05%) Bichon Frise (3.95%) Cocker Spaniel (3.95%) Miniature Schnautzer (3.95%) Shih Tzu (2.63%) Basset Hound(2.63%) Boston terrier(2.63%) Golden Retriever (2.63%) Other Purbreeds of n=1 (31.58 2 5 .0 0 % M ix e d 2 1 .0 5 % L a b ra d o r R e 3 .9 5 % B ic h o n F rise 3 .9 5 % C o c k e r S p a n i 3 .9 5 % M in ia tu re S ch n 2 .6 3 % S h ih T zu 2 .6 3 % B a ss e t H o u n d 2 .6 3 % B o sto n T e rrie 2 .6 3 % G o ld e n R e trie 3 1 .5 8 % O th e r P u re b T o ta l= 7 6 A ffe c te d B re e d s M ix e d B re e d L a b ra d o r R e trie v e r Total=76 Left cranial lobe (10.53%) Left caudal lobe (28.95%) Right cranial lobe (9.21%) Right middle lobe(5.26%) Right caudal lobe (17.11%) Unspecified (28.95%) T o ta l= 7 6 Shih Tzu (2.63%) Basset Hound(2.63%) Boston terrier(2.63%) Golden Retriever (2.63%) Other Purbreeds of n=1 ( Sex Distribution Adenocarcinoma Subtype Spayed Female (48.68%) Unknown Female (1.32%) Castrated Male (42.11%) Intact Male (6.58%) Unknown (1.32%) Papillary (61.84%) Tubulopapillary (11.84%) Mucinous Papillary( 6.58%) Acinar(10.53%) Solid (3.95%) Unespecified Subtype (5.26) Total=76 Total=76 4 8 .6 8 % S p a y e d F e m a le 1 .3 2 % U n k n o w n F e m a le 4 2 .1 1 % C a s tra te d M a le 6 .5 8 % In ta c t M a le T o ta l= 7 6 S e x D is trib u tio n 1 .3 2 % U n k n o w n S p a y e d F e m a le Sex Distribution Spayed Female (48.68%) Unknown Female (1.32%) Castrated Male (42.11%) Intact Male (6.58%) Unknown (1.32%) Total=76 T o ta l= 7 6 S e x D is trib u tio n S p a y e d F e m a le Sex Distribution S e x D is trib u tio n Adenocarcinoma Subtype Treatment Figure S1 Lung lobectomy (71.05%) Vinorelbine only (1.32%) Lobectomy and radiation(1.32%) Lobectomy, cytotoxic chemo or toceranib (14.47%) None(2.63%) Unknown (7.89%) Other (1.32) Total=76 T o ta l= 7 6 T re a tm e n t L u n g L o b e c to m y Adenocarcinoma Subtype Papillary (61.84%) Tubulopapillary (11.84%) Mucinous Papillary( 6.58%) Acinar(10.53%) Solid (3.95%) Unespecified Subtype (5.26) Total=76 Spayed Female (48.68%) Unknown Female (1.32%) Castrated Male (42.11%) Intact Male (6.58%) Unknown (1.32%)
W4391025794.txt	https://journals.wlb-stuttgart.de/ojs/index.php/wfr/article/download/9693/9583	de		Rezension von: Diefenbacher, Karl (Hrsg.), Schweizer Einwanderer in den Kraichgau nach dem Dreißigjährigen Krieg	Württembergisch Franken	2,024	cc-by	1,080	Neue Bücher 307 Claus Peter Clasen: Die Augsburger Weber. Leistungen und Krisen des Textilgewerbes um 1600. (= Abhandlungen zur Geschichte der Stadt Augsburg, Schriftenreihe des Stadtarchivs Augsburg, 27). Augsburg: Mühlberger 1981. 460 S. Der Verfasser, gebürtiger Berliner, jetzt Professor an der Universität in Los Angeles, untersucht in mustergültiger Weise alles, was das Augsburger Weberhandwerk im 16. Jahrhundert betrifft: das Material durch den Rat, der Weber, die Technik der Verarbeitung, die Regulierung Wirtschaftsgeschichte und Absatz, das Verlagswesen, die Sozialstruktur. Die Weber waren, obwohl sie die Grundlage des Wohlstands der Stadt erarbeiteten, »ein armes Volk«. Woran das lag und wie es sich auswirkte, wird in diesem Buch deutlich. Dabei ergibt sich eine Fülle von Einsichten in die Wirtschafts- und Sozialgeschichte weit über Augsburg hinaus. G. Wunder Wolfgang von Hippel: Auswanderung aus Südwestdeutschland. Studien zur württembergischen Auswanderung und Auswanderungspolitik im 18. und 19. Jahrhundert. (=lndustrielle Welt, 36). Stuttgart: Klett-Cotta 1984. 352 S. Im 18. und 19. Jahrhundert stellte Südwestdeutschland während der großen Massenemigration aus Deutschland das größte Kontingent an Auswanderern. Bevorzugte Gebiete zunächst Ost- und Südosteuropa, dann Professor für vor allem Nordamerika. Geschichte an der Universität Mannheim, neuere Buch die institutionellen waren Wolfgang von Hippel, untersucht nun in diesem Rahmenbedingungen, die Transportmöglichkeiten, die Aus- und Einwanderungspolitik der einzelnen Staaten, die Triebkräfte und Motive für die Auswanderung, die Altersgruppen und Berufe. Demographische und sozialgeschichtliche Fragestellun- gen stehen dabei im Vordergrund. Zentrale chung aber bilden Orientierungspunkte in der gesamten Untersu- die Fragen nach Verlauf, Ausmaß, Struktur und Gründen für die Auswanderung. Die Arbeit behandelt in einem ersten Teil im Überblick die Auswanderung und Auswanderungspolitik im Herzogtum Württemberg während einem zweiten Teil die Massenauswanderung aus (1815-1870). Es wird des 18. Jahrhunderts, in Württemberg im Zeitalter des Pauperismus versucht, mit Statistiken und karthographischen Schaubildern die Forschungsfragen zu beantworten und das umfangreiche Archivmaterial auszubreiten. Mit einem Quellenanhang von 18. Jahrhunderts aus 15 Dokumenten soll zudem die Auswanderungsproblematik des staatlicher Sicht veranschaulicht werden. Württembergisch Franken wird fast nur mit seinen Oberämtern in den Statistiken berücksichtigt, dazu finden sich Bemerkungen aus dem Gebiet um Öhringen, aus Hall keine. Otto Uhlig: Die Schwabenkinder aus Tirol und einige A. Zieger Vorarlberg. Innsbruck: Wagner, Stuttgart: Theiss 1983. 384 S. Uhlig beschreibt in diesem Buch, das die Vorlage zu dem Film des ZDF »Die vergessenen Kinder« lieferte, die Geschichte Tiroler Jugendlicher, die vom Anfang des 19. Jh. bis in die ersten Jahrzehnte des 20. Jh. in den Sommermonaten als Wanderarbeiter nach Süddeutschland zogen. Er macht damit auf eine oft vergessene Schicht - die Kinder - aufmerksam und zeigt, daß sie zu dem von den wirtschaftlichen und sozialen Mißständen in der Frühphase der Industrialisierung am härtesten betroffenen Personenkreis gehörten. Seine Ausführungen über die Ursachen der Armut, die Einreisebestimmungen, die Entlohnung und die Arbeits- aufnahme geben einen hervorragenden Einblick in die Verhältnisse jener Zeit. Die Lektüre dieses faktenreich und spannend geschriebenen Buches, das dem kürzlich verstorbenen Autor - wie er einleitend sagte — zum »Erlebnis« wurde und dem Leser jederman nur wärmstens empfohlen werden. zur Freude wird, kann O. Windmüller Schweizer Einwanderer in den Kraichgau nach dem Dreißigjährigen Krieg. Mit ausgewählter Ortsliteratur. Hrsg, von Karl Diefenbacher, Hans Ulrich Pfister [u. .a.]. (= Sonder- drucks des Heimatvereins Kraichgaue. V.). HeimatvereinKraichgaue. V., Sinsheim, Verein für Familien- und Wappenkunde in Württemberg und Baden e. V., Stuttgart, Heimatstelle Pfalz e.V., Kaiserslautern, Schweizerische Gesellschaft für Familienforschung. 1983. 296 S. 308 Neue Bücher Der vorliegende Band nennt (auf S. 17-198) 5185 Namen von Schweizern, in 30jährigen Krieg die entvölkerten Gebiete des wie Kraichgaus (Pfalz eingewandert sind. Orts- und Literaturverzeichnisse ergänzen den Band. meisten Schweizer Kantone als Heimatland die nach dem Ritterschaft) Es sind dabei die vertreten, besonders Zürich und Bern, dann Thurgau und Aargau. Es fehlen auch wechseln nur nicht einige Namen aus der welschen Schweiz. Viele häufig den Ort, andere sind weitergezogen oder gar zurückgekehrt. Deshalb kann genealogische Einzelforschung den Tatbestand der Einwanderung genau erfassen. Aber diese aus vielen Quellen zusammengetragenen Listen sind doch eine wesentliche Hilfe, um den Vorgang im ganzen zu überschauen. G. Wunder Wurzeln des Wohlstands. Bilder und Dokumente südwestdeutscher Wirtschaftsgeschichte. Hrsg, von den Industrie- und Handelskammern in Baden-Württemberg. Stuttgart: Theiss 1984. 239 S. Das Bändchen will »keine geschlossene Darstellung..., keine Dokumentation mit wissen- schaftlichem Anspruch« sein. Das ist es gewiß nicht. Was geboten wird, ist vielmehr ein sehr reichhaltiges Bildmaterial mit relativ sparsamem Text. Insbesondere von den Bildern her ist das Werk eine Fundgrube für jeden, der an Wirtschaftsgeschichte interessiert ist. Freilich kann das Buch nirgends verleugnen, daß es primär eine Public-Relations-Schrift der heutigen Industrie-Organisationen ist. Soziale Konflikte, gewiß ein wichtiger Faktor auch dersüdwestdeutschen Wirtschaftsgeschichte, kommen nicht vor. Von den Gewerkschaften liest man praktisch nichts, dafür um so mehr von den im Einzelfall durchaus anerkennenswerten Sozialleistungen und -einrichtungen der Unternehmer. Zwar bemüht sich der Text um - - Zurückhaltung und Objektivität, an vielen Stellen dringt aber doch eine eindeutige Richtung durch: Es ist die Ideologie eines ganz naiven, ungebrochenenFortschrittsglaubens, den man nach den Erfahrungen des 20. Jahrhunderts eigentlich längst als überholt ansehen müßte. So klingt etwa der Straßenbau ganz im Stil einer modernen Heldengeschichte an, zur Zerstörung unserer Flüsse durch Kanalisierung, Hafen- und Pipelinebau fällt den Herausgebern gleich- falls nichts anderes ein, als daß diese eben Fortschritt sei, und Atomkraft schließlich (man meidet übrigens das Reizwort »Atom«, das erinnert zu sehr an die fatale Bombe, und redet von neutraler »Kernkraft«) ist eine ganz harmlose Sache, die nur die »Abhängigkeit mildert» und die »Strompreise in Grenzen« hält. G. Fritz 7. Rechts- und Verwaltungsgeschichte Lateinische Rechtsregeln und Rechtssprichwörter. Zsgest., übers, u. erl. unter Mitarb. von Hannes Lehmann u. von Detlef Liebs Gallus Strobel. 2. durchges. Aufl. München: Beck 1982. 277 S. Mit einer Auswahl von mehr als 1600 lateinischen Rechtssätzen und Rechtsmaximen haben der Freiburger Ordinarius D. Liebs und seine Mitarbeiter erstmals eine kritische dieser Art vorgelegt und bereits ein entstammen vor dienten Sammlung vielfältiges dankbares Echo ausgelöst. Die Rechtsregeln allem dem spätmittelalterlichen und frühneuzeitlichen Lehrbetrieb und didaktischen Zwecken. Es sind »Merkregeln gewissermaßen aus der juristischen Subkultur« (S. 13), gleichwohl spiegeln sie einen wesentlichen Teil der europäischen juristischen Tradition. In die nicht nach Sachgruppen, sondern alphabetisch nach Satzanfängen geordnete Sammlung wurden grundsätzlich nur Rechtsregeln aufgenommen, die auch heute noch von aktueller Bedeutung sind oder sein können. Dem lateinischen Originaltext sind eine Übersetzung und Erläuterungen beigefügt. Nicht nur die Fundstellen in Quellen der Antike bis zur frühen Neuzeit sind angegeben, es fehlt auch nicht der Bezug zu den derzeit geltenden Rechtsnormen. Verschiedene ausführliche Register (insbesondere Gesetzesregister, Sachregister, lateinisches Stichwortregister und Urheberregister) erschließen die Sammlung -, eine Fundgrube für jeden, der sich für die Entwicklung des Rechts interessiert. K. K. Finke
https://openalex.org/W4320493458	https://anthor.org/index.php/anthor/article/download/113/79	Indonesian	null	Meningkatkan Kemampuan Moral dan Agama Anak Melalui Metode Demonstrasi di Taman Kanak-Kanak	Anthor	2,023	cc-by-sa	4,263	Keywords: Ability, Morals, Religion, Methods of Demonstration, Kindergarten. Abstract The background of this research is that children are not yet able to be polite to fellow friends. The purpose of this study was to improve the moral and religious abilities of children through the demonstration method at Mutiara Bunda Kindergarten in Palung Raya for the 2022 academic year. This research was conducted on Wednesday mornings from 07.35 to 11.00 on October 12 2022. This research included classroom action research (PTK) carried out in 2 cycles, which consists of planning, implementing, observing, and reflecting on the body. As the subject of this study were the children of Mutiara Bunda Kindergarten, Tambang District, Kampar Regency. Data collection techniques used observation and assignments in core activities, in order to obtain information about the activeness of children in participating in the teaching and learning process. The results of research on children's morals and religion through the demonstration method at Mutiara Bunda Kindergarten, Tambang District, Kampar Regency, can be improved using the demonstration method. Improving moral and religious abilities using the demonstration method in cycle I and cycle II developed very well. This is illustrated by the results of observations on moral and religious abilities in children. The first cycle of research and discussion results obtained children who were able to develop well and easily understand the material given by 55% and the second cycle developed very well and experienced an increase in morals and religion through demonstration methods, and cards or equal to 77%. Corresponding Author: Rinah Pendidikan Agama Islam, Sekolah Tinggi Agama Islam Diniyah Pekanbaru, Riau, Indonesia: rinahmpd@gmail.com Copyright © 2023 Rinah This work is licensed under Attribution-ShareAlike 4.0 International (CC BY-SA 4.0) Corresponding Author: Rinah Copyright © 2023 Rinah This work is licensed under Attribution-ShareAlike 4.0 International (CC BY-SA 4.0) Copyright © 2023 Rinah This work is licensed under Attribution-ShareAlike 4.0 International (CC BY-SA 4.0) py g This work is licensed under Attribution-ShareAlike 4.0 International (CC BY-SA 4.0) Abstrak Latar belakang penelitian ini anak belum mampu bersikap sopan santun yang baik kepada sesama teman. Tujuan penelitian ini untuk meningkatkan Kemampuan Moral Dan Agama Anak Melalui Metode Demonstrasi pada TK Mutiara Bunda di Palung Raya Tahun Ajaran 2022. Penelitian ini dilakukan pada hari Rabu pagi jam 07.35 sampai 11.00 pada tanggal 12 Oktober 2022. Penelitian ini termasuk penelitian tindakan kelas (PTK) yang dilaksanakan pada 2 siklus,yg terdiri dari perencanaan,pelaksanaan,observasi,dan refleksi badan. Sebagai subjek penelitian ini adalah anak TK Mutiara Bunda Kecamatan Tambang Kabupaten Kampar,teknik pengumpulan data menggunakan observasi dan pemberian tugas dalam kegiatan inti,guna untuk mendapatkan informasi tentang keaktifan anak dalam mengikuti proses belajar mengajar. Hasil penelitian moral dan agama anak melalui metode demonstrasi pada TK Mutiara Bunda Kecamatan Tambang Kabupaten Kampar dapat di tingkatkan dengan menggunakan metode demonstrasi,peningkatan kemampuan moral dan agama mengg8nakan metode demonstrasi pada siklus I dan siklus II berkembang sangat baik. Hal ini tergambar dari hasil observasi terhadap kemampuan moral dan agama pada anak. siklus I hasil penelitian dan pembahasan memperoleh anak yang mampu berkembang secara baik dan mudah memahami materi yang diberikan sebesar 55% dan siklus II berkembang sangat baik dan mengalami peningkatan dalam moral dan agama melalui metode demonstrasi ,dan kartu atau sama dengan 77%. Kata Kunci: Kemampuan, Moral, Agama, Metode Demonstrasi, Taman Kana- kanak. First Receive First Received: 02 Februari 2023 First Received First Received: 02 Februari 2023 MENINGKATKAN KEMAMPUAN MORAL DAN AGAMA ANAK MELALUI METODE DEMONSTRASI DI TAMAN KANAK-KANAK Rinah Pendidikan Agama Islam, FTIK, Sekolah Tinggi Agama Islam Diniyah Pekanbaru rinahmpd@gmail.com 140 \| ANTHOR: Education and Learning Journal Volume 2 Nomor 1, 2023 PENDAHULUAN Pendidikan Anak Usia Dini merupakan salah satu bentuk penyelenggaraan pendidikan yang menitik beratkan pada peletakan dasar ke arah pertumbuhan dan perkembangan fisik (koordinasi motorik halus dan kasar), kecerdasan (daya pikir, daya cipta, kecerdasan emosi, kecerdasan spiritual), sosio emosional (sikap 140 \| ANTHOR: Education and Learning Journal Volume 2 Nomor 1, 2023 e-ISSN: 2963 – 198X p-ISSN: 2963 – 2498 Meningkatkan Kemampuan Moral dan Agama Anak Melalui Metode Demonstrasi di Taman Kanak-Kanak dan perilaku serta beragama), bahasa dan komunikasi, serta dengan keunikan dan tahap-tahap perkembangan yang dilalui oleh anak usia dini (Sujiono, 2013). Suyanto (2005) Pendidikan Anak Usia Dini dinyatakan dalam Undang-undang Nomor 20 Tahun 2003 tentang Sistem Pendidikan Nasional Bab 1 Pasal 1 Ayat 14. Currie (2001) Pendidikan anak usia dini adalah suatu upaya pembinaan yang ditujukan kepada anak sejak lahir sampai dengan usia enam tahun yang dilakukan melalui pemberian rangsangan pendidikan untuk membantu pertumbuhan dan perkembangan jasmani dan rohani agar anak memiliki kesiapan dalam memasuki pendidikan lanjut” (Kementrian Pendidikan dan Kebudayaan, 2014). Pendidikan nilai-nilai moral agama pada program PAUD merupakan fondasi yang kokoh dan sangat penting keberdaanya, dan jika hal itu telah tertanam dengan baik dalam setiap insan sejak dini, hal tersebut merupakan awal yang baik bagi pendidikan anak bangsa untuk menjalani pendidikan selanjutnya (Safitri et al., 2019). Bangsa indonesia sangat menjunjung tinggi nilai-nilai moral agama (Sardila, 2015). Nilai-nilai luhur ini pun dikehendaki menjadi motivasi spiritual bagi bangsa ini dalam rangka melaksanakan sila-sila lainnya dalam pancasila (Saleh, 2022). p ( ) Anak usia dini adalah seorang individu yang sedang mengalami pertumbuhan dan perkembangan (Maghfiroh & Shofia Suryana, 2021). Pertumbuhan berkaitan dengan bagian tubuh yang dapat diukur misalnya berat badan, tinggi badan, lingkar kepada (Hijriati, Putri Rahmi, 2021) Sedangkan perkembangan merupakan perubahan yang terjadi dalam hidupnya. Usia dini merupakan masa yang tepat untuk memberikan dasar pengembangan kemampuan fisik, bahasa, sosial emosional, konsep diri, seni moral dan nilai-nilai agama. (Farhurohman, 2017) Oleh karena itu anak memiliki potensi besar untuk mengoptimalkan perkembangannya. Pertumbuhan dan perkembangan berjalan secara beriringan. Salah satu sikap dasar yang harus dimiliki seorang anak untuk menjadi seorang manusia yang baik dan benar adalah memiliki sikap dan nilai moral yang baik dalam berperilaku sebagai umat Tuhan, anak, anggota keluarga dan anggota masyarakat (Maghfiroh & Shofia Suryana, 2021). Usia taman kanak-kanak adalah saat paling baik bagi guru taman kana-kanak untuk meletakkan dasardasar pendidikan nilai, moral dan agama kepada anak taman kanak-kanak (Ananda, 2017). 141 \| ANTHOR: Education and Learning Journal Volume 3 Nomor 1, 2023 Meningkatkan Kemampuan Moral dan Agama Anak Melalui Metode Demonstrasi di Taman Kanak-Kanak Meningkatkan Kemampuan Moral dan Agama Anak Melalui Metode Demonstrasi di Taman Kanak-Kanak Meningkatkan Kemampuan Moral dan Agama Anak Melalui Metode Demonstrasi di Taman Kanak-Kanak Pendidikan keagamaan merupakan awal yang baik bagi pendidikan anak untuk menjalani kehidupan selanjutnya (Fabiana, 2019). Pendidikan harus berprinsip pada pengembangan nilai agama sebagai upaya untuk melaksanakan ibadah dan berperilaku terpuji sesuai dengan ajuran agama. Nilai agama untuk anak usia dini ditanamkan melalui keteladanan dan pembiasaan dari guru maupun orang tua (Kohlberg, 1974). Jika orang tua dan guru membiasakan dan memberikan teladan yang baik untuk anak dengan melakukan kebaikan-kebaikan dan peribadahan yang baik maka kemungkinan besar akan berkembang menjadi individu yang berakhlak mulia (Kjeldsen, 2019). Keteladanan yang dilakukan oleh orang tua maupun guru mengharuskan untuk mempelajari, memahami dan mampu mengimplementasikan dan mengarahkan pada aspek perkembangan nilai agama untuk anak usia dini (Macmullen, 2004). Menurut Hakim dalam agama Islam nilai agama mencakup aqidah, ibadah dan akhlak (Chidester, 2003) Penanaman nilai-nilai agama merupakan suatu upaya mengenalkan dan mengajarkan ajaran agama kepada agama kepada anak agar anak dapat mengetahui dan memahaminya dan kelak melaksanakan ajaran agama dengan baik (Chidester, 2003). Pendidikan nilai agama Islam sejak dini merupakan tanggungjawab bersama semua pihak, baik guru maupun orang tua (Imam Tabroni & Rahmania, 2022). Pengembangan moral agama sangat erat kaitannya dengan budi pekerti, sikap sopan santun, dan kemauan melaksanakan ajaran agama dalam kehidupan sehari-hari (Imam Tabroni & Rahmania, 2022). Pendidikan moral akan terus berkembang dengan berbagai pendapat pakar dalam aspek budi pekerti, nilai moral dan keagamaan (Anik Lestariningrum, 2014). Juhriati & Rahmi (2021) lebih menekankan pendidikan moral diarahkan kepada tahap-tahap pembentukannya, sehingga pendidikan moral di dasarkan untuk membentuk setiap tahap-tahap peserta didik. Disamping tahapan perkembangan moralnya, (Samsuri, 2018) juga menawarkan konsep keadilan sebagai dasar pelaksanaan pendidikan moral di Barat. Prinsip tersebut merupakan suatu kondisi iman dalam jaringan reasi social atau suatu hukum yang mengatur keseimbangan semua relasi social tersebut (Nasution, 2016) Oleh karena itu, Kholbergh memberikan cerita kepada orang-orang yang memiliki umur yang berbeda dan budaya yang menempatkan seseorang dalam posisi dan situasi tertentu yang di konfrontaris dengan masalah moral dalam standar tertentu. Piaget menyatakan bahwa anak anak berfikir dengan 2 cara yang sangat berbeda tentang moralitas tergantung pada kedewasaan perkembangan mereka (Kusrahmadi, 2007). Perilaku baik dihubungkan dengan pemuasan kinginan dan kebutuhan sendiri tanpa mempertimbangkan kebutuhan orang lain (Nawawi, 2018). Meningkatkan Kemampuan Moral dan Agama Anak Melalui Metode Demonstrasi di Taman Kanak-Kanak Menurut Syaodih menyatakan bahwa perkembangan nilai-nilai agama dan moral anak usia dini antara lain: anak besikap imitasi (imitation) yakni mulai menirukan sikap, cara pandang serta tingkah laku orang lain, anak bersikap inernalisasi yakni anak sudah mulai bergaul dengan lingkungan sosialnya dan mulai terpengaruh dengan keadaan di lingkungan tersebut, anak bersikap introvert dan ekstrovert yakni reaksi yang ditunjukkan anak berdasarkan pengalaman (Athfal, 2021). Menurut John Dewey, tahapan perkembangan moral sesorang berada pada fase pra konvensional yang memiliki karakteristik sikap dan perilaku anak dilandasi oleh implus biologis dan social (Fabiana , 2019). Menurut (Depi, 2022) perkembangan moral dan agama anak usia 5–6 tahun adalah suatu kemampuan untuk berinteraksi dengan tingkah laku yang baik sesuai dengan norma-norma, sehingga menimbulkan perilaku yang baik dan buruk. g gg p y g Untuk memberikan kesempatan kepada peserta didik (anak, generasi penerus) menanamkan ketuhanan, nilai-nilai estetik dan etik, nilai baik dan buruk, benar dan salah, mengenai perbuatan sikap dan kewajiban akhlak mulia, budi pekerti luhur agar mencapai kedewasaannya dan bertanggung jawab (Fabiana, 2019). Peningkatan potensi spiritual anak melalui pengalaman agar menjadi kebiasaan, baik di dalam maupun di luar sekolah. Adapun penanaman nilai-nilai moral dan agama yang diberikan oleh guru di kelas yaitu dengan metode bercerita, demonstrasi, pemberian tugas, karyawisata, pembiasaan dan bercakap- cakap. Nilai moral dan agama sangat berperan dalam membentuk perilaku anak sehingga anak mampu berinteraksi dan bersikap sesuai pertumbuhan dan perkembangannya (Tamuri, 2007). Untuk itu diperlukan pengawasan serta pemeliharaan yang berlangsung secara terus menerus untuk pembentukan kebiasaan dan sikap anak. Pada dasarnya, penanaman nilai-nilai moral dan agama sejak dini membentuk naluri anak untuk menerima sikap keutamaan dan kemuliaan, dan akan terbiasa melakukan akhlak mulia. Dengan masalah tersebut peneliti ingin meneliti tentang Penanaman Nilai- nilai Moral dan Agama Anak Usia Dini melalui metode Demonstrasi. 142 \| ANTHOR: Education and Learning Journal Volume 3 Nomor 1, 2023 PENDAHULUAN Walaupun peran orang tua sangatlah besar dalam membangun dasar moral dan agama bagi anak-anaknya, peran guru taman kanak-kanak juga tidaklah kecil dalam meletakkan dasar moral dan agama bagi seorang anak, karena biasanya anakanak taman kanak-kanak senang menuruti perintah gurunya.oleh karena itu (Rahmat, 2018). Seorang guru taman kanak-kanak harus selalu berupaya dengan berbagai cara agar dapat membimbing anak seusia taman kanak-kanak agar mempunyai kepribadian yang baik, yang dilandasi dengan nilai moral dan agama (Feblyna & Wirman, 2020). Setiap aspek perkembangan anak harus dikembangkan secara optimal, karena antara aspek satu dan lainnya saling berkaitan dan mempengaruhi (Sulaiman et al., 2019). Pendidikan mencakup proses hidup dalam rangka mengembangkan potensi yang dimilikinya supaya dapat berjalan secara optimal. Pendidikan anak usia dini harus menjadi proses awal pertumbuhan dan perkembangan seseorang sebelum memasuki umur dewasa (Ita et al., 2020). Pendidikan anak usia dini merupakan pemberian upaya untuk membimbing, mengasuh, dan menstimulasi anak sehingga akan menghasilkan kemampuan dan keterampilan bagi anak (Fauzi, 2018). Selain itu, anak usia dini harus mempunyai hak untuk mendapatkan pendidikan, perawatan, pengasuhan, kesehatan serta kebutuhan gizinya (Husna et al., 2019). Tujuan pendidikan bagi taman kanak-kanak yaitu membantu meletakkan dasar untuk mengembangkan sikap, perilaku, pengakuan, keterampilan, dan kreativitas yang nantinya nantinya akan diperlukan anak untuk pertumbuhan dan perkembangannya (StudyCha, 2013). Salah satu bagian penting yang harus mendapatkan perhatian terkait dengan pendidikan anak usia dini yaitu pendidikan nilai agama Islam (StudyCha, 2013). Pendidikan nilai agama berkaitan dengan kemauan melaksanakan ajaran agama dalam kehidupan sehari- hari (Ardiansari & Dimyati, 2021). Anak harus diberikan bimbingan dan arahan yang tepat dalam memahami tentang nilai keagamaan anak usia dini. Kegiatan keagamaan anak usia dini berkaitan dengan kegiatan berdoa, beribadah dan berperilaku sesuai ajaran agama. Dalyono & Enny Dwi Lestariningsih (2017) Manfaat kegiatan keagamaan yang dilakukan anak usia dini diharapkan anak kelak akan menjadi individu yang taat beribadan dan berperilaku sesuai ajaran agamnya. Apabila anak secara terus menerus dilatih dengan cara yang kurang tepat maka ketika mereka berusia dewasa tidak memiliki kepedulian yang tinggi pada kehidupan beragama dalam kesehariannya (Ardiansari & Dimyati, 2021). Pendidikan nilai keagamaan pada anak usia dini merupakan pondasi yang kokok dan sangat penting sehingga perlu ditanamkan sejak dini. Apabila nilai keagamaan tidak berikan sejak dini akan menghambat perkembangan nilai agama dan moral anak dalam keberlangsungan kehidupan selanjutnya (Safitri et al., 2019). e-ISSN: 2963 – 198X p-ISSN: 2963 – 2498 141 \| ANTHOR: Education and Learning Journal Volume 3 Nomor 1, 2023 METODE PENELITIAN Metode dalam penelitian ini merupakan penelitian tindakan kelas (PTK) (Wardani e-ISSN: 2963 – 198X p-ISSN: 2963 – 2498 e-ISSN: 2963 – 198X p-ISSN: 2963 – 2498 142 \| ANTHOR: Education and Learning Journal Volume 3 Nomor 1, 2023 Meningkatkan Kemampuan Moral dan Agama Anak Melalui Metode Demonstrasi di Taman Kanak-Kanak 2018), subjek penelitian adalah anak di TK Mutiara Bunda. Penelitian tindakan kelas ini dilaksanakan melalui dua siklus. Dimana pada masing-masing siklus ini terdiri dari 4 tahapan yang dilakukan, yaitu : Tahapan Perencanaan, Tahapan Pelaksanaan Tindakan, Tahapan pengamatan dan tahapan reflesi. Tahapan-tahapan tersebut dijelaskan sebagai berikut: Gambar I.Tahapan Penelitian Tindakan Kelas Gambar I.Tahapan Penelitian Tindakan Kelas Pelaksanan tindakan ini dilaksanakan dalam siklus berulang. Setiap siklus dilaksanakan sesuai dengan desain yang telah dikemukakan di atas yang dengan melihat perubahan yang ingin dicapai dalam tindakan. Jenis data yang digunakan dalam penelitian ini yaitu data kualitatif terkait peningkatan penerapan metode demonstrasi yang diperoleh dari hasil pengamatan berdasarkan lembar observasi anak serta aktivitas guru (peneliti). Adapun cara pengumpulan data yaitu dengan melakukan observasi, dengan merekam seluruh aktivitas baik yang dilakukan oleh anak dengan menggunakan lembar observasi, pemberian tugas, dan tanya jawab yang telah dipersiapkan. Analisis data kualitatif dilakukan selama dan sesudah penelitian dilakukan dikelas dan dilakukan melalui tiga tahap, yatu reduksi data, paparan data dan penyimpulan atau verifikasi data. Gambar I.Tahapan Penelitian Tindakan Kelas Pelaksanan tindakan ini dilaksanakan dalam siklus berulang. Setiap siklus dilaksanakan sesuai dengan desain yang telah dikemukakan di atas yang dengan melihat perubahan yang ingin dicapai dalam tindakan. Jenis data yang digunakan dalam penelitian ini yaitu data kualitatif terkait peningkatan penerapan metode demonstrasi yang diperoleh dari hasil pengamatan berdasarkan lembar observasi anak serta aktivitas guru (peneliti). Adapun cara pengumpulan data yaitu dengan melakukan observasi, dengan merekam seluruh aktivitas baik yang dilakukan oleh anak dengan menggunakan lembar observasi, pemberian tugas, dan tanya jawab yang telah dipersiapkan. Analisis data kualitatif dilakukan selama dan sesudah penelitian dilakukan dikelas dan dilakukan melalui tiga tahap, yatu reduksi data, paparan data dan penyimpulan atau verifikasi data. 143 \| ANTHOR: Education and Learning Journal Volume 3 Nomor 1, 2023 HASIL DAN PEMBAHASAN Dari hasil penelitian ini, peneliti mendeskripsikan tentang perencanaan dalam kegiatan demonstrasi yang telah peneliti dibuat sebelumnya yaitu perencanaan pembelajaran untuk meningkatkan keterampilan berwudhu pada siklus 1 dan siklus 2 adalah sebagai berikut. Dalam perencanaan di siklus satu peneliti mengalami kesulitan dalam meningkatkan keterampilan berwudhu oleh karena itu disusun rencana perbaikan pembelajaran pada siklus 2 dengan harapan adanya peningkatan keterampilan berwudhu anak. (Moeslichatoen, 2004:121) mengemukakan persiapan yang matang mutlak diperlukan, agar memperoleh hasil yang diharapan, terdapat beberapa langkah yang harus diperhatikan yaitu: a) Menetapkan rancangan tujuan dan tema kegiatan demonstrasi, b) Menetapkan rancangan bentuk demonstrasi yang dipilih, c) Menetapkan rancangan bahan dan alat yang diperlukan untuk demonstrasi, d) Menetapkan rancangan langkah kegiatan demonstrasi, dan e) Menetapkan rancangan penilaian kegiatan demonstrasi. Pada sub bab ini akan di paparkan tentang hasil yang diperoleh peneliti saat penelitian tindakan kelas di TK Mutiara Bunda. Penelitian tindakan kelas ini dilaksanakan melalui dua siklus. Dimana pada masing-masing siklus ini terdiri dari 4 tahapan yang dilakukan, yaitu : Tahapan Perencanaan, Tahapan Pelaksanaan Tindakan, Tahapan Observasi dan Tahap Refleksi. Dapat ditemukan hasil dari penelitian dengan judul Meningkatkan Kemampuan Moral dan Agama anak melalui metode Demonstrasi di TK Mutiara Bunda. Dari data hasil penelitian yang terjadi pada siklus I, peneliti memperoleh metode gambaran keberhasilan anak dalam perkembangan peningkatan moral dan agama anak melalui metode Demostrasi, yang di tuangkan penulis dalam grafik nilai kemampuan anak sebagaimana pada gambar 2. Dari data grafik diatas, Kemampuan anak dalam peningkatan moral dan agama anak melalui Demontrasi melalui penggunaan media demonstrasi pada siklus I Indikator 1 terdapat 6 orang anak yang mampu berkembang secara baik dan mudah memahami metode demonstrasi, atau sama dengan 55%. Pada indikator 2 terdapat 4 anak yang mampu memahami materi dengan mudah atau sama dengan 35%. Pada indikator 3 terdapat 2 anak yang mulai berkembang dengan materi demonstrasi e-ISSN: 2963 – 198X p-ISSN: 2963 – 2498 e-ISSN: 2963 – 198X p-ISSN: 2963 – 2498 143 \| ANTHOR: Education and Learning Journal Volume 3 Nomor 1, 2023 Meningkatkan Kemampuan Moral dan Agama Anak Melalui Metode Demonstrasi di Taman Kanak-Kanak artinya mereka cukup memahami metode tersebut, atau sama dengan 20%. Pada Indikator 4 terdapat anak belum berkembang atau mengerti materi metode demontrasi yang di berikan yaitu 1 orang atau sama dengan 15%. Gambar 2. Data Peningkatan Moral dan Agama melalui metode Demonstrasi Siklus I Gambar 2. 144 \| ANTHOR: Education and Learning Journal Volume 3 Nomor 1, 2023 DAFTAR RUJUKAN Ananda, R. (2017). Implementasi Nilai-nilai Moral dan Agama pada Anak Usia Dini. Jurnal Obsesi: Jurnal Pendidikan Anak Usia Dini, 1(1), 19. https://doi.org/10.31004/obsesi.v1i1.28 p g Anik, L. (2014). Pengaruh Penggunaan Media Vcd Terhadap Nilai-Nilai Agama Dan Moral Anak. Jurnal Pendidikan Anak Usia Dini, 8(2), 3. Ardiansari, B. F., & Dimyati, D. (2021). Identifikasi Nilai Agama Islam pada Anak Usia Dini. Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 6(1), 420–429. https://doi.org/10.31004/obsesi.v6i1.926 Athfal R (2021) Kiat-Kiat Mengembangkan Perilaku Baik (Akhlakul Karimah) Pada Anak Usia Dini Ardiansari, B. F., & Dimyati, D. (2021). Identifikasi Nilai Agama Islam pada Anak Usia Dini. Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 6(1), 420–429. https://doi.org/10.31004/obsesi.v6i1.926 Athfal, R. (2021). Kiat-Kiat Mengembangkan Perilaku Baik (Akhlakul Karimah) Pada Anak Usia Dini. Jurnal Asghar 1(2) 40–51 Athfal, R. (2021). Kiat-Kiat Mengembangkan Perilaku Baik (Akhlakul Karimah) Pada Anak Usia Dini. Jurnal Asghar, 1(2), 40–51. Chidester, D. (2003). Religion education in South Africa: Teaching and learning about religion, religions, and religious diversity. British Journal of Religious Education, 25(4), 261–278. https://doi.org/10.1080/0141620030250402 Currie, J. (2001). Early childhood education programs. Journal of Economic Perspectives, 15(2), 213–238. https://doi.org/10.1257/jep.15.2.213 Dalyono, B., & Enny Dwi Lestariningsih. (2017). Implementasi penguatan pendidikan karakter di sekolah. Bangun Rekaprima, 3(3), 33–42. Depi, S.H., F. (2022). Analisis Dampak Menonton Serial Kartu Upin dan Ipin Terhadap Perkembangan Moral Agama Anak Usia 5-6 Tahun. Journal of Education and Teaching, 3(1), 39–56. http://ejournal.stainkepri.ac.id/index.php/tanjak Fabiana, M. F.. (2019). Peranan Pendidikan Agama Dalam Keluarga Terhadap Pembentukan Kepribadian Anak-anak. Jurnal Al-Abyadh, 3(2), 8–25. Farhurohman, O. (2017). Kata Kunci: pendidikan anak usia dini. Jurnal Fakultas Ilmu Pendidikan, Universitas Negeri Yogyakarta, 2(1), 27–36. Fauzi, F. (2018). Hakikat Pendidikan bagi Anak Usia Dini. Jurnal Pemikiran Alternatif Kepen 15(3), 386–402. https://doi.org/10.24090/insania.v15i3.1552 Feblyna, T., & Wirman, A. (2020). Penggunaan Reward untuk Meningkatkan Pembiasaan Disiplin Anak di Taman Kanak-Kanak. Jurnal Pendidikan Tambusai, 4(2), 1132–1141. https://jptam.org/index.php/jptam/article/view/576 Hijriati, P. R. (2021). Proses Belajar Anak Usia 0 Sampai 12 Tahun Berdasarkan Karakteristik Perkembangannya. Jurnal Pendidikan Anak, 7(1), 152. https://doi.org/10.22373/bunayya.v7i1.9295 Husna, F., Yunus, N. R., & Gunawan, A. (2019). Hak Mendapatkan Pendidikan Bagi Anak Berkebutuhan Khusus Dalam Dimensi Politik Hukum Pendidikan. Jurnal Sosial Dan Budaya Syar-I, 6(2), 207– 222. https://doi.org/10.15408/sjsbs.v6i1.10454 Imam, T, & Rahmania, S. (2022). Implementation of Akhlaqul Karimah Through Islamic Religious Education Approach In Early Children. Journal of Multidisciplinary Research, 1(1), 33–40. https://doi.org/10.54259/eajmr.v1i1.454 Ita, E., Wewe, M., & Go.o, E. (2020). Analisis Perkembangan Kemampuan Bahasa Anak Kelompok A Taman Kanak-Kanak. HASIL DAN PEMBAHASAN Data Peningkatan Moral dan Agama melalui metode Demonstrasi Siklus I Berdasarkan hasil penelitian pada siklus I terlihat anak sudah tertarik dengan proses belajar melalui sholat zuhur, moral dan agama dengan menggunakan metode Demonstrasi. Proses pembelajaran ini dilakukan dengan menggunakan metode demonstrasi,yakni anak dapat mampu menjabarkan kembali pembelajaran sholat zuhur. Berdasarkan hasil penelitian pada siklus I terlihat anak sudah tertarik dengan proses belajar melalui sholat zuhur, moral dan agama dengan menggunakan metode Demonstrasi. Proses pembelajaran ini dilakukan dengan menggunakan metode demonstrasi,yakni anak dapat mampu menjabarkan kembali pembelajaran sholat zuhur. Gambar 3. Data Peningkatan Moral dan Agama melalui metode Demonstrasi Siklus II Gambar 3. Data Peningkatan Moral dan Agama melalui metode Demonstrasi Siklus II Hasil pengamatan pada indikator 1 terdapat 10 orang anak yang berkembang sangat baik dan mengalami peningkatan dalam moral dan agama melalui metode demonstrasi ,dan kartu atau sama dengan 77% .Pada indikator 2 terdapat 1 anak yang berkembang sesuai harapan atau mampu menggambarkan atau sama dengan 15%.Pada indikator 3 terdapat 1 anak yang mulai berkembang atau sama dengan 15%.Pada siklus II ini kemampuan berkembang moral dan agama anak telah mencapai indikator keberhasilan yang telah di tentukan oleh peneliti.Untuk itu peneliti menghentikan tindakan sampai pada siklus II. Sehingga dapat di simpulkan bahwa penerapan metode bercerita pada TK Mutiara Bunda Kecamatan tambang kabupaten bangkinang Tahun ajaran 2023 dapat meningkatan perkembangan moral dan agama anak.Adapun terjadi nya peningkatan kemampuan anak tiap siklusnya karena cara penyampian media kartu yang di berikan pada waktu proses pembelajaran sesuai dengan semua sumber yang di perlukan untuk melakukan komunikasi dengan anak.Sehubungan dengan hal tersebut maka untuk meningkatan kemapuan moral dan agama anak menggunakan alat peraga yang telah disiapkan guru melalui strategi pembelajaran dengan cara bercerita dan bermain.Sehingga sangat berkesan dan merik lagi anak usia dini Hasil penelitian tersebut menunjukan bahwa penanaman nilai moral dan agama di TK Mutiara Bunda Kecamatan tambang kabutpaten bangkinang sudah diterapkan dengan baik menggunakan beberapa metode.hal ini sejalan dengan pendapat Plato bahwa perkembangan moral agama anak usia dini dapat dikembangkan pada awal kehidupan individu untuk dapat mengembangkan moral, anak dapat membedakan yang baik dan yang buruk, anak terbiasa dalam antrian, kebajukan, keadilan, kesederhanaan, dan keberanian (Orangtua et al., 2019). e-ISSN: 2963 – 198X p-ISSN: 2963 – 2498 144 \| ANTHOR: Education and Learning Journal Volume 3 Nomor 1, 2023 Meningkatkan Kemampuan Moral dan Agama Anak Melalui Metode Demonstrasi di Taman Kanak-Kanak Meningkatkan Kemampuan Moral dan Agama Anak Melalui Metode Demonstrasi di Taman Kanak-Kan KESIMPULAN Berdasarkan uraian di atas maka peneliti dapat menyimpulkan bahwa penerapan metode demonstrasi dapat meningkatkan moral anak pada anak di TK Mutiara Bunda Kecamatan tambang kabutpaten bangkinang, Hasil penelitian moral dan agama anak melalui metode demonstrasi pada TK Mutiara Bunda Kecamatan Tambang Kabupaten Kampar dapat di tingkatkan dengan menggunakan metode demonstrasi,peningkatan kemampuan moral dan agama menggunakan metode demonstrasi pada siklus I dan siklus II berkembang sangat baik. Hal ini tergambar dari hasil observasi terhadap kemampuan moral dan agama pada anak. siklus I hasil penelitian dan pembahasan memperoleh anak yang mampu berkembang secara baik dan mudah memahami materi yang diberikan sebesar 55% dan siklus II berkembang sangat baik dan mengalami peningkatan dalam moral dan agama melalui metode demonstrasi ,dan kartu atau sama dengan 77%. 145 \| ANTHOR: Education and Learning Journal Volume 3 Nomor 1, 2023 DAFTAR RUJUKAN Jurnal Ilmiah Pendidikan Anak Usia Dini, 3(2), 174–186. https://doi.org/10.24042/ajipaud.v3i2.7317 p g j p Juhriati, I., & Rahmi, A. (2021). Implementasi Nilai Agama dan Moral melalui Metode Esensi Pembinaan Perilaku pada Anak Usia Dini. Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 6(2), 1070–1076. https://doi.org/10.31004/obsesi.v6i2.1147 p g Kjeldsen, K. (2019). A study-of-religion(S)-based religion education: Skills, knowledge, and aims. Journal Center for Educational Policy Studies, 9(4), 11–29. https://doi.org/10.26529/cepsj.678 K hlb L (1974) Ed ti M l D l t d F ith J l f M l Ed i 4(1) 5 16 e-ISSN: 2963 – 198X p-ISSN: 2963 – 2498 145 \| ANTHOR: Education and Learning Journal Volume 3 Nomor 1, 2023 Meningkatkan Kemampuan Moral dan Agama Anak Melalui Metode Demonstrasi di Taman Kanak-Kanak 146 \| ANTHOR: Education and Learning Journal Volume 3 Nomor 1, 2023 Meningkatkan Kemampuan Moral dan Agama Anak Melalui Metode Demonstrasi di Taman Kanak-Kanak https://doi.org/10.1080/0305724740040102 p g Kusrahmadi, S. (2007). Pentingnya Pendidikan Moral Bagi Anak Sekolah Dasar. Jurnal Dinamika Pendidikan, 2(1), 118–129. http://staff.uny.ac.id/sites/default/files/Pendidikan Moral Anak SD ABC 21 April sangat penting.pdf Macmullen, I. (2004). Parochial elementary Schools. Journal of Philosophy of Education, 38(4). Maghfiroh, & Shofia Suryana, D. (2021). Pembelajaran di Pendidikan Anak Usia Dini. Jurnal Pendidikan Tambusai, 05(01), 1561. Nasution, A. R. (2016). Urgensi Pendidikan Kewarganegaraan sebagai Pendidikan Karakter Bangsa Indonesia melalui Demokrasi, HAM dan Masyarakat Madani. Jurnal Pendidikan Ilmu-Ilmu Sosial, 8(2), 201–212. https://doi.org/10.24114/jupiis.v8i2.5167 Nawawi, A. (2018). Pentingnya Pendidikan Nilai Moral Bagi Generasi Penerus. Jurnal Pemikiran Alternatif Kependidikan, 16(2), 119–133. https://doi.org/10.24090/insania.v16i2.1582 f p p g Orangtua, P., Menanamkan, D., Agama, N., Moral, D., Anak, P., Dini, U., Islam, P., & Usia, A. (2019). Peran Orang Dalam Menamkan Nilai Agama Dan Moral Pada Anak Usia Dini. Jurnal Anak Usia Dini, 2(3), 80–86. Rahmat, S. T. (2018). Pola Asuh yang Efektif dalam Mendidik Anak di Era Digital. Journal Education and Culture Missio, 10(2), 143. https://repasitory.stikipsantupaulus.ac.id/122/1/Artikel-jurnal-missio Safitri, N., Kuswanto, C. W., & Alamsyah, Y. A. (2019). Metode Penanaman Nilai-Nilai Agama Dan Moral Anak Usia Dini. Journal of Early Childhood Education (JECE), 1(2), 29–44. https://doi.org/10.15408/jece.v1i2.13312 Saleh, R. (2022). Kerja Sama Orang Tua dan Pendidik dalam Mengenalkan Nilai-Nilai Moral Anak. Jurnal Pendidikan Anak Usia Dini, 3(1), 24–33. https://doi.org/10.37985/murhum.v3i1.70 Samsuri, M. (2018). Konsep Keadilan Hukum (Konsep Barat, Konsep Islam, Dan Konsep Pancasila). Jurnal Mamba’ul ’Ulum, 14(2), 45–60. https://doi.org/10.54090/mu.40 Sardila, V. (2015). Implementasi Pengembangan Nilai-Nilai Etika Dan Estetika Dalam Pembentukan Pola Prilaku Anak Usia Dini. Jurnal RISALAH, 26(2), 86–93. StudyCha, L. (2013). Pengembangan Tema Pembelajaran Untuk Taman Kanak-Kanak. Jurnal Agama Dan Ilmu Pengetahuan, 2(3), 41–47. Sulaiman, U., Ardianti, N., & Selviana, S. (2019). Tingkat Pencapaian Pada Aspek Perkembangan Anak Usia Dini 5-6 Tahun Berdasarkan Strandar Nasional Pendidikan Anak Usia Dini. Journal of Early Childhood Education, 2(1), 52. https://doi.org/10.24252/nananeke.v2i1.9385 Suyanto, S. (2005). Dasar-Dasar Pendidikan Anak Usia Dini (Hikayat Publising. (ed.)). Tamuri, A. H. (2007). Islamic Education teachers’ perceptions of the teaching of akhlāq in Malaysian secondary schools. Journal of Moral Education, 36(3), 371–386. https://doi.org/10.1080/03057240701553347 Wardani IGAK, W. K. (2018). Penelitian Tindakan Kelas. (Universitas Terbuka (ed.)). e-ISSN: 2963 – 198X p-ISSN: 2963 – 2498
https://openalex.org/W2971181140	https://zenodo.org/record/3372134/files/Paper%20PPM%20Mathlaul%20Anwar%20Agustus%202019.pdf	Indonesian	null	Pelatihan Internet Pembuatan Blog bagi Guru-Guru HIMAPAUDI Kecamatan Kemayoran, Jakarta	Jurnal pengabdian pada masyarakat	2,019	cc-by	2,446	JURNAL PENGABDIAN PADA MASYARAKAT ISSN 2540-8739 (print) \|\| ISSN 2540-8747 (online) LEMBAGA PENELITIAN, PENGABDIAN, DAN PENGEMBANGAN MASYARAKAT UNIVERSITAS MATHLA’UL ANWAR BANTEN http://ppm.ejournal.id 1 Corresponding author: Program Studi Sistem Informasi, STMIK Nusa Mandiri Jakarta; Jl. Damai No. 8 Warung Jati Barat Margasatwa Jakarta Selatan; Telp: (021) 98839513,; Email: akmaludin.akm@nusamandiri.ac.id Article History ABSTRACT Received 27.04.2019 Received in revised form 20.06.2019 Accepted 26.07.2019 Available online 20.08.2019 INTERNET TRAINING OF BLOG MAKING FOR TEACHERS OF HIMAPAUDI OF KEMAYORAN DISTRICT, CENTRAL JAKARTA. In the current era of Digitization 4.0, all data and information must use technology based on a fully computerized system, where output is displayed and produced in electronic form. Thus the need for internet for HIMAPAUDI Teachers is in dire need and necessary for Internet training. Especially in the case of making BLOGs. Blog creation training using Blogspot by following the rules for making Blogs. Before the training process, the pretest test was conducted, the results of which were decidedly less than expected. After going through the training process, participants were asked to fill out instrumentation in the form of a questionnaire, and the results were processed using the direct rating method, method combined with pure mathematics, using five variables and 10 question indicators, it turns out the results obtained using a combination of these two methods give the predicate value very well and through the graphic line the results are scoring 87.27. Thus it can be said that there is an increase in learning outcomes through this training. Thus the training process can provide a significant improvement in the achievement of the progress of HIMAPAUDI teachers in the Kemayoran District, Central Jakarta. KEYWORDS:1Blogspot, Direct rating method, Internet, Matematika sederhana, Pelatihan. KEYWORDS:1Blogspot, Direct rating method, Internet, Matematika sederhana, Pelatihan. DOI: 10.30653/002.201942.144 Open Access \| Url: http://ppm.ejournal.id/index.php/pengabdian/article/view/144 Open Access \| Url: http://ppm.ejournal.id/index.php/pengabdian/article/view/144 This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2019 Akmaludin, Popon Handayani, Laila Septiana. PENDAHULUAN Internet merupakan infrastruktur yang paling sukses dalam pengembangan informasi dengan membantu kemajuan dibidang-bidang lainnya yang menjalar hingga seluruh dunia (Tronco, 2010). Keberhasilan ini terbukti begitu banyak para pengguna internet diseluruh dunia dan berlomba-lomba membangun jaringan internet. Teknologi pembelajaran menggunakan internet telah menjadi salah satu instrumentasi yang banyak JURNAL PENGABDIAN PADA MASYARAKAT is published by LP3M of Universitas Mathla’ul Anwar Banten 111 Jurnal Pengabdian Pada Masyarakat 2019, 4(2), 111-118 digunakan oleh kalangan masyarakat saat ini (Hapsari, Novitasari, & Wahyuningsih, 2019). Guru guru PAUD kelurahan Sunter Kecamatan Kemayoran Jakarta Pusat, merupakan sekolah pendidikan anak usia dini khusus untuk taman pendidikan qur’an (TPQ), dengan demikian para pendidik harus mampu mengantarkan peserta didik dalam aspek pengetahuan (cognitive) pemahaman dan penerapan nilai-nilai keislaman ( Setiawati, Netriwati, & Nasution, 2018). Berdasarkan hasil wawancara yang dilakukan, membutuhkan pelatihan tentang penggunaan Internet, untuk meningkatkan kemampuan guru-guru paud (HIMAPAUDI) dalam mengaplikasikan beberapa kegiatan yang berhubungan dengan Internet seperti pembuatan blog, pembuatan email, dan fasilitas penggunaan internet lainnya. Oleh sebab itu, Para dosen dan mahasiswa bersama-sama bermaksud memenuhi permintaan tersebut untuk mengadakan kegiatan pengabdian kepada masayarakat bagi guru-guru paud untuk melakukan pembelajaran dan meningkatkan ilmu pengetahuan di bidang Internet. Pada awalnya dilakukan pretest terhadap guru-guru paud (HIMAPAUDI) dan hasil yang didapat dapat disimpulkan memang benar guru-guru paud tersebut sangat membutuhkan proses pembelajaran terhadap penggunaan fasilitas internet yang pada khususnya adalah kebutuhan pembuatan blog yang diiringi dengan pembuatan email sebagai prasyarat pembuatan blog tersebut. Diakhir proses pelatihan ini, akan dilakukan penilaian tehadap apa yang telah didapat dari proses pelatihan ini dengan menggunakan metode direct rating method yang dikombinasi dengan metode matematika sederhana. Bagaimana penilaian dari hasil pelatihan dengan penentuan nilai scoring dari lima variable dan sepuluh indikator penilaian. Dalam pembelajaran, peran media pengajaran memiliki amanat penting dalam pengembangan pengetahuan disegala bidang ilmu pengetahuan dan teknologi (Vebrianto & Kamisah, 2011). Teknologi internet merupakan salah satu media atau instrumentasi yang ampuh dan dapat digunakan sebagai sarana dan prasarana dalam pengembangan berbagai bidang ilmu (Riyana, 2008). Peranan internet dalam kemajuan proses pembelajaran akan menghabiskan waktu dikalangan para remaja dan orang tua, tetapi lama kemudian penggunaan fasilitas internet akan berubah menjadi komunikasi yang terupdate untuk menghabiskan waktunya di kalangan remaja dan orang tua, karena fungsi dan kegunaanya mampu memfasilitasi kemampuan komunikasi kepada seluruh lapisan masyarakat di dunia (Shim, 2006). METODE PELAKSANAAN Metode yang digunakan untuk pelatihan internet dalam hal pembuatan e-mail dan pembuatan blog dengan fasilitas melalui blogspot.com dilakukan secara in house training dan terjun langsung praktik untuk mengetahui secara langsung sambil memberikan penjelasan terhadap tahapan dan cara penggunaanya, hal ini dilakukan agar para peserta pelatihan khususnya guru-guru PAUD (HIMAPAUDI) agar dapat secara cepat menerima dan mempraktikan secara implisit untuk keberhasilan transfer knowledge. Adapun tahapan metode yang dilakukan adalah: 1) Memberikan pre-test, sebagai awal untuk mengetahui kemampuan dari peserta pelatihan dengan sejumlah indikator pertanyaan. 2) Para tim tutor yang dibantu para mahasiswa untuk memberikan pengetahuan materi internet dan pemahaman pembuatan e-mail dan blog kepada guru-guru PAUD (HIMAPAUDI), disesi ini juga diberikan kebebasan para peserta untuk bertanya secara langsung kepada tim tutor. 3) Para tutor yang dibantu para mahasiwa untuk memberikan bimbingan dan tuntunan kepada para peserta dengan pendampingan satu mahasiswa untuk satu peserta, agar secara cepat peserta bertanya dan secara cepat memahaminya. 4) Para mahasiswa memberikan tuntunan dengan mengacu kepada tim tutor, agar proses pembelajaran berjalan sesuai materi dan waktu pemberian tahap pembelajaran. 5) Para peserta guru-guru PAUD yang sudah memahami atas penyampaian materi diberikan tips dan trik lain oleh para mahasiswa sebagai pendampingan yang dapat dikembangkan oleh peserta, agar tidak jenuh menunggu peserta lain yang belum memahaminya. Pengisian lembar post-test, digunakan untuk mengetahui hasil akhir dari proses pelatihan dan untuk memberikan takaran penilaian secara keseluruhan bagi pelaksanaan pelatihan ini. PENDAHULUAN Guru-guru paud ini juga masih dikatakan level remaja dan beberapa orang tua yang masih mau menggunakan dan belajar tentang internet, mereka memang belum tau sebenarnya fasilitas yang ada melalui jalur internet, tetapi mungkin dengan internet ini mereka akan dapat lebih untuk menggunakan pemanfaatannya secara lebih jauh lagi. Mereka menggunakan internet dengan harapan dapat menunjang proses pembelajaran dan juga pemerintah telah memberikan fasilitas untuk para guru paud untuk dapat menggunakan website khusus bagi guru-guru PAUD dengan alamat anggunpaud.kemdikbud.go.id yang dirancang dalam bentuk networking, terdapat beberapa fitur seperti ada ruang video, ruang lagu, ruang cerita, dan ruang permainan. Dalam pelaksanaan kegiatan pelatihan untuk meningkatkan kemampuan pada guru-guru PAUD membuat Blog melalui template Blogspot yang ditunjang secara wajib untuk pembuatan e-mail sekaligus sebagai bahan pembelajaran juga, karena hasil pretest ditemukan belum banyak yang memiliki e-mail dari mereka dan masih minim sekali pengetahuan tentang penggunaan fasilitas yang ada dalam penggunaan internet, 112 Akmaludin, Popon Handayani, Laila Septiana sehingga perlu diberikan pelatihan penggunaan internet dan fasilitasnya. Pembuatan blog ini adalah cara yang termudah untuk mengembangkan pemikiran anda sesuai keinginan sendiri dan ada situs gratis yang menyediakan fasilitas pembuatan blog seperti blogspot.com, wordpress.com dan dapat menggunakan fasilitas disain sesuai keinginan sendiri (Gordon, 1995). HASIL DAN PEMBAHASAN Pelatihan yang dilakukan pada guru-guru PAUD (HIMAPAUDI) bertujuan untuk memberikan dukungan kepada mereka untuk dapat mengembangkan keinginannya menggunakan fasilitas internet baik berupa e-mail, pembuatan blog, komunikasi via fitur chatting dan fasilitas lainnya (Said, 2015), tentunya akan terjadi perubahan besara dalam setiap diri manusia. Dalam kondisi kerja di organisasi setelah kedatangan teknologi IT menjadi konsep yang terkenal dibanyak pasar vertical dan horizontal kehidupan sehari- hari orang sudah biasa memili aplikasi sendiri-sendiri untuk memprediktabilitas kemampuan mereka sendiri (Madakam, Ramaswamy, & Tripathi, 2015). 113 Jurnal Pengabdian Pada Masyarakat 2019, 4(2), 111-118 Di lain hal diharapkan proses pembelajaran di tingkat sekolah PAUD lebih menekankan kepada penggunaan ICT yang dapat dan memungkinkan untuk menggunakan fasilitas internet dari segala keguanaan dan fungsinya (Puteh & Salam, 2011), terutama pengembangan ilmu para guru-guru PAUD (HIMAPAUDI) dalam hal penggunaan internet terlebih dahulu dapat menyiapkan bekal untuk mampu mengarah kepada penggunaan ICT di masa yang akan datang, sehingga akan memberikan dampak gelobal yang dapat dikenal oleh seluruh peserta didiknya, terutama pengembangan para guru-guru PAUD yang mampu mengexplorasi kemampuan secara pribadinya. Proses pelatihan diberikan satu peserta didampingi oleh satu mahasiswa sebagai peran pendukung tim tutor dalam menyampaikan materi pelatihan dalam proses belajar transfer knowledge. Bebarapa variable yang digunakan sebagai barometer ada lima (Afektif, Cognitif, Psikomotorik, Teknologi, dan Manfaat) dan indicator yang digunakan sebanyak sepuluh pertanyaan dengan jumlah peserta sebagai responden sebanyak 30 orang. Beberapa formulasi yang digunakan untuk pengolahan hasil pre-test yaitu: Pengolahan pre-test menggunakan skala lingkert dengan jangkauan 1-5, sehingga interval dapat dilakukan dengan menggunakan formula-1. 1) 1) Dalam pelatihan ini terdapat 30 responden dan untuk menentukan prosenstasi responden dapat digunakan formula-2. 2) Dari jumlah responden yang telah ditentukan, karena menggunakan sampling jenus, artinya semua responden yang berjumlah 30 orang, digunakan seluruhnya untuk menentukan apakah terjadi kemajuan atau tidak, yang awalnya peserta pelatihan mendominasi kurang mengetahui penggunaan internet khususnya 3) 114 114 Akmaludin, Popon Handayani, Laila Septiana Dalam pembuatan e-mail dan blog, ingin diketahui peningkatan yang terjadi dengan pemberikan scoring dari lima variable sebagai barometer pengukuran. Adapun rumus yang dapat digunakan terlihat pada formula-3. Adapun hasil dari masing-masing variabel yang telah diukur, setelah melakukan pelatihan sebagai hasil post-test yang diolah dengan menggunakan kolaborasi metode direct rating dan matematika sederhana adalah: Gambar 1. Variabel Afektif Gambar 2. Variabel Cognitif Gambar 3. Variabel Psikomotorik Gambar 4. Variabel Teknologi Gambar 5. Variabel Manfaat Gambar 6. HASIL DAN PEMBAHASAN Basis Skala Tiap Variabel dan Hasil Total Score Dari hasil yang diperoleh terhadap kegiatan pelatihan internet pembuatan blog pada guru-guru PAUD (HIMAPAUDI) kecamatan Kemayoran Jakarta Pusat, dapat dijelaskan secara kuantitatif dan kualitatif adalah sebagai berikut: Gambar 1. Variabel Afektif Gambar 1. Variabel Afektif Gambar 2. Variabel Cognitif Gambar 1. Variabel Afektif Gambar 2. Variabel Cognitif Gambar 3. Variabel Psikomotorik Gambar 4. Variabel Teknologi Gambar 3. Variabel Psikomotorik Gambar 4. Variabel Teknologi Gambar 6. Basis Skala Tiap Variabel dan Hasil Total Score Gambar 5. Variabel Manfaat Gambar 6. Basis Skala Tiap Variabel dan Hasil Total Score Gambar 5. Variabel Manfaat Dari hasil yang diperoleh terhadap kegiatan pelatihan internet pembuatan blog pada guru-guru PAUD (HIMAPAUDI) kecamatan Kemayoran Jakarta Pusat, dapat dijelaskan secara kuantitatif dan kualitatif adalah sebagai berikut: 115 Jurnal Pengabdian Pada Masyarakat 2019, 4(2), 111-118 Pertama, penilaian terhadap variabel Afektif yang terlihat pada (Gambar 1), bahwa perolehan jangkauan nilai skala yang didapat dengan bobot 4.33 dengan hasil konversi direct rating bernilai 17,33, hal ini kemampuan semangat terhadap hasil pelatihan dikategorikan sangat baik. Kedua, penilaian terhadap variabel Cognitif yang terlihat pada (Gambar 2), bahwa perolehan terhadap jangkauan nilai skala dengan bobot 4,50 dan hasil konversi dengan direct rating sebesar 18,00, hal ini pemahaman terhadap penerimaan materi yang disampaikan dapat diterima dengan sangat baik. Ketiga, penilaian terhadap variabel Psikomotorik yang terlihat hasilnya pada (Gambar 3), bahwa hasil nilai yang diperoleh dengan jangkauan skala sebesar 4,30 dan besaran nilai konversi direct rating sebesar 17,20, hal ini menjelaskan bahwa penggunaan fasilitas peralatan baik mouse maupun fungsi tombol dapat digunakan dengan sangat baik. Keempat, penilaian terhadap variabel Teknologi yang dapat dilihat pada (Gambar 4), bahwa perolehan nilai jangkauan skala dengan bobot 4,57 dan nilai konversi direct rating dengan nilai 18,27, hal ini memberikan penilai terhadap pemahaman akan teknologi sangat baik sekali. Kelima, penilaian terhadap variabel manfaat yang dapat dilihat pada (Gambar 5) dengan nilai skala jangkauan memiliki bobot 4,53 dan nilai konversi direct rating dengan nilai 18,13, hal ini memberikan arti bahwa manfaat dari pelatihan internet untuk pembuatan blog bagi peserta sangat baik sekali dan kalau bisa dilanjutkan untuk tahap yang lebih mahir. Gambar 7. Dokumentasi Kegiatan Pelatihan Internet Pembuatan Blog Gambar 7. HASIL DAN PEMBAHASAN Dokumentasi Kegiatan Pelatihan Internet Pembuatan Blog Dengan demikian jika dilakukan penjumlahan tehadap perolehan nilai konversi direct rating dengan total score 88,93 hal ini memberikan gambaran terhadap hasil seluruh kegiatan pelatihan internet pembuatan blog untuk guru-guru PAUD (HIMAPAUDI) sangat merespon dengan baik dan mereka memiliki keinginan yang besar untuk 116 Akmaludin, Popon Handayani, Laila Septiana mempelajari lebih dalam tentang dunia internet dengan segala fungsinya. Dengan demikian pelatihan internet pembuatan blog dapat memberikan kepada mereka untuk melek internet dengan dukungan ICT yang memadai mampu memberikan masukan pengetahuan dan informasi yang berkualitas yang didukung infrastruktur yang baik. SIMPULAN Proses pelatihan internet bagi guru-guru PAUD (HIMAPAUDI) memberikan minat yang tiggi tentunya harus didukung oleh teknologi yang memadai dengan adanya koneksi internet, infrastruktur yang baik, tutor yang baik dan tidak kalah pentingnya semangat belajar bagi pesertanya. Hasil yang didapat berdasarkan lima variable dan sepuluh indicator yang diolah menggunakan metode direct rating yang dikombinasi dengan metode matematika sederhana memberikan nilai total score 88,93 hal ini terbukti bahwa seluruh aspek afektif, kognitif, psikomotorik, teknologi, dan manfaat memberikan bagi peserta kegiatan pelatihan internet pembuatan blog mendapat respon yang sangat baik dan perlu dilanjutkan ke jenjang pelatihan yang lebih jauh lagi terhadap penggunaan fasilitas yang ada di internet seperti media on the internet, streaming, online chat, social networking, social book marking dan lainnya sesuai dengan era digitalisasi 4.0. REFERENSI Gordon, S. (1995). Internet 101. Computers and Security, 14(7), 599–604. doi: 10.1016/0167- 4048(96)81666-X Hapsari, A., Novitasari, R., & Wahyuningsih, H. (2019). Pelatihan Literasi Sumber dan Bahan Belajar di Internet bagi Guru PAUD di Kecamatan Ngaglik, Sleman. Jurnal Pengabdian Pada Masyarakat, 3(2), 135–140. doi: 10.30653/002.201832.61 Madakam, S., Ramaswamy, R., & Tripathi, S. (2015). Internet of Things (IoT): A literature review. Journal of Computer and Communications, 3(5), 164. doi: 10.4236/jcc.2015.35021 Puteh, S. N., & Salam, K. A. A. (2011). Tahap kesediaan penggunaan ICT dalam pengajaran dan kesannya terhadap hasil kerja dan tingkah laku murid prasekolah (level of readiness in using ICT for teaching and ıts effect on the work and behaviour of preschool pupils). Jurnal Pendidikan Malaysia, 36(1), 23–54. Riyana, C. (2008). Peranan teknologi dalam pembelajaran. Depok: Universitas Indonesia. iyana, C. (2008). Peranan teknologi dalam pembelajaran. Depok: Universitas Indonesia. Said, H. (2015). Penggunaan ınternet sebagai media pembelajaran pada mahasiswa ıain Palu. HUNAFA: Jurnal Studia Islamika, 12(1), 95–119. Setiawati, R., Netriwati, & Nasution, S. P. (2018). Desain model pembelajaran gerlach dan ely yang berciri nilai-nilai ke-islaman untuk meningkatkan kemampuan komunikasi matematis. AKSIOMA: Jurnal Program Studi Pendidikan Matematika, 7(3), 371-379. Shim, Y. S. (Southern I. U. C. (2006). The Impact of the Internet on Teenagers’ Face -to-Face Communication Young Soo Shim Southern Illinois University Carbondale July 2006. Global Media Journal, (July), 1–32. 117 Jurnal Pengabdian Pada Masyarakat 2019, 4(2), 111-118 Tronco, T. R. (2010). A brief history of the internet. Studies in Computational Intelligence, 297(5), 1–11. doi: 10.1007/978-3-642-13247-6_1 Tronco, T. R. (2010). A brief history of the internet. Studies in Computational Intelligence, 297(5), 1–11. doi: 10.1007/978-3-642-13247-6_1 Tronco, T. R. (2010). A brief history of the internet. Studies in Computational Intelligence, 297(5), 1–11. doi: 10.1007/978-3-642-13247-6_1 Vebrianto, R., & Osman, K. (2011). The effect of multiple media instruction in improving students’ science process skill and achievement. Procedia-Social and Behavioral Sciences, 15, 346-350. Vebrianto, R., & Osman, K. (2011). The effect of multiple media instruction in improving students’ science process skill and achievement. Procedia-Social and Behavioral Sciences, 15, 346-350. 118 118
https://openalex.org/W2987506717	https://www.nature.com/articles/s41467-019-13022-7.pdf	English	null	Unassisted solar lignin valorisation using a compartmented photo-electro-biochemical cell	Nature communications	2,019	cc-by	12,105	1 School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea. 2 Department of Energy Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea. 3 UNIST Central Research Facilities & School of Natural Science, Ulsan National Institute of Science and Technology (UNIST), 50 UNIST-gil, Ulsan 44919, Republic of Korea. 4Present address: Department of Chemistry, Kwangwoon University, 20 Gwangwoon-ro, Nowon-gu, Seoul 01897, Republic of Korea. 5These authors contributed equally: Myohwa Ko, Le Thanh Mai Pham, Young Jin Sa. email: shjoo@unist.ac.kr; metalkim@unist.ac.kr; jiwjang@unist.ac.kr ARTICLE ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 produced in the conduction band, and these are then utilised for O2 reduction, producing H2O2. Recently, several groups have combined this photocatalytic H2O2 system with biocatalysts and applied them for various chemical reactions. For example, a modiﬁed powder-type TiO2 photocatalyst was utilised for H2O2 generation, and it was integrated with a biocatalytic system of peroxygenases for the selective oxyfunctionalisations of carbon–hydrogen bonds, which has been one of the major chal- lenges in organic synthesis32–35. However, this integrated system (biocatalytic system with photocatalytically generated H2O2) has been rarely applied for lignin valorisation, and most of the research has been focused on developing an efﬁcient powder-type photocatalyst, such as Nb2O5/TiO2 heterojunction, metal-doped TiO2 or graphene–TiO2 nanocomposite for direct lignin con- version rather than for H2O2 production12,36. One of the important limitations of the integrated system is that, for efﬁcient H2O2 generation using powder-type photocatalysts, additional chemicals are required to scavenge the remaining hole in the valence band. Most crucially, the reactive oxygen species (ROS) generated during the photochemical reaction not only destabilise the biocatalyst but also cause the random cracking/breaking of the lignin structure, thus decreasing the selectivity of lignin conversion dramatically. B B iomass is considered a promising replacement for fossil fuels because it is the most abundant carbon source in nature and is carbon neutral1,2. There has been remarkable progress on biomass conversion technologies, especially the conversion of sugar or starch crops to biofuels and various che- micals. However, the use of edible biomass as a feedstock is controversial in terms of ethics and cost3. Hence, waste biomass, so-called lignocellulosic biomass, such as wood residues, straw, and crop stover, have recently emerged as promising carbon sources3. Lignin is one of the three major components of lignocellulosic biomass, together with cellulose and hemicellulose. It is nature’s most abundant source of aromatic carbon compounds and can be potentially transformed into high-value products4–11. However, because of its complex/irregular chemical structure and currently limited processing technology, more than 99% of lignin is abandoned or burned12. To make lignocellulosic biomass a more compatible renewable carbon source, an effective method for lignin valorisation is vital. ARTICLE The key issue here is ﬁnding effective strategies for the selective cleavage of carbon–oxygen bonds (C–O–C), more speciﬁcally, the β-ether (β-O-4) bond connecting the three basic aromatic units (coumaryl alcohol, coniferyl alco- hol, and sinapyl alcohol); this is a critical step for the depoly- merisation of lignin to yield valuable aromatic chemicals and feedstocks13–17. Here, we propose a compartmented photo-electro-biochemical cell, in which three catalytic systems (a photocatalyst for photo- voltage generation, an electrocatalyst for H2O2 production, and a biocatalyst for lignin valorisation) are integrated for selective lignin valorisation without the need for electrical energy or additional chemicals (Fig. 1). Importantly, the compartmentali- sation of the three catalytic systems using Naﬁon and cellulose membranes stabilises the biocatalyst from enzyme-damaging elements, such as ROS, the high concentration of H2O2, shear stress from gas (O2) bubbles, and light. Moreover, because the lignin and photocatalyst are separated from each other, the dark- coloured native lignin does not reduce the light adsorption efﬁ- ciency of the photocatalyst, a problem that has plagued the use of a photocatalyst for lignin valorisation (Fig. 1). The compart- mented photo-electro-biochemical cell can catalyse the depoly- merisation of lignin dimer with high selectivity (>95%) and the polymerisation of a benchmark lignin monomer, coniferyl alco- hol, in 73.3% yield. The catalytic performance of the three- compartment system is superior to those of single-compartment and two-compartment catalytic systems. Various treatment technologies including physical (ball mil- ling, ultrasonication, plasma irradiation, and microwave heating), chemical (using an organic or inorganic acid), or catalytic (with a heterogeneous catalyst) methods have been used for lignin valorisation18–21. However, these processes are usually energy- intensive, requiring high temperatures and pressures, and envir- onmentally unfriendly, producing chemical waste. Most impor- tantly, selective carbon–oxygen bond breakage is not plausible using these conventional methods. However, microorganisms including fungi and bacteria have been selectively degrading lig- nin for more than 300 million years, although the detailed mechanism remains elusive22. In particular, the lignin peroxidase isozyme H8 (LiPH8) biocatalyst from white rot fungi has received much attention because of its exceptional ability for the selective cleavage of β-O-4 bonds in lignin23–25. However, a critical lim- itation of biocatalyst systems is that hydrogen peroxide (H2O2), which acts as an electron acceptor, must be provided from an external source, and its high concentration is detrimental to enzyme stability, limiting the scale-up of these systems. ARTICLE The integration of the H2O2 generation system with the bio- catalytic conversion system allows for continuous production and utilisation of H2O2, which would allow the use of low con- centrations of H2O2 and the production of large-scale devices. The current method of producing H2O2 is predominantly based on the anthraquinone process, which consists of multi-step pro- cesses and requires additional separation steps in the presence of high pressure H2 and expensive precious metal catalysts26. Direct reaction of H2 and O2 on a catalyst is a simple and clean process, but this requires high-pressure gases (H2 and O2)27,28. Therefore, the anthraquinone process and direct H2O2 synthesis are not compatible with biocatalytic systems. In contrast, electrochemical H2O2 production via two-electron O2 reduction is a simple, clean, safe, and low-cost process29–31. Furthermore, this process oper- ates under ambient pressure and temperatures, enabling its facile integration with the biocatalytic system. However, a critical lim- itation remains: additional electrical energy must be supplied to operate this integrated system. NATURE COMMUNICATIONS \| (2019) 10:5123 \| https://doi.org/10.1038/s41467-019-13022-7 \| www.nature.com/naturecommunications Unassisted solar lignin valorisation using a compartmented photo-electro-biochemical cell 1 5 1 5 1 4 5 1 2 1 Myohwa Ko1,5, Le Thanh Mai Pham1,5, Young Jin Sa 1,4,5, Jinwoo Woo1,2, Trang Vu Thien Nguyen1, Jae Hyung Kim1,2, Dongrak Oh1, Pankaj Sharma1, Jungki Ryu 1,2, Tae Joo Shin 3, Sang Hoon Joo1,2, Yong Hwan Kim1* & Ji-Wook Jang 1* Lignin is a major component of lignocellulosic biomass. Although it is highly recalcitrant to break down, it is a very abundant natural source of valuable aromatic carbons. Thus, the effective valorisation of lignin is crucial for realising a sustainable bioreﬁnery chain. Here, we report a compartmented photo-electro-biochemical system for unassisted, selective, and stable lignin valorisation, in which a TiO2 photocatalyst, an atomically dispersed Co-based electrocatalyst, and a biocatalyst (lignin peroxidase isozyme H8, horseradish peroxidase) are integrated, such that each system is separated using Naﬁon and cellulose membranes. This cell design enables lignin valorisation upon irradiation with sunlight without the need for any additional bias or sacriﬁcial agent and allows the protection of the biocatalyst from enzyme- damaging elements, such as reactive radicals, gas bubbles, and light. The photo-electro- biochemical system is able to catalyse lignin depolymerisation with a 98.7% selectivity and polymerisation with a 73.3% yield using coniferyl alcohol, a lignin monomer. 1 School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea. 2 Department of Energy Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea. 3 UNIST Central Research Facilities & School of Natural Science, Ulsan National Institute of Science and Technology (UNIST), 50 UNIST-gil, Ulsan 44919, Republic of Korea. 4Present address: Department of Chemistry, Kwangwoon University, 20 Gwangwoon-ro, Nowon-gu, Seoul 01897, Republic of Korea. 5These authors contributed equally: Myohwa Ko, Le Thanh Mai Pham, Young Jin Sa. *email: shjoo@unist.ac.kr; metalkim@unist.ac.kr; jiwjang@unist.ac.kr 1 NATURE COMMUNICATIONS \| (2019) 10:5123 \| https://doi.org/10.1038/s41467-019-13022-7 \| www.nature.com/naturecommunications TURE COMMUNICATIONS \| (2019) 10:5123 \| https://doi.org/10.1038/ NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 The schematic of the second and third compartments shows the gradual increase in the concentration of photogenerated H2O2 and continuous ﬂow to the biocatalytic system (third compartment), which are ideal conditions for selective lignin conversion catalysts are incompatible with photocatalysis cells because of their instability41. In order to endow catalytic stability while preserving the intrinsic activity of the Co-porphyrin-based molecular catalysts, we used a silica-protective-layer-assisted synthesis42,43 (see the “Methods” section for detail). In the resulting catalyst, atomically dispersed Co–Nx sites are homo- geneously generated on carbon nanotubes (denoted Co–N/CNT) without the aggregation of the Co species (Supplementary Fig. 5a, b). For comparison, a cobalt porphyrin molecular catalyst immobilised on the CNTs was also prepared (CoTMPP/CNT). The Co–N/CNT catalyst was found to contain 0.8 and 1.3 wt% of Co and N, respectively, as determined by inductively coupled plasma optical emission spectrometry and combustion elemental analysis, respectively. Co 2p and N 1s X-ray photoelectron spectroscopy (XPS) scans (Supplementary Fig. 6) suggest the presence of oxidised Co species (satellite peaks) and four types of N species on the surface of the Co–N/CNT catalyst. Extended X- ray absorption ﬁne structure analysis (Supplementary Fig. 7) reveals that the Co–N coordination bonds (Co–Nx sites) of the cobalt porphyrin precursor were retained in the Co–N/CNT catalyst after the high-temperature treatment, and there is no observable Co–Co bond peak, indicating that the aggregation of Co metal was minimised by the silica-protective-layer strategy. protection of incident light by a back cover of the photoelectrode (Supplementary Fig. 1a, b), respectively. These features of the photo-electro-biochemical process allow for a continuous and stable lignin valorisation (Fig. 1). Unassisted photo-electrochemical H2O2 production. As a photoanode for water oxidation, a rutile TiO2 nanowire ﬁlm was used. This ﬁlm was hydrothermally grown on a ﬂuorine-doped tin oxide (FTO) glass substrate37 and was subsequently annealed in a hydrogen atmosphere to improve the charge transfer prop- erties of TiO2 (denoted H:TiO2)38. The X-ray diffraction (XRD) pattern of H:TiO2 (Supplementary Fig. 2a) shows two diffraction peaks at 36.1° and 62.8°, which is consistent with that of rutile TiO2. There was no signiﬁcant change in the UV–vis spectra of the TiO2 ﬁlms upon hydrogen treatment (Supplementary Fig. 2b). The scanning electron microscopy (SEM) image of H:TiO2 (Supplementary Fig. 3) reveals that a homogeneous ﬁlm was formed on the FTO substrate, consisting of vertically aligned nanowire arrays of 100–200 nm in diameter. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 The photoanode is connected to the Co–N/carbon nanotube (CNT) cathode through a copper wire for in situ H2O2 generation in the second compartment (middle part). A Naﬁon proton exchange membrane separates the anodic and cathodic parts of the photo-electrochemical cell. The use of a Naﬁon membrane also helps to prevent the oxidation of photogenerated H2O2 on the highly photoactive H:TiO2 photoanode surface. To carry out the continuous O2 reduction to H2O2 on the Co–N/CNT cathode surface, the second compartment is also equipped with continuous O2 gas supply. However, the presence of •O2−radicals, a high H2O2 concentration, and continuous O2 bubbling have adverse effects on the biocatalytic activity of enzymes. Therefore, the photo-electrochemical and biochemical cells are separated by a cellulose membrane. The schematic of the second and third compartments shows the gradual increase in the concentration of photogenerated H2O2 and continuous ﬂow to the biocatalytic system (third compartment), which are ideal conditions for selective lignin conversion Fig. 1 Schematic illustration of the three-compartment photo-electro-biochemical reactor. The photo-electro-biochemical reactor consists of three separate compartments. The ﬁrst compartment (left-hand side) is the anodic part of the photo-electrochemical cell where water oxidation takes place. The cell comprises a cubic reactor with a quartz window on the left-hand side for the photochemical reactions on the TiO2 photoanode surface. Rutile TiO2 nanowire ﬁlms grown on ﬂuorine-doped tin oxide (FTO) glass substrate are used as the photoanode. Hydrogen treatment was performed to enhance the photocatalytic activity of TiO2 photoanode (H:TiO2). The photoanode is connected to the Co–N/carbon nanotube (CNT) cathode through a copper wire for in situ H2O2 generation in the second compartment (middle part). A Naﬁon proton exchange membrane separates the anodic and cathodic parts of the photo-electrochemical cell. The use of a Naﬁon membrane also helps to prevent the oxidation of photogenerated H2O2 on the highly photoactive H:TiO2 photoanode surface. To carry out the continuous O2 reduction to H2O2 on the Co–N/CNT cathode surface, the second compartment is also equipped with continuous O2 gas supply. However, the presence of •O2−radicals, a high H2O2 concentration, and continuous O2 bubbling have adverse effects on the biocatalytic activity of enzymes. Therefore, the photo-electrochemical and biochemical cells are separated by a cellulose membrane. Results Design of the photo-electro-biochemical system. The designed photo-electro-biochemical system is composed of three com- partments (photocatalyst anode, electrocatalyst cathode, and biocatalyst part), as shown in Fig. 1. Each cell is ﬁlled with an appropriate solution, and the cells are separated by Naﬁon and cellulose membranes. In the ﬁrst compartment, a semiconductor photocatalyst receives the solar energy, and photoexcitation generates charge carriers. The photogenerated hole in the valence band oxidises water to O2, and the electrons move to the second compartment, where the electrocatalyst selectively reduces O2 to H2O2. The produced H2O2 is transferred through the size- selective cellulose membrane to the third compartment, where lignin valorisation via the biocatalyst occurs with the aid of the permeated H2O2. Importantly, the two membranes between each catalytic system prevent the permeation of O2 bubbles and ROS, such as hydroxyl radicals (•OH) and superoxide anions (•O2−), thus protecting the biocatalyst. In addition, the compartmented design ensures that the biocatalyst is not exposed to a high concentration of H2O2 and intense light because of the gradual permeation of H2O2 through the cellulose membrane and the On the other hand, photocatalysis can also produce H2O2 in a straightforward, clean manner and can be operated under ambient conditions like electrocatalytic H2O2 production. On the irradiation of the photocatalyst with light, excited electrons are URE COMMUNICATIONS \| (2019) 10:5123 \| https://doi.org/10.1038/s41467-019-13022-7 \| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 NATURE COMMUNICATIONS \| (2019) 10:5123 \| https://doi.org/10.1038/s41467-019-13022-7 \| www.nature.com/naturecommunicatio NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 Nafion membrane Cellulose membrane Co–N/CNT Electrocatalyst Photocatalyst H:TiO2 Lignin Flavor Plant Cosmetics Plastic Biocatalyst hv O2 O2 O2+2H+ H2O2 Nafion membrane Cellulose membrane Co–N/CNT Electrocatalyst Photocatalyst H:TiO2 Lignin Flavor Plant Cosmetics Plastic Biocatalyst hv O2 O2 O2+2H+ H2O2 Nafion membrane Cellulose membrane Co–N/CNT Electrocatalyst Photocatalyst H:TiO2 Lignin Flavor Plant Cosmetics Plastic Biocatalyst hv O2 O2 O2+2H+ H2O2 Fig. 1 Schematic illustration of the three-compartment photo-electro-biochemical reactor. The photo-electro-biochemical reactor consists of three separate compartments. The ﬁrst compartment (left-hand side) is the anodic part of the photo-electrochemical cell where water oxidation takes place. The cell comprises a cubic reactor with a quartz window on the left-hand side for the photochemical reactions on the TiO2 photoanode surface. Rutile TiO2 nanowire ﬁlms grown on ﬂuorine-doped tin oxide (FTO) glass substrate are used as the photoanode. Hydrogen treatment was performed to enhance the photocatalytic activity of TiO2 photoanode (H:TiO2). The photoanode is connected to the Co–N/carbon nanotube (CNT) cathode through a copper wire for in situ H2O2 generation in the second compartment (middle part). A Naﬁon proton exchange membrane separates the anodic and cathodic parts of the photo-electrochemical cell. The use of a Naﬁon membrane also helps to prevent the oxidation of photogenerated H2O2 on the highly photoactive H:TiO2 photoanode surface. To carry out the continuous O2 reduction to H2O2 on the Co–N/CNT cathode surface, the second compartment is also equipped with continuous O2 gas supply. However, the presence of •O2−radicals, a high H2O2 concentration, and continuous O2 bubbling have adverse effects on the biocatalytic activity of enzymes. Therefore, the photo-electrochemical and biochemical cells are separated by a cellulose membrane. The schematic of the second and third compartments shows the gradual increase in the concentration of photogenerated H2O2 and continuous ﬂow to the biocatalytic system (third compartment), which are ideal conditions for selective lignin conversion Fig. 1 Schematic illustration of the three-compartment photo-electro-biochemical reactor. The photo-electro-biochemical reactor consists of three separate compartments. The ﬁrst compartment (left-hand side) is the anodic part of the photo-electrochemical cell where water oxidation takes place. The cell comprises a cubic reactor with a quartz window on the left-hand side for the photochemical reactions on the TiO2 photoanode surface. Rutile TiO2 nanowire ﬁlms grown on ﬂuorine-doped tin oxide (FTO) glass substrate are used as the photoanode. Hydrogen treatment was performed to enhance the photocatalytic activity of TiO2 photoanode (H:TiO2). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 RHE) 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 d c E (V vs. RHE) Fig. 2 Photo-electrocatalytic production of H2O2 in the three-compartment reactor. a Photocurrent density of the H:TiO2 electrode under illumination (orange line) and in the dark (black line) in 0.1 M phosphate borate solution at pH 4.5. Inset: SEM image of H:TiO2. Sacle bar is 1 µm. b Linear sweep voltammetry (LSV) curves of Co–N/CNT (sky blue), CoTMPP/CNT (pink), and CNT (grey) catalysts before (solid line) and after (dashed line) potential cycling tests. The current densities for H2O2 production are shown and were obtained via rotating ring disk electrode measurements at an electrode rotation speed of 1,600 rpm in O2-saturated 0.1 M phosphate borate solution at pH 4.5. c LSV curves of H:TiO2 photoanode (orange line) under simulated 1 sun (AM1.5 G) illumination in 0.1 M phosphate borate solution at pH 4.5 and LSV curves of Co–N/CNT cathode (sky blue line) in O2-saturated 0.1 M phosphate borate solution at pH 4.5. d Amount of H2O2 generated over time in 0.1 M phosphate borate solution at pH 4.5 during the operation of the integrated three-compartment reactor. Error bars indicate the standard deviation Koutecky–Levich analyses show the consistently high H2O2 selectivity (48–62%) of the Co–N/CNT catalyst (Supplementary Fig. 8b). The molecular CoTMPP/CNT catalyst shows a better H2O2 selectivity of 60–64% than Co–N/CNT, which is attributed to its well-deﬁned Co–N4 structure. However, after being subjected to prolonged potential cycling tests, CoTMPP/CNT showed a decline in the activity for H2O2 production of around 20%, whereas the Co–N/CNT catalyst mostly maintained its activity (Fig. 2b). Scanning transmission electron microscopy (STEM) images of Co–N/CNT before and after the stability test were nearly the same, indicating that atomically dispersed Co species are not agglomerated or detached from CNT support (Supplementary Fig. 5). The Co–N/CNT catalyst is also active for electrochemical H2O2 production at low pH (in 0.1 M HClO4), extending the potential applicability of this catalyst to photo- catalytic H2O2 production, as well as use with other photoanodes and biocatalysts (Supplementary Fig. 9). To verify the stability of our integrated photo-electrochemical H2O2 production system, the amount of H2O2 generated on the Co–N/CNT cathode was estimated as a function of time using a colorimetric method with N,N-diethyl-p-phenylenediamine (DPD) (Fig. 2d). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 Phosphate borate solution at pH 4.5 was utilised as an electrolyte. Under sunlight illumination, the photocurrent density of the bare TiO2 nanowire photoanode at 1.23 V (vs. reversible hydrogen electrode, RHE) was 0.97 mA cm−2, which is far higher than that of planar-type TiO2 electrode (0.11 mA cm−2) because of the improved charge transfer rate and increased surface area due to the nanowire morphology (Supplementary Fig. 4a). Upon hydrogen treatment of TiO2 (H:TiO2), the photocurrent density was further increased to 1.25 mA cm−2 with almost the same onset potential of 0.40 V (vs. RHE) and there was no sign of a decrease in its performance for 12 h (Fig. 2a and Supplementary Fig. 4). The ORR activity and selectivity of the Co–N/CNT and CoTMPP/CNT catalysts were investigated using the rotating ring disk electrode (RRDE) technique (Fig. 2b and Supplementary Fig. 8). The ORR polarisation curve shows that the Co–N/CNT catalyst started to generate the H2O2 production current from 0.78 V (vs. RHE) in phosphate borate solution at pH 4.5 (Fig. 2b). This onset potential is very close to thermodynamic equilibrium potential for two-electron ORR with an overpotential of only 0.04 V (Supplementary Note 1), indicating the excellent catalytic activity of the Co–N/CNT catalyst. In contrast, CoTMPP/CNT and pristine CNT required much larger overpotentials of 0.33 and 0.47 V (Fig. 2b), respectively, suggesting that the heat- treated Co–Nx structure results in high activity. RRDE and g pp y g The photovoltage generated at the anode is used to catalyse O2 reduction at the cathode, completing the unassisted production of H2O2. As H2O2 production electrocatalysts, cobalt porphyrins have shown high activity and selectivity for the two-electron oxygen reduction reaction (ORR)39,40. However, these molecular URE COMMUNICATIONS \| (2019) 10:5123 \| https://doi.org/10.1038/s41467-019-13022-7 \| www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 a b c d Cathode Co–N/CNT Photoanode H: TiO2 0.62 mA 0.55 V \| i \| (mA) 0.0 CNT 1000 cycles Co–N/CNT 1000 cycles CoTMPP/CNT –0.3 –0.6 –0.9 –1.2 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 jperoxide (mA cm–2) E–iR (V vs. RHE) E (V vs. RHE) 0.9 20 15 10 5 0 0 1 2 Time (h) 3 4 5 6 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.5 0.5 1.0 1.5 2.0 j (mA cm–2) E (V vs. RHE) 1.2 0.9 0.6 0.3 0.0 Dark Light Generated H2O2 (µmol) Fig. 2 Photo-electrocatalytic production of H2O2 in the three-compartment reactor. a Photocurrent density of the H:TiO2 electrode under illumination (orange line) and in the dark (black line) in 0.1 M phosphate borate solution at pH 4.5. Inset: SEM image of H:TiO2. Sacle bar is 1 µm. b Linear sweep voltammetry (LSV) curves of Co–N/CNT (sky blue), CoTMPP/CNT (pink), and CNT (grey) catalysts before (solid line) and after (dashed line) potential cycling tests. The current densities for H2O2 production are shown and were obtained via rotating ring disk electrode measurements at an electrode rotation speed of 1,600 rpm in O2-saturated 0.1 M phosphate borate solution at pH 4.5. c LSV curves of H:TiO2 photoanode (orange line) under simulated 1 sun (AM1.5 G) illumination in 0.1 M phosphate borate solution at pH 4.5 and LSV curves of Co–N/CNT cathode (sky blue line) in O2-saturated 0.1 M phosphate borate solution at pH 4.5. d Amount of H2O2 generated over time in 0.1 M phosphate borate solution at pH 4.5 during the operation of the integrated three-compartment reactor. Error bars indicate the standard deviation a 1.5 0.5 1.0 1.5 2.0 j (mA cm–2) E (V vs. RHE) 1.2 0.9 0.6 0.3 0.0 Dark Light b 0.0 CNT 1000 cycles Co–N/CNT 1000 cycles CoTMPP/CNT –0.3 –0.6 –0.9 –1.2 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 jperoxide (mA cm–2) E–iR (V vs. RHE) b a E–iR (V vs. RHE) d 20 15 10 5 0 0 1 2 Time (h) 3 4 5 6 Generated H2O2 (µmol) c Cathode Co–N/CNT Photoanode H: TiO2 0.62 mA 0.55 V \| i \| (mA) E (V vs. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 Error bars indicate the standard ation URE COMMUNICATIONS \| https://doi.org/10.1038/s41467 019 13022 7 ARTICLE 100 Conversion (%) Selectivity (%) Three-compartment 80 60 40 20 0 100 80 60 40 20 0 0 1 2 3 Time (h) 4 5 6 b c OH + – – – Co–N/CNT Nafion membrane Cellulose membrane Value-added products Lignin H2O2 O2+2H+ OH H:TiO2 FTO hv O2 O2 – •O2 – a b a c 100 Conversion (%) Single-compartment 80 60 40 20 0 0 1 2 3 Time (h) 4 5 6 Selectivity (%) 100 80 60 40 20 0 e f OH OH O O O MeO MeO OMe OMe OMe HO O O OMe HO TiO2 Random conversion Enzyme deactivation + – Lignin hv O2 – O2 – d d e Selectivity (%) 100 80 60 40 20 0 i OH OH Enzyme deactivation Nafion membrane + – – – Co–N/CNT H:TiO2 FTO hv O2 Value-added products Lignin H2O2 O2+2H+ O2 – O2 – g 100 Conversion (%) Two-compartment 80 60 40 20 0 0 1 2 3 Time (h) 4 5 6 h i h g Fig. 3 Reactor conﬁgurations and photo-electro-biochemical lignin dimer conversion. a, d, g Schematic representations of the three-compartment photo- electro-biochemical system (a), single-compartment powder-based photo-biochemical system (d), and two-compartment photo-electrochemical system (g) used for lignin dimer conversion. b, e, h Conversion and c, f, i selectivity of the three-compartment photo-electro-biochemical system (b, c), single- compartment powder-based photo-biochemical system (e, f), and two-compartment photo-electrochemical system (h, i). Error bars indicate the standard deviation For comparison, we also tested lignin depolymerisation in single-compartment and two-compartment systems. In the single-compartment system, the powder-type TiO2 photocatalyst, the LiPH8 biocatalyst, and the lignin dimer were placed in the same compartment (Fig. 3d and Supplementary Fig. 1c). In this environment, lignin conversion can take place in two possible ways: direct lignin conversion by the charge carriers generated by the TiO2 photocatalyst or biocatalytic lignin conversion using LiPH8 with the aid of the photogenerated H2O2. The obtained lignin dimer conversion and selectivity were 37.3% and 34.8%, respectively (Fig. 3e, f). To investigate the role of the enzyme in the single-compartment system, we performed the lignin conversion experiment in the absence of LiPH8. Interestingly, a similar conversion and selectivity were obtained without LiPH8 (Supplementary Fig. 16) and very little H2O2 was generated in the single compartment (Supplementary Fig. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 We observed continuous production of H2O2 over 6 h of reaction, and found that the H2O2 production rate can be easily increased by scaling up the size of electrodes and the reactor (Supplementary Fig. 11). In addition, the H2O2 produced at the cathode freely diffused into the biocatalyst cell through the cellulose membrane (Supplementary Fig. 12), enabling the utilisation of H2O2 by the biocatalyst for lignin depolymerisation and biopolymer synthesis. Photo-electro-biochemical lignin dimer depolymerisation. There are many reported lignin model compounds, such as ferulic acid, vanillyl alcohol isoeugenol, and benzyl alcohol12,44. We selected lignin dimer as a representative model compound to observe the speciﬁc cleavage of β-O-4. For the selective cleavage of the lignin dimer into 3,4-dimethoxybenzaldehyde (a derivative of vanillin), the LiPH8 biocatalyst was used, along with the photo- electrochemically generated H2O2 (Supplementary Fig. 13). Because our three-compartment cell design protects the bioca- talyst from deactivation, the enzyme activity was maintained throughout the reaction (Fig. 3a and Supplementary Fig. 14). The depolymerisation conversion efﬁciency and selectivity for the lignin dimer were 93.7% and 98.7%, respectively, in the three- compartment photo-electro-biochemical system (Fig. 3b, c). A control reaction carried out in the absence of the LiPH8 enzyme in the three-compartment cell indicated low conversion and selectivity, suggesting that the biocatalyst is essential for selective lignin valorisation (Supplementary Fig. 15). y pp y g Next, a large-area cathode composed of the Co–N/CNT catalyst was prepared on a piece of carbon paper for photo- electrocatalytic H2O2 production. The photo-electrocatalytic activities of the photoanode and cathode for water oxidation and the ORR, respectively, were tested in phosphate borate solution using linear sweep voltammetry (LSV) measurements. The operating current of the integrated photo-electrochemical cell could be estimated from the intersection of the LSV curves of the H:TiO2 photoanode and Co–N/CNT cathode (Fig. 2c). The intersection point in the LSV of H:TiO2 and Co–N/CNT was 0.62 mA, which far exceeds that of H:TiO2 and CoTMPP/CNT (0.09 mA), as well as those of H:TiO2 and pristine CNT (0.07 mA), highlighting the importance of an effective electrocatalyst for unassisted H2O2 production (Fig. 2c and Supplementary Fig. 10). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 NATURE COMMUNICATIONS \| (2019) 10:5123 \| https://doi.org/10.1038/s41467-019-13022-7 \| www.nature.com/naturecommunications 4 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 OH 100 Conversion (%) Selectivity (%) Three-compartment 80 60 40 20 0 100 80 60 40 20 0 0 1 2 3 Time (h) 4 5 6 100 Conversion (%) Single-compartment 80 60 40 20 0 0 1 2 3 Time (h) 4 5 6 100 Conversion (%) Two-compartment 80 60 40 20 0 0 1 2 3 Time (h) 4 5 6 OH OH OH OH O O O MeO MeO OMe OMe OMe HO O O OMe HO TiO2 Random conversion Enzyme deactivation Enzyme deactivation Nafion membrane + – + – + – – – – – Co–N/CNT Nafion membrane Cellulose membrane Value-added products Lignin Lignin H2O2 O2+2H+ OH H:TiO2 FTO Co–N/CNT H:TiO2 FTO hv hv hv O2 O2 O2 – •O2 – Value-added products Lignin H2O2 O2+2H+ O2 – O2 – O2 – O2 – Selectivity (%) 100 80 60 40 20 0 Selectivity (%) 100 80 60 40 20 0 d e f a b c g h i 3 Reactor conﬁgurations and photo-electro-biochemical lignin dimer conversion. a, d, g Schematic representations of the three-compartment photo- tro-biochemical system (a), single-compartment powder-based photo-biochemical system (d), and two-compartment photo-electrochemical system used for lignin dimer conversion. b, e, h Conversion and c, f, i selectivity of the three-compartment photo-electro-biochemical system (b, c), single- partment powder-based photo-biochemical system (e, f), and two-compartment photo-electrochemical system (h, i). NATURE COMMUNICATIONS \| (2019) 10:5123 \| https://doi.org/10.1038/s41467-019-13022-7 \| www.nature.com/naturecommunications ARTICLE In the case of the two-compartment cell, even though •OH radicals do not interrupt the biochemical reactions, the presence of •O2−radicals and O2- coordinating cobalt porphyrin form a superoxide-like structure51, which can disrupt the polymerisation reaction/biopolymer formation (Fig. 4g). This leads to the formation of a signiﬁcant amount of soluble by-products, although the amount produced is lower than that produced in the single-compartment system (Supplementary Figs. 32 and 33). A variety of by-products were detected in the GC–MS analysis (Supplementary Figs. 32 and 33). This indicates that the formation of undesirable highly reactive •O2−and •OH radicals in these systems resulted in low- molecular-weight by-products rather than selective polymerisa- tion. However, in the three-compartment photo-electro- biochemical system, the interaction between ROS radicals and the substrate was limited by separating the electrocatalytic (cathodic) and biocatalytic compartments with a cellulose membrane, as experimentally demonstrated earlier (Supplemen- tary Figs. 25 and 26). This results in the selective oxidation of phenolic compounds, and successive radical coupling leads to a high polymer yield. Control reactions performed in the absence of the HRP biocatalyst using all three systems showed no polymer product formation, demonstrating the pivotal role of biocatalyst for biopolymer synthesis (Supplementary Figs. 35–37). In the three-compartment photo-electro-biochemical system, we veriﬁed that the concentrations of •OH and •O2−radicals were very low in the enzyme cell compared to those in the anode and cathode cells, respectively (Supplementary Figs. 25 and 26), resulting in high conversion and selectivity of lignin dimer depolymerisation. The gradual increase of H2O2 concentration in the three-compartment cell is also a reason for the high performance, as this helps to maintain low concentrations of H2O2 in the enzyme cell. When the initial concentrations were 1 and 10 mM, the conversion efﬁciencies were 82% and 21%, respectively. These values are lower than that of three- compartment system (93.7%) because of enzyme deactivation due to high H2O2 concentration (Supplementary Fig. 27). When we fed a small amount of H2O2 every 15 min, the conversion efﬁciency was increased, indicating that the continuous and low concentration of H2O2 supply is preferred for the efﬁcient biocatalytic lignin degradation (Supplementary Fig. 28). Turnover frequency (TOF) of the enzyme was 0.036 s−1, which is relatively lower than that of the LiPH8 enzyme reported in the literature48, due to the rate of lignin conversion being limited by H2O2 concentration (Supplementary Fig. 29). ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 light, and O2 gas bubbling) are the main reasons for the low selectivity of the single-compartment system. number average molecular weight of 1103, which were conﬁrmed by two-dimensional-nuclear magnetic resonance spectroscopy (2D-NMR, Supplementary Fig. 31), and gel permeation chroma- tography, respectively. y g p y In the two-compartment system, the photoanode (H:TiO2) and electrocatalyst cathode (Co–N/CNT) were separated by a Naﬁon proton exchange membrane, but the cellulose membrane was removed between the cathode and biocatalyst parts (Fig. 3g and Supplementary Fig. 1d). This conﬁguration prevents the direct photochemical lignin conversion observed in the single- compartment system and restrains the deactivation of LiPH8 biocatalyst by the photogenerated holes and •OH radicals in the anodic part. Furthermore, the remaining area was blackened and the H:TiO2 photoanode was covered from behind, as shown in Supplementary Fig. 1e, to shield the biocatalyst from UV light and inhibit its photo-deactivation as in the three-compartment system. The two-compartment cell showed increased selectivity of over 48% (Fig. 3i), which mainly arises from biocatalytic lignin conversion with the help of the in situ photogenerated H2O2 (Supplementary Fig. 23), because only 3% selectivity was obtained when parallel reaction was carried out in the absence of the LiPH8 biocatalyst (Supplementary Fig. 24). However, lignin dimer conversion was still very low (<25%) because the LiPH8 was exposed to O2 bubbles, which are detrimental to enzyme activity (Fig. 3h and Supplementary Fig. 14). Moreover, •O2− radical intermediate generated during the O2 reduction also has an adverse effect on the LiPH8 enzyme activity. g p y p y Unlike the three-compartment system, the single-compartment and two-compartment systems yielded negligible amounts of biopolymers at the end of the reaction (Fig. 4f, i) but a large number of by-products were formed (Supplementary Figs. 32 and 33), although > 90% of the monomeric substrate was consumed during the dehydrogenative polymerisation reaction (Fig. 4e, h). Because the HRP biocatalyst shows good stability against O2 bubbling and solar irradiation (Supplementary Fig. 34), the low selectivity for polymerisation may originate from the generation of highly reactive •OH and •O2−reaction intermediates on the TiO2 photocatalyst surface (Fig. 4d). These ROS can lead to further oxidation or the disproportionation of phenolic radicals rather than radical coupling and selective polymerisation (Supplemen- tary Fig. 30)50. Thus, the majority of the products in the single- compartment reactor are soluble organic compounds rather than biopolymers (Supplementary Figs. 32 and 33). ARTICLE These results show that TOF can be controlled in a stable manner by adjusting the H2O2 generation speed of the photo-electro-biochemical system. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 17), which indicates that the reaction mainly proceeds via direct photocatalytic lignin conversion by the TiO2 photocatalyst. The presence of many unidentiﬁed peaks using gas chromatography–mass spectroscopy (GC–MS) analysis also revealed that many side reactions take place upon the direct photocatalytic conversion of lignin (Supplementary Figs. 18–22). In the single-compartment system, solar light ﬁrst excites the photocatalyst, generating holes and electrons that initiate the photochemical reaction and generate intermediate radical species (•OH and •O2−, respectively). These intermediate radicals can directly attack the biocatalyst, leading to a signiﬁcant drop in activity. As for other proteins/enzymes, such as chymotrypsin, lysozyme, ribonuclease, and formate dehydro- genase, it was found that LiPH8 in this study also rapidly lost its activity upon irradiation and in the presence of bubbles of O2 gas (Supplementary Fig. 14)45–47. In particular, ultraviolet (UV) light directly destroys the protein structures45. Furthermore, gas bubbling also alters the protein structure upon adsorption of the biocatalyst at gas–liquid interfaces46,47. These factors (ROS, 5 Discussion In summary, we have demonstrated that a compartmented photo- electro-biochemical system that integrates a photocatalyst, an electrocatalyst, and a biocatalyst, is effective for selective and stable lignin dimer valorisation under solar irradiation without the need for any additional bias or sacriﬁcial agent. In this design, the placement of appropriate membranes as separators between cells protects the biocatalyst from detrimental conditions generated during the reaction, thus preserving its stability and activity. This photo-electro-biochemical system can catalyse lignin dimer clea- vage with a 93.7% conversion efﬁciency and 98.7% selectivity, which far surpasses those of single-compartment (37.3% and 34.8%) and two-compartment (25.0%, 48.1%) systems. The system was further applied for sustainable polymer synthesis using a lignin monomer, coniferyl alcohol, with a 73.3% yield and 98.3% of conversion efﬁciency; however, the polymer yields of the single- compartment and the two-compartment systems were only ca. 0% and 8.6%, respectively. This unassisted selective lignin valorisation technology could convert waste lignin to value-added aromatics and polymer without the need for any additional energy and chemicals, possibly overcoming the problems associated with Photo-electro-biochemical biopolymer synthesis. The three different types of reactors were also applied for the biopolymer synthesis using coniferyl alcohol, which is one of the three major units of lignin (Supplementary Fig. 30)49. Because current methods of polymer production depend on petrochemicals and, thus, have unavoidable negative environmental effects, polymer synthesis using the abundant, carbon neutral, and recalcitrant lignin biomass is a promising alternative. In the three- compartment photo-electro-biochemical system, biopolymer synthesis reaction was performed with horseradish peroxidase (HRP) biocatalyst with in situ solar H2O2 generation at ambient temperature and pressure (Fig. 4a). The conversion efﬁciency of coniferyl alcohol monomer and the yield of the biopolymer were 98.3% (Fig. 4b) and 73.3% (Fig. 4c), respectively. Discussion This polymer was comprised of the common linkages between coniferyl alcohol molecules, such as β-O-4, β-β and β-5, and has a NATURE COMMUNICATIONS \| (2019) 10:5123 \| https://doi.org/10.1038/s41467-019-13022-7 \| www.nature.com/naturecommunications 6 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 100 80 Conversion (%) Three-compartment 60 40 20 0 100 80 Polymer yield (%) 60 40 20 0 100 80 Polymer yield (%) 60 40 20 0 100 80 Polymer yield (%) 60 40 20 0 0 1 2 3 Time (h) 4 5 6 100 80 Conversion (%) Single-compartment 60 40 20 0 0 1 2 3 Time (h) 4 5 6 100 80 Conversion (%) Two-compartment 60 40 20 0 0 1 2 3 Time (h) 4 5 6 O2+2H+ Co–N/CNT Nafion membrane Cellulose membrane OH hv hv OH OH Lignin monomer Biopolymer Lignin monomer Interruption Interruption Biopolymer O2 – O2 – O2 – H2O2 H:TiO2 FTO O2 + – – – O2+2H+ Co–N/CNT Nafion membrane OH hv OH Lignin monomer Biopolymer O2 – H2O2 H:TiO2 FTO O2 + – – – – + – TiO2 a d e f g h i b c Reactor conﬁgurations and photo-electro-biochemical biopolymer synthesis. a, d, g Schematic representations of the: (a) three-compartment photo- o-biochemical system, (d) single-compartment powder-based photo-biochemical system, and (g) two-compartment photo-electrochemical system for biopolymer synthesis. b, e, h Conversion percentage of coniferyl alcohol over time and c, f, i polymer yield after 6 h of reaction of the three- artment photo-electro-biochemical system (b, c), single-compartment powder-based photo-biochemical system (e, f), and two-compartment photo- ochemical system (h, i). Discussion Error bars indicate the standard deviation URE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 ARTICLE 100 80 Conversion (%) Three-compartment 60 40 20 0 100 80 Polymer yield (%) 60 40 20 0 0 1 2 3 Time (h) 4 5 6 b c O2+2H+ Co–N/CNT Nafion membrane Cellulose membrane OH hv OH Lignin monomer Biopolymer O2 – O2 – H2O2 H:TiO2 FTO O2 + – – – a b a c 100 80 Polymer yield (%) 60 40 20 0 100 80 Conversion (%) Single-compartment 60 40 20 0 0 1 2 3 Time (h) 4 5 6 e f hv OH Lignin monomer Interruption Biopolymer O2 – – + – TiO2 d d e 100 80 Polymer yield (%) 60 40 20 0 100 80 Conversion (%) Two-compartment 60 40 20 0 0 1 2 3 Time (h) 4 5 6 h i i Interruption O2+2H+ Co–N/CNT Nafion membrane OH hv OH Lignin monomer Biopolymer O2 – H2O2 H:TiO2 FTO O2 + – – – g i h g Fig. 4 Reactor conﬁgurations and photo-electro-biochemical biopolymer synthesis. a, d, g Schematic representations of the: (a) three-compartment photo- electro-biochemical system, (d) single-compartment powder-based photo-biochemical system, and (g) two-compartment photo-electrochemical system used for biopolymer synthesis. b, e, h Conversion percentage of coniferyl alcohol over time and c, f, i polymer yield after 6 h of reaction of the three- compartment photo-electro-biochemical system (b, c), single-compartment powder-based photo-biochemical system (e, f), and two-compartment photo- electrochemical system (h, i). Error bars indicate the standard deviation current biomass upgradation, such as its low cost effectiveness and limited processing technology. We believe that the development and scaling-up of this technology will be a milestone for the replacement of petrochemicals with biochemicals. Characterisation of TiO2/FTO. XRD patterns of TiO2 and H:TiO2 ﬁlms were recorded using a PANalytical X’Pert PRO diffractometer from 10° to 70° in 2θ at a scanning speed of 1° min−1. SEM images were obtained using a Cold FE-SEM (SU8220, Hitach High-Technologies). Optical properties were determined by UV–vis diffuse reﬂectance spectroscopy using a UV/Vis/NIR spectrophotometer (UV-3600, Shimadzu). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 The absorbance of mixed solution at λ = 551 nm was measured using a UV/visible spectrophotometer (UV-2600, Shimadzu). X-ray absorption spectroscopy. X-ray absorption spectroscopy (XAS) was per- formed at beamline 6D of the Pohang Accelerator Laboratory. The storage ring was operated at an energy of 3 GeV and a beam current of 360 mA. The incident photons were monochromated by Si(111) double crystal, detuned by 30% to remove high-order spectral contamination, and calibrated using a standard Co foil. Co–N/CNT powder sample was pressed using a hand-held pelletiser. Finally, XAS spectra were obtained in transmission detection mode. We also measured the XAS spectrum of CoTMPP. The resulting XAS data were treated using the Athena programme to remove the background and for normalisation52. Enzyme stability investigation. To determine the stability of the enzymes against light irradiation and O2 gas purging, LiPH8 and HRP enzymes in 0.1 M phosphate borate solution at pH 4.5 and 6.0 were subject to the same light irradiation and O2 gas purging as the actual lignin conversion experiments. Three sets of experiments (viz., without light irradiation and O2 purging, with light irradiation, and with O2 bubbling) were performed. The residual activity of the enzyme was measured at several points during the 3-h experiment using the oxidation activity of 189 µM 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) in the presence of 250 μM H2O2 and 0.1 M Britton–Robinson buffer of pH 3.0. The formation of the product was recorded at 420 nm within 1 min with Ɛ420 nm = 36.7 mM−1 cm−1 using the UV/visible spectrophotometer. Electrochemical characterisation (RRDE). Electrochemical measurements were conducted using an electrochemical workstation (CHI760E, CH Instruments). The three-electrode system was built for measurement using a graphite rod counter electrode, an Ag/AgCl (saturated KCl ﬁlled, RE-1B, ALS) reference electrode, and a catalyst-loaded RRDE (AFE7R9GCPT, Pine Research Instrumentation). Before each use, the RRDE was polished with aqueous 1.0 and 0.3 μm alumina suspensions and a microcloth. The Co–N/CNT catalyst ink was prepared by mixing 10 mg of cat- alyst, 100 μL of DI water, 13 μL of Naﬁon dispersion (5 wt%, D521, Dupont), and 1085 μL of anhydrous ethanol. The CoTMPP/CNT catalyst ink was prepared in the same manner but diluted by addition of 300 μL of DI water and 3300 μL of ethanol. The catalyst inks were homogenised using an ultrasonic bath. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 LSV curves of the H:TiO2 were recor- ded using a digital multimeter (Ivium-n-Stat Multichannel potentiostat) with the Ag/AgCl reference electrode and a Pt wire counter electrode in 0.1 M phosphate borate solution at pH 4.5 from 0 to 2.00 V (vs. RHE) at a scan rate of 5 mV s−1. The front side of the photoanode (1.33 cm2) was illuminated with the solar simulator (10500, Abet Technologies), and the light intensity was adjusted to 100 mW cm−2 (AM1.5 G) at the sample position using a standard Si cell (PEC-SI01, Peccell Technologies, Inc.). To measure photoanode stability, the chronoamperometric current of H:TiO2 was measured at a constant applied voltage of 1.23 V (vs. RHE) during 12 h. LSV curves of the Co–N/CNT-based cathode (2.0 cm2) were also recorded from 1.2 to 0.05 V (vs. RHE) at a scan rate of 5 mV s−1 in O2-saturated 0.1 M phosphate borate solution at pH 4.5. Characterisation of Co–N/CNT. XPS measurements were performed with a K- Alpha X-ray photoelectron spectrometer (Thermo Fisher Scientiﬁc) with a monochromatic Al Kα X-ray source (1486.6 eV). Co 2p and N 1s XPS spectra were deconvoluted using the XPSPeak41 software with the mixed (Gaussian 70, Lor- entzian 30)-function after a Shirley-type background correction. The Co content in Co–N/CNT catalyst was analysed using an inductively coupled plasma-optical emission spectrometer (700-ES, Varian). STEM images were obtained using a JEOL JEM-2100F microscope at an acceleration voltage of 200 kV. Hydrogen peroxide detection. The concentration of H2O2 was estimated using the DPD method55. Depending on the concentration of produced H2O2, the samples were diluted with 0.1 M phosphate borate solution at pH 4.5 or 6.0 in the cases of lignin degradation and biopolymer synthesis, respectively, to avoid exceeding the detection limit of the DPD method. In the case of the powder system, the sample was ﬁltered using a 0.45-μm polytetraﬂuoroethylene (PTFE) ﬁlter (SLCR013NL, Millipore). Furthermore, 0.05 g of N,N-diethyl-1,4-phenylene-dia- mine sulphate (DPD, ≥98.0%, Aldrich) was dissolved in 5 mL of 0.1 N H2SO4 and stored in the dark at 5 °C and 5 mg of peroxidase (POD, horseradish, Sigma) was dissolved in 5 mL DI water and kept at 5 °C. Finally, 2.7 mL of a 0.1 M sodium phosphate buffer (pH 6.0), 0.05 mL of DPD solution, 0.05 mL of POD solution, and 0.2 mL of sample were mixed. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 Then, 6 μL of the catalyst ink was deposited onto the RRDE disk (24 μL for the CoTMPP/CNT cat- alyst). The resulting catalyst loading was 0.2 mg cm−2. Electrochemical measure- ments were made both in the 0.1 M phosphate borate solution and 0.1 M HClO4 (diluted from 70% Veritas double distilled, GFS chemicals). After soaking the catalyst-loaded RRDE into a N2-saturated electrolyte, cyclic voltammetry (CV) was performed between 0.05 and 1.20 V (vs. RHE) at a scan rate of 100 mV s−1. Steady CV responses were observed within 20 cycles. The Pt ring of the RRDE was elec- trochemically cleaned in the same potential range with a scan rate of 500 mV s−1 for 10 cycles. Electrochemical impedance spectroscopy measurement was carried out at 0.68 V (vs. RHE) from 100,000 to 1 Hz at an electrode rotation speed of 1600 rpm in an O2-saturated electrolyte. The series resistance was determined at the high-frequency tail of the Nyquist plot for iR-compensation. The ORR polarisation curve was obtained by LSV from 1.2 to 0.2 V (vs. RHE) at a scan rate of 5 mV s−1 and at different electrode rotation speeds of 2025, 1600, 1225, and 900 rpm for Koutecky–Levich analysis, as shown below. Detection of hydroxyl/superoxide radicals. The formation of •OH was mea- sured using a ﬂuorescence probe method with coumarin56. 0.2 mM coumarin (Aldrich) was added to the 0.1 M phosphate borate solution at the anode and enzyme cell in the three-compartment reactor, and the ﬂuorescence spectra were measured using a ﬂuorescence spectrophotometer (Cray Eclipse, Varian) with the excitation wavelength at 332 nm. Coloration of XTT reduction to XTT–formazam was used for •O2−detection57,58. For this, 0.1 M phosphate borate solution con- taining 0.1 mM 2,3-bis(2-methoxy-4-nitro-5-sulfophehyl)-2H-tetrazolium-5-car- boxanilide (XTT, Aldrich) was added in the cathode and enzyme cell, and the absorbance spectra were recorded from 650 nm to 350 nm using the UV/visible spectrophotometer. Enzyme preparation. The LiPH8 synthetic gene, including the seven-residue pro- sequence, was synthesised by Bioneer Company (South Korea). The gene coding protein sequence was retrieved from UniProtKB database (P06181). LiPH8 was expressed as inclusion body in Escherichia coli BL21 (DE3) and reactivated through in vitro refolding procedure as previously reported59 with slight modiﬁcation. The inclusion body was added into the refolding solution containing 100 mM Tris–HCl pH 8.0, CaCl2 2 mM, guanidine hydrochloride 0.36 M, L-glutathione oxidised form 0.7 mM and stirred overnight at 4 °C. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 To measure H2O2 yield, the Pt ring potential was held at 1.3 V (vs. RHE) during the LSV measurements, and the H2O2 selectivity was then calculated according to the following equation, Aldrich) were mixed in the mortar for 5 min. The same volume of formic acid (99.5%, Samchun Chemical) was added to the mixture to initiate TEOS poly- merisation. The mixture was dried at 60 °C for 3 h. The dried solid was ground to ﬁne powder and a part (0.60 g) was pyrolyzed at 900 °C under 1 L min−1 N2 ﬂow for 3 h (ramping rate: ca. 2.1 °C min−1). To etch the silica and acid-soluble Co- species, the pyrolyzed powders were added to an acid solution of 4 M HF + 2 M HCl (diluted from 50 wt% HF (JT Baker) and 36 wt% HCl) with a similar volume of ethanol (94.5%, Samchun Chemical) and stirred at RT for 30 min. The sus- pension was ﬁltered and washed with ethanol and DI water. Acid leaching was repeated once more in the same manner. Finally, the product was dried at 60 °C and collected. Then, the Co–N/CNT-based cathode was prepared by drop-casting catalyst ink on both sides of a carbon paper (0.2 mg cm−2). H2O2 selectivity % ð Þ ¼ 200 1 þ N ´ id ir ð2Þ ð2Þ where id, ir, and N indicate the disk current, the ring current, and the collection efﬁciency (37%, provided by the manufacturer), respectively. To assess the durability of the catalysts, potential cycling tests were performed between 0.6 and 1.0 V (vs. RHE) with an electrode rotation of 1600 rpm at a scan rate of 50 mV s−1 for 1000 cycles in the O2-saturated electrolyte. After cycling, the ORR activity was measured in a fresh electrolyte. Synthesis of the CoTMPP/CNT catalyst. First, 75 mg of AT-CNT was dispersed in 50 mL of N,N-dimethylformamide (DMF, 99.5%, Samchun Chemical) and ultrasonicated for 1 h. During the ultrasonication, CoTMPP in a DMF solution was prepared (10 mg mL−1). The CoTMPP solution (2.3 mL; 23 mg of CoTMPP) was added to the AT-CNT suspension, ultrasonicated for 1 h, and stirred at RT for 12 h. The suspension was centrifuged at 8000 rpm and the supernatant was decanted. The precipitate was washed with DMF twice and anhydrous ethanol and dried at 60 °C. The nominal Co loading was 1 wt%. (Photo-)electrochemical measurements. Methods P ti Synthesis of acid-treated carbon nanotubes (AT-CNT). Before catalyst synth- esis, the CNTs were acid-treated to remove metallic impurities. First, 10.0 g of multi-walled CNTs (MR99, Carbon Nanotech Co., LTD) and 715 g of 6 M HNO3 (diluted from 60 wt% HNO3, Samchun Chemical) were mixed and stirred at 80 °C for 12 h. The CNT slurry was ﬁltered, washed with copious amounts of DI water, and dried at 60 °C. The HNO3-treated CNTs were subsequently washed with 700 g of 6 M HCl (diluted from 36 wt% HCl, Samchun Chemical) in the same manner. Preparation of TiO2 photoanode. A rutile TiO2 nanowire ﬁlm was hydrothermal grown on FTO glass37. First, 15 mL of hydrochloric acid (35%, Samchun Chemical) was diluted with 15 mL deionised (DI) water and mixed with 0.5 mL titanium (IV) butoxide (97%, Aldrich) in a 100 mL beaker. This clear solution and a clean FTO glass substrate were transferred to a Teﬂon-lined stainless-steel autoclave (125 mL). The sealed autoclave was heated in an oven at 150 °C for 5 h and then cooled to room temperature (RT) slowly. After rinsing with DI water, calcination was per- formed at 550 °C for 5 h to increase the crystallinity. Finally, the sample was annealed in a hydrogen atmosphere at 350 °C for 30 min38. In addition, an epoxy resin was added behind the TiO2 electrode to block the light transmitted through the TiO2 photoanode. A planar TiO2 ﬁlm was made on FTO substrate using a radiofrequency sputtering device (SRN-120, SORONA) and calcination was per- formed at 550 °C for 5 h. Synthesis of the Co–N/CNT catalyst. First, 0.75 g of AT-CNT and 1.50 g of 5,10,15,20-tetrakis(4-methoxyphenyl)-21H,23H-porphine cobalt(II) (CoTMPP, 98%, Porphyrin Systems) were mixed in an agate mortar for 15 min. The mixture was heated at 400 °C under 1 L min−1 N2 ﬂow for 3 h (ramping rate: ca. 2.1 °C min−1). The heat-treated powder and 3.75 mL of tetraethyl orthosilicate (TEOS, 98%, Synthesis of the Co–N/CNT catalyst. First, 0.75 g of AT-CNT and 1.50 g of 5,10,15,20-tetrakis(4-methoxyphenyl)-21H,23H-porphine cobalt(II) (CoTMPP, 98%, Porphyrin Systems) were mixed in an agate mortar for 15 min. The mixture was heated at 400 °C under 1 L min−1 N2 ﬂow for 3 h (ramping rate: ca. 2.1 °C min−1). The heat-treated powder and 3.75 mL of tetraethyl orthosilicate (TEOS, 98%, 7 7 NATURE COMMUNICATIONS \| (2019) 10:5123 \| https://doi.org/10.1038/s41467-019-13022-7 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 Before injection, the sample was ﬁltered through a hydrophilic 0.2-μm PTFE membrane ﬁlter. The products were identiﬁed relative to an authentic library of standards based on their retention times and UV absorption spectra. The quantiﬁcation of the reaction was performed by HPLC based on a linear external standard curve (R2 > 0.95) of the respective compound. For the by-products, the phenolic derivatives were extracted with dichloromethane and analysed using a GC–MS (Agilent) with a DB-5MS (60 m × 250 μm × 0.25 μm) column. The oven temperature was elevated from 50 to 280 °C. The product mass spectrum was identiﬁed using an authentic library of standards. 15. Van den Bosch, S. et al. Reductive lignocellulose fractionation into soluble lignin-derived phenolic monomers and dimers and processable carbohydrate pulps. Energy Environ. Sci. 8, 1748–1763 (2015). p p gy 16. Tien, M. & Kirk, T. K. Lignin-degrading enzyme from the hymenomycete Phanerochaete chrysosporium burds. Science 221, 661–663 (1983). 17. Shuai, L. et al. Formaldehyde stabilization facilitates lignin monomer production during biomass depolymerization. Science 354, 329–333 (2016). 18. Silva, G. G. D., Couturier, M., Berrin, J.-G., Buléon, A. & Rouau, X. Effects of grinding processes on enzymatic degradation of wheat straw. Bioresour. Technol. 103, 192–200 (2012). 19. Li, H., Qu, Y., Yang, Y., Chang, S. & Xu, J. Microwave irradiation—a green and efﬁcient way to pretreat biomass. Bioresour. Technol. 199, 34–41 (2016). 20. Yuan, Z. et al. Process intensiﬁcation effect of ball milling on the hydrothermal pretreatment for corn straw enzymolysis. Energy Convers. Manag. 101, 481–488 (2015). g 21. Wu, M. et al. Separation and characterization of lignin obtained by catalytic hydrothermal pretreatment of cotton stalk. Ind. Crops Prod. 66, 123–130 (2015). Characterisation of the polymer. The polymer was collected by centrifugation at 13,000 rpm within 10 min, washed three times with 2 N HCl solution, and dried under vacuum conditions. The polymer yield was determined by UV analysis at 280 nm based on a linear external standard curve (R2 > 0.95) of the respective synthesised dehydrogenative polymer from coniferyl alcohol. The dried polymer was dissolved in tetrahydrofuran (THF) at a concentration of 3 mg mL−1. The solution was ﬁltered using a 0.2 μm PTFE ﬁlter and analysed by gel permeation chromatography, for which THF was used as eluent with a ﬂow rate of 1 mL min−1. Samples were detected at 280 nm and polystyrene standard used for calibration curves. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 sulfate–polyacrylamide gel electrophoresis and used for this study. HRP type VI was purchased from Aldrich Co. (USA) and used without any further puriﬁcation. sulfate–polyacrylamide gel electrophoresis and used for this study. HRP type VI was purchased from Aldrich Co. (USA) and used without any further puriﬁcation. 4. Calvo-Flores, F. G. & Dobado, J. A. Lignin as renewable raw material. Ch S Ch 3 1227 1235 (2010) lvo-Flores, F. G. & Dobado, J. A. Lignin as renewable raw mater 4. Calvo-Flores, F. G. & Dobado, J. A. Lignin as renewable raw material. ChemSusChem 3, 1227–1235 (2010). emSusChem 3, 1227–1235 (2010). ChemSusChem 3, 1227–1235 (2010). 5. Zakzeski, J., Bruijnincx, P. C. A., Jongerius, A. L. & Weckhuysen, B. M. The catalytic valorization of lignin for the production of renewable chemicals. Chem. Rev. 110, 3552–3599 (2010). Photo-electro-biochemical lignin conversion. The overall reaction was per- formed in an acryl reactor composed of an anode cell with a quartz window on one side, a cathode cell, and an enzyme cell. In the case of the three-compartment reactor, the anode cell was separated from cathode cell through a Naﬁon mem- brane (Naﬁon® 117, 0.18 mm thick, Aldrich), and the cathode cell was separated from an enzyme cell through a cellulose membrane (Spectra/Por®, 6–8 kDa, Spectrum). For the lignin dimer conversion, 0.1 M phosphate borate solution of pH 4.5 was used as the electrolyte. The anode cell, cathode cell, and enzyme cell consisted of H:TiO2 photoanode (1.33 cm2) with 8 mL electrolyte, Co–N/CNT- based cathode (2.0 cm2) with 4 mL electrolyte, and 0.5 mM of lignin dimer and 0.8 μM of LiPH8 in 8 mL electrolyte, respectively. The photoanode and cathode were connected to each other with alligator clips and copper wire as an external circuit. In the case of the two-compartment reactor without an enzyme cell, the cathode cell was composed of the Co–N/CNT cathode, 0.5 mM of lignin dimer, and 0.8 μM of the LiPH8 in 4 mL electrolyte. In the single cell, Degussa P25 TiO2 powder (0.5 mg mL−1) used as a photocatalyst for H2O2 production, 0.5 mM of lignin dimer, and 0.8 μM of the LiPH8 enzyme were added together in 8 mL electrolyte. The photoanode (or P25 photocatalyst) was illuminated using a solar simulator (10500, Abet Technologies) at 100 mW cm−2 (AM1.5 G). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 O2 gas was continuously bubbled into the cathode electrolyte from 20 min before the reaction until the reaction was completed. The biopolymer synthesis was performed in the three kinds of the reactors as depolymerisation of lignin with slight modiﬁcations. The reactions were carried out with 0.5 mM of coniferyl alcohol as a substrate and 1.4 μM of the HRP as an enzyme in 0.1 M phosphate borate solution at pH 6.0. 6. Azadi, P., Inderwildi, O. R., Farnood, R. & King, D. A. Liquid fuels, hydrogen and chemicals from lignin: a critical review. Renew. Sustain. Energy Rev. 21, 506–523 (2013). 7. Ragauskas, A. J. et al. Lignin valorization: improving lignin processing in the bioreﬁnery. Science 344, 1246843 (2014). 8. Beckham, G. T., Johnson, C. W., Karp, E. M., Salvachúa, D. & Vardon, D. R. Opportunities and challenges in biological lignin valorization. Curr. Opin. Biotechnol. 42, 40–53 (2016). 9. Rinaldi, R. et al. Paving the way for lignin valorisation: recent advances in bioengineering, bioreﬁning and catalysis. Angew. Chem. Int. Ed. 55, 8164–8215 (2016). 10. Schutyser, W. et al. Chemicals from lignin: an interplay of lignocellulose fractionation, depolymerisation, and upgrading. Chem. Soc. Rev. 47, 852–908 (2018). 11. Sun, Z., Fridrich, B., de Santi, A., Elangovan, S. & Barta, K. Bright side of lignin depolymerization: toward new platform chemicals. Chem. Rev. 118, 614–678 (2018). 12. Li, S.-H., Liu, S., Colmenares, J. C. & Xu, Y.-J. A sustainable approach for lignin valorization by heterogeneous photocatalysis. Green Chem. 18, 594–607 (2016). 13. Rahimi, A., Azarpira, A., Kim, H., Ralph, J. & Stahl, S. S. Chemoselective metal-free aerobic alcohol oxidation in lignin. J. Am. Chem. Soc. 135, 6415–6418 (2013). 14. Rahimi, A., Ulbrich, A., Coon, J. J. & Stahl, S. S. Formic-acid-induced depolymerization of oxidized lignin to aromatics. Nature 515, 249–252 (2014). Identiﬁcation of phenolic compounds. The completed reaction mixture was analysed by high-performance liquid chromatography (HPLC). The HPLC pro- cedure was performed by injecting fractions using an Agilent 1200 HPLC system onto a reverse-phase Eclipse XDB-C18 column (4.6 × 150 mm, 5 μm, Agilent). Gradient separation was performed using 0.1% aqueous triﬂuoroacetic acid (sol- vent A) to methanol/acetonitrile (25:75; v/v; solvent B) with the following condi- tions: analysis time of 15 min ﬂow of 1.5 mL min−1 and column temperature of 30 °C. The gradient programme was as follows: 0 min—15% B, 6 min—60% B, 11.5 min—100% B, and 13 min—0% B. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 For 2D 13C–1H heteronuclear single quantum correlation (HSQC) analysis, the polymer was dissolved in 0.5 mL DMSO-d6 before NMR analysis. NMR spectra from the polymer were acquired on a 400 MHz FT-NMR (Bruker) instrument with a 5 mm BBO NMR probe. The central DMSO solvent peak was used as an internal reference (δH/δC 2.50/39.52 ppm). The HSQC parameters and NMR chemical assignments followed previously published methods60. 22. Floudas, D. et al. The Paleozoic origin of enzymatic lignin decomposition reconstructed from 31 fungal genomes. Science 336, 1715–1719 (2012). 23. Smith, A. T., Doyle, W. A., Dorlet, P. & Ivancich, A. Spectroscopic evidence for an engineered, catalytically active Trp radical that creates the unique reactivity of lignin peroxidase. Proc. Natl Acad. Sci. USA 106, 16084–16089 (2009). 24. Saez-Jimenez, V. et al. Demonstration of lignin-to-peroxidase direct electron transfer: a transient-state kinetics, directed mutagenesis, EPR, and NMR study. J. Biol. Chem. 290, 23201–23213 (2015). y 25. Sáez-Jiménez, V. et al. Role of surface tryptophan for peroxidase oxidation of nonphenolic lignin. Biotechnol. Biofuels 9, 198–210 (2016). 26. Campos-Martin, J. M., Blanco-Brieva, G. & Fierro, J. L. G. Hydrogen peroxide synthesis: an outlook beyond the anthraquinone process. Angew. Chem. Int. Ed. 45, 6962–6984 (2006). Data availability ll d l bl f 27. Edwards, J. K. et al. Switching off hydrogen peroxide hydrogenation in the direct synthesis process. Science 323, 1037–1041 (2009). All data are available from the corresponding author upon request. 28. Liu, Y., Chen, F., Wang, Q., Wang, J. & Wang, J. Direct unassisted hydrogen peroxide generation from oxygen and water on plasmonic Ag–graphene–Cu nanosandwitch. Appl. Catal. B 224, 940–950 (2018). Received: 3 April 2019; Accepted: 10 October 2019; Received: 3 April 2019; Accepted: 10 October 2019; Received: 3 April 2019; Accepted: 10 October 2019; 29. Fukuzumi, S., Yamada, Y. & Karlin, K. D. Hydrogen peroxide as a sustainable energy carrier: electrocatalytic production of hydrogen peroxide and the fuel cell. Electrochim. Acta 82, 493–511 (2012). 30. Seh, Z. W. et al. Combining theory and experiment in electrocatalysis: insights into materials design. Science 355, eaad4998 (2017). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 The refolded solution was concentrated and buffer-exchanged with sodium acetate buffer 100 mM at pH 4.0 then pH 6.0 before subjected for puriﬁcation. The anion-exchange chromatography with Mono Q 5/50 GL column (GE Healthcare Life Sciences Co., USA) was used for puriﬁcation step in this study. The column was equilibrated with sodium acetate 10 mM, pH 6.0 (buffer A) and eluted with a linear gradient of buffer B—sodium acetate 500 mM, pH 6.0. The highest activity fraction of LiPH8 was tested with sodium dodecyl 1 i ¼ 1 ik þ 1 0:62nFAD2=3 O v1=6COω1=2 ¼ 1 ik þ 1 B ´ ω1=2 ð1Þ Here, i, ik, n, F, A, DO, v, CO, and ω represent the measured current, the kinetic current, the electron transfer number, the Faraday constant (96,485 C mol−1), the diffusion coefﬁcient of O2, the kinematic viscosity, the O2 concentration, and the electrode rotation speed, respectively. The plot of i−1 as a function of ω−1/2 gives a line with a slope of B−1, which is used to calculate n. Note that DO, v, and CO values vary with the electrolyte: DO (1.90 × 10−5 and 1.93 × 10−5 cm2 s−1), v (0.010 and 0.0101 cm2 s−1), and CO (1.21 and 1.26 × 10−6 mol cm−3) for 0.1 M phosphate borate solution and 0.1 M HClO4, respectively53,54. Here, i, ik, n, F, A, DO, v, CO, and ω represent the measured current, the kinetic current, the electron transfer number, the Faraday constant (96,485 C mol−1), the diffusion coefﬁcient of O2, the kinematic viscosity, the O2 concentration, and the electrode rotation speed, respectively. The plot of i−1 as a function of ω−1/2 gives a line with a slope of B−1, which is used to calculate n. Note that DO, v, and CO values vary with the electrolyte: DO (1.90 × 10−5 and 1.93 × 10−5 cm2 s−1), v (0.010 and 0.0101 cm2 s−1), and CO (1.21 and 1.26 × 10−6 mol cm−3) for 0.1 M phosphate borate solution and 0.1 M HClO4, respectively53,54. 8 NATURE COMMUNICATIONS \| (2019) 10:5123 \| https://doi.org/10.1038/s41467-019-13022-7 \| www.nature.com/naturecommunications ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 Author contributions 41. Bezerra, C. W. B. et al. A review of Fe–N/C and Co–N/C catalysts for the oxygen reduction reaction. Electrochim. Acta 53, 4937–4951 (2008). J.-W.J., Y.H.K., and S.H.J. proposed, designed, and directed the research. J.-W.J., M.K., L.T.M.P., and Y.J.S. conceived the concept of the compartmented photo-electro- biocatalyst system. M.K. and L.T.M.P. prepared the photocatalysts with D.O. and the biocatalysts with T.V.T.N., respectively, and measured the conversion efﬁciency and selectivity for lignin valorisation. Y.J.S. prepared and characterised the electrocatalysts and measured their performances with the help of J.W. and J.H.K. P.S., J.R., and T.J.S. helped to analyse the data and provided valuable input. M.K., L.T.M.P., Y.J.S., S.H.J., Y.H.K., and J.-W.J. co-wrote the manuscript. All authors read and commented on the manuscript. 42. Sa, Y. J. et al. A general approach to preferential formation of active Fe–Nx sites in Fe–N/C electrocatalysts for efﬁcient oxygen reduction reaction. J. Am. Chem. Soc. 138, 15046–15056 (2016). 43. Sa, Y. J. et al. Heterogeneous Co–N/C Electrocatalysts with controlled cobalt site densities for the hydrogen evolution reaction: structure–activity correlations and kinetic insights. ACS Catal. 9, 83–97 (2019). 44. Augugliaro, V. et al. Synthesis of vanillin in water by TiO2 photocatalysis. Appl. Catal. B 111-112, 555–561 (2012). pp 45. McLaren, A. D. & Luse, R. A. Mechanism of inactivation of enzyme proteins by ultraviolet light. Science 134, 836–837 (1961). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13022-7 33. Zhang, W. et al. Selective aerobic oxidation reactions using a combination of photocatalytic water oxidation and enzymatic oxyfunctionalizations. Nat. Catal. 1, 55–62 (2018). 57. Sutherland, M. W. & Learmonth, B. A. The tetrazolium dyes MTS and XTT provide new quantitative assays for superoxide and superoxide dismutase. Free Radic. Res. 27, 283–289 (1997). 34. van Schie, M. M. C. H. et al. Cascading g-C3N4 and peroxygenases for selective oxyfunctionalization reactions. ACS Catal. 9, 7409–7417 (2019). 58. Li, Y., Zhang, W., Niu, J. & Chen, Y. Mechanism of photogenerated reactive oxygen species and correlation with the antibacterial properties of engineered metal-oxide nanoparticles. ACS Nano 6, 5164–5173 (2012). 35. Willot, S. J. P. et al. Expanding the spectrum of light-driven peroxygenase reactions. ACS Catal. 9, 890–894 (2019). 59. Doyle, W. A. & Smith, A. T. Expression of lignin peroxidase H8 in Escherichia coli: folding and activation of the recombinant enzyme with Ca2+ and haem. Biochem. J. 315, 15–19 (1996). 36. Zhang, N., Yang, M.-Q., Liu, S., Sun, Y. & Xu, Y.-J. Waltzing with the versatile platform of graphene to synthesize composite photocatalysts. Chem. Rev. 115, 10307–10377 (2015). 60. Kim, H. & Ralph, J. Solution-state 2D NMR of ball-milled plant cell wall gels in DMSO-d6/pyridine-d5. Org. Biomol. Chem. 8, 576–591 (2010). 37. Liu, B. & Aydil, E. S. Growth of oriented single-crystalline rutile TiO2 nanorods on transparent conducting substrates for dye-sensitized solar cells. J. Am. Chem. Soc. 131, 3985–3990 (2009). Acknowledgements 38. Wang, G. et al. Hydrogen-treated TiO2 nanowire arrays for photoelectrochemical water splitting. Nano Lett. 11, 3026–3033 (2011). 8. Wang, G. et al. Hydrogen-treated TiO2 nanowire arrays for This research was supported by the National Research Foundation (NRF) funded by the Ministry of Science and ICT (nos. NRF-2017M1A2A2087630, NRF- This research was supported by the National Research Foundation (NRF) funded by the Ministry of Science and ICT (nos. NRF-2017M1A2A2087630, NRF- This research was supported by the National Research Foundation (NRF) funded by the Ministry of Science and ICT (nos. NRF-2017M1A2A2087630, NRF- 2017R1D1A1B03035450, and NRF-2017R1A2B2008464) and the 2018 Research Fund (1.180006.01) of Ulsan National Institute of Science and Technology (UNIST). The X-ray absorption spectroscopy experiments performed at beamlines 6D and 10C of the Pohang Accelerator Laboratory (PAL) were supported in part by the Ministry of Science and ICT, POSTECH, and UNIST. This research was supported by the National Research Foundation (NRF) funded by the Ministry of Science and ICT (nos. NRF-2017M1A2A2087630, NRF- 2017R1D1A1B03035450, and NRF-2017R1A2B2008464) and the 2018 Research Fund (1.180006.01) of Ulsan National Institute of Science and Technology (UNIST). The X-ray absorption spectroscopy experiments performed at beamlines 6D and 10C of the Pohang Accelerator Laboratory (PAL) were supported in part by the Ministry of Science and ICT, POSTECH, and UNIST. 39. Anson, F. C., Ni, C. L. & Saveant, J. M. Electrocatalysis at redox polymer electrodes with separation of the catalytic and charge propagation roles. reduction of dioxygen to hydrogen peroxide as catalyzed by cobalt(II) tetrakis (4-N-methylpyridyl)porphyrin. J. Am. Chem. Soc. 107, 3442–3450 (1985). 40. Claude, E., Addou, T., Latour, J.-M. & Aldebert, P. A new method for electrochemical screening based on the rotating ring disc electrode and its application to oxygen reduction catalysts. J. Appl. Electrochem. 28, 57–64 (1998). The authors declare no competing interests. The authors declare no competing interests. The authors declare no competing interests. Additional information 47. Bommarius, A. S. & Karau, A. Deactivation of formate dehydrogenase (FDH) in solution and at gas–liquid interfaces. Biotechnol. Prog. 21, 1663–1672 (2005). Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 019-13022-7. Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 019-13022-7. 48. Pham, L. T. M., Kim, S. J. & Kim, Y. H. Improvement of catalytic performance of lignin peroxidase for the enhanced degradation of lignocellulose biomass based on the imbedded electron-relay in long-range electron transfer route. Biotechnol. Biofuels 9, 247 (2016). Correspondence and requests for materials should be addressed to S.H.J., Y.H.K. or J.- W.J. Peer review information Nature Communications thanks Jonathan Bloh, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Peer review information Nature Communications thanks Jonathan Bloh, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. 49. Li, Q. et al. Dehydrogenative polymerization of coniferyl alcohol in artiﬁcial polysaccharides matrices: effects of xylan on the polymerization. J. Agric. Food Chem. 63, 4613–4620 (2015). 50. Awungacha Lekelefac, C., Busse, N., Herrenbauer, M. & Czermak, P. Photocatalytic based degradation processes of lignin derivatives. Int. J. Photoenergy 2015, 1–18 (2015). Reprints and permission information is available at http://www.nature.com/reprints Reprints and permission information is available at http://www.nature.com/reprints References 1. Hoffert, M. I. et al. Advanced technology paths to global climate stability: energy for a greenhouse planet. Science 298, 981–987 (2002). 1. Hoffert, M. I. et al. Advanced technology paths to global climate stability: energy for a greenhouse planet. Science 298, 981–987 (2002). 31. Sa, Y. J., Kim, J. H. & Joo, S. H. Active edge-site-rich carbon nanocatalysts with enhanced electron transfer for efﬁcient electrochemical hydrogen peroxide production. Angew. Chem. Int. Ed. 58, 1100–1105 (2019). 2. Ragauskas, A. J. et al. The path forward for biofuels and biomaterials. Science 311, 484–489 (2006). 2. Ragauskas, A. J. et al. The path forward for biofuels and biomaterials. Science 311, 484–489 (2006). 32. Zhang, W. et al. Selective activation of C−H bonds in a cascade process combining photochemistry and biocatalysis. Angew. Chem. Int. Ed. 56, 15451–15455 (2017). 32. Zhang, W. et al. Selective activation of C−H bonds in a cascade process combining photochemistry and biocatalysis. Angew. Chem. Int. Ed. 56, 15451–15455 (2017). 3. Thompson, P. The agricultural ethics of biofuels: the food vs. fuel debate. Agriculture 2, 339–358 (2012). 3. Thompson, P. The agricultural ethics of biofuels: the food vs. fuel debate. Agriculture 2, 339–358 (2012). NATURE COMMUNICATIONS \| (2019) 10:5123 \| https://doi.org/10.1038/s41467-019-13022-7 \| www.nature.com/naturecommunications 9 Competing interests 46. Caussette, M., Gaunand, A., Planche, H. & Lindet, B. in Progress in Biotechnology, Vol. 15 (eds Ballesteros, A., Plou, F. J., Iborra, J. L. & Halling, P. J.) 393–398 (Elsevier, 1998). Reprints and permission information is available at http://www.nature.com/reprints Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 51. Zhao, Z., Ozoemena, K. I., Maree, D. M. & Nyokong, T. Synthesis and electrochemical studies of a covalently linked cobalt(II) phthalocyanine–cobalt (II) porphyrin conjugate. Dalton Trans. 0, 1241–1248 (2005). 52. Ravel, B. & Newville, M. ATHENA, ARTEMIS, HEPHAESTUS: data analysis for X-ray absorption spectroscopy using IFEFFIT. J. Synchrotron Radiat. 12, 537–541 (2005). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. 53. Xing, W., Yin, G. & Zhang, J. Rotating Electrode Methods And Oxygen Reduction Electrocatalysts (Elsevier, 2014). 54. Marković, N. M., Gasteiger, H. A., Grgur, B. N. & Ross, P. N. Oxygen reduction reaction on Pt(111): effects of bromide. J. Electroanal. Chem. 467, 157–163 (1999). 55. Bader, H., Sturzenegger, V. & Hoigné, J. Photometric method for the determination of low concentrations of hydrogen peroxide by the peroxidase catalyzed oxidation of N,N-diethyl-p-phenylenediamine (DPD). Water Res. 22, 1109–1115 (1988). 56. Zhang, J. & Nosaka, Y. Quantitative detection of OH radicals for investigating the reaction mechanism of various visible-light TiO2 photocatalysts in aqueous suspension. J. Phys. Chem. C 117, 1383–1391 (2013). 10 NATURE COMMUNICATIONS \| (2019) 10:5123 \| https://doi.org/10.1038/s41467-019-13022-7 \| www.nature.com/naturecommunications
https://openalex.org/W3168627514	https://zenodo.org/record/6784383/files/FEDSM2020-10212.pdf	English	null	Diversion of Pulsatile Flow Over a Rectangular Sidewall Cavity Using Superhydrophobic Mesh	null	2,020	cc-by	4,912	ABSTRACT loading on the cavity walls [6]–[8]. Consequently it reduces the risk of rupture and allows gradual intra-aneurysmal embolization and resorption to occur [9]. Despite being a promising clinical intervention strategy, endovascular treatments currently face some technical barriers. First, stents/grafts are sensitive to platelet activation and protein binding, in part because of the induced shear stress on the device’s surfaces, which lead to thrombus and plaque formation and increased arterial stenosis [1], [10]. Patients who received endovascular treatments require life-long anticoagulant medication to mitigate their risk of arterial stenosis and stroke [11]. Second, the deployment of diverters sometimes causes significantly altered local hemodynamics, which may result in life-threatening rupture events during or after procedures [12]. The lack of systematic quantitative studies of pulsatile flow over stented aneurysm cavities causes ambiguity in terms of its efficacy under varied patient conditions. In a recent CFD study, Dholakia et al. [4] claimed that “there is poor standardization of study variables” in terms of interpreting the hemodynamic effects of the mesh flow diverters. In addition to the aneurysm flow diversion, pulsatile flow over cavity and mesh structures can also be found in the left atrial appendage closure devices (WATCHMANTM, Boston Scientific), which is designed to prevent leakage of blood clots and stroke in patients with atrial fibrillation [13]. Pulsatile flow over open cavity represents one type of physiological phenomenon related to a few common cardiovascular diseases, such as cerebral sidewall aneurysm and arrhythmia-induced thromboembolism in the left atrium appendage (LAA). In recent years, endovascular treatments using mesh-based implants have become increasingly popular. In this paper, we study the characteristics of pulsatile flow over a simplified sidewall cavity under two Reynolds/Womersley number conditions using Particle Image Velocimetry. The impacts of a regular mesh and a superhydrobobically-coated mesh on the cavity flow are investigated. Our results quantify the phase-to-phase changes of the flow fields and reveal the formation and the transport of the primary vortex over the ostium of the rectangular cavity. Results suggest the meshes diverted the main flow away from the cavity and prohibited the development of the primary vortex. A penetrated jet flow was formed near the front side of the cavity due to the presence of the mesh. The superhydrophobic mesh dramatically reduced the kinetic energy of the penetrated jet into the cavity. Proceedings of the ASME 2020 Fluids Engineering Division Summer Meeting FEDSM2020 July 12-16, 2020, Orlando, Florida, USA Proceedings of the ASME 2020 Fluids Engineering Division Summer Meeting FEDSM2020 July 12-16, 2020, Orlando, Florida, USA Proceedings of the ASME 2020 Fluids Engineering Division Summer Meeting FEDSM2020 July 12-16, 2020, Orlando, Florida, USA ABSTRACT It indicates the mesh flow diversion is effective because of the destruction of the shear- induced vortex dynamics that causes flow stagnation on the rear cavity wall. Our results also indicate the superhydrophobic coating is potentially beneficial in terms of reducing the hemodynamic loading inside the cavity. Keywords: PIV, pulsatile flow, cavity, flow diverter, superhydrophobic mesh FIGURE 1: ILLUSTRATION OF ENDOVASCULAR FLOW DIVERSION Benjamin Eichholz, Ruihang Zhang, Yan Zhang1 North Dakota State University, Fargo, USA 1Corresponding author: yan.zhang.4@ndsu.edu North Dakota State University, Fargo, USA 1Corresponding author: yan.zhang.4@ndsu.edu 1. INTRODUCTION Endovascular flow diversion is an increasingly popular treatment option for large cerebral aneurysms because of its effectiveness and minimally invasive nature [1]–[4], compared with traditional surgical clippings and coil embolization [5]. This procedure deploys an expandable mesh structure through catheter over the neck of the aneurysm to divert the blood flow away from the sac, as illustrated in Fig.1. Early experimental results suggest the placement of a mesh redirected most of the flow away from the cavity and reduced the hemodynamic FIGURE 1: ILLUSTRATION OF ENDOVASCULAR FLOW DIVERSION 1 © 2019 by ASME 1 shear stress on the mechanical heart valve, indicating its potential for reducing the thrombotic risk caused by blood- material interactions. The purpose of this paper is to quantitatively study the impact of a mesh structure on the pulsatile flow over a simplified sidewall cavity under two Reynolds/Womersley number conditions using Particle Image Velocimetry. We also aim to test a hypothesis that a diverter with superhydrophobic coatings could improve the flow diversion outcomes by reducing the flow penetration into the cavity. The proposed superhydrophobic (SHB) mesh is motivated by the superior biological (anti-platelet [14]) and fluid dynamic (drag-reducing [15]) properties of SHB surfaces and their potential to provide transformative solutions, overcoming the current technical barriers of flow diversion treatments. Super-hydrophobic surfaces repel water and water- based liquids because of the micro-surface structures. The low surface energy creates slip wall boundaries, which dramatically reduce surface friction and flow drag [15]. In recent years, SHB materials have been studied in a variety of in vitro biomedical applications. The anti-adhesion property of SH surfaces made them attractive in applications in which protein and bacteria adsorption to biomaterial surfaces need to be inhibited [16]–[18]. SH surfaces are also incorporated in microfluidic devices to control flow in lab-on-a-chip diagnostics [19] and in drug delivery platforms to enable more efficient delivery of therapeutic agents [20]. Novel flexible tubes made from SH- materials show significant drag reduction and excellent self- cleaning properties: in a sliding and free falling water droplet experiments, SH tubes showed an up to 5000 times increase in droplet acceleration and 99% friction reduction [15]. SH polydimethylsiloxane (PDMS) tubes with titanium-oxide layers also showed excellent blood repellency, and sliding blood droplets on an inclined tube did not leave any blood stain [15]. 1. INTRODUCTION Recently, the potential application of SHB coating on mechanical bi-leaflet prosthetic heart valves have been investigated [21] via a bioengineering experiment. The work showed dramatically reduced cell adhesion and minimized fluid In this study, we performed a particle image velocimetry (PIV) experiment to investigate the pulsatile flow characteristics over a rectangular sidewall cavity model and implanted meshes with and without SHB properties. The cavity is a simplified scaled-up model from the actual aneurysm/LAA sizes while the dynamic similarity parameters were matched. The result analysis of the study will focus on the phase-to-phase variations of flow characteristics over the open cavity and the impact of a regular mesh and a superhydrophobically-coated mesh on the cavity flow. 2. MATERIALS AND METHODS 2.1 The Pulsatile Flow Simulator and Cavity Model A closed-loop cardiovascular flow simulator, as illustrated in Fig. 2a, was used to provide flow conditions for this experiment. The pulsatile flow was generated by a programmable piston pump (PD-1100, BDC Laboratories) through a user-defined skewed sinusoidal function. A pump head module equipped with a left ventricular diastole module (BDC Laboratories) was connected to the pump to generate the tuned flow outputs. Compliance and resistance units were used downstream of the test model to tune pressure-flow conditions. A fluid tank was used upstream of the pump head to receive the return flow and a magnetic stirrer was used to mix the PIV seeding particles. g p In the experiments, a time ratio of 40% between systolic (contraction) and diastolic (relaxation) period was programmed to the pump to generate a physiological flow waveform [22]. The working fluid is a mixture of water and glycerin with a FIGURE 2: SCHEMATIC OF THE CARDIOVASCULAR SIMULATOR AND THE CAVITY MODEL FIGURE 2: SCHEMATIC OF THE CARDIOVASCULAR SIMULATOR AND THE CAVITY MODEL © 2019 by ASME 2 2 first tested on a flat solid surface. The static contact angle was measured using an optical goniometer, as shown in Fig. 3b-d. After the coating was applied, the static contact angle is >150 deg. Fig.3c shows the results of contact angle measurements of the coated surface using different volumetric ratios of water- glycerin mixture. It suggests that the coating exhibited superhydrophocity to varied mixtures and was relative insensitive to the mixing ratio. Fig.3d shows the contact angle of the coated surface remained relatively stable after being pulled out from prolonged water immersion. volumetric ratio of 0.67:0.33. The mixture has a density 𝜌= 1085 𝑘𝑔/𝑚3 and a dynamic viscosity 𝜇= 0.0035 𝑃𝑎∙𝑠 , which closely resemble the properties of the blood plasma. Non- Newtonian property of blood was not considered in the present study. The flow rate and frequency of the flow was set to provide appropriate Reynolds and Womersley numbers for the dynamic similarity of large cerebral aneurysm flow and the left atrial flow [23]. 2.2 Mesh Structure and Superhydrophobic Coating FIGURE 3: THE MESH MODEL AND SUPERHYDROPHOBIC COATING An aluminum wire mesh with diamond-shaped pores was used to model the flow condition induced by flow diverters (Fig.3a). The mesh has a porosity (total pore areas/total area) of 70% and a pore density of 9.4 pores/cm2. The weave pattern and porosity of the mesh closely resemble those in commercial flow diverters [4]. The detailed dimensions of the mesh pores are shown in Table 1. Note that these dimensions were measured from digital images using ImageJ and each dimension was measured from ten sample pores to calculate the average and relative errors. To apply the mesh over the cavity, a square piece of the mesh was cut and inserted into the cavity to cover the cavity ostium. 2. MATERIALS AND METHODS The two dimensionless numbers are defined as 𝑅𝑒= 𝜌𝐷u/𝜇 , and 𝛼= 0.5D√𝜔𝜌/𝜇 , respectively, where D is the width of the channel (0.025 m); u represents the average velocity of the flow; 𝜌 represents the density of the working liquid; 𝜇 is the dynamic viscosity and 𝜔 is the angular frequency of the pulsatile flow. The list of similarity parameters is presented in Table 2 in the Results section. More details regarding the simulator can also be found in [24] FIGURE 3: THE MESH MODEL AND SUPERHYDROPHOBIC COATING A square channel with a rectangular sidewall cavity model with aspect ratio of 1.5 (depth/width) was constructed using acrylic sheets, as shown in Fig. 2b. The width (D) of the channel and the cavity (L) are both 1 inch (25.4 mm) and the depth (H) of the cavity was 1.5 inches (38.1 mm). The geometric scale of the model is approximately 10:1 if considering an aneurysm ostium size of 2-3mm and approximately 2:1 if considering a left atrial appendage ostium size of 12-16 mm. The aspect ratio was selected according to the clinical finding of the statistical threshold [26], [27] regarding aneurysms rupture, which suggests that an aspect ratio greater than 1.6 is associated with increased risk of aneurysm rupture. 2.3 Particle Image Velocimetry A PIV system with a double-pulsed Nd:YAG laser (NewWave Gemini 200, 100mJ, 532nm) was used in the experiments. A set of concave and cylindrical lenses were applied to convert the laser beams into thin sheets with a thickness of approximately 1mm. The laser sheets pass thought the mid-plane of the test acrylic section vertically from the side wall. A 2M-pixel CCD (charge-coupled device) camera was placed on the top of the test section. The lasers and camera were synchronized by a delay generator (BNC Model 577, Berkeley Nucleonic Corp.), which was externally triggered by the pump digital output signal. The delay to the trigger signals was then controlled specifically for each test case to obtain phase shifts. The uncertainty of the synchronizing (based on the rise time of TTL signal) was less than 3 nanoseconds. The working fluid was seeded with hollow glass microspheres with a seeding density of 0.1 ppm. These particles have an average diameter of 50 μm and a density of 1100 𝑘𝑔/𝑚3, which was very close to the density of the liquid mixture. The Stokes number of the particle flow was much less than 1, indicating that the particle could follow the Table 1. Detailed dimensions of the mesh Pore Density (#/cm2) Porosity (%) Pore Area (mm) Length L1 (mm) Length L2 (mm) Angle 𝜶 (deg.) Angle 𝜷 (deg.) 9.4 70.0 7.4 ±0.2 3.4 ±0.1 3.1 ±0.1 70.3 ±2.6 115.9 ±2.2 Table 1. Detailed dimensions of the mesh The superhydrophobic mesh was prepared by applying a commercial superhydrophobic spray coating (Rust-Oleum® NeverWet®). The spray consists of a base coat of hydrocarbon resin and a topcoat of acetone. The effectiveness of coating was © 2019 by ASME © 2019 by ASME 3 Fig.4 shows the variations of the centerline inlet velocity at the inlet of the channel within one cardiac cycle. The time was normalized by the pulsation period for each case and was redefined with the percentage of a complete cycle. The velocity variation shows a 40% of systolic time when the valve upstream is open and 60% of diastolic time when the valve is closed, corresponding well with the programmed pump motion. It should be noted that the peak flow phase (peak systole) occurs at near 20% of the cycle for both flow conditions. The mainstream flow accelerates during 0-20% of the cycle and decelerates 20- 40% of the cycle. 2.3 Particle Image Velocimetry The mainstream flow quickly dissipates after 40% of the period as the cycle goes into diastolic phases. flow streamline very well. The raw PIV image is shown in Fig.2b. For the post-processing, instantaneous PIV velocity vectors were computed using cross-correlation algorithms provided in LaVision Davis software. The computation of cross-correlation involved successive pairs of raw images in a multiple-pass process with interrogation window sizes of from 32×32 to 16×16 pixels. An overlap of 50% was used for the cross-correlation and the Nyquist criterion was satisfied. We captured 20 and 40 phases per cycle for the aneurysm and LAA flow conditions, respectively, with a sampling rate of 10 frames/sec. A hundred instantaneous velocity samples were captured and used for averaging flow field information for each case. Fig.5 presents the normalized velocity magnitude and normalized out-of-plane vorticity of Case #1 (sidewall aneurysms) averaged at multiple phases during 10%-60% of the cycle. The vorticity magnitude was calculated using the equation 𝜔𝑍= 𝜕𝑣 𝜕𝑥− 𝜕𝑢 𝜕𝑦. The velocity magnitude was normalized by the bulk average velocity of a cycle (𝑈/𝑈𝑎𝑣𝑔), while the vorticity magnitude was normalized using the width of the channel and the bulk average velocity (𝜔𝑧𝐿/𝑈𝑎𝑣𝑔). The results show that at the beginning of the systolic phase (10%), a primary vortex with a clockwise rotation was developed due to the shear force between the accelerating main flow and the flow inside the cavity. The vortex then was transported towards the rear side of the cavity and impinged on the rear wall (20%-30%), causing a downwash flow on the back side. At the end of the systole (40%), the primary vortex was enlarged, strengthened, moved out of the cavity due to the deceleration and decreasing pressure in the main flow. The strengthened vortex was evidenced by the increased velocity magnitude near both front and rear side of the cavity ostium. The enlarged vortex persisted but started to dissipate during the early diastolic phases (50%-60%). 3.1 Pulsatile flow over open cavity Two flow conditions were simulated in the present study to establish the dynamic similarity for the cerebral aneurysm (Case #1) and left atrium appendage flows (Case #2), respectively. Calculated from the PIV results, the bulk average velocity of a cycle (𝑈𝑎𝑣𝑔) for the two conditions are 0.027 m/s and 0.108 m/s, respectively. The peak systolic mean velocity are 0.09 m/s and 0.22 m/s, respectively. The characteristic length was the width of the channel, i.e. 0.025m. The corresponding dimensionless number for the two study cases are listed in Table 2. Note that the average Reynolds number was calculated based on the bulk average flow velocity, while the peak flow Reynolds number was calculated based on the mean flow velocity at the peak systole. FIGURE 4: THE CENTERLINE VELOCITY WAVEFORM OF ONE CYCLE Table 2. Dimensionless numbers of the two flow conditions Case # Average Re Peak flow Re Wo 1 (Aneurysm) 206 725 6.2 2 (LAA) 826 1697 12.3 Table 2. Dimensionless numbers of the two flow conditions Fig. 6 shows the same normalized quantities for Case #2 (left atrial appendage case) which was performed under a greater Reynolds and Womersley number. Overall, the process of vortex formation and transportation was quite similar to the case discussed above. However, a few differences were noticed: first, the downwash flow caused by the impingement of the primary vortex at the rear wall was obviously stronger around the peak systolic phases (20%-30%) due to the higher Re number of the main flow. As a result, the flow momentum was able to reach much deeper into the cavity in Case #2, as evidenced by the velocity magnitude contours during 40%-60% of the cycle. Second, the upward motion of the primary vortex at the end of the systole (40%) was less apparent compared to that occurred in Case #1. The strength of the vortex did not appear to increase in this phase, as shown by the decreased velocity magnitude. The results reveal different vortex dynamics in a sidewall cavity under different Re and Wo numbers, particularly during the late systolic and early diastolic phases. While the low Re and Wo aneurysm flow favors long-lasting primary vortex, higher Re and Wo number flow of the LAA case tends to transport stronger momentum towards the bottom of the cavity. 3.1 Pulsatile flow over open cavity FIGURE 4: THE CENTERLINE VELOCITY WAVEFORM OF ONE CYCLE © 2019 by ASME © 2019 by ASME 4 FIGURE 5: NORMALIZED VELOCITY AND VORTICITY CONTOURS OF THE ANEURYSM FLOW CASE (CASE #1) ALIZED VELOCITY AND VORTICITY CONTOURS OF THE ANEURYSM FLOW CASE (CASE #1) FIGURE 5: NORMALIZED VELOCITY AND VORTICITY CONTOURS OF THE ANEURYSM FLOW C FIGURE 5: NORMALIZED VELOCITY AND VORTICITY CONTOURS OF THE ANEURYSM FLOW CASE (CASE #1) FIGURE 6: NORMALIZED VELOCITY AND VORTICITY CONTOURS OF THE LAA FLOW CASE (CASE #2) FIGURE 6: NORMALIZED VELOCITY AND VORTICITY CONTOURS OF THE LAA FLOW CASE (C © 2019 by ASME © 2019 by ASME 5 5 FIGURE 7: THE FLOW DIVERSION EFFECTS (NORMALIZED VORTICITY FIELDS) OF TWO MESH MODELS (CASE #1) FIGURE 7: THE FLOW DIVERSION EFFECTS (NORMALIZED VORTICITY FIELDS) OF TWO MESH MODELS (CASE #1) KE was concentrated near the rear wall, the regular mesh caused a strong downward jet flow that contributed to an increased KE near the center of the cavity and away from the rear cavity wall. The location of the high KE varied under the two flow conditions. Furthermore, the results consistently show that the KE in the cavity with SHB mesh is of order-of-magnitude weaker than the regular mesh and no mesh case. 3.2 Effects of the mesh flow diverter The effects of the regular mesh and the SHB mesh on the cavity flow are examined. Fig. 7 shows the normalized vorticity contours and streamlines near the ostium of the cavity during the systole phases of Case #1. The white dashed line shows the location of the mesh over the ostium. The slight curvature was caused by the force applied to the mesh in order to firmly attached it into the cavity. The results show that both mesh diverters significantly altered the hemodynamics around the cavity. The mesh over the ostium diverted the main stream away as evidenced by the altered streamlines and vorticity layers and prohibited the formation of the primary vortex in the meantime. The destruction of shear-induced vortex is particularly beneficial for the aneurysm hemodynamics as it would reduce the pressure and shear stresses on the real wall of the cavity. The presence of the mesh also caused a penetrated jet flow near the front side of the cavity due to the interaction between the main flow and the mesh struct. The penetrated jet also caused a vortex that rotates in counterclockwise direction between the mesh and the jet. Furthermore, the SHB-coated mesh reduced such jet flow penetration as shown by the decreased vorticity magnitudes. The flow fields of Case #2 are not shown here for brevity as they show similar results. FIGURE 8: THE SPECIFIC KINETIC ENERGY DISTRIBUTION 0.2H BELOW THE CAVITY OSTIUM (CASE #1) FIGURE 8: THE SPECIFIC KINETIC ENERGY DISTRIBUTION 0.2H BELOW THE CAVITY OSTIUM (CASE #1) The reduction of jet flow penetration can be seen more clearly in terms of specific kinetic energy profiles shown in Fig. 8-9. These profiles were extracted in X direction (horizontally) at 0.2H (depth) below the ostium of the cavity from the PIV results of Case #1 and Case 2, respectively. The specific kinetic energy was defined as the kinetic energy (KE) per unit mass of the flow, i.e. 0.5𝑈2, where U is the local velocity magnitude. The results show that in contrast to the no-mesh case where the © 2019 by ASME © 2019 by ASME © 2019 by ASME 6 FIGURE 9: THE SPECIFIC KINETIC ENERGY DISTRIBUTION 0.2H BELOW THE CAVITY OSTIUM (CASE #2) studied by previous research. Future studies are needed to quantify the effects of pore density, porosity, and realistic geometries on the flow diversion outcomes of superhydrophobic diverters. ACKNOWLEDGEMENTS The project is supported by the American Heart Association under the Institutional Research Enhancement Award (AIREA) award 19AIREA34470008. REFERENCES [1] L. Pierot, “Flow diverter stents in the treatment of intracranial aneurysms: Where are we?,” J. Neuroradiol., vol. 38, no. 1, pp. 40–46, Mar. 2011, doi: 10.1016/j.neurad.2010.12.002. [1] L. Pierot, “Flow diverter stents in the treatment of intracranial aneurysms: Where are we?,” J. Neuroradiol., vol. 38, no. 1, pp. 40–46, Mar. 2011, doi: 10.1016/j.neurad.2010.12.002. FIGURE 9: THE SPECIFIC KINETIC ENERGY DISTRIBUTION 0.2H BELOW THE CAVITY OSTIUM (CASE #2) [2] P. I. D’Urso, G. Lanzino, H. J. Cloft, and D. F. Kallmes, “Flow Diversion for Intracranial Aneurysms,” Stroke, Aug. 2011. Overall, the results of the mesh flow diverter suggest the presence of the mesh prohibits the formation of the primary vortex and therefore prevents the flow stagnation and downwash flow at the real cavity wall. Instead, the mesh struct promotes the formation of penetration jets near the front side of the cavity, causing the increase of flow kinetic energy near the middle of the cavity ostium. The SHB mesh significantly reduces the penetrated jet flow energy compared to the regular mesh, which might be beneficial to further mitigate the risks of hemodynamic loading immediately after the deployment of the diverter. [3] D. F. Kallmes, Y. H. Ding, D. Dai, R. Kadirvel, D. A. Lewis, and H. J. Cloft, “A New Endoluminal, Flow- Disrupting Device for Treatment of Saccular Aneurysms,” Stroke, Aug. 2007. [4] R. Dholakia, C. Sadasivan, D. J. Fiorella, H. H. Woo, and B. B. Lieber, “Hemodynamics of Flow Diverters,” J. Biomech. Eng., vol. 139, no. 2, pp. 021002-021002–10, Jan. 2017, doi: 10.1115/1.4034932. [5] [5] C. Groden, J. Laudan, S. Gatchell, and H. Zeumer, “Three- Dimensional Pulsatile Flow Simulation before and after Endovascular Coil Embolization of a Terminal Cerebral Aneurysm,” J. Cereb. Blood Flow Metab., vol. 21, no. 12, pp. 1464–1471, Dec. 2001, doi: 10.1097/00004647- 200112000-00011. 4. CONCLUSION We performed an experimental study of pulsatile flow over a rectangular cavity with different mesh diverters using a cardiovascular flow simulator and particle image velocimetry. Two flow conditions studied here are 1) average Re=206, Wo=6.2; 2) average Re=824, Wo=12.3, to reach dynamic similarity to aneurysm flow and left atrial appendage flow, respectively. A scaled-up rectangular cavity was used, and aluminum meshes with a porosity of 70% with and without superhydrophobic coatings were deployed over the ostium of the cavity during the experiments. [6] [6] B. B. Lieber, A. P. Stancampiano, and A. K. Wakhloo, “Alteration of hemodynamics in aneurysm models by stenting: influence of stent porosity,” Ann. Biomed. Eng., vol. 25, no. 3, pp. 460–469, Jun. 1997. [7] B. B. Lieber, V. Livescu, L. N. Hopkins, and A. K. Wakhloo, “Particle Image Velocimetry Assessment of Stent Design Influence on Intra-Aneurysmal Flow,” Ann. Biomed. Eng., vol. 30, no. 6, pp. 768–777, Jun. 2002, doi: 10.1114/1.1495867. PIV results revealed the formation and transport of the primary vortex over the open cavity and different flow patterns at different phases of the pulsatile flow. The aneurysm flow case showed a persisting primary vortex that was intensified and elevated from the cavity towards the end of the systole and into the early diastole phases. The left atrial appendage flow case showed a stronger downwash flow near the rear wall at the peak systolic phase. The presence of the mesh diverted the main flow and prohibited the formation of the primary vortices. With mesh implanted, a penetrated jet flow was developed near the front end of the cavity. The superhydrophobic mesh significantly decreased the kinetic energy of the jet flow penetrating the cavity. The results imply potential benefits of superhydrophobic mesh for reducing hemodynamic loading inside the aneurysm cavities after the deployment, besides other anticoagulation and anti- platelet properties of superhydrophobic surfaces that has been PIV results revealed the formation and transport of the primary vortex over the open cavity and different flow patterns at different phases of the pulsatile flow. The aneurysm flow case showed a persisting primary vortex that was intensified and elevated from the cavity towards the end of the systole and into the early diastole phases. The left atrial appendage flow case showed a stronger downwash flow near the rear wall at the peak systolic phase. 4. CONCLUSION The presence of the mesh diverted the main flow and prohibited the formation of the primary vortices. With mesh implanted, a penetrated jet flow was developed near the front end of the cavity. The superhydrophobic mesh significantly decreased the kinetic energy of the jet flow penetrating the cavity. The results imply potential benefits of superhydrophobic mesh for reducing hemodynamic loading inside the aneurysm cavities after the deployment, besides other anticoagulation and anti- platelet properties of superhydrophobic surfaces that has been [8] A. L. Trager, C. Sadasivan, J. Seong, and B. B. Lieber, “Correlation between angiographic and particle image velocimetry quantifications of flow diverters in an in vitro model of elastase-induced rabbit aneurysms,” J. Biomech. Eng., vol. 131, no. 3, p. 034506, Mar. 2009, doi: 10.1115/1.3049528. [9] B. Lubicz et al., “Flow-Diverter Stent for the Endovascular Treatment of Intracranial Aneurysms,” Stroke, Oct. 2010. [10] G. K. C. Wong, M. C. L. Kwan, R. Y. T. Ng, S. C. H. Yu, and W. S. Poon, “Flow diverters for treatment of intracranial aneurysms: Current status and ongoing 7 © 2019 by ASME © 2019 by ASME clinical trials,” J. Clin. Neurosci., vol. 18, no. 6, pp. 737– 740, Jun. 2011, doi: 10.1016/j.jocn.2010.10.011. [22] O. Bazan and J. P. Ortiz, “Duration of Systole and Diastole for Hydrodynamic Testing of Prosthetic Heart Valves: Comparison Between ISO 5840 Standards and in vivo Studies,” Braz. J. Cardiovasc. Surg., vol. 31, no. 2, pp. 171–173, 2016, doi: http://dx.doi.org/10.5935/1678- 9741.20160036. clinical trials,” J. Clin. Neurosci., vol. 18, no. 6, pp. 737– 740, Jun. 2011, doi: 10.1016/j.jocn.2010.10.011. [11] [11] S. O. Kim, Y. G. Chung, Y. S. Won, and M. H. Rho, “Delayed Ischemic Stroke after Flow Diversion of Large Posterior Communicating Artery Aneurysm,” J. Cerebrovasc. Endovasc. Neurosurg., vol. 18, no. 1, pp. 19–26, Mar. 2016, doi: 10.7461/jcen.2016.18.1.19. [23] Y. C. Fung, Biomechanics: Circulation. Springer Science & Business Media, 2013. [12] J. R. Cebral et al., “Aneurysm Rupture Following Treatment with Flow-Diverting Stents: Computational Hemodynamics Analysis of Treatment,” Am. J. Neuroradiol., vol. 32, no. 1, pp. 27–33, Jan. 2011, doi: 10.3174/ajnr.A2398. [24] R. Zhang and Y. Zhang, “An Experimental Study of Pulsatile Flow in a Compliant Aortic Root Model under Varied Cardiac Outputs,” Fluids, vol. 3, no. 4, p. 71, Dec. 2018, doi: 10.3390/fluids3040071. j [13] D. R. Holmes et al., “Left Atrial Appendage Closure as an Alternative to Warfarin for Stroke Prevention in Atrial Fibrillation: A Patient-Level Meta-Analysis,” J. 4. CONCLUSION Am. Coll. Cardiol., vol. 65, no. 24, pp. 2614–2623, Jun. 2015, doi: 10.1016/j.jacc.2015.04.025. [25] H. Ujiie et al., “Effects of size and shape (aspect ratio) on the hemodynamics of saccular aneurysms: a possible index for surgical treatment of intracranial aneurysms,” Neurosurgery, vol. 45, no. 1, pp. 119–129; discussion 129- 130, Jul. 1999. [14] X. Hou et al., “Preparation of polypropylene superhydrophobic surface and its blood compatibility,” Colloids Surf. B Biointerfaces, vol. 80, no. 2, pp. 247–250, Oct. 2010, doi: 10.1016/j.colsurfb.2010.06.013. [26] H. Ujiie, Y. Tamano, K. Sasaki, and T. Hori, “Is the Aspect Ratio a Reliable Index for Predicting the Rupture of a Saccular Aneurysm?,” Neurosurgery, vol. 48, no. 3, pp. 495–503, Mar. 2001, doi: 10.1097/00006123-200103000- 00007. [15] S. Hoshian, E. Kankuri, R. H. A. Ras, S. Franssila, and V. Jokinen, “Water and Blood Repellent Flexible Tubes,” Sci. Rep., vol. 7, no. 1, p. 16019, Nov. 2017, doi: 10.1038/s41598-017-16369-3. [16] D. R. Schmidt, H. Waldeck, and W. J. Kao, “Protein Adsorption to Biomaterials,” in Biological Interactions on Materials Surfaces, Springer, New York, NY, 2009, pp. 1– 18. [17] A. Vaterrodt, B. Thallinger, K. Daumann, D. Koch, G. M. Guebitz, and M. Ulbricht, “Antifouling and Antibacterial Multifunctional Polyzwitterion/Enzyme Coating on Silicone Catheter Material Prepared by Electrostatic Layer-by-Layer Assembly,” Langmuir, vol. 32, no. 5, pp. 1347–1359, Feb. 2016, doi: 10.1021/acs.langmuir.5b04303. [18] J. Zhao, L. Song, J. Yin, and W. Ming, “Anti-bioadhesion on hierarchically structured, superhydrophobic surfaces,” Chem. Commun., vol. 49, no. 80, pp. 9191–9193, 2013, doi: 10.1039/C3CC44971H. [19] E. Gogolides, K. Ellinas, and A. Tserepi, “Hierarchical micro and nano structured, hydrophilic, superhydrophobic and superoleophobic surfaces incorporated in microfluidics, microarrays and lab on chip microsystems,” Microelectron. Eng., vol. 132, pp. 135–155, Jan. 2015, doi: 10.1016/j.mee.2014.10.002. [20] J. B. Wolinsky, Y. L. Colson, and M. W. Grinstaff, “Local drug delivery strategies for cancer treatment: Gels, nanoparticles, polymeric films, rods, and wafers,” J. Controlled Release, vol. 159, no. 1, pp. 14–26, Apr. 2012, doi: 10.1016/j.jconrel.2011.11.031. j j [21] D. L. Bark et al., “Hemodynamic Performance and Thrombogenic Properties of a Superhydrophobic Bileaflet Mechanical Heart Valve,” Ann. Biomed. Eng., vol. 45, no. 2, pp. 452–463, Feb. 2017, doi: 10.1007/s10439-016- 1618-2. © 2019 by ASME © 2019 by ASME 8
https://openalex.org/W2493459217	https://europepmc.org/articles/pmc4949886?pdf=render	English	null	Associations between health literacy and preventive health behaviors among older adults: findings from the health and retirement study	BMC public health	2,016	cc-by	7,183	* Correspondence: dfernand@umich.edu 1 Department of Internal Medicine, Division of General Medicine, University of Michigan, 1500 E. Medical Center Drive, Ann Arbor, MI 48109-2029, USA 2Department of Health Behavior and Biological Sciences, University of Michigan School of Nursing, 400 North Ingalls Building, Ann Arbor, MI 48109-5482, USA Full list of author information is available at the end of the article Associations between health literacy and preventive health behaviors among older adults: findings from the health and retirement study Dena M. Fernandez1,2*, Janet L. Larson2 and Brian J. Zikmund-Fisher1,3,4 © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: While the association between inadequate health literacy and adverse health outcomes has been well documented, less is known about the impact of health literacy on health perceptions, such as perceptions of control over health, and preventive health behaviors. Methods: We identified a subsample of participants (N = 707) from the Health and Retirement Study (HRS), a nationally representative sample of older adults, who participated in health literacy testing. Self-reported health literacy was measured with a literacy screening question, and objective health literacy with a summed score of items from the Test of Functional Health Literacy. We compared answers on these items to those related to participation in health behaviors such as cancer screening, exercise, and tobacco use, as well as self-referencing health beliefs. Results: In logistic regression models adjusted for gender, education, race, and age, participants with adequate self-reported health literacy (compared to poorer levels of health literacy) had greater odds of participation in mammography within the last 2 years (Odds ratio [OR] = 2.215, p = 0.01) and participation in moderate exercise two or more times per week (OR = 1.512, p = 0.03). Participants with adequate objective health literacy had reduced odds of participation in monthly breast self-exams (OR = 0.369, p = 0.004) and reduced odds of current tobacco use (OR = 0.456, p = 0.03). In adjusted linear regression analyses, self-reported health literacy made a small but significant contribution to explaining perceived control of health (β 0.151, p = <0.001) and perceived social standing (β 0.112, p = 0.002). Conclusion: In a subsample of older adult participants of the HRS, measures of health literacy were positively related to several health promoting behaviors and health-related beliefs and non-use of breast self-exams, a screening behavior of questionable benefit. These relationships varied however, between self-reported and objectively-measured health literacy. Further investigation into the specific mechanisms that lead higher literacy people to pursue health promoting actions appears clearly warranted. Keywords: Health literacy, Perceptions, Health behaviors, Preventive health behaviors, Mammography, Perceived control, Tobacco, Physical activity, Discrimination © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Fernandez et al. BMC Public Health (2016) 16:596 DOI 10.1186/s12889-016-3267-7 Fernandez et al. BMC Public Health (2016) 16:596 DOI 10.1186/s12889-016-3267-7 Sample In the present study, data from the HRS 2009 Internet Survey were linked to the HRS 2008 Core Survey to ob- tain a more comprehensive list of covariates on the same respondents. Household and individual identification numbers were used in order to distinguish persons who participated in both waves. For analysis purposes, we focus on those individuals who were administered the PLQ and offered all of the health literacy questions in the internet survey. Of 5,742 HRS respondents invited to participate in the internet survey, 4,433 participated (yielding a response rate of 77.2 %). Of the 4,433 initial participants that started the internet survey, a total of 4,351 completed the survey. The PLQ was completed by 1,902 of those 4,351, and of those, 707 underwent health literacy testing with both reading comprehension and numeracy items. The relationship between health literacy and health out- comes is well established, but the mechanisms underlying this relationship are not yet fully understood. Commonly cited theoretical models suggest that health literacy influ- ences health outcomes at least partially through its effect on patients’ self-efficacy, knowledge, and health behaviors [23–25] as well as their health-related perceptions, health- related experiences, and familiarity with health concepts [26, 27]. However, these models have not yet been tested in full, and further work is needed to empirically validate the proposed relationships in these frameworks [28]. The purpose of this study is to examine relationships between health literacy and health perceptions, and between health literacy and health behaviors in a sample of older adults, a population segment growing in size with a greater rate of chronic conditions, utilization of medical services, and limited health literacy than other age groups. Background To complement the core surveys, supplemental ques- tionnaires have been administered to both random and purposefully selected respondents. Internet based surveys are one type of supplemental questionnaire for which HRS participants who report regular use of the internet in the core survey are eligible. Internet based surveys are conducted in the alternate years and cover topics includ- ing health behaviors and health-related knowledge. In the HRS 2009 Internet Survey, health literacy was additionally assessed; however, only a random subsample was selected to undergo health literacy testing with both reading comprehension and numeracy items. The Psychosocial and Lifestyle Questionnaire (PLQ) is a supplemental ques- tionnaire that was designed to elicit participants’ views of their life, health, and well-being. This questionnaire is given to a random 50 % of the core participants who are interviewed face to face; the questionnaire is left behind with them to complete and mail-in. g Health literacy, the capacity of individuals to obtain and understand health information needed to make health decisions, is recognized by the Institute of Medicine and the federal government as fundamental to quality health care [1–3]. However, findings from the National Assess- ment of Adult Literacy (a nationally representative assess- ment of literacy among adults in the United States) suggest that health literacy is limited in a third of adults and in a markedly larger portion of older adults [4, 5]. Reports from health literacy research estimate rates of limited health liter- acy in 33–60 % of adults in outpatient and inpatient settings [6–11]. Limited health literacy has consistently been shown to be a strong predictor of poorer health out- comes [12]. Individuals with limited health literacy are prone to greater use of emergency services [13–15], higher rates of hospitalization [16, 17], and higher rates of mortal- ity [18, 19]. Limited health literacy is also associated with less use of preventive services [14, 20], and poorer adher- ence to medication regimens [21, 22]. Abstract The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Fernandez et al. BMC Public Health (2016) 16:596 Fernandez et al. BMC Public Health (2016) 16:596 Page 2 of 8 Page 2 of 8 Variables and measurements Health literacy Health literacy was measured in the HRS using items from the Test of Functional Health Literacy (TOFHLA) [31] and a literacy screener [32]. The TOFHLA is a measure of an individual’s ability to read and understand two passages, and to understand and use quantitative informa- tion [31]. The HRS employed seven TOFHLA reading comprehension items and seven TOFHLA numeric items. In the present study, respondents are given one point for each correct answer. The data were not normally distrib- uted, so we followed the procedures of Parker et al. [31] and labeled participants with < 60 correct as having inad- equate health literacy, those with 60–75 correct as having marginal health literacy, and those with greater than 75 % correct as having adequate health literacy. We dichoto- mized responses as either adequate or inadequate by mer- ging inadequate and marginal categories [33–35]. Health Analyses To examine the associations between health literacy and health perceptions and between health literacy and health behaviors, we first conducted bivariate analyses. Chi-squared tests were used for categorical outcomes and the Yates correction for continuity was applied as recommended for each condition in which there were two categories per variable [49]. The Mann–Whitney U test was used for the two continuous health perception variables (perceived control over health and perceived social standing). We selected this test because scores were skewed for perceived control over health and per- ceived social standing. We selected perceived control over health and perceived healthcare discrimination as measures of health percep- tions plausibly related to health literacy based on positive relationships demonstrated in health literacy research between limited health literacy and healthcare-related patient dissatisfaction, reduced participation, and shame [38–40]. We additionally examined the outcome of perceived social standing because of the well-documented relationships between social standing and the incidence and severity of disease and access to care in the U.S. and other countries [41–43]. We assessed perceived control over health with a single item which asks participants to “Think about a 0 to 10 scale where ‘0’ means ‘no control at all’ and ‘10’ means ‘very much control.’ How would you rate the amount of control you have these days over your health?” [44]. We assessed perceived social standing with the MacArthur Scale of Subjective Social Status [45], which asks participants to select a rung on a ladder that represents where they believe they stand in society, with the top representing those who are doing the best (0–10). We assessed perceived healthcare discrimination with a single item that asked “In day-to-day life, how often have any of the following things happened to you?” [You receive poorer service or treatment than other people from doctors or hospitals] (almost every day, at least once a week, a few times a month, a few times a year, less than once a year, never) [46, 47]. Study design and data sources We conducted a secondary analysis of cross-sectional data from supplemental questionnaires administered as part of the Health and Retirement Study (HRS). The HRS is a biennial longitudinal interview survey of U.S. adults over the age of 50 sponsored by the National Institute on Aging and conducted by the Institute for Social Research (ISR) at the University of Michigan ([29, 30], The Health and Retirement Study). Employ- ing a multi-stage area probability sampling design, HRS researchers began collecting data on issues of aging, health, and retirement in 1992. Since then, core interviews have been repeated on the original sample, along with newly added cohorts, every two years. Fernandez et al. BMC Public Health (2016) 16:596 Page 3 of 8 Page 3 of 8 Page 3 of 8 literacy determined by these TOFHLA items is termed from here on as objective health literacy. self-examination (BSE), prostate exam, current tobacco use, and moderate and vigorous physical activity. Partici- pants were asked if they had participated in flu immunization in the last 2 years, if they had cholesterol screening within the last 2 years, if they currently smoked, and how often they performed moderate and vigorous physical activity (daily, more than once per week, once per week, 1–3 times per month, hardly ever, or never). Female participants were asked if they had a mammogram within the last 2 years and whether they performed BSE monthly (yes/no); male participants were asked whether they had a prostate exam in the last 2 years. The Chew health literacy screener is a single item screen- ing question that asks “How confident are you filling out medical forms by yourself?” [32]. This screening question has been shown to be a valid and reliable predictor of func- tional health literacy [32, 36]. Possible responses are on a Likert style scale and range from 0–4, zero being “extremely” and 4 being “not at all.” We also dichotomized this variable based on the recommended cut-off of 2 or less to identify adequate health literacy [36, 37]. Health literacy determined by this screener is termed from here on as self- reported health literacy. Analyses We additionally measured perceptions of risks of colon cancer, risks of colon cancer death, and benefits of colon cancer screening with the following three items: “Out of every 100 people, about how many do you think will die of colon cancer?” (0–100), “Out of every 100 people, about how many will be diagnosed with colon cancer at some time in their lives?” (0–100), and “Do you think regular colon cancer screening for people over age 50 does or does not reduce the risk of dying from colon cancer?” (does/does not/don’t know) [48]. We conducted multiple regression analyses to evaluate the influence of health literacy on health perceptions and health behaviors. To examine relationships between health literacy and health perceptions, we conducted linear regressions for perceived control of health and perceived social standing. In preliminary analyses we examined normality, linearity, multicollinearity, and homoscedasticity. We checked normal probability plots of the regression standardized residual for normality as well as a scatterplot of the standardized residuals for homoscedasticity and outliers. We also reviewed the correlations tables closely for any strong correlations between the independent variables. We conducted logis- tic regressions for each categorical health perception variable (including perceived healthcare discrimination and perceived colon cancer risks) and to examine rela- tionships between health literacy and health behaviors, regressing the dichotomous health behaviors (including flu immunization, cholesterol testing, mammography, BSE, prostate exam, current tobacco use, and physical activity) against first objective health literacy and then self-reported health literacy. Each multivariable analysis controlled for the socio-demographic characteristics of gender, education, race, and age because these variables have been previously identified as confounding variables in the relationship between health literacy and health outcomes [4, 50–53]. We also measured 8 specific health behaviors: flu immunization, cholesterol testing, mammography, breast Page 4 of 8 Fernandez et al. BMC Public Health (2016) 16:596 Page 4 of 8 Results respectively, (z = −4.15, p = <0.0005). Also, compared to participants with inadequate self-reported health literacy, participants with adequate self-reported health literacy were more likely to correctly respond that colon cancer screen- ing does reduce the risk of dying from colon cancer, 89.1 % versus 81.9 %, (x 2 = 4.94, p = 0.026). Sample characteristics Participants in the final sample (N = 707) were predomin- antly white (92), and approximately half were female (57 %). Participants had a mean age of 66, and most were married (81 %). The sample was well educated with almost 95 % of participants having at least a high school education. College educated and post-college educated participants made up 45 and 24 % of the sample respectively. Self- reported health literacy scores ranged from 0–4, with a mean of 0.84 (and standard deviation of 1.02). Objective health literacy scores ranged from 0–14, with a mean score of 12.45 (and standard deviation of 1.72) Table 1. p In adjusted linear regression analyses that evaluated the influence of self-reported health literacy on per- ceived control over health and perceived social standing, self-reported health literacy made a statistically signifi- cant contribution to explaining perceived control of health (β 0.151, p = <0.0005) and perceived social stand- ing (β 0.112, p = 0.002), however the R-Square values were weak (0.053 and 0.169 respectively). In adjusted lo- gistic regression models, self-reported health literacy did not significantly influence the likelihood of reporting healthcare discrimination or correctly responding to the colon cancer items (Table 2). Associations of health literacy with health perceptions Associations of health literacy with health perceptions In bivariate analyses, there were significant differences between participants with adequate and inadequate self- reported health literacy in perceived control over health, Md = 8 versus Md = 7 respectively, (z = −4.91, p = <0.0005), and in perceived social standing, Md = 7 versus Md = 6 Bivariate associations between objective health literacy and health perceptions were not significant. In adjusted linear regression analyses that evaluated the influence of objective health literacy on perceived control over health and perceived social standing, the models were statisti- cally significant but weak, and did not identify objective health literacy as a statistically significant or unique con- tributor in explaining the rates of perceived control over health or perceived social standing. In adjusted logistic regression models, objective health literacy did not sig- nificantly influence the likelihood of reporting healthcare discrimination or correctly responding to the colon can- cer items (Table 3). Table 1 Characteristics of Participants (N = 707) Characteristic Mean +/−SD or % Age (years) 65.80 +/−9.05 Gender (Female) 57.1 Race/Ethnicity White 92.1 Black 5.2 Othera 2.7 Hispanic 4.4 Education 0–8 years 1.0 9–11 years 4.6 12 years 25.3 College 45.2 Post-college 23.9 Marital Status Married or Live-in Partner 80.8 Unmarriedb 19.2 Employment Employed 36.4 Unemployedc 12.4 Retired 46.8 Homemaker 4.4 Note: Final sample N = 707. Valid percentages reported due to missing data. HTN = hypertension; SD = standard deviation aOther race includes American Indian, Alaskan Native, Asian, and Pacific Islander bUnmarried includes separated, divorced, never married, widowed, and one refusal to respond cUnemployed includes unemployed and looking for work, laid off, disabled, sick or other leave, and one refusal to respond Associations of health literacy with behavior variables Associations of health literacy with behavior variables In bivariate analyses, significant associations were demon- strated between self-reported health literacy and mam- mography, moderate physical activity, and tobacco use. Comparing the two groups, participants with adequate self-reported health literacy were more likely to report hav- ing a mammogram within the last 2 years (85 % versus 69 %) x ð 2 = 8.67, p = 0.003), more likely to report perform- ing moderate physical activity two or more times per week (66 % versus 54 %) x ð 2 = 6.76, p = 0.009), and less likely to report current tobacco use (8 % versus 14 %) x ð 2 = 5.08, p = 0.024). In adjusted logistic regression analyses, par- ticipants with adequate self-reported health literacy had greater odds (compared to participants with inadequate self-reported health literacy) of participation in mammog- raphy (OR = 2.215, p = 0.010) and moderate physical activ- ity (OR = 1.512, p = 0.033), and a tendency toward lower odds of current tobacco use (OR = 0.550, p = 0.054), although this did not reach statistical significance (Table 2). Bivariate analyses between objective health literacy Note: Final sample N = 707. Valid percentages reported due to missing data. HTN = hypertension; SD = standard deviation Bivariate analyses between objective health literacy and health behaviors revealed a significant association between objective health literacy and BSE. Women with bUnmarried includes separated, divorced, never married, widowed, and one refusal to respond cUnemployed includes unemployed and looking for work, laid off, disabled, sick or other leave, and one refusal to respond Fernandez et al. Associations of health literacy with behavior variables BMC Public Health (2016) 16:596 Page 5 of 8 Table 2 Multivariate adjusted association of adequate self-reported health literacy with health perceptions and behaviors Health perceptions Adjusteda ORb CI P Perceived control over health β(SE) 0.759 ± 0.188 βs 0.148 < 0.0005 Perceived social standing β(SE) 0.460 ± 0.147 βs 0.112 0.002 Perceived healthcare discrimination 0.69 (0.43–1.12) 0.13 Perceptions of colon cancer (correct) Perceived risk of diagnosis 1.39 (0.78–2.50) 0.27 Perceived risk of death 1.68 (0.89–3.16) 0.11 Perceived benefit of screen 1.63 (0.97–2.72) 0.064 Health Behaviors Flu immunization 0.94 (0.62–1.43) 0.78 Cholesterol testing 1.41 (0.77–2.59) 0.26 Mammography 2.22 (1.21–4.66) 0.010 Self-breast exam 0.66 (0.39–1.13) 0.13 Prostate examination 1.27 (0.68–2.40) 0.46 Current tobacco use 0.55 (0.30–1.01) 0.054 Moderate Physical activity 1.51 (1.03–2.21) 0.033 Vigorous Physical activity 1.21 (0.81–1.80) 0.35 aAdjusted for age, gender, race, and education bLogistic regression unless otherwise noted with a β (SE). β; unstandardized slope coefficient. Bs; standardized slope coefficient. SE; Standard Error = 0.369, p = 0.004) and reduced odds of reporting current tobacco use (OR = 0.456, p = 0.025) (Table 3). = 0.369, p = 0.004) and reduced odds of reporting current tobacco use (OR = 0.456, p = 0.025) (Table 3). adequate objective health literacy were less likely to report monthly BSE compared to those with inadequate objective health literacy (49.4 % versus 72 %) x ð 2 = 8.056, p = 0.005). In adjusted logistic regression models, women with adequate objective health literacy had reduced odds of reporting participation in monthly BSE (Odds ratio [OR] Adjusted for age, gender, race, and education bLogistic regression unless otherwise noted with a β (SE). β; unstandardized slope coefficient. βs; standardized slope coefficient. SE; Standard Error justed for age, gender, race, and education gistic regression unless otherwise noted with a β (SE). β; unstandardized slope coefficient. βs; standardized slope coefficient. SE; Stand aAdjusted for age, gender, race, and education b Discussion To the best of our knowledge, this secondary analysis is the first study to investigate the relationships between Table 3 Multivariate adjusted association of adequate objective health literacy with health perceptions and behaviors Health perceptions Adjusteda ORb CI P Perceived control over health β(SE)–0.14 ± 0.21 βs–0.02 0.52 Perceived social standing β(SE)–0.12 ± 0.17 βs–0.03 0.47 Perceived healthcare discrimination 1.04 (0.59–1.84) 0.90 Perceptions of colon cancer (correct) Risk of diagnosis 1.89 (0.90–3.96) 0.09 Perceived risk of death 1.75 (0.83–3.68) 0.14 Perceived benefit of screen 1.72 (0.98–3.02) 0.06 Health Behaviors Flu immunization 0.93 (0.58–1.51) 0.78 Cholesterol testing 1.72 (0.86–3.44) 0.12 Mammography 1.54 (0.74–3.21) 0.25 Breast self-exam 0.37 (0.19–0.73) 0.004 Prostate examination 1.02 (0.50–2.10) 0.95 Current tobacco use 0.46 (0.23–0.91) 0.025 Moderate physical activity 0.94 (0.61–1.46) 0.79 Vigorous physical activity 0.93 (0.59–1.46) 0.75 aAdjusted for age, gender, race, and education bLogistic regression unless otherwise noted with a β (SE) β; unstandardized slope coefficient βs; standardized slope coefficient SE; Standard Error Multivariate adjusted association of adequate objective health literacy with health perceptions and behaviors eptions Adjusteda ORb CI Fernandez et al. BMC Public Health (2016) 16:596 Page 6 of 8 Page 6 of 8 health literacy and health behaviors and perceptions in a subsample of a nationally representative sample of adults. The principal findings include significant positive relation- ships between self-reported health literacy and health behaviors of mammography and physical activity, and per- ceptions of social standing and control over health. Additionally, significant inverse relationships were noted between objective health literacy and participation in monthly BSE and current use of tobacco. health literacy and perception of social standing by describing the negative self-perceptions held by indi- viduals with inadequate health literacy. In one study, participants with inadequate health literacy described feeling bad about themselves, worthless, or lazy for not having obtained health literacy [39]. In another study, participants with inadequate health literacy revealed worries that they may be seen by others as a bad people or incompetent members of society [60]. Findings from this study confirm those of previous studies by demonstrating that women with inadequate self-reported health literacy were less likely to have had a mammogram in the last 2 years [20, 54–56]. However the negative relationship observed between objective health literacy and monthly BSE practices in the women in this sample, to our knowledge, has not been identified in previous literature. Discussion This finding was unexpected, but is possible that women with lower health literacy are utilizing BSE to replace mammog- raphy due to barriers accessing health services, which has been identified as especially challenging for those with inadequate health literacy [1]. Alternatively, it is possible that women with higher levels of health liter- acy are aware of the limitations of BSE, which at this time is actually not recommended as a breast cancer screening method by the World Health Organization [57]. Because mammography is known to be effective in reducing breast cancer mortality, and BSE is not [58], future research is needed to explore the know- ledge, beliefs, and breast cancer screening practices of women of all health literacy levels. This study has limitations that must be considered. The first set of limitations is related to external validity. Although sampling methods employed by the HRS were designed to obtain a nationally representative sample, there was some reading involved in all modes used by the HRS to recruit and collect data, which may have led to self-selection bias (those with the lowest literacy refusing to participate in the HRS). The HRS subsample used for this study was invited from the core sample based on their report of regular internet access and use, which may have led to another recruitment bias. Either of these biases may have contributed to the overrepre- sentation of more educated adults in this sample. A second set of limitations is related to the measurement of health literacy. Health literacy as a concept is comprised of not only reading and quantitative ability, but an inter- action between knowledge, societal, and cultural influences that are difficult to measure [1]. In fact, experts agree that all existing measures of health literacy are inadequate or incomplete [61] and that none comprehensively assess the capacity of an individual [26]. In this study, objective health literacy was measured by performance on a limited number of available TOFHLA items. While the Short Test of Func- tional Health Literacy in Adults (STOFHLA), which is made up 36 TOFHLA items, has had reliability and validity supported in diverse populations [18, 22, 62], in this study a health literacy index of 14 TOFHLA items was used. This may make comparisons with the TOFHLA or STOFHLA difficult. Innovation, Center for Bioethics and Social Sciences in Medicine, 2800 Plymouth Road, Ann Arbor, MI 48109-2800, USA. Innovation, Center for Bioethics and Social Sciences in Medicine, 2800 Plymouth Road, Ann Arbor, MI 48109-2800, USA. Abbreviations S b lf BSE, breast self-exam; HRS, health and retirement study; OR, odds ratio; PLQ, psychological and Lifestyle Questionnaire; STOFHLA, short test of functional health literacy in adults; TOFHLA, test of functional health literacy in adults 6. Gazmararian JA, Baker DW, Williams MV, et al. Health literacy among Medicare enrollees in a managed care organization. JAMA. 1999;281:545–51 Received: 30 July 2015 Accepted: 23 June 2016 Received: 30 July 2015 Accepted: 23 June 2016 Conclusion The purpose of this study was to examine relationships between health literacy and health behaviors and percep- tions in a sample of older adults. Results provided evidence for significant relationships between health lit- eracy and breast screening behaviors, physical activity, and current tobacco use, as well significant relationships between health literacy and perceived control over health and perceived social standing. Future research is needed to further examine the impact of health literacy on these important health behaviors and to describe the psychosocial experience of individuals with inadequate health literacy. Consent for publication Not applicable. 16. Baker DW, Parker RM, Williams MV, Clark WS. Health literacy and the risk of hospital admission. J Gen Intern Med. 1998;13:791–8. Availability of data and materials 11. Morris NS, Grant S, Repp A, Maclean C, Littenberg B. Prevalence of limited health literacy and compensatory strategies used by hospitalized patients. Nurs Res 2011;60;361–366. HRS data are available to registered users at http://hrsonline.isr.umich.edu/ index.php. 12. Berkman ND, Sheridan SL, Donahue KE, Halpern DJ, Crotty K. Low health literacy and health outcomes: an updated systematic review. Ann Intern Med. 2011;155(2):97–107. Funding Study funding was provided by the University Of Michigan School Of Nursing New Investigator Award. Support for DMF was generously provided by the University of Michigan Rackham Merit Fellowship Program. Neither program had any role in the design of the study; in the collection, analysis, or interpretation of data; or in the preparation or approval of the manuscript. 9. Wolf MS, Gazmararian JA, Baker DW. Health literacy and health risk behaviors among older adults. Am J Prev Med. 2007;32:19–24. 9. Wolf MS, Gazmararian JA, Baker DW. Health literacy and health risk behaviors among older adults. Am J Prev Med. 2007;32:19–24. 10. Williams MV, Baker DW, Parker RM, Nurss JR. Relationship of functional health literacy to patients’ knowledge of their chronic disease: a study of patients with hypertension and diabetes. Arch Intern Med. 1998;158:166–72. Acknowledgements h k h 7. Schillinger D, Grumbach K, Piette J, et al. Association of health literacy with diabetes outcomes. JAMA. 2002;288:475–82. Thank you to Beth Simmert, PhD, for counsel and training on the HRS data, and to Laura Fernandes, PhD, for advice regarding statistical analyses. Thank you to Beth Simmert, PhD, for counsel and training on the HRS data, and to Laura Fernandes, PhD, for advice regarding statistical analyses. 8. Walker J, Pepa C, Gerard PS. Assessing the health literacy levels of patients using selected hospital services. Clinical Nurse Specialist. 2010;24:31–7. 8. Walker J, Pepa C, Gerard PS. Assessing the health literacy levels of patients using selected hospital services. Clinical Nurse Specialist. 2010;24:31–7. Ethics approval and consent to participate 17. Baker DW, Gazmararian JA, Williams MV, Scott T, Parker RM, Green D, et al. Functional health literacy and the risk of hospital admission among medicare managed care enrollees. Am J Public Health. 2002;92: 1278–83. The HRS has been approved by the Institutional Review Board at the University of Michigan. The HRS obtains informed verbal consent from voluntary participants and follows strict procedures to protect study participants from disclosure (including maintaining a Federal Certificate of Confidentiality). The public data, made available to registered researchers and used in this study, is de-identified. 18. Baker DW, Wolf MS, Feinglass J, Thompson JA, Gazmararian JA, Huang J. Health literacy and mortality among elderly persons. Arch Intern Med. 2007;167(14):1503–9. References 1. Nielsen-Bohlman L, Panzer AM, Kindig DA. Institute of Medicine Committee on Health Literacy. In: Health Literacy: A Prescription to End Confusion. Washington, DC: National Academies Press; 2004. g 2. U.S. Department of Health and Human Services. Office of Disease Prevention and Health Promotion. In: National Action Plan to Improve Health Literacy. Washington, DC: National Institutes of Health; 2010. 3. U.S. Department of Health and Human Services. Office of Disease Prevention and Health Promotion. In: Healthy People 2020. Washington, DC: National Institutes of Health; 2011. g 2. U.S. Department of Health and Human Services. Office of Disease Prevention and Health Promotion. In: National Action Plan to Improve Health Literacy. Washington, DC: National Institutes of Health; 2010. 3. U.S. Department of Health and Human Services. Office of Disease Prevention and Health Promotion. In: Healthy People 2020. Washington, DC: National Institutes of Health; 2011. 4. Baker DW, Gazmararian JA, Sudano J, Patterson M. The association between age and health literacy among elderly persons. The Journals of Gerontology. 2000;55(6):S368–74. 4. Baker DW, Gazmararian JA, Sudano J, Patterson M. The association between age and health literacy among elderly persons. The Journals of Gerontology. 2000;55(6):S368–74. 5. Kutner M, Greenberg E, Jin Y, Paulsen C (2006). The Health Literacy of America’s Adults: Results From the 2003 National Assessment of Adult Literacy (Report no. NCES 2006–483). U.S. Department of Education. Washington, DC: US Department of Education, National Center for Education Statistics. Retrieved March 29, 2013 from National Center of Education Statistics website: http://nces.ed.gov/naal. Authors’ contributions d f h DMF conceived of the study, performed analyses, and drafted the manuscript. JLL and BJZ-F assisted in designing the methodology and revising the manuscript for publication. All authors have read and approved the final version of the manuscript. 13. Baker DW, Gazmararian JA, Williams MV. Health literacy and use of outpatient physician services by medicare managed care enrollees. J Gen Intern Med. 2004;19:215–20. 14. Cho YI, Lee SY, Arozullah AM, Crittenden KS. Effects of health literacy on health status and health service utilization amongst the elderly. Soc Sci Med. 2008;66:1809–16. Competing interests Competing interests The authors declare that they have no competing interests. p g The authors declare that they have no competing interests. 15. Murray MD, Tu W, Wu J, et al. Factors associated with exacerbation of heart failure include treatment adherence and health literacy skills. Clinical Pharmacology & Therapeutics. 2009;85:651–8. Discussion In addition, the different predictive abilities of the self-report and objective measures demonstrated in this study suggest that they may be assessing related but differ- ent constructs. Further studies are needed to closely exam- ine and compare the construct validity of these commonly used measures of health literacy. Prior research regarding the relationship between health literacy and smoking status is limited and inconsistent. Our results support the findings of Von Wagner et al. [25] that lower health literacy increased the odds of reporting being a current smoker. Our results are not consistent with findings of Baker et al. [18] who found negative asso- ciations between health literacy and smoking behaviors, but the relationship lost significance in adjusted analyses. Future research is needed to confirm and describe the relationship between health literacy and tobacco use, and to further explore its underlying mechanisms. Other important results from the present study in- clude positive relationships between health literacy and perceived social standing. This finding is consistent with those of Van De Heide et al. [59], who examined the health literacy of the general population of the Netherlands using the Dutch data from the European Health Literacy Survey (HLS-EU) (N = 925). They dem- onstrated significant positive relationships between three of four domains of health literacy and perceived social status. Additionally, the findings from qualitative studies of adults with limited health literacy provide theoretical support for a positive relationship between Finally, given data limitations, several single item scales were employed in this study. While single literacy screening items have been shown to correlate strongly with well accepted multiple-item health literacy measures [32, 36, 37, 63], other single item variables in this study have unknown validity and utility. Perceived healthcare discrimination was measured with a question from the multiple-item Everyday Discrimination Scale that was modified to inquire about discrimination in the healthcare setting [47]. Further testing for correlation of this item with validated measures of healthcare discrimination Page 7 of 8 Page 7 of 8 Fernandez et al. BMC Public Health (2016) 16:596 Page 7 of 8 (such as the Discrimination in Medical Settings Scale; [64]) is needed to determine the validity of this question. (such as the Discrimination in Medical Settings Scale; [64]) is needed to determine the validity of this question. Author details 1 The meaning and measure of health literacy. J Gen Intern Med. 2006;21:878–83. The prevalence of limited health literacy. J Gen Intern Med. 20 52. Paasche-Orlow MK, Cheng DM, Palepu A, Meli S, Faber V, Samet JH. Health literacy, antiretroviral adherence, and HIV-RNA suppression: a longitudinal perspective. J Gen Intern Med. 2006;21(8):835–40e. 27. Speros C. Health literacy: concept analysis. J Adv Nurs. 2005;50(6):633–40. 28. Osborn CY, Paasche-Orlow MK, Bailey SC, Wolf MS. The mechanisms linking health literacy to behavior and health status. Am J Health Behav. 2011;35(1):118–28. 53. Ussher M, Ibrahim S, Reid F, Shaw A, Rowlands G. Psychosocial correlates of health literacy among older patients with coronary heart disease. J Health Commun. 2010;15(7):788–804. 29. Juster FT, Suzman RM. An overview of the health and retirement study. J Hum Resour. 1995;30:S7–S56. 54. Garbers S, Chiasson MA. Inadequate functional health literacy in Spanish as a barrier to cervical cancer screening among immigrant Latinas in New York City. Prev Chronic Dis. 2004;1:A07. 30. U.S. Department of Health and Human Services. National Institute on Aging. In: Growing Older in America: The Health and Retirement Study. Washington, DC: National Institutes of Health; 2007. 55. Guerra CE, Krumholz M, Shea JA. Literacy and knowledge, attitudes and behavior about mammography in Latinas. Journal of Health Care for the Poor Underserved. 2005;16:152–66. 31. Parker RM, Baker DW, Williams MV, Nurss JR. The test of functional health literacy in adults: a new instrument for measuring patients’ literacy skills. J Gen Intern Med. 1995;10:537–41. 56. Pagán J, Brown C, Asch D, Armstrong K, Bastida E, Guerra C. Health literacy and breast cancer screening among Mexican American women in south Texas. J Cancer Educ. 2012;27(1):132–7. 32. Chew LD, Bradley KA, Boyko EJ. Brief questions to identify patients with inadequate health literacy. Fam Med. 2004;36(8):588–94. 33. Gazmararian JA, Baker DW, Parker R, Blazer DG. A multivariate analysis of factors associated with depression: evaluating the role of health literacy as a potential contributor. Arch Intern Med. 2000;160(21):3307–14. 57. World Health Organization. (2016). Breast cancer: prevention and control. Retrieved July 11, 2016 from: http://www.who.int/cancer/detection/ breastcancer/en/index3.html. 34. Lee SD, Gazmararian JA, Arozullah AM. Health literacy and social support among elderly medicare enrollees in a managed care plan. J Appl Gerontol. 2006;25:324–37. 58. Nelson HD, Tyne K, Naik A, et al. Screening for Breast Cancer: Systematic Evidence Review Update for the US Preventive Services Task Force. Rockville, MD: Agency for Healthcare Research and Quality; 2009. Author details 1 Retrieved from: http://www.ncbi.nlm.nih.gov/books/NBK36392/. 35. Scott TL, Gazmararian JA, Williams MV, Baker DW. Health literacy and preventive health care use among medicare enrollees in a managed care organization. Med Care. 2002;40(5):395–404. 59. Van De Heide I, Rademakers J, Schipper M, Droomers M, Sørensen K, Uiters E. Health literacy of Dutch adults: a cross sectional survey. BMC Public Health. 2013;13:179. doi:10.1186/1471-2458-13-179. 36. Chew LD, Griffin JM, Partin MR, Noorbaloochi S, Grill JP, Snyder A, et al. Validation of screening questions for limited health literacy in a large veterans outpatient population. J Gen Intern Med. 2008;23(5):561–6. 60. Brez SM, Taylor M. Assessing literacy for patient teaching: perspectives of adults with low literacy skills. J Adv Nurs. 1997;25:1040–7. 37. Sarkar U, Schillinger D, López A, Sudore R. Validation of self-reported health literacy questions among diverse English and Spanish-speaking populations. J Gen Intern Med. 2011;26(3):265–71. 61. Pleasant A, McKinney J. Coming to consensus on health literacy measurement: an online discussion and consensus-gauging process. Nurs Outlook. 2011;59(2):95–106. 38. Aboumatar HJ, Carson KA, Beach MC, Roter DL, Cooper LA. The impact of health literacy on desire for participation in healthcare, medical visit communication, and patient reported outcomes among patients with hypertension. J Gen Intern Med. 2013;28(11):1469–76. 62. Baker DW, Williams MV, Parker RM, Gazmararian JA, Nurss J. Development of a brief test to measure functional health literacy. Patient Educ Couns. 1999;38(1):33–42. 63. Bishop WP, Craddock Lee SJ, Skinner CS, Jones TM, McCallister K, Tiro JA. Validity of single-item screening for limited health literacy in English and Spanish speakers. Am J Public Health. 2016;106(5):889–92. 39. Baker DW, Parker RM, Williams MV, Pitkin K, Parikh NS, Coates W, Imara M. The health care experience of patients with low literacy. Arch Fam Med. 1996;5:329–44. 40. Parikh NS, Parker RM, Nurss JR, Baker DW, Williams MV. Shame and health literacy: the unspoken connection. Patient Educ Couns. 1996;27:33–9. 64. Peek ME, Nunez-Smith M, Drum M, Lewis TT. Adapting the everyday discrimination scale to medical settings: reliability and validity testing in a sample of African American patients. Ethn Dis. 2011;21(4):502–9. 64. Peek ME, Nunez-Smith M, Drum M, Lewis TT. Adapting the everyday discrimination scale to medical settings: reliability and validity testing in a sample of African American patients. Ethn Dis. 2011;21(4):502–9. 41. Berkman LF. Social epidemiology: social determinants of health in the United States: Are we losing ground? Annual Reviews of Public Health. 2009;30:27–41. 42. Author details 1 19. Sudore RL, Yaffe K, Satterfield S, Harris TB, Mehta KM, Simonsick EM, et al. Limited literacy and mortality in the elderly: the health, aging, and body composition study. J Gen Intern Med. 2006;21:806–12. 1 Department of Internal Medicine, Division of General Medicine, University of Michigan, 1500 E. Medical Center Drive, Ann Arbor, MI 48109-2029, USA. 2Department of Health Behavior and Biological Sciences, University of Michigan School of Nursing, 400 North Ingalls Building, Ann Arbor, MI 48109-5482, USA. 3Department of Health Behavior & Health Education, School of Public Health, University of Michigan, 1415 Washington Heights, Ann Arbor, MI 48109-2029, USA. 4Institute for Healthcare Policy and 20. White S, Chen J, Atchison R. Relationship of preventive health practices and health literacy: a national study. Am J Health Behav. 2008;32(3):227–42. 21. Kripilani S, Gatti ME, Jacobsen TA. Association of age, health literacy, and medication management strategies with cardiovascular medication adherence. Patient Educ Couns. 2010;81:177–81. Page 8 of 8 Fernandez et al. BMC Public Health (2016) 16:596 Page 8 of 8 Page 8 of 8 22. Murray MD, Wu J, Tu W, Clark DO, Weiner M, Morrow DG, Brater DC. Health literacy predicts medication adherence. American Society for Clinical Pharmacology and Therapeutics. Clinical Pharmacology & Therapeutics. 2004;76. 48. Couper MP, Zikmund-Fisher BJ, Singer E, Fagerlin A, Ubel PA, Fowler Jr, FA, Levin C (2006). National Survey of Medical Decisions. Inter-university Consortium for Political and Social Research [distributor]: Ann Arbor, MI. Retrieved from: http://doi.org/10.3886/ICPSR25983.v1. 23. Nutbeam D. The evolving concept of health literacy. Soc Sci Med. 2008;67(12):2072–8. Retrieved from: http://doi.org/10.3886/ICPSR25983.v1. 49. Sheskin DJ. Handbook of parametric and nonparametric stati procedures. Danvers, MA: Chapman & Hall; 2011. 49. Sheskin DJ. Handbook of parametric and nonparame procedures. Danvers, MA: Chapman & Hall; 2011. 24. Paasche-Orlow MK, Wolf M. The causal pathways linking health literacy to health outcomes. The American Journal of Health Behavior. 2007;31(1):19–26. 50. Morrow D, Clark D, Wanzu T, Wu J, Weiner M, Steinley D, Murray MD. Correlates of health literacy in patients with chronic heart failure. The Gerontologist. 2006;46(5):669–76. 25. Von Wagner C, Steptoe A, Wolf MS, Wardle J. Health literacy and health actions: a review and a framework from health psychology. Health Education and Behavior. 2009;36(5):860–77. 51. Paasche-Orlow MK, Parker RM, Gazmararian JA, Nielsen-Bohlman L, Rudd R. The prevalence of limited health literacy. J Gen Intern Med. 2005;20(2):175–84 e prevalence of limited health literacy. J Gen Intern Med. 2005;20(2):175– 26. Baker DW. Author details 1 Braveman PA, Cubbin C, Egerter S, Williams DR, Pamuk E. Socioeconomic disparities in health in the United States: what the patterns tell us. Am J Public Health. 2010;100 suppl 1:S186–96. 43. Kunst AE, Bos V, Lahelma E, Bartley M, Lissau I, et al. Trends in socioeconomic inequalities in self-assessed health in 10 European countries. Int J Epidemiol. 2005;34:295–305. Fernandez et al. BMC Public Health (2016) 16:596 Submit your next manuscript to BioMed Central and we will help you at every step: 44. Brim OG, Baltes PB, Bumpass LL, Cleary PD, Featherman DL, et al. (1995). National Survey of Midlife Development in the United States (MIDUS). Inter-university Consortium for Political and Social Research [distributor]: Ann Arbor, MI. Retrieved from: http://doi.org/10.3886/ICPSR02760.v8. • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit and we will help you at every step: • We accept pre-submission inquiries 45. Adler N, Stewart J. The MacArthur Scale of Subjective Social Status. (2007). Summary prepared in collaboration with the Psychosocial Working Group. 2007. Retrieved from: http://www.macses.ucsf.edu/research/psychosocial/ subjective.php. 46. Williams DR, Yu Y, Jackson JS, Anderson NB. Racial differences in physical and mental health: socioeconomic status, stress and discrimination. J Health Psychol. 1997;2:335–51. 46. Williams DR, Yu Y, Jackson JS, Anderson NB. Racial differences in physical and mental health: socioeconomic status, stress and discrimination. J Health Psychol. 1997;2:335–51. 47. Williams DR, Neighbors HW, Jackson JS. Racial/ethnic discrimination and health: findings from community studies. Am J Public Health. 2003;93:200–8.
https://openalex.org/W3201670717	https://periodicos.ufersa.edu.br/acta/article/download/10155/10778	English	null	Seminoma and sertolioma in non-cryptorchid dog – case report	Acta Veterinaria Brasilica	2,021	cc-by	4,523	Seminoma and sertolioma in non-cryptorchid dog – case report Guilherme Mantuani Silva1 , Isabela Cristina de Oliveira1 , Rodrigo Samuel de Toledo1 , Juliana Evangelista Bezerril2 , Gabriela Maria Benedetti Vasques1 , Beatriz Gasser3 , Paulo Fernandes Marcusso4 , Isaac Romani1* ABSTRACT: The most common testicular neoplasms in dogs are seminoma, leydigocytoma and sertolioma, affecting middle- -aged and elderly dogs, where cryptorchidism is a predisposing factor, as well as some breeds. It can occur alone or, less fren- quently, concurrently, generally affecting the same testicle. This study aimed to report the case of a non-cryptorchid 14-year- -old mixed breed dog diagnosed with seminoma and sertolioma, each in a testicle. The animal showed an increase in scrotal volume, with no changes in other clinical parameters on physical examination. On ultrasound examination, it was possible to observe alterations suggestive of neoplasia in both testicles and prostatic alteration suggestive of benign prostatic hyperplasia. Complementary blood count and biochemical tests were performed and, as treatment, orchiectomy was performed. Fragments were collected from both testicles and sent for histopathological examination. Microscopy of the left testicle showed the presence of round cells, multiple and evident nucleoli, cells in different phases of mitosis and binucleation, these changes being compa- tible with seminoma. In contrast, in the right testicle, spindle and elongated cells (pseudo-lobular) were observed, presence of long cytoplasmic projections with rounded ovoid nucleus, spindle cells and degeneration of seminiferous tubules, compatible with sertolioma. It was observed that physical examination associated with ultrasound was efficient to detect the presence of neoplasms, being validated by histopathological examination. Orchiectomy was an assertive treatment indicated for this case. KEYWORDS: Histopathological; Neoplasia; Reproductive system; Testicle. RESUMO: As neoplasias testiculares mais comuns em cães são o seminoma, leydigocitoma e o sertolioma, acometendo cães de meia idade e idosos, com predisposição racial e animais criptorquidas, podendo ocorrer isoladamente, ou concomitantemente, porém com menor frequência. Esse trabalho objetivou relatar o caso de um cão, SRD, 14 anos, não criptorquida diagnosticado com seminoma e sertolioma em testículos distintos. O animal apresentou aumento do volume testicular bilateral, sem alterações nos demais parâmetros clínicos ao exame físico. Ao exame ultrassonográfico foi possível observar alterações sugestivas de neoplasia para ambos os testículos e alteração prostática sugestiva de hiperplasia prostática benigna. Foram realizados exames complemen- tares de hemograma e bioquímico e como tratamento a realização de orquiectomia. Fragmentos foram coletados de ambos os tes- tículos e enviados para análise histopatológica. Seminoma and sertolioma in non-cryptorchid dog – case report A microscopia do testículo esquerdo, evidenciou a presença de células arredonda- das, nucléolos múltiplos e evidentes, células em diferentes fases de mitose e binucleação, alteração compatíveis de seminoma. Em contrapartida, no testículo direito foi observado a presença de células fusiformes e alongadas (pseudo-lobular), presença de longas projeções citoplasmáticas com núcleo ovóide arredondado, células fusiformes e degeneração dos túbulos seminíferos compatíveis de sertolioma. Observou-se que o exame físico associado ao ultrassonográfico foi eficiente para detectar a presença das neopla- sias e isto foi validado pelo exame histopatológico. A realização de orquiectomia foi tratamento assertivo indicado para este caso. PALAVRAS-CHAVE: Histopatológico; Neoplasia; Sistema Reprodutor; Testículo. CLINICAL REPORTS CLINICAL REPORTS http://dx.doi.org/10.21708/avb.2021.15.4.10155 INTRODUCTION of histopathological exams, lack of periodic veterinary fol- low-up and the fact that they are usually necropsy findings, it is difficult to assess the real incidence of these neoplasms (FOSTER, 2016). Testicular neoplasms are the most common disorders in the reproductive tract of male dogs, corresponding to approxi- mately 90% of the reports of reproductive neoplasms and representing the second most common location observed in the species. Due to factors such as early orchiectomy, absence Testicular tumors are classified mainly according to their cellular origin, in: germ cell tumors (seminomas, teratomas 281 Acta Veterinaria Brasilica December 15 (2021) 281-286 1UNINGÁ – Ingá University Center, Maringá/PR, Brazil 2Mineiros University Center, Trindade/GO, Brazil 3São Paulo State University, Jaboticabal/SP, Brazil 4Federal University of the Valleys of Jequitinhonha and Mucuri, Unaí/MG, Brazil Corresponding author: prof.isaacromani@uninga.edu.br Received: 08/02/2021. Accepted: 24/04/2021 1UNINGÁ – Ingá University Center, Maringá/PR, Brazil 2Mineiros University Center, Trindade/GO, Brazil 3São Paulo State University, Jaboticabal/SP, Brazil 4Federal University of the Valleys of Jequitinhonha and Mucuri, Unaí/MG, Brazil Corresponding author: prof.isaacromani@uninga.edu.br Received: 08/02/2021. Accepted: 24/04/2021 281 Acta Veterinaria Brasilica December 15 (2021) 281-286 Silva et al. and embryonic carcinomas), tumors of stroma and cord sex cells (Sertoli cell tumor, Leydig cell tumor and undifferen- tiated intermediary tumors), vascular tumors, mesothelio- mas and multiple primary tumors (CATOI et al., 2008). Testicular neoplasms affect middle-aged and elderly dogs, with an average age of 11.46 ± 3.47 years (SANTOS et al. 2000), the most predisposing breeds are Boxer, German shep- herd, Weimaraner, Afghan hound and Shetland sheepdog and the least predisposing are Dachshund and mixed breed dogs (STAUT; ASSEF; CIRIO, 2007). serum concentration of alanine aminotransferase (ALT) and creatinine. The blood test showed that all results were within the normal range, however, in the biochemical anal- ysis there was an increase in the hepatic enzyme ALT with a value of 157.0 IU/L (21.0 to 73.0 IU/L), yet the animal did not show any clinical symptoms associated with the increase in this enzyme. The ultrasound evaluation was performed at the Diagnostic Imaging Sector of the Veterinary Clinic of UNINGÁ using the equipment Toshiba Istyle - Nemio XG. INTRODUCTION The ultrasound evaluation of the abdominal cavity did not reveal any changes, except for the prostate, presenting regular contours, bilobed aspect, measuring approximately 4.40 cm in length and 2.54 cm in height in longitudinal plane, with slightly heterogeneous parenchyma with the presence of cysts, suggestive of benign prostatic hyperplasia as can be seen in (Figure 1). However, in the testicular ultrasound examination (Figure 2b) decreased dimensions were observed in the right testicle (length: 2.61 cm, width: 1.32 cm, depth: 5.37 cm), with slightly heterogeneous parenchyma, suggestive of nodules / neoplasia and regular testicular mediastinum. In the left testicle (Figure 2a), mark- edly increased dimensions (length: 8.90 cm, width: 4.90 cm, depth: 1.37 cm), heterogeneous parenchyma with hypoecho- genic areas and absence of testicular mediastinum were noted, suggestive of neoplasia. Testicular neoplasms in the canine species can develop in normal testicles, but they are more prone to appear in cryptorchid testicles (GRIECO et al., 2008). The most common are Leydig cell tumors, seminomas and sertolio- mas, the first being the most frequent. It is also observed that approximately 40% of dogs that develop testicular neoplasms present more than one type of testicular tumor (STAUT; ASSEF; CIRIO, 2007). However, it is not com- monly reported in the literature the presence of different neoplasms affecting different testicles in a non-cryptorchid animal. In view of the above, the present study aimed to report the case of a non-cryptorchid dog presenting bilat- eral testicular neoplasms. Acta Veterinaria Brasilica December 15 (2021) 281-286 CASE REPORT Biochemical assessments were performed using the semi-automatic biochemical analyzer model Bio-200S (Bioplus®) using commercial kits (Analisa®) to assess the 282 282 Acta Veterinaria Brasilica December 15 (2021) 281-286 Seminoma and sertolioma in non-cryptorchid dog – case report Figure 2. Sonogram (A - 8 MHZ, B - 12 MHZ, B-mode) of testicles of mixed breed dog, 14 years old. (A) LT - Left testicle with markedly increased dimensions, length: 8.90 cm, width: 4.90 cm, depth: 1.37 cm; (B) RT - Right testicle with reduced dimensions, length: 2.61 cm, width: 1.32 cm, depth: 5.37 cm. Figure 2. Sonogram (A - 8 MHZ, B - 12 MHZ, B-mode) of testicles of mixed breed dog, 14 years old. (A) LT - Left testicle with markedly increased dimensions, length: 8.90 cm, width: 4.90 cm, depth: 1.37 cm; (B) RT - Right testicle with reduced dimensions, length: 2.61 cm, width: 1.32 cm, depth: 5.37 cm. Table 1. Testicular measurements obtained after orchiectomy of a non-cryptorchid 14-year-old mixed breed dog with testicular neoplasia. After the surgical procedure, testicular measurements were taken using a caliper and digital precision scale. The data obtained are shown in Table 1. Table 1. Testicular measurements obtained after orchiectomy of a non-cryptorchid 14-year-old mixed breed dog with testicular neoplasia. Variables evaluated Left Testicle Right Testicle Lenght (cm) 8.29 2.48 Width (cm) 5.91 1.56 Thickness (cm) 5.55 1.21 Volume (cm3) 142.21 2.44 Mass (g) 159.26 3.73 Once these characteristics were collected, incisional biop- sies in the testicles were performed, obtaining fragments with both macroscopically normal and altered testicular tissue, being packed in previously identified vials containing 10% form- aldehyde for fixation for 48 hours, respecting the volume of 1 part of tissue for 9 parts of formalin. Subsequently, the testicle fragments were stored in cas- settes for processing, where they were dehydrated, diaphanized, included in paraffin, cut between three and five micrometers (μm), laminated and stained using hematoxylin and eosin (HE) techniques for later evaluation under an optical microscope Eclipse E200 - Nikon ®, at the UNINGÁ Animal Pathology Sector. The slides containing the testicular fragments were evaluated and the histopathological changes found were clas- sified (FOSTER, 2016). to be within normal limits, and further complementary tests were requested: blood count, creatinine and ALT, abdominal ultrasound and chest radiographic examination to search for possible metastasis. CASE REPORT After the ultrasound diagnosis, the animal was referred for surgical treatment by pre-scrotal orchiectomy at the Small Animal Surgery Sector of UNINGÁ. As pre-anesthetic medi- cation, acepromazine (0.05 mg/kg) and morphine (0.3 mg/kg) were administered intramuscularly, followed by induction with ketamine (5 mg/kg) and diazepam (0.2 mg/kg) intra- venously and local anesthetic in the incision line, with lidocaine (1 ml). As postoperative medication, meloxicam (0.1 mg/kg) was administered subcutaneously and dipyrone (25mg/kg) intramuscularly. A 14-year-old male mixed breed dog weighing 17.5 kg was attended at the Veterinary Clinic of UNINGÁ – Ingá University Center. On physical examination, the animal presented all vital parameters within the normal range, on inspection and palpa- tion an increase in volume of hard consistency was noted in the left testicle. According to the owners, the increase occurred within 6 to 8 months progressively, a circumference of 24.8 cm of the scrotum was measured, the right testicle did not pres- ent any noteworthy changes. Complementary blood tests, biochemical tests and ultrasound assessment of the abdominal region and scro- tum were requested. 3 ml of blood were collected through the jugular venipuncture, in which 0.5 ml were stored in a micro tube of dipotassium ethylene diamine tetra acetic acid (EDTA K2) and 2.5 ml in a dry tube for biochemi- cal analyzes. The hematological evaluation was carried out at the Laboratory of Veterinary Clinical Pathology at UNINGÁ. The erythrogram and total leukocyte and platelet count were performed using the BC2800Vet automatic counter (Mindray United States). For differ- ential leukocyte count and qualitative assessment of cells, stained blood smears were made using the quick dye for hematology (Instant-Prov - NEWPROV), being analyzed under 1000x magnification light microscopy (Eclipse E200MV - NIKON). Figure 1. Sagittal sonogram (8 MHZ, B-mode) of the prostate of a mixed breed dog, 14 years old, with regular contours, bilobed aspect, slightly heterogeneous parenchyma, with the presence of cysts and measuring approximately 4.40 cm length and 2.54 cm height in longitudinal plane. Figure 1. Sagittal sonogram (8 MHZ, B-mode) of the prostate of a mixed breed dog, 14 years old, with regular contours, bilobed aspect, slightly heterogeneous parenchyma, with the presence of cysts and measuring approximately 4.40 cm length and 2.54 cm height in longitudinal plane. CASE REPORT The blood count and biochemical exams showed normal values, noting the return to normal levels of the liver enzyme ALT of 54.0 IU/L. In the ultrasound (Figure 4) and radio- graphic exams, no significant alterations were found, except for the same prostatic alterations observed previously compat- ible with benign prostatic hyperplasia, however a reduction in size was observed when compared to the previous exam, with the measurements being 2.20 cm in length and 1.68 cm in height in longitudinal plane. The histopathological examination showed, in the left testicle, rounded cells, multiple and evident nucleoli, cells in different phases of mitosis and binucleation (Figure 3a), these histological characteristics found are suggestive of seminoma. The right testicle, on the other hand, presented fusiform and elongated cells, which were arranged in a pseudo-lobular struc- ture, in addition to the presence of long cytoplasmic projec- tions with rounded ovoid nucleus, spindle cells and degen- eration of seminiferous tubules (Figure 3b), these changes are suggestive of sertolioma. Acta Veterinaria Brasilica December 15 (2021) 281-286 DISCUSSION After the histopathological examination of the testicles, two distinct neoplasms were observed, the seminoma being observed in the left testicle and sertolioma in the right testicle. Testicular neoplasms are common in domestic dogs; however, After six months, the animal returned to the Veterinary Clinic of UNINGÁ for a new evaluation and monitoring of the case. On physical examination, all parameters were found Acta Veterinaria Brasilica December 15 (2021) 281-286 Acta Veterinaria Brasilica December 15 (2021) 281-286 283 Silva et al. Figure 3. a) Seminoma, presence of round cells (continuous arrow), binucleation (arrowhead), different phases of mitosis (, ), evident multiple nucleoli (discontinuous arrow) (HE, 100x objective) are observed. b) Sertolioma, spindle cells (arrow with tip in eclipse), ovoid nucleus (eclipse) and degeneration of seminiferous tubules (short arrow) (HE, 40x objective). Silva et al. Figure 3. a) Seminoma, presence of round cells (continuous arrow), binucleation (arrowhead), different phases of mitosis (, **), evident multiple nucleoli (discontinuous arrow) (HE, 100x objective) are observed. b) Sertolioma, spindle cells (arrow with tip in eclipse), ovoid nucleus (eclipse) and degeneration of seminiferous tubules (short arrow) (HE, 40x objective). Figure 4. Longitudinal sonogram (8 MHZ, B-mode) of the prostate of a mixed breed dog, 14 years old, presenting small cysts in parenchyma and measuring 2.20 cm length and 1.68 cm height in longitudinal plane. with enlarged dimensions, heterogeneous and vascularized parenchyma, suggestive of neoplasia and prostatic hyperplasia, with heterogeneous parenchyma and anechoic areas (COSTA, 2016). After orchiectomy and histopathological evaluation, the presence of seminoma in the right testicle and sertolioma in the left was verified. Differing in the macroscopic presen- tation of this report, in which the testicle affected by semi- noma increased in size, while the contralateral affected by sertolioma maintained its dimensions. Therefore, the macro- scopic presentation of neoplasms does not appear to have a well-defined pattern. Bilateral seminoma and leydigocytoma have been reported in non-cryptorchid dogs (SILVA et al., 2014; VISCONE et al., 2016). Both cases, as in the case presented, were mixed breed dogs, advanced age, who came to the service with tes- ticular enlargement. Figure 4. Longitudinal sonogram (8 MHZ, B-mode) of the prostate of a mixed breed dog, 14 years old, presenting small cysts in parenchyma and measuring 2.20 cm length and 1.68 cm height in longitudinal plane. Acta Veterinaria Brasilica December 15 (2021) 281-286 Seminoma and sertolioma in non-cryptorchid dog – case report change in size was observed in the left testicle. At follow-up, after complementary examinations, the dog did not present any metastasis due to testicular neoplasms, which corrobo- rates with other reports (CIAPUTA et al., 2012; GRIECO et al., 2008). These authors describe that seminomas are rarely malignant, do not produce hormones, and usually do not show clinical signs, however in some cases pain is noticed due to the pressure created by the growth of the tumor. present in hepatocytes such as cytochromes P450 are essential for estrogen metabolism (SANTIAGO, 2003), it is common to observe in sertoliomas the serum increase in estrogenic hor- mones (MARQUES et al., 2020), therefore hyperestrogenism could cause an exaggerated enzymatic activity, leading to liver damage and increased ALT, an extravasation enzyme. As soon as the tumor was removed, the enzyme levels returned to the reference values (GIANNINI et al., 2005). The prostate enlargement secondary to sertolioma occurs due to the influence of estrogen on the prostate gland caus- ing squamous metaplasia of the epithelium and obstruction of the prostatic ducts, predisposing to the formation of cysts, as observed in the case reported (COSTA, 2016). Sertoliomas originate from support cells located in seminif- erous tubules, being the third most common neoplasm in males of the canine species, with reports in other species as well. They occur more frequently in cryptorchid animals, unilaterally, and may be bilateral in cases of bilateral cryptorchidism (COSTA, 2016; GRIECO et al., 2008; MADUREIRA et al., 2017) in contrast to that described in this report. Around 24% to 57% of animals affected by sertolioma have a hormonal imbalance, developing hyperestrogenism, resulting in paraneoplastic fem- inization syndrome. The most common clinical signs are apa- thy, depression, gynecomastia, alopecia, contralateral testicular atrophy, hypopigmentation, penile atrophy, pendulous foreskin, prostatic cysts and abscesses and even granulocytic anemia and thrombocytopenia (CIAPUTA et al., 2012; MADUREIRA et al., 2017; MARQUES et al., 2020). However, the animal in this report did not present any of these clinical signs. Benign prostatic hyperplasia is an abnormal accumulation of dihydrotestosterone due to a high conversion of testoster- one (CRUZ; ZIMMERMANN, 2017; MACPHAIL, 2014). Even with a low production of androgens, which occurs with aging, this hyperplasia can develop as long as there is an increase in the production of estrogens. Although the size increases, the secretory function of the prostate decreases (MACPHAIL, 2014). Seminoma and sertolioma in non-cryptorchid dog – case report It is considered the most common prostatic disease of male dogs, not neutered, over the age of six (BAUZAITE; ANIULIENE, 2003). Therefore, it is possible that the pros- tatic alterations observed in this report are due to the age of the animal and the fact that he was not neutered. Despite causing problems, sertolioma has a benign behavior and its metastasis rate is less than 10%, being more common in lumbar and iliac lymph nodes, however cases of metastasis in organs such as liver, lung, spleen have also been reported, and may also occur in the skin (MARQUES et al., 2020). Sertolioma presents itself as a proliferation of elongated cells, perpendicular to the basal membrane, forming tubular structures and supported by a large amount of fibrous connec- tive tissue (ARGENTA et al., 2016; GRIECO et al., 2008). Regarding the treatment of testicular neoplasia, surgical treatment by bilateral orchiectomy and retroperitoneal lymphad- enectomy in histologically malignant cases is suggested (COSTA, 2016). Blood transfusion is also recommended in cases of ane- mia and thrombocytopenia, antibiotic therapy to protect against infections and the use of anti-inflammatory drugs, if these treat- ments are needed (HENRIQUE et al., 2016). However, as the animal was in good clinical condition, it was directly submitted to the orchiectomy procedure, using anti-inflammatory in the postoperative period and this proved to be efficient. Despite causing problems, sertolioma has a benign behavior and its metastasis rate is less than 10%, being more common in lumbar and iliac lymph nodes, however cases of metastasis in organs such as liver, lung, spleen have also been reported, and may also occur in the skin (MARQUES et al., 2020). Sertolioma presents itself as a proliferation of elongated cells, perpendicular to the basal membrane, forming tubular structures and supported by a large amount of fibrous connec- tive tissue (ARGENTA et al., 2016; GRIECO et al., 2008). Orchiectomy is the treatment of choice for benign pros- tatic hyperplasia, causing no side effects, resulting in a 70% decrease in prostate size. The gland begins to shrink in days, and a palpable decrease in size is expected within 7 to 14 days (CRUZ; ZIMMERMANN, 2017). As described above in the case, where there was a reduction in prostate size from 4.40 cm to 2,20 cm in length and from 2.54 cm to 1.68 cm in height in longitudinal plane, after 6 months of performing orchiectomy. CONCLUSION Based on the aforementioned content, it is possible to conclude that the thorough physical examination of the reproductive system associated with complementary exams such as ultra- sound and histopathology were essential for the conclusion of the diagnosis of neoplasms and the appropriate treatment. In this report we observed the presence of two different neo- plasms – seminoma and sertolioma in a non-cryptorchid dog. In cases where there is metastatic development, oncologi- cal follow-up and chemotherapy or radiotherapy are indicated, however this treatment was not necessary in this report, since the animal did not present metastases in the follow-up exams (COSTA, 2016). Regarding the ALT enzyme, even observing an increase of about 3 times above the reference value, this information is not significant, due to its presence in other tissues of the organism, such as heart, kidneys, muscles and erythrocytes (GIANNINI et al., 2005). However, microsomal enzymes DISCUSSION Seminomas have their origin in the germ cells of the tes- ticular sperm epithelium and are common in dogs, as well as sertolioma. The authors mention that, among the predispos- ing factors are age and cryptorchidism, however, in approxi- mately 70% of seminoma cases, its development occurs in scrotal testicles, as in the reported case, and only one third in cryptorchid testicles. (BOMFIM et al., 2016; COSTA, 2016; LOPES et al., 2019; NETA et al., 2019). their incidence is difficult to estimate due to early orchiecto- mies and the low frequency of histopathological exams. Some predisposing factors are well defined, such as advanced age, breed, environmental carcinogens and unilateral and bilat- eral cryptorchidism, with seminoma and sertolioma being the most common neoplasms (COSTA, 2016; HENRIQUE et al., 2016; LOPES et al., 2019; NETA et al., 2019). In this report, the animal presented the two most common types of testicular neoplasms, age of 14 years as an important predis- posing factor, but did not presented cryptorchidism. Seminomas usually occur unilateral and uninodular, however 10% to 18% are bilateral and multinodular, being more common in the right than in the left testicle (MORRIS; DOBSON, 2001), in this report the seminoma was unilat- eral and uninodular in the left testicle. It was observed that, in the first visit, the dog did not show clinical signs that indi- cated a possible testicular neoplasia, however, on palpation, a The occurrence of sertolioma and seminoma in a bilat- eral cryptorchid dog has been reported which the ultrasound examination revealed the right testicle with preserved dimen- sions, parenchyma suggestive of normality, the left testicle Acta Veterinaria Brasilica December 15 (2021) 281-286 284 284 Seminoma and sertolioma in non-cryptorchid dog – case report Seminoma and sertolioma in non-cryptorchid dog – case report Clinical treatment can also be carried out using finasteride, a drug responsible for blocking 5-alpha-reductase leading to atrophy of the prostate parenchyma, and preventing the accu- mulation of dihydrotestosterone (CRUZ; ZIMMERMANN, 2017). However, as it was a case of neoplasms, this option would not be effective in treating the patient. Regarding the treatment of testicular neoplasia, surgical treatment by bilateral orchiectomy and retroperitoneal lymphad- enectomy in histologically malignant cases is suggested (COSTA, 2016). Blood transfusion is also recommended in cases of ane- mia and thrombocytopenia, antibiotic therapy to protect against infections and the use of anti-inflammatory drugs, if these treat- ments are needed (HENRIQUE et al., 2016). However, as the animal was in good clinical condition, it was directly submitted to the orchiectomy procedure, using anti-inflammatory in the postoperative period and this proved to be efficient. Acta Veterinaria Brasilica December 15 (2021) 281-286 MACPHAIL, C. M. Surgery of the reproductive and genital systems. In: FOSSUM, T. W. Small animal surgery. 4. ed. Rio de Janeiro: Elsevier, 2014. BAUZAITE, N.; ANIULIENE, A. Enlarged prostate lesions of purê- bred and mongrel dogs. Medycyna Weterynaryjna, v. 59, n. 8, p. 686-690, 2003. MADUREIRA, R. et al. Seminoma and sertolioma in a male pseudohermaphrodite dog with unilateral cryptorchidism. Semina: Ciências Agrárias, v. 38, p. 2-7, 2017. BOMFIM, E. M. O. et al. Seminoma em um cão com testículo ectópico–Relato de caso. Jornal Interdisciplinar de Biociências, v. 1, p. 36-39, 2016. MARQUES, B. A. S. et al. Sertolioma in dog associated with cryptoquidism: report case. Revista de Educação Continuada em Medicina Veterinária e Zootecnia do CRMV-SP, v. 18, p. 1-6, 2020. CATOI, C. et al. Morphological study of testicular tumors in dogs. Lucrări ştiinłifice medicină veterinară, v. 41, p. 2-6, 2008. CIAPUTA, R. et al. Seminoma, sertolioma, and leydigoma in dogs: clinical and morphological correlations. Bulletin of the Veterinary Institute in Pulawy, v. 56, p. 361-367, 2012. MORRIS, J.; DOBSON, J. Trato Genital p. 175-178. In: MORRIS, J.; DOBSON, J. Oncologia em pequenos animais. São Paulo: Roca, 2001. NETA, J. H. et al. Sertolioma in a Canadian Husky: Relationship between Tumor, Hormones, Neurons and Skin. Acta Scientiae Veterinariae, v. 47, n. 416, p. 1-6, 2019. COSTA, T. R. Neoplasia Testicular: Relato de Caso. 2016. 20 f. Monografia (Graduação em Medicina Veterinária) – Fundação Educacional Jayme de Altavila, Salvador, 2016. SANTIAGO, L. M. A. Metabolism in the cytochrome P450 system and its importance in general practice. Revista Portuguesa de Clínica Geral, v. 19, p. 121-129, 2003. CRUZ, B. P. A.; ZIMMERMANN, M. Comparação entre o tratamento clínico e cirúrgico da hiperplasia prostática benigna em cães. i REVET – Revista Científica do Curso de Medicina Veterinária, v. 4, p. 13-27, 2017. SANTOS, R. L. et al. Testicular tumors in dogs: frequency and age distribution. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, v. 52, n. 1, p. 25-26, 2000. Foster, R. A. Male Genital System. In: JUBB; KENNEDY; PALMER´S. Pathology of domestic animal. 6. ed. Elsevier, 2016. SILVA, W. P. N. et al. Concomitant leydigocytoma and seminoma in a dog - Case Report. Acta Veterinaria Brasilica, v. 8, p. 132-135, 2014. GIANNINI, E. G. et al. Liver enzyme alteration: a guide for clinicians. Canadian Medical Association or its licensors, v. 172, n. 3, p. 367–379, 2005. STAUT, J. L.; ASSEF, M. L. M.; CIRIO, S. M. Estudo histoquímico da matriz extracelular de neoplasias testiculares de cães (Canis Familiares Linnaeus, 1758). Estudos de Biologia, v. REFERENCES LOPES, C. E. B. Diffuse sertolioma in a criptorquidic dog. Ars Veterinaria, v. 35, p. 021-025, 2019. ARGENTA, F. F. et al. Testicular Neoplasms in Dogs in Rio Grande do Sul, Brazil. Acta Scientiae Veterinariae, v. 44, p. 1-6, 2016. LOPES, C. E. B. Diffuse sertolioma in a criptorquidic dog. Ars Veterinaria, v. 35, p. 021-025, 2019. VISCONE, E. A. et al. Seminoma and interstitial cell tumor in the same testicle as a dog – Case Report. Enciclopédia biosfera, Centro Científico Conhecer, v. 13, p. 919-923, 2016. © 2021 Universidade Federal Rural do Semi-Árido This is an open access article distributed under the terms of the Creative Commons license. MACPHAIL, C. M. Surgery of the reproductive and genital systems. In: FOSSUM, T. W. Small animal surgery. 4. ed. Rio de Janeiro: Elsevier, 2014. 29, n. 68-69, p. 242-247, 2007. GRIECO, V. et al. Canine testicular tumours: a study on 232 dogs. Journal of comparative pathology, v. 138, n. 2-3, p. 86-89, 2008. HENRIQUE, F. V. Sertoli cell tumor and diffuse seminoma in dog with bilateral cryptorchidism - Case report. Brazilian Journal of Veterinary Medicine, v. 38, p. 217-221, 2016. VISCONE, E. A. et al. Seminoma and interstitial cell tumor in the same testicle as a dog – Case Report. Enciclopédia biosfera, Centro Científico Conhecer, v. 13, p. 919-923, 2016. 286 286 ACKNOWLEDGMENTSh The authors thank the UNINGÁ – Ingá University Center, for the financing of the project, and everyone who partici- pated directly and indirectly in the project. Acta Veterinaria Brasilica December 15 (2021) 281-286 285 Silva et al. Acta Veterinaria Brasilica December 15 (2021) 281-286 Acta Veterinaria Brasilica December 15 (2021) 281-286 Acta Veterinaria Brasilica December 15 (2021) 281-286
https://openalex.org/W2059257796	https://zenodo.org/records/1141950/files/CheckList_article_19043.pdf	English	null	Range extension and geographic distribution of Amphisbaena mitchelli Procter, 1923 in the state of Pará, Brazil	Check list	2,014	cc-by	1,482	Journal of species lists and distribution Chec List Journal of species lists and distribution Chec List Check List 10(5): 1229–1230, 2014 © 2014 Check List and Authors ISSN 1809-127X (available at www.checklist.org.br) Check List 10(5): 1229–1230, 2014 © 2014 Check List and Authors Notes on Geographic Distribution Notes on Geographic Distribution Range extension and geographic distribution of Amphisbaena mitchelli Procter, 1923 in the state of Pará, Brazil Síria Ribeiro 1, 2, Alfredo P. Santos-Jr 1, Lívia Carla Chagas 3 and Hipócrates de Menezes Ribeiro 1, 2, Alfredo P. Santos-Jr 1, Lívia Carla Chagas 3 and Hipócrates de Menezes Chalkidis 1 Universidade Federal do Oeste do Pará, Laboratório de Ecologia e Comportamento Animal do Instituto de Ciências da Educação – ICED, Ca Tapajós. Rua Vera Paz, s/n, Salé. 68015-110, Santarém, PA, Brazil. 3 Universidade Federal do Oeste do Pará, Curso de Licenciatura Plena em Ciências Biológicas, Campus Rondon. Rua Marechal Rondon s/n, Aparecida. 68040-070, Santarém, PA, Brazil. 4 Faculdades Integradas do Tapajós, Curso de Ciências Biológicas. Rua Rosa Vermelha 335, Aeroporto Velho. 68010-200, Santarém, PA, Brazil. * Corresponding author. E-mail: siherp@hotmail.com 4 Faculdades Integradas do Tapajós, Curso de Ciências Biológicas. Rua Rosa Vermelha 335, Aeroporto Velho. 68010-200, Santarém, PA, Brazil. * Corresponding author. E-mail: siherp@hotmail.com 4 Faculdades Integradas do Tapajós, Curso de Ciências Biológicas. Rua Rosa Vermelha 335, Aeroporto Velho. 68010 200, Santarém, PA, Brazil. * Corresponding author. E-mail: siherp@hotmail.com Abstract: We present here a new record for Amphisbaena michelli for the western of Pará State, northern Brazil. The specimen was found in the municipality of Belterra, on the right bank of the Tapajós River, and represents an expansion ca. 350 km west of the previously known geographic distribution of the species. DOI: 10.15560/10.5.1229 Amphisbaena mitchelli Procter, 1923 was described from Ilha do Marajó, state of Pará, Brazil, based on a specimen deposited in the British Museum of Natural History (BMNH 1946.8.2.31) (Gans 1963). This relatively small species is characterized by two elongated (oval) pre-cloacal pores, 203–220 body annuli, 26–29 caudal annuli, autotomic site in the sixth and seventh tail annuli, 12–14 dorsal and 14–16 ventral segments on a midbody annulus. After its description, four publications have presented new distribution data for A. mitchelli (Gans 1963, 1964; Hoogmoed and Avila-Pires 1991; Mott et al. 2011; Teixeira Jr. et al. 2014) and all the known records have been from the northwestern portion of the state of Maranhão to the east of the state of Pará, Brazil (Figure 1). Here we present a new record for A. mitchelli, which was collected in the western portion of the state of Pará. This new record contributes to the knowledge of the geographical distribution of this poorly known species. to the nearest millimeter. Range extension and geographic distribution of Amphisbaena mitchelli Procter, 1923 in the state of Pará, Brazil The voucher specimen (LPHA 4942) has the following morphological characteristics: 145 mm snout-vent length, an autotomized tail of 7.5 mm, 5.3 mm head length (3.7% of snout-vent length), 3.9 mm midbody diameter, two elongate precloacal Figure 1. Geographical distribution of Amphisbaena mitchelli. Black symbols represent records from literature (Hoogmoed and Avila-Pires 1991; Mott et al. 2011; and Teixeira Jr. et al. 2014); star represents the type-locality; red circle represents the new record for the municipality of Belterra, state of Pará, Brazil. Localities: 1, Ilha do Marajó (type locality); 2, Belterra; 3, Rio Tocantins (cf. Hoogmoed and Avila-Pires 1991); 4, Belém; 5, Ananindeua; 6, Santo Antônio do Tauá; 7, Peixe-boi; 8, Ourém; 9, Santa Luzia, Capitão Poço; 10, Viseu; 11, Colônia Nova; 12, Paruá; 13, Puraqueú; 14, Carajás; 15, Parauapebas; and 16, Juruá, Rio Xingu. On 20 October 2010, a specimen of Amphisbaena mitchelli (Figures 2 and 3) was captured by local residents of Vila Americana, an urban area of the municipality of Belterra, on the right margin of the Tapajós River and was delivered to one of the authors (LCC). The locality is in the west of the state of Pará, Brazil (02°38′19.78″ S; 54°56′30.86″ W; center of the urban area). The specimen was deposited in the collection of the Linha de Pesquisa em Herpetologia da Amazônia (LPHA), Laboratório de Pesquisas Zoológicas, Faculdades Integradas do Tapajós, municipality of Santarém, state of Pará, Brazil. Meristic data follow Gans and Alexander (1962) and morphometric data follows Perez et al. (2012). The measurements were taken with digital calipers having 0.01 mm precision, except for the snout- vent length, which was measured using a flexible ruler Figure 1. Geographical distribution of Amphisbaena mitchelli. Black symbols represent records from literature (Hoogmoed and Avila-Pires 1991; Mott et al. 2011; and Teixeira Jr. et al. 2014); star represents the type-locality; red circle represents the new record for the municipality of Belterra, state of Pará, Brazil. Localities: 1, Ilha do Marajó (type locality); 2, Belterra; 3, Rio Tocantins (cf. Hoogmoed and Avila-Pires 1991); 4, Belém; 5, Ananindeua; 6, Santo Antônio do Tauá; 7, Peixe-boi; 8, Ourém; 9, Santa Luzia, Capitão Poço; 10, Viseu; 11, Colônia Nova; 12, Paruá; 13, Puraqueú; 14, Carajás; 15, Parauapebas; and 16, Juruá, Rio Xingu. 1229 1229 Ribeiro et al. \| Geographic distribution of Amphisbaena mitchelli in the state of Pará, Brazil Figure 3. Literature Cited Gans, C. 1963. Notes on amphisbaenids (Amphisbaenia, Reptilia). 7. Redescription and redefinition of Amphisbaena mitchelli Procter and Amphisbaena slevini Schmidt from the Middle and Lower Amazon, Brazil. American Museum Novitates 2127: 1–22 (http://hdl.handle. net/2246/3375). Gans, C. 1964. Amphisbaena mitchelli Procter recorded from Belém, Pará, Brazil. Herpetologica 20(3): 192–194 (doi: 10.2307/3891042). Gans C. and A.A. Alexander 1962. Studies on amphisbaenids (Amphisbaenia, Reptilia). 2. On the amphisbaenids of the Antilles. Bulletin of the Museum of Comparative Zoology 128(3): 65–158 (http://www.biodiversitylibrary.org/page/4284282). Figure 2. Specimen of Amphisbaena mitchelli (LPHA 4942) from municipality of Belterra, Pará state, Brazil. (A) Dorsal, (B) lateral and (C) ventral view of head. Hoogmoed, M.S. and T.C.S. Avila-Pires. 1991. A new species of small Amphisbaena (Reptilia: Amphisbaenia: Amphisbaenidae) from western Amazonian, Brazil. Boletim do Museu Paraense Emilio Goeldi, Serie Zoologia 7(1): 77–94. pores (Figure 3), 213 body annuli, three lateral annuli and seven tail annuli, 13 dorsal and 16 ventral segments in an annuli at a midbody. Mott T., D.D.S. Carvalho Neto and K.D.S. Carvalho Filho. 2011. Amphis baena miringoera Vanzolini, 1971 (Squamata: Amphisbaenidae): New state record. Check List 7(5): 594–595 (http://www.checklist. org.br/getpdf?NGD108-11). This is the first record of Amphisbaena mitchelli for the lower Tapajós River and extends the known distribution ca. 350 km west of the nearest localities (Figure 1). This new record suggests that the area of occurrence of A. mitchelli is broader than previously recorded, and that its occurrence in the lower Amazon region has been underestimated. Additionally, considering the importance of the zoogeographic information for conservation planning and systematic studies (Nogueira et al. 2011), new surveys are still needed to add to the knowledge of the geographical distribution of many poorly known South American amphisbaenian species [e.g., Amphisbaena acrobeles (Ribeiro, Castro-Mello & Nogueira, 2009); A. arda Rodrigues, 2003; A. carli Pinna, Mendonça, Bocchiglieri & Fernandes, 2010; A. cuiabana (Strüssmann & Carvalho, 2001); A. kraoh Vanzolini, 1971; A. neglecta Dunn & Piatt, 1936; A. Nogueira, C., S. Ribeiro, G.C. Costa and G.R. Colli. 2011. Vicariance and endemism in a Neotropical savanna hotspot: distribution patterns of Cerrado squamate reptiles. Journal of Biogeography 38: 1907–1922 (doi: 10.1111/j.1365-2699.2011.02538.x) Perez, R., S. Ribeiro and S. and M. Borges-Martins. 2012. Reappraisal of the taxonomic status of Amphisbaena prunicolor (Cope 1885) and Amphisbaena albocingulata Boettger 1885 (Amphisbaenia: Amphisbaenidae). Zootaxa 3550: 1–25. p ) Procter, J. 1923. On new and rare reptiles from South America. Range extension and geographic distribution of Amphisbaena mitchelli Procter, 1923 in the state of Pará, Brazil Ventral view of tail, cloaca and precloacal pores of Amphisbaena mitchelli (LPHA 4942) from municipality of Belterra, Pará state, Brazil. Ribeiro et al. \| Geographic distribution of Amphisbaena mitchelli in the state of Pará, Brazil Figure 2. Specimen of Amphisbaena mitchelli (LPHA 4942) from municipality of Belterra, Pará state, Brazil. (A) Dorsal, (B) lateral and (C) ventral view of head. Figure 3. Ventral view of tail, cloaca and precloacal pores of Amphisbaena mitchelli (LPHA 4942) from municipality of Belterra, Pará state, Brazil. talisae Vanzolini, 1995; Mesobaena huebneri Mertens, 1925; Mesobaena rhachicephala Hoogmoed, Pinto, Rocha & Pereira, 2009]. Acknowledgements: We thank E. Xavier for delivering the captured specimen. We are thankful of P.H. Pinna, P.M.S. Nunes and anonymous reviewer for comments and suggestions; and C.S. Oliveira for help with photographs. Received: February 2014 Accepted: September 2014 Published online: October 2014 Editorial responsibility: Pedro Nunes Literature Cited Proceedings of the Zoological Society of London for 1923 93(4): 1061–1067 (doi: 10.1111/j.1096-3642.1923.tb02220.x). Teixeira Jr, M., F. Dal Vechio, A.M Neto and M.T. Rodrigues. 2014. A new two-pored Amphisbaena Linnaeus, 1758, from Western Amazonia, Brazil (Amphisbaenia: Reptilia). South American Journal of Herpetology 9(1): 62–74 (doi: 10.2994/SAJH-D-14-00004.1). Received: February 2014 Accepted: September 2014 Published online: October 2014 Editorial responsibility: Pedro Nunes Received: February 2014 Accepted: September 2014 Published online: October 2014 Editorial responsibility: Pedro Nunes 1230
https://openalex.org/W2899893323	https://www.repository.cam.ac.uk/bitstream/1810/287947/5/ece3.4746.pdf	English	null	Linking warming effects on phenology, demography, and range expansion in a migratory bird population	Ecology and evolution	2,019	cc-by	9,742	Abstract Changing climatic conditions have been linked to changes in phe- nology (Parmesan & Yohe, 2003; Visser, Holleman, & Gienapp, 2006), distribution (Chen, Hill, Ohlemüller, Roy, & Thomas, 2011; This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2019 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. José A. Alves1,2 \| Tómas G. Gunnarsson2 \| William J. Sutherland3 \| Peter M. Potts4 \| Jennifer A. Gill1 1School of Biological Sciences, University of East Anglia, Norwich, UK 2South Iceland Research Centre, University of Iceland, Laugarvatn, Iceland 3Department of Zoology, University of Cambridge, Cambridge, UK 4Farlington Ringing Group, Southampton, UK Received: 5 March 2018 \| Revised: 21 June 2018 \| Accepted: 18 August 2018 Received: 5 March 2018 \| Revised: 21 June 2018 \| Accepted: 18 August 2018 DOI: 10.1002/ece3.4746 Linking warming effects on phenology, demography, and range expansion in a migratory bird population José A. Alves1,2 \| Tómas G. Gunnarsson2 \| William J. Sutherland3 \| Peter M. Potts4 \| Jennifer A. Gill1 Abstract Abstract Phenological changes in response to climate change have been recorded in many taxa, but the population‐level consequences of these changes are largely unknown. If phenological change influences demography, it may underpin the changes in range size and distribution that have been associated with climate change in many species. Over the last century, Icelandic black‐tailed godwits (Limosa limosa islandica) have increased 10‐fold in numbers, and their breeding range has expanded throughout lowland Iceland, but the environmental and demographic drivers of this expansion remain unknown. Here, we explore the potential for climate‐driven shifts in phenol- ogy to influence demography and range expansion. In warmer springs, Icelandic black‐tailed godwits lay their clutches earlier, resulting in advances in hatching dates in those years. Early hatching is beneficial as population‐wide tracking of marked in- dividuals shows that chick recruitment to the adult population is greater for early hatched individuals. Throughout the last century, this population has expanded into progressively colder breeding areas in which hatch dates are later, but temperatures have increased throughout Iceland since the 1960s. Using these established relation- ships between temperature, hatching dates and recruitment, we show that these warming trends have the potential to have fueled substantial increases in recruitment throughout Iceland, and thus to have contributed to local population growth and expansion across the breeding range. The demographic consequences of tempera- ture‐mediated phenological changes, such as the advances in lay dates and increased recruitment associated with early hatching reported here, may therefore be key pro- cesses in driving population size and range changes in response to climate change. K E Y W O R D S Correspondence José A. Alves, School of Biological Sciences, University of East Anglia, Norwich, UK. Email: j.alves@uea.ac.uk Correspondence José A. Alves, School of Biological Sciences, University of East Anglia, Norwich, UK. Email: j.alves@uea.ac.uk Funding information Fundação para a Ciência e a Tecnologia, Grant/Award Number: SFRH/ BPD/91527/2012; Arcadia; Natural Environment Research Council, Grant/ Award Number: NE/H008527/1; Icelandic Centre for Research, Grant/Award Number: 130412-051 climate change, population dynamics, species distribution, temperature, waders Ecology and Evolution. 2019;9:2365–2375. \| 2365 www.ecolevol.org 1 \| INTRODUCTION Understanding biological responses to global climatic and envi- ronmental change is among the most urgent of challenges facing ecologists and conservationists (Lebreton, 2011; Parmesan, 2006). 1 \| INTRODUCTION 1 ecologists and conservationists (Lebreton, 2011; Parmesan, 2006). Changing climatic conditions have been linked to changes in phe- nology (Parmesan & Yohe, 2003; Visser, Holleman, & Gienapp, 2006), distribution (Chen, Hill, Ohlemüller, Roy, & Thomas, 2011; Understanding biological responses to global climatic and envi- ronmental change is among the most urgent of challenges facing \| 2365 www.ecolevol.org \| www.ecolevol.org Ecology and Evolution. 2019;9:2365–2375. 2366 ALVES et al. traits and demographic rates across a population (McLean, Lawson, Leech, & Pol, 2016). Hickling, Roy, Hill, Fox, & Thomas, 2006; Hill et al., 2002), and demography (Both, Bouwhuis, Lessells, & Visser, 2006; Hansen, Aanes, Herfindal, Kohler, & Sæther, 2011; Post & Forchhammer, 2008) across many taxa (Root et al., 2003; Thackeray et al., 2010; Walther et al., 2002), but the mechanistic processes driv- ing these changes in free‐ranging animals, and their population‐ level consequences, remain poorly understood (but see Lane, Kruuk, Charmantier, Murie, & Dobson, 2012; Ozgul et al., 2010). Predicting species responses to future climatic scenarios requires a mechanistic understanding of the ecological, behavioral, and historical factors that influence species demographic and distribu- tional responses to changing environments (Mustin, Sutherland, & Gill, 2007; Norris, 2004). Quantifying the mechanisms through which changing climatic conditions can influence changes in pop- ulation size and distribution is therefore key to predicting future responses to environmental change and identifying associated conservation actions (Beale, Lennon, & Gimona, 2008; Guisan et al., 2013). Among the most rapid and severe changes in climatic conditions to have occurred over the last century are the warming tempera- tures in arctic and subarctic zones (IPCC, 2007; Robinson, 2009). Iceland lies mostly within the subarctic climate zone and has expe- rienced a general increasing trend in temperatures since records began in 1845 (Einarsson, 1984; Jónsson, 2006), with rapid tem- perature increases in the 20th century during which annual mean temperature at the longest running weather station rose ~1.2°C (Jónsson, 2006). Iceland hosts internationally important breed- ing populations of many migratory bird species (Gunnarsson, Gill, Appleton, et al., 2006a), for which changing climatic conditions could have important implications. For example, the breeding and wintering range of Icelandic black‐tailed godwits (Limosa limosa islandica) has expanded over the last century, concomitant with this warming period (Gill et al., 2001; Gunnarsson, Gill, Newton, Potts, & Sutherland, 2005a). FI G U R E 1 Map of Iceland with 200 m asl contour line below which most suitable habitat for breeding Icelandic black‐tailed godwits occurs. Circles show areas occupied by the species before the 1900s (white) or colonized during the 1950s (very light gray), 1960s (light gray), 1970s (gray), 1980s (dark gray), or 1990s (black). All these areas have an operational weather station collecting data for each lowland basin (see Supporting Information Table S3 for details) 1 \| INTRODUCTION In the early 1900s, this species was restricted to the southern lowlands of Iceland but since then it has gradually colonized coastal lowland areas throughout the country (Figure 1) with larger areas closer to occupied sites being colonized first (Gunnarsson, Gill, Petersen, Appleton, & Sutherland, 2005b). The population now numbers ~50,000 individuals, which is likely to be an approximately 10‐fold increase in numbers over the last cen- tury (Gill et al., 2007; Gunnarsson, Gill, Potts, et al., 2005c), but the environmental and demographic changes underpinning this popula- tion increase and range expansion are not known (Gill et al., 2007). Icelandic godwits are long‐lived migratory shorebirds (Alves et al., 2012; Gunnarsson, Gill, Atkinson, et al., 2006b) with a typical lifes- pan of ~15–20 years (Gill et al., 2007), that breed almost exclusively in Iceland and winter in coastal zones of north and west Europe (Gill, Hatton, & Potts, 2002). They have a modal clutch size of four eggs, nesting in lowland wetlands dominated by grasses or by dwarf birch (Betula nana) and sedges (Carex spp.) (Gunnarsson, Gill, Newton, et al., 2005a) and are among the largest nest‐concealing species of the Scolopacidae (Cramp & Simmons, 1983), thus requiring a suitable Phenological changes in response to climate change (e.g., Amano, Smithers, Sparks, & Sutherland, 2010; Gordo, 2007) will only directly affect population demography (Pettorelli, Pelletier, Hardenberg, Festa‐Bianchet, & Côté, 2007) and distribution (Van der Jeugd et al., 2009) if fitness parameters are influenced by changes in the timing of events across the annual cycle (Chuine, 2010; Visser et al., 2006). Phenological mismatches with resource availability have been linked to local population trends in several species (Both et al., 2006; Gaillard et al., 2013; Hansen et al., 2011), but the extent to which these patterns can drive large‐scale population and range expansion or contraction in free‐ranging animals remains poorly understood. In addition, recent studies have found that climate‐ mediated changes in phenology may not always lead to changes in population size (Dunn & Møller, 2014), even when fitness effects are apparent (Reed, Grøtan, Jenouvrier, Sæther, & Visser, 2013a; Reed, Jenouvrier, & Visser, 2013b). Understanding how local climate ef- fects can potentially scale up to population‐level changes requires quantification of the magnitude of climate effects on individual FI G U R E 1 Map of Iceland with 200 m asl contour line below which most suitable habitat for breeding Icelandic black‐tailed godwits occurs. 1 \| INTRODUCTION Circles show areas occupied by the species before the 1900s (white) or colonized during the 1950s (very light gray), 1960s (light gray), 1970s (gray), 1980s (dark gray), or 1990s (black). All these areas have an operational weather station collecting data for each lowland basin (see Supporting Information Table S3 for details) FI G U R E 1 Map of Iceland with 200 m asl contour line below which most suitable habitat for breeding Icelandic black‐tailed godwits occurs. Circles show areas occupied by the species before the 1900s (white) or colonized during the 1950s (very light gray), 1960s (light gray), 1970s (gray), 1980s (dark gray), or 1990s (black). All these areas have an operational weather station collecting data for each lowland basin (see Supporting Information Table S3 for details) FI G U R E 1 Map of Iceland with 200 m asl contour line below which most suitable habitat for breeding Icelandic black‐tailed godwits occurs. Circles show areas occupied by the species before the 1900s (white) or colonized during the 1950s (very light gray), 1960s (light gray), 1970s (gray), 1980s (dark gray), or 1990s (black). All these areas have an operational weather station collecting data for each lowland basin (see Supporting Information Table S3 for details) ALVES et al. 2367 FI G U R E 2 Changes in (a) mean (± SE) annual nest laying date (days from 1st of May); and (b) length of the period from hatching to fledging, of Icelandic black‐tailed godwits in relation to: (a) mean daily temperature in June between 2001 and 2013 (laying date = −5.49 * temperature + 90.7, r2 = 0.75, F(1,6) = 18.32, p = 0.005), and (b) cumulative daily temperature in 2012 (gray) and 2013 (black) during the 21 days post‐hatching (time to fledge = −0.013 * cumulative temperature + 45.91, r2 = 0.40, F(1,16) = 10.69, p = 0.005) vegetation height in order to initiate nesting. As the onset and rate of vegetation growth in subarctic ecosystems (Thorvaldsson, Björnsson, & Hermansson, 2005), as well as the timing of emergence of invertebrate prey for wader chicks (Halldórsson et al., 2013; Tulp & Schekkerman, 2008), are strongly temperature‐dependent, timing of nesting, and chick growth rate are likely to be influenced by local temperatures. 1 \| INTRODUCTION Over eight years during which temperatures in Iceland varied substantially, we quantified (a) the influence of temperature on nest cup vegetation heights, laying dates and the duration of the pre‐ fledging period of Icelandic godwits, and (b) the subsequent influ- ence of hatching dates on recruitment of chicks to the wintering or subsequent breeding population. We then use these relationships to investigate the potential contribution of warming‐derived changes in phenology and recruitment to the rapid population increase, by quantifying (c) variation in spring temperature and hatch dates across breeding areas colonized at different times during the 20th century, and (d) the magnitude of temperature changes and conse- quent estimated rates of change in godwit recruitment within these areas during the period of range expansion. 2.1.1 \| Timing of nesting and vegetation growth FI G U R E 2 Changes in (a) mean (± SE) annual nest laying FI G U R E 2 Changes in (a) mean (± SE) annual nest laying date (days from 1st of May); and (b) length of the period from hatching to fledging, of Icelandic black‐tailed godwits in relation to: (a) mean daily temperature in June between 2001 and 2013 (laying date = −5.49 * temperature + 90.7, r2 = 0.75, F(1,6) = 18.32, p = 0.005), and (b) cumulative daily temperature in 2012 (gray) and 2013 (black) during the 21 days post‐hatching (time to fledge = −0.013 * cumulative temperature + 45.91, r2 = 0.40, F(1,16) = 10.69, p = 0.005) The breeding success of godwits was intensively monitored annu- ally between 2001–2003 and 2005, and 2010–2013, with 11 study sites in South Iceland being surveyed 1–2 times per week during the breeding season (Supporting Information Table S1). For each godwit nest found, the incubation stage of the clutch (all eggs) was measured using standard egg floatation method, allowing the laying date (day when the last egg was laid) and hatch date to be estimated. Categorical classification following (Liebezeit et al., 2007) was adapted to our study species (Supporting Information Table S2): firstly the average length of incubation was determined in successfully hatched nests which were found during egg‐laying (22.7 days ±0.3 SE, n = 7); then two additional floatation levels were added to the five described in Figure 2 of Liebezeit et al. (2007) to allow greater accuracy during the later stages of incuba- tion, based on field observations (J. A. Alves, personal observa- tion). When eggs of the same clutch varied in incubation stage, the middle value between categories (in days) was assigned to the clutch. Every nest was visited regularly, and successful nests were revisited at the estimated hatching date in order to cap- ture and mark chicks and adults with individual combinations of color‐rings. Vegetation height around the rim of the nest cup was quantified when nests were first located, using a tape measure at four equidistant points from the center of the nest, and also at 15 randomly located points in the vicinity of the nests (within 100 m) throughout the breeding season (May to July) in 2011–2013. 2.3 \| Data analyses The influence of temperature on annual variation in mean godwit lay- ing dates across the 11 study areas in South Iceland was explored in regression analyses using (a) mean June temperature (the month in which the majority of godwit clutches are laid; although some clutches were laid during the last week of May, temperatures during late May and June were very strongly correlated (rs = 0.87, n = 8, p < 0.005) in these years) and (b) mean daily temperature during the laying period (from the laying date of the first nest to the laying date of the last nest located) in each year. The former analysis facilitated use of histori- cal mean June temperatures in subsequent analyses while the latter ensured that the patterns were consistent with more highly resolved temperature data which are not available for the historical time series. Mean temperature was chosen as predictor of laying dates as this pa- rameter is strongly related to the growth rates of grasses in Iceland (Thorvaldsson et al., 2005). Temperature data were recorded at the closest weather monitoring station (Eyrarbakki, 63°52′N, 21°09′W; which is within 7.5 km of all the nests found and broods tracked). 2.2.2 within 1–2 days of chick hatching (J. A. Alves, personal observation, 25 June & 3 July 2012). Models of the length of the pre‐fledging period were constructed both including and excluding these two families. Brood mortality was defined as two consecutive failures to relocate tagged families (at 2 and 4 days) after the last confirmed ob- servation (when adults still displayed aggressive defensive behavior). When families were not located at the last known position, scans for tag transmissions were taken at vantage points (minimum of 3) in a triangular shape surrounding the last known position and cover- ing a range of more than 1 km from that position (a distance that exceeds the distance over which godwit broods have been recorded moving; Kentie, Hooijmeijer, Trimbos, Groen, & Piersma, 2013; J. A. Alves, personal observation). Godwit chicks typically fledge at ~25 days (Kentie et al., 2013); however, some families fledged young prior to that age and, for these, visual confirmation of at least one of the fledged young was attained by reading its individual color‐ring combination. To explore the potential influence of temperature changes in recent decades on recruitment rates in the Icelandic godwit population, average monthly temperatures were extracted from the Icelandic Meteorological Office (available at www.vedur.is) for 14 lowland areas around Iceland in which the average year of colonization by godwits during the past century had previously been compiled (Supporting Information Table S3; Figure 1). The average year of colonization has been reconstructed by collating reports and re- cords of new breeding species within each lowland basin provided by local residents which are encouraged by and submitted to the Icelandic Institute of Natural History (see details in Gunnarsson, Gill, Petersen, et al., 2005b; Supporting Information Table S3). None of the weather stations provided temperature data prior to 1949 (Supporting Information Table S3). et al., 2006b), allowing recruitment to the wintering or subsequent breeding population (i.e., any record after the first autumn period) of the color‐marked chicks to be established. As the vast majority of godwit recruits were first recorded within their first 2 years of life (mean no. of years to first observation = 1.5 ± 2.1 SD, N = 394), comparison of chicks that recruited (observed after the first autumn migration period) or did not (never recorded since ringing) was re- stricted to chicks ringed up to and including the summer of 2011 and using sightings up to and including those in summer 2013. More recently ringed individuals (since 2012) that may not yet have been observed in the breeding or nonbreeding sites at the time when this analysis was executed were excluded. TA B LE 1 Results of binomial GLMMs of: (a) the influence of hatching date on the probability of Icelandic black‐tailed godwit chicks recruiting to the wintering population; (b) the effect of laying date on daily nest survival probabilities Estimate SE z value p (a) Intercept 2.53 0.62 4.05 <0.001 Hatch date −0.04 0.01 −4.55 <0.001 (b) Intercept −2.71 0.46 −5.9 <0.001 Lay date −0.01 0.01 −0.55 0.58 Annual variation (in a and b) is included as random factor (see methods for details). Significant effects are highlighted in bold. Annual variation (in a and b) is included as random factor (see methods for details). Significant effects are highlighted in bold. 2.1.2 \| Length of the pre‐fledging period In 2012 and 2013, 32 godwit families were tracked during chick‐ rearing, from hatching to fledging (n = 18) or brood loss (n = 14). The length of the pre‐fledging period could therefore be measured for 18 families (nine in each year). For each family, one of the adults was captured using either a nest‐trap or a hand‐held net‐gun (details in Edwards & Gilchrist, 2011) and fitted with an individual combi- nation of color‐rings and a back‐mounted radio‐tag weighing 2.4 g with a 1.5‐month lifespan (PIP3, Ag393 Biotrack Ltd). The position of each radio‐tagged individual was recorded every two days using a hand‐held Yagi antenna and receiver (Sika, Biotrack Ltd) and a GPS (eTrex Garmin). Exact hatch dates were unknown for the two broods of adults trapped with the net‐gun, but the highly aggressive behavior of the adults (adult godwits stay very close to their chicks and defend them during the pre‐fledging period, Gunnarsson, Gill, Petersen, et al., 2005b) indicated that these had both been captured 2368 ALVES et al. ALVES et al. 3.1 \| Effects of temperature on the phenology of breeding season events response variable. Cumulative temperature was considered a proxy of time available for chicks to forage, as low temperatures often re- quire parental brooding thus reducing foraging time. As cumulative temperatures will inevitably be higher for chicks that take longer to fledge, we restricted the calculation of cumulative temperatures to the first 21 days after hatching (the age at which the earliest brood fledged). Icelandic godwits lay their clutches earlier and rear their chicks faster when temperatures are warmer (Figure 2). In years with warmer mean June temperatures (~11°C, Figure 2a), laying dates are ~11 days earlier than in colder years (~9°C, Figure 2a). These patterns are also appar- ent when using mean daily temperature during the sampled laying pe- riods in each year (measured as the mean daily temperature between the start and end of laying; laying date = −3.07 * temperature + 66.90, r2 = 0.67, F(1,6) = 12.28, p = 0.013). The estimation of laying dates using categorical data for large waders has a reported absolute mean (± SE) deviation ranging from 0.2 (± 0.2) to 2.5 (± 0.5) days (Liebezeit et al., 2007); however, there is no reason to expect systematic an- nual bias in this error and the measured differences in laying dates are considerably larger than the associated error (Figure 2a). Broods that experienced warmer cumulative daily mean temperatures dur- ing the pre‐fledging period also had significantly shorter pre‐fledging periods (Figure 2b). This relationship persisted when the two broods with unknown hatching date were excluded (time to fledge = −0.011 * cumulative temperature + 43.53, r2 = 0.31, F(1,14) = 6.28, p = 0.025). To estimate the seasonal effect of laying date on nest survival, we used a formulation of Mayfield's method (Mayfield, 1961, 1975) as a logistic model and a logit link function (Crawley, 2007) in which success or failure over a given number of days (as a binary variable) was modeled with the number of days over which the nest was monitored as the binomial denominator (Aebischer, 1999; Morrison, Robinson, Leech, Dadam, & Toms, 2014), laying date as a predic- tor and year as a random effect. For predated nests, the number of exposure days was considered to be the mid‐point between the maximum and minimum possible number of exposure days, given the timing of nest visits. 3.1 \| Effects of temperature on the phenology of breeding season events In order to investigate the effect of hatch date on the probabil- ity of recruiting into the wintering population, and as the data were too over‐dispersed for a capture‐mark‐recapture analysis, we con- structed a GLMM with binomial error distribution and a logit link function (Crawley, 2007), with recruited (observed after the first au- tumn migration) or not (not recorded since ringing) as the response variable, hatch date as a fixed factor and year of capture as random factor. Variation in resighting probability of marked juveniles is not included in these analyses but systematic variation in resighting probability in relation to hatch date is unlikely. 2.2.1 \| Determining recruitment rates In addition to the chicks marked at the 11 study sites (see above), Icelandic godwit chicks have been caught, measured, and individu- ally color‐ringed at locations all around Iceland since 1999 (n = 966; yearly average = 69 ± 56.2 SD). The majority of these chicks were captured, marked, and measured (mass to nearest 0.5 g using a spring balance or a digital scale) after leaving the nest and so their age was unknown. As very few chicks are repeatedly recaptured during the pre‐fledging period, we used a previously published growth curve established for this species (Beintema & Visser, 1989) to estimate the age of each chick at capture (and thus hatch date, used in the model below) from their body mass at capture. Although uncertainty in age estimation increases with body mass (Kentie et al., 2013), the vast majority (>75%) of the chicks in this study weighed 150 g or less at ringing. A network of hundreds of volunteer observers regularly report observations of color‐ringed Icelandic godwits from across the migratory range (Alves et al., 2013; Gunnarsson, Gill, Atkinson, To explore the effect of temperature on the length of the chick‐ rearing period, we calculated the daily cumulative temperature sum since hatching for each brood and used this as a predictor in a regres- sion model with the length of the pre‐fledging period (days) as the ALVES et al. 2369 FI G U R E 3 Variation in the probability (solid line ± SE, dashed lines) of Icelandic godwit chicks recruiting to the wintering or subsequent breeding population in relation to their hatch date which temperature data were not available were not included in this analysis. We then used the relationships established between (a) local temperature and hatch dates, and (b) hatch dates and recruitment probability, to estimate the magnitude of change in recruitment probabilities for the breeding areas during the decades following colonization, given average June temperatures in each area in each decade (see Supporting Information for details). These estimates conservatively assume that the relationships between temperature and timing of breeding season events are consistent at the lower temperatures that occur elsewhere in lowland Iceland. All analyses were performed in R 2.15.0 (R Development Core Team, 2011). 2.2.1 \| Determining recruitment rates FI G U R E 3 Variation in the probability (solid line ± SE, dashed lines) of Icelandic godwit chicks recruiting to the wintering or subsequent breeding population in relation to their hatch date \| TA B LE 2 Results of a GLM of variation in mean June temperatures over five decades (1950s–1990s) in 14 breeding areas around Iceland colonized in different years 3.3 \| The influence of temperature, phenology and demography on range expansion df F p Estimate (± SE) Colonization year 1 81.66 <0.001 −0.06 (±0.01) Decade 4 3.88 0.008 Col. year*Decade 53 0.68 0.609 Error 62 Significant effects are highlighted in bold. The sequence of colonization of lowland areas around Iceland by breeding godwits over the last century follows a tempera- ture gradient, with warmer areas being colonized first (Table 2; Figure 4a), and godwit chicks hatch earlier in regions that were colonized first (F4,686 = 12.8, p < 0.001; Figure 4b; Supporting Information Table S3). Following relatively warm average June temperatures in the nine areas around Iceland with available temperature data in the 1950s (and when few sites had been colonized; Figure 1), subsequent temperatures were lower in the 1960s but have generally increased since then (Table 2; Figure 4c). Significant effects are highlighted in bold. Significant effects are highlighted in bold. SE) of chicks hatched early (before day 57), indicating the substantial effect that early hatching can have on the overall numbers of chicks recruiting from warmer than colder years. SE) of chicks hatched early (before day 57), indicating the substantial effect that early hatching can have on the overall numbers of chicks recruiting from warmer than colder years. 3.2 \| Effect of breeding season temperatures and phenology on demography For Icelandic godwit chicks, the probability of successfully recruit- ing to the wintering population is strongly influenced by hatch date (Table 1a). Chicks hatched during the first two weeks of June (before day 47 on Figure 3) have, on average, >60% probability of recruiting, whereas those hatching after 12 July (day 73 on Figure 3) have, on average, <35% probability of recruiting. The benefits of early lay- ing in warmer years could be reduced if hatching success was lower early in the season, but we found no seasonal effect of laying date on daily nest survival (Table 1b). In colder years (2001, 2011), when average June temperatures were below 9.5°C, the mean percent of chicks hatched before day 57 (when recruitment probability exceeds 50%, Figure 3) was 45% (± 1 SE). By contrast, in warmer years (aver- age June temperature≥11°C; 2010, 2012), an average of 64% (± 5 Over the last century, godwits have expanded into progressively colder regions of Iceland. Regional variation in chick hatch dates were explored with an ANOVA test, and rates of spring tempera- ture change during the population expansion in colonized areas were explored in a GLM, with mean decadal June temperature modeled as function of decade (1950s to 1990s), mean year of colonization (from Gunnarsson, Gill, Newton, et al., 2005a), and their interaction. Areas colonized by Icelandic godwits during the past century but for 2370 ALVES et al. 3.3 \| The influence of temperature, phenology and demography on range expansion FI G U R E 4 Variation in mean (± SE): (a) June temperature in 14 lowland areas in Iceland colonized by godwits during the second half of the 20th century (lowland areas colonized in different decades colored as in Figure 1; see Supporting Information Table S3 for details of temperature ranges); (b) hatch dates (ordinal days since 1st of May) of godwit chicks between 1999 and 2011 across regions of Iceland for which temperature and colonization dates are available (number of chicks per region given within bars and bar color reflects the predominant decade of colonization within each region); (c) June temperatures in five decades during the range expansion period (see Table 2 for statistics); and (d) the potential consequences of spatial and annual variation in June temperatures and chick hatch dates for estimated annual recruitment probability in areas colonized in different decades (number of areas with available temperature data shown in parentheses, details in Supporting Information Table S3) during the 1960s (light gray bars), 1970s (gray bars), 1980s (dark gray bars), and 1990s (black bars) across Iceland FI G U R E 4 Variation in mean (± SE): (a) June temperature in 14 lowland areas in Iceland colonized by godwits during the second half of the 20th century (lowland areas colonized in different decades colored as in Figure 1; see Supporting Information Table S3 for details of temperature ranges); (b) hatch dates (ordinal days since 1st of May) of godwit chicks between 1999 and 2011 across regions of Iceland for which temperature and colonization dates are available (number of chicks per region given within bars and bar color reflects the predominant decade of colonization within each region); (c) June temperatures in five decades during the range expansion period (see Table 2 for statistics); and (d) the potential consequences of spatial and annual variation in June temperatures and chick hatch dates for estimated annual recruitment probability in areas colonized in different decades (number of areas with available temperature data shown in parentheses, details in Supporting Information Table S3) during the 1960s (light gray bars), 1970s (gray bars), 1980s (dark gray bars), and 1990s (black bars) across Iceland ALVES et al. ALVES et al. 2371 \| 2 The changes in June temperatures around Iceland since the 1950s (Figure 4a,c) can be used to assess the potential magnitude of temperature‐driven variation in hatch dates and recruitment of Icelandic godwits. 3.3 \| The influence of temperature, phenology and demography on range expansion In every decade, the areas of Iceland that were colonized during the 1950s were warmest and estimated recruit- ment is therefore consistently highest in these areas (Figure 4d). In the 1990s, when all areas had been colonized, recruitment probabil- ity was estimated to be highest in the earlier occupied (warmer) areas and lowest in the most recently colonized (colder) areas (Figure 4d— black bars). Rapid warming between the 1980s and 1990s (Figure 4c) could potentially therefore have driven a widespread increase in re- cruitment rates of up to ~3.0% (Figure 4d) across most of lowland Iceland during this later decade (with the exception of areas colo- nized during the 1960s, for which temperature data in the 1990s are only available for one site). Warming temperatures facilitating earlier nesting and associated increases in chick recruitment therefore have the potential to have contributed quite substantially to the post‐col- onization population growth around Iceland. 4.1 \| Effect of temperature on timing of breeding season events The timing of nest laying and the length of the pre‐fledging pe- riod varied in relation to temperature, with warmer conditions being associated with earlier and shorter breeding periods. In this system, mean laying dates varied by ~11 days between the warm- est (11.2°C, estimated laying = 29.2 ± 2.7 days) and coldest years (9.2ºC, estimated laying = 40.2 ± 2.7 days), and this was the domi- nant driver of annual variation in mean hatching dates. In contrast, the lengths of pre‐fledging period varied by ~3.6 days between the warmest and coldest years. The annual variation in nest laying dates could be also influenced by carry‐over effects of adult pre‐ breeding condition, nesting site availability, or suitability of con- ditions for chick‐rearing. Icelandic godwits are known to migrate with sufficient reserves (Alves et al., 2012) and typically arrive in Iceland 2–3 weeks before nest initiation (Gill et al., 2014). This sub- stantial amount of time to improve body condition prior to nesting may reduce the effects of any carry‐over from winter conditions (Harrison, Blount, Inger, Norris, & Bearhop, 2011). However, faster vegetation growth rates in warmer springs may facilitate earlier nesting because Icelandic godwits are among the largest‐bodied nest‐concealing species of their family (Cramp & Simmons, 1983) and, while average vegetation height in the vicinity of godwit nests on our study areas was ~9–10 cm by mid May, godwit nests have on average a vegetation height of 23.3 cm (±1.2 SE, n = 45). The spring onset and rate of growth of the grasses and sedges in these wet- lands are positively influenced by warm temperatures, particularly by the length of warm periods (Thorvaldsson et al., 2005). Thus, in years with warmer average temperatures, vegetation will reach suitable heights faster, removing potential limitations on nesting site availability (i.e., patches with tall enough vegetation to con- ceal nests), which may allow godwits to nest earlier than in colder years. In addition to suitable vegetation heights for nesting, food resources for chicks are also likely to be more abundant earlier (and generally) in warmer years (Tulp & Schekkerman, 2008), and this association may also contribute to the success of early breeding attempts. 4 \| DISCUSSION Phenological changes in response to changing climatic conditions have been widely reported across many taxa in recent years, but the evidence for population‐level consequences of these shifts for free‐ranging species varies considerably (e.g., Both et al., 2006; Wright et al., 2009). Although climate‐driven changes in phenol- ogy may have little effect on population‐level processes in some cases (Dunn & Møller, 2014; Reed, Grøtan, et al., 2013a), pheno- logical changes that influence demography have the potential to alter population and range size, particularly if these changes facili- tate local population growth and range expansion into previously unoccupied areas in which density‐dependent constraints may be weak (McLean et al., 2016). Icelandic black‐tailed godwits have colonized and become established in breeding locations around lowland Iceland over the last century, concomitant with decades of sustained warming. In south Iceland, earlier nesting occurs in years with warmer temperatures, resulting in advances in hatching dates. As early hatching confers substantial increases in chick recruitment probability, the widespread temperature increases around Iceland may have contributed substantially to the sustained population increase and range expansion of Icelandic godwits during the last century, if the temperature effects on breeding phenology occur throughout the range. As godwits conceal their nests from preda- tors in tall vegetation, more rapid vegetation growth in recent decades in response to warming could have facilitated earlier (and perhaps more successful) nesting across the breeding range. Given the consistently high adult survival rates and philopatry of godwits (Alves et al., 2013; Gill et al., 2001; Gunnarsson, Sutherland, Alves, Potts, & Gill, 2012), increases in recruitment rates as a consequence of earlier nesting are likely to have contributed substantially to the post‐colonization establishment and growth of local populations around Iceland. Phenological changes in response to changing climatic conditions have been widely reported across many taxa in recent years, but the evidence for population‐level consequences of these shifts for free‐ranging species varies considerably (e.g., Both et al., 2006; Wright et al., 2009). Although climate‐driven changes in phenol- ogy may have little effect on population‐level processes in some cases (Dunn & Møller, 2014; Reed, Grøtan, et al., 2013a), pheno- logical changes that influence demography have the potential to alter population and range size, particularly if these changes facili- tate local population growth and range expansion into previously unoccupied areas in which density‐dependent constraints may be weak (McLean et al., 2016). 4.2 \| Cascading effects of temperature‐mediated phenological changes on demography and distribution The ~11‐day delay in hatch dates re- sulting solely from laying dates in warm and cold years equates to a difference in recruitment probability of ~10% (Figure 3), with the additional benefits of earlier fledging in warmer years likely contrib- uting to the higher probability of recruitment. The decline in recruit- ment probability with hatch date means that in cold years, when most nests are laid late, very few chicks are likely to recruit to the adult population. For example, in 2011, the coldest year recorded during our study, only ~10% of 101 ringed chicks recruited into the wintering population. Benefits of early hatching for recruitment are likely to be manifest through advantages associated with suc- cessful and early fledging, such as increased time to improve body condition prior to migration, increased probability of traveling in adult‐dominated migratory flocks, and earlier departure for winter grounds allowing more time in which to select a favorable winter- ing location (Alves et al., 2013; Gunnarsson, 2006). Given that adult survival is relatively high (0.91 ± 0.02 SE) and with little annual vari- ability (Alves et al., 2013), recruitment into the adult population is likely to be a major driver of population growth. Positive associa- tions between temperature and productivity have been reported in migratory common sandpiper Actitis hypoleucos but these appear to have been offset by declines in adult survival rates (Pearce‐Higgins, Yalden, Dougall, & Beale, 2009). Iceland's position in the North Atlantic, between the Atlantic and Arctic oceans, means that the south and west of the country typi- cally experience warmer temperatures than the north and east. Over the last century, the godwit breeding range has expanded from the warmer south and west regions to colonize new sites in the north and east (Gunnarsson, Gill, Petersen, et al., 2005b). Estimated re- cruitment probability is consistently higher in traditionally occupied areas (Figure 4d), primarily as a consequence of the earlier laying and hatching that is possible in these warmer areas (Figure 4b), and potential additional benefits of early fledging. The estimations of recruitment probability assume that the relationships between temperature and timing of breeding season events persist at lower temperatures than those recorded on our study sites. 4.2 \| Cascading effects of temperature‐mediated phenological changes on demography and distribution 4.2 \| Cascading effects of temperature‐mediated phenological changes on demography and distribution Increased recruitment in new areas could also be influenced by increases in food resources or habitat availability, or by reduced lev- els of nest predation. During the early 19th century, both avian and terrestrial nest predator species increased in Iceland, with gull (Larus fuscus and L. argentatus) and owl (Asio flameus) species colonizing the country in ~1920 to 1930 and increasing in numbers until ~1990 to 2010 (Skarphéðinsson, Katrínardóttir, Guðmundsson, & Auhage, 2016), and mink (Mustela vision) spreading around the country after escaping captivity in 1937 (Bonesi & Palazon, 2007). Population trends of native nest predators as raven (Corvus corax) and arctic skua (Stercorarius parasiticus) are unknown, but arctic foxes (Vulpes lagopus) which declined in the 1960s, have increased since to numbers above those recorded in the late 1950s (Unnsteinsdottir, Hersteinsson, Pálsson, & Angerbjörn, 2016). For species breeding at the northern and colder areas of the distribution range, warming temperatures likely relax environmental constraints (e.g., vegetation growth, food abundance) and can positively influence productivity and population growth. The range expansion in this system could have been driven by increased productivity and continuous dispersal from traditionally colonized areas and/or by increased productivity within newly colonized areas. Given the high levels of natal philo- patry in waders, and specifically in this population (Gunnarsson et al., 2012), it seems likely that improved breeding conditions follow- ing colonization of these areas have contributed substantially to the population increase, particularly given the consistently high adult survival rates in this population in recent decades (Alves et al., 2013). Although some recent studies report that climate‐driven changes in phenology might not always lead to population‐level consequences (Dunn & Møller, 2014; Reed, Jenouvrier, et al., 2013b), in Icelandic godwits the links between temperature effects on breeding phenol- ogy and subsequent juvenile recruitment may be apparent because they have occurred during a phase of population expansion and col- onization of previously unoccupied breeding areas, in which density‐ dependent constraints on growth are likely to have been relatively weak (McLean et al., 2016). Overall, the additive effects of an ~11‐day difference in timing of laying, and a ~3.6‐day difference in pre‐fledging period can result in an average difference of up to ~14.6 days at fledging between warm (~11°C) and cold (~9°C) years. 4 \| DISCUSSION Icelandic black‐tailed godwits have colonized and become established in breeding locations around lowland Iceland over the last century, concomitant with decades of sustained warming. In south Iceland, earlier nesting occurs in years with warmer temperatures, resulting in advances in hatching dates. The pre‐fledging period of godwits was slightly longer in colder temperatures. Variation in the length of the pre‐fledging period is a function of chick growth rate, which is also influenced by tempera- ture, with the sum of daily temperature during the pre‐fledging pe- riod likely to provide a proxy for chick foraging time. At low ambient temperatures, wader chicks need to be brooded by their parents for longer (Schekkerman & Boele, 2009), which can limit foraging opportunities and result in depressed growth rates and, in extreme cases, death by starvation (Beintema & Visser, 1989). Greater abun- dance of invertebrate prey resources for chicks in warmer conditions (Halldórsson et al., 2013) is also likely to translate into faster chick growth rates (Eglington et al., 2010). In addition, the trade‐off in al- locating resources between somatic growth and body temperature (Price & Dzialowski, 2018) is likely more biased toward the latter in colder years. 2372 ALVES et al. suggesting a contribution of warming‐driven changes in recruitment to the population expansion. ACKNOWLEDGMENTS States of America, 105, 14908–14912. https://doi.org/10.1073/ pnas.0803506105 States of America, 105, 14908–14912. https://doi.org/10.1073/ pnas.0803506105 We thank Lilja Jóhannesdóttir, Borgný Katrínardóttir, Verónica Méndez, Sara Pardal, Juan Carlos Illera, Graham Appleton and Ruth Croger for help in the field, Catriona Morrison and Maria Dias for help with analyses, members of Farlington Ringing Group for chick ring- ing in Iceland and volunteer observers across the range for reporting ringed godwits. Funding was provided by NERC (NE/H008527/1), Arcadia, The Icelandic Research Fund (130412‐051) and FCT (SFRH/ BPD/91527/2012). The Icelandic Meteorological Office provided the temperature data. Beintema, A. J., & Visser, G. H. (1989). The effect of weather on the time budgets and development of chicks of meadow birds. Ardea, 77, 181–192. Bonesi, L., & Palazon, S. (2007). The American mink in Europe: Status, impacts, and control. Biological Conservation, 134, 470–483. https:// doi.org/10.1016/j.biocon.2006.09.006 doi.org/10.1016/j.biocon.2006.09.006 Both, C., Bouwhuis, S., Lessells, C. M., & Visser, M. E. (2006). Climate change and population declines in a long‐distance migratory bird. Nature, 441, 81–83. https://doi.org/10.1038/nature04539 Chen, I.‐C., Hill, J., Ohlemüller, R., Roy, D., & Thomas, C. (2011). Rapid range shifts of species associated with high levels of climate warming. Science, 333, 1024–1026. https://doi.org/10.1126/science.1206432 Chuine, I. (2010). Why does phenology drive species distribution? Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences, 365, 3149–3160. https://doi.org/10.1098/ rstb.2010.0142 CONFLICT OF INTEREST None declared. Cramp, S., & Simmons, K. E. L. (1983). Birds of the Western Palearctic. Oxford, UK: Oxford University Press. AUTHORS' CONTRIBUTIONS Crawley, M. J. (2007). The R book. Chichester, UK: John Wiley & Sons Ltd. Dunn, P. O., & Møller, A. P. (2014). Changes in breeding phenology and population size of birds (ed S Griffith). Journal of Animal Ecology, 83, 729–739. https://doi.org/10.1111/1365-2656.12162 JAA, JAG, TGG and WJS formulated the idea and designed the re- search program. JAA, TGG, JAG and PMP collected and compiled the data. JAA developed the analysis with support from JAG. JAA, JAG and TGG wrote the article with contributions from WJS. Edwards, D. B., & Gilchrist, H. G. (2011). A new means to catching shore- birds: The Super Talon Net Gun. Wader Study Group Bulletin, 118, 134–136. Eglington, S. M., Bolton, M., Smart, M. A., Sutherland, W. J., Watkinson, A. R., & Gill, J. A. (2010). Managing water levels on wet grasslands to improve foraging conditions for breeding northern lapwing Vanellus vanellus. Journal of Applied Ecology, 47, 451–458. ORCID José A. Alves https://orcid.org/0000-0001-7182-0936 Tómas G. Gunnarsson https://orcid.org/0000-0001-7692-0637 William J. Sutherland https://orcid.org/0000-0002-6498-0437 Jennifer A. Gill https://orcid.org/0000-0003-0167-6857 José A. Alves https://orcid.org/0000-0001-7182-0936 Tómas G. Gunnarsson https://orcid.org/0000-0001-7692-0637 William J. Sutherland https://orcid.org/0000-0002-6498-0437 Jennifer A. Gill https://orcid.org/0000-0003-0167-6857 Gill, J. A., Alves, J. A., Sutherland, W. J., Appleton, G. F., Potts, P. M., & Gunnarsson, T. G. (2014). Why is timing of bird migration advancing when individuals are not? Proceedings. Biological sciences/The Royal Society, 281, 20132161. https://doi.org/10.1098/rspb.2013.2161 Gill, J. A., Hatton, L., & Potts, P. M. (2002). Black‐tailed Godwit. In C. V. Wernham, M. P. Toms, J. H. Marchant, J. A. Clark, G. M. Siriwardena, & S. R. Baillie (Eds.), The Migration Atlas: Movements of the birds of Britain and Ireland (pp. 323–325). London, UK: T & A D Poyser. DATA ACCESSIBILITY All data used in this manuscript are presented in the figures and sup- porting information. Einarsson, M. Á. (1984). Climate of Iceland. In H. van Loon (Ed.), World sur- vey of climatology: Climates of the oceans (pp. 673–697). Amsterdam: Elsevier. Gaillard, J.‐M., Hewison, A. J. M., Klein, F., Plard, F., Douhard, M., Davison, R., & Bonenfant, C. (2013). How does climate change in- fluence demographic processes of widespread species? Lessons from the comparative analysis of contrasted populations of roe deer. EcologyLetters, 16(Suppl. 1), 48–57. https://doi.org/10.1111/ ele.12059 4.2 \| Cascading effects of temperature‐mediated phenological changes on demography and distribution These as- sumptions are likely to be conservative, as colder conditions may well delay nesting even more than a linear extrapolation of Figure 2a, and it is extremely unlikely that laying dates would advance at such temperatures. Population size estimates for Icelandic godwits on the wintering grounds are available for the United Kingdom since the late 1970s, through the British Trust for Ornithology Wetland Bird Survey. The lack of change in estimated recruitment rates between the 1970s and 1980s, followed by widespread increases from the 1980s to the 1990s, matches counts on the UK wintering grounds, where a period of stability in the 1980s was followed by a sustained increase in numbers during the 1990s (Holt et al., 2015), again Identifying the mechanisms through which environmental changes influence population size and distribution is key to predict- ing the future status of species and designing appropriate conserva- tion strategies (McLean et al., 2016). The phenological changes that are being widely recorded across taxa at present have the potential to influence fitness and may therefore be a key component of many of the demographic and range shifts being reported. Projections of future distributions of species rely largely on associations between their current distribution and the climatic conditions that they expe- rience, but these associations are often weak (Beale et al., 2008), and lack the mechanistic basis required for robust predictions (Norris, 2004). Understanding relationships between phenology and fitness, the environmental processes that underpin these relationships, and the conditions in which these changes can influence population dy- namics and distribution are likely to greatly improve projections of future distributions of species. ALVES et al. 2373 Managing threatened species: The ecological toolbox, evolutionary theory and declining‐population par- adigm. Journal of Applied Ecology, 41, 413–426. https://doi. org/10.1111/j.0021-8901.2004.00910.x Gunnarsson, T. G., Gill, J. A., Potts, P. M., Atkinson, P. W., Croger, R. E., Gélinaud, G., … Sutherland, W. J. (2005c). Estimating population size in Black‐tailed Godwits Limosa limosa islandica by colour‐marking. Bird Study, 52, 153–158. Ozgul, A., Childs, D. Z., Oli, M. K., Armitage, K. B., Blumstein, D. T., Olson, L. E., … Coulson, T. (2010). Coupled dynamics of body mass and pop- ulation growth in response to environmental change. Nature, 466, 482–485. https://doi.org/10.1038/nature09210 Halldórsson, G., Sigurdsson, B. D., Hrafnkelsdóttir, B., Oddsdóttir, E. S., Eggertsson, Ó., & Ólafsson, E. (2013). New arthropod herbivores on trees and shrubs in Iceland and changes in pest dynamics: A review. Icelandic Agricultural Sciences, 26, 69–84. Parmesan, C. (2006). Ecological and evolutionary responses to re- cent climate change. Annual Review of Ecology, Evolution, and Systematics, 37, 637–669. https://doi.org/10.1146/annurev. ecolsys.37.091305.110100 Hansen, B. B., Aanes, R., Herfindal, I., Kohler, J., & Sæther, B.‐E. (2011). Climate, icing, and wild arctic reindeer: Past relation- ships and future prospects. Ecology, 92, 1917–1923. https://doi. org/10.1890/11-0095.1 Parmesan, C., & Yohe, G. (2003). A globally coherent fingerprint of cli- mate change impacts across natural systems. Nature, 421, 37–42. https://doi.org/10.1038/nature01286 Harrison, X. A., Blount, J. D., Inger, R., Norris, D. R., & Bearhop, S. (2011). Carry‐over effects as drivers of fitness differences in animals. Journal of Animal Ecology, 80, 4–18. https://doi. org/10.1111/j.1365-2656.2010.01740.x Pearce‐Higgins, J. W., Yalden, D. W., Dougall, T. W., & Beale, C. M. (2009). Does climate change explain the decline of a trans‐Saharan Afro‐Palaearctic migrant? Oecologia, 159, 649–659. https://doi. org/10.1007/s00442-008-1242-4 Hickling, R., Roy, D. B., Hill, J. K., Fox, R., & Thomas, C. D. (2006). The distributions of a wide range of taxonomic groups are expand- ing polewards. Global Change Biology, 12, 450–455. https://doi. org/10.1111/j.1365-2486.2006.01116.x Pettorelli, N., Pelletier, F., von Hardenberg, A., Festa‐Bianchet, M., & Côté, S. D. (2007). Early onset of vegetation growth vs. rapid green‐ up: Impacts on juvenile mountain ungulates. Ecology, 88, 381–390. https://doi.org/10.1890/06-0875 Hill, J. K., Thomas, C. D., Fox, R., Telfer, M. G., Willis, S. G., Asher, J., & Huntley, B. (2002). Responses of butterflies to twentieth cen- tury climate warming: Implications for future ranges. Proceedings of the Royal Society B: Biological Sciences, 269, 2163–2171. https://doi. org/10.1098/rspb.2002.2134 Post, E., & Forchhammer, M. C. (2008). Gunnarsson, T. G. (2006). Monitoring wader productivity during autumn passage in Iceland. Wader Study Group Bulletin, 110, 21–29. Liebezeit, J. R., Smith, P. A., Lanctot, R. B., Schekkerman, H., Tulp, I., Kendall, S. J., … Zack, S. W. (2007). Assessing the development of shorebird eggs using the flotation method: Species‐specific and gen- eralized regression models. Condor, 109, 32–47. https://doi.org/10.1 650/0010-5422(2007)109[32:ATDOSE]2.0.CO;2 Gunnarsson, T. G., Gill, J. A., Appleton, G. F., Gíslason, H., Gardarsson, A., Watkinson, A. R., & Sutherland, W. J. (2006a). Large‐scale habitat as- sociations of birds in lowland Iceland: Implications for conservation. Biological Conservation, 128, 265–275. 650/0010-5422(2007)109[32:ATDOSE]2.0.CO;2 Mayfield, H. F. (1961). Nesting success calculated from exposure. The Wilson Bulletin, 73, 255–261. Gunnarsson, T. G., Gill, J. A., Newton, J., Potts, P. M., & Sutherland, W. J. (2005a). Seasonal matching of habitat quality and fitness in a mi- gratory bird. Proceedings of the Royal Society of London B: Biological Sciences, 272, 2319–2323. Mayfield, H. F. (1975). Suggestions for calculating nest success. The Wilson Bulletin, 87, 456–466. McLean, N., Lawson, C. R., Leech, D. I., & van de Pol, M. (2016). Predicting when climate‐driven phenotypic change affects population dynam- ics. Ecology Letters, 19, 595–608. https://doi.org/10.1111/ele.12599 McLean, N., Lawson, C. R., Leech, D. I., & van de Pol, M. (2016). Predicting when climate‐driven phenotypic change affects population dynam- Gunnarsson, T. G., Gill, J. A., Petersen, A., Appleton, G. F., & Sutherland, W. J. (2005b). A double buffer effect in a migratory shorebird popu- lation. Journal of Animal Ecology, 74, 965–971. ics. Ecology Letters, 19, 595–608. https://doi.org/10.1111/ele.12599 Morrison, C. A., Robinson, R. A., Leech, D. I., Dadam, D., & Toms, M. P. (2014). Using citizen science to investigate the role of productivity in House Sparrow Passer domesticus population trends. Bird Study, 61, 91–100. Gunnarsson, T. G., Sutherland, W. J., Alves, J. A., Potts, P. M., & Gill, J. A. (2012). Rapid changes in phenotype distribution during range expansion in a migratory bird. Proceedings. Biological sci- ences/The Royal Society, 279, 411–416. https://doi.org/10.1098/ rspb.2011.0939 Mustin, K., Sutherland, W. J., & Gill, J. A. (2007). The complexity of pre- dicting climate‐induced ecological impacts. Climate Research, 35, 165–175. https://doi.org/10.3354/cr00723 Gunnarsson, T. G., Gill, J. A., Atkinson, P. W., Gélinaud, G., Potts, P. M., Croger, R. E., … Sutherland, W. J. (2006b). Population‐scale drivers of individual arrival times in migratory birds. The Journal of Animal Ecology, 75, 1119–1127. Norris, K. (2004). REFERENCES Aebischer, N. J. (1999). Multi‐way comparisons and generalized linear models of nest success: Extensions of the Mayfield method. Bird Study, 46, S22–S31. https://doi.org/10.1080/00063659909477228 Gill, J. A., Norris, K., Potts, P. M., Gunnarsson, T. G., Atkinson, P. W., & Sutherland, W. J. (2001). The buffer effect and large‐scale popula- tion regulation in migratory birds. Nature, 412, 436–438. https://doi. org/10.1038/35086568 Alves, J. A., Gunnarsson, T. G., Hayhow, D. B., Appleton, G. F., Potts, P. M., Sutherland, W., & Gill, J. A. (2013). Costs, benefits and fitness consequences of different migratory strategies. Ecology, 94, 11–17. https://doi.org/10.1890/12-0737.1 Gill, J. A., Langston, R. H. W., Alves, J. A., Atkinson, P. W., Bocher, P., Vieira, N. C., … Piersma, T. (2007). Contrasting trends in two Black‐ tailed Godwit populations: A review of causes and recommenda- tions. Wader Study Group Bulletin, 114, 43–50. Alves, J. A., Gunnarsson, T. G., Potts, P. M., Gélinaud, G., Sutherland, W. J., & Gill, J. A. (2012). Overtaking on migration: Does longer distance migration always incur a penalty? Oikos, 121, 464–470. https://doi. org/10.1111/j.1600-0706.2011.19678.x Gordo, O. (2007). Why are bird migration dates shifting? A review of weather and climate effects on avian migratory phenology. Climate Research, 35, 37–58. https://doi.org/10.3354/cr00713 Amano, T., Smithers, R. J., Sparks, T. H., & Sutherland, W. J. (2010). A 250‐year index of first flowering dates and its response to tempera- ture changes. Proceedings of the Royal Society B Biological Sciences, 277, 2451–2457. https://doi.org/10.1098/rspb.2010.0291 Guisan, A., Tingley, R., Baumgartner, J. B., Naujokaitis‐Lewis, I., Sutcliffe, P. R., Tulloch, A. I. T., … Buckley, Y. M. (2013). Predicting species dis- tributions for conservation decisions. Ecology Letters, 1424–1435. https://doi.org/10.1111/ele.12189 Beale, C. M., Lennon, J. J., & Gimona, A. (2008). Opening the climate en- velope reveals no macroscale associations with climate in European birds. Proceedings of the National Academy of Sciences of the United 2374 ALVES et al. Visser, M. E., Holleman, L. J. M., & Gienapp, P. (2006). Shifts in caterpil- lar biomass phenology due to climate change and its impact on the breeding biology of an insectivorous bird. Oecologia, 147, 164–172. https://doi.org/10.1007/s00442-005-0299-6 Schekkerman, H., & Boele, A. (2009). Foraging in precocial chicks of the black‐tailed godwit Limosa limosa: Vulnerability to weather and prey size. Journal of Avian Biology, 40, 369–379. Skarphéðinsson, K. H., Katrínardóttir, B., Guðmundsson, G. A., & Auhage, S. N. V. (2016). Mikilvæg fuglasvæði á Íslandi.Fjölrit Náttúrufræðistofnunar. Nr. 55 Walther, G.‐R., Post, E., Convey, P., Menzel, A., Parmesan, C., Beebee, T. J. C., … Bairlein, F. (2002). Ecological responses to recent climate change. Nature, 416, 389–395. https://doi.org/10.1038/416389a Thackeray, S. J., Sparks, T. H., Frederiksen, M., Burthe, S., Bacon, P. J., Bell, J. R., … Wanless, S. (2010). Trophic level asynchrony in rates of phenological change for marine, freshwater and terrestrial en- vironments. Global Change Biology, 16, 3304–3313. https://doi. org/10.1111/j.1365-2486.2010.02165.x Wright, L. J., Hoblyn, R. A., Green, R. E., Bowden, C. G. R., Mallord, J. W., Sutherland, W. J., & Dolman, P. M. (2009). Importance of climatic and environmental change in the demography of a multi‐brooded passerine, the woodlark Lullula arborea. Journal of Animal Ecology, 78, 1191–1202. Thorvaldsson, G., Björnsson, H., & Hermansson, J. (2005). The influence of weather on early growth rate of grasses. Icelandic Agricultural Studies, 4, 65–73. Climate change reduces repro- ductive success of an Arctic herbivore through trophic mismatch. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences, 363, 2369–2375. Price, E. R., & Dzialowski, E. M. (2018). Development of endothermy in birds: Patterns and mechanisms. Journal of Comparative Physiology B, 188, 373–391. https://doi.org/10.1007/s00360-017-1135-0 Holt, C., Austin, G., Calbrade, N., Mellan, H., Hearn, R., Stroud, D., … Musgrove, A. (2015). Waterbirds in the UK 2013/14: The Wetland Bird. Thetford: Survey. R Development Core Team. (2011). R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing. Retrieved from https://www.R-project.org/ IPCC. (2007). Intergovernmental Panel on Climate Change: Fourth. Assessment Report. Jónsson, T. (2006). Hitafar á Íslandi eftir 1800 (Temperatures in Iceland from 1800). www.vedur.is/loftslag/loftslag/fra1800/hitafar/. Reed, T. E., Grøtan, V., Jenouvrier, S., Sæther, B.‐E., & Visser, M. E. (2013a). Population Growth in a Wild Bird Is Buffered Against Phenological Mismatch. Science, 340, 488–491. Kentie, R., Hooijmeijer, J. C. E. W., Trimbos, K. B., Groen, N. M., & Piersma, Kentie, R., Hooijmeijer, J. C. E. W., Trimbos, K. B., Groen, N. M., & Piersma, T. (2013). Intensified agricultural use of grasslands reduces growth and survival of precocial shorebird chicks (ed J Wilson). Journal of Applied Ecology, 50, 243–251. https://doi.org/10.1111/1365-2664.12028 Reed, T. E., Jenouvrier, S., & Visser, M. E. (2013b). Phenological mismatch strongly affects individual fitness but not population demography in a woodland passerine. Journal of Animal Ecology, 82, 131–144. woodland passerine. Journal of Animal Ecology, 82, 131–144 Lane, J. E., Kruuk, L. E. B., Charmantier, A., Murie, J. O., & Dobson, F. S. (2012). Delayed phenology and reduced fitness associated with cli- mate change in a wild hibernator. Nature, 489, 554–557. https://doi. org/10.1038/nature11335 Robinson, S. A. (2009). Introduction: Climate change biology at the ends of the Earth‐International Polar year special issue. Global Change Biology, 15, 1615–1617. https://doi.org/10.1111/j.1365-2486.2009.01977.x Root, T. L., Price, J. T., Hall, K. R., Schneider, S. H., Rosenzweig, C., & Pounds, J. A. (2003). Fingerprints of global warming on wild an- imals and plants. Nature, 421, 57–60. https://doi.org/10.1038/ nature01333 Lebreton, J.‐D. (2011). The impact of global change on terrestrial Vertebrates. Comptes Rendus Biologies, 334, 360–369. https://doi. org/10.1016/j.crvi.2011.01.005 ALVES et al. 2375 \| SUPPORTING INFORMATION Tulp, I., & Schekkerman, H. (2008). Weather‐ and season‐related vari- ation in the abundance of tundra arthropods: Hindcasting the ef- fect of climate change on prey availability for arctic birds. Arctic, 61, 48–60. Additional supporting information may be found online in the Supporting Information section at the end of the article. Unnsteinsdottir, E. R., Hersteinsson, P., Pálsson, S., & Angerbjörn, A. (2016). The fall and rise of the Icelandic Arctic fox (Vulpes la- gopus): A 50‐year demographic study on a non‐cyclic Arctic fox population. Oecologia, 181, 1129–1138. https://doi.org/10.1007/ s00442-016-3635-0 How to cite this article: Alves JA, Gunnarsson TG, Sutherland WJ, Potts PM, Gill JA. Linking warming effects on phenology, demography, and range expansion in a migratory bird population. Ecol Evol. 2019;9:2365–2375. https://doi. org/10.1002/ece3.4746 How to cite this article: Alves JA, Gunnarsson TG, Sutherland WJ, Potts PM, Gill JA. Linking warming effects on phenology, demography, and range expansion in a migratory bird population. Ecol Evol. 2019;9:2365–2375. https://doi. org/10.1002/ece3.4746 Van der Jeugd, H. P., Eichhorn, G., Litvin, K. E., Stahl, J., Larsson, K., Vand der Graaf, A. J., & Drent, R. H. (2009). Keeping up with early springs: Rapid range expansion in an avian herbivore incurs a mismatch be- tween reproductive timing and food supply. Global Change Biology, 15, 1057–1071. https://doi.org/10.1111/j.1365-2486.2008.01804.x
https://openalex.org/W3000258742	https://zenodo.org/records/3537628/files/Artigo_Gustavo_e_Deyla_-_Advances.pdf	English	null	Risking development by prioritizing the possession of firearms in Brazil: development is not achieved by arming the population!	Advances in social sciences research journal	2,019	cc-by	2,523	Deyla Paula de Oliveira Independent Researcher, Palmas, Brazil Although Brazil is a country with enormous potential for economic development and international research, these areas have been neglected by the current government. This article analyzes the possession of firearms in Brazil as a means of inhibiting the country’s development. It concludes that investing in the above-mentioned areas can reduce violence and poverty among other problems that are present in countries where governments invest little in such crucial sectors. Keywords: Development; Firearms; Social Sciences; Brazil; Violence; Public Safety. Deyla Paula de Oliveira Independent Researcher, Palmas, Brazil ABSTRACT The information in this article problematizes and broadens the debate on the importance of science to the development of Brazil, which is currently in crisis due to cuts in research investments in the country, making it impossible for scientists to continue carrying out ground-breaking research. We argue that this is due to two main factors: the approval of the Constitutional Amendment Project no. 55/2016, popularly known as the “death PEC,” which freezes public investments for twenty years and heightens the precariousness of education, health, science, security, and other sectors key to the country’s development along with the lack of interest in investing in national science by President Jair Bolsonaro who, among other things, prioritizes the possession of firearms as well as relaxation in the control of pesticides, which can further increase deforestation and deteriorate Brazil’s environment. The development of a nation is dependent on many factors such as investment in health, education, public safety as well as science, technology, and innovation. Although Brazil is a country with enormous potential for economic development and international research, these areas have been neglected by the current government. This article analyzes the possession of firearms in Brazil as a means of inhibiting the country’s development. It concludes that investing in the above-mentioned areas can reduce violence and poverty among other problems that are present in countries where governments invest little in such crucial sectors. ABSTRACT The information in this article problematizes and broadens the debate on the importance of science to the development of Brazil, which is currently in crisis due to cuts in research investments in the country, making it impossible for scientists to continue carrying out ground-breaking research. We argue that this is due to two main factors: the approval of the Constitutional Amendment Project no. 55/2016, popularly known as the “death PEC,” which freezes public investments for twenty years and heightens the precariousness of education, health, science, security, and other sectors key to the country’s development along with the lack of interest in investing in national science by President Jair Bolsonaro who, among other things, prioritizes the possession of firearms as well as relaxation in the control of pesticides, which can further increase deforestation and deteriorate Brazil’s environment. The development of a nation is dependent on many factors such as investment in health, education, public safety as well as science, technology, and innovation. Risking development by prioritizing the possession of firearms in Brazil: development is not achieved by arming the population! Gustavo Cunha de Araújo Department of Education, Universidade Federal do Tocantins, Tocantinópolis, Brazil Deyla Paula de Oliveira Independent Researcher, Palmas, Brazil t t THREATS TO THE DEVELOPMENT OF BRAZIL Bolsonaro is a threat to Brazil’s development not only because of his extremely conservative style but also because he defends controversial ideas, such as being in favor of the military dictatorship that ruled the country from 1964 to 1985, that the solution to end the violence in the country is arming the population, being in favor of making half of the penal code a heinous crime, and defending the death penalty and summary execution in the national prisons. Bolsonaro also does not support indigenous causes and those of minorities, such as the LGBT (lesbian, gay, bisexual, and transgender) community or women, and is reportedly homophobic and misogynist. This goes against NGOs that “preach” human and environmental rights. In particular, with respect to environmental issues, in addition to the scrapping of the Brazilian Institute of Environment and Natural Resources (IBAMA) and the Chico Mendes Institute for Biodiversity Conservation (ICMBio), this lack of support from the Federal Government is a significant threat to nature, especially to the world’s largest rainforest, the Amazon, which has been plagued with deforestation and declining biodiversity over the years. Many Brazilian scientists estimate that almost 7,000 square kilometers of the Amazon were deforested by 2017 (Tollefson, 2018), and this may worsen under the Bolsonaro government. In addition, such deforestation will further reduce the protected environmental and indigenous areas (Tollefson, 2018). Moreover, the approval of PEC no. 55/2016 puts the country in a situation of economic and social vulnerability, regressing its development (Brazil, 2016). In addition to that, Bolsonaro’s pro-firearm defense is extremely worrying, given that in Brazil, the data on violence—mostly homicides—is alarming and could increase with the approval for firearm possession in society. In 2016, more than 62,000 homicides were recorded in the country, and more than 500,000 Brazilians have lost their lives in the last 10 years (IPEA, 2018). To establish the seriousness of this problem, the death rate is 30.3 per 100,000 inhabitants; 30 times that of Europe (IPEA, 2018). Other statistics are even scarier; between 1980 and 2016, around 910,000 people were killed by firearms in Brazil, and in 2003, firearms accounted for 71.1% deaths in the country; homicide rates in other countries with a serious history of violence like El Salvador and Honduras were 76.9% and 83.4% respectively, far higher than the European average of 19.3% (IPEA, 2018). INTRODUCTION In our opinion, the victory of the extreme right-wing politician Jair Bolsonaro is likely to further reduce investments in several strategic sectors in Brazil as they are not a priority for his government (Editorial, 2018). In addition, President Bolsonaro approved Decree no. 9685 in January 2019, enabling Brazilians to own firearms, so that they may have the right to “defend themselves” against any threat. This demonstrates that instead of investing in Science, Technology and Innovation (C,T&I) along with education, public safety, and other segments that are important for national development and increasing Brazilians’ quality of life, the President is concerned about making the possession of firearms more flexible in a country with a worrying history of violence aided by firearms—they account for approximately 70% of violent deaths in Brazil (IPEA, 2018). However, this is not the first time that “Tropical Trump,” as he is popularly called, has startled many segments in the society—the president was one of the Brazilian politicians who voted in favor of the Constitutional Amendment Project – PEC no. 55/2016, known in the country as Copyright © Society for Science and Education, United Kingdom 65 Araújo, G. C., & Oliveira, D. P. (2019). Risking development by prioritizing the possession of firearms in Brazil: development is not achieved by arming the population!. Advances in Social Sciences Research Journal, 6(11) 65-69. “death PEC,” which paralyzes public investments in several sectors for 20 years (Brazil, 2016), including C,T&I, health, public safety, and education. When this PEC was approved by Brazil’s politicians, their justification for it was “reducing” expenses that could allow the country’s economic growth. In fact, this reduction only favors the politicians. In this sense, we argue that this Project (PEC) as well as the flexibility of possession of firearms, defended by the Bolsonaro Government, end up regressing the development of the country and several of its sectors. THREATS TO THE DEVELOPMENT OF BRAZIL Also, in the last ten years, the age group of 15 to 19 years among men has seen the highest homicide rate in Brazil due to firearms—56.5%. The victimization rate of the black population has increased by 23.1%, while that of the non-black population has seen a 6.8% decrease (IPEA, 2018). This indicates that 71.5% of the homicides by firearms in Brazil kill black and/or brown people (IPEA, 2018). Brazil is a country with enormous potential for national and international research and has been developing high-quality science in many areas. Yet, the Bolsonaro government will not prioritize C,T&I; this is a cause for concern since these are strategic areas vital to further the development of a nation, and lack of investments herein and/or dilutions in such sectors could affect Brazil for years. In 2018, the two most important research and graduate development agencies in the country, namely Coordination for the Improvement of Higher Education Personnel (CAPES) and the National Council for Scientific and Technological Development (CNPq), published open letters to the community and Michel Temer, the president at the time, expressing concern over the financial cuts in their budgets for the year 2019. This decrease will URL: http://dx.doi.org/10.14738/assrj.611.7295. 66 Advances in Social Sciences Research Journal (ASSRJ) Vol.6, Issue 11 Nov-2019 greatly affect scientific research in the country in all areas of knowledge including the primordial ones such as biotechnology, which deals directly with the manufacture of medicines and vaccines essential for public health. There is a greater risk of the continuity of these researches getting significantly affected if such reduction in the federal budget for science occurs under Bolsonaro’s government as well. Research grants in Brazil have great relevance and are fundamental for the development of C,T&I, since the knowledge production in Brazil is based mostly on the work of researchers who get scholarships for their fields of study. Decrease in the C,T&I budget may affect this availability of scholarships for researchers and postgraduate programs in the country. Furthermore, female scientists may lose important positions in the management of the country’s C,T&I—for instance in public university rectors—keeping in mind Bolsonaro’s misogynistic views and statements. The fact that only 30% of these organs are occupied by women (Benício, 2019) worsens the scenario. THREATS TO THE DEVELOPMENT OF BRAZIL However, the creation of the Prize for Women in Science by the Institute of Pure and Applied Mathematics (IMPA, 2019)—which awards research scholarships to promising Brazilian scientists in different knowledge areas—is a relevant way of valuing women’s work for the country’s C,T&I development; it also helps recognize gender equality (IMPA, 2019) within Brazilian scientific spaces, which are thoroughly devalued under the current government. CONCLUSION From this summary of the conservative and polemical vision of President Jair Bolsonaro, we can foresee the immeasurable regression in the development of the country. The federal government needs to recognize that the flexibility afforded to the arms industry will not bring great benefits to Brazilian society. Along with the fact that firearm homicide numbers in Brazil are alarming, it should be noted that our country was recently caught in a troublesome situation when two adolescents entered a public school in São Paulo with firearms and killed ten people—students and staff (Fantástico, 2019). Such tragic situations only reinforce the point that arming the population is not the solution. Also, with Decree no. 9685 of January 2019 (Brazil, 2019), the Disarmament Statute that has contributed to stop the arms race in the country now loses strength and relevance (Brazil, 2003). Nevertheless, it is important to clarify that we are not affirming that possession of firearms is the main reason behind increasing violence in the country; there are other contributing factors, such as social and economic inequality, the precariousness of the public safety system, the presence of numerous illicit markets that sell firearms, and criminal factions that devastate the country (IPEA, 2018). However, it is essential to propose strategies that have a responsible control over firearms in Brazil, failing which, homicide rates in the country—especially under the current government that supports the possession of weapons—will increase significantly. That is why, we believe it does not make sense to arm the population for their defense and that there are other factors more relevant to the country’s development, which deserve increased investments and attention from the government—C,T&I, health, public safety, and education are fundamental pillars for economic and social growth of any nation, besides reducing violence, poverty, and other problems that may plague countries where governments do not invest in the primary areas. List of abbreviations Institute for Applied Economic Research - IPEA Constitutional Amendment Project - PEC 67 Copyright © Society for Science and Education, United Kingdom Araújo, G. C., & Oliveira, D. P. (2019). Risking development by prioritizing the possession of firearms in Brazil: development is not achieved by arming the population!. Advances in Social Sciences Research Journal, 6(11) 65-69. Author’s Contribution Gustavo was responsible for designing, delineating, analyzing, and interpreting the data produced in the manuscript. Deyla was responsible for the critical revision of the content and interpretation of the data. The authors are responsible for the approval of the final version. Competing interests Funding Not applicable Availability of data and material Not applicable y Not applicable Consent for publication We declare that the present work is unpublished, original, and not submitted for publication to another international journal. It does not present any conflicts of interest, complies with all the ethical procedures of scientific research, and has our authorization for publication in Advances in Social Sciences Research Journal (ASSRJ). Copyright © Society for Science and Education, United Kingdom Advances in Social Sciences Research Journal (ASSRJ) Vol.6, Issue 11 Nov-2019 Institute for Applied Economic Research. 2018. Retrieved from: http://www.ipea.gov.br/portal/images/stories/PDFs/relatorio_institucional/180604_atlas_da_violencia_2018.pd f (Accessed on: May 21th, 2019.) Vol.6, Issue 11 Nov-2019 g Not applicable Not applicable Benício, RA. 2019. Uncertain future for women scientists in Brazil. Nature 567 (31). DOI: 10.1038/d41586-019- 00762-1 Brazil. 2016. Constitutional Amendment n. 95 of December 15, 2016. It amends the Transitory Constitutional Provisions Act, to establish the New Tax Regime, and provides other measures. Brasília: Câmara dos Deputados. Retrieved from: http://www.planalto.gov.br/ccivil_03/Constituicao/Emendas/Emc/emc95.htm (Accessed on: May 23th, 2019.) Brazil. 2016. Constitutional Amendment n. 95 of December 15, 2016. It amends the Transitory Constitutional Provisions Act, to establish the New Tax Regime, and provides other measures. Brasília: Câmara dos Deputados. Retrieved from: http://www.planalto.gov.br/ccivil_03/Constituicao/Emendas/Emc/emc95.htm (Accessed on: May 23th, 2019.) Brazil. 2019. Decree no. 9,685, dated January 15, 2019. Changes Decree No. 5,123, dated July 1, 2004, which regulates Law No. 10,826, dated December 22, 2003, which provides for the registration, possession and sale of firearms and ammunition, on the National System of Arms - SINARM and defines crimes. Retrieved from: Brazil. 2019. Decree no. 9,685, dated January 15, 2019. Changes Decree No. 5,123, dated July 1, 2004, which regulates Law No. 10,826, dated December 22, 2003, which provides for the registration, possession and sale of firearms and ammunition, on the National System of Arms - SINARM and defines crimes. Retrieved from: http://www.planalto.gov.br/ccivil 03/ Ato2019-2022/2019/Decreto/D9685.htm (Accessed on: May 20th, 2019). http://www.planalto.gov.br/ccivil_03/_Ato2019-2022/2019/Decreto/D9685.htm (Accessed on: May 20th, 2019). Editorial. 2018. Brazil’s new president adds to global threat to science. Nature 563: 5-6, DOI: 10.1038/d41586- 018-07236-w Editorial. 2018. Brazil’s new president adds to global threat to science. Nature 563: 5-6, DOI: 10.1038/d41586- 018-07236-w Fantástico. 2019. Understand the step by step of the Suzano school attack with exclusive images. Retrieved from: https://g1.globo.com/fantastico/noticia/2019/03/17/entenda-o-passo-a-passo-do-ataque-em-escola-de-suzano- com-imagens-exclusivas.ghtml (Accessed on: May 15th, 2019). IMPA. 2019. Institute of Pure and Applied Mathematics. Prize for Women in Science is with open enrollment. Retrieved from: https://impa.br/page-noticias/premio-para-mulheres-na-ciencia-esta-com-as-inscricoes- abertas/ (Accessed on: May 20th, 2019). URL: http://dx.doi.org/10.14738/assrj.611.7295. 68 Vol.6, Issue 11 Nov-2019 Tollefson, J. 2018. ‘Tropical Trump’ victory in Brazil stuns scientists. Nature. DOI: 10.1038/d41586-018-07220-4 Copyright © Society for Science and Education, United Kingdom 69
https://openalex.org/W4220818053	https://pure.eur.nl/ws/files/54163598/PaG_10_2_China_and_Climate_Multilateralism_A_Review_of_Theoretical_Approaches.pdf	English	null	China and Climate Multilateralism: A Review of Theoretical Approaches	Politics and governance	2,022	cc-by	9,451	Issue This review is part of the issue “Developing Countries and the Crisis of the Multilateral Order” edited by Wil Hout (Erasmus University Rotterdam) and Michal Onderco (Erasmus University Rotterdam / Peace Research Center Prague). © 2022 by the author(s); licensee Cogitatio (Lisbon, Portugal). This review is licensed under a Creative Commons Attribu‐ tion 4.0 International License (CC BY). connects it to its western counterparts by land (Davidson & Wang, 2021). It is thus evident that the way in which China positions itself in multilateral negotiations is cru‐ cial for determining the capacity of the international community in the face of climate change (Stalley, 2015). In recognition of China’s unique leverage to either halt or drive multilateral climate change mitigation efforts, scholars in recent years have sought to understand the complex politics affecting its international positioning. Various theoretical frameworks have discussed China’s climate change policy, including regime theory, two‐ level game theory, the global leadership and cham‐ pionship literature, the advocacy coalition framework, rational‐choice theory, multi‐level governance (MLG), consultative authoritarianism (Teets, 2013), fragmented authoritarianism (Mertha, 2009), and authoritarian envi‐ ronmentalism (Kwon & Hanlon, 2016). Though all fixated on China, these approaches have taken on different per‐ spectives to frame their insights. For instance, the discus‐ sion by MLG scholars studied the vertical organization Hao Zhang ernational Institute of Social Studies, Erasmus University of Rotterdam, The Netherlands; h.zhang@iss.nl Submitted: 29 September 2021 \| Accepted: 26 January 2022 \| Published: 21 April 2022 Submitted: 29 September 2021 \| Accepted: 26 January 2022 \| Published: 21 April 2022 Abstract China’s approach to multilateral climate negotiation has shifted greatly over the past decades. From being an obstacle to a follower, and now a potential leader, China has attracted academic attention. This article surveys the literature on China’s role in climate multilateralism as examined by scholars through different lenses. The article asks whether analyses at different policymaking levels can explain China’s changing position. I review studies addressing the international level and the nexus between the complementary international and domestic levels to offer a comprehensive understanding of China’s strategic moves and choices in multilateral discussions on climate change. The review finds that factors at the inter‐ national level are influencing China’s climate ambitions and goals, and even to some extent are determining its strategies toward climate multilateralism; however, for China to deliver its international climate commitments, its enhanced ability will need domestic support. While these insights are valuable to understand China’s international behavior, an emerging framework needs to be included in this discussion, as transnational governance scholarship might be able to explain how new actors may unlock China’s position on climate change in the future. Keywords y China; Chinese climate policy; climate change; global climate governance; multilateralism Politics and Governance, 2022, Volume 10, Issue 2, Pages 50–60 Politics and Governance (ISSN: 2183–2463) 2022, Volume 10, Issue 2, Pages 50–60 https://doi.org/10.17645/pag.v10i2.4920 Politics and Governance (ISSN: 2183–2463) 2022, Volume 10, Issue 2, Pages 50–60 https://doi.org/10.17645/pag.v10i2.4920 2. The Puzzle: China’s Changing Positions and Roles in UN‐Led Multilateral Climate Talks China has participated in multilateral climate negotia‐ tions led by the UN since the 1990s. The country’s role in the negotiations had always been controversial because it used to strongly oppose legally binding agreements. In recent years, China’s opposition has given way to a more favorable view of multilateral climate governance. Thus, it is safe to conclude that China’s view of climate change has evolved greatly over the past years (Cao, 2015; Stalley, 2015; Zhang, 2013). This can be ascribed to the international pressure that China received and its domestic interest in taking a more sustainable approach to economic development. This article reviews the recent academic literature on China’s international positioning in multilateral cli‐ mate change mitigation efforts that focus on the inter‐ national arena and the nexus between the international and domestic levels. I do not include analyses that focus on the domestic level since a proper discussion would require a separate analysis solely concentrating on vari‐ ables at this level. This article draws on three theoretical streams that are frequently used to study this topic (inter‐ national regime theory, global leadership and champi‐ onship literature, and two‐level game theory); it con‐ cludes that factors in the international political arena can influence China’s climate ambitions and goals and even to some extent determine its strategies toward multilat‐ eral climate governance. However, for China to deliver its international climate commitments, it will need to mobilize the strategies it has applied domestically. This discussion may be particularly relevant as China’s poten‐ tial leadership position in climate affairs has elicited both support and opposition. In light of a multilateral political system in crisis, as is widely proclaimed, a better under‐ standing of China’s willingness and ability to participate in multilateral climate change mitigation efforts could foretell the success of such efforts. Two summits signified the turn in China’s approach to climate diplomacy: the Copenhagen summit (2009), which marked the softening of China’s position, and the Paris summit (2015), after which China began to take a more active role in global climate governance. These two summits also divided China’s engagement in UN‐centric multilateral diplomatic efforts into three stages, during which China occupied different roles: as a passive fol‐ lower, an active participant, and a potential leader as illustrated in Table 1 below. Throughout the negotiations, three key issues have been central to discussions involving China. 1. Introduction Climate change is considered one of the most press‐ ing global issues at present and has become a top pri‐ ority in the international arena following widespread recognition of the need for urgent collective efforts to tackle it. After decades of participating in climate gov‐ ernance at both the international and domestic lev‐ els, China, as the largest developing country, is set to become a global leader in multilateral climate change mitigation and response efforts for at least two rea‐ sons. First, China can play an important role in spear‐ heading multilateral efforts to limit global warming to the 1.5°C pathways proposed by the UNFCCC (IPCC, 2018), as demonstrated by Chinese President Xi Jinping’s pledge to the UN General Assembly to reach peak car‐ bon emissions by 2030 and carbon neutrality by 2060 (McGrath, 2020). Second, China is already investing in green energy, which through multilateral agreements can be diffused through its Belt and Road Initiative that 50 Politics and Governance, 2022, Volume 10, Issue 2, Pages 50–60 of the governance functions within China; they stated that China’s domestic environmental governance can be fragmented and is subject to complex decision‐making processes (Hensengerth, 2015; Hensengerth & Lu, 2019; Liu et al., 2021; Schreurs, 2017). Some researchers have argued that MLG applications to China need to reflect more on its local context (Westman et al., 2019) and how the Chinese authorities recognize it (Wu, 2018). However, this article is not the place to discuss this perspective in detail. of the governance functions within China; they stated that China’s domestic environmental governance can be fragmented and is subject to complex decision‐making processes (Hensengerth, 2015; Hensengerth & Lu, 2019; Liu et al., 2021; Schreurs, 2017). Some researchers have argued that MLG applications to China need to reflect more on its local context (Westman et al., 2019) and how the Chinese authorities recognize it (Wu, 2018). However, this article is not the place to discuss this perspective in detail. to questions about the applicability of Western theories for studying non‐Western contexts, as it is important to evaluate the explanatory value of existing theories in the Chinese context and to consider where they need to be adapted to better understand China. This article first provides a brief overview of China’s multilateral climate governance engagement. Section 2 reviews the main theoretical approaches put forward by scholars to understand China’s international position in mitigating climate change. 1. Introduction Section 3 proposes transna‐ tional climate governance as a useful approach to study the future direction that China could take in leading the global effort to fight climate change. The final section concludes the article. Based on the above work, three dominant perspec‐ tives to studying China’s evolving positions on climate change can be discerned: those that focus the expla‐ nation on the international arena; those that empha‐ size China’s domestic politics, suggesting that domestic interests and political institutions are at the root of any explanation; and those that look at the nexus of interna‐ tional and domestic factors. However, perceptions still differ on China’s contribution to climate issues (Zhang, 2013). The question then arises whether current analy‐ ses of China’s position toward climate change at different policy‐making levels are sufficient to explain its changing behaviors and roles. Politics and Governance, 2022, Volume 10, Issue 2, Pages 50–60 2. The Puzzle: China’s Changing Positions and Roles in UN‐Led Multilateral Climate Talks The first is whether China should commit to a legally binding cli‐ mate agreement. The second issue regards climate mit‐ igation or, in other words, the reduction of emissions, which has been core to all intergovernmental climate negotiations to date. In the debate about the responsibil‐ ities of industrialized and developing countries for man‐ aging climate change, China has always been a staunch promoter of the “common but differentiated respon‐ sibilities principle.” This emphasizes that industrialized countries are responsible for historical emissions and have the primary obligation for global warming while developing countries should still have the right to pro‐ duce emissions to enable development. However, as China replaced the US in 2014 as the world’s largest greenhouse gas emitter, accounting for more than one‐ third of global emissions (Williams, 2014) and became Those studies that have applied the theoretical approaches discussed in this article have accurately cap‐ tured the current evolution of China’s international posi‐ tioning on climate mitigation. The study of these various approaches shows that China’s approach to global gov‐ ernance is not static. The evolution of its position and attitudes on multilateralism may have implications for the perception of China as a strategic player; it raises the important empirical question of how China’s strate‐ gic decisions in international climate politics should be viewed and responded to. More generally, it also leads 51 Politics and Governance, 2022, Volume 10, Issue 2, Pages 50–60 Table 1. China’s changing positions across three participation stages. Stages/issues Prior to Copenhagen 2009 Copenhagen 2009—Paris 2015 After Paris 2015 Commitment to a legally A passive follower/spoiler Softened position Active player/investor binding agreement (Kastner et al., 2020, p. 166) (Kastner et al., 2020, p. 167) Climate mitigation Reluctant player Positive transition Potential leader/champion (Engels, 2018) Green finance and Positive transition Investor technology transfer Table 1. China’s changing positions across three participation stages. Positive transition Green finance and technology transfer the world’s second‐largest economy, some industrialized countries began pushing China to cut carbon emissions more aggressively and even started questioning whether China should still be considered a developing country. This dispute has continued for years in multilateral cli‐ mate negotiations. The third issue that China has some‐ times been criticized for is that of the Green Climate Funds and technology transfer. 2. The Puzzle: China’s Changing Positions and Roles in UN‐Led Multilateral Climate Talks While negotiating for the establishment of the Green Climate Funds, China argued that industrialized countries should provide financial and technological transfers to developing countries to allow them to combat climate change. Industrialized countries, however, argue that China should be at the giving rather than the receiving end, considering China’s current rising emissions and rapid economic development. on or implications for the current international order (Wu, 2018), Kastner et al. (2020) modeled China’s approach to climate diplomacy and sought to reveal whether China should be considered a “revisionist” or a “status‐quo” force in global governance, using two inde‐ pendent variables in their analysis. The first factor was the balance of external options, while the second was whether contributions made by a rising power like China are viewed as indispensable for the regime’s success (Kastner et al., 2018). This model is, to some extent, consistent with the empirical evidence from the period when China was hold‐ ing up the negotiation process. When external options were extremely strong, meaning that other countries were paying for the cost of mitigation, China was able to become a spoiler, using its bargaining power to push for the restructuring of the international regime to bet‐ ter suit China’s interests (Kastner et al., 2018, p. 2). For instance, during the Copenhagen summit in 2009, China and the industrialized countries wrangled over the seven emission reduction plans, which include the United Nations IPCC program, the G8 country program, the UNDP program, the OECD program, the Garnaut program, the CCCPST program, and the Srensen pro‐ gram. China criticized all seven plans because they were mostly based on experts’ work from industrialized coun‐ tries while largely neglecting the reality in developing countries; China received strong opposition from indus‐ trialized countries in response. British climate minister Ed Miliband accused China of “trying to hijack the UN cli‐ mate summit” and “hold the world to ransom” to pre‐ vent an agreement from being reached (Vidal, 2009). The Copenhagen conference ended with no substan‐ tive progress. 3. Unpacking China’s Approach to Climate Multilateralism China’s evolving attitude toward climate change has attracted much academic attention. International Relations scholars are the major contributors to this dis‐ cussion: regime theory, the global leadership and cham‐ pionship literature, and two‐level game theory focus on unpacking China’s (changing) climate positions at the international level. Politics and Governance, 2022, Volume 10, Issue 2, Pages 50–60 3.1. Regime Theory The idea of con‐ structing a community of shared future for humankind was first brought up by President Xi in 2014, then reit‐ erated by him on multiple occasions regarding interna‐ tional cooperation. It is interesting to consider the defi‐ nition of leadership; scholars from China are optimistic about China’s potential leadership role, but they empha‐ size that China is a “torchbearer” (Zhuang et al., 2018), which implies a guiding function rather than structural leadership. At the same time, they also recognize the potential conflict between the EU and the US regarding structural leadership on climate change, while China can work to set better roles for other developing countries (Zhou & Zhuang, 2021). Nonetheless, China’s willingness to be a climate leader is the fruit of its domestic environ‐ mental interests and global political strategy. certain multilateral decision‐making across different issues. However, their position also leaves room for some reflection. Importantly, in the issue area of climate change, we need to reflect further on how to better account for the variation of positions over time. As mentioned ear‐ lier, Kastner et al. (2020, p. 167) argue that China’s posi‐ tion dramatically impacted the 2009 Copenhagen sum‐ mit because of the country’s strong external options. However, the situation where there are external options and China is indispensable under the climate change regime is evolving. For instance, when external options are relatively strong, and China’s contribution is consid‐ ered essential to the regime’s success, China is still very likely to be cooperative. Based on empirical observa‐ tions, the year 2009 witnessed the beginning of China’s transformation, as the country showed both intran‐ sigence and softening. Former Chinese President Hu Jintao’s speech at the opening session in Copenhagen demonstrated China’s “strong determination to assume responsibility for global climate governance” (Gao, 2018). Furthermore, China also released its climate pledges— more specifically, an emission intensity reduction tar‐ get for 2020—which was welcomed by the interna‐ tional community. China’s pledges were also praised by President Obama as ambitious and impressive (Gao, 2018). Another example of the impact of external options would be the US’s entry and exit from the mul‐ tilateral agreement. Before and after US withdrawal, China was actively pushing for ratification of the Paris Agreement. In this case, external options did not seem to impact China’s willingness to cooperate. A second question is whether China is able to take the leadership role. 3.1. Regime Theory Regime theory argues that international regimes exist on many levels. A regime consists of “a set of implicit or explicit principles, norms, rules, and decision‐making procedures” (Krasner, 1982, p. 186). Regime theory has been applied to provide explanations of Chinese domes‐ tic policymaking from external factors (Kim, 2018; Moore & Yang, 2001; Robinson, 1994), with the attempts mainly concentrated on the economic and military spheres (Botchway, 2011; Hu, 1997; Ramezani & Kamali, 2021). Scholars have identified policymakers’ perceptions of the international regime, the foreign policies of super‐ powers, the structure of the international system, and China’s calculation of its relative power as key vari‐ ables in understanding Chinese policymaking (Kim, 2018; Robinson, 1994). Regime theorists argue that external pressures from the negotiating table explain China’s willingness to coop‐ erate. The work of Kastner et al. (2018, 2020) shows how external factors influence China’s international behav‐ ior, particularly in relation to China’s willingness to con‐ tribute to global governance, although the authors do not give much attention to how its behavior may affect the regime’s success. Kastner et al. (2018, 2020) con‐ sidered only external factors, viewing China as a ris‐ ing power in the international regime. This enables the theoretical framework to explain China’s behav‐ iors, specifically, when China will support or undermine Inspired by the question of how China’s rise and expanding role on the world stage has had impacts Politics and Governance, 2022, Volume 10, Issue 2, Pages 50–60 52 been addressing at a very early stage (Wu, 2017), as it is likely for China to realize its global strategy. Chinese lead‐ ers recognize that an active response to climate change should not only be based on environmental considera‐ tions and China’s sustainable development but should also improve its standing in the international community, as this is related to China’s long‐term political interests (Buzan, 2021). One of China’s diplomatic strategies under President Xi’s leadership pictures China as a responsible nation that should participate in the international rule‐ making process and help shape the international order. Xi’s vision for China’s future is to “stand on the cen‐ tral stage of global affairs, make greater contributions to humanity, and construct a global community with a com‐ mon destiny” (Shen & Xie, 2018, p. 709). 3.1. Regime Theory Empirical observations suggest that China’s leadership goals on climate change may be achieved through making institutional, moral, and finan‐ cial contributions (Shen & Xie, 2018). Institutionally, China has demonstrated some entrepreneurial spirit in its negotiations. Its efforts to reach bilateral agree‐ ments with the US and the EU have helped build a consensus between industrialized countries and devel‐ oping countries and establish the Green Climate Fund (Shen & Xie, 2018). At the same time, China has forged new partnerships with the BASIC countries (Brazil, South Africa, India, and China), seeking to further the nego‐ tiating interests of developing countries. On the finan‐ cial side, China has pledged to establish a $3.1 billion South‐South climate fund under the UN framework for mitigation and adaptation projects in the most vulnera‐ ble and least developed countries (“China South‐South Climate Cooperation Fund,” 2015), which not only pro‐ motes the development of green finance but also shows the world that China is willing to be a responsible player as the biggest developing country. China has also incor‐ porated a green development strategy into the Belt and Road Initiative, a $900 billion international initia‐ tive on infrastructure proposed by China. The United Nations Environment Program and the Chinese Ministry of Ecology and Environment announced the formation of an international coalition to ensure that the Belt and Another question for further reflection is whether a theory can be applied to understand China’s international behavior without considering the country’s domestic con‐ text, or at least the connection to domestic factors? This will be probed in the following discussion section. Politics and Governance, 2022, Volume 10, Issue 2, Pages 50–60 between China and the industrialized countries may con‐ tinue, but this will not involve who should take more responsibility as was the issue over the past two decades but will rather concern competition for more voice and influence. If China’s interest in taking on climate change leadership were only due to domestic priorities (Engels, 2018), it is obvious that these domestic inter‐ ests and international commitments would need to con‐ verge for China to become the next champion or leader (Wu, 2017). Chinese researchers have confirmed this trend. It has shown that over 100 countries have commit‐ ted to carbon neutrality (Motive Power, 2021) and that China has also begun to map its low‐carbon development plan to reach carbon neutrality by 2060—suggesting that policymakers in China have recognized the importance of a green transition and the potential for a new interna‐ tional order based on the new rules relating to carbon neutrality (Chai et al., 2020, p. 37). President Xi’s con‐ cept of building a human community for a shared future is consistent with the idea of global climate governance leadership (Li & Liu, 2019), reinforcing high‐level convic‐ tions and setting a milestone in the history of China’s cli‐ mate governance. Road Initiative leads to green growth. Morally, China has been a strong supporter of UN‐centered climate mul‐ tilateralism, protecting the values and ideas related to addressing climate change and curbing climate skepti‐ cism (Shen & Xie, 2018). However, scholars also have reservations, arguing that China’s domestic interest is key to understanding its actual ability to assume a lead‐ ership role. Engels (2018, p. 5) believes that China’s championship of climate mitigation only happens to coin‐ cide with important domestic priorities rather than being intentional and could pose a risk for the continuation of China’s support of the global climate mitigation regime. pp g g g Lastly, we ask whether China is expected to become a leader? In terms of climate leadership, the Annex I coun‐ tries have always been expected to play this role (Hurri, 2020). The topic of climate leadership has always been associated with highly industrialized countries such as the US or the EU (Bäckstrand & Elgström, 2013; Dai & Diao, 2010). Given China’s volume of emissions, its invest‐ ments in renewable energy, and its large economy and population size, it is expected to take on more respon‐ sibility. However, how much leadership power do the industrialized countries expect China to hold? Would the Annex I countries, which have always taken a leading role in setting the rules and agendas, controlling the direction of international negotiations, et cetera, be will‐ ing to hand some of these prerogatives to the Chinese? Rethinking China’s expected leadership, China may be far from becoming an actual leader. According to Hurri (2020), industrialized countries want China to take more weight onto its shoulders. Over the last decade, China has taken over some responsibilities to assist the global progress on climate change, especially in helping other developing countries confront climate change. However, China remains in conflict with industrialized countries as it attempts to seize more institutional power, related, for instance, to setting the rules and agendas, steering the direction of negotiations, or mediating international cooperative efforts. In other words, China certainly has the potential to be a leader, but acknowledging the coun‐ try as a climate leader creates a role conflict for the cur‐ rent leaders (Hurri, 2020). 3.2. Global Leadership and Mitigation Championship Literature The global leadership and championship literature con‐ tains a very recent discussion of China’s approach to cli‐ mate multilateralism. Although it addresses the same question as regime theory, this line of inquiry builds on China’s active attitude toward climate issues and its increased willingness and capacity. However, some schol‐ ars have shown reservations about claims that China is leading international climate negotiations (Engels, 2018), while others have expressed optimism, especially in the absence of leadership in climate change governance today (Buzan, 2021; Zhuang et al., 2018). Hence, it is cru‐ cial to consider the potential for Chinese leadership on climate change. First, we should ask whether China is willing to take the leadership role? China is perceived as aiming to play a more constructive role in shaping global governance, and climate change is one of the issue areas that China has 53 Politics and Governance, 2022, Volume 10, Issue 2, Pages 50–60 Politics and Governance, 2022, Volume 10, Issue 2, Pages 50–60 3.3. Two‐Level Game Theory The two‐level game theory was originally derived by Putnam (1988) from game theory. The two‐level game approach focuses specifically on the negotiating posi‐ tions of state actors in relation to international and national interest. This analytical framework is considered appropriate for researchers to interpret China’s interna‐ tional climate negotiation strategy because it provides a baseline logic (as illustrated in Figure 1) to look at differ‐ ent actors and issues across the two levels (Cao, 2012; G. Chan et al., 2008; Gunter & Rosen, 2011; Hsu & Jiang, 2015; Wang, 2018; Zhang, 2013). The application of the theory in Chinese studies has inherited certain elements from Putnam’s experimen‐ tal work. This logic of interaction between international negotiation and domestic interests is reflected in the application by Chinese scholars. First, researchers agree that China’s behavior at the international level is influ‐ enced by domestic interests. There is much empirical evi‐ dence to support the idea that the roots of China’s shift‐ ing positions at the international level can be traced to Returning to President Xi’s vision on China’s future contribution to global governance, it is quite clear that China aspires to take on institutional leadership on certain issues. Regarding climate change, the conflict Level 1 Level 2 Shape Two-level game Results of interna onal climate nego a ons Poli cal ins tu on + distribu on of preferences = China’s climate policy Inﬂuence Figure 1. The logic of two‐level games analysis. Figure 1. The logic of two‐level games analysis. Politics and Governance, 2022, Volume 10, Issue 2, Pages 50–60 54 analysis. Moreover, these researchers concluded that fac‐ tors at either the domestic or the international level evi‐ dently have different weights in terms of their explana‐ tory power and that the different levels are not equally important. Empirically, during a certain period, interna‐ tional factors might play a decisive role, while at other times, domestic factors would be critical. Therefore, it is explicitly clear that factors at the international and domestic levels are not equally influential in shaping China’s strategic climate choices. domestic causes. For instance, China’s aggressive invest‐ ment in renewable technologies has led to a proactive stance at the negotiating table. Various factors are considered when applying two‐ level game theory to the study of China. G. Chan et al. 4. Fully Answered? External Factors, Interplay Between Two Levels and China in Climate Multilateralism Regime theory, the leadership and championship litera‐ ture, and two‐level game theory share certain common‐ alities. First, this research aims to understand China’s (evolving) international position on climate change; in other words, the dependent variables are the same in general terms, with leadership literature focusing on more recent changes. Even though domestic interests are raised in some discussions, the phenomenon that is explained remains China at the international level. For this reason, certain prevalent theories, such as advo‐ cacy coalition framework, MLG, consultative multilater‐ alism (Teets, 2013), and authoritarian environmentalism (Kwon & Hanlon, 2016), which are used to shed light on domestic factors and dynamics of China’s national cli‐ mate governance, are not included in the discussion in this article. The application of two‐level game theory in the study of China is no longer state‐centric. International insti‐ tutions, national and local governments, domestic and international NGOs, and the media are all considered key actors in mobilizing China’s climate multilateralism (G. Chan et al., 2008; Gunter & Rosen, 2011; Hsu & Jiang, 2015; Wang, 2018). One application of two‐level game theory to the situation in China shows that the Chinese government interacts with international institu‐ tions such as the UNFCCC to engage in setting activi‐ ties, rule negotiations, and rule compliance at the inter‐ national level, while Chinese media and local govern‐ ments focus more on shaping China’s domestic climate policy. NGOs, on the other hand, can be active at either level, using inside‐out strategies to advocate for pol‐ icy change. These two levels then interact to influence China’s approach to global climate governance. The three theoretical approaches that were dis‐ cussed in Section 2 all note that external factors, such as the geopolitical dynamics at the international level and the systemic flaws in the global climate regime (Buzan, 2021; Kastner et al., 2020; Zhuang et al., 2018), impact China’s strategy toward climate multilateralism. In fact, external factors may enjoy more explanatory power than traditionally thought. In practice, China is a highly strate‐ gic player, and its approach to multilateralism should not be considered a consistent feature (Kastner et al., 2020, p. 165) but as dynamically evolving in response to inter‐ national influences. However, some researchers have noted that the orig‐ inal theory was based on the US model of policymak‐ ing and thus has “greater relevance in the context of American politics” (Zhang, 2013, p. 3). 3.3. Two‐Level Game Theory (2008) pointed out that China’s domestic environmen‐ tal crisis, the ecological cost of its rapid economic devel‐ opment, incidents of environment‐related protest and unrest, and the recognized need for sustainable develop‐ ment have all clearly impacted China’s diplomacy. This can explain why China is willing to participate in talks on fighting global warming while, at the same time, it guards the principle of common but differentiated responsibil‐ ity, for its own benefit as well as for that of the develop‐ ing world (G. Chan et al., 2008). Likewise, international social engagement through participation in international treaties or trade can help China integrate with the world politically and raise its environmental standards to meet the higher standards set by the industrialized countries (G. Chan et al., 2008). Similarly, Zhang (2013) also recog‐ nizes that decision‐makers are under pressure from both international negotiations and domestic politics. To clar‐ ify, Zhang (2013) further hypothesizes that if the cost of abatement were high, China would be less willing to participate in international climate change negotiations. If climate change caused high levels of ecological vulnera‐ bility, and the principle of equity was accepted by parties at the international level, then China would be likely to take a more cooperative attitude in international climate change negotiations (Zhang, 2013). In sum, the three pieces of literature discussed in this section collectively highlight the importance of factors at the international level to examinations of China’s inter‐ national behavior regarding climate change. The leader‐ ship literature and two‐level game theory also consider domestic factors. The next question is to what extent these theoretical approaches adequately address China’s changing international position on climate governance. Politics and Governance, 2022, Volume 10, Issue 2, Pages 50–60 4. Fully Answered? External Factors, Interplay Between Two Levels and China in Climate Multilateralism The leader‐ ship and championship approach is proposed because China has taken a proactive stance on climate change in recent years. China’s turn to become a potential cli‐ mate leader is arguably shaped not only by its expe‐ rience as a participant in the UNFCCC processes but also the fact that as its economic wealth and emissions have continued to increase, it has gradually gained suffi‐ cient structural power to participate in international cli‐ mate governance. An example of this is China’s leadership of the institu‐ tional arrangement of the “bottom‐up” approach under the Paris Agreement (Li et al., 2021). In addition, discus‐ sions of global leadership and championship may stim‐ ulate the study of how China might use climate issues as an opportunity to advance its political stance (Buzan, 2021) and further expand its political influence, espe‐ cially in the Global South. Two‐level game theory enjoys the advantage of harnessing factors at both international and domestic levels to account for China’s international climate strategies. While some scholars in the global championship literature have also examined domestic influences, they have not analyzed the interplay between the two levels. Two‐level game theory has been applied in different contexts and to various issues, but when it comes to China, it is confronted by the question of how to adapt to the domestic context to attain an in‐depth analysis. Another valuable contribution that emerges from these analyses is that they help position China’s role in climate multilateralism relative to that of other coun‐ tries. When China is reluctant to participate, other coun‐ tries exert pressure to give China the impetus to change. As China makes progress, other countries could coop‐ erate with China in addressing global climate change. From the current perspective, China and the industrial‐ ized countries of the EU have the competence and the willingness to “lead” or, in other words, to seize more institutional power in climate‐related international insti‐ tutions. This situation might lead to conflict between China and industrialized countries, not about who should take more responsibility for having caused global warm‐ ing, but about who should have a greater voice in the negotiation process. Secondly, we need to take into account the differ‐ ences in civil society actors when applying theories orig‐ inally developed in Western democracies to the Chinese context, especially in the field of environment and cli‐ mate change. 4. Fully Answered? External Factors, Interplay Between Two Levels and China in Climate Multilateralism Therefore, ele‐ ments of the analysis such as “win‐sets,” “strategies of negotiators,” and the “ratification process,” which are all prevalent in democratic contexts, may take on dif‐ ferent forms or meanings when applied to the Chinese context given the differences in the political and cultural system. Instead, researchers have expanded their expla‐ nations by adding more participants and factors into the The three theoretical approaches all have their own unique features and merits in terms of contribu‐ tions. International regime theory examines only exter‐ nal factors to comprehend China’s international behav‐ ior, and it attempts to make general statements on China’s strategy toward multilateralism in the context of a relatively long period of changing external condi‐ tions. The global leadership and championship literature 55 Politics and Governance, 2022, Volume 10, Issue 2, Pages 50–60 (Westman et al., 2019, p. 14) means that democratic concepts such as competing interests (between polit‐ ical parties) and ratification processes would have to be replaced by Chinese indigenous governance con‐ cepts. For instance, regarding the environment and climate‐related issues, the Chinese decision‐making pro‐ cess may involve coordination between the Ministry of Ecology and Environment, which supervises and guides environment‐related plans and decision‐making; the China Meteorological Administration, which pro‐ vides the scientific environment and climate evidence; the Ministry of Foreign Affairs, which coordinates inter‐ national negotiations; and other government agencies. When applying Western theories to the Chinese con‐ text, the potential for conflict between the functional hierarchies (such as those mentioned above), as well as territorial (kuài) and vertical (tiáo) chains of command (Hensengerth, 2015, p. 313), need to be considered, rather than conflicts of interest between political parties, as in democracies. The term tiáo (vertical) refers to the vertical lines of authority over various sectors reaching down from the ministries of the central government. Kuài (horizontal) refers to the horizontal level of authority of the territorial government at the provincial or local level. The ministries and government bodies mentioned above may share overlapping functions which should be consid‐ ered when transplanting theories: For instance, both the Ministry of Ecology and Environment and the Ministry of Foreign Affairs are responsible for the supervision of China’s participation in the UN Conferences of Parties. investigates China’s potential as a climate leader more than it reflects on China’s past strategies. 4. Fully Answered? External Factors, Interplay Between Two Levels and China in Climate Multilateralism NGOs in the West have played a signifi‐ cant role in advocacy throughout the policymaking pro‐ cess. In China, the definition and functions of civil society actors are very different from those in Western contexts. Policy entrepreneurs such as international NGOs often undertake the role of policy translators (Stone, 2012), while in China, international NGO operations are lim‐ ited by local management schemes, one of which is the overseas NGO law. On the other hand, local NGOs in China lack the capacity to establish direct global inter‐ connections due to the relatively strict regulatory regime imposed on Chinese civil society. Therefore, for inter‐ national organizations or international NGOs to be able to disseminate norms, set standards, or advocate poli‐ cies, they often need to target local actors such as Chinese NGOs, rather than the Chinese state (Gunter & Rosen, 2011). Politics and Governance, 2022, Volume 10, Issue 2, Pages 50–60 5. A New Question? Transnational Climate Governance and China One of the major changes in the current global system to address climate issues is the rising importance of non‐state actors (Andonova et al., 2009; S. Chan et al., 2015; Hale, 2020; Kuyper & Bäckstrand, 2016). Given that the traditional state‐centric climate system, which evolves around the UNFCCC regime, has been under con‐ testation academically (Keohane & Victor, 2011; Lederer, 2015), transnational, non‐state actors’ efforts to influ‐ ence state‐centric global responses to climate change beyond the multilateral system are intended to create a new form of governance. This is also referred to as transnational climate governance (Hale, 2020). According to Andonova et al. (2009, p. 56), “transna‐ tional governance occurs when networks operating in the transnational sphere authoritatively steer con‐ stituents towards public goals.” NGOs, private compa‐ nies, subnational governments, et cetera, can all par‐ ticipate in the process of transnational governance. Polycentrism (Ostrom, 2009), fragmentation (Biermann et al., 2009; Zelli & van Asselt, 2015), and regime com‐ plexity (Keohane & Victor, 2011) are all conceptual frames to capture the changing landscape of climate governance. Despite the different positions of these concepts, they are all based on the growing empirical process of multi‐actor and multi‐level (both horizontal and vertical) processes of governance efforts (Dorsch & Flachsland, 2017). Furthermore, researchers also seem to agree that the current international system for man‐ aging climate change is in transition, with more actors engaged in more activities at significantly more levels of governance (Jordan et al., 2018). This transnational system may fill some governance gaps and may be able to co‐exist with the traditional state‐centric system, yet there is no consensus on whether it is a supplement to or an alternative for the traditional one. Chinese NGOs, subnational governments, busi‐ nesses, and other non‐state actors can still enjoy some political space to engage in transnational schemes. As research on China’s domestic climate governance indi‐ cated, “although China remains authoritarian, it is never‐ theless responsive to the increasingly diverse demands of Chinese society” (Mertha, 2009, p. 995). As climate change has become a policy priority, the barriers to entry into the policymaking process have been lowered for certain “policy entrepreneurs” (Mertha, 2009), as Chinese leaders see the need to incorporate Chinese civil society actors into climate decision‐making to fill governance gaps. Collective action in rural China “often hinges on locat‐ ing and exploiting divisions within the state by using the rhetoric and regulations of the central government to resist misconduct by local officials” (Zhang, 2017, p. 286). The concept of rightful resistance is a case where Chinese practices could inform existing Western theories on con‐ tentious politics. In the context of studying Chinese cli‐ mate policy, a new idea like “a community of shared future” (renlei mingyun gongtongti), which is inspired by the concept of “all‐under‐heaven” (tianxia) from tradi‐ tional Chinese philosophy, could provide opportunities for such innovation. As argued by Buzan (2021), climate change is a profound threat to “everything in the world,” which is difficult to describe and interpret using Western concepts of international relations (Buzan, 2021). ical context may condition actors’ engagement with transnational governance, and this informs the study of China in transnational governance (Hale & Roger, 2018). Empirically, given China’s domestic political context, the question is whether China can be or has been associated with transnational climate governance and what factors affect the scale, form, and shape of its engagement. Transnational climate governance remains rather a novel concept in China. While Chinese officials have made statements welcoming the efforts of subnational and non‐state actors openly, in practice, the regula‐ tion of civil society at home does not seem to be loos‐ ening up. A distinctive feature of the Chinese engage‐ ment with transnational climate governance is that the authority (the Chinese government) remains at the cen‐ ter, challenging the traditional understanding of the con‐ cept at the global level where non‐state and sub‐state actors are the main actors. According to Hale and Roger (2018), domestic factors are the main drivers of China’s response to climate change. Beyond that, Chinese offi‐ cials have made it clear that China is a firm supporter of UN‐centered multilateralism regarding climate change. And global climate governance has always been the main narrative that they use to describe the global efforts to address climate change. Empirical evidence confirms this, as China’s participation in transnational governance initiatives remains relatively shallow and uneven, with national government and subnational gov‐ ernment units being the major participants (Hale & Roger, 2018). However, does this imply that the participa‐ tion of other Chinese actors in transnational governance will be limited as well? 4.1. Adaptation to the Chinese Context It is challenging to transplant theories and concepts from political science, such as regime theory and two‐level game theory, to China, as the approaches have been primarily applied in Western and democratic contexts. The direct application of Western theories to China’s circumstances or to use China’s case to contribute to Western theories requires further exploration. In order to reconcile Western theories with the unique case of China, it seems inevitable that Western theories will merge with indigenous Chinese theories or concepts (Zhang, 2017). Conceptual innovation may pro‐ vide an answer. For instance, consider the concept of rightful resistance developed by O’Brien and Li (2006) to conceptualize Chinese farmers’ contentious practices. First, it is important to consider the differences in parameters of the political systems. Specifically, the lack of deliberative democracy in the Chinese system Politics and Governance, 2022, Volume 10, Issue 2, Pages 50–60 56 Politics and Governance, 2022, Volume 10, Issue 2, Pages 50–60 5. A New Question? Transnational Climate Governance and China Although most Chinese actors are involved in transnational governance as followers, and many grassroots environmental NGOs are still unfamil‐ iar with the concept, transnational climate governance may be a particularly attractive strategy for NGOs. As an increasing number of foreign NGOs and transnational NGO networks join hands with local NGOs, they may be able to lobby or initiate local‐level activism (Hale & Roger, 2018) or even become more embedded in the policymaking process. This transnational policy diffusion is empirically observed, as in the case of the low‐carbon economy agenda, which was introduced by transnational actors, backed by foreign funding, promoted by policy entrepreneurs from domestic research institutes, pro‐ The involvement of China, as a key player in mul‐ tilateral climate negotiations, with the transnational trend of climate governance has attracted academic attention. Researchers acknowledge that domestic polit‐ Politics and Governance, 2022, Volume 10, Issue 2, Pages 50–60 57 Another gap may be the limited range of actors included. With the introduction of the transnational gov‐ ernance framework, non‐state actors have become an integral part of the discussion, and as a result, the form of international cooperation is transforming. The current literature on climate change in China focuses on the state but rarely mentions civil society actors. Similar to the Chinese state in its participation in global climate governance, Chinese NGOs are also constrained by their domestic political constituency (Wang, 2018). However, they are subject to international and transnational influ‐ ence from foreign peers regarding their approach to climate policy advocacy (Hofem & Heilmann, 2013). Attention to this element would require further work on how plural theories can be employed in the discussion. pelled by top‐level attention, and finally adopted by the government bureaucracy (Hofem & Heilmann, 2013). Further work needs to be carried out on the conditions for and the kind of transnational governance initiatives that can be accepted in China. 6. Conclusion Departing from analyses at different policymaking lev‐ els, this article presents a review of approaches that help understand China’s shifting positions toward cli‐ mate multilateralism. This article has shown that it is essential to consider a range of theories that can pro‐ vide additional insights into understanding China’s inter‐ national climate position. Conceptually, it is crucial to focus on a variety of the‐ oretical approaches to examine how China’s climate mul‐ tilateralism has evolved. At different levels of analysis, different sets of variables are considered to be impor‐ tant influences on China’s position, and they lead to different conclusions. The analysis at the international level focuses on China’s external options and the urgency of the issue itself. However, to fully explain the vari‐ ation in China’s position, factors at the international and domestic levels have played different roles at dif‐ ferent times. China’s domestic willingness and capabili‐ ties impact China’s global expansion. The analysis of the international–domestic nexus reflected on the interna‐ tional context or the “strategic landscape” of the climate change issue, as well as China’s political willingness and ability. It emphasized the interaction between the two levels, which may add additional insights to the analy‐ ses of regime theory and global leadership and champi‐ onship literature. Acknowledgments I thank my supervisor Wil Hout for all his guidance and constructive feedback during the process of improving the quality of this article. I also thank my colleague Lize Swartz, Dennis Penu, as well as journal editors and aca‐ demic reviewers for their suggestions on the earlier ver‐ sion of the article. References Andonova, L. B., Betsill, M. M., & Bulkeley, H. (2009). Transnational climate governance. Global Environ‐ mental Politics, 9(2), 52–73. What appears to be missing from the current ana‐ lysis is how “changes” in the trajectory of China’s cli‐ mate multilateralism may be interpreted. As Kastner et al. (2020, p. 165) have argued that analysts “should not treat China’s approach to multilateralism as a constant feature of the country’s general disposition” (Kastner et al., 2020, p. 165), it is relevant to focus on the dynam‐ ics and conditions of the change process as a poten‐ tial area for further research. The conceptual framework of “critical junctures,” derived from historical institution‐ alism, can be invoked to discover patterns of political changes (Capoccia & Kelemen, 2007; Collier & Collier, 2002). In general, this framework can be used to explain various development processes, including the organiza‐ tional decision‐making process (Capoccia & Kelemen, 2007, p. 343). Some scholars have argued that China’s cli‐ mate policy has reached a critical juncture, whether seek‐ ing energy self‐sufficiency by burning coal or promoting “ecological civilization” to gain a green reputation inter‐ nationally (Oxford Analytica, 2020). Nevertheless, it is yet to observe whether a period of significant change as demonstrated in Chinese climate policy can produce dis‐ tinct legacies (Capoccia & Kelemen, 2007, p. 347) in the study of development and change. Bäckstrand, K., & Elgström, O. (2013). The EU’s role in climate change negotiations: From leader to “lea‐ diator.” Journal of European Public Policy, 20(10), 1369–1386. Biermann, F., Pattberg, P., van Asselt, H., & Zelli, F. (2009). The fragmentation of global governance architec‐ tures: A framework for analysis. Global Environmen‐ tal Politics, 9(4), 14–40. Botchway, F. N. (2011). Regime theory and China–Africa economic relations: New ideas in old paradigms? In F. N. Botchway (Ed.), Natural resource investment and Africa’s development (pp. 368–394). Edward Elgar. Buzan, B. (2021). China and climate change governance: A golden opportunity. Guoji Zhanwang. Cao, H. (2012). European Union climate change policy: In the nexus of internal policy‐making and interna‐ tional negotiations [Doctoral dissertation, University of Catania]. ArchivIA. http://archivia.unict.it:8080/ handle/10761/1120 Cao, H. (2015). China and the European Union in global climate governance: Ideas, actions, differences and cooperation. Chinese Journal of European Studies, 5, 50–65. Capoccia, G., & Kelemen, R. D. (2007). The study of crit‐ Capoccia, G., & Kelemen, R. D. (2007). Politics and Governance, 2022, Volume 10, Issue 2, Pages 50–60 References The study of crit‐ Politics and Governance, 2022, Volume 10, Issue 2, Pages 50–60 58 ical junctures: Theory, narrative, and counterfactu‐ als in historical institutionalism. World Politics, 59(3), 341–369. Hensengerth, O., & Lu, Y. (2019). Emerging environmen‐ tal multi‐level governance in China? Environmental protests, public participation and local institution‐ building. Public Policy and Administration, 34(2), 121–143. Chai, L., Guo, H., Liu, C., Dong, L., Ju, L., Liu, C., Chen, Y., Chen, H., & Zhuang, G. (2020). Global climate change and China’s action plan: China’s climate governance during the 14th five‐year plan. Yuejiang Academic Journal, 2020(6), 36–58. Hofem, A., & Heilmann, S. (2013). Bringing the low‐ carbon agenda to China: A study in transnational pol‐ icy diffusion. Journal of Current Chinese Affairs, 42(1), 199–215. Chan, G., Lee, P. K., & Chan, L. (2008). China’s envi‐ ronmental governance: The domestic–international nexus. Third World Quarterly, 29(2), 291–314. Hsu, C. L., & Jiang, Y. (2015). An institutional approach to Chinese NGOs: State alliance versus state avoid‐ ance resource strategies. The China Quarterly, 221, 100–122. Chan, S., van Asselt, H., Hale, T., Abbott, K. W., Beisheim, M., Hoffmann, M., Guy, B., Höhne, N., Hsu, A., & Pat‐ tberg, P. (2015). Reinvigorating international climate policy: A comprehensive framework for effective non‐ state action. Global Policy, 6(4), 466–473. Hu, R. W. (1997). Play by international rules: The develop‐ ment of China’s nuclear export controls. The Monitor. Hurri, K. (2020). Rethinking climate leadership: Annex I countries’ expectations for China’s leadership role in the post‐Paris UN climate negotiations. Environ‐ mental Development, 35. https://doi.org/10.1016/ j.envdev.2020.100544 China South‐South Climate Cooperation Fund bene‐ fits developing countries. (2015, November 30). Chinadaily. https://www.chinadaily.com.cn/world/ XiattendsParisclimateconference/2015‐11/30/ content_22557413.htm IPCC. (2018). Global Warming of 1.5 °C. https://www. ipcc.ch/sr15 Collier, R. B., & Collier, D. (2002). Shaping the politi‐ cal arena: Critical junctures, the labor movement, and regime dynamics in Latin America. University of Notre Dame Press. Jordan, A., Huitema, D., van Asselt, H., & Forster, J. (2018). Governing climate change: Polycentricity in action? Cambridge University Press. Dai, X., & Diao, Z. (2010). Towards a new world order for climate change: China and the European Union’s lead‐ ership ambition. Routledge. Kastner, S. L., Pearson, M. M., & Rector, C. (2018). China’s strategic multilateralism: Investing in global gover‐ nance. Cambridge University Press. Kastner, S. L., Pearson, M. M., & Rector, C. (2020). China and global governance: Opportunistic multilateral‐ ism. Global Policy, 11(1), 164–169. Davidson, M. R., & Wang, Y. References (2021, February 27). Climate cooperation with China can power the world sustain‐ ably. The Diplomat. https://thediplomat.com/2021/ 02/climate‐cooperation‐with‐china‐can‐power‐the‐ world‐sustainably Keohane, R. O., & Victor, D. G. (2011). The regime com‐ plex for climate change. Perspectives on Politics, 9(1), 7–23. Dorsch, M. J., & Flachsland, C. (2017). A polycentric approach to global climate governance. Global Envi‐ ronmental Politics, 17(2), 45–64. Kim, S. S. (2018). Chinese foreign policy in theory and practice. In S. S. Kim (Ed.), China and the world: Chi‐ nese foreign policy faces the new millennium (pp. 3–33). Routledge. Engels, A. (2018). Understanding how China is champi‐ oning climate change mitigation. Palgrave Communi‐ cations, 4(1), 1–6. Krasner, S. D. (1982). Structural causes and regime con‐ sequences: Regimes as intervening variables. Interna‐ tional organization, 36(2), 185–205. Gao, X. (2018). China’s evolving image in international cli‐ mate negotiation: From Copenhagen to Paris. China Quarterly of International Strategic Studies, 4(2), 213–239. Kuyper, J. W., & Bäckstrand, K. (2016). Accountability and representation: Nonstate actors in UN climate diplo‐ macy. Global Environmental Politics, 16(2), 61–81. Gunter, M. M., Jr., & Rosen, A. C. (2011). Two‐level games of international environmental NGOs in China. William & Mary Policy Review, 3, 270–294. Kwon, K. L., & Hanlon, R. J. (2016). A comparative review for understanding elite interest and climate change policy in China. Environment, Development and Sus‐ tainability, 18(4), 1177–1193. Hale, T. (2020). Transnational actors and transnational governance in global environmental politics. Annual Review of Political Science, 23, 203–220. Lederer, M. (Ed.). (2015). Global governance. Edward Elgar. Hale, T., & Roger, C. (2018). Domestic politics and Chinese participation in transnational climate governance. In S. Kennedy (Ed.), Global governance and China: The dragon’s learning curve (pp. 250–271). Routledge. Li, B., & Liu, C. (2019). Global climate governance from the perspective of the community with a shared future for mankind—China’s plan and practice path. Contem‐ porary World and Socialism, 2019(5), 170–177. Hensengerth, O. (2015). Multi‐level governance of hydropower in China? The problem of transplanting a western concept into the Chinese governance con‐ text. In E. Ongaro (Ed.), Multi‐level governance: The missing linkages (pp. 295–320). Emerald. Li, Z., Dong, L., & Zhang, H. (2021). China’s 30‐year partic‐ ipation in international climate governance in retro‐ spect. China Population, Resources and Environment, 31(9), 202–210. Politics and Governance, 2022, Volume 10, Issue 2, Pages 50–60 59 (pp. 202–212). Edward Elgar. Liu, Z., Guo, J., Zhong, W., & Gui, T. (2021). References Multi‐level governance, policy coordination and subnational responses to Covid‐19: Comparing China and the US. Journal of Comparative Policy Analysis: Research and Practice, 23(2), 204–218. Stone, D. (2012). Transfer and translation of policy. Policy Studies, 33(6), 483–499. Teets, J. C. (2013). Let many civil societies bloom: The rise of consultative authoritarianism in China. The China Quarterly, 213, 19–38. McGrath, M. (2020, September 22). Climate change: China aims for “carbon neutrality by 2060.” BBC. https://www.bbc.com/news/science‐environment‐ 54256826 Vidal, J. (2009, December 20). Ed Miliband: China tried to hijack Copenhagen climate deal. The Guardian. https://www.theguardian.com/environment/2009/ dec/20/ed‐miliband‐china‐copenhagen‐summit Mertha, A. (2009). “Fragmented authoritarianism 2.0”: Political pluralization in the Chinese policy process. The China Quarterly, 200, 995–1012. Wang, B. (2018). China’s transition on climate change communication and governance: From zero to hero. Social Sciences Academic Press (China). Moore, T. G., & Yang, D. (2001). Empowered and restrained: Chinese foreign policy in the age of eco‐ nomic interdependence. In D. M. Lampton (Ed.), The making of Chinese foreign and security policy in the era of reform, 1978–2000 (pp. 191–229). Stanford University Press. Westman, L. K., Broto, V. C., & Huang, P. (2019). Revisit‐ ing multi‐level governance theory: Politics and inno‐ vation in the urban climate transition in Rizhao, China. Political Geography, 70, 14–23. Williams, L. (2014). China’s climate change policies: Actors and drivers. The Lowy Institute for Interna‐ tional Policy. https://www.lowyinstitute.org/ publications/chinas‐climate‐change‐policies‐actors‐ and‐drivers Motive Power. (2021). Carbon neutral goals by country. https://www.motive‐power.com/npuc‐resource/ carbon‐neutral‐goals‐by‐country O’Brien, K. J., & Li, L. (2006). Rightful resistance in rural China. Cambridge University Press. Wu, F. (2017, March 30). From Paris to Beijing: China, next champion on climate change? Eurasia Review. https://www.eurasiareview.com/30032017‐from‐ paris‐to‐beijing‐china‐next‐champion‐on‐climate‐ change‐analysis Ostrom, E. (2009). A polycentric approach for coping with climate change. The World Bank. Oxford Analytica. (2020). China’s climate policy reaches critical juncture. Emerald. https://www.emerald. com/insight/content/doi/10.1108/OXAN‐ DB253910/full/html Wu, X. (2018). China in search of a liberal partner‐ ship international order. International Affairs, 94(5), 995–1018. Putnam, R. D. (1988). Diplomacy and domestic politics: The logic of two‐level games. International Organiza‐ tion, 43(3), 427–460. Zelli, F., & van Asselt, H. (2015). Fragmentation. In K. Bäckstrand & E. Lövbrand (Eds.), Research handbook on climate governance (pp. 121–131). Edward Elgar. Ramezani, A., & Kamali, Y. (2021). China–Japan eco‐ nomic cooperation with ASEAN: Keohane’s interna‐ tional regime theory. East Asian Policy, 13(1), 80–93. Zhang, C. (2017). Review essay: How to merge western theories and Chinese indigenous theories to study Chinese politics? References Journal of Chinese Political Science, 22(2), 283–294. Robinson, T. W. (1994). Chinese foreign policy from the 1940s to the 1990s. In T. W. Robinson & D. Sham‐ baugh (Eds.), Chinese foreign policy: Theory and prac‐ tice (pp. 555–602). Clarendon Press. Zhang, H. (2013). China and international climate change negotiations. WeltTrends Online. http://welttrends. de/res/uploads/Zhang_China‐and‐International‐ climate‐change‐negotiations.pdf Schreurs, M. (2017). Multi‐level climate governance in China. Environmental Policy and Governance, 27(2), 163–174. Zhou, W., & Zhuang, G. (2021). The global climate gover‐ nance after the United States officially rejoining the Paris Agreement: Competing for leadership or seek‐ ing global consensus? Pacific Journal, 9, 17–29. Shen, W., & Xie, L. (2018). Can China lead in multilateral environmental negotiations? Internal politics, self‐ depiction, and China’s contribution in climate change regime and Mekong governance. Eurasian Geogra‐ phy and Economics, 59(5‐6), 708–732. Zhuang, G., Bo, F., & Zhang, J. (2018). China’s role and strategic choice in global climate governance. World Economics and Politics, 4, 4–27. Stalley, P. (2015). China. In K. Bäckstrand & E. Lövbrand (Eds.), Research handbook on climate governance About the Author Hao Zhang is a PhD candidate at the International Institute of Social Studies (ISS), Erasmus University Rotterdam (EUR). She is also a consultant to the China Civil Climate Action Network (CCAN). Before joining ISS, she was a master’s student majoring in international affairs at the School of Global Policy and Strategy at the University of California, San Diego. Her current research focuses on the policy advocacy of Chinese NGOs in global climate governance. Her research interests lie in Chinese politics, international climate politics, and diplomacy. Politics and Governance, 2022, Volume 10, Issue 2, Pages 50–60 60
https://openalex.org/W2956108627	https://europepmc.org/articles/pmc6606812?pdf=render	English	null	Allergic Airway Disease Prevents Lethal Synergy of Influenza A Virus-Streptococcus pneumoniae Coinfection	MBio	2,019	cc-by	12,611	July/August 2019 Volume 10 Issue 4 e01335-19 RESEARCH ARTICLE Host-Microbe Biology RESEARCH ARTICLE Host-Microbe Biology crossm Allergic Airway Disease Prevents Lethal Synergy of Inﬂuenza A Virus-Streptococcus pneumoniae Coinfection Received 22 May 2019 Accepted 28 May 2019 Published 2 July 2019 ® mbio.asm.org 1 Citation Roberts S, Salmon SL, Steiner DJ, Williams CM, Metzger DW, Furuya Y. 2019. Allergic airway disease prevents lethal synergy of inﬂuenza A virus-Streptococcus pneumoniae coinfection. mBio 10:e01335-19. https://doi .org/10.1128/mBio.01335-19. Editor Keith P. Klugman, Emory University Copyright © 2019 Roberts et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Yoichi Furuya, furuyay@amc.edu. Received 22 May 2019 Accepted 28 May 2019 Published 2 July 2019 Citation Roberts S, Salmon SL, Steiner DJ, Williams CM, Metzger DW, Furuya Y. 2019. Allergic airway disease prevents lethal synergy of inﬂuenza A virus-Streptococcus pneumoniae coinfection. mBio 10:e01335-19. https://doi .org/10.1128/mBio.01335-19. Editor Keith P. Klugman, Emory University Copyright © 2019 Roberts et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Yoichi Furuya, furuyay@amc.edu. Received 22 May 2019 Accepted 28 May 2019 Published 2 July 2019 ® bi 1 KEYWORDS Streptococcus pneumoniae, coinfection, inﬂuenza, interferon gamma Allergic Airway Disease Prevents Lethal Synergy of Inﬂuenza A Virus-Streptococcus pneumoniae Coinfection aDepartment of Immunology and Microbial Disease, Albany Medical College, Albany, New York, USA ABSTRACT Fatal outcomes following inﬂuenza infection are often associated with secondary bacterial infections. Allergic airway disease (AAD) is known to inﬂuence se- vere complications from respiratory infections, and yet the mechanistic effect of AAD on inﬂuenza virus-Streptococcus pneumoniae coinfection has not been investigated previ- ously. We examined the impact of AAD on host susceptibility to viral-bacterial coinfec- tions. We report that AAD improved survival during coinfection when viral-bacterial chal- lenge occurred 1 week after AAD. Counterintuitively, mice with AAD had signiﬁcantly deceased proinﬂammatory responses during infection. Speciﬁcally, both CD4 and CD8 T cell interferon gamma (IFN-) responses were suppressed following AAD. Resis- tance to coinfection was also associated with strong transforming growth factor 1 (TGF-1) expression and increased bacterial clearance. Treatment of AAD mice with IFN- or genetic deletion of TGF- receptor II expression reversed the protective effects of AAD. Using a novel triple-challenge model system, we show for the ﬁrst time that AAD can provide protection against inﬂuenza virus-S. pneumoniae coinfection through the production of TGF- that suppresses the inﬂuenza virus-induced IFN- response, thereby preserving antibacterial immunity. IMPORTANCE Asthma has become one of the most common chronic diseases and has been identiﬁed as a risk factor for developing inﬂuenza. However, the impact of asthma on postinﬂuenza secondary bacterial infection is currently not known. Here, we developed a novel triple-challenge model of allergic airway disease, primary in- ﬂuenza infection, and secondary Streptococcus pneumoniae infection to investigate the impact of asthma on susceptibility to viral-bacterial coinfections. We report for the ﬁrst time that mice recovering from acute allergic airway disease are highly re- sistant to inﬂuenza-pneumococcal coinfection and that this resistance is due to inhi- bition of inﬂuenza virus-mediated impairment of bacterial clearance. Further charac- terization of allergic airway disease-associated resistance against postinﬂuenza secondary bacterial infection may aid in the development of prophylactic and/or therapeutic treatment against coinfection. Citation Roberts S, Salmon SL, Steiner DJ, Williams CM, Metzger DW, Furuya Y. 2019. Allergic airway disease prevents lethal synergy of inﬂuenza A virus-Streptococcus pneumoniae coinfection. mBio 10:e01335-19. https://doi .org/10.1128/mBio.01335-19. Editor Keith P. Klugman, Emory University Copyright © 2019 Roberts et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Yoichi Furuya, furuyay@amc.edu. RESULTS Allergic airway disease confers protection against postinﬂuenza secondary bacterial infection. To investigate the impact of AAD on viral-bacterial coinfection, we developed a triple-challenge mouse model of AAD, primary inﬂuenza infection, and secondary bacterial infection (Fig. 1A). Using this model, it was ﬁrst established that inﬂuenza H1N1 A/California/4/2009 (CA04) virus-infected, non-AAD mice had defective A66.1 pneumococcal clearance at day 1 after bacterial infection (Fig. 1B), an observation consistent with our previous publications (4, 10, 16–18) and those of others (5–7, 9, 19, 20). In contrast, bacterial burden was undetectable in coinfected OVA-AAD mice and was comparable to that in control mice that were infected with S. pneumoniae alone (Fig. 1B). However, no differences in viral burden were observed in non-AAD versus OVA-AAD mice (Fig. 1C). Survival analysis showed that coinfection was lethal in non-AAD mice but not in OVA-AAD mice (Fig. 1D). This enhanced survival was not unique to the CA04 and A66.1 challenge strains, as OVA-AAD also provided protection against coinfection involving H1N1 A/Puerto Rico/8/1934 (PR8) viral or D39 bacterial strains (Fig. 1E and F). Additionally, OVA-AAD had a positive, albeit not statistically signiﬁcant, effect on survival during secondary methicillin-resistant Staphylococcus aureus (MRSA) infection (Fig. 1G). The improved survival against viral-bacterial coinfec- tion was also not dependent on the order of viral and bacterial infections, as OVA-AAD mice were also resistant to bacterial-viral coinfection (see Fig. S1 in the supplemental material). Consistent with the OVA-AAD model, HDM-AAD mice were also resistant to postinﬂuenza secondary bacterial challenge (Fig. S2). Importantly, the C57BL/6 mouse strain likewise exhibited improved survival during viral-bacterial coinfection following induction of OVA-AAD (Fig. S3). Thus, the positive effect of AAD was not restricted to a particular mouse strain. Based on these results, we conclude that AAD mice are resistant to inﬂuenza-bacterial coinfection and that this resistance is due to intact bacterial clearance. AAD mice have reduced inﬂammatory cytokine responses during infection. AAD is a predominantly T helper 2 (Th2) cell-driven disease, and it is widely recognized that Th2-type cytokines play a critical role in initiating and amplifying the inﬂammatory response during AAD (21). Given that Th2-type cytokine production during inﬂuenza infection is linked to asthma exacerbation (22), we investigated if infection superim- posed on AAD mice would have an additive or synergistic effect on inﬂammation (Fig. 2A and Fig. S4A). KEYWORDS Streptococcus pneumoniae, coinfection, inﬂuenza, interferon gamma B B oth Streptococcus pneumoniae and inﬂuenza virus infections are leading causes of morbidity and mortality worldwide. A synergistic relationship between these two pathogens is well documented, as the majority of deaths during the 1918 inﬂuenza pandemic were attributed to secondary complications from S. pneumoniae infection (1, 2). Recent studies have begun to address why inﬂuenza patients become more sus- ceptible to secondary bacterial infections (3–10), but we are far from having a complete understanding of these superinfections. Even less understood are how we can prevent increased susceptibility and what immune responses are required for protection. Editor Keith P. Klugman, Emory University Copyright © 2019 Roberts et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. 2). Recent studies have begun to address why inﬂuenza patients become more sus- ceptible to secondary bacterial infections (3–10), but we are far from having a complete understanding of these superinfections. Even less understood are how we can prevent increased susceptibility and what immune responses are required for protection. The effect of asthma on mucosal immunity against respiratory pathogens has not been adequately addressed. During the inﬂuenza pandemic of 2009, asthma was the most common risk factor associated with morbidity among patients hospitalized with ® mbio.asm.org 1 ® Roberts et al. inﬂuenza (11). Interestingly, although asthma was associated with a higher hospital admission rate during inﬂuenza, hospitalized asthmatics were less likely to develop severe disease or to die than nonasthmatics (12, 13). It is possible that in the clinical setting, other factors, such as earlier hospital admission due to asthma exacerbation and the preadmission use of inhaled corticosteroids, positively inﬂuenced the disease outcome in asthmatic patients during the 2009 inﬂuenza pandemic (12). In addition, we recently reported using a mouse model of allergic airway disease (AAD) followed by inﬂuenza virus infection that AAD can be either beneﬁcial or detrimental depending on the timing of viral and allergic challenge (14, 15). However, the susceptibility of AAD mice was assessed in the absence of secondary bacterial infection. As mentioned above, secondary bacterial infection is an important cause of mortality and morbidity during inﬂuenza pandemics. In this work, we examined the inﬂuence of AAD on viral-bacterial coinfections, an approach that has not been documented previously. Our results demonstrate that ovalbumin (OVA)- or house dust mite (HDM)-mediated AAD confers resistance against inﬂuenza-S. pneumoniae coinfection in a mouse model. KEYWORDS Streptococcus pneumoniae, coinfection, inﬂuenza, interferon gamma Counterintuitively, preceding AAD was associated with suppressed inﬂuenza-induced inﬂammation, and protection was dependent upon AAD-induced transforming growth factor (TGF-) production. July/August 2019 Volume 10 Issue 4 e01335-19 RESULTS As expected, in the absence of infection, Th2- but not Th1-type cytokines were highly upregulated at day 0 after the last allergen inoculation (Fig. S4B). By day 7 after the last HDM challenge, the levels of all cytokines were below the limit of detection (Fig. S4B). In the presence of viral-bacterial coinfection, both Th1-type and July/August 2019 Volume 10 Issue 4 e01335-19 mbio.asm.org 2 ® Impact of Allergic Airway Disease on Coinfection FIG 1 AAD mice are resistant to inﬂuenza-S. pneumoniae coinfection. (A) Schematic diagram of the OVA-induced AAD and inﬂuenza-S. pneumoniae (S.p) coinfection protocol. (B) BALF was harvested, and bacterial burden was assessed 1 day after S. pneumoniae A66.1 infection (n 4 to 5 mice/group). The horizontal dotted line is the limit of detection. (C) The CA04 viral burden in BALF was assessed 1 day after secondary bacterial challenge by a standard plaque assay (n 5 mice/group). The dotted line is the limit of detection. (D to F) OVA-AAD and non-AAD mice were singly infected or coinfected with 10 PFU of CA04 or PR8 and 2 102 CFU of S. pneumoniae A66.1 or 1.5 104 CFU of S. pneumoniae D39. The vertical dotted line indicates bacterial infection. (G) Non-AAD and OVA-AAD mice were coinfected with 10 PFU of CA04 and 1.7 108 CFU of MRSA. Infected mice were monitored for survival for 20 days (n 3 to 13 mice). The vertical dotted line indicates bacterial infection. , P 0.01; , P 0.001. FIG 1 AAD mice are resistant to inﬂuenza-S. pneumoniae coinfection. (A) Schematic diagram of the OVA-induced AAD and inﬂuenza-S. pneumoniae (S.p) coinfection protocol. (B) BALF was harvested, and bacterial burden was assessed 1 day after S. pneumoniae A66.1 infection (n 4 to 5 mice/group). The horizontal dotted line is the limit of detection. (C) The CA04 viral burden in BALF was assessed 1 day after secondary bacterial challenge by a standard plaque assay (n 5 mice/group). The dotted line is the limit of detection. (D to F) OVA-AAD and non-AAD mice were singly infected or coinfected with 10 PFU of CA04 or PR8 and 2 102 CFU of S. pneumoniae A66.1 or 1.5 104 CFU of S. pneumoniae D39. The vertical dotted line indicates bacterial infection. (G) Non-AAD and OVA-AAD mice were coinfected with 10 PFU of CA04 and 1.7 108 CFU of MRSA. July/August 2019 Volume 10 Issue 4 e01335-19 RESULTS Infected mice were monitored for survival for 20 days (n 3 to 13 mice). The vertical dotted line indicates bacterial infection. , P 0.01; , P 0.001. FIG 1 AAD mice are resistant to inﬂuenza-S. pneumoniae coinfection. (A) Schematic diagram of the OVA-induced AAD and inﬂuenza-S. pneumoniae (S.p) coinfection protocol. (B) BALF was harvested, and bacterial burden was assessed 1 day after S. pneumoniae A66.1 infection (n 4 to 5 mice/group). The horizontal dotted line is the limit of detection. (C) The CA04 viral burden in BALF was assessed 1 day after secondary bacterial challenge by a standard plaque assay (n 5 mice/group). The dotted line is the limit of detection. (D to F) OVA-AAD and non-AAD mice were singly infected or coinfected with 10 PFU of CA04 or PR8 and 2 102 CFU of S. pneumoniae A66.1 or 1.5 104 CFU of S. pneumoniae D39. The vertical dotted line indicates bacterial infection. (G) Non-AAD and OVA-AAD mice were coinfected with 10 PFU of CA04 and 1.7 108 CFU of MRSA. Infected mice were monitored for survival for 20 days (n 3 to 13 mice). The vertical dotted line indicates bacterial infection. , P 0.01; **, P 0.001. Th2-type cytokines were highly upregulated in non-AAD mice (Fig. 2B and Fig. S4C). In contrast, with the exception of interleukin-5 (IL-5), these cytokine responses were signiﬁcantly reduced in OVA- and HDM-AAD mice. Recent studies have demonstrated that inﬂuenza-induced inﬂammatory cytokines are central mediators of immune sup- pression after inﬂuenza infection (4–6, 8, 9, 23–25). The altered inﬂammatory response mbio.asm.org 3 July/August 2019 Volume 10 Issue 4 e01335-19 ® Roberts et al. FIG 2 Cytokine responses are suppressed in AAD mice. (A) Experimental setup. (B) Pulmonary cytokine levels in WT BALB/c mice were quantiﬁed day 1 after secondary bacterial challenge (n 4 to 10 mice/group). , P 0.05; , P 0.01; , P 0001; **, P 0.0001. FIG 2 Cytokine responses are suppressed in AAD mice. (A) Experimental setup. (B) Pulmonary cytokine levels in WT BALB/c mice were quantiﬁed day 1 after secondary bacterial challenge (n 4 to 10 mice/group). , P 0.05; , P 0.01; , P 0001; **, P 0.0001. during inﬂuenza infection may explain why AAD mice were protected against second- ary bacterial infection. AAD suppresses detrimental IFN- responses. July/August 2019 Volume 10 Issue 4 e01335-19 RESULTS Interferon gamma (IFN-) has been implicated in inﬂuenza virus-induced immune suppression during secondary S. pneu- moniae infection (4, 16, 26, 27). We therefore measured pulmonary IFN- protein concentrations. IFN- responses to viral single infection or viral-bacterial coinfection were signiﬁcantly suppressed in AAD mice (Fig. 3A). In agreement with IFN- protein levels in bronchoalveolar lavage ﬂuid (BALF), numbers of IFN--positive (IFN-) T cells were also suppressed in AAD mice (Fig. 3B and Fig. S5A). Similarly, reduced granzyme B-positive (GzmB) T cell responses were observed in AAD mice (Fig. S5B). We next investigated the potential roles of IFN- in our triple-challenge model of AAD and coinfection. IFN-/ but not non-AAD wild-type (WT) mice were protected against coinfection (Fig. 3C and D and Fig. S5C), consistent with previous reports (4, 16). IFN- deﬁciency had no effect on the survival of AAD mice (Fig. 3C and D and Fig. S5). These observations suggest that while IFN- exerts detrimental effects on non-AAD mice during coinfection, it does not play a protective role in AAD mice. Consistent with the survival data, IFN- deﬁciency prevented inhibition of A66.1 and D39 bacterial clear- ance in coinfected non-AAD mice (Fig. 3E and F). The protected IFN-/ non-AAD mice still expressed higher levels of various cytokines than did IFN-/ AAD mice (Fig. 3G), suggesting that IFN- plays an important role in predisposing the host to secondary bacterial infections. Taken together, these data suggest that AAD mice were protected against secondary bacterial challenge due to suppression of IFN- responses that otherwise disabled antibacterial immunity. To directly demonstrate that suppres- sion of IFN- responses was responsible for resistance against secondary bacterial challenge in AAD mice, we treated AAD mice intranasally (i.n.) with recombinant IFN- to increase pulmonary IFN- levels during coinfection (Fig. 4A). Exogenous IFN- signiﬁcantly impaired early bacterial clearance in AAD mice (Fig. 4B and C). Therefore, the protective mechanism that exists in AAD mice may entail inhibition of detrimental IFN- responses. July/August 2019 Volume 10 Issue 4 e01335-19 mbio.asm.org 4 Alveolar macrophage-mediated bacterial clearance is intact in AAD mice. Since innate immune cells are involved in early bacterial clearance we next characterized the FIG 3 Detrimental IFN- is suppressed during infection in AAD mice. (A) IFN- levels in BALF samples harvested 1 day after secondary bacterial challenge (n 4 to 10 mice/group). RESULTS (B) Numbers of pulmonary IFN- CD4 or CD8 T cells at day 8 after inﬂuenza infection (n 4 to 5 mice/group). (C and D) Survival analysis of WT BALB/c and IFN-/ mice singly infected or coinfected with CA04 and D39 (C) or A66.1 (D) (n 4 to 14 mice/group). Vertical dotted lines indicate bacterial infection. (E and F) Pulmonary bacterial burdens 1 day after single infection or coinfection in WT BALB/c or IFN-/ mice (n 3 to 5 mice/group). Horizontal dotted lines indicate the limit of detection. (G) Pulmonary cytokine levels in IFN-/ mice at day 1 after secondary bacterial challenge (n 7 to 9 mice/group). The dotted line is the limit of detection. , P0.05; , P0.01; *, P 0.0001; NS, not signiﬁcant. Impact of Allergic Airway Disease on Coinfection ® Impact of Allergic Airway Disease on Coinfection FIG 3 Detrimental IFN- is suppressed during infection in AAD mice. (A) IFN- levels in BALF samples harvested 1 day after secondary bacterial challenge (n 4 to 10 mice/group). (B) Numbers of pulmonary IFN- CD4 or CD8 T cells at day 8 after inﬂuenza infection (n 4 to 5 mice/group). (C and D) Survival analysis of WT BALB/c and IFN-/ mice singly infected or coinfected with CA04 and D39 (C) or A66.1 (D) (n 4 to 14 mice/group). Vertical dotted lines indicate bacterial infection. (E and F) Pulmonary bacterial burdens 1 day after single infection or coinfection in WT BALB/c or IFN-/ mice (n 3 to 5 mice/group). Horizontal dotted lines indicate the limit of detection. (G) Pulmonary cytokine levels in IFN-/ mice at day 1 after secondary bacterial challenge (n 7 to 9 mice/group). The dotted line is the limit of detection. , P0.05; , P0.01; *, P 0.0001; NS, not signiﬁcant. July/August 2019 Volume 10 Issue 4 e01335-19 Alveolar macrophage-mediated bacterial clearance is intact in AAD mice. Since innate immune cells are involved in early bacterial clearance, we next characterized the pulmonary cellular environment to elucidate the role of innate immune cells in AAD-mediated resistance to secondary bacterial infection. Flow cytometric analysis Alveolar macrophage-mediated bacterial clearance is intact in AAD mice. Since innate immune cells are involved in early bacterial clearance, we next characterized the pulmonary cellular environment to elucidate the role of innate immune cells in AAD-mediated resistance to secondary bacterial infection. Flow cytometric analysis mbio.asm.org 5 July/August 2019 Volume 10 Issue 4 e01335-19 ® Roberts et al. FIG 4 IFN- treatment compromises bacterial clearance in AAD mice. (A) Amount of IFN- in BALF 4 h after secondary bacterial challenge. Mice were i.n. treated with PBS or recombinant IFN- prior to a bacterial infection (n 5 mice/group). (B and C) Pulmonary bacterial load, expressed as CFU per milliliter of BALF (B) or as a percentage of the input (C), 4 h after secondary bacterial challenge (n 5 mice/group). The dotted line is the limit of detection , P 0.05; *, P 0.001; *, P 0.0001. FIG 4 IFN- treatment compromises bacterial clearance in AAD mice. (A) Amount of IFN- in BALF 4 h after secondary bacterial challenge. Mice were i.n. treated with PBS or recombinant IFN- prior to a bacterial infection (n 5 mice/group). (B and C) Pulmonary bacterial load, expressed as CFU per milliliter of BALF (B) or as a percentage of the input (C), 4 h after secondary bacterial challenge (n 5 mice/group). The dotted line is the limit of detection , P 0.05; *, P 0.001; *, P 0.0001. showed that on day 7 after AAD, monocyte numbers were marginally increased in uninfected OVA-AAD mice (Fig. S6 and Fig. S7). However, upon CA04 infection, the monocyte numbers were higher in non-AAD mice than in in OVA-AAD mice. Similarly, increased numbers of neutrophils were observed in CA04-infected non-AAD mice. In contrast, levels of eosinophils were consistently higher in OVA-AAD mice on day 7 after AAD and day 8 after CA04 infection. However, eosinophil depletion using anti-IL-5 neutralizing monoclonal antibody (mAb) did not impact the survival of coinfected AAD mice (data not shown), thus eliminating the role of eosinophils in our observed protection in AAD mice. Finally, no striking differences in alveolar macrophage numbers were observed among non-AAD and OVA-AAD mice before or after CA04 infection. Based on these ﬂow cytometry analyses, we conclude that AAD does not increase pulmonary cell numbers that may contribute to the resistance of AAD mice. July/August 2019 Volume 10 Issue 4 e01335-19 Alveolar macrophage-mediated bacterial clearance is intact in AAD mice. Since innate immune cells are involved in early bacterial clearance, we next characterized the pulmonary cellular environment to elucidate the role of innate immune cells in AAD-mediated resistance to secondary bacterial infection. Flow cytometric analysis We have previously reported that alveolar macrophages play an important role in early bacterial clearance (4, 28, 29). Indeed, depletion of alveolar macrophages during coinfection of AAD mice signiﬁcantly reduced survival (Fig. 5A and Fig. S8A). Consistent with the survival data, alveolar macrophage depletion signiﬁcantly increased the bacterial burden in coinfected OVA-AAD mice (Fig. 5B). It is important to note that the bacterial burden measured in clodronate-treated AAD mice was comparable to that in non-AAD mice treated with clodronate. This suggests that the observed protection in AAD mice was dependent on alveolar macrophages. The data described above dem- onstrated that IFN- played a detrimental role during coinfection and that AAD suppressed this response. We next determined if macrophages are the primary target of IFN--mediated immune suppression by utilizing mice insensitive to IFN- (MIIG) (30). MIIG mice have a truncation in the IFN- receptor gene in CD68 cells, rendering macrophages nonresponsive to IFN-. IFN- signaling deﬁciency in macrophages sig- niﬁcantly improved the survival of coinfected non-AAD mice but had no deleterious effect on AAD mice (Fig. 5C and D and Fig. S8B). Similarly, IFN- signaling deﬁciency in macrophages resulted in fewer pulmonary bacteria in non-AAD mice although not to a statistically signiﬁcant extent (Fig. 5E). We next assessed whether the difference in bacterial clearance was due to differences in macrophage-dependent phagocytosis. We measured the expression levels of mannose receptor (MR), a pattern recognition receptor that mediates nonopsonic phagocytosis by macrophages (31). CA04 infection signiﬁcantly reduced mannose receptor expression in non-AAD mice (Fig. 5F to H). This reduction was absent in IFN-/ mice, indicating that inﬂuenza-induced IFN- medi- ates the downregulation of mannose receptor expression. OVA-AAD mice also main- tained baseline expression of mannose receptor following CA04 infection, likely due to the absence of IFN- responses. Consistent with these phenotypic data, an in vivo phagocytosis assay revealed that alveolar macrophages of OVA-AAD mice maintained intact phagocytic capacity during inﬂuenza infection, while non-AAD alveolar macro- mbio.asm.org 6 ® Impact of Allergic Airway Disease on Coinfection FIG 5 The inﬂuenza-induced defect in alveolar macrophages is absent in AAD mice. (A and B) CA04-infected mice were i.n. treated with clodronate liposomes (L-Clon) or PBS liposomes (L-PBS) on day 7 after inﬂuenza infection. CA04-infected, liposome-treated mice were coinfected with A66.1 on day 8 after inﬂuenza infection. (A) Mice were monitored for survival (n 5 to 8 mice/group). The vertical dotted line indicates bacterial infection. Alveolar macrophage-mediated bacterial clearance is intact in AAD mice. Since innate immune cells are involved in early bacterial clearance, we next characterized the pulmonary cellular environment to elucidate the role of innate immune cells in AAD-mediated resistance to secondary bacterial infection. Flow cytometric analysis (B) Additional mice were euthanized for assessment of bacterial burden (n 8 mice/group). The limit of detection was 1 102 CFU/ml. (C and D) WT and MIIG mice were OVA treated and coinfected as described in the legend of Fig. 1A, using A66.1 (C) or D39 (D) (n 5 to 9 mice/group). Vertical dotted lines indicate bacterial infection. (E) Bacterial numbers in BALF harvested at day 1 after secondary bacterial infection (n 4 to 10 mice/group). The limit of detection was 1 102 CFU/ml. (F to H) Flow cytometric analysis of mannose receptor (MR) expression on BALF CD11chi CD11blo Ly6G cells. (F to H) Representative histograms (F), median ﬂuores- cence intensity (MFI) (G), and fold change in MFI over the PBS control (H) of MR expression at day 8 after CA04 infection (n 5 to 7 mice/group). (I and J) Mock-infected or CA04-infected mice were i.n. inoculated with FITC-labeled latex beads on day 8 after inﬂuenza infection and BALF cells were harvested for ﬂow cytometric analysis Representative histograms FIG 5 The inﬂuenza-induced defect in alveolar macrophages is absent in AAD mice. (A and B) CA04-infected mice were i.n. treated with clodronate liposomes (L-Clon) or PBS liposomes (L-PBS) on day 7 after inﬂuenza infection. CA04-infected, liposome-treated mice were coinfected with A66.1 on day 8 after inﬂuenza infection. (A) Mice were monitored for survival (n 5 to 8 mice/group). The vertical dotted line indicates bacterial infection. (B) Additional mice were euthanized for assessment of bacterial burden (n 8 mice/group). The limit of detection was 1 102 CFU/ml. (C and D) WT and MIIG mice were OVA treated and coinfected as described in the legend of Fig. 1A, using A66.1 (C) or D39 (D) (n 5 to 9 mice/group). Vertical dotted lines indicate bacterial infection. (E) Bacterial numbers in BALF harvested at day 1 after secondary bacterial infection (n 4 to 10 mice/group). The limit of detection was 1 102 CFU/ml. (F to H) Flow cytometric analysis of mannose receptor (MR) expression on BALF CD11chi CD11blo Ly6G cells. (F to H) Representative histograms (F), median ﬂuores- cence intensity (MFI) (G), and fold change in MFI over the PBS control (H) of MR expression at day 8 after CA04 infection (n 5 to 7 mice/group). (I and J) Mock-infected or CA04-infected mice were i.n. July/August 2019 Volume 10 Issue 4 e01335-19 Alveolar macrophage-mediated bacterial clearance is intact in AAD mice. Since innate immune cells are involved in early bacterial clearance, we next characterized the pulmonary cellular environment to elucidate the role of innate immune cells in AAD-mediated resistance to secondary bacterial infection. Flow cytometric analysis inoculated with FITC-labeled latex beads on day 8 after inﬂuenza infection, and BALF cells were harvested for ﬂow cytometric analysis. Representative histograms (C i d ) (Continued on next page) mbio.asm.org 7 July/August 2019 Volume 10 Issue 4 e01335-19 ® Roberts et al. phages had reduced bead uptake after CA04 infection (Fig. 5I and J). Furthermore, to evaluate the inﬂuence of inﬂuenza virus on S. pneumoniae killing activities, BALF cells were harvested on day 8 after CA04 infection and incubated with live bacteria. Bacterial burdens in culture supernatants showed that BALF cells from OVA-AAD mice can better clear S. pneumoniae in vitro than BALF cells from non-AAD mice (Fig. 5K). Collectively, these data suggest that inﬂuenza virus disables antibacterial functions of alveolar macrophages via IFN- signaling and that this detrimental immune pathway is absent in AAD mice due to suppression of IFN- responses. Correlation between upregulation of anti-inﬂammatory TGF-1 and absence of detrimental IFN- during AAD. We previously showed that TGF- promotes survival of AAD mice during lethal inﬂuenza infection (14). To investigate if TGF- also plays a role during coinfection, we determined whether there was a correlation between upregulation of TGF-, suppression of IFN-, and improved survival. For this, AAD mice were examined at two time points after induction of AAD: week 1 versus week 6 after AAD (Fig. 6A and Fig. S9A). Consistent with our previous report (14), pulmonary TGF-1 expression was highly upregulated at week 1 after AAD but returned to baseline levels at week 6 (Fig. 6B and Fig. S9B). A slight increase in TGF-2 expression was observed in HDM-AAD mice at week 6 but not in OVA-AAD mice. TGF-3 levels were unchanged in both OVA- and HDM-AAD mice. Following inﬂuenza infection, TGF-1 levels were comparable between non-AAD and OVA-AAD mice (Fig. 6C). Next, AAD mice were infected with CA04 virus at week 1 or 6 for IFN- analysis. Robust pulmonary IFN- T cell responses were observed in non-AAD mice as well as in week 6 post-OVA-AAD mice but were absent in week 1 post-OVA-AAD mice (Fig. 6D and E). These ﬂow cytometry data were conﬁrmed by a cytokine enzyme-linked immunosorbent assay (ELISA): CA04 infection elicited strong BALF IFN- expression in non-AAD mice and week 6 post-OVA- AAD mice but not in week 1 post-OVA-AAD mice (Fig. 6F). As expected, AAD-mediated protection against coinfection was completely lost at week 6 (Fig. 6G and Fig. S9C). July/August 2019 Volume 10 Issue 4 e01335-19 Alveolar macrophage-mediated bacterial clearance is intact in AAD mice. Since innate immune cells are involved in early bacterial clearance, we next characterized the pulmonary cellular environment to elucidate the role of innate immune cells in AAD-mediated resistance to secondary bacterial infection. Flow cytometric analysis This loss of protection correlated with increased bacterial burden but not viral burden (Fig. 6H). The above-described data indicate that TGF-1 is responsible for the sup- pressed IFN- cytokine milieu in the lungs of AAD mice, which in turn decreases susceptibility to secondary bacterial infections after inﬂuenza infection. TGF-RII signaling mediates suppression of IFN- and protection against sec- ondary bacterial challenge. To determine whether there was a causal relationship between the above-described observations, conditional TGF- receptor II (TGF-RII)- deﬁcient mice were used. Loss of TGF-RII during coinfection signiﬁcantly increased the expression of various cytokines, including IFN-, in OVA-AAD mice (Fig. 7A). While TGF-RII deﬁciency had a minimal impact on survival during single infection, a signif- icant reduction in survival of coinfected AAD mice was observed (Fig. 7B and Fig. S9D). TGF-RII deﬁciency also caused a signiﬁcant increase in the bacterial burden during coinfection in OVA-AAD mice (Fig. 7C), which likely accounts for the observed mortality. (I) and MFI (J) of the FITC signal on BALF CD11chi CD11blo Ly6G cells are shown. (K) Bacterial burden in vitro after 4 h of incubation with 5 104 BALF cells at an MOI of 1 CFU of A66.1/cell. BALF cells were harvested from non-AAD and OVA-AAD mice at day 8 after inﬂuenza infection (n 4 to 5 mice/group). , P 0.05; , P 0.01; , P 0.001; **, P 0.0001. DISCUSSION This is the ﬁrst study to examine the impact of AAD on viral-bacterial coinfection, using a novel triple-challenge mouse model of AAD, primary inﬂuenza infection, and secondary S. pneumoniae infection. Here, we provide evidence for a novel ﬁnding in AAD mice whereby the host becomes resistant to postinﬂuenza bacterial infection. We conclude that AAD can transiently prevent viral-bacterial lethal synergy and thereby provide a survival advantage during secondary bacterial infection. Using mice with a conditional deletion of TGF-RII, we identiﬁed TGF- as a central mediator of the observed protection. The data strongly suggest, however, that TGF- is not an effector cytokine that directly or indirectly facilitates bacterial clearance. FIG 5 Legend (Continued) (I) and MFI (J) of the FITC signal on BALF CD11chi CD11blo Ly6G cells are shown. (K) Bacterial burden in vitro after 4 h of incubation with 5 104 BALF cells at an MOI of 1 CFU of A66.1/cell. BALF cells were harvested from non-AAD and OVA-AAD mice at day 8 after inﬂuenza infection (n 4 to 5 mice/group). , P 0.05; , P 0.01; , P 0.001; *, P 0.0001. mbio.asm.org 8 ® Impact of Allergic Airway Disease on Coinfection FIG 6 Suppressed IFN- responses are inversely associated with TGF-1 responses. (A) Diagram depicting the timeline of OVA treatment and viral-bacterial coinfection. (B) TGF- levels in BALF at week 1 or 6 after OVA challenge (n 4 to 5 mice/group). (C) TGF-1 levels in BALF in PBS-treated or CA04-infected mice at day 8 after inﬂuenza infection (n 9 mice/group). (D and E) Flow cytometric analysis of IFN- CD4 T cells (D) and IFN- CD8 T cells (E) in BALF at day 8 after inﬂuenza infection (n 5 to 8 mice/group). (F) Amount of IFN- in BALF at day 8 after inﬂuenza infection (n 5 to 8 mice/group). (G) Survival analysis of OVA-AAD mice coinfected at week 1 or 6 after OVA challenge (n 5 mice/group). The vertical dotted line indicates bacterial infection. (H) Viral and bacterial loads in OVA-AAD mice coinfected at week 1 or 6 after OVA challenge (n 5 mice/group). The dotted line is the limit of detection. , P 0.01; *, P 0.001; , P 0.0001. FIG 6 Suppressed IFN- responses are inversely associated with TGF-1 responses. (A) Diagram depicting the timeline of OVA treatment and viral-bacterial coinfection. (B) TGF- levels in BALF at week 1 or 6 after OVA challenge (n 4 to 5 mice/group). (C) TGF-1 levels in BALF in PBS-treated or CA04-infected mice at day 8 after inﬂuenza infection (n 9 mice/group). (D and E) Flow cytometric analysis of IFN- CD4 T cells (D) and IFN- CD8 T cells (E) in BALF at day 8 after inﬂuenza infection (n 5 to 8 mice/group). (F) Amount of IFN- in BALF at day 8 after inﬂuenza infection (n 5 to 8 mice/group). (G) Survival analysis of OVA-AAD mice coinfected at week 1 or 6 after OVA challenge (n 5 mice/group). FIG 5 Legend (Continued) The vertical dotted line indicates bacterial infection. (H) Viral and bacterial loads in OVA-AAD mice coinfected at week 1 or 6 after OVA challenge (n 5 mice/group). The dotted line is the limit of detection. , P 0.01; *, P 0.001; *, P 0.0001. Rather, TGF- reverses or prevents inﬂuenza virus-induced inhibition of pulmonary bacterial clearance. In support of this, i.n. treatment with recombinant mouse IFN- renders AAD mice unable to effectively control bacterial replication despite having high levels of TGF-. Additionally, AAD-associated protection was lost following depletion of alveolar macrophages, a primary effector cell type responsible for early bacterial clearance. It is important to note that the bacterial burden measured in macrophage- depleted AAD mice was comparable to that in macrophage-depleted or -undepleted non-AAD mice. This suggests that the observed protection in AAD mice is dependent on intact alveolar macrophages and that AAD does not enhance other antibacterial pathways that could compensate for the loss of macrophages. In support of this, numbers of other phagocytic cell types, such as monocytes and neutrophils, were found to be reduced in AAD mice. July/August 2019 Volume 10 Issue 4 e01335-19 mbio.asm.org 9 ® Roberts et al. FIG 7 TGF- mediates resistance of AAD mice against secondary bacterial challenge. TRIIf/f-Cre mice and wild-type littermates were OVA treated and coinfected as described in the legend of Fig. 1A. (A) Cytokine analysis at day 1 after secondary bacterial challenge (n 7 to 9 mice/group). GM-CSF, granulocyte-macrophage colony-stimulating factor. (B) Survival analysis of mice with viral infection, bacterial infection, or coinfection (4 to 10 mice/group). The vertical dotted line represents PBS or A66.1 challenge. (C) Bacterial burden at day 1 after secondary bacterial challenge (n 7 to 9 mice/group). The dotted line is the limit of detection. , P 0.05; , P 0.01; , P 0.001. FIG 7 TGF- mediates resistance of AAD mice against secondary bacterial challenge. TRIIf/f-Cre mice and wild-type littermates were OVA treated and coinfected as described in the legend of Fig. 1A. (A) Cytokine analysis at day 1 after secondary bacterial challenge (n 7 to 9 mice/group). GM-CSF, granulocyte-macrophage colony-stimulating factor. (B) Survival analysis of mice with viral infection, bacterial infection, or coinfection (4 to 10 mice/group). The vertical dotted line represents PBS or A66.1 challenge. (C) Bacterial burden at day 1 after secondary bacterial challenge (n 7 to 9 mice/group). July/August 2019 Volume 10 Issue 4 e01335-19 mbio.asm.org 10 FIG 5 Legend (Continued) Given that AAD-induced TGF-1 responses were transient, it was not surprising that AAD-mediated protection was also transient. By week 6 after AAD, TGF-1 levels returned to baseline, and protection against coinfection was completely lost. Similar to our mouse model, TGF- upregulation is inducible and transient in human asthmatics (37). One longitudinal clinical study demonstrated that the concentration of TGF-1 in BALF returns to baseline within 1 week after allergen exposure (38). Thus, it can be predicted that asthmatic episodes must precede viral infection for TGF- to be up- regulated and provide survival beneﬁts against secondary bacterial infection. In support of this, Avila et al. (39) demonstrated that allergic subjects with experimental rhinovirus infection exhibit a signiﬁcantly delayed onset of cold symptoms and a reduced duration of illness if high-dose allergen exposure preceded viral inoculation. Consistent with our previous publication (14), this study also showed that pulmo- nary viral load was not exacerbated by AAD. Other investigators that have relied on a comorbidity mouse model of asthma and inﬂuenza have also reported that a preceding acute allergen challenge does not impair viral clearance (40–44). In fact, it was shown that AAD reduces pulmonary inﬂuenza viral burden (40–44), although the proposed immunological mechanisms are inconsistent among various studies. In support of these ﬁndings, a recent human study using an ex vivo inﬂuenza infection model of bronchial tissue explants demonstrated that viral load was reduced in bronchial biopsy speci- mens derived from asthmatic subjects (45). Thus, our ﬁnding that viral clearance was not impeded by prior AAD is consistent with the literature. Of note, cytolytic T cell responses were detected in our mouse model of AAD following inﬂuenza infection. It is likely that detectable, albeit reduced, levels of antiviral GzmB T cells were sufﬁcient to clear the viral infection given that a low dose of inﬂuenza virus (10 PFU) was used in our coinfection model. Suppression of not only IFN- but also the cytolytic protein GzmB in AAD mice suggests that a preceding AAD leads to a general inhibition of T cell effector functions. Our results indicate that TGF- is playing a role in suppressing effector functions of T cells in AAD mice. FIG 5 Legend (Continued) The dotted line is the limit of detection. , P 0.05; , P 0.01; , P 0.001. It has been shown that proinﬂammatory cytokine responses directed against inﬂu- enza viruses are a key driver of secondary bacterial infections. In particular, IFN- appears to play a central role in suppressing antibacterial immunity during inﬂuenza infection (23). This detrimental IFN- response was found to be signiﬁcantly suppressed during infection of AAD mice. However, deletion of TGF-RII signaling unleashed IFN- responses in AAD mice. This was associated with the outgrowth of S. pneumoniae and the loss of a survival advantage in AAD mice. Based on these observations, it was concluded that AAD-induced TGF- promotes survival during coinfection by suppress- ing detrimental IFN- responses. Furthermore, we investigated the downstream dele- terious effects of IFN- signaling. The use of MIIG mice provided evidence that inﬂuenza-induced IFN- directly interacts with phagocytic cells to suppress antibacte- rial immunity. Thus, we have identiﬁed the protective immune pathway triggered by AAD: transiently heightened levels of TGF- result in suppression of IFN- expression, mbio.asm.org 10 July/August 2019 Volume 10 Issue 4 e01335-19 ® Impact of Allergic Airway Disease on Coinfection and this in turn prevents IFN-–alveolar macrophage interactions that would otherwise inhibit antibacterial immunity and cause enhanced susceptibility to secondary bacterial infections. TGF- is known to be directly activated by microbial enzymes such as neuramini- dase (NA) of inﬂuenza A virus (32–35) and of S. pneumoniae (36). One group recently reported that inﬂuenza A virus NA enhances in vitro bacterial adherence to cultured A549 human lung carcinoma cells, in a TGF- signaling-dependent manner (32). Therefore, it was hypothesized that upregulation of TGF- during inﬂuenza infection promotes secondary bacterial infection in vivo. Indeed, the same group reported that primary inﬂuenza infection enhances group A Streptococcus bacterial burden in the lungs of WT mice but not in the lungs of mice deﬁcient in TGF- signaling (32). Whether inﬂuenza virus NA-activated TGF- could play a similar role in inﬂuenza-S. pneumoniae coinfection is unknown. Of note, in our model, TGF- activation was triggered by AAD and preceded inﬂuenza infection. Thus, activation of TGF- prior to inﬂuenza infection may be necessary to exert its protective effect during viral-bacterial coinfection. Our ﬁndings also suggest that the beneﬁt of suppressing IFN- by AAD-induced TGF- outweighs any potential detrimental effect of TGF--mediated bacterial adherence for coinfections involving S. pneumoniae. July/August 2019 Volume 10 Issue 4 e01335-19 FIG 5 Legend (Continued) Given that 29 to 55% of deaths during the 2009 H1N1 pandemic were due to complications from secondary bacterial infections (60–62), it is possible that less severe inﬂuenza outcomes among asthmatics are due to prevention of secondary bacterial infections. Nonetheless, no data are available to support or refute this prediction, since the majority of clinical studies into the role of asthma in infection severity have focused on single pathogens. Only a limited number of studies have investigated the potential interaction between asthma and viral-bacterial coinfection. For example, Kloepfer et al. (63) recently exam- ined coinfection in school-age children with and without asthma and concluded that asthma is not a risk factor for rhinovirus-S. pneumoniae coinfection. Unfortunately, patient samples that were positive for other viruses were excluded from their analysis. Thus, whether asthma is a risk or protective factor for inﬂuenza-S. pneumoniae coin- fection is currently unknown. y A lower threshold of hospitalization for asthmatics has been proposed to explain why asthma was associated with less severe outcomes among hospitalized patients. However, Myles et al. (12) concluded that there was not a lower threshold for hospital admission for asthmatic patients since asthmatic and nonasthmatic patients presented with pneumonia at the time of admission in equal proportions. It was also noted in that clinical report that asthmatics were in fact more likely to exhibit features of severe respiratory compromise at the time of hospital admission. Thus, the improved clinical outcomes of asthmatic patients are unlikely to be the result of milder illness at the time of hospital admission. The same authors further concluded that preadmission steroid use contributed to the association of asthma with less severe clinical outcomes; however, preadmission steroid use was found to be beneﬁcial only in asthmatic patients but not in nonasthmatic patients. This suggests that the preadmission steroid is not inherently protective against inﬂuenza. In addition, the beneﬁt of in- hospitalization systemic steroid therapy is controversial, as some recent studies re- ported that steroid administration results in higher incidences of hospital-acquired bacterial pneumonia and of mortality (64–67). Thus, it is plausible that the immuno- suppressive effects of preadmission steroid use may have predisposed asthmatics to inﬂuenza infections, which would explain why asthma was found to be a risk factor for increased hospitalization due to inﬂuenza. We propose that future studies should investigate the role of corticosteroids, in the context of asthma, in inﬂuencing suscep- tibility to coinfections. FIG 5 Legend (Continued) This hypothesis is based on observations that deletion of TGF-RII signaling signiﬁcantly increased IFN- levels in the lungs of coinfected AAD mice at day 9 after viral infection, a time point that corresponds to the peak of the T cell response in inﬂuenza-infected mice. Furthermore, intracellular staining revealed that the major sources of IFN- during coinfection are CD4 and CD8 T cells in susceptible non-AAD mice. The suppressive effects of TGF- on T cells are well established in cancer immunology (46–49). Substantial evidence exists that TGF- can directly regulate activation, proliferation, differentiation, and survival of T cells. TGF- signaling in T cells is mediated by TGF-RI and -II, and the eventual activation of downstream transcription factors, such as Smad, regulates the T cell phenotype (50). This pleiotropic cytokine can mbio.asm.org 11 July/August 2019 Volume 10 Issue 4 e01335-19 ® Roberts et al. also promote CD4 regulatory T cell (Treg) responses. Whether the suppression of IFN- T cell responses in AAD mice is a direct effect of TGF- on effector T cells or of Treg induction remains to be elucidated. Like CA04 viral infection, preexisting AAD also conferred protection in PR8 virus- infected mice against secondary S. pneumoniae infection. This observation suggests that our result was not due to a unique phenotype of the CA04 virus but rather reﬂected a general phenomenon associated with inﬂuenza A virus infection in AAD mice. Furthermore, CA04-infected AAD mice were also resistant to secondary methicillin-resistant Staphylococcus aureus infection. MRSA is an emerging bacterial pathogen associated with recent seasonal and pandemic inﬂuenza. Thus, our ﬁndings may have broad application to other secondary bacterial pathogens. A better under- standing of the protective immune mechanisms that exist in AAD mice is a signiﬁcant ﬁrst step that could eventually lead to the development of immunomodulation strat- egies to ameliorate detrimental immune responses. The long-standing dogma that asthma is a risk factor for severe inﬂuenza has been challenged by recent clinical studies. Veerapandian et al. (51) conducted a systematic literature review of clinical reports on asthmatic patients during the 2009 pandemic of H1N1 virus and conﬁrmed that asthma was a risk factor for hospitalization. However, the same authors also concluded, based on an overwhelming amount of clinical data (12, 13, 52–59), that asthmatics were less likely to develop severe inﬂuenza, as deﬁned as intensive care unit (ICU) admittance and/or death (51). July/August 2019 Volume 10 Issue 4 e01335-19 FIG 5 Legend (Continued) Such studies could provide a deﬁnitive answer for the contro- versial role of corticosteroids in inﬂuenza-infected asthmatic patients. While epidemiological data derived from the H1N1 pandemic of 2009 support our mbio.asm.org 12 ® Impact of Allergic Airway Disease on Coinfection current ﬁndings, caution is needed in extrapolating data from mice to human disease. Since mice do not naturally develop asthma, the applicability of mouse models of asthma has long been debated. Of particular concern are the lack of irreversible airway remodeling and the lack of chronicity in the acute asthma model (68, 69). As such, short-term models can be used to investigate the impact of severe acute allergic inﬂammation on subsequent respiratory infection but are inadequate for investigation of the relationship between chronic inﬂammation and host susceptibility to pulmonary pathogens. In an attempt to overcome some of the limitations of acute mouse models of asthma, a number of investigators have developed mouse models of chronic asthma by extending the period of allergen challenge (70–73). These chronic mouse models better mimic various features of human airway remodeling and therefore make it possible to study host susceptibility during the chronic phase of asthma in mice. The impact of chronic allergic inﬂammation on inﬂuenza-induced susceptibility to second- ary bacterial infection is under investigation. While informative epidemiological data are greatly lacking, it has been reported by various investigators that asthmatic patients exhibit defective type I IFN (IFN-I) and IFN-II responses during viral infections, as characterized in vivo and ex vivo (74–77). Coincidently, numerous mouse studies of viral-bacterial coinfection have identiﬁed IFN-I and -II as mediators of heightened sensitivity to secondary bacterial challenges (4, 6, 8, 9, 16). Thus, if these cytokines are indeed responsible for predisposing the host to secondary bacterial infections in humans, it can be extrapolated that decreased levels of IFN-I and -II in asthmatic patients would confer some level of protection during coinfection. Further research will be needed to conﬁrm our hypothesis on the role of asthma during viral-bacterial coinfection. The synergistic mechanisms of viral-bacterial coinfections have been investigated by a number of researchers, with the ultimate goal of developing therapeutic approaches to prevent mortality and morbidity. FIG 5 Legend (Continued) Most, if not all, investigators have relied on a mouse model of primary inﬂuenza infection and secondary bacterial infections (4, 6–9, 19, 20, 78–80), with the principal aims of identifying detrimental immune responses in coin- fected mice and of understanding how inﬂuenza virus predisposes mice to secondary bacterial infections. The rationale behind this approach is that understanding the nature of the disadvantageous immune response may enable reversal of the immuno- compromised state. In contrast, the present study focused on understanding protective elements of the immune response by examining mice that are resistant to secondary bacterial infections. Our data showing a remarkable resistance of AAD mice to coin- fection were surprising and now offer a unique opportunity to understand a beneﬁcial immune pathway that may render the host transiently resistant to secondary bacterial infections. To the best of our knowledge, a triple-challenge mouse model of asthma, primary inﬂuenza infection, and secondary pneumococcal infection has not been previously documented in the literature. Further characterization of AAD-associated resistance against viral-bacterial coinfection may aid in the development of prophylac- tic and/or therapeutic treatment against coinfection. July/August 2019 Volume 10 Issue 4 e01335-19 MATERIALS AND METHODS Mice. Adult 6- to 8-week-old BALB/c and C57BL/6 mice were purchased from Charles River Labora- tories through a contract with the National Cancer Institute. BALB/c IFN-/ mice were obtained from Jackson Laboratories (Bar Harbor, ME). C57BL/6 mice with macrophages insensitive to IFN- (MIIG) were previously generated at Cincinnati Children’s Hospital Medical Center (30). TRIIf/f-Cre mice were generated by crossing Floxed TRII (TRIIf/f) and Ubc-CreERT2 (Cre) mice (14). To induce conditional deletion of TRII, mice were injected intraperitoneally (i.p.) with 2 mg of tamoxifen (Sigma-Aldrich) in corn oil (Sigma-Aldrich) once daily for ﬁve consecutive days. Mice were treated with tamoxifen prior to intranasal (i.n.) allergen challenge. Animal care and experimental protocols were in accordance with the NIH Guide for the Care and Use of Laboratory Animals (81) and were approved by the Institutional Animal Care and Use Committee at Albany Medical College (protocol number 17-03006). A triple-challenge mouse model of AAD, primary inﬂuenza infection, and secondary S. pneu- moniae infection. For the OVA-AAD model, mice were immunized i.p. twice with 10 g of OVA in 4 mg of aluminum hydroxide (General Chemical). The sensitized mice were anaesthetized with isoﬂurane and challenged i.n. with 100 g of OVA in phosphate-buffered saline (PBS) once daily for 5 days. For induction of HDM-AAD, mice were anaesthetized and i.n. treated with 50 g of HDM extract (Dermatophagoides mbio.asm.org 13 ® Roberts et al. pteronyssinus; Greer Laboratories) in PBS for three consecutive days every 3 weeks. Control non-AAD mice received 50 l PBS. The AAD or non-AAD mice were infected i.n. with 10 PFU of H1N1 A/California/4/2009 (CA04) virus or H1N1 A/Puerto Rico/8/1934 (PR8) virus and subsequently infected with 2 102 CFU of the S. pneumoniae serotype 3 A66.1 strain, 1.5 104 CFU of the S. pneumoniae serotype 2 D39 strain, or 2 108 CFU of methicillin-resistant Staphylococcus aureus (MRSA) strain USA300 at day 8 after inﬂuenza infection. Coinfection was routinely performed on week 1 after the last i.n. treatment, unless otherwise stated. This time point was chosen to minimize the unintended effects of i.n. PBS treatment while allowing investigation of the impact of AAD on host susceptibility to coinfection. Pulmonary viral and bacterial burdens. Bronchoalveolar lavage ﬂuid (BALF) was harvested by lavaging the lungs with 1 ml of PBS. Serial dilutions of cell-free BALF were added to MDCK cell monolayers and blood agar plates to enumerate viral PFU and bacterial CFU, respectively. MATERIALS AND METHODS The culture supernatants were harvested and added to blood agar plates to enumerate bacterial CFU. In vitro bacterial burden assay. BALF cells were harvested from non-AAD and AAD mice on day 8 after CA04 infection and cultured in 96-well plates with S. pneumoniae A66.1 at an MOI of 1. The culture supernatants were harvested and added to blood agar plates to enumerate bacterial CFU. Recombinant IFN- treatment. Mice were given 20 g of recombinant IFN- (BioLegend) i.n. on day 8 after inﬂuenza infection. One hour later, mice were i.n. inoculated with 105 CFU of D39 with or without 20 g of IFN-. The bacterial burden was then determined 4 h after D39 infection. Statistical analysis. Results were analyzed using GraphPad Prism 6 software, with a P value of 0.05 considered to be statistically signiﬁcant. Survival data were analyzed with a log rank (Mantel-Cox) test. All other data were analyzed by unpaired Student’s t test with Welch’s correction for comparison of two groups and by one- or two-way analysis of variance (ANOVA) with Bonferroni correction for comparison of multiple groups. SUPPLEMENTAL MATERIAL Supplemental material for this article may be found at https://doi.org/10.1128/mBio .01335 19. FIG S1, PDF ﬁle, 0.04 MB. FIG S2, PDF ﬁle, 0.04 MB. FIG S3, PDF ﬁle, 0.02 MB. FIG S4, PDF ﬁle, 0.05 MB. FIG S5, PDF ﬁle, 0.04 MB. FIG S6, PDF ﬁle, 0.1 MB. FIG S7, PDF ﬁle, 0.04 MB. FIG S8, PDF ﬁle, 0.03 MB. FIG S9, PDF ﬁle, 0.05 MB. .01335 19. FIG S1, PDF ﬁle, 0.04 MB. FIG S2, PDF ﬁle, 0.04 MB. FIG S3, PDF ﬁle, 0.02 MB. FIG S4, PDF ﬁle, 0.05 MB. FIG S5, PDF ﬁle, 0.04 MB. FIG S6, PDF ﬁle, 0.1 MB. FIG S7, PDF ﬁle, 0.04 MB. FIG S8, PDF ﬁle, 0.03 MB. FIG S9, PDF ﬁle, 0.05 MB. FIG S1, PDF ﬁle, 0.04 MB. MATERIALS AND METHODS Cytokine analysis. Protein levels of IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, tumor necrosis factor alpha (TNF-), and IFN- in cell-free BALF samples were analyzed using Bio-Plex mouse cytokine assays (Bio-Rad, Hercules, CA). Flow cytometric analysis. The BALF cells were harvested in 1 ml of PBS. Live cells were enumerated based on trypan blue staining. Dead cells were labeled with ﬁxable viability dye (FVD; eBioscience). Fc receptors were blocked by incubation with mouse 2.4G2 (FcIII/II receptor) antibody. Fc receptor-blocked cells were then stained with mixtures of anti-mouse surface antigen mAbs: Alexa Fluor 488-conjugated anti-CD11b (clone M1/70; BioLegend), brilliant violet 421-conjugated anti-Ly6C (clone HK1.4; BioLegend), ﬂuorescein isothiocyanate (FITC)-conjugated anti-CD4 (clone GK1.5; BD Pharmingen), phycoerythrin (PE)-conjugated anti-SiglecF (clone E50-2440; BD Pharmingen), PE-Cy7-conjugated anti-CD8 (clone 53- 6.7; BD Pharmingen), FITC-conjugated anti-macrophage mannose receptor (clone C068C2; BioLegend), allophycocyanin-conjugated anti-CD11c (clone N418; BioLegend), peridinin chlorophyll protein (PerCP)- Cy5.5-conjugated anti-CD11b (clone M1/70; eBioscience), PerCP-Cy5.5-conjugated anti-F4/80 (clone BM8; BioLegend), and PE-Cy7-conjugated anti-Ly6G (clone 1A8; BioLegend). Stained cells were analyzed using a FACSCanto ﬂow cytometer. Intracellular staining. To enumerate GzmB T cells and IFN- T cells, 5 105 live BALF cells were restimulated with CA04 virus at a multiplicity of infection (MOI) of 1 (5 105 PFU/well) for 1 h, followed by 1 h of incubation with 10 g/ml of brefeldin A (Sigma). Cells were then stained with FVD, FcR blocked, and cell surface stained as described above. This was followed by incubation with BD ﬁxation/permea- bilization solution. After washing with BD Perm/Wash buffer, the cells were intracellularly stained with a PE-conjugated anti-IFN- mAb (clone XMG1.2; BioLegend) or FITC-conjugated anti-granzyme B mAb (clone NGZB; eBioscience). Rat IgG1-PE and rat IgG2a-FITC were used as isotype controls. Stained cells were quantitated using a FACSCanto ﬂow cytometer. In vivo phagocytosis assay. FITC-labeled ﬂuorescent beads were i.n. administered on day 7 after inﬂuenza infection. BALF phagocytic cells were analyzed for FITC ﬂuorescence intensity 24 h later by ﬂow cytometric analysis. In vivo phagocytosis assay. FITC-labeled ﬂuorescent beads were i.n. administered on day 7 after inﬂuenza infection. BALF phagocytic cells were analyzed for FITC ﬂuorescence intensity 24 h later by ﬂow cytometric analysis. In vitro bacterial burden assay. BALF cells were harvested from non-AAD and AAD mice on day 8 after CA04 infection and cultured in 96-well plates with S. pneumoniae A66.1 at an MOI of 1. REFERENCES Type 2 inﬂammation in asthma—present in most, absent in many. Nat Rev Immunol 15:57–65. https://doi.org/10.1038/nri3786. 6. Shahangian A, Chow EK, Tian X, Kang JR, Ghaffari A, Liu SY, Belperio JA, Cheng G, Deng JC. 2009. Type I IFNs mediate development of postin- ﬂuenza bacterial pneumonia in mice. J Clin Invest 119:1910–1920. https://doi.org/10.1172/JCI35412. 6. Shahangian A, Chow EK, Tian X, Kang JR, Ghaffari A, Liu SY, Belperio JA, Cheng G, Deng JC. 2009. Type I IFNs mediate development of postin- ﬂuenza bacterial pneumonia in mice. J Clin Invest 119:1910–1920. https://doi.org/10.1172/JCI35412. 22. Chang YJ, Kim HY, Albacker LA, Baumgarth N, McKenzie AN, Smith DE, Dekruyff RH, Umetsu DT. 2011. Innate lymphoid cells mediate inﬂuenza- induced airway hyper-reactivity independently of adaptive immunity. Nat Immunol 12:631–638. https://doi.org/10.1038/ni.2045. at Immunol 12:631–638. https://doi.org/10.1038/ni.2045. 7. Kudva A, Scheller EV, Robinson KM, Crowe CR, Choi SM, Slight SR, Khader SA, Dubin PJ, Enelow RI, Kolls JK, Alcorn JF. 2011. Inﬂuenza A inhibits Th17-mediated host defense against bacterial pneumonia in mice. J Immunol 186:1666–1674. https://doi.org/10.4049/jimmunol.1002194. 7. Kudva A, Scheller EV, Robinson KM, Crowe CR, Choi SM, Slight SR, Khader SA, Dubin PJ, Enelow RI, Kolls JK, Alcorn JF. 2011. Inﬂuenza A inhibits Th17-mediated host defense against bacterial pneumonia in mice. J Immunol 186:1666–1674. https://doi.org/10.4049/jimmunol.1002194. 23. Duvigneau S, Sharma-Chawla N, Boianelli A, Stegemann-Koniszewski S, Nguyen VK, Bruder D, Hernandez-Vargas EA. 2016. Hierarchical effects of pro-inﬂammatory cytokines on the post-inﬂuenza susceptibility to pneumococcal coinfection. Sci Rep 6:37045. https://doi.org/10.1038/ srep37045. 8. Nakamura S, Davis KM, Weiser JN. 2011. Synergistic stimulation of type I interferons during inﬂuenza virus coinfection promotes Streptococcus pneumoniae colonization in mice. J Clin Invest 121:3657–3665. https:// doi.org/10.1172/JCI57762. 24. Robinson KM, Lee B, Scheller EV, Mandalapu S, Enelow RI, Kolls JK, Alcorn JF. 2015. The role of IL-27 in susceptibility to post-inﬂuenza Staphylo- coccus aureus pneumonia. Respir Res 16:10. https://doi.org/10.1186/ s12931-015-0168-8. 9. Li W, Moltedo B, Moran TM. 2012. Type I interferon induction during inﬂuenza virus infection increases susceptibility to secondary Strepto- coccus pneumoniae infection by negative regulation of gammadelta T cells. J Virol 86:12304–12312. https://doi.org/10.1128/JVI.01269-12. 25. Rynda-Apple A, Harmsen A, Erickson AS, Larson K, Morton RV, Richert LE, Harmsen AG. 2014. Regulation of IFN-gamma by IL-13 dictates suscep- tibility to secondary postinﬂuenza MRSA pneumonia. Eur J Immunol 44:3263–3272. https://doi.org/10.1002/eji.201444582. 10. Sun K, Metzger DW. 2014. Inﬂuenza infection suppresses NADPH oxidase-dependent phagocytic bacterial clearance and enhances sus- ceptibility to secondary methicillin-resistant Staphylococcus aureus in- fection. J Immunol 192:3301–3307. https://doi.org/10.4049/jimmunol .1303049. REFERENCES 26. Breslow-Deckman JM, Mattingly CM, Birket SE, Hoskins SN, Ho TN, Garvy BA, Feola DJ. 2013. Linezolid decreases susceptibility to secondary bacterial pneumonia postinﬂuenza infection in mice through its effects on IFN-gamma. J Immunol 191:1792–1799. https://doi.org/10 .4049/jimmunol.1300180. 11. Gilca R, De Serres G, Boulianne N, Ouhoummane N, Papenburg J, Douville-Fradet M, Fortin E, Dionne M, Boivin G, Skowronski DM. 2011. Risk factors for hospitalization and severe outcomes of 2009 pandemic H1N1 inﬂuenza in Quebec, Canada. Inﬂuenza Other Respir Viruses 5:247–255. https://doi.org/10.1111/j.1750-2659.2011.00204.x. 27. Hang DTT, Choi EJ, Song JY, Kim SE, Kwak J, Shin YK. 2011. Differential effect of prior inﬂuenza infection on alveolar macrophage phagocytosis of Staphylococcus aureus and Escherichia coli: involvement of interferon-gamma production. Microbiol Immunol 55:751–759. https:// doi.org/10.1111/j.1348-0421.2011.00383.x. 12. Myles P, Nguyen-Van-Tam JS, Semple MG, Brett SJ, Bannister B, Read RC, Taylor BL, McMenamin J, Enstone JE, Nicholson KG, Openshaw PJ, Lim WS, Inﬂuenza Clinical Information Network FLU-CIN. 2013. Differences between asthmatics and nonasthmatics hospitalised with inﬂuenza A infection. Eur Respir J 41:824–831. https://doi.org/10.1183/09031936 .00015512. 28. Sun K, Gan Y, Metzger DW. 2011. Analysis of murine genetic predispo- sition to pneumococcal infection reveals a critical role of alveolar mac- rophages in maintaining the sterility of the lower respiratory tract. Infect Immun 79:1842–1847. https://doi.org/10.1128/IAI.01143-10. 29. Sanﬁlippo AM, Furuya Y, Roberts S, Salmon SL, Metzger DW. 2015. Allergic lung inﬂammation reduces tissue invasion and enhances sur- vival from pulmonary pneumococcal infection in mice, which correlates with increased expression of transforming growth factor beta1 and SiglecF(low) alveolar macrophages. Infect Immun 83:2976–2983. https:// doi.org/10.1128/IAI.00142-15. 13. Louie JK, Acosta M, Samuel MC, Schechter R, Vugia DJ, Harriman K, Matyas BT, California Pandemic (H1N1) Working Group. 2011. A novel risk factor for a novel virus: obesity and 2009 pandemic inﬂuenza A (H1N1). Clin Infect Dis 52:301–312. https://doi.org/10.1093/cid/ciq152. 14. Furuya Y, Furuya AK, Roberts S, Sanﬁlippo AM, Salmon SL, Metzger DW. 2015. Prevention of inﬂuenza virus-induced immunopathology by TGF- beta produced during allergic asthma. PLoS Pathog 11:e1005180. https://doi.org/10.1371/journal.ppat.1005180. 30. Lykens JE, Terrell CE, Zoller EE, Divanovic S, Trompette A, Karp CL, Aliberti J, Flick MJ, Jordan MB. 2010. Mice with a selective impairment of IFN-gamma signaling in macrophage lineage cells demonstrate the critical role of IFN-gamma-activated macrophages for the control of protozoan parasitic infections in vivo. J Immunol 184:877–885. https:// doi.org/10.4049/jimmunol.0902346. 15. Furuya Y, Roberts S, Hurteau GJ, Sanﬁlippo AM, Racine R, Metzger DW. 2014. REFERENCES 17. Califano D, Furuya Y, Metzger DW. 2018. Effects of inﬂuenza on alveolar macrophage viability are dependent on mouse genetic strain. J Immunol 201:134–144. https://doi.org/10.4049/jimmunol.1701406. 1. Brundage JF, Shanks GD. 2008. Deaths from bacterial pneumonia during 1918-19 inﬂuenza pandemic. Emerg Infect Dis 14:1193–1199. https://doi .org/10.3201/eid1408.071313. 1. Brundage JF, Shanks GD. 2008. Deaths from bacterial pneumonia during 1918-19 inﬂuenza pandemic. Emerg Infect Dis 14:1193–1199. https://doi .org/10.3201/eid1408.071313. 201:134–144. https://doi.org/10.4049/jimmunol.1701406. g 2. Morens DM, Taubenberger JK, Fauci AS. 2008. Predominant role of bacterial pneumonia as a cause of death in pandemic inﬂuenza: impli- cations for pandemic inﬂuenza preparedness. J Infect Dis 198:962–970. https://doi.org/10.1086/591708. 2. Morens DM, Taubenberger JK, Fauci AS. 2008. Predominant role of bacterial pneumonia as a cause of death in pandemic inﬂuenza: impli- cations for pandemic inﬂuenza preparedness. J Infect Dis 198:962–970. https://doi.org/10.1086/591708. 18. Sun K, Yajjala VK, Bauer C, Talmon GA, Fischer KJ, Kielian T, Metzger DW. 2016. Nox2-derived oxidative stress results in inefﬁcacy of antibiotics against post-inﬂuenza S. aureus pneumonia. J Exp Med 213:1851–1864. https://doi.org/10.1084/jem.20150514. 3. Metzger DW, Sun K. 2013. Immune dysfunction and bacterial coinfec- tions following inﬂuenza. J Immunol 191:2047–2052. https://doi.org/10 .4049/jimmunol.1301152. 19. Ellis GT, Davidson S, Crotta S, Branzk N, Papayannopoulos V, Wack A. 2015. TRAIL monocytes and monocyte-related cells cause lung dam- age and thereby increase susceptibility to inﬂuenza-Streptococcus pneumoniae coinfection. EMBO Rep 16:1203–1218. https://doi.org/10 .15252/embr.201540473. j 4. Sun K, Metzger DW. 2008. Inhibition of pulmonary antibacterial defense by interferon-gamma during recovery from inﬂuenza infection. Nat Med 14:558–564. https://doi.org/10.1038/nm1765. j 4. Sun K, Metzger DW. 2008. Inhibition of pulmonary antibacterial defense by interferon-gamma during recovery from inﬂuenza infection. Nat Med 14:558–564. https://doi.org/10.1038/nm1765. 20. Jamieson AM, Yu S, Annicelli CH, Medzhitov R. 2010. Inﬂuenza virus- induced glucocorticoids compromise innate host defense against a secondary bacterial infection. Cell Host Microbe 7:103–114. https://doi .org/10.1016/j.chom.2010.01.010. p g 5. van der Sluijs KF, van Elden LJ, Nijhuis M, Schuurman R, Pater JM, Florquin S, Goldman M, Jansen HM, Lutter R, van der Poll T. 2004. IL-10 is an important mediator of the enhanced susceptibility to pneumococ- cal pneumonia after inﬂuenza infection. J Immunol 172:7603–7609. https://doi.org/10.4049/jimmunol.172.12.7603. g 5. van der Sluijs KF, van Elden LJ, Nijhuis M, Schuurman R, Pater JM, Florquin S, Goldman M, Jansen HM, Lutter R, van der Poll T. 2004. IL-10 is an important mediator of the enhanced susceptibility to pneumococ- cal pneumonia after inﬂuenza infection. J Immunol 172:7603–7609. https://doi.org/10.4049/jimmunol.172.12.7603. 21. Fahy JV. 2015. ACKNOWLEDGMENTS This work was supported by American Lung Association biomedical research grant RG341974 to Y.F. and by U.S. NIH grant RO1 HL140496 to D.W.M. The funders had no This work was supported by American Lung Association biomedical research grant RG341974 to Y.F. and by U.S. NIH grant RO1 HL140496 to D.W.M. The funders had no mbio.asm.org 14 July/August 2019 Volume 10 Issue 4 e01335-19 ® Impact of Allergic Airway Disease on Coinfection role in study design, data collection and interpretation, or the decision to submit the work for publication. We have no conﬂicting ﬁnancial interests. We have no conﬂict of interest. July/August 2019 Volume 10 Issue 4 e01335-19 REFERENCES PLoS One 12:e0173008. https://doi.org/10.1371/journal.pone.0173008. 57. Louie JK, Acosta M, Winter K, Jean C, Gavali S, Schechter R, Vugia D, Harriman K, Matyas B, Glaser CA, Samuel MC, Rosenberg J, Talarico J, Hatch D, California Pandemic Working Group. 2009. Factors associated with death or hospitalization due to pandemic 2009 inﬂuenza A(H1N1) infection in California. JAMA 302:1896–1902. https://doi.org/10.1001/ jama.2009.1583. 43. Lee DCP, Tay NQ, Thian M, Prabhu N, Furuhashi K, Kemeny DM. 2018. Prior exposure to inhaled allergen enhances anti-viral immunity and T cell priming by dendritic cells. PLoS One 13:e0190063. https://doi.org/ 10.1371/journal.pone.0190063. 58. Nguyen-Van-Tam JS, Openshaw PJ, Hashim A, Gadd EM, Lim WS, Semple MG, Read RC, Taylor BL, Brett SJ, McMenamin J, Enstone JE, Armstrong C, Nicholson KG, Inﬂuenza Clinical Information Network (FLU-CIN). 2010. Risk factors for hospitalisation and poor outcome with pandemic A/H1N1 inﬂuenza: United Kingdom ﬁrst wave (May-September 2009). Thorax 65:645–651. https://doi.org/10.1136/thx.2010.135210. 44. An S, Jeon YJ, Jo A, Lim HJ, Han YE, Cho SW, Kim HY, Kim HJ. 2018. Initial inﬂuenza virus replication can be limited in allergic asthma through rapid induction of type III interferons in respiratory epithelium. Front Immunol 9:986. https://doi.org/10.3389/ﬁmmu.2018.00986. 45. Nicholas B, Dudley S, Tariq K, Howarth P, Lunn K, Pink S, Sterk PJ, Adcock IM, Monk P, Djukanovic R, U-BIOPRED Study Group. 2017. Susceptibility to inﬂuenza virus infection of bronchial biopsies in asthma. J Allergy Clin Immunol 140:309.e4–312.e4. https://doi.org/10.1016/j.jaci.2016.12.964. 59. Myles PR, Semple MG, Lim WS, Openshaw PJ, Gadd EM, Read RC, Taylor BL, Brett SJ, McMenamin J, Enstone JE, Armstrong C, Bannister B, Nich- olson KG, Nguyen-Van-Tam JS, Inﬂuenza Clinical Information Network (FLU-CIN). 2012. Predictors of clinical outcome in a national hospitalised cohort across both waves of the inﬂuenza A/H1N1 pandemic 2009-2010 in the UK. Thorax 67:709–717. https://doi.org/10.1136/thoraxjnl-2011 -200266. 46. Nakamura S, Yaguchi T, Kawamura N, Kobayashi A, Sakurai T, Higuchi H, Takaishi H, Hibi T, Kawakami Y. 2014. TGF-beta1 in tumor microenviron- ments induces immunosuppression in the tumors and sentinel lymph nodes and promotes tumor progression. J Immunother 37:63–72. https://doi.org/10.1097/CJI.0000000000000011. 60. Weinberger DM, Simonsen L, Jordan R, Steiner C, Miller M, Viboud C. 2012. Impact of the 2009 inﬂuenza pandemic on pneumococcal pneu- monia hospitalizations in the United States. J Infect Dis 205:458–465. https://doi.org/10.1093/infdis/jir749. 47. Vanpouille-Box C, Diamond JM, Pilones KA, Zavadil J, Babb JS, Formenti SC, Barcellos-Hoff MH, Demaria S. 2015. TGFbeta is a master regulator of radiation therapy-induced antitumor immunity. Cancer Res 75: 2232–2242. https://doi.org/10.1158/0008-5472.CAN-14-3511. 61. REFERENCES Halwani R, Al-Muhsen S, Al-Jahdali H, Hamid Q. 2011. Role of transform- ing growth factor-beta in airway remodeling in asthma. Am J Respir Cell Mol Biol 44:127–133. https://doi.org/10.1165/rcmb.2010-0027TR. 38. Batra V, Musani AI, Hastie AT, Khurana S, Carpenter KA, Zangrilli JG, Peters SP. 2004. Bronchoalveolar lavage ﬂuid concentrations of trans- forming growth factor (TGF)-beta1, TGF-beta2, interleukin (IL)-4 and IL-13 after segmental allergen challenge and their effects on alpha- smooth muscle actin and collagen III synthesis by primary human lung ﬁbroblasts. Clin Exp Allergy 34:437–444. https://doi.org/10.1111/j.1365 -2222.2004.01885.x. 53. Bramley AM, Dasgupta S, Skarbinski J, Kamimoto L, Fry AM, Finelli L, Jain S, 2009 Pandemic Inﬂuenza A (H1N1) Virus Hospitalizations Investiga- tion Team. 2012. Intensive care unit patients with 2009 pandemic inﬂuenza A (H1N1pdm09) virus infection—United States, 2009. Inﬂu- enza Other Respir Viruses 6:e134–e142. https://doi.org/10.1111/j .1750-2659.2012.00385.x. 39. Avila PC, Abisheganaden JA, Wong H, Liu J, Yagi S, Schnurr D, Kishiyama JL, Boushey HA. 2000. Effects of allergic inﬂammation of the nasal mucosa on the severity of rhinovirus 16 cold. J Allergy Clin Immunol 105:923–932. https://doi.org/10.1067/mai.2000.106214. 54. Eriksson CO, Graham DA, Uyeki TM, Randolph AG. 2012. Risk factors for mechanical ventilation in U.S. children hospitalized with seasonal inﬂu- enza and 2009 pandemic inﬂuenza A. Pediatr Crit Care Med 13: 625–631. https://doi.org/10.1097/PCC.0b013e318260114e. 40. Samarasinghe AE, Woolard SN, Boyd KL, Hoselton SA, Schuh JM, McCull- ers JA. 2014. The immune proﬁle associated with acute allergic asthma accelerates clearance of inﬂuenza virus. Immunol Cell Biol 92:449–459. https://doi.org/10.1038/icb.2013.113. 55. von der Beck D, Seeger W, Herold S, Günther A, Löh B. 2017. Character- istics and outcomes of a cohort hospitalized for pandemic and seasonal inﬂuenza in Germany based on nationwide inpatient data. PLoS One 12:e0180920. https://doi.org/10.1371/journal.pone.0180920. 41. Ishikawa H, Sasaki H, Fukui T, Fujita K, Kutsukake E, Matsumoto T. 2012. Mice with asthma are more resistant to inﬂuenza virus infection and NK cells activated by the induction of asthma have potentially protective effects. J Clin Immunol 32:256–267. https://doi.org/10.1007/s10875-011 -9619-2. 56. McKenna JJ, Bramley AM, Skarbinski J, Fry AM, Finelli L, Jain S, 2009 Pandemic Inﬂuenza A (H1N1) Virus Hospitalizations Investigation Team. 2013. Asthma in patients hospitalized with pandemic inﬂuenza A(H1N1)pdm09 virus infection—United States, 2009. BMC Infect Dis 13:57. https://doi.org/10.1186/1471-2334-13-57. 42. Kawaguchi A, Suzuki T, Ohara Y, Takahashi K, Sato Y, Ainai A, Nagata N, Tashiro M, Hasegawa H. 2017. Impacts of allergic airway inﬂammation on lung pathology in a mouse model of inﬂuenza A virus infection. REFERENCES Asthma increases susceptibility to heterologous but not homolo- gous secondary inﬂuenza. J Virol 88:9166–9181. https://doi.org/10.1128/ JVI.00265-14. 31. Stahl PD, Ezekowitz RA. 1998. The mannose receptor is a pattern rec- ognition receptor involved in host defense. Curr Opin Immunol 10: 50–55. https://doi.org/10.1016/S0952-7915(98)80031-9. 16. Sun K, Ye J, Perez DR, Metzger DW. 2011. Seasonal FluMist vaccination induces cross-reactive T cell immunity against H1N1 (2009) inﬂuenza and secondary bacterial infections. J Immunol 186:987–993. https://doi .org/10.4049/jimmunol.1002664. 32. Li N, Ren A, Wang X, Fan X, Zhao Y, Gao GF, Cleary P, Wang B. 2015. Inﬂuenza viral neuraminidase primes bacterial coinfection through TGF- mbio.asm.org 15 ® Roberts et al. beta-mediated expression of host cell receptors. Proc Natl Acad Sci U S A 112:238–243. https://doi.org/10.1073/pnas.1414422112. 33. Carlson CM, Turpin EA, Moser LA, O’Brien KB, Cline TD, Jones JC, Tumpey TM, Katz JM, Kelley LA, Gauldie J, Schultz-Cherry S. 2010. Transforming growth factor-beta: activation by neuraminidase and role in highly pathogenic H5N1 inﬂuenza pathogenesis. PLoS Pathog 6:e1001136. https://doi.org/10.1371/journal.ppat.1001136. 50. Li MO, Flavell RA. 2008. TGF-beta: a master of all T cell trades. Cell 134:392–404. https://doi.org/10.1016/j.cell.2008.07.025. 34. Dutta A, Huang CT, Chen TC, Lin CY, Chiu CH, Lin YC, Chang CS, He YC. 2015. IL-10 inhibits neuraminidase-activated TGF-beta and facilitates Th1 phenotype during early phase of infection. Nat Commun 6:6374. https:// doi.org/10.1038/ncomms7374. 51. Veerapandian R, Snyder JD, Samarasinghe AE. 2018. Inﬂuenza in asthmatics: for better or for worse? Front Immunol 9:1843. https://doi .org/10.3389/ﬁmmu.2018.01843. g 52. Van Kerkhove MD, Vandemaele KA, Shinde V, Jaramillo-Gutierrez G, Koukounari A, Donnelly CA, Carlino LO, Owen R, Paterson B, Pelletier L, Vachon J, Gonzalez C, Hongjie Y, Zijian F, Chuang SK, Au A, Buda S, Krause G, Haas W, Bonmarin I, Taniguichi K, Nakajima K, Shobayashi T, Takayama Y, Sunagawa T, Heraud JM, Orelle A, Palacios E, van der Sande MA, Wielders CC, Hunt D, Cutter J, Lee VJ, Thomas J, Santa-Olalla P, Sierra-Moros MJ, Hanshaoworakul W, Ungchusak K, Pebody R, Jain S, Mounts AW, WHO Working Group for Risk Factors for Severe H1N1pdm Infection. 2011. Risk factors for severe outcomes following 2009 inﬂu- enza A (H1N1) infection: a global pooled analysis. PLoS Med 8:e1001053. https://doi.org/10.1371/journal.pmed.1001053. 35. Schultz-Cherry S, Hinshaw VS. 1996. Inﬂuenza virus neuraminidase acti- vates latent transforming growth factor beta. J Virol 70:8624–8629. 36. Gratz N, Loh LN, Mann B, Gao G, Carter R, Rosch J, Tuomanen EI. 2017. Pneumococcal neuraminidase activates TGF-beta signalling. Microbiol- ogy 163:1198–1207. https://doi.org/10.1099/mic.0.000511. 37. July/August 2019 Volume 10 Issue 4 e01335-19 REFERENCES https://doi.org/10 .1186/s13054-015-0764-5. 74. Sykes A, Edwards MR, Macintyre J, del Rosario A, Bakhsoliani E, Trujillo- Torralbo MB, Kon OM, Mallia P, McHale M, Johnston SL. 2012. Rhinovirus 16-induced IFN-alpha and IFN-beta are deﬁcient in bronchoalveolar lavage cells in asthmatic patients. J Allergy Clin Immunol 129: 1506.e6–1514.e6. https://doi.org/10.1016/j.jaci.2012.03.044. 65. Lansbury L, Rodrigo C, Leonardi-Bee J, Nguyen-Van-Tam J, Lim WS. 2019. Corticosteroids as adjunctive therapy in the treatment of in- ﬂuenza. Cochrane Database Syst Rev 2:CD010406. https://doi.org/10 .1002/14651858.CD010406.pub3. 1506.e6–1514.e6. https://doi.org/10.1016/j.jaci.2012.03.044. 75. Baraldo S, Contoli M, Bazzan E, Turato G, Padovani A, Marku B, Calabrese F, Caramori G, Ballarin A, Snijders D, Barbato A, Saetta M, Papi A. 2012. Deﬁcient antiviral immune responses in childhood: distinct roles of atopy and asthma. J Allergy Clin Immunol 130:1307–1314. https://doi .org/10.1016/j.jaci.2012.08.005. 66. Lee N, Leo YS, Cao B, Chan PK, Kyaw WM, Uyeki TM, Tam WW, Cheung CS, Yung IM, Li H, Gu L, Liu Y, Liu Z, Qu J, Hui DS. 2015. Neuraminidase inhibitors, superinfection and corticosteroids affect survival of inﬂuenza patients. Eur Respir J 45:1642–1652. https://doi.org/10.1183/09031936 .00169714. 76. Gill MA, Bajwa G, George TA, Dong CC, Dougherty II, Jiang N, Gan VN, Gruchalla RS. 2010. Counterregulation between the FcepsilonRI pathway and antiviral responses in human plasmacytoid dendritic cells. J Immu- nol 184:5999–6006. https://doi.org/10.4049/jimmunol.0901194. 67. Brun-Buisson C, Richard J-CM, Mercat A, Thiébaut ACM, Brochard L, REVA-SRLF A/H1N1v 2009 Registry Group. 2011. Early corticosteroids in severe inﬂuenza A/H1N1 pneumonia and acute respiratory distress syn- drome. Am J Respir Crit Care Med 183:1200–1206. https://doi.org/10 .1164/rccm.201101-0135OC. 77. Rupani H, Martinez-Nunez RT, Dennison P, Lau LC, Jayasekera N, Have- lock T, Francisco-Garcia AS, Grainge C, Howarth PH, Sanchez-Elsner T. 2016. Toll-like receptor 7 is reduced in severe asthma and linked to an altered microRNA proﬁle. Am J Respir Crit Care Med 194:26–37. https:// doi.org/10.1164/rccm.201502-0280OC. 68. Aun MV, Bonamichi-Santos R, Arantes-Costa FM, Kalil J, Giavina-Bianchi P. 2017. Animal models of asthma: utility and limitations. J Asthma Allergy 10:293–301. https://doi.org/10.2147/JAA.S121092. 69. Kumar RK, Foster PS. 2012. Are mouse models of asthma appropriate for investigating the pathogenesis of airway hyper-responsiveness? Front Physiol 3:312. https://doi.org/10.3389/fphys.2012.00312. 78. Bosch AA, Biesbroek G, Trzcinski K, Sanders EA, Bogaert D. 2013. Viral and bacterial interactions in the upper respiratory tract. PLoS Pathog 9:e1003057. https://doi.org/10.1371/journal.ppat.1003057. 70. Temelkovski J, Hogan SP, Shepherd DP, Foster PS, Kumar RK. 1998. REFERENCES Gill JR, Sheng ZM, Ely SF, Guinee DG, Beasley MB, Suh J, Deshpande C, Mollura DJ, Morens DM, Bray M, Travis WD, Taubenberger JK. 2010. Pulmonary pathologic ﬁndings of fatal 2009 pandemic inﬂuenza A/H1N1 viral infections. Arch Pathol Lab Med 134:235–243. https://doi.org/10 .1043/1543-2165-134.2.235. 48. Mariathasan S, Turley SJ, Nickles D, Castiglioni A, Yuen K, Wang Y, Kadel EE, III, Koeppen H, Astarita JL, Cubas R, Jhunjhunwala S, Banchereau R, Yang Y, Guan Y, Chalouni C, Ziai J, S¸enbabaog˘lu Y, Santoro S, Sheinson D, Hung J, Giltnane JM, Pierce AA, Mesh K, Lianoglou S, Riegler J, Carano RAD, Eriksson P, Höglund M, Somarriba L, Halligan DL, van der Heijden MS, Loriot Y, Rosenberg JE, Fong L, Mellman I, Chen DS, Green M, Derleth C, Fine GD, Hegde PS, Bourgon R, Powles T. 2018. TGFbeta attenuates tumour response to PD-L1 blockade by contributing to exclusion of T cells. Nature 554:544–548. https://doi.org/10.1038/nature25501. 62. Centers for Disease Control and Prevention. 2009. Bacterial coinfections in lung tissue specimens from fatal cases of 2009 pandemic inﬂuenza A (H1N1)—United States, May-August 2009. MMWR Morb Mortal Wkly Rep 58:1071–1074. 63. Kloepfer KM, Lee WM, Pappas TE, Kang TJ, Vrtis RF, Evans MD, Gangnon RE, Bochkov YA, Jackson DJ, Lemanske RF, Jr, Gern JE. 2014. Detection of 63. Kloepfer KM, Lee WM, Pappas TE, Kang TJ, Vrtis RF, Evans MD, Gangnon RE, Bochkov YA, Jackson DJ, Lemanske RF, Jr, Gern JE. 2014. Detection of 49. Tauriello DVF, Palomo-Ponce S, Stork D, Berenguer-Llergo A, Badia- 49. Tauriello DVF, Palomo-Ponce S, Stork D, Berenguer-Llergo A, Badia- mbio.asm.org 16 mbio.asm.org 16 ® Impact of Allergic Airway Disease on Coinfection pathogenic bacteria during rhinovirus infection is associated with in- creased respiratory symptoms and asthma exacerbations. J Allergy Clin Immunol 133:1301–1307, 1307.e1–1307.e3. https://doi.org/10.1016/j.jaci .2014.02.030. pathogenic bacteria during rhinovirus infection is associated with in- creased respiratory symptoms and asthma exacerbations. J Allergy Clin Immunol 133:1301–1307, 1307.e1–1307.e3. https://doi.org/10.1016/j.jaci .2014.02.030. exposure to house dust mite elicits chronic airway inﬂammation and structural remodeling. Am J Respir Crit Care Med 169:378–385. https:// doi.org/10.1164/rccm.200308-1094OC. 73. Kumar RK, Herbert C, Webb DC, Li L, Foster PS. 2004. Effects of anticy- tokine therapy in a mouse model of chronic asthma. Am J Respir Crit Care Med 170:1043–1048. https://doi.org/10.1164/rccm.200405-681OC. 64. Zhang Y, Sun W, Svendsen ER, Tang S, MacIntyre RC, Yang P, Zhang D, Wang Q. 2015. Do corticosteroids reduce the mortality of inﬂuenza A (H1N1) infection? A meta-analysis. Crit Care 19:46. July/August 2019 Volume 10 Issue 4 e01335-19 REFERENCES An improved murine model of asthma: selective airway inﬂammation, epi- thelial lesions and increased methacholine responsiveness following chronic exposure to aerosolised allergen. Thorax 53:849–856. https:// doi.org/10.1136/thx.53.10.849. 79. Jamieson AM, Pasman L, Yu S, Gamradt P, Homer RJ, Decker T, Medzhitov R. 2013. Role of tissue protection in lethal respiratory viral-bacterial coinfection. Science 340:1230–1234. https://doi.org/10 .1126/science.1233632. 80. Wong SM, Bernui M, Shen H, Akerley BJ. 2013. Genome-wide ﬁtness proﬁling reveals adaptations required by Haemophilus in coinfection with inﬂuenza A virus in the murine lung. Proc Natl Acad Sci U S A 110:15413–15418. https://doi.org/10.1073/pnas.1311217110. 71. Kumar RK, Herbert C, Yang M, Koskinen AM, McKenzie AN, Foster PS. 2002. Role of interleukin-13 in eosinophil accumulation and airway remodelling in a mouse model of chronic asthma. Clin Exp Allergy 32:1104–1111. https://doi.org/10.1046/j.1365-2222.2002.01420.x. 81. National Research Council. 2011. Guide for the care and use of labora- tory animals, 8th ed. National Academies Press, Washington, DC. 72. Johnson JR, Wiley RE, Fattouh R, Swirski FK, Gajewska BU, Coyle AJ, Gutierrez-Ramos JC, Ellis R, Inman MD, Jordana M. 2004. Continuous mbio.asm.org 17
https://openalex.org/W1970430253	https://jmedicalcasereports.biomedcentral.com/track/pdf/10.1186/1752-1947-5-75	English	null	High origin of a testicular artery: a case report and review of the literature	Journal of medical case reports	2,011	cc-by	3,106	CASE REPORT Open Access © 2011 Paraskevas et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Discussion Anatomical variations of TAs are common. Variants were noticed in 4.7 percent of cases in a study of 150 cadavers [2]. Another study of 90 fetuses revealed a frequency of 8.8 percent [7]. TA variations include variations in the origin, course and even the number of arteries presented. This can include double arteries, a common origin of both arteries, the absence of one artery, a higher origin than normal and origin from the lumbar artery, renal or polar renal, middle or superior suprarenal, common or internal iliac, or superior epigastric artery [2,5,7-11]. Introduction death was cardiovascular ischemic disease. Following dis- section of the retro-peritoneum and preparation of the abdominal aorta and its branches, an unusual high origin of the left TA was observed. The artery had a diameter of 32 mm and arose from the anterolateral surface of the abdominal aorta, 2 cm proximal to the ipsilateral renal artery. At 1 cm distal to its origin, it branched off into the inferior suprarenal artery that supplied the left adre- nal gland. The left TA then progressed in an oblique course outwards and caudally, crossing anterior to the left renal artery (Figure 1 and Figure 2). His right TA and both the left and right testicular veins were normal. The testis mainly receives its blood supply from the testi- cular artery (TA) and drains into the testicular vein [1]. Testicular vessels have an important role in testis ther- moregulation [2]. Variations of these arteries and veins have been extensively studied due to their importance in testicular physiology. Moreover, this knowledge has a practical implication during renal and testicular surgery. Anomalies in the origin, course, and number of TAs were observed in 4.7 percent of cases in a study of 150 cadavers [2]. A high origin of the TA from the abdom- inal aorta, as in our case report, has been noted in only a few instances in the literature [3-6]. We report on such a case and review the relative literature about the macroscopic anatomy, embryology and likely physiologi- cal and surgical implications of this variant. Abstract Introduction: Although variations in the origin of the testicular artery are not uncommon, few reports about a high origin from the abdominal aorta exist in the literature. We discuss the case of a high origin of the testicular artery, its embryology, classification systems, and its clinical significance. Case presentation: We report a very rare case of high origin of the left testicular artery in a 68-year-old Caucasian male cadaver. The artery originated from the anterolateral aspect of the abdominal aorta, 2 cm cranially to the ipsilateral renal artery. Approximately 1 cm after its origin, it branched off into the inferior suprarenal artery. During its course, the artery crossed anterior to the left renal artery. Conclusions: A knowledge of the variant origin of the testicular artery is important during renal and testicular surgery. The origin and course must be carefully identified in order to preserve normal blood circulation and prevent testicular atrophy. A reduction in gonadal blood flow may lead to varicocele under circumstances. A knowledge of this variant anatomy may be of interest to radiologists and helpful in avoiding diagnostic errors. Open Access Open Access * Correspondence: g_paraskevas@yahoo.gr Department of Anatomy, Medical School of Aristotle University of Thessaloniki, PO Box 300, Postal Code 54124, Thessaloniki, Greece Paraskevas et al. Journal of Medical Case Reports 2011, 5:75 http://www.jmedicalcasereports.com/content/5/1/75 Paraskevas et al. Journal of Medical Case Reports 2011, 5:75 http://www.jmedicalcasereports.com/content/5/1/75 Paraskevas et al. Journal of Medical Case Reports 2011, 5:75 http://www.jmedicalcasereports.com/content/5/1/75 JOURNAL OF MEDICAL CASE REPORTS High origin of a testicular artery: a case report and review of the literature George K Paraskevas*, Orestis Ioannidis, Athanasios Raikos, Basileios Papaziogas, Konstantinos Natsis, Ioannis Spyridakis, Panagiotis Kitsoulis Case presentation We identified a variation in the origin of the TA in a 68- year-old Caucasian male formalin-embalmed cadaver used for educational and research purposes. His cause of Page 2 of 4 Page 2 of 4 Paraskevas et al. Journal of Medical Case Reports 2011, 5:75 http://www.jmedicalcasereports.com/content/5/1/75 Paraskevas et al. Journal of Medical Case Reports 2011, 5:75 http://www.jmedicalcasereports.com/content/5/1/75 vertebra below the renal artery [1]. Each TA passes inferolaterally under the parietal peritoneum and over the psoas major muscle. The right TA lies anterior to the inferior vena cava and posterior to the third portion of the duodenum, while the left lies posterior to the lower part of the descending colon [12]. In rare instances, the right TA passes posterior to the inferior vena cava [13]. Figure 1 The left testicular artery (TA) arose from the abdominal aorta (AA), superior to the left renal artery (LRA). After its origin, it branched off to the inferior suprarenal artery (SA) and then descended inferiorly, passing over the left renal artery (SG: suprarenal gland, LK: left kidney, U: ureter). In both men and women, the abdominal portion of the TA (ovarian in females) seems to have the same topographical relationship. Along its course, the TA supplies anatomical structures such as the peritoneum and profound inguinal ring, perirenal fat, ureter, iliac lymph, retroperitoneum, spermatic cord and cremaster muscle. Sometimes the TA branches off to the inferior pole of the ipsilateral adrenal gland [1,12,13]. There are few reports of a high TA origin in the lit- erature. Shinohara et al. found a TA originating 1 cm superior to the origin of the inferior phrenic artery [3]. After a short course, it branched off and subdivided into a supernumerary inferior phrenic artery and a superior suprarenal artery. In another case, Onderoglu et al. reported the case of a high origin of the right TA located at the level of the right renal artery lineage [4]. It branched off and was subdivided into an inferior phrenic artery and a superior suprarenal artery. In another study, Brohi et al. described the case of a high origin of the left TA which originated from the left renal artery [5]. The artery branched off and was subse- quently subdivided into three branches that supplied the left suprarenal gland. Two more cases of a higher origin of the TA were reported by Ozan et al. [6]. Further- more, Xue et al. Case presentation found a right TA artery arising from the anterior surface of the abdominal aorta at the level of the left renal artery [14]. Figure 1 The left testicular artery (TA) arose from the abdominal aorta (AA), superior to the left renal artery (LRA). After its origin, it branched off to the inferior suprarenal artery (SA) and then descended inferiorly, passing over the left renal artery (SG: suprarenal gland, LK: left kidney, U: ureter). TAs are paired and usually originate from the antero- lateral or lateral aspect of the abdominal aorta. The TA is a long, thin vessel that arises at an acute angle from the abdominal aorta, at the level of the second lumbar Figure 2 A schematic representation of Figure 1 (SG: suprarenal gland, LK: left kidney, RA: renal artery). The first attempt at classification of TA variations was made by Machnicki et al. [15]. Their study included TAs from both fetuses and adults grouped according to their origin from the aorta or renal artery. Four major types were observed: Type A - a single TA originating from the aorta; Type B - a single TA originating from the renal artery; Type C - two TAs originating from the aorta that supplied the same gonad; Type D - two TAs supplying the same gonad, one arising from the aorta and the other from the renal artery [15]. Some years later, Çiçekcibasi et al. classified the variations into four alternative types: Type I - TA arising from the suprare- nal artery; Type II - TA originating from the renal artery; Type III - TA of high-positional origin from the abdominal aorta, close to the renal artery lineage; Type IV - TA duplication originating from the aorta or from various vessels [7]. Our case report is Type A, according to classification by Machnicki et al. [15] and Type III, according to classification by Çiçekcibasi et al. [7]. Figure 2 A schematic representation of Figure 1 (SG: suprarenal gland, LK: left kidney, RA: renal artery). Paraskevas et al. Journal of Medical Case Reports 2011, 5:75 http://www.jmedicalcasereports.com/content/5/1/75 Paraskevas et al. Journal of Medical Case Reports 2011, 5:75 http://www.jmedicalcasereports.com/content/5/1/75 Page 3 of 4 Notkovich described the relationship of the TA to the renal vein [16]. Case presentation In his study, the anatomical variations are divided into three types: Type I - TA arising from the aorta, passing posterior or inferior to the renal vein but without making contact with it; Type II - TA origi- nating from the aorta, superior to the renal vein and crossing in front of it; Type III - TA arising from the aorta and passing posterior or inferior to the renal vein and coursing superiorly and around the renal vein [16]. Our case report is classified as Type II according to Notkovich classification. disappearing. This leads to anatomical variations of the celiac, superior and inferior mesenteric arteries. Like- wise, various disappearing phases of the lateral meso- nephric arteries and their longitudinal anastomotic channels can take place during the embryonic develop- ment of the gonads. These modifications can lead to variants of the suprarenal, renal and testicular arteries. The persistence of many mesonephric arteries may lead to multiple testicular arteries [24]. The anatomical variations of TAs are of clinical importance as well as embryological and anatomical interest. Practical implications can be found in the kidney and gonad blood flow. Such conditions could lead to varicocele under circumstances [16]. The variant becomes more significant in light of the fact that testi- cular arterial blood flow was found to be significantly decreased in men with varicocele [25]. Additionally, anomalous TA origin may affect the testicular perfusion and testicular function. Since age-related disturbances in spermiogenesis are well described in the literature, it would be wise for the clinician to differentially diagnose age-related impaired spermiogenesis from perfusion- induced spermiogenesis. The ratio of common origin for the TA and the suprarenal artery is approximately 1:26 [17]. The super- ior suprarenal artery usually arises from the inferior phrenic artery, the middle suprarenal artery arises from the abdominal aorta and the inferior suprarenal artery from the renal artery [1,10]. Although anatomical varia- tions of the middle suprarenal artery are common [18], reports of variations of the inferior and superior suprar- enal arteries are rare [2,19]. The phenomenon of a com- mon origin for both the testicular and suprarenal artery has also been described [20,21]. Variations in the origin, course and branches of TAs are attributed to their embryologic derivation. Conclusions l k Anatomical knowledge of the origin and course of the TA is of great importance during renal and testicular surgery. The origin and course of the TA must be care- fully identified and demarcated in order to preserve and prevent testicular atrophy. Aside from surgical interest, the trait is of clinical value because anomalies in arterial and venous perfusion may have severe consequences for the thermoregulation of the testicular glands and may therefore influence spermiogenesis. Furthermore, radiol- ogists should be familiar with TA variants in order to provide an accurate diagnosis during pre-clinical studies. Case presentation Felix pro- posed that there are nine lateral mesonephric arteries in an 18 mm embryo and that they are grouped as follows: 1) the cranial group, which is made up of the first and second mesonephric arteries that are located proximal to the celiac trunk of the abdominal aorta and directed posterior to the suprarenal gland; 2) the middle group, which is made up of the third to fifth mesonephric arteries which run along the ventral surface of the suprarenal gland; 3) the caudal group, which is made up of the sixth to ninth mesonephric arteries which run along to the ventral surface of the suprarenal gland [22]. The caudal group forms the arterial plexus of the uro- genital system [22,23]. Authors’ contributions GKP id ifi d h i Authors contributions GKP identified the variant, performed the anatomical dissection, created the schematic drawing and reviewed the final version of the manuscript. OI and AR prepared the draft of the manuscript. AR improved the image presented in this report. BP, KN, IS, and PK performed the final edit of the manuscript. All authors read and approved the final manuscript. During developmental modifications of the gastroin- testinal tract, the celiac splanchnic arteries and their longitudinal anastomotic channels are gradually Consent Despite the fact that any of the nine mesonephric arteries can evolve to become the TA, Felix reported that the TA usually derives from the caudal group. In the same study, Felix claimed that the TA rarely derives from the cranial group. When such a case occurs, the TA is brought posteriorly to the renal artery, which ori- ginates from the middle group. In our case report, the TA corresponds to the cranial group as it is located superior to the celiac trunk [22]. However, in our case report, and contrary to Felix’s report, the TA is located anterior to the renal artery. This means that the cranial and caudal groups are not necessarily independent of each other but connected by longitudinal anastomotic channels located ventrally to the developing renal artery. Written informed consent was obtained from the patient’s next-of-kin for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Paraskevas et al. Journal of Medical Case Reports 2011, 5:75 http://www.jmedicalcasereports.com/content/5/1/75 Paraskevas et al. Journal of Medical Case Reports 2011, 5:75 http://www.jmedicalcasereports.com/content/5/1/75 References 1. Tsikaras P, Paraskevas G, Natsis K: [Abdominal aorta. Textbook of anatomy Vol 2: Circulatory System] Thessaloniki: University Studio Press; 2005, 160-177. 1. Tsikaras P, Paraskevas G, Natsis K: [Abdominal aorta. Textbook of anatomy Vol 2: Circulatory System] Thessaloniki: University Studio Press; 2005, 160-177. 2. Asala S, Chaudhary SC, Masumbuko-Kahamba N, Bidmos M: Anatomical variations in the human testicular blood vessels. Ann Anat 2001, 183:545-549. 3. Shinohara H, Nakatani T, Fukuo Y, Morisawa S, Matsuda T: Case with high- positioned origin of the testicular artery. Anat Rec 1990, 226:264-265. 3. Shinohara H, Nakatani T, Fukuo Y, Morisawa S, Matsuda T: Case with high- positioned origin of the testicular artery. Anat Rec 1990, 226:264-265. g y 4. Onderoğlu S, Yüksel M, Arik Z: Unusual branching and course of the testicular artery. Ann Anat 1993, 175:541-544. 4. Onderoğlu S, Yüksel M, Arik Z: Unusual branching and course of the testicular artery. Ann Anat 1993, 175:541-544. y 5. Brohi RA, Sargon MF, Yener N: High origin and unusual sup of a testicular artery. Surg Radiol Anat 2001, 23:207-208. y g 6. Ozan H, Gümüşalan Y, Önderoğlu S, Simşek C: High origin of gonadal arteries associated with other variations. Ann Anat 1995, 177:156-160. 7. Çiçekcibaşi AE, Salbacak A, Seker M, Ziylan T, Büyükmumcu M, Uysal II: The origin of gonadal arteries in human fetuses: anatomical variations. Ann Anat 2002, 184:275-279. 7. Çiçekcibaşi AE, Salbacak A, Seker M, Ziylan T, Büyükmumcu M, Uysal II: The origin of gonadal arteries in human fetuses: anatomical variations. Ann Anat 2002, 184:275-279. 8. Acar HI, Yazar F, Ozan H: Unusual origin and course of the testicular arteries. Surg Radiol Anat 2007, 29:601-603. 8. Acar HI, Yazar F, Ozan H: Unusual origin and course of the testicular arteries. Surg Radiol Anat 2007, 29:601-603. 9. Bhaskar PV, Bhasin V, Kumar S: Abnormal branch of the testicular artery. Clin Anat 2006, 19:569-570. 9. Bhaskar PV, Bhasin V, Kumar S: Abnormal branch of the testicular artery. Clin Anat 2006, 19:569-570. 10. Nayak BS: Multiple variations of the right renal vessels. Singapore Med J 2008, 49:e153-155. 11. Tanyeli E, Uzel M, Soyluoğlu AI: Complex renal vascular variation: A case report. Ann Anat 2006, 188:455-458. 12. In Gray’s Anatomy. 37 edition. Edited by: Williams PL, Warwick R, Dyson M, Bannister LH. Edinburgh, London: Churchill Livingston; 1989:774-776. 13. Competing interests Th h d l h Competing interests The authors declare that they have no competing interests. Page 4 of 4 Page 4 of 4 References Kocabiyik N, Yalcin B, Kiliç C, Kirici Y, Ozan H: Accessory renal arteries and an anomalous testicular artery of high origin. Gülhane Tip Dergisi 2005, 47:141-143. 14. Xue HG, Yang CY, Ishida S, Ishizaka K, Ishihara A, Ishida A, Tanuma K: Duplicate testicular veins accompanied by anomalies of the testicular arteries. Ann Anat 2005, 187:393-398. 15. Machnicki A, Grzybiak M: Variations in testicular arteries in fetuses and adults. Folia Morphol (Warsz) 1997, 56:277-285. 16. Notkovich H: Variations of the testicular and ovarian arteries in relation to the renal pedicle. Surg Gynecol Obstet 1956, 103:487-495. p g y 17. Adachi B: Das arteriensystem der Japaner. Volume II Kyoto: Maruzen; 1928, 73-74. 18. Manso JC, DiDio LJ: Anatomical variations of the human suprarenal arteries. Ann Anat 2000, 182:483-488. 19. Bordei P, St Antohe D, Sapte E, Iliescu D: Morphological aspects of the inferior suprarenal artery. Surg Radiol Anat 2003, 25:247-251. 20. Bergman RA, Thompson SA, Afifi AK: Catalog of Human Variation Baltimore, Munich: Urban and Schwartzenberg; 1983, 119. 21. In Development and Structure of the Cardiovascular System. Edited by: Luisada AA. New York: McGraw-Hill; 1961:145. 22. Felix W: Mesonephric arteries. In Manual of Human Embryology. Volume 2. Edited by: Keibel F, Mall FP. Philadelphia, London: J.B. Lippincott; 1912:820-825. 23. Arey LB: Developmental Anatomy. A Textbook and Laboratory Manual of Embryology. 6 edition. Philadelphia, London: WB Saunders; 1960, 373-374. 24. Kitamura S, Nishiguchi T, Sakai A, Kumamoto K: Rare case of the inferior mesenteric artery arising from the superior mesenteric artery. Anat Rec 1987, 217:99-102. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 25. Tarhan S, Gümüs B, Gündüz I, Ayyildiz V, Göktan C: Effect of varicocele on testicular artery blood flow in men - color Doppler investigation. Scand J Urol Nephrol 2003, 37:38-42. doi:10.1186/1752-1947-5-75 Cite this article as: Paraskevas et al.: High origin of a testicular artery: a case report and review of the literature. Journal of Medical Case Reports 2011 5:75.
https://openalex.org/W2114744546	https://europepmc.org/articles/pmc3439827?pdf=render	English	null	Impact strength of denture base and reline acrylic resins: An in vitro study	Journal of dental biomechanics	2,012	cc-by	5,980	Introduction The proper functioning of hard chairside reline resins depends to a great extent on their characteristics and mechanical prop- erties. Therefore, previous studies of autopolymerizing reline resins have examined their chemical compositions and resid- ual monomer content1,2; physical and mechanical properties such as hardness,3,4 flexural strength,4,5 water sorption and solubility,1 and porosity6,7; and bond strength to the heat- polymerized denture base resins.8–11 Impact strength (IS) is also a desirable property because it is a measure of the energy required to initiate and propagate a crack through the mate- rial. Thus, it can reflect the contact force needed to cause frac- ture in a denture under situations such as accidental dropping.12 The occurrence of fracture, observed in maxillary and man- dibular removable prostheses,13 results in additional costs, as well as discomfort to patients14 as they must be without the dentures during the laboratory procedures required to repair or replace the broken denture. from the changes in temperature. The bond between reline materials and denture base resins is obtained by interpene- tration and interpenetrating polymer network formation at the interface.10,17 Archadian et al.5 evaluated different com- binations between denture base and reline resins and found that some of the polymer combinations showed a decrease in the flexural strength after thermocycling, demonstrating that the thermal effect caused weakening of the bond between the materials. Besides, some adhesive failures were observed. Similar results were obtained in the study by Minami et al.,11 which investigated the bond strength between a high-impact denture base resin and two autopo- lymerizing resins, one used as a denture repair material and the other marketed as a relining material. The bond strength of the reline resin was significantly lower than that of the repair resin, under all conditions evaluated. In addition, the 1Department of Dental Materials and Prosthodontics, Araraquara Dental School, UNESP—Univ Estadual Paulista, Araraquara, SP, Brazil 2Department of Materials Engineering, São Carlos Federal University, São Carlos, SP, Brazil 1Department of Dental Materials and Prosthodontics, Araraquara Dental School, UNESP—Univ Estadual Paulista, Araraquara, SP, Brazil 2Department of Materials Engineering, São Carlos Federal University, São Carlos, SP, Brazil Because dentures are exposed to temperature changes as a result of ambient temperature changes and the intake of hot/cold foods,15 resistibility to thermal stresses is also another important consideration clinically. Article Article Journal of Dental Biomechanics 3: 1758736012459535 © The Author(s) 2012 Reprints and permission: sagepub. co.uk/journalsPermissions.nav DOI: 10.1177/1758736012459535 dbm.sagepub.com Keywords I Impact strength, denture base, acrylic resins Abstract This study evaluated the impact strength of a denture base resin (Lucitone 550—L) and four reline resins (Tokuyama Rebase II—T; Ufi Gel Hard—U; New Truliner—NT, and Kooliner—K), both intact and in a reline combination (L/L, L/T, L/U, L/NT, and L/K). For each group (n = 20), half of the specimens were thermocycled before testing. Charpy tests were performed, and the impact strengths were calculated. Data were analyzed by two-way analyses of variance and Tukey’s test (p = 0.05). For the intact groups, mean impact strength values for L (1.65 and 1.50) were significantly higher than those of the reline resins (0.38–1.17). For the relined groups, the highest mean impact strength values were produced by L/T (5.76 and 5.12), L/NT (6.20 and 6.03), and L/K (5.60 and 5.31) and the lowest by L/U (0.76 and 0.78). There were no significant differences between L and L/L. Thermocycling reduced the impact strength of T (from 0.73 to 0.38) and L/L (from 1.82 to 1.56). Journal of Dental Biomechanics 3: 1758736012459535 © The Author(s) 2012 Reprints and permission: sagepub. co.uk/journalsPermissions.nav DOI: 10.1177/1758736012459535 dbm.sagepub.com 1Department of Dental Materials and Prosthodontics, Araraquara Dental School, UNESP—Univ Estadual Paulista, Araraquara, SP, Brazil 2Department of Materials Engineering, São Carlos Federal University, São Carlos, SP, Brazil Impact strength of denture base and reline acrylic resins: An in vitro study Ana L Machado1, Bruna C Bochio1, Amanda F Wady1, Janaina H Jorge1, Sebastião V Canevarolo Jr2 and Carlos E Vergani1 Introduction Therefore, stud- ies have used thermal cycling to simulate the clinical condi- tions of the oral cavity.5,11,16 The presence of an interface between the denture base resin/relining material may also have an influence on the magnitude of the effects resulting Corresponding author: Ana L Machado, Department of Dental Materials and Prosthodontics, Araraquara Dental School, UNESP—Univ Estadual Paulista, Rua Humaitá, no. 1680, CEP: 14801-903, Araraquara, SP, Brazil. Email: cucci@foar.unesp.br Journal of Dental Biomechanics 2 Material Code Batch no. Manufacturers Composition Powder/liquid ratio (g/mL) Polymerization cycles Powder Liquid Lucitone 550 L P-303758 and L-213457 Dentsply Indústria e Comércio Ltda., Petrópolis, Rio de Janeiro, Brazil PMMA MMA and EDGMA 2.1/1.0 90 min at 73°C and 30 min at 100°C Tokuyama Rebase II T UF64145 Tokuyama Dental Corp., Tokyo, Japan PEMA AAEM and 1,9-ND 2.4/1.0 5.5 min at room temperature Ufi Gel Hard U 631742 VOCO, Cuxhaven, Germany PEMA 1,6-HDMA 2.12/1.2 7 min at room temperature New Truliner NT 0310528 The Bosworth Co., Skokie, IL, USA PEMA IBMA and DBP 1.34/1.0 20 min at room temperature Kooliner K 0508187 GC America Inc., Alsip, IL, USA PEMA IBMA 2.1/1.0 10 min at room temperature PMMA: poly(methyl methacrylate); PEMA: poly(ethyl methacrylate); MMA: methyl methacrylate; EDGMA: ethylene glycol dimethacrylate; AAEM: 2-(acetoacetoxy) ethyl methacrylate; 1,9-ND: 1,9-nonanediol dimethacrylate; 1,6-HDMA: 1,6-hexanediol dimethacrylate; IBMA: isobutyl methacrylate; DBP: di-n-butyl phthalate. bond strength of the reline resin significantly decreased after thermal cycling, regardless of the conditions of sur- face treatment and water content. These findings were attributed to the high-molecular-weight monomers used in the reline resins, which may not penetrate into the polymer structure of the denture base resin, thus resulting in a poorly interpenetrating network.5,11 In addition, if there is a differ- ence in the integration between the reline and the denture base polymers, microcracks are likely to occur at their interface after thermocycling.5 On the other hand, the results from Azevedo et al.18 indicated that for some relin- ing materials, the bond strength could increase after storage in water at 37°C, suggesting a continued polymerization reaction of both the interpenetrating polymer formed at the interfacial region and the relining material located close to the interface. A review of the available literature reveals that, to date, little information is available on the IS of autopolymerizing reline materials.19–21 Moreover, the effect of thermal stresses on the IS of denture base and reline resins has not been investigated. Introduction Thus, the aim of this study was to evalu- ate the IS of one denture base acrylic resin and four hard chairside reline resins, tested alone (bulk specimens) or in combination (denture base/reline materials). The effect of thermocycling on the IS of bulk and relined specimens of each resin was also evaluated. The null hypothesis was that IS was not determined by the (a) choice of intact material, (b) presence of a reline, and (c) thermocycling, within the parameters of the experiment. Material and methods One heat-polymerized denture base acrylic resin and four autopolymerizing reline resins were selected for this study (Table 1). Initially, rectangular bars (60 × 6 × 2 mm) from Lucitone 550 (L) material were prepared. The material was proportioned, and after the mixture reached the dough stage, it was inserted into the mold in a dental flask and packed. Molds were prepared by investing silicone patterns (Zetaplus Putty; Zhermack, Rovigo, Italy), placed between two glass slides, in type IV stone (Troquel Quatro; Polidental Manufacturing and Trade Ltd, SP, Brazil). The denture base acrylic resin was polymerized in a thermo- statically controlled water bath (Termotron P-100; Termotron, Piracicaba, SP, Brazil) according to the manu- facturer’s recommendations (Table 1). After polymeriza- tion, the flasks were left on the bench to cool at room temperature for 30 min and then placed for 15 min under running water before opening, following the manufactur- er’s instructions. Thereafter, the L rectangular bars were removed from the flasks, the edges were finished with 400- grit silicon carbide paper, and the accuracy of the dimen- sions was verified at three locations for each dimension. A tolerance of ±0.03 mm was accepted. The L rectangular bars were stored in distilled water at 37°C ± 1°C for 50 ± 2 h 3 Machado et al. before relining.22 After water storage, the denture base resin surfaces to be bonded with the reline materials were ground wet in an automatic grinding machine (Metaserv 2000, model 95-2829; Buehler UK Ltd, Coventry, England) using silicon carbide paper (240 grit).18,20 The surfaces were brushed with liquid detergent for 20 s, rinsed with distilled water, and dried with absorbent paper. Before testing, the specimens were notched with a notching cutter (Notchvis; Ceast, Pianezza, Italy). The V-notches were cut into the reline materials, across the width of the specimens with a depth of 0.8 mm leaving an effective depth under the notch of 3.2 mm.20 The ISs were evaluated by the Charpy impact tester (RESIL 25R; Ceast, Pianezza, Italy), with the unnotched surface of the specimens facing the pendulum. The test was performed with 0.5-J pendulum and a 150° lifting angle. The IS (in kJ/m2) was calculated as IS = EC/(hbA), where EC is the corrected energy absorbed by breaking the test specimen, bA is the remaining thickness at the notch tip, and h is the width of the specimen. Material and methods Before the L rectangular bars are relined with the autopolymerizing materials New Truliner (NT), Tokuyama Rebase II (T), and Ufi Gel Hard (U), the bond surfaces were treated with the bonding agents recom- mended by the manufacturers, whereas for the reline resin Kooliner (K), methyl methacrylate (MMA) mono- mer was applied for 180 s.9 For experimental conditions L/T, L/U, L/NT, and L/K, delamination between the denture base and reline resin was observed for the majority of the samples, and the results were distinct from those of the other conditions evaluated. Thus, it was considered appropriate to analyze their data separately, using a two-way analysis of vari- ance (ANOVA) with the factors material and group. A separate two-way ANOVA was also used to test for dif- ferences among the other experimental conditions (L, T, U, NT, K, and L/L). Levene and Shapiro–Wilk tests (Statistica 6.0; Statsoft, Tulsa, OK, USA) were applied to test homogeneity of variance and normality, respectively. Tukey’s honestly significant difference (HSD) post hoc test was applied to the results to determine whether sig- nificant differences existed among the means. A signifi- cance level of p ≤ 0.05 was established. The relined specimens were fabricated using a stainless steel mold with a cavity of 60 × 6 × 4 mm. The L rectangu- lar bars were placed into the cavity, and the reline resins were inserted to fill the remainder of the mold. The manu- facturers’ instructions of each reline material for mixing and processing were followed (Table 1). An acetate sheet and a glass plate were placed over the reline material, and pressure was applied until polymerization was complete. The edges of the specimens were finished, and the accuracy of the dimensions (width and thickness) was verified with a caliper (Mitutoyo Sul Americana, Suzano, SP, Brazil) at three locations of each dimension to within 0.03 mm tolerance.20 To evaluate the IS of the specimens made following laboratory reline procedures, L rectangular bars were relined with the same material. Initially, silicone patterns (60 × 6 × 4 mm) were obtained, placed between two glass slides, and then flasked to create molds for packing the L–L specimens. The L rectangular bars (2.0 mm) were then adapted in the lower portion of the stone mold, and the remaining 2.0 mm was filled with L acrylic resin dough. Processing, finishing, and verification of the accuracy of the specimens were performed as described. Results The mean IS values and the standard deviations for all con- ditions evaluated are shown in Table 2. For the intact groups, the mean IS of L was higher than that of K, and both were higher than those of T, U, and NT. For the relined groups, the highest IS values were seen for L/T, L/NT, and L/K, and the lowest IS values were seen for L/U specimens. There were no significant differences between L bulk and L/L specimens. For comparison purposes, the bulk specimens of all materials were prepared to the thickness of the relined specimens (4 mm) and tested. Half of the bulk (n = 20) and half of the relined (n = 20) specimens of each material were thermocycled before testing. Thermal cycles were made in a thermocycling machine (model MSCT-3; Marcelo Nucci—ME, São Carlos, SP, Brazil) and consisted of 5000 cycles at 5°C and 55°C with a 30-s dwell time.16 Table 2 also shows that thermocycling reduced the IS of T, while the other intact materials (L, U, NT, and K) were not significantly affected. For the relined groups, the results demonstrated that L/L specimens exhibited a small but sig- nificant reduction in the IS after thermocycling. No signifi- cant differences were found between nonthermocycled and thermocycled specimens for the other reline combinations (L/T, L/U, L/NT, and L/K). For K, NT, T, and U reline resins, the specimens (bulk and relined) were subjected to the impact tests, or to the thermal cycling prior to the impact tests, within 30 min after polymerization. This time period was used as the patients will be wearing the relined denture bases soon after polymerization. For the L denture base material, the speci- mens (bulk and relined with the same material) were sub- jected to the impact tests, or to the thermal cycling prior to the impact tests, after storage in distilled water at 37°C ± 1°C for 50 ± 2 h.22 All specimens broken with a sharp fracture typical of the brittle fracture behavior, characterized by a lack of distor- tion of the broken parts. In addition, several specimens of the experimental conditions L/T, L/NT, and L/K showed delamination between the denture base resin and the relin- ing material. Journal of Dental Biomechanics 4 Table 2. Results Impact strength mean values (kJ/m2) and standard deviations (SDs) of acrylic resins and groups evaluated Materials Groups Nonthermocycled Thermocycled L 1.65 (0.14)Ca 1.50 (0.19)Ea T 0.73 (0.06)Aa 0.38 (0.15)Ab U 0.58 (0.11)Aa 0.67 (0.12)BCa NT 0.61 (0.06)Aa 0.52 (0.08)ABa K 1.17 (0.14)Ba 1.02 (0.06)Da L/L 1.82 (0.27)Ca 1.56 (0.26)Eb L/T 5.76 (1.33)Da 5.12 (1.82)Fa L/U 0.76 (0.16)Aa 0.78 (0.16)Ca L/NT 6.20 (1.74)Da 6.03 (2.23)Fa L/K 5.60 (3.34)Da 5.31 (2.53)Fa Horizontally, identical superscripted small letters denote no significant dif- ferences among groups (p > 0.05). Vertically, identical superscripted capital letters denote no significant differences among materials (p > 0.05). Table 2. Impact strength mean values (kJ/m2) and standard deviations (SDs) of acrylic resins and groups evaluated acrylic resin,25 and an increase in the cross-linking reaction decreased the flowability of polymer, resulting in the reduc- tion of the IS.20,26 Thus, the lower IS values of T and U com- pared to L and K resins could be related to their higher concentration of cross-linking agents. It is likely that during the polymerization reaction of T and U materials, a highly cross-linked network was formed, thus resulting in polymers with reduced IS. In spite of the composition of resin NT being similar to that of K, the IS value of the former was significantly lower than the latter. A possible explanation could be the presence of 8% plasticizer agent (di-n-butyl phthalate (DBP)) in the liquid of material NT,1 which may have an influence on its resistance to impact forces. The mean values of IS for L/L were greater than the cor- responding values for the intact material L, indicating that when relining is performed with the same heat-polymerized resin used for denture base fabrication, the IS of the denture is not detrimentally affected. A possible explanation for these results could be the bond between the two materials. Some researchers have looked at solvent bonding since it gives rela- tively strong bonding, without introducing a foreign adhesive material. Results The mechanism for solvent bonding is through the dissolution of the polymer from both bonding surfaces fol- lowed by the interdiffusion of polymer chains.27,28 Similar mechanism has been used for bonding in denture repair.11,29–32 In the relining procedure, the bond between resins is due to the diffusion and polymerization of monomer across the reline resins–denture base interface to form interpenetrating polymer networks.10,17 For interlacing, the solubility parame- ter of the solvent monomer should match the solubility parameter of the polymer.33 With the basic principle of “like dissolves like,” polymers will be soluble in liquids that have solubility parameters not too different from theirs.27 The solu- bility parameter of MMA (8.8 (cal/cm3)1/2) is similar to that of PMMA (9.45 (cal/cm3)1/2).33 In addition, the resin L remained under pressure for 30 min in contact with the polymerized denture base resin, before polymerization, and was processed at higher temperatures, with a terminal boil of 30 min. All these factors may have enhanced the diffusion of the mono- mer MMA, present in the dough mixture, from the reline material into the denture base PMMA polymer,17,28 promot- ing an adequate bond and strong interface between the mate- rials. During the impact tests of L/L specimens, no delamination between the two materials was observed. This further suggests that the interfacial bonding was strong enough and that the cracks may have propagated in the relined specimens as if through a homogeneous material.20 As a result, no significant differences were found between L bulk intact specimens and L relined specimens. The mean values of IS for L/L were greater than the cor- responding values for the intact material L, indicating that when relining is performed with the same heat-polymerized resin used for denture base fabrication, the IS of the denture is not detrimentally affected. A possible explanation for these results could be the bond between the two materials. Some researchers have looked at solvent bonding since it gives rela- tively strong bonding, without introducing a foreign adhesive material. The mechanism for solvent bonding is through the dissolution of the polymer from both bonding surfaces fol- lowed by the interdiffusion of polymer chains.27,28 Similar mechanism has been used for bonding in denture repair.11,29–32 Horizontally, identical superscripted small letters denote no significant dif- ferences among groups (p > 0.05). Vertically, identical superscripted capital letters denote no significant differences among materials (p > 0.05). Discussion The null hypothesis that the IS was not determined by the (a) choice of intact material, (b) presence of a reline, and (c) thermocycling, within the parameters of the experiment, was rejected. It is clear that the mean values of IS for resin L were the highest of the intact materials tested. A study2 reported the residual monomer content of some denture base and reline resins. Resin L, when polymerized by a short cycle (which was also used in the present investigation), showed a lower percentage of residual monomer (0.08%) than the reline resins K (1.52%) and U (0.45%). The residual mono- mer content may have an adverse effect on mechanical prop- erties such as hardness4 and tensile strength.23 However, a direct relationship between the quantity of residual monomer and IS could not be observed in the present study. The differ- ences in composition of the materials evaluated could help explain, at least partly, their different behaviors when sub- jected to the impact test. The powder of resin L consists of polymethyl methacrylate (PMMA), while the liquid contains MMA monomer and the cross-linking agent ethylene glycol dimethacrylate (EDGMA). Although the manufacturer does not provide information on the exact concentration of EDGMA in the resin L, the percentage of this cross-linking agent in denture base acrylic resins usually is limited to 15%.1,24 For the reline resin K, the powder is essentially pol- yethyl methacrylate (PEMA), and the liquid contains isobu- tyl methacrylate (IBMA), without a cross-linking agent. The powder of resins U and T also consists of PEMA. However, the liquid of material U is mainly composed of the cross- linking agent 1,6-hexanediol dimethacrylate (1,6-HDMA). For material T, the liquid consists of 59% acetoacetoxy ethyl methacrylate (AAEM) monomer and 39% of 1,9-nonanediol dimethacrylate (1,9-ND) as the cross-linking agent. Discussion As a result, the IS, which is the energy needed to cause a material to fracture when struck, was higher for the L/T, L/NT, and L/K specimens than for the L/U specimens. The results observed for the L/U specimens could also be attributed to the lower IS of the autopolymer- izing reline resin U compared to the heat-polymerized den- ture base acrylic resin L. L liquid used for treating the bonding surfaces during the fabrication of the L/K specimens. Despite the similarity of the solubility parameters of MMA and PMMA, the contact time between the solvent and the bonding surfaces was much lower compared to that of the L/L specimens (30 min of dough time). In addition, the molecular weight of IBMA (142.19 g/mol),34 the main component of NT and K liquids, is higher than that of MMA (100.18 g/mol).34 Larger mole- cules may act slower with respect to swelling PMMA and penetrating the PMMA surface. For the L/T specimens, the L bond surfaces were treated with the adhesive supplied by the reline resin T manufacturer, which contains the nonpo- lymerizable solvents, ethyl acetate (EA) and acetone (A).29 Their solubility parameters are close to that of PMMA (EA: 9.1 (cal/cm3)1/2; A: 9.9 (cal/cm3)1/2).33,35 The surfaces to be bonded with the reline resin U were treated with the bond- ing agent recommended by the manufacturer, which con- tains a monomer (2-hydroxyethyl methacrylate (HEMA)) and a solvent (dichloromethane (DCH)), with solubility parameters of 11.4 and 9.68 (cal/cm3)1/2, respectively.35 Although the solubility parameter of HEMA is higher than that of PMMA, bonding agents containing both solvents and monomers have been reported to have a positive effect on the bond strength of denture base to reline resins,10,18 and the use of a DCH-based primer was more effective than the EA-based primer.30 The application of MMA and Tokuyama Rebase II adhesive on heat-polymerized acrylic resin resulted only in shallow pits and small crests, respectively, in contrast with the three-dimensional (3D) pores pro- duced by other solvents.29 In addition, it has been found that the reline resin U exhibited higher mean shear bond strength values to the denture base acrylic resin L than other hard reliners,36 including K and NT materials.18 The molecular weight of the monomers may also have played a role. Discussion Cross- linking agent concentration influenced the IS of denture base In the relining procedure, the bond between resins is due to the diffusion and polymerization of monomer across the reline resins–denture base interface to form interpenetrating polymer networks.10,17 For interlacing, the solubility parame- ter of the solvent monomer should match the solubility parameter of the polymer.33 With the basic principle of “like dissolves like,” polymers will be soluble in liquids that have solubility parameters not too different from theirs.27 The solu- bility parameter of MMA (8.8 (cal/cm3)1/2) is similar to that of PMMA (9.45 (cal/cm3)1/2).33 In addition, the resin L remained under pressure for 30 min in contact with the polymerized denture base resin, before polymerization, and was processed at higher temperatures, with a terminal boil of 30 min. All these factors may have enhanced the diffusion of the mono- mer MMA, present in the dough mixture, from the reline material into the denture base PMMA polymer,17,28 promot- ing an adequate bond and strong interface between the mate- rials. During the impact tests of L/L specimens, no delamination between the two materials was observed. This further suggests that the interfacial bonding was strong enough and that the cracks may have propagated in the relined specimens as if through a homogeneous material.20 As a result, no significant differences were found between L bulk intact specimens and L relined specimens. As stated for the L/L specimens, the bond between the denture base L and the hard reline materials may have also contributed to the results of the present study. For the L/NT specimens, the L bond surfaces were treated with the adhe- sive supplied by the reline resin NT manufacturer, which contains MMA that is also the main constituent of the resin 5 Machado et al. between the reinforcements and the polymer, suggesting that the chemical bonding of the denture base polymer to the fiber reinforcements was not adequate.37 Delamination consumes energy and causes a crack to deviate from the initial direction, and the energy is not used to drive cracks through the next material but to drive the deflected crack along the interface between the denture base and reline res- ins.20 It is likely that the cracks must have been propagated through the interface between the L and U materials with- out deflection. Discussion Reline resin U contains 1,6-HDMA with a molecular weight of 254.33 g/mol.34 For material T, although the molecular weight of the monomer AAEM is 214.22 g/mol,34 this reline resin also contains 39% of the cross- linking agent 1,9-ND, which has a higher molecular weight of 296.4 g/mol.34 Thus, it is likely that for the hard chair- side reline T, NT, and K, the interfacial bonding with the denture base resin L was not as strong as the bond achieved when the relining material was the same as that of the den- ture base (L/L combinations) or when the specimens were relined with resin U (L/U). During the tests, a large number of L/T, L/NT, and L/K specimens showed delamination between the reline resin For the majority of the experimental conditions evalu- ated, thermocycling did not affect the IS of the materials. However, for the reline resin T bulk specimens, thermocy- cling resulted in significantly lower IS. Although one could argue that the reduced IS of T for the thermocycled group was due to the effect of water absorption alone and that thermocycling may not be a factor, it has been observed that storage in water at 37°C for up to 6 months did not affect the IS of reline resin T.21 In addition, Archadian et al.5 also observed that thermocycling led to a decrease in the flexural strength of the reline polymer-only specimens. These results suggest that for some materials, besides the plasticizing effect of the absorbed water, the increased tem- perature may also alter their mechanical properties. It is important to emphasize, however, that the reduction observed when resin T was tested alone was not observed when combined with resin L (L/T specimens). Given that during clinical use, the forces would be applied to the relined denture base as a whole, the results from the L/T specimens can be considered more clinically meaningful. The results of this study also showed that the L/L speci- mens exhibited a small but significant decrease after ther- mocycling. Despite this reduction, the mean value of the L/L thermocycled specimens was not significantly different from that of L intact thermocycled specimens, which in turn did not differ from that obtained for L intact nonthermocy- cled specimens. In addition, the statistical analysis did not show significant difference between L and L/L nonthermo- cycled specimens. Discussion In a previous study,16 it was observed that the microleakage, which can be an early indication of debonding, between hard chairside relines and denture base acrylic resins, including the combinations L/L, L/T, L/U, and L/K, was not significantly influenced by thermal cycling. Thus, it can be supposed that the decrease in the IS of L/L specimens after thermocycling would probably not significantly affect the clinical performance of the relined dentures. During the tests, a large number of L/T, L/NT, and L/K specimens showed delamination between the reline resin and the denture base resin, while for L/U specimens, no delamination was observed. Studies on composites have shown that the propagation of fracture during loading initi- ates from microdamage of the polymer matrix and results in transverse matrix cracking, delamination, and finally to fiber failure.37 It has been observed that when the fiber-rich area was covered with the polymer, the fracture propagated until it reached the fibers and continued as delamination The results on the strength of intact and relined den- ture-shaped specimens, loaded in compression until Journal of Dental Biomechanics 6 7. Wyatt CC, Harrop TJ and Macentee MI. A comparison of physical characteristics of six hard denture reline materials. J Prosthet Dent 1986; 55: 343–346. fracture, have suggested that the thermal and mechani- cal stresses are mechanisms that act independently and exhibit differences in the degree to which they contrib- ute to the degradation of denture bases.38 Therefore, the use of a simple rectangular-shaped specimen rather than a complex denture base and the absence of cyclic loading to simulate the masticatory forces are limita- tions of this in vitro study and should be taken into consideration in further investigations on the IS of these materials. fracture, have suggested that the thermal and mechani- cal stresses are mechanisms that act independently and exhibit differences in the degree to which they contrib- ute to the degradation of denture bases.38 Therefore, the use of a simple rectangular-shaped specimen rather than a complex denture base and the absence of cyclic loading to simulate the masticatory forces are limita- tions of this in vitro study and should be taken into consideration in further investigations on the IS of these materials. 8. Arima T, Nikawa H, Hamada T, et al. Composition and effect of denture base resin surface primers for reline acrylic resins. J Prosthet Dent 1996; 75: 457–462. 9. Discussion Leles CR, Machado AL, Vergani CE, et al. Bonding strength between a hard chairside reline resin and a denture base material as influenced by surface treatment. J Oral Rehabil 2001; 28: 1153–1157. 10. Takahashi Y and Chai J. Assessment of shear bond strength between three denture reline materials and a denture base acrylic resin. Int J Prosthodont 2001; 14: 531–535. Conclusions 11. Minami H, Suzuki S, Minesaki Y, et al. In vitro evaluation of the influence of repairing condition of denture base resin on the bonding of autopolymerizing resins. J Prosthet Dent 2004; 91: 164–170. Within the limitations of this in vitro study, the following conclusions were drawn: 12. Jagger DC and Harrison A. The effect of continuous poly(methyl methacrylate) fibres on some properties of acrylic resin denture base material. Eur J Prosthodont Restor Dent 2000; 8: 135–138. 1. The mean ISs of the intact resin L specimens were significantly greater than those of the other intact materials. 2. There were no significant differences between the mean ISs of the intact L and relined L/L specimens. 13. Prombonas AE and Vlissidis DS. Comparison of the midline stress field in maxillary and mandibular complete dentures: a pilot study. J Prosthet Dent 2006; 95: 63–70. 3. The highest mean ISs overall were observed for L/T, L/NT, and L/K and the lowest for L/U. 14. Vallittu PK, Vojtkova H and Lassila VP. Impact strength of den- ture polymethyl methacrylate reinforced with continuous glass fibers or metal wire. Acta Odontol Scand 1995; 53: 392–396. 4. Thermocycling did not significantly affect the IS of corresponding groups, with the exception of T and 15. Palmer DS, Barco MT and Billy EJ. Temperature extremes produced orally by hot and cold liquids. J Prosthet Dent 1992; 67: 325–327. L/L for which a significant reduction was observed. Funding 16. Giampaolo ET, Jorge JH, Machado AL, et al. Effect of ther- mal cycling on microleakage between hard chairside relines and denture base acrylic resins. Gerodontology 2011; 28: 121–126. This investigation was supported by Brazilian Council for Scientific and Technological Development (CNPq—Grant 301042/2004-7) and Sao Paulo State Research Foundation (FAPESP—Grant 05/04236-2). 17. Vallittu PK and Ruyter IE. The swelling phenomenon of acrylic resin polymer teeth at the interface with denture base polymers. J Prosthet Dent 1997; 78: 194–199. References 18. Azevedo A, Machado AL, Giampaolo ET, et al. The effect of water immersion on the shear bond strength between chair- side reline and denture base acrylic resins. J Prosthodont 2007; 16: 255–262. 1. Arima T, Murata H and Hamada T. Properties of highly cross- linked autopolymerizing reline acrylic resins. J Prosthet Dent 1995; 73: 55–59. 19. Latta MA and Tateosian LH. Comparison of two light cured reline systems. J Clin Dent 1989; 1: 116–120. 2. Urban VM, Machado AL, Oliveira RV, et al. Residual mon- omer of reline acrylic resins. Effect of water-bath and micro- wave post-polymerization treatments. Dent Mater 2007; 23: 363–368. 20. Perez LE, Machado AL, Canevarolo SV, et al. Effect of reline material and denture base surface treatment on the impact strength of a denture base acrylic resin. Gerodontology 2010; 27: 62–69. 3. Haywood J, Wood DJ, Gilchrist A, et al. A comparison of three hard chairside denture reline materials. Part II. Changes in col- our and hardness following immersion in three commonly used denture cleansers. Eur J Prosthodont Restor Dent 2003; 11: 165–169. 21. Wady AF, Machado AL, Vergani CE, et al. Impact strength of denture base and reline acrylic resins sub- jected to long-term water immersion. Braz Dent J 2011; 22(1): 56–61. 4. Urban VM, Machado AL, Vergani CE, et al. Effect of water-bath post-polymerization on the mechanical proper- ties, degree of conversion, and leaching of residual com- pounds of hard chairside reline resins. Dent Mater 2009; 25: 662–671. 22. ISO 1567:1999/Amd 1: 2003. Dentistry-denture base poly- mers. Amendment, http://www.iso.ch/iso/en/prods-services/ ISOstore/store.html (accessed 8 May 2009). 23. Dogan A, Bek B, Cevik NN, et al. The effect of preparation conditions of acrylic denture base materials on the level of residual monomer, mechanical properties and water absorp- tion. J Dent 1995; 23: 313–318. 5. Archadian N, Kawano F, Ohguri T, et al. Flexural strength of rebased denture polymers. J Oral Rehabil 2000; 27: 690–696. 24. Price CA. The effect of cross-linking agents on the impact resistance of linear poly(methyl methacrylate) denture- base polymer. J Dent Res 1986; 65: 987–992. 6. Novais PM, Giampaolo ET, Vergani CE, et al. The occur- rence of porosity in reline acrylic resins. Effect of microwave disinfection. Gerodontology 2009; 26: 65–71. Machado et al. 7 25. Caycik S and Jagger RG. The effect of cross-linking chain length on mechanical properties of a dough molded poly(methyl methacrylate) resin. Dent Mater 1992; 8: 153–157. References 32. Vallittu PK, Lassila VP and Lappalainen R. Wetting the repair surface with methyl methacrylate affects the trans- verse strength of repaired heat-polymerized resin. J Prosthet Dent 1994; 72: 639–643. 26. Oku J. Impact properties of acrylic denture base resin. 3. Impact properties of cross-linked polymers. Dent Mater J 1989; 8: 215–222. 33. Vallittu PK. Interpenetrating polymer networks (IPNS) in dental polymers and composites. J Adhes Sci Technol 2009; 23: 961–972. 27. Ng SH, Tjeung RT, Wang ZF, et al. Thermally activated solvent bonding of polymers. Microsyst Technol 2008; 14: 753–759. 34. ChemExper Inc. 2012, https://www.chemexper.com/ 28. Arena CA, Evans DB and Hilton TJ. A comparison of bond strengths among chairside hard reline materials. J Prosthet Dent 1993; 70: 126–131. 35. Berek D. Separation of parent homopolymers from diblock copolymers by liquid chromatography under limiting condi- tions of desorption. 4. Role of eluent and temperature. J Sep Sci 2010; 33: 3476–3493. 29. Thunyakitpisal N, Thunyakitpisal P and Wiwatwarapan C. The effect of chemical surface treatments on the flexural strength of repaired acrylic denture base resin. J Prosthodont 2011; 20: 195–199. 36. Vergani CE, Seó RS, Reis JM, et al. Effect of water stor- age on the shear strength and fatigue limit of the reline resin bond to denture base resins. J Adhes Dent 2010; 12: 319–327. 30. Ohkubo T, Oizumi M and Kobayashi T. Influence of meth- ylmercaptan on the bonding strength of autopolymerizing reline resins to a heat-polymerized denture base resin. Dent Mater J 2009; 28: 426–432. 37. Narva KK, Lassila LVJ and Vallittu PK. Flexural fatigue of denture base polymer with fiber-reinforced composite rein- forcement. Composites Part A 2005; 36: 1275–1281. 31. Shimizu H, Kakigi M, Fujii J, et al. Effect of surface prepara- tion using ethyl acetate on the shear bond strength of repair resin to denture base resin. J Prosthodont 2008; 17: 451–455. 38. Seo RS, Murata H, Hong G, et al. Influence of thermal and mechanical stresses on the strength of intact and relined den- ture bases. J Prosthet Dent 2006; 96: 59–67.
https://openalex.org/W4392852108	https://pubs.rsc.org/en/content/articlepdf/2024/su/d3su00398a	English	null	Mycomining: Perspective on fungi as scavengers of scattered metal, mineral, and rare earth element resources	RSC sustainability	2,024	cc-by	6,776	PERSPECTIVE Open Access Article. Published on 15 March 2024. Downloaded on 10/24/2024 5:03:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Cite this: RSC Sustainability, 2024, 2, 1350 Received 1st November 2023 Accepted 13th March 2024 DOI: 10.1039/d3su00398a rsc.li/rscsus d on 15 March 2024. Downloaded on 10/24/2024 5:03:26 AM. nsed under a Creative Commons Attribution 3.0 Unported Licence. Mycomining: perspective on fungi as scavengers of scattered metal, mineral, and rare earth element resources ess Article. Published on 15 March 2024. Downloaded on 10/24/2024 5:03:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Cite this: RSC Sustainability, 2024, 2, 1350 Cite this: RSC Sustainability, 2024, 2, 1350 Mitchell P. Jones a and Alexander Bismarck ab Mining provides raw materials critical to our energy, agriculture, infrastructure, and technology but is associated with many environmental challenges. Resource recovery alternatives like urban mining rely on inconsistent supply streams and complicated disassembly and sorting, while extreme mining alternatives such as deep sea and space mining are potentially even less sustainable than traditional mining. This perspective investigates biological mining with emphasis on the potential of fungi for scavenging metals, minerals, and rare earth elements. “Mycomining” produces only biomass-based organic waste and can oﬀer more versatile growth conditions than phytomining using hyperaccumulating plants including substrates ranging from soil, wood, water, and rock to living organisms and dark, space-restricted, or extreme i.e., pH levels, high salt, acidic, radioactive environments. This concept could represent a useful supplement to urban and phytomining to oﬀset demand for traditional mining and is particularly viable when conventional mining may be ineﬃcient or uneconomical i.e., with low-grade ores and sites unsuited to traditional mining for geographical, political, or social reasons. Sustainability spotlight Mining is essential for many sectors but is associated with considerable environmental challenges. Urban mining supply streams are inconsistent and asso- ciated disassembly and sorting complicated, while deep sea and space mining pose severe environmental risks. Biological mining using fungi can oﬀset demand for terrestrial mining, representing environmental benet through this energy- and waste-eﬃcient process and remediate contaminated land. It can be applied in diverse growth environments with low-grade ores and sites unsuited to traditional mining for geographical, political, or social reasons. This work aligns with UN SDG 9 (Industry, Innovation, and Infrastructure), SDG 11 (Sustainable Cities and Communities), SDG 12 (Responsible Consumption and Production) and SDG 15 (Life on Land). other products are reliant on mined metals, such as lithium, cobalt, and rare earth elements (REE).5 aPolymer & Composite Engineering (PaCE) Group, Institute of Materials Chemistry and Research, Faculty of Chemistry, University of Vienna, W¨ahringer Straße 42, 1090, Vienna, Austria. E-mail: mitchell.jones@univie.ac.at; alexander.bismarck@univie.ac. at bDivision of Materials Science, Department of Engineering Sciences and Mathematics, Lule˚a University of Technology, SE-97187 Lule˚a, Sweden RSC Sustainability View Article Online View Journal \| View Issue bDivision of Materials Science, Department of Engineering Sciences and Mathematics, Lule˚a University of Technology, SE-97187 Lule˚a, Sweden Open Access Article. Published on 15 March 2024. Downloaded on 10/24/2024 5:03:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 15 March 2024. Downloaded on 10/24/2024 5:03:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Deep sea mining at depths >200 m can yield gold, silver and copper but primarily targets polymetallic nodules and sulphides, and cobalt-rich ferromanganese crusts.20,21 Potential deep sea mining sites can be identied by autonomous underwater vehicles or remotely operated vehicles, which can then collect polymetallic nodules from the seabed using a vacuum-like system or scrape cobalt-rich crusts from underwater formations.22,23 Polymetallic sulphides occur near hydrothermal vent systems and require more traditional mining techniques, similar to terrestrial mining.24–26 Deep sea mining operations have serious environmental concerns, such as habitat destruction,27 threat to biodiversity, sediment plumes through disruption of the seabed,28 and could release toxic substances trapped in the seabed and impact carbon sequestra- tion.29 Space mining i.e., the harvesting of resources from aster- oids, the moon or other celestial bodies has also been proposed but is still in a conceptual phase.30 1. Introduction 1. Traditional mining practices typically produce overburden (soil and rock removed to access ore beneath) and tailings (material leaer the minerals are extracted from the ore that can contain chemicals and are usually disposed of in “tailings dams” or ponds).6 They are also associated with many envi- ronmental challenges: mine water contaminated with chem- icals, heavy metals and other pollutants that can contaminate local waterways and groundwater,7 chemical wastes from mineral extraction, such as cyanide, sulfuric acid and mercury,8 used oil and fuel from mining machinery9 and waste rock that can produce acid when exposed to water and air leading to “acid mine drainage”.10 Smelting can also lead to emissions and dust including sulphur dioxide, heavy metals and other pollutants, and production of slag.11 Terrestrial mining is a complex process comprising exploration, mine planning and design, site preparation, extraction, ore processing, waste management and transportation of essential raw materials like metals (e.g., iron, gold, copper, and aluminium), minerals (e.g., gypsum, limestone, and salt), and fossil fuels (e.g., coal, oil, and natural gas).1,2 Fossil fuels have historically been primary sources of energy,3 while minerals like potash and phosphate are extracted for use in fertilisers used in food production.4 Metals like iron (for steel) and copper are vital for building infrastructure and much technological advance- ment in consumer products, medicine, energy storage and Alternatives to traditional mining can help to oﬀset demand for extraction of new metals and minerals and consequently improve environmental outcomes. Urban mining constitutes the collection and sorting of e-waste and old infrastructure,12 © 2024 The Author(s). Published by the Royal Society of Chemistry 1350 \| RSC Sustainability, 2024, 2, 1350–1357 RSC Sustainability View Article Online RSC Sustainability View Article Online © 2024 The Author(s). Published by the Royal Society of Chemistry Perspective RSC Sustainability typically through a process of manual disassembly, shredding, and magnetic separation13 prior to metal recovery using hydro- (metal recovery using aqueous chemistry or dissolving in a strong acid followed by precipitation)14 or pyrometallurgical (melted metals are separated from the slag or dissolved metals in solution are deposited onto a substrate using a current) processes,15 which can then be further rened for reuse. Pro- cessing 2 tonnes of spent car catalysts can prevent the mining of 150 kg of platinum group element (PGE) ores.16 That said, urban mining suﬀers from inconsistent supply streams due to its reliance on discarded products as feedstock,17 requires complicated and intricate sorting processes to separate complex, miniaturized, and fused components18 and requires the handling of toxic substances, such as lead, mercury, cadmium, and brominated re retardants,19 all of which result in high costs and regulatory and policy challenges. in a more environmentally friendly fashion than traditional mining. We map the research space linking mycoremediation (rehabilitation of a polluted environment through degradation or accumulation and disposal of contaminants e.g., oil, plastics, heavy metals), phytomining and so-called “mycomining” (scav- enging dispersed valuable resources using plants and fungi, respectively, and recovery of said resources from the biomass). The perspective serves as a white paper with a broad applied scope based on the similarities, diﬀerences, interplay, and ultimately cohesion of these themes. In this vein, we collect, contrast, and extrapolate relevant points from each of these topics, to demon- strate in a single manuscript how principles from each topic can support each other and in doing so provide some insight into the potential of fungi in the active collection and winning of valuable resources, i.e., “mycomining”. Where possible, we also provide vision for how these concepts could be applied and the associated potential benet and challenges. 2. Phytomining Phytomining is the growth, harvesting and processing of hyperaccumulating plant species to extract valuable metals, such as Ni, Zn, Cd, Mn, As, and Se, and REEs from contami- nated soils or subgrade ore bodies.31–34 Metal hyper- accumulation in plants is a genetically controlled trait that is thought to have evolved as a method of preventing herbivores or pests feeding on the plants,35 to alter soil chemistry to provide a competitive advantage over other plants,36 or enhance toler- ance to drought or other environmental stresses.37 Over 500 taxa of plants,32 including Berkheya coddii (especially eﬃcient at accumulating Ni)38 possess this ability to absorb high quantities of metals without experiencing detrimental eﬀects to their own health.39 Such plants take specic metals and nutrients from the soil up through their roots using specialised transport proteins40 before binding potentially toxic metals to metal- This perspective focuses on an oen-overlooked alternative mining technique: biological mining i.e., the use of plants or microorganisms to recover scattered metals, minerals, and REEs Fig. 1 Metal hyperaccumulation in plants comprising uptake of nutrients and metals through the roots using specialized transport proteins prior to binding metals to organic molecules causing high metal concentrations in cell organelles, older tissues and trichomes (hair-like structures on the surface of the plant). Fig. 1 Metal hyperaccumulation in plants comprising uptake of nutrients and metals through the roots using specialized transport proteins prior to binding metals to organic molecules causing high metal concentrations in cell organelles, older tissues and trichomes (hair-like structures on the surface of the plant). 2. Phytomining Hyperaccumulator plants oen have enhanced DNA repair mechanisms,47 detoxication pathways,45 and increased anti- oxidant production to counteract metal-induced oxidative stress.48 Open Access Article. Published on 15 March 2024. Downloaded on 10/24/2024 5:03:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The phytomining process commences with a soil composi- tion assessment at the site to determine the type and concen- tration of metals available followed by pairing with a suitable hyperaccumulating plant e.g., Alyssum spp. for Ni, Brassica juncea for Pb,49 or Fagopyrum esculentum and Cannabis sativa for the simultaneous phytoextraction of potentially toxic and REEs (Fig. 2).50 The soil may then be prepared by tilling, addition of fertilizer or pH adjustment, and traditional agricultural tech- niques to sow the site with seed or plant seedlings.51,52 While in place these plants act as a carbon sink,53 oﬀsetting the envi- ronmental impact associated with their later incineration and making the process close to carbon neutral. The plants are harvested, either manually or using machinery,54 dried using sunlight or industrial dryers, and burned to reduce the plant biomass to ash55 (the accumulated metal compounds have a higher melting point than the combustion temperature and consequently remain in the ash). Incineration typically occurs at 450–850 °C over a period ranging from a few minutes to hours. These temperatures are considerably lower (and hence more energy eﬃcient) than those associated with traditional ore processing, such as smelting, which oen occurs at tempera- tures exceeding 1200 °C but have much lower metal yields.56 Phytomining has a lesser physical disruption to the land- scape than traditional mining techniques, which commonly cause deforestation, erosion, and contamination of water sources.33 The main waste produced is also organic i.e., plant Fig. 2 The phytomining process typically starts with soil assessment to ascertain the metal type and concentration followed by pairing with a suitable hyperaccumulator plant. The soil is then prepared by tilling, fertilisation, and pH adjustment prior to sowing with seeds or planting of seedlings. Metals are accumulated and in the pre-harvest phase the plants act as carbon sinks. Plants are harvested manually or using machinery, dried and incinerated prior to recovery of the metal from the plant biomass ash using leaching, electrolysis, or precipitation. Fig. 2 The phytomining process typically starts with soil assessment to ascertain the metal type and concentration followed by pairing with a suitable hyperaccumulator plant. 2. Phytomining RSC Sustainability, 2024, 2, 1350–1357 \| 1351 Perspective View Article Online Perspective View Article Online RSC Sustainability Plant biomass ash is then processed to remove the metals through leaching (treated with a suitable solvent, such as an acid, that dissolves the desired metal and can then be separated from the solid residue),57 electrolysis (a current is passed through the solution and the metal accumulates at one of the electrodes),58 or precipitation (chemicals are added to the solution to precipitate the metal out and it is then ltered from the solution).59 Electrolysis is arguably the most environmen- tally friendly of these processes since it does not introduce new chemicals, can be precise and yield high-purity metal deposits.60 Electrolytes used in this process may, however, degrade over time and need to be replaced and since the process is energy intensive, its environmental footprint depends heavily on the source of the energy.58,61 Precipitation can be eﬃcient in recovering certain metals from solution and does not require as much energy as electrolysis but does result in the addition of new chemicals (precipitating agents) that may pose environ- mental or disposal risks and may produce sludge or solid wastes that require management.59,62 The precipitated metal may also require further processing to improve purity. Leaching is arguably the least environmentally friendly process due to the use of strong chemicals, the potential for spills or leaks and the environmental burden associated with the production and disposal of these chemicals, which have energy intensive production routes and produce waste that can contaminate water sources.6,8 Rening processes, specic to each metal, may then be used to remove impurities from the extracted metal. specic organic molecules called “chelators”.41,42 This prevents the metals from harming the plant's cellular machinery.43 High concentrations of metals are stored in organelles within the cell,44 older tissues,45 or trichomes (hair-like structures on the surface of the plant)46 to prevent metabolic disruption (Fig. 1). Hyperaccumulator plants oen have enhanced DNA repair mechanisms,47 detoxication pathways,45 and increased anti- oxidant production to counteract metal-induced oxidative stress.48 specic organic molecules called “chelators”.41,42 This prevents the metals from harming the plant's cellular machinery.43 High concentrations of metals are stored in organelles within the cell,44 older tissues,45 or trichomes (hair-like structures on the surface of the plant)46 to prevent metabolic disruption (Fig. 1). 3. Mycoremediation Like plants, some fungi can also absorb and concentrate metals, minerals and REEs, which they typically bind to proteins, peptides, and other organic molecules within their cellular structures.68 Unlike plants, however, fungi are heterotrophic (not photosynthetic), meaning that they excrete enzymes, such as laccase and peroxidase, which can break down complex organic molecules, including oil spills,69 pesticides,70 and dyes71 into water, carbon dioxide and basic mineral components. Startling examples of fungi being able to degrade or bio- accumulate pollutants are as radical as observations of Crypto- coccus neoformans and Cladosporium growing towards radioactive hotspots at Chernobyl in 1986, seemingly feeding on the radiation in something known as “radiosynthesis”.72 Open Access Article. Published on 15 March 2024. Downloaded on 10/24/2024 5:03:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 15 March 2024. Downloaded on 10/24/2024 5:03:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 2. Phytomining The soil is then prepared by tilling, fertilisation, and pH adjustment prior to sowing with seeds or planting of seedlings. Metals are accumulated and in the pre-harvest phase the plants act as carbon sinks. Plants are harvested manually or using machinery, dried and incinerated prior to recovery of the metal from the plant biomass ash using leaching, electrolysis, or precipitation. 1352 \| RSC Sustainability, 2024, 2, 1350–1357 © 2024 The Author(s). Published by the Royal Society of Chemistry RSC Sustainability View Article Online Perspective RSC Sustainability biomass, which can also be used for energy production through biofuel synthesis63 or anaerobic digestion for biogas produc- tion.64,65 Most notably, it has the advantage of simultaneously remediating contaminated soils and harvesting valuable metals, minerals and REEs.55 However, it is not directly comparable with traditional mining processes that extract and process high grade ores, which have high energy requirements and environmental ramications but also produce high yields.66 It is instead particularly useful in situations where conventional mining may be ineﬃcient or uneconomical, such as when dealing with low-grade ores and sites inaccessible to traditional mining for geographical, political, or social reasons.67 down oil spills and rejuvenate mine soil74 and port sediment health.75 Fungi have also been used to treat run-oﬀfrom roads,73 oen containing toxic chemicals and heavy metals, and to treat soils contaminated with herbicides. Mycoremediation revolves around the use of mycelium (root- like growth of lamentous fungi), which is introduced at the contaminated site in the form of fully colonised grain, straw or wood chip spawn.70,76 Mycelium can be directly introduced through mixing into the soil using tilling equipment or by hand,70 in the form of a mycelium mat, which is laid directly over surface contaminations,73 as pellets that are spread across the treatment area, as ‘socks’ or ‘baﬄes’ i.e., mesh tubes lled with colonised substrate that can be placed in contaminated water bodies, as inoculated wood chips or mulch, or can be sprayed over large-scale or uneven terrains as liquid mycelium culture. Species are selected based on the type of contaminant (e.g., hydrocarbons, heavy metals, pesticides), soil type, pH and moisture content, and existing microbial communities.69,77–79 Genera commonly used in mycoremediation include Pleurotus, Phanerochaete, and Trametes.71,80,81 The fungal growth and reduction in contaminants at the site are monitored, with the attainment of acceptable contaminant levels signalling the end of the process. Further restoration, include planting vegetation, soil amendments and reintroduction of native species may also be necessary at this stage. RSC Sustainability Mycoremediation provides insight into processes that can be used to achieve open environment inoculation of large natural areas but has little emphasis on the recovery of the accumulated material as it focusses on the rehabilitation of a polluted envi- ronment and relevant contaminant degradation or accumula- tion mechanisms. Phytomining processes utilise diﬀerent bioaccumulation mechanisms but provide insight into recov- ering valuable resources from biomass. environments, fungi have a smaller physical footprint than plants.68 Some fungi also have a high tolerance for heavy metals and can accumulate metals at concentrations much higher than many plants, and organic acids produced by some fungi can mobilise metals from the environment making them more available for uptake.84,91 It is also worth noting that endophytic fungi live inside plants, and like endophytic bacteria could help them to accumulate metals, and subsequently be implemented into phytomining to enhance yields.92,93 Open Access Article. Published on 15 March 2024. Downloaded on 10/24/2024 5:03:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The use of fungal mycelium to scavenge valuable metals, minerals and REEs holds some key advantages over the use of hyperaccumulating plants (Fig. 3). Some fungal species grow very quickly, producing a larger amount of biomass in a shorter time than plants (although this is, of course, species and growth environment dependent with obvious exceptions e.g., bamboo is a plant with a very fast growth rate) and, hence accumulating more metals, minerals or REEs.77,83,84 Fungi can also grow in a wider range of environmental conditions than most plants, including low-light, varied pH levels85 and on a range of substrates from soil, wood, water,86 and rock to living organ- isms87 and extreme e.g., high salt, acidic,88 radioactive envi- ronments89 (although it should be noted that species that exhibit growth versatility in such environments may not necessarily accumulate the desired, or any metal, mineral or REE, in suﬃcient quantities to be suitable for mycomining). 4. Mycomining The success of phytomining and mycoremediation hint at the potential of so-called “mycomining” i.e., the intentional scav- enging of dispersed valuable resources (e.g., metals, minerals, and REEs) from the environment in a fashion impossible using conventional mining.82 Mycomining is essentially a combina- tion of mycoremediation and phytomining principles. This ability of fungi to not only accumulate scattered heavy metal contaminants from soils but also to degrade hydrocarbon pollutants inspired the concept of ‘mycoremediation’ as early as 1997, where oyster mushrooms were used to break down hydrocarbons from petroleum products at “The Bioneers” conference.73 Since then, fungi have been employed to break Fig. 3 Key advantages, disadvantages, and limitations of traditional (terrestrial) mining, phytomining and “mycomining”. Fig. 3 Key advantages, disadvantages, and limitations of traditional (terrestrial) mining, phytomining and “mycomining”. antages, disadvantages, and limitations of traditional (terrestrial) mining, phytomining and “mycomining”. © 2024 The Author(s). Published by the Royal Society of Chemistry © 2024 The Author(s). Published by the Royal Society of Chemistry RSC Sustainability, 2024, 2, 1350–1357 \| 1353 Perspective View Article Online Perspective View Article Online RSC Sustainability Since fungi do not need light to grow, they may be less aﬀected by pests and diseases and their lower nutrient requirements may make them lower maintenance than plants.90 In space- limited environments, such as underground mines or urban The environmental impact of mycomining compared to alternatives, such as terrestrial mining, urban mining and phytomining is challenging to accurately predict, however, Folchi matrices (a common environmental impact assessment in mining operations) can provide broad insight into the rela- tive environmental impact of the diﬀerent mining approaches (Table 1).94,95 Mycomining would likely improve soil quality and positively impact local ora and fauna through the bio- accumulation process. This is opposed to terrestrial mining, which, as discussed, can severely degrade soil quality, lead to considerable water, and air pollution, and disrupt local ora and fauna. Mycomining likely would not require use of fertil- isers or pesticides that can lead to water pollution and soil degradation in phytomining, and is not associated with the manual disassembly or disposal of non-recoverable e-waste residues of urban mining, or the excavation and ore process- ing stages of terrestrial mining, both of which can release toxic materials into the environment and expose workers to health hazards. Like other mining methods, mycomining also oﬀers Table 1 Folchi matrices for terrestrial, urban, phyto- and mycomining Mining activities Soil quality Water quality Air quality Flora and fauna Human health Landscape Socio-economic Terrestrial mining Excavation − − − − − − − Ore processing − − − − − − + Waste disposal − − − − − − − Tailings management − − − − − − − Transportation − − − + Urban mining E-waste collection − + Disassembly − − − − − + Processing − − − − − + Material recovery + + + + + + Residue disposal − − − − − − − Phytomining Planting + − − − + + Maintenance + − − − + + Harvesting + − − − + + Processing − − − − + Waste disposal − − − − − − − Mycomining Inoculation + − + − + + Maintenance + − + − + + Harvesting + + − + + Processing − − − − − − + Waste disposal − − − − − − − Table 1 Folchi matrices for terrestrial, urban, phyto- and mycomining © 2024 The Author(s). © 2024 The Author(s). Published by the Royal Society of Chemistry 5. Conclusion socio-economic benets, including job opportunities and the promotion of green technology. However, it is worth noting that the extraction process associated with mycomining might release spores or dust, aﬀecting air quality and posing health risks. Bioprocesses, including bioreduction, bioleaching, bio- precipitation and similar, based on plants and fungi are well established and documented and have wide industry utiliza- tion. On-location, open environment bioprocesses, such as the use of plants to scavenge dispersed valuable resources e.g., metals, minerals, and rare earth elements, which are then recovered from the biomass (phytomining), and fungi to degrade or accumulate environmental contaminants e.g., oil, plastics, heavy metals to then be disposed of with the biomass (mycoremediation), have also received considerable interest. The success of phytomining and mycoremediation hint at the potential of so-called “mycomining”, which could oﬀer key benets, such as faster and more versatile growth conditions, including in substrates ranging from soil, wood, water, and rock to living organisms and dark, space-restricted, or extreme i.e., pH levels, high salt, acidic, radioactive environments (although not all species that exhibit growth versatility in such environ- ments may necessarily be suitable for mycomining). The exist- ing eld of mycoremediation can provide insight into biological processes that can be used to achieve open environment inoc- ulation of large natural areas, such as the tilling of fully colonised grain, straw or wood chip spawn into soil, application as a surface mat, pellets, or spraying of uneven terrains using liquid spore medium. Mycoremediation also provides insight into bioaccumulation mechanisms and achieved concentra- tions, albeit for environmental contaminants rather than valu- able resources, such as metals, minerals, and REEs, and with limited emphasis on resource recovery. Conversely, established phytomining processes utilise diﬀerent bioaccumulation mechanisms but provide insight into recovering valuable resources from biomass through ashing followed by leaching, electrolysis, or precipitation and the relative environmental impact of these processes. Contrasting and extrapolating on phytomining and mycoremediation bioaccumulation processes, “mycomining” would seem conceptually viable, with broad estimates suggesting comparable recovery potential and environmental impact to phytomining. Although not directly comparable with traditional mining processes, mycomining could be a useful supplement to urban mining to oﬀset demand for mining or used in situations where conventional mining may be ineﬃcient or uneconomical. Access Article. Published on 15 March 2024. Downloaded on 10/24/2024 5:03:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 15 March 2024. Downloaded on 10/24/2024 5:03:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. © 2024 The Author(s). Published by the Royal Society of Chemistry RSC Sustainability Published by the Royal Society of Chemistry 1354 \| RSC Sustainability, 2024, 2, 1350–1357 Perspective Table 2 Concentration of rare earth elements obtained from saprobic macro fungi,102 Phytolacca americana plant biomass (control sample and assuming REE distribution is consistent across samples),103 and size-reduced e-waste dissolved using nitric acid104 Table 2 Concentration of rare earth elements obtained from saprobic macro fungi,102 Phytolacca americana plant biomass (control sample and assuming REE distribution is consistent across samples),103 and size-reduced e-waste dissolved using nitric acid104 Source Rare earth element (mg kg−1) La Ce Nd Pr Sm Eu Gd Tb Dy Saprobic macrofungi 207 357 139 40 37 13 30 9 58 P. americana plant biomass 522 224 322 — — — — — — Ground sieved undersize e-waste 5144 7851 91 235 9475 10 558 4061 4061 4602 25 719 Ground un-sieved e-waste 4286 9286 60 714 5000 7857 3571 3571 3571 19 286 Ground sieved oversize e-waste 3134 5445 23 643 2942 3026 1653 2830 1462 4899 Open Access Article. Published on 15 March 2024. Downloaded on 10/24/2024 5:03 This article is licensed under a Creative Commons Attribution 3.0 Unp 33 R. R. Brooks, M. F. Chambers, L. J. Nicks and B. H. Robinson, Trends Plant Sci., 1998, 3, 359–362. 34 T. Dinh, Z. Dobo and H. Kovacs, Chemosphere, 2022, 297, 134259. 5 S. H. Ali, D. Giurco, N. Arndt, E. Nickless, G. Brown, A. Demetriades, R. Durrheim, M. A. Enriquez, J. Kinnaird and A. Littleboy, Nature, 2017, 543, 367–372. 35 N. Rascio and F. Navari-Izzo, Plant Sci., 2011, 180, 169–181. 6 B. Lottermoser and B. G. Lottermoser, Mine Wastes: Characterization, Treatment and Environmental Impacts, 2010, 205–241. 36 A. J. Pollard, R. D. Reeves and A. J. Baker, Plant Sci., 2014, 217, 8–17. 37 G. Potters, T. P. Pasternak, Y. Guisez, K. J. Palme and M. A. Jansen, Trends Plant Sci., 2007, 12, 98–105. 7 P. L. Younger, S. A. Banwart, R. S. Hedin, P. L. Younger, S. A. Banwart and R. S. Hedin, Mine Water Hydrology, Springer, 2002. 38 J. Mesjasz-Przybyłowicz, M. Nakonieczny, P. Migula, M. Augustyniak, M. Tarnawska, W. Reimold, C. Koeberl, W. Przybyłowicz and E. Głowacka, Acta Biol. Cracov., Ser. Bot., 2004, 46, 75–85. 8 A. G. Smith and T. Mudder, The Chemistry and Treatment of Cyanidation Wastes, Mining journal books limited, 1991. 9 G. Kodilinye, Commonwealth Caribbean Tort Law, Routledge-Cavendish, 2009. 39 A. van Der Ent, A. J. Baker, R. D. Reeves, R. L. Chaney, C. W. Anderson, J. A. Meech, P. D. Erskine, M.-O. Simonnot, J. Vaughan and J. L. Morel, Environ. Sci. Technol., 2015, 49(8), 4773–4780. 10 A. Akcil and S. Koldas, J. Cleaner Prod., 2006, 14, 1139–1145. 11 T. Norgate and N. Haque, J. Cleaner Prod., 2010, 18, 266–274. 40 T. Kobayashi and N. K. Nishizawa, Annu. Rev. Plant Biol., 2012, 63, 131–152. 12 V. Balaram, Geosci. Front., 2019, 10, 1285–1303. 13 C. Hagelüken and C. W. Corti, Gold Bull., 2010, 43, 209–220. 41 J. ´a. Hall, J. Exp. Bot., 2002, 53, 1–11. 14 A. Mecucci and K. Scott, J. Chem. Technol. Biotechnol., 2002, 77, 449–457. 42 S. Clemens, M. G. Palmgren and U. Kr¨amer, Trends Plant Sci., 2002, 7, 309–315. 15 J. Cui and L. Zhang, J. Hazard. Mater., 2008, 158, 228–256. 43 A. Manara, in Plants and Heavy Metals, ed. A. Furini, Springer Netherlands, Dordrecht, 2012, pp. 27–53, DOI: 10.1007/978-94-007-4441-7_2. 16 A. Fornalczyk and M. Saternus, Metalurgija, 2009, 48, 133. 17 X. Zeng, J. A. Mathews and J. Li, Environ. Sci. Technol., 2018, 52, 4835–4841. 44 N. Verbruggen, C. 5. Conclusion Published by the Royal Society of Chemistry Perspective View Article Online View Article Online View Article Online RSC Sustainability Perspective The authors have no conicts of interest to declare. 28 D. O. Jones, S. Kaiser, A. K. Sweetman, C. R. Smith, L. Menot, A. Vink, D. Trueblood, J. Greinert, D. S. Billett and P. M. Arbizu, PLoS One, 2017, 12, e0171750. Conﬂicts of interest 27 L. A. Levin, K. Mengerink, K. M. Gjerde, A. A. Rowden, C. L. Van Dover, M. R. Clark, E. Ramirez-Llodra, B. Currie, C. R. Smith and K. N. Sato, Mar. Pol., 2016, 74, 245–259. References 29 D. J. Amon, J. Gobin, C. L. Van Dover, L. A. Levin, L. Marsh and N. A. Raineault, Front. mar. sci., 2017, 4, 342. © 2024 The Author(s). Published by the Royal Society of Chemistry 5. Conclusion It is challenging to accurately predict how viable mycomin- ing would be commercially, however, it could be eﬀective in bioaccumulation of metals including Au,96,97 Cu,98,99 Ni,97 Pt97 and Zn.100 Metal recovery potential of fungi cultivated on soil or rock substrates from former mine sites is challenging to esti- mate as a dearth in the literature exists for fungi-based metal bioaccumulation on such substrates. However, phytomining is known to accumulate up to 304 mg kg−1 Au from gold-enriched silica sand using Brassica campestris and 22 000 mg kg−1 Ni from Ni-contaminated soil using Alyssum spp.101 Mycomining is also likely suitable for use with REEs: ectomycorrhizal and saprobic macrofungi grown in “unpolluted sites with diﬀering bedrock chemistry” i.e., normal sites not especially conducive to mycomining, can exhibit concentrations as high as 276 and 357 mg kg−1, respectively, of 14 REEs (i.e., La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu).102 These values are competitive with available data on phytoextraction of La, Ce and Nd using Phytolacca americana plant biomass grown on mine tailings from a former mining site i.e., a much more favourable substrate for bioaccumulation, suggesting that mycomining at a similar site could potentially recover similar or even greater concentrations of REEs.103 The described phytomining example recovers up to 16.6% of the REE concentration associated with e-waste (urban mining), while the described “mycomining” example (on poorly suited substrate) recovers up to 6.6% (Table 2).102–104 Selective accumulation of specic REEs is also likely possible with Mucor javanicus, accumulating approxi- mately three times more Sm than it does Y, La, Er and Lu.105 Oth t bl f t hi ti t th i bilit f i i Other notable factors hinting at the viability of mycomining include cost estimations competitive with biohydrometallurgy (i.e., 10–70 US $ per t) based on an example of Ni accumulation using Trichoderma harzianum with a soil penetration depth of 1 m and yield of 1.1% Ni per kg dry weight (11 kg Ni per t biomass)86 and the fact that the accumulated resources e.g., metals, tend to be concentrated in the fruiting body of the fungus rather than the young or old mycelium;106,107 harvesting of protruding fruiting bodies rather than interconnected mycelial sheets may be more straightforward. RSC Sustainability, 2024, 2, 1350–1357 \| 1355 © 2024 The Author(s). icle. Published on 15 March 2024. Downloaded on 10/24/2024 5:03:26 AM. s article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 30 I. A. Crawford, Prog. Phys. Geogr., 2015, 39, 137–167. 2 P. Darling, SME Mining Engineering Handbook, SME, 2011. 31 V. Sheoran, A. S. Sheoran and P. Poonia, J. Geochem. Explor., 2013, 128, 42–50. 3 V. Smil, Energy at the Crossroads: Global Perspectives and Uncertainties, Cambridge, Mass, MIT Press, 2003. 32 A. Van der Ent, A. J. Baker, R. D. Reeves, A. J. Pollard and H. Schat, Plant Soil, 2013, 362, 319–334. 4 J. Havlin, S. L. Tisdale, W. L. Nelson and J. D. Beaton, Soil Fertility and Fertilizers: An Introduction to Nutrient Management, Pearson, 2014. Open Access Article. Published on 15 March 2024. Downloaded on 10/24/2024 5:03:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 1 H. L. Hartman and J. M. Mutmansky, Introductory Mining Engineering, John Wiley & Sons, 2002. Open Access Article. Published on 15 March 2024. Downloaded on 10/24/2024 5:03:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 86 G. Cecchi, E. Roccotiello, S. Di Piazza, A. Riggi, M. G. Mariotti and M. Zotti, J. Environ. Sci. Health, Part B, 2017, 52, 166–170. 58 M. L. Free, Hydrometallurgy: Fundamentals and applications, John Wiley and Sons, Hoboken, New Jersey, 2013, vol. 199, p. 222. 87 G. Strobel, B. Daisy, U. Castillo and J. Harper, J. Nat. Prod., 2004, 67, 257–268. 59 F. Habashi, Principles of Extractive Metallurgy, Routledge, 2017. 88 G. Cecchi, A. Ceci, P. Marescotti, A. M. Persiani, S. Di Piazza and M. Zotti, Mycol. Prog., 2019, 18, 415–423. 60 G. Chen, Sep. Purif. Technol., 2004, 38, 11–41. 61 A. P. Abbott, G. Frisch, J. Hartley and K. S. Ryder, Green Chem., 2011, 13, 471–481. 89 S. Onofri, L. Selbmann, G. S. de Hoog, M. Grube, D. Barreca, S. Ruisi and L. Zucconi, Adv. Space Res., 2007, 40, 1657– 1664. 62 F. Fu and Q. Wang, J. Environ. Manage., 2011, 92, 407–418. 90 G. N. Agrios, Plant Pathology, Elsevier, 2005. 63 S. N. Naik, V. V. Goud, P. K. Rout and A. K. Dalai, Renewable Sustainable Energy Rev., 2010, 14, 578–597. 91 P. Baldrian, Enzyme Microb. Technol., 2003, 32, 78–91. 64 N. Zaﬀar, E. Ferchau, U. Feuerstein, H. Heilmeier, C. Moschner and O. Wiche, Carpathian J. Earth Environ. Sci., 2020, 15, 43–48. 92 M. Rajkumar, N. Ae and H. Freitas, Chemosphere, 2009, 77, 153–160. 93 Y. Ma, M. Rajkumar, Y. Luo and H. Freitas, J. Hazard. Mater., 2011, 195, 230–237. 65 Y. Tian and H. Zhang, Int. J. Green Energy, 2016, 13, 1556– 1563. 94 R. Folchi, Environmental Impact Statement for Mining with Explosives: A Quantitative Method, I.S.E.E. 29th Annual Conference on Explosives and Blasting Technique, Nashville, Tennessee, U.S.A., 2003. 66 R. L. Chaney, J. S. Angle, C. L. Broadhurst, C. A. Peters, R. V. Tappero and D. L. Sparks, J. Environ. Qual., 2007, 36, 1429–1443. 67 C. W. Anderson, R. R. Brooks, R. B. Stewart and R. Simcock, Nature, 1998, 395, 553–554. 95 M. Monjezi, K. Shahriar, H. Dehghani and F. Samimi Namin, Environ. Geol., 2009, 58, 205–216. 68 G. M. Gadd, Mycol. Res., 2007, 111, 3–49. 96 F. Reith, M. F. Lengke, D. Falconer, D. Craw and G. Southam, ISME J., 2007, 1, 567–584. 69 A. Haritash and C. Kaushik, J. Hazard. Mater., 2009, 169, 1– 15. 97 B. A. Moore, J. R. Duncan and J. E. Burgess, Miner. Eng., 2008, 21, 55–60. 70 H. Open Access Article. Published on 15 March 2024. Downloaded on 10/24/2024 5:03 This article is licensed under a Creative Commons Attribution 3.0 Unp Hermans and H. Schat, New Phytol., 2009, 181, 759–776. 18 S. Gupta, G. Modi, R. Saini and V. Agarwala, Int. Refereed J. Eng. Nat. Appl. Sci., 2014, 3, 05–17. 45 U. Kr¨amer, Annu. Rev. Plant Biol., 2010, 61, 517–534. 19 K. Lundgren, The Global Impact of E-Waste: Addressing the Challenge, International Labour Organization, 2012. 46 D. E. Salt, I. Baxter and B. Lahner, Annu. Rev. Plant Biol., 2008, 59, 709–733. 20 J. R. Hein, K. Mizell, A. Koschinsky and T. A. Conrad, Ore Geol. Rev., 2013, 51, 1–14. 47 G. DalCorso, S. Farinati and A. Furini, Plant Signaling Behav., 2010, 5, 663–667. 21 J. R. Hein, T. A. Conrad and R. E. Dunham, Mar. Georesour. Geotechnol., 2009, 27, 160–176. 48 P. Sharma, A. B. Jha, R. S. Dubey and M. Pessarakli, J. Bot., 2012, 2012, 217037. 22 R. Sharma, Deep-sea Mining: Resource Potential, Technical and Environmental Considerations, Springer, 2017. 49 P. N. Kumar, V. Dushenkov, H. Motto and I. Raskin, Environ. Sci. Technol., 1995, 29, 1232–1238. 23 J. Halfar and R. M. Fujita, Science, 2007, 316, 987. 50 P. U. Okoroafor, C. O. Ogunkunle, H. Heilmeier and O. Wiche, Int. J. Phytorem., 2022, 24, 1310–1320. 24 P. M. Herzig and M. D. Hannington, Ore Geol. Rev., 1995, 10, 95–115. 51 B. Robinson, A. Chiarucci, R. Brooks, D. Petit, J. Kirkman, P. Gregg and V. De Dominicis, J. Geochem. Explor., 1997, 59, 75–86. 25 S. Petersen, A. Kr¨atschell, N. Augustin, J. Jamieson, J. R. Hein and M. D. Hannington, Mar. Pol., 2016, 70, 175–187. 26 P. Hoagland, S. Beaulieu, M. A. Tivey, R. G. Eggert, C. German, L. Glowka and J. Lin, Mar. Pol., 2010, 34, 728– 732. 52 I. Raskin and B. D. Ensley, Phytoremediation of Toxic Metals, John Wiley and Sons, 2000. 53 R. Lal, Science, 2004, 304, 1623–1627. 1356 \| RSC Sustainability, 2024, 2, 1350–1357 © 2024 The Author(s). Published by the Royal Society of Chemistry View Article Online Perspective RSC Sustainability 54 V. Sheoran, A. Sheoran and P. Poonia, Miner. Eng., 2009, 22, 1007–1019. 82 X. Liang and G. M. Gadd, Microb. Biotechnol., 2017, 10, 1199–1205. 55 A. J. Baker and R. Brooks, Biorecovery, 1989, 1, 81–126. 83 B. Volesky, Water Res., 2007, 41, 4017–4029. 56 W. J. Rankin, Minerals, Metals and Sustainability: Meeting Future Material Needs, CSIRO publishing, 2011. 84 M. Fomina and G. M. Gadd, Bioresour. Technol., 2014, 160, 3–14. 85 G. M. Gadd, Fungi in Biogeochemical Cycles, Cambridge University Press, 2006. 57 J. O. Marsden and C. I. House, Chemistry of Gold Extraction, Society for Mining, Metallurgy, and Exploration, Colorado, USA, 2006. Open Access Article. Published on 15 March 2024. Downloaded on 10/24/2024 5:03:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Singh, Mycoremediation: Fungal Bioremediation, John Wiley & Sons, 2006. 98 K. Bosecker, FEMS Microbiol. Rev., 1997, 20, 591–604. 71 D. Wesenberg, I. Kyriakides and S. N. Agathos, Biotechnol. Adv., 2003, 22, 161–187. 99 S. Zapotoczny, A. Jurkiewicz, G. Tylko, T. Anielska and K. Turnau, Microbiol. Res., 2007, 162, 219–228. 72 E. Dadachova and A. Casadevall, Curr. Opin. Microbiol., 2008, 11, 525–531. 100 E. Mohammadian, A. Babai Ahari, M. Arzanlou, S. Oustan and S. H. Khazaei, Chemosphere, 2017, 185, 290–296. 73 P. Stamets, Mycelium Running: How Mushrooms Can Help Save the World, Ten Speed Press, Berkeley, CA, 2005. 101 LAB Novo, P. M. L. Castro, P. Alvarenga and E. F. da Silva, in Phytoremediation: Management of Environmental Contaminants, ed. A. A. Ansari, S. S. Gill, R. Gill, G. R. Lanza and L. Newman, Springer International Publishing, Cham, 2017, vol. 5, pp. 469–486, DOI: 10.1007/978-3-319- 52381-1_18. 74 T. M. Palanivel, B. Pracejus and L. A. B. Novo, Chemosphere, 2023, 314, 137688. 75 G. Cecchi, G. Vagge, L. Cutroneo, G. Greco, S. Di Piazza, M. Faga, M. Zotti and M. Capello, Environ. Sci. Pollut. Res., 2019, 26, 35602–35609. 102 J. Boroviˇcka, J. Kubrov´a, J. Rohovec, Z. ˇRanda and C. E. Dunn, BioMetals, 2011, 24, 837–845. 76 V. ˇSaˇsek, T. Cajthaml and M. Bhatt, Water, Air, Soil Pollut.: Focus, 2003, 3, 5–14. 103 W.-S. Liu, Y.-Y. Chen, H. Huot, C. Liu, M.-N. Guo, R.-L. Qiu, J. L. Morel and Y.-T. Tang, J. Cleaner Prod., 2020, 275, 122959. 77 G. M. Gadd, Geoderma, 2004, 122, 109–119. 78 R. M. Atlas and J. Philp, Bioremediation. Applied Microbial Solutions for Real-World Environmental Cleanup, ASM press, 2005. 104 A. Yuksekdag, B. Kose-Mutlu, B. Zeytuncu-Gokoglu, M. Kumral, M. R. Wiesner and I. Koyuncu, Environ. Sci. Pollut. Res., 2022, 1–10. 79 S. Kuppusamy, P. Thavamani, K. Venkateswarlu, Y. B. Lee, R. Naidu and M. Megharaj, Chemosphere, 2017, 168, 944– 968. 05 T. Tsuruta, J. Rare Earths, 2007, 25, 526–532. 106 H. Ghahremani-Majd and F. Dashti, Hortic., Environ. Biotechnol., 2015, 56, 376–382. 80 S. Pointing, Appl. Microbiol. Biotechnol., 2001, 57, 20–33. 107 J. Falandysz, A. R. Fernandes and D. Meloni, Trends Food Sci. Technol., 2022, 119, 338–347. 81 P. Baldrian, FEMS Microbiol. Ecol., 2004, 50, 245–253. © 2024 The Author(s). Published by the Royal Society of Chemistry RSC Sustainability, 2024, 2, 1350–1357 \| 1357
https://openalex.org/W4390621817	https://jurnal.untirta.ac.id/index.php/JoCSE/article/download/21410/pdf_24	English	null	Implementation of hygiene and sanitation in processing food products for catering businesses in Ciora Village, Cilegon	Journal of Community Service in Science and Engineering	2,023	cc-by-sa	3,840	JOURNAL OF COMMUNITY SERVICE IN SCIENCE AND ENGINEERING Homepage jurnal: http://jurnal.untirta.ac.id/index.php/JoCSE/ A B S T R A C T A R T I C L E I N F O Article history: Submitted 8 July 2023 Reviewed 20 July 2023 Received 20 August 2023 Accepted 25 August 2023 Available online on 1 October 2023 Keywords: Clean, hygiene, catering, food, sanitation. Kata kunci: Bersih, higienis, jasaboga, makanan, sanitasi. Catering services are business activities that order food for certain banquet events. More and more people are interested in this business, one of which is the food service business in Ciora Village, Grogol District, Cilegon City, which is multiplying. Hygiene and sanitation elements are essential but have not been given special attention to food service industry owners, "Dapoer Umi Ewi" has several problems that need to be considered, including the layout of the production house and placement of equipment that has not paid attention to the elements of hygiene and sanitation. Hence, it is still possible for this to occur. Contamination of food products can harm consumers. Modifications made to the "Clean Production House Model" in Ciora village include a "Processing Room," which helps carry out the production process, and an "Equipment and Raw Material Washing Area" for cleaning cooking utensils and raw materials which require clean water and a suitable place. Away from the bathroom and keep it clean. In this case, the human factor or the element of food handlers is also crucial because humans are the actors in the production house process. The human factor as a workforce must be clean. Changes to the space layout, where a raw material storage area was added next to the dishwasher. This area has facilities such as storage shelves with a minimum height of 15 cm from the floor and 60 cm from the ceiling. Akbar Gunawan a, 1, Dhena Ria Barleany b, c, Erlin Filiandini b, Shera Dwi Andini b, Rifan Maulana a, Retno Sulistyo Dhamar Lestari b, Denni Kartika Sari b strial Engineering, Faculty of Engineering, Universitas Sultan Ageng Tirtayasa, Jln. Jendral Sudirman Km. 03, Cilegon, Bante b Department of Chemical Engineering, Faculty of Engineering, Universitas Sultan Ageng Tirtayasa, Jln. Jendral. Sudirman Km. 03, Cilegon, Banten 42435, Indonesia. , c Centre of Excellence for Local Food Innovation, Universitas Sultan Ageng Tirtayasa, Jln. Raya Jakarta Km. 04, Serang 42124, Indonesia. c Centre of Excellence for Local Food Innovation, Universitas Sultan Ageng Tirtayasa, Jln. Raya Jakarta Km. 04, Serang 42124, Indonesia. 1E-mail: a68ar@untirta.ac.id Implementation of hygiene and sanitation in processing food products for catering businesses in Ciora Village, Cilegon Akbar Gunawan a, 1, Dhena Ria Barleany b, c, Erlin Filiandini b, Shera Dwi Andini b, Rifan Maulana a, Retno Sulistyo Dhamar Lestari b, Denni Kartika Sari b JOURNAL OF COMMUNITY SERVICE IN SCIENCE AND ENGINEERING VOL 02 NO 02 (2023): 52–55 JOURNAL OF COMMUNITY SERVICE IN SCIENCE AND ENGINEERING VOL 02 NO 02 (2023): 52–55 Available online at http://dx.doi.org/10.36055/jocse.v2i2.21410. 1. Introduction Cilegon City is famous as a steel city because it contains the largest steel factory in Southeast Asia, PT Krakatau Steel. The factory had a significant influence on economic development in Cilegon City. Cilegon City has eight subdistricts, one of which is the Grogol Subdistrict. Grogol Subdistrict has several villages, including Ciora, Cipinang, Kubangkoru, and Cikebel. Ciora Village has many branch names: Ciora Kwista, Ciora Tengah, Ciora Jaya, Ciora Wetan, and Ciora Gede. This village is about 5.7 km from the center of Cilegon city. Ciora Village has an area of 1,734.28 km² [1] and a population of 1,435,003 people. Most Ciora Village residents are household assistants, farm laborers, and factory workers. Geographically, Ciora Village has fertile land and a large plantation area. The location supports Ciora Village in increasing agricultural and plantation production. However, production results can be of low quality due to the influence of the economic crisis experienced by Indonesia. Most residents in Ciora Village are farmers, and some use it to open small businesses. Some people in Ciora Village open food business opportunities, such as opening food stalls and opening stalls at home. Ciora village has a well-known catering business among the people around Cilegon called "Dapoer Umi Ewi," located in central Ciora. "Dapoer Umi Ewi" is a business in the food sector that has been established for many years. "Dapoer Umi Ewi" produces and markets traditional food products. Most of the "Dapoer Umi Ewi" consumers come from the Cilegon area because the sales system is done at home to produce food and utilize social media to sell. "Dapoer Umi Ewi" has a lot of food menus and follows the development of the times where the food is made very creatively and full of inspiration and follows the story of modern times, various kinds of food menus including rice bowls, shredded chicken, fried eggs, sambal matah, baso aci, melted brownies, green banana ice, and many other menus. "Dapoer Umi Ewi" also provides food in the form of tumpeng rice for Aqiqah events, thanksgiving events, and birthday events. Figure 1 below shows some food products at "Dapoer Umi Ewi." Figure 2. Food packaging at "Dapoer Umi Ewi". Figure 1.Several products from "Dapoer Umi Ewi". Figure 1.Several products from "Dapoer Umi Ewi". Figure 2. Food packaging at "Dapoer Umi Ewi". 1. Introduction "Dapoer Umi Ewi" and several other catering providers in Ciora Village have a food production site at the owner's residence. Therefore, this production site has a dual function. Like a residence, the parts of the house have a bathroom, kitchen, sink, and adequate drainage facilities. Analysis occurs at every process stage, from production to packaging and marketing. The catering industry in Ciora Village is relatively clean but needs to meet hygiene and sanitation standards according to government regulations. Figure 1 shows a small part of the documentation for presenting processed food products in the "Dapoer Umi Ewi" catering industry. Figure 2 shows that the packaging process is carried out on the floor, and the workers prepare the product presentation in a sitting posture, which could be more ergonomic. Placing it on the floor allows physical contaminants such as fine dust and hair to enter. The floor used visually looks clean, but fine dirt can be blown by the wind and get into food packaging. The condition could be more desirable for consumers who order. Hygiene and sanitation elements are important but have yet to be a particular concern for food service industry owners [2]. Therefore, more comprehensive knowledge is needed among the public in general and catering business actors regarding the impacts that arise if these standards are met. Food products, a primary human need, should be processed carefully and free from all physical, chemical, and biological contaminants [3]. Contamination of food products at the production, storage, and distribution stages will have fatal consequences, including nausea, vomiting, and diarrhea. In the long term, it can even cause damage to vital organs in the human body [4]. The negative impact can be felt by consumers and producers, who will also experience losses due to the potential for reduced buyer interest and decreased turnover on future occasions. The industry could even go out of business. Seeing the increasing increase in the catering industry, especially in Ciora Village, it is necessary to carry out community service activities focusing on food processing hygiene and sanitation issues. Increasing the knowledge of the village community is hoped to provide benefits in developing food service businesses while maintaining food safety and public health. A B S T R A K Jasaboga merupakan kegiatan usaha yang melayani pemesanan berbagai macam makanan untuk keperluan jamuan acara tertentu. Usaha ini semakin banyak peminatnya, salah satunya adalah usaha jasaboga di Desa Ciora Kecamatan Grogol Kota Cilegon yang berkembang sangat pesat. Unsur higiene dan sanitasi merupakan hal yang sangat penting tetapi belum menjadi perhatian khusus bagi pemilik industri jasaboga, “Dapoer Umi Ewi” memiliki beberapa permasalahan yang perlu diperhatikan diantaranya adalah tata letak rumah produksi dan penempatan peralatan belum memperhatikan unsur higiene dan sanitasi sehingga masih dimungkinkan terjadinya kontaminasi produk makanan dan dapat merugikan konsumen. Modifikasi yang dilakukan pada “Model Rumah Produksi Bersih” di desa Ciora diantaranya terdiri dari “Ruang Pengolahan” yang berguna untuk melakukan proses produksi, dan “Area Pencucian Peralatan dan Bahan Baku” untuk membersihkan alat masak dan bahan baku yang memerlukan air bersih dan tempat yang jauh dari kamar mandi serta tetap terjaga kebersihannya. Dalam hal ini faktor manusia atau unsur penjamah makanan juga sangat krusial karena manusia sebagai pelaku dalam proses rumah produksi tersebut. Faktor manusia sebagai tenaga kerja wajib dalam keadaan bersih. Perubahan tata letak ruang, dimana ditambahkan area penyimpanan bahan mentah yang berada di sebelah tempat cuci piring. Area ini dilengkapi dengan fasilitas seperti rak penyimpanan dengan ketinggian minimal 15 cm dari lantai dan 60 cm dari langit-langit ruangan. Available online at http://dx.doi.org/10.36055/jocse.v2i2.21410. Journal of Community Service in Science and Engineering (JoCSE) is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License. JOURNAL OF COMMUNITY SERVICE IN SCIENCE AND ENGINEERING VOL 02 NO 02 (2023): 52-55 53 1. Introduction Based on the situation analysis that has been carried out, it can be concluded that the people of Ciora Village still have various problems that need to be resolved in the fields of education, social culture, economics, and entrepreneurship. The following is a description of the issues experienced by Ciora Village: 1. Education Sector. Some of the people in Ciora Village have a low level of education. This low level of education results in people's mindset being a little behind even though the location is close to the city. 1. Education Sector. Some of the people in Ciora Village have a low level of education. This low level of education results little behind even though the location is close to the city. 2. Economic Sector. Most heads of families in Ciora Village work as farm laborers and factory workers. At the same time, mothers seek additional income as household assistants to meet their daily needs, and some do small business. 2. Economic Sector. Most heads of families in Ciora Village work as farm laborers and factory workers. At the same time, mothers seek additional income as household assistants to meet their daily needs, and some do small business. 3. Entrepreneurship. Some residents of Ciora Village still do not have high motivation in terms of entrepreneurship, although there are a few who have started small businesses with limited capital. The people in this village still need to be given sufficient motivation and knowledge in entrepreneurship to raise their standard of living. The business sector that the people of Ciora Village are starting to start is culinary. 3. Entrepreneurship. Some residents of Ciora Village still do not have high motivation in terms of entrepreneurship, although there are a few who have started small businesses with limited capital. The people in this village still need to be given sufficient motivation and knowledge in entrepreneurship to raise their standard of living. The business sector that the people of Ciora Village are starting to start is culinary. 4. Socio-Cultural Sector. Low levels of education also have an impact on the social and cultural life of society. Few people still hold old thoughts that are difficult to eradicate, making it challenging to provide innovation or change. 2.3. Technology applied In this case, development technology focuses on the production system and layout in the production house. In production systems, it is recommended to use a production layout to minimize disease so that food hygiene is better maintained. The public is also introduced to the sterilization system for equipment and maintaining the cleanliness of human workers to ensure the safety of the food products produced. 3.1. Production house model Clean production aims to increase productivity by increasing the efficiency of using raw materials and energy, as well as reducing sources of pollution, according to [6-10]. In industrial processes, clean production means increasing the efficiency of using raw materials energy, preventing or replacing the use of hazardous materials and toxic, and reducing the amount and poisonous level of all emissions and waste before leaving the process. In the production process at DS Ciora, a model was implemented to achieve a clean, hygienic production house. This model was formed after conducting interviews and observations within one month to get maximum results. The model depicted above explains that the production house model at DS Ciora includes a processing Room, which helps carry out the production process and requires clean cooking utensils and raw materials. Tool and raw material washing facilities allow clean cooking utensils and raw materials that are hygienic and free from bacteria requiring clean water and a place far from the bathroom to remain clean. The human factor is needed because humans are the actors in the production house's running process. The human factor as a workforce must be clean. In human labor, clean clothing is required, washing hands every time you carry out activities, work safety is needed in the production process, and a clean culture is necessary to implement the production process. he production process. Figure 3. Clean production house model. Figure 4. The old layout of the "Dapoer Umi Ewi" production house. Figure 4. The old layout of the "Dapoer Umi Ewi" production house. Figure 4. The old layout of the "Dapoer Umi Ewi" production house. p p Figure 3. Clean production house model. Figure 4. The old layout of the "Dapoer Umi Ewi" production house. Figure 3. Clean production house model. 1. Introduction The people of Ciora Village are still reluctant to participate in community activities such as youth and young women's associations, PKK women, or other meetings, and tend to live their daily lives in a matter- of-fact and very static way. 4. Socio-Cultural Sector. Low levels of education also have an impact on the social and cultural life of society. Few people still hold old thoughts that are difficult to eradicate, making it challenging to provide innovation or change. The people of Ciora Village are still reluctant to participate in community activities such as youth and young women's associations, PKK women, or other meetings, and tend to live their daily lives in a matter- of-fact and very static way. Especially for "Dapoer Umi Ewi," there are several problems that need to be considered, including: The layout of the production house and the placement of equipment do not pay attention to hygiene and sanitation elements, so it is still possible for food product contamination to occur, which can be detrimental to consumers. Not knowing the standard standards of cleanliness in the production house can confuse workers and disrupt the production JOURNAL OF COMMUNITY SERVICE IN SCIENCE AND ENGINEERING VOL 02 NO 02 (2023): 52-55 54 process. The aim is to provide an excellent understanding to food service business actors to pay more attention to health elements in producing food products. Implementing hygiene and sanitation standards by government regulations can significantly contribute to public health. It will further advance the food service business "Dapoer Umi Ewi" and other industries operating in the food sector. 2.2. Stages of activity This activity was carried out in several stages, namely starting with collecting information data using a direct survey of food service production houses in Ciora Village and interviews with business owners, followed by making a model of a hygienic food service production house and a production house model that is clean from contamination. The last is socialization and assistance in creating a healthy catering production house. 2. Method The application of hygiene and sanitation in the food processing industry in Ciora Cilegon Village is carried out by combining several appropriate methods. In this program, business actors are given a persuasive approach through socialization and counseling regarding hygienic food processing. After business actors understand the existing standards, assistance is provided to implement the applicable rules according to their respective places of business. Specifically for "Dapoer Umi Ewi" and the catering industry in Ciora Village, the spatial layout in the production house area was re-arranged, and a model of the production house was made using descriptive methods through sketches/models in the form of pictures so that they were easier to understand [5]. 2.1. Society target The target of this program is catering business actors or industries providing food or similar foodstuffs, especially those in Ciora Village, Grogol District, and Cilegon City. The target market for catering businesses is people in the Cilegon area and surrounding areas as consumers, especially households and offices that often hold meetings or banquets, as well as several industries that provide daily consumption for factory employees. 3.2. Spatial planning New layout of the "Dapoer Umi Ewi" production house. In Figure 5, the latest design has quite significant changes, including: Addition of trash bins and fire extinguisher, addition of hand washing facilities, a place for washing dishes and raw materials that are separated from the toilet/bathroom, a further distance for food processing is around 5 meters from the bathroom, separate storage area for raw materials and finished materials, add ventilation to circulate air in the room, attach health and safety information, and bright lighting and diffused lighting in all directions. 4. Conclusion The conclusion that can be drawn from this activity is that implementing a hygienic and sanitary production house in Ciora Village, Cilegon city, is implemented with a clean production house model, and the layout of the production house is according to government regulations. The results obtained in this activity can be used as evaluation material for food service entrepreneurs to understand more about cleanliness in production. 3.2. Spatial planning The principle of food and drink sanitation hygiene is the control of places/buildings, equipment, people, and food ingredients that can or may cause health problems or food poisoning [3]. Many efforts can be made to meet the Standard of Food Processing Sanitation Hygiene to avoid various sources of JOURNAL OF COMMUNITY SERVICE IN SCIENCE AND ENGINEERING VOL 02 NO 02 (2023): 52-55 55 contamination that can cause food damage, one of which is spatial planning. For example, the case of a catering business in Ciora Village is "Dapoer Umi Ewi," which already has building facilities or premises used for food processing. The layout of the rooms at "Dapoer Umi Ewi" is shown in Figure 4. The building facilities owned by the catering business "Dapoer Umi Ewi" have two floors. The 2nd floor is for living quarters, and the 1st floor is a catering production area. contamination that can cause food damage, one of which is spatial planning. For example, the case of a catering business in Ciora Village is "Dapoer Umi Ewi," which already has building facilities or premises used for food processing. The layout of the rooms at "Dapoer Umi Ewi" is shown in Figure 4. The building facilities owned by the catering business "Dapoer Umi Ewi" have two floors. The 2nd floor is for living quarters, and the 1st floor is a catering production area. Based on Figure 4, the production area (cooking food) is close to the equipment washing area and bathroom. It is necessary to pay attention to the placement of the toilet facing the food processing area to prevent contamination. At "Dapoer Umi Ewi," fresh raw materials and frozen products are stored in the refrigerator, and dry ingredients with a long shelf life are placed in cardboard boxes on the edge of the production area. Food materials should not be placed on the floor or against walls because this will increase humidity and make it easier for animals such as ants, mice, and cockroaches to enter. To improve food sanitation hygiene at "Dapoer Umi Ewi," changes were made to the layout of the space, where a raw material storage area was added next to the dishwasher. This area has facilities such as storage shelves with a minimum height of 15 cm from the floor and 60 cm from the ceiling. Figure 5. New layout of the "Dapoer Umi Ewi" production house. Figure 5. Acknowledgement Profound thanks are given to the Institute for Research and Community Service (LPPM) Sultan Ageng Tirtayasa University (UNTIRTA) for the funding support provided in the 2023 Research and Community Service (PPM) Grant scheme. [9] Masruroh, M., Fernanda, F., & Wibowo, T. (2014). Analisis efisiensi biaya terhadap keputusan penggunaan outsourcing bidang cleaning service dan catering pada PT Kuwera Jaya Jakarta. The Winners, vol. 15, no. 1, pp. 34-46. Widyastuti, N., & Almira, V. G. (2019). Higiene dan Sanitasi dalam Penyelenggaraan Makanan. Yogyakarta: K-Media. [5] Irawati, R. & Hardiastuti, E. B. W. (2016). Perancangan Standard Operating Procedure (SOP) proses pembelian bahan baku, proses produksi dan pengemasan pada industri jasa boga (Studi kasus pada PT. KSM Catering & Bakery Batam). Jurnal Akuntansi, Ekonomi dan Manajemen Bisnis, vol. 4, no. 2, pp. 186-193. [10] Karnita, I., & Yurianto, Y. Y. (2020). Hygiene pengelolaan makanan di CV Mitra Jaya Katering Bandung. Jurnal Pariwisata Vokasi, vol. 1, no. 1, pp. 1-10. g g gy [8] Sari, T. P. (2021). Pengaruh kualitas penyajian, harga dan kebersihan terhadap keputusan konsumen dalam membeli makanan pada CV. Epen Catering. Prosiding: Ekonomi dan Bisnis, vol. 1, no. 1, pp. 275-285. [6] Jalmi, S., Ahmad, S., Broerie, P., & Sjamsiwarni, R. S. (2020). Penerapan sistem produksi bersih di sentra IKM Kepala Terpadu Kota Bitung. Jurnal Penelitian Teknologi Industri, vol. 12, no. 1, pp. 41-51. [2] Purnawita, W., Rahayu, W. P., & Nurjanah, S. (2020). Praktik higiene sanitasi dalam pengelolaan pangan di sepuluh industri jasa boga di Kota Bogor. Jurnal Ilmu Pertanian Indonesia (JIPI), vol. 25, no. 3, pp. 426-433. Indraswati, D. (2016). Kontaminasi Makanan (Food Contamination). Ponorogo: Forum Ilmiah Kesehatan (Forikes). REFERENCE [1] Badan Pusat Statistik Kota Cilegon. (2020). Kecamatan Grogol dalam Angka 2020. Cilegon: Badan Pusat Statistik Kota Cilegon. [1] Badan Pusat Statistik Kota Cilegon. (2020). Kecamatan Grogol dalam Angka 2020. Cilegon: Badan Pusat Statistik Kota Cilegon. [2] Purnawita, W., Rahayu, W. P., & Nurjanah, S. (2020). Praktik higiene sanitasi dalam pengelolaan pangan di sepuluh industri jasa boga di Kota Bogor. Jurnal Ilmu Pertanian Indonesia (JIPI), vol. 25, no. 3, pp. 426-433. [4] Indraswati, D. (2016). Kontaminasi Makanan (Food Contamination). Ponorogo: Forum Ilmiah Kesehatan (Forikes). [5] Irawati, R. & Hardiastuti, E. B. W. (2016). Perancangan Standard Operating Procedure (SOP) proses pembelian bahan baku, proses produksi dan pengemasan pada industri jasa boga (Studi kasus pada PT. KSM Catering & Bakery Batam). Jurnal Akuntansi, Ekonomi dan Manajemen Bisnis, vol. 4, no. 2, pp. 186-193. [6] Jalmi, S., Ahmad, S., Broerie, P., & Sjamsiwarni, R. S. (2020). Penerapan sistem produksi bersih di sentra IKM Kepala Terpadu Kota Bitung. Jurnal Penelitian Teknologi Industri, vol. 12, no. 1, pp. 41-51. [7] Nugraheni, M. (2018). Kemasan Pangan. Yogyakarta: Plantaxia. [8] Sari, T. P. (2021). Pengaruh kualitas penyajian, harga dan kebersihan terhadap keputusan konsumen dalam membeli makanan pada CV. Epen Catering. Prosiding: Ekonomi dan Bisnis, vol. 1, no. 1, pp. 275-285. [9] Masruroh, M., Fernanda, F., & Wibowo, T. (2014). Analisis efisiensi biaya terhadap keputusan penggunaan outsourcing bidang cleaning service dan catering pada PT Kuwera Jaya Jakarta. The Winners, vol. 15, no. 1, pp. 34-46. [10] Karnita, I., & Yurianto, Y. Y. (2020). Hygiene pengelolaan makanan di CV Mitra Jaya Katering Bandung. Jurnal Pariwisata Vokasi, vol. 1, no. 1, pp. 1-10.
https://openalex.org/W4382397666	https://digitalsociology.guu.ru/jour/article/download/245/139	Russian	null	Agricultural workers’ labour satisfaction dependence in Russia with the use of the internet in professional activities	Cifrovaâ sociologiâ	2,023	cc-by	6,553	Ключевые слова Цифровизация, информационно-коммуникационные технологии, сельское хозяйство, работники, Интернет, удовлетво- ренность трудом, профессиональный рост, условия труда, оплата труда Былина Светлана Геннадиевна Канд. экон. наук, ст. науч. сотр. лаборатории социального развития агропромышленного комплекса и сельских территорий ORCID: 0000-0002-5179-7721 E-mail: svbylina@rambler.ru Институт аграрных проблем – обособленное структурное подразделение Федерального исследовательского центра «Саратовский научный центр Российской академии наук», г. Саратов, Россия © Былина С.Г., 2023. Статья доступна по лицензии Creative Commons «Attribution» («Атрибуция») 4.0. всемирная (http://creativecommons.org/licenses/by/4.0/). АННОТАЦИЯ уровня удовлетворенности трудом. На основе результатов корреляционного анализа определена степень влияния профессионального использования Интернета на уровень удовлетворенности трудом; осуществлено моделирование зависимости различных аспектов удовлетворенности трудом от интенсивности использования Интернета в производ- ственных целях. Установлено, что использование информа- ционно-коммуникационных технологий в работе повышает степень удовлетворенности различными аспектами трудовой деятельности, особое влияние оказывая на возможности про- фессионального роста. Однако рост цифровых компетенций приводит к увеличению уровня притязаний работника и, соответственно, к снижению степени его удовлетворенности трудом. Для закрепления высокопрофессиональных кадров в отрасли необходим многоаспектный подход, включающий всевозможные механизмы социальной и экономической поддержки, способствующие повышению уровня удовлет- воренности работника сельского хозяйства результатами своей трудовой деятельности. уровня удовлетворенности трудом. На основе результатов корреляционного анализа определена степень влияния профессионального использования Интернета на уровень удовлетворенности трудом; осуществлено моделирование зависимости различных аспектов удовлетворенности трудом от интенсивности использования Интернета в производ- ственных целях. Установлено, что использование информа- ционно-коммуникационных технологий в работе повышает степень удовлетворенности различными аспектами трудовой деятельности, особое влияние оказывая на возможности про- фессионального роста. Однако рост цифровых компетенций приводит к увеличению уровня притязаний работника и, соответственно, к снижению степени его удовлетворенности трудом. Для закрепления высокопрофессиональных кадров в отрасли необходим многоаспектный подход, включающий всевозможные механизмы социальной и экономической поддержки, способствующие повышению уровня удовлет- воренности работника сельского хозяйства результатами своей трудовой деятельности. Процесс цифровизации агропромышленного комплекса России сопровождается ростом потребности в высококва- лифицированных кадрах, обладающих компетенциями в области информационно-коммуникационных технологий. Одним из важнейших стимулов закрепления высокопро- фессиональных кадров на селе является высокая степень удовлетворенности работника результатами своей трудовой деятельности. Цель исследования заключается в определе- нии степени влияния использования работниками сельско- го хозяйства России сети «Интернет» (далее – Интернет) в производственных целях на различные аспекты их удов- летворенности трудом. На базе данных Российского мони- торинга экономического положения и здоровья населения Национального исследовательского университета «Высшая школа экономики» (РМЭЗ НИУ ВШЭ) за 2010–2021 гг. осу- ществлен сравнительный динамический анализ различных аспектов уровня удовлетворенности трудом пользователей и непользователей Интернета в производственных целях, а также динамики использования Интернета работника- ми сельского хозяйства с динамикой различных аспектов Зависимость удовлетворенности трудом работников сельского хозяйства России от использования сети «Интернет» в профессиональной деятельности 23 Доработано после рецензирования 06.05.2023 Принято 19.05.2023 Цифровая социология / / Т. 6 , № 2 2023 г. Цифровая социология / / Т. 6 , № 2 2023 г. Былина С.Г. Зависимость удовлетворенности трудом работников сельского хозяйства России от использования сети «Ин- тернет» в профессиональной деятельности//Цифровая социология. 2023. Т. 6, № 2. С. 40–50. ABSTRACT analysis determined the influence degree of professional Internet use on the level of labour satisfaction; a dependence modeling between various aspects of labour satisfaction on the using the Internet intensity for professional purposes was accomplished. It is established that the information and communication tech- nologies’ use increases the degree of satisfaction with various aspects of labor activity, having a special impact on professional growth opportunities. However, the growth of digital compe- tencies raises the employees’ ambition level and, accordingly, decreases the level of his or her labour satisfaction. In order to consolidate highly professional personnel in the industry, a multi-faceted approach is needed, including all possible mech- anisms of social and economic support that contribute to growth the satisfaction level of an agricultural employee with the results of his or her labor activity. analysis determined the influence degree of professional Internet use on the level of labour satisfaction; a dependence modeling between various aspects of labour satisfaction on the using the Internet intensity for professional purposes was accomplished. It is established that the information and communication tech- nologies’ use increases the degree of satisfaction with various aspects of labor activity, having a special impact on professional growth opportunities. However, the growth of digital compe- tencies raises the employees’ ambition level and, accordingly, decreases the level of his or her labour satisfaction. In order to consolidate highly professional personnel in the industry, a multi-faceted approach is needed, including all possible mech- anisms of social and economic support that contribute to growth the satisfaction level of an agricultural employee with the results of his or her labor activity. Digitalization process in the Russian agro-industrial complex is connected with an increase in the need for highly qualified personnel with competencies in the field of information and communication technologies. One of the most important incen- tives for consolidation highly professional personnel in rural areas is a high degree of employee satisfaction with the results of their work. The article purpose is to determine the influence degree of the Internet use by Russian agricultural workers for professional purposes on various aspects of their labour satis- faction. Agricultural workers’ labour satisfaction dependence in Russia with the use of the internet in professional activities Revised 06.05.2023 Accepted 19.05.2023 Svetlana G. Bylina Cand. Sci. (Econ.), Senior Researcher at the Social Development Laboratory of the Agro-Industrial complex and Rural areas ORCID: 0000-0002-5179-7721 E-mail: svbylina@rambler.ru Institute of Agrarian Problems – Economically autonomous structural subdivision of the Federal Research Center “Saratov Scientif Center of the Russian Academy of Sciences“, Saratov, Russia ABSTRACT Based on data from The Russia Longitudinal Monitoring Survey – Higher School of Economics (RLMS-HSE) from 2010 to 2021, a various aspects’ comparative dynamic analysis of the labour satisfaction level of Internet users and non-users for production purposes, as well as the dynamics analysis of the Internet use by agricultural workers with the dynamics of job satisfaction level various aspects was carried out. Correlation Keywords d communication technologies, agriculture, employees, Internet, labour satisfaction, professional wages Digitalization, information and communication technologies, agriculture, employees, Internet, labour satisfaction, professional growth, working conditions, wages Для цитирования Былина С.Г. Зависимость удовлетворенности трудом работников сельского хозяйства России от использования сети «Ин- тернет» в профессиональной деятельности//Цифровая социология. 2023. Т. 6, № 2. С. 40–50. 40 Цифровая социология: направления исследований Bylina S.G. (2023) Agricultural workers’ labour satisfaction dependence in Russia with the use of the internet in professional activities, Digital Sociology, vol. 6, no. 2, pp. 40–50. DOI: 10.26425/2658-347X-2023-6-2-40-50 © Bylina S.G., 2023. This is an open access article under the CC BY 4.0 license (http://creativecommons.org/licenses/by/4.0/). ВВЕДЕНИЕ / INTRODUCTION Становление цифровой экономики предъявля- ет все новые требования к уровню компетенций работников по степени владения средствами со- временных информационно-коммуникационных технологий (далее – ИКТ). В Стратегии развития агропромышленного и рыбохозяйственного ком- плексов Российской Федерации (далее – Стратегия) на период до 2030 г. одним из ключевых ориенти- ров развития обозначена «цифровизация отраслей и подотраслей агропромышленного и рыбохозяй- ственного комплексов»1. В Стратегии подчеркивает- ся, что в современных условиях существует острая необходимость увеличения доли высококвалифи- цированных кадров, занятых в агропромышлен- ном комплексе (далее – АПК). Однако, привлече- ние квалифицированных кадров с определенным уровнем компетенций, прежде всего цифровых, для работы в АПК должно базироваться на опре- деленных социальных преимуществах, выража- ющихся в оплате, условиях труда, возможностях профессионального роста, в удовлетворенности своим трудом в целом. Соответствие ожиданий реальным возможностям получения удовлетво- рения результатами своей профессиональной де- ятельности является важнейшим стимулом закре- пления высокопрофессиональных кадров на селе. Поэтому исследования в данном направлении яв- ляются весьма актуальными. For citation Bylina S.G. (2023) Agricultural workers’ labour satisfaction dependence in Russia with the use of the internet in professional activities, Digital Sociology, vol. 6, no. 2, pp. 40–50. DOI: 10.26425/2658-347X-2023-6-2-40-50 © Bylina S.G., 2023. This is an open access article under the CC BY 4.0 license (http://creativecommons.org/licenses/by/4.0/). 41 Цифровая социология / / Т. 6 , № 2 2023 г. в производственных целях; определение тесноты связи между профессиональным использованием Интернета и уровнем удовлетворенности трудом; моделирование зависимости различных аспектов удовлетворенности трудом от интенсивности ис- пользования Интернета для работы. 1 Правительство Российской Федерации. Стратегия развития аг- ропромышленного и рыбохозяйственного комплексов Российской Федерации. Утверждена распоряжением Правительства Россий- ской Федерации от 8 сентября 2022 г. № 2567-р. Режим доступа: http://government.ru/docs/46497/ (дата обращения: 09.03.2023). ОБСУЖДЕНИЕ ПРОБЛЕМЫ / PROBLEM DISCUSSION 42 Цифровая социология: направления исследований Уровень оплаты сельского труда как один из фак- торов удовлетворенности трудом отражен в иссле- дованиях M. Hobbs, E. Klachky, M. Cooper [2020]. Довольно широко используются также методы корреляционно-регрессионного анализа, а также сочетание социологических и экономико-мате- матических методов анализа данных. Например, С.М. Юсуповой [2020] с использованием кластер- ного анализа исследован уровень удовлетворен- ности по группам занятий в гендерном аспекте. S.Anastasiou и др. [2014] на основе опроса руково- дителей производства греческих рыбных хозяйств и с использованием методов корреляционного анализа установили, что удовлетворенность ра- ботой увеличивается с повышением следующих аспектов: уровня участия в принятии решений, заработной платы и возраста. И в меньшей сте- пени – с производительностью рыбной фермы. В работе S.S. Maican и др. [2021] выявлен характер корреляций между мотивацией румынских фер- меров к работе в сельском хозяйстве и их удов- летворенностью работой. В условиях перехода к цифровой экономике из- учение связи между использованием ИКТ в про- фессиональной деятельности и уровнем удовлет- воренности различными аспектами трудовой дея- тельности приобретает особое значение на рынке труда. Между тем исследований по данной тема- тике крайне мало. А.С. Смирновой на базе дан- ных 29-ой волны результатов Российского мони- торинга экономического положения и здоровья населения Национального исследовательского университета «Высшая школа экономики» (далее – РМЭЗ НИУ ВШЭ) оценены социальные преиму- щества, получаемые пользователями Интернета в рабочих целях от тех, кто не является пользова- телем [Смирнова, 2021]. P. DiMaggio, B.Bonikowski определили эффект влияния пользования Ин- тернетом для работы на уровень оплаты труда [DiMaggio et al., 2008]. А.Л. Лукьяновой в про- цессе исследования воздействия цифровизации на уровень занятости и заработной платы муж- чин и женщин сделан вывод, что «цифровизация сама по себе оказывает позитивное воздействие на заработные платы» [Лукьянова, 2021, с. 114]. В настоящем исследовании использовались методы сравнительного анализа, а также эконо- мико-математические методы анализа данных. Расчеты многомерных распределений, корреля- ционный и регрессионный анализы осуществля- лись в системе SPSS.17.0. Фактологической основой настоящего иссле- дования послужили материалы Федеральной службы государственной статистики (далее –Рос- стат)2,3, а также результаты РМЭЗ НИУ ВШЭ, вол- ны с 19 по 30, соответствующие 2010–2021 гг4. Ис- пользованы данные репрезентативной выборки. Из общего массива данных выделена группа ра- ботников сельского хозяйства, составившая от 177 до 410 человек. Уровень удовлетворенности раз- личными аспектами труда рассчитывался, исхо- дя из ответов на вопросы анкеты РМЭЗ, отража- ющих удовлетворенность работой в целом, ус- ловиями труда, оплатой труда и возможностями профессионального роста. 3 Абдрахманова Г.И., Васильковский С.А., Вишневский К.О. и др. (2022). Цифровая экономика: краткий статистический сборник / Национальный исследовательский университет «Высшая шко- ла экономики». Режим доступа: https://issek.hse.ru/mirror/pubs/ share/552091260.pdf (дата обращения: 12.03.2023). ОБСУЖДЕНИЕ ПРОБЛЕМЫ / PROBLEM DISCUSSION Существует несколько подходов к определе- нию понятия «удовлетворенность трудом». Часть исследователей делает упор на субъективное вос- приятие работником результатов своей трудовой активности, которое приводит к внутреннему чув- ству удовлетворения, другая часть ученых уделяет особое внимание объективным факторам, таким как качество жизни на работе, или социальная за- щищенность со стороны работодателя [Witt et al., 2020; E.Asiedu-Darko et al., 2016]. Примем понятие «удовлетворенность трудом», сформулированное Ф.Р.Хагуром как «состояние сбалансированности требований (запросов), предъявляемых работни- ком к содержанию, характеру и условиям труда, и субъективной оценки возможностей реализа- ции этих запросов» [Хагур, 2011, с. 83]. Проблеме удовлетворенности трудом посвя- щено огромное количество публикаций. Изна- чально данная проблема подробно изучалась психологами и социологами, затем она привлек- ла внимание экономистов и стала соотноситься ими с функцией полезности и функцией сча- стья [Монусова, 2008; Freeman, 1978; Blanchflower et al., 2004]. В настоящее время такая проблема продолжает широко изучаться, особый интерес вызывают анализ конкретных причин удовлет- воренности людей определенных профессий, влияние социально-экономических, демогра- фических, психологических и других факторов на удовлетворенность трудом в условиях разви- тия цифровой экономики, а также выявление ро- ли современных информационных технологий в повышении уровня и качества жизни населе- ния [Bayona et al., 2020; Чабелкова, 2015; Юсупо- ва, 2020; Каримов и др., 2022]. Цель настоящего исследования – определить влияние использования сети «Интернет» (далее – Интернет) в профессиональных целях работни- ками сельского хозяйства Российский Федерации (далее – РФ) на различные аспекты удовлетворен- ности трудом. Объектом исследования являются работники в сельском хозяйстве РФ, использую- щие Интернет для работы. р р Ключевой гипотезой исследования является предположение о наличии положительного влия- ния профессионального использования работни- ками сельского хозяйства Интернета на уровень их удовлетворенности работой в целом, условия- ми и оплатой труда, а также возможностями про- фессионального роста. В задачи исследования вхо- дят: сравнительный анализ динамики использова- ния Интернета работниками сельского хозяйства с динамикой различных аспектов уровня удовлет- воренности трудом; также сравнительный дина- мический анализ уровня удовлетворенности тру- дом пользователей и непользователей Интернета Однако, несмотря на значительное число публи- каций, посвященных данной проблеме, научных работ по различным аспектам удовлетворенности трудом работников сельского хозяйства немного. Так, связь между удовлетворенностью работой в сельском хозяйстве и качеством жизни изучалась следующими учеными: А. Янч [2021], A.Jantsch, T.Weirowski, N.Hirschauer [2019]. Зависимость уровня удовлетворенности от условий сельского труда затронута в работах О.Я. Старковой [2020]. 1 Правительство Российской Федерации. Стратегия развития аг- ропромышленного и рыбохозяйственного комплексов Российской Федерации. Утверждена распоряжением Правительства Россий- ской Федерации от 8 сентября 2022 г. № 2567-р. Режим доступа: http://government.ru/docs/46497/ (дата обращения: 09.03.2023). Цифровая социология / / Т. 6 , № 2 2023 г. Цифровая социология / / Т. 6 , № 2 2023 г. произошли существенные изменения. По срав- нению с 2010 г. снизилась доля пользователей для работы у законодателей, крупных чиновни- ков и руководителей (с 25 % до 8,3 %), специа- листов высшего и среднего уровня квалифика- ции (с 33 % до 31,3 % и с 27,8 % до 20,8% соответ- ственно), а также квалифицированных рабочих, занятых ручным трудом (с 5,6 % до 2,1 %). Дан- ные снижения произошли за счет роста доли остальных квалификационных групп работни- ков сельского хозяйства, особенно выросла доля квалифицированных работников сельского, лес- ного хозяйства и рыбоводства (с 5,6 % до 14,6 %). Тем не менее в 2021 г., так же как и в 2010 г., мак- симальная доля пользователей Интернета для производственных целей в квалификационной структуре работников сельского хозяйства при- ходится на специалистов высшего уровня квали- фикации, к которым относятся и IT-специалисты. произошли существенные изменения. По срав- нению с 2010 г. снизилась доля пользователей для работы у законодателей, крупных чиновни- ков и руководителей (с 25 % до 8,3 %), специа- листов высшего и среднего уровня квалифика- ции (с 33 % до 31,3 % и с 27,8 % до 20,8% соответ- ственно), а также квалифицированных рабочих, занятых ручным трудом (с 5,6 % до 2,1 %). Дан- ные снижения произошли за счет роста доли остальных квалификационных групп работни- ков сельского хозяйства, особенно выросла доля квалифицированных работников сельского, лес- ного хозяйства и рыбоводства (с 5,6 % до 14,6 %). Тем не менее в 2021 г., так же как и в 2010 г., мак- симальная доля пользователей Интернета для производственных целей в квалификационной структуре работников сельского хозяйства при- ходится на специалистов высшего уровня квали- фикации, к которым относятся и IT-специалисты. Удовлетворенными считались респонденты, выбравшие варианты ответа «полностью удов- летворены» и «скорее удовлетворены». Неудов- летворенными – ответившие «скорее неудовлет- воренны» и «совсем не удовлетворены». РЕЗУЛЬТАТЫ И ОБСУЖДЕНИЕ / RESULTS AND DISCUSSION Согласно данным Росстата за 2020 г., лишь 57,8 % сельскохозяйственных организаций из об- щего их числа имели широкополосный доступ к Интернету, что на 20,8 % ниже, чем по эконо- мике в целом. К сожалению, нет возможности отследить динамику процесса внедрения циф- ровых технологий в сельскохозяйственной от- расли или в сельском, лесном хозяйстве, охоте, рыболовстве и рыбоводстве как виде экономи- ческой деятельности, так как в сборниках Рос- стата по развитию информационного общества в РФ соответствующие данные появились лишь за 2019 г. и 2020 г. Поэтому их можно сопоста- вить лишь с ситуацией по экономке в целом. Результаты сопоставления показывают, что до- ля организаций, относящихся к данному виду экономической деятельности, использующих средства ИКТ и ИКС (информационно-ком- муникационных систем), от общего их числа существенно меньше, чем по экономике в це- лом. Так, по данным за 2019 г. процент отста- вания по наличию серверов составляет 17,8 %, локальных вычислительных сетей – 22,4 %, Ин- тернета – 10,4 %, в том числе широкополосно- го доступа – 14,2 %, веб-сайтов – 51,6 %. Доля работников организаций данного вида эконо- мической деятельности, использующих ком- пьютеры, составляет 15,4 %, Интернет – 13,5 %, портативные устройства, обеспечивающие до- ступ к Интернету по мобильной связи, предо- ставленные организацией – 2 %, что на 54,5– 65,8 % ниже, чем по экономике в целом. Сле- дует отметить, что данное положение связано как с особенностями процесса цифровизации агропромышленного комплекса, так и со специ- фикой сельскохозяйственной деятельности. Тем не менее, согласно результатам РМЭЗ, с 2010 г. по 2021 г. доля пользователей Интернета сре- ди работников сельского хозяйства выросла в 4,5 раза с 16,8 % до 75,7 % (рис. 1). При этом в 3 раза выросла доля сельскохозяйственных ра- ботников, использующих Интернет в профес- сиональных целях (с 8,8 % до 27,1 %). В структуре пользователей Интернета для работы среди работников сельского хозяй- Составлено автором по материалам исследования / Compiled by the author on the materials of the study Рис. 1. Динамика использования Интернета работниками сельского хозяйства Fig. 1. Dynamics of the Internet use by agricultural workers 0 10 20 30 40 50 60 70 80 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 Год Доля от числа респондентов, % Пользователи Интернета (1) Использование Интернета для работы (2) 1 2 Составлено автором по материалам исследования / Compiled by the author on the materials of the study Рис. 1. Динамика использования Интернета работниками сельского хозяйства Рис. 1. Динамика использования Интернета работниками сельского хозяйства МАТЕРИАЛЫ И МЕТОДЫ ИССЛЕДОВАНИЯ / MATERIALS AND RESEARCH METHODS В исследованиях влияния различных факто- ров на удовлетворенность трудом используют- ся в большинстве своем социологические мето- ды. Так, Н.Э.Соболевой на основании региональ- ного репрезентативного опроса, проведенного в 38 российских регионах, установлено, что «роль удовлетворенности работой в удовлетворенности жизнью усиливает наличие внутренней трудовой мотивации» [Соболева, 2020, с. 386]. J.A. Bayona и др. [2020] по результатам опроса 531 колум- бийского работника из 20 секторов экономики пришли к выводу о высокой значимости уров- ня знаний у работника для определения его вов- леченности в работу. M.Hobbs и др [2020] обсле- довали рабочих виноградников Напы (Калифор- ния) в выборке из 665 респондентов. Установлено, что на проблему текучести кадров наибольшее влияние оказывают такие параметры удовлет- воренности трудом, как общение, оплата тру- да, характер сельскохозяйственных работ, обя- зательства перед семьей. Улучшение этих па- раметров, по мнению авторов, может повысить удовлетворенность работой и удержание суще- ствующих работников для стабилизации сель- скохозяйственной рабочей силы. 2 Федеральная служба государственной статистики, Национальный исследовательский университет «Высшая школа экономики» (2020). Ин- формационное общество в Российской Федерации: статистический сборник. Режим доступа: https://rosstat.gov.ru/storage/mediabank/ lqv3T0Rk/info-ob2020.pdf (дата обращения: 11.03.2023). 4 «Российский мониторинг экономического положения и здоровья населения НИУ-ВШЭ (RLMS-HSE)», проводимый Национальным исследовательским университетом «Высшая школа экономики» и ООО «Демоскоп» при участии Центра народонаселения Уни- верситета Северной Каролины в Чапел Хилле и Института социо- логии Федерального научно-исследовательского социологическо- го центра РАН. (Режим доступа сайтов обследования RLMS-HSE: https://rlms-hse.cpc.unc.edu и http://www.hse.ru/rlms)» (дата об- ращения: 15.03.2023). 43 Рис. 1. Динамика использования Интернета работниками сельского хозяйства Fig. 1. Dynamics of the Internet use by agricultural workers Согласно данным Росстата за 2019 г., на 10 тыс. работников организаций данного вида эконо- мической деятельности приходилось 60 специ- алистов по ИКТ, что в 3,8 раза меньше, чем по экономике в целом [Цифровая экономи- ка, 2022]. Из них специалистов высшего уров- ня квалификации меньше в 6,6 раза, среднего уровня квалификации меньше в 2,2 раза. Учи- тывая серьезный разрыв в обеспеченности сель- скохозяйственной отрасли в квалифицирован- ных кадрах, обладающих достаточно высоким уровнем цифровых компетенций по отноше- нию к экономике в целом, следовало бы пред- положить, что владение средствами ИКТ и их использование в своей профессиональной де- ятельности дает работнику ряд конкурентных В структуре пользователей Интернета для работы среди работников сельского хозяй- ства за исследуемый период времени также 44 Цифровая социология: направления исследований преимуществ, что позволяет надеяться на более высокий уровень их удовлетворенности сво- им трудом. преимуществ, что позволяет надеяться на более высокий уровень их удовлетворенности сво- им трудом. деятельности (кроме оплаты труда). Значитель- ней всего у работников сельского хозяйства, ис- пользующих средства ИКТ, снизился уровень удовлетворенности возможностями професси- онального роста – на 19,4 %, и уровень оплаты труда – на 16,6 %. У работников, не использу- ющих Интернет в профессиональной деятель- ности, за указанный период времени отмечен рост удовлетворенности возможностями профес- сионального роста на 46,4 %, работой в целом на 20 %, условиями труда – на 10,5 %. Лишь до- ля «непользователей», удовлетворенных опла- той труда, снизилась на 11,9 %. Однако результаты исследования показыва- ют несколько иную картину. В табл. 1 представ- лен сравнительный анализ степени удовлетво- ренности различными аспектами труда работ- ников сельского хозяйства, которые пользуются и не пользуются средствами ИКТ в своей про- фессиональной деятельности. ф Очевидно, что специалисты с более высоким уровнем цифровых компетенций демонстриру- ют и более высокий уровень удовлетворенности различными аспектами своей трудовой деятель- ности. Наибольший уровень удовлетворенно- сти выявлен у работников сельского хозяйства, независимо от использования Интернета, да- лее удовлетворенность работой в целом, затем условиями труда. Наименьшая степень удов- летворенности сельских тружеников отмеча- ется уровнем оплаты труда и возможностями профессионального роста. Если же рассматри- вать данные процессы в динамике, то следует отметить, что в 2021 г. по отношению к 2010 г. Рис. 1. Динамика использования Интернета работниками сельского хозяйства Сравнительная динамика использования Интернета работниками сельского хозяйства в профессиональных целях и различных аспектов уровня их удовлетворенности трудом Fig. 2. Comparative dynamics of the Internet use by agricultural workers for professional purposes and various aspects of their level of job satisfaction 90 80 70 60 50 40 30 20 90 80 70 60 50 40 30 20 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 Годы Доля от числа использующих Интернет для работы, % Доля ответов респондентов, % Пользователи Интернета в течение последних 12 месяцев для работы Степень удовлетворенности условиями труда Степень удовлетворенности оплатой труда Степень удовлетворенности возможностями профессионального роста о автором по материалам исследования / Compiled by the author on the materials of the study Составлено автором по материалам исследования / Compiled by the author on the materia Рис. 2. Сравнительная динамика использования Интернета работниками сельского хозяйства в профессиональных целях и различных аспектов уровня их удовлетворенности трудом Fig. 2. Comparative dynamics of the Internet use by agricultural workers for professional purposes and various aspects of their level of job satisfaction Результаты исследования показывают, что с 2010 г. по 2014 г. наблюдается снижающий- ся тренд доли пользователей Интернета в про- фессиональных целях, который практически со- впадает по направленности с трендами различ- ных аспектов уровня удовлетворенности трудом (за исключением условий труда). Как известно, в 2014 г. США и Европейский союз (далее – ЕС) ввели антироссийские санкции в связи с при- соединением Республики Крым, затем после- довал ответный запрет на импорт продоволь- ствия РФ из этих стран. В октябре 2014 г. принят трехлетний план мероприятий по содействию импортозамещения в сельском хозяйстве. Мож- но предположить, что данные внешние условия способствовали резкому увеличению в 2015 г. до- ли пользователей средствами ИКТ в производ- ственных целях среди работников сельского хо- зяйства, что сопровождается небольшим ростом доли удовлетворенных работой в целом, возмож- ностями профессионального роста и условиями труда. Однако доля пользователей, удовлетворен- ных оплатой труда, продолжает уменьшаться. Интересна дальнейшая взаимная динамика ис- следуемых факторов. Как показывают результа- ты, 2016 г. характеризуется минимальными зна- чениями рассматриваемых показателей уровня удовлетворенности трудом среди работников сельского хозяйства, использующих Интернет в производственных целях. В следующем 2017 г. напротив, на фоне минимально наблюдаемого снижения доли пользователей Интернета для работы произошел резкий одиночный всплеск показателей удовлетворенности трудом данной группы работников сельского хозяйства с после- дующим снижением всех индикаторов, кроме удовлетворенности работой в целом. Рис. 1. Динамика использования Интернета работниками сельского хозяйства уровень удовлетворенности обозначенными аспектами труда снижается у пользователей, но растет у непользователей Интернета в трудовой Следовательно, необходимо отметить, что, несмотря на более высокий уровень удовлет- воренности трудом работников сельского хо- зяйства, использующих средства ИКТ, работа в сельскохозяйственной отрасли не способству- ет росту их удовлетворенности от профессио- нальной деятельности. Еще более интересную картину дает анализ совместной динамики доли пользователей Ин- тернета среди работников сельского хозяйства, использующих средства ИКТ для работы, с ди- намикой уровня их удовлетворенности тру- дом (рис. 2). Таблица 1. Сравнительная динамика уровня удовлетворенности трудом работников сельского хозяйства, использующих и не использующих Интернет в профессиональной деятельности Table 1. Comparative dynamics of the satisfaction level with the work of agricultural workers who use and do not use the Internet in their professional activities Годы Респонденты 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 Доля респондентов, % Насколько Вы удовлетворены или не удовлетворены Вашей работой в целом? пользователи 80,6 80,6 80,0 81,6 78,1 78,6 65,9 75,0 77,3 76,0 74,0 74,0 «непользователи» 54,5 54,5 63,8 60,0 59,6 51,2 52,5 54,9 60,0 62,2 65,4 65,4 Насколько Вы удовлетворены или не удовлетворены условиями Вашего труда? пользователи 75,0 75,0 81,8 83,7 75,0 78,6 56,8 83,3 68,2 70,3 72,0 72,0 «непользователи» 54,5 54,5 48,3 52,3 51,9 34,1 44,1 53,5 50,8 55,2 60,3 60,3 Насколько Вы удовлетворены или не удовлетворены оплатой Вашего труда? пользователи 52,8 52,8 54,5 55,1 46,9 40,5 34,1 50,0 40,9 42,1 44,0 44,0 «непользователи» 36,4 36,4 27,6 32,3 32,7 17,1 22,0 26,8 30,8 31,4 32,1 32,1 Насколько Вы удовлетворены или не удовлетворены возможностями Вашего профессионального роста? пользователи 69,4 69,4 69,1 65,3 59,4 64,3 61,4 72,2 54,5 55,1 56,0 56,0 «непользователи» 39,4 39,4 39,7 43,1 34,6 29,3 32,2 52,1 41,5 50,3 57,7 57,7 Составлено автором по материалам исследования / Compiled by the author on the materials of the study 45 Цифровая социология / / Т. 6 , № 2 2023 г. 90 80 70 60 50 40 30 20 90 80 70 60 50 40 30 20 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 Годы Доля от числа использующих Интернет для работы, % Пользователи Интернета в течение последних 12 месяцев для работы Степень удовлетворенности своей работой в целом Степень удовлетворенности условиями труда Степень удовлетворенности оплатой труда Степень удовлетворенности возможностями профессионального роста Доля ответов респондентов, % Составлено автором по материалам исследования / Compiled by the author on the materials of the study Рис. 2. Рис. 1. Динамика использования Интернета работниками сельского хозяйства у р р Для объяснения подобной динамики проана- лизированы исходные данные по численности респондентов и доли пользователей Интернета среди них, а также основные экономические по- казатели развития сельского, лесного хозяйства с 2014 г. по 2019 г.: индекс производства продук- ции, инвестиций, величины валовой добавлен- ной стоимости и численности занятых. Уста- новлено, что в численности репрезентативной группы работников сельского хозяйства за рас- сматриваемый период времени не произошло су- щественных количественных изменений. Анализ динамики экономических показателей позволил установить, что параметром, динамика которого могла повлиять на уровень использования средств ИКТ в сельском хозяйстве, а также долю работ- ников отрасли, удовлетворенных своей трудо- вой деятельностью, может быть индекс инвести- ций в данный вид экономической деятельности. 46 Цифровая социология: направления исследований Так, согласно данным Росстата [Российский ста- тистический сборник, 2017], в 2014 г. и 2015 г. на- блюдалось существенное падение уровня инве- стиций в сельское и лесное хозяйство, ниже 100 % по отношению к предыдущему периоду, однако в 2016 г. объем инвестиций составил 112,5 % от- носительно уровня 2015 г. Дальнейший рост ин- вестиций продолжался гораздо меньшими тем- пами. Следует предположить, что реакция ра- ботников сельского хозяйства в виде изменения уровня удовлетворенности трудом и активно- сти использования средств ИКТ в производствен- ных целях, вызвана экономическими причина- ми с временным лагом в 1 год. выми компетенциями и использующими их на рабочем месте, приводит к сокращению уровня их удовлетворенности достижениями в профес- сии и размерами денежного вознаграждения, что вызывает снижение удовлетворенности работой в целом и в меньшей степени условиями труда. Таким образом, у работника сельского хозяй- ства, овладевшего средствами ИКТ для профес- сиональной деятельности, растет уровень при- тязаний и снижается степень удовлетворенности различными аспектами своего труда. р ру Высокие значения коэффициентов корреля- ции позволяют предположить о наличии зависи- мости между отдельными параметрами удовлет- воренности трудом и активностью использования средств ИКТ для работы. Построены регрессион- ные зависимости показателей уровня удовлетво- ренности трудом работников сельского хозяйства от доли пользователей Интернета для работы. Для определения тесноты связи между ак- тивностью использования Интернета для рабо- ты и уровнем удовлетворенности различными аспектами труда осуществлен корреляционный анализ между долей работников сельского хозяй- ства, использующих Интернет для работы, и до- лей данной же группы респондентов, удовлетво- ренных работой в целом, условиями труда, опла- той труда и возможностями профессионального роста (табл. 2). Обратимся к табл. 2. Таблица 3. Параметры регрессионных зависимостей показателей уровня удовлетворенности трудом от доли работников сельского хозяйства, использующих Интернет для работы Table 3. Рис. 1. Динамика использования Интернета работниками сельского хозяйства Regression dependences’ parameters of the job satisfaction level indicators on the share of agricultural workers using the Internet for work Зависимый параметр Кон- станта Коэффи- циент Коэффициент множествен- ной детерминации Доля удовлет- воренных ра- ботой в целом 82,956 -0,143 0,343 Доля удов- летворенных оплатой труда 59,296 -0,731 0,473 Доля удов- летворенных возможностями профессио- нального роста 77,198 -0,817 0,618 Составлено автором по материалам исследования / Compiled by the author on the materials of the study Таблица 3. Параметры регрессионных зависимостей показателей уровня удовлетворенности трудом от доли работников сельского хозяйства, использующих Интернет для работы Таблица 2. Коэффициенты корреляции между долей работников сельского хозяйства, использующих Интернет для работы, Таблица 2. Коэффициенты корреляции между долей работников сельского хозяйства, использующих Интернет для работы, и уровнем удовлетворенности трудом Table 2. Correlation coefficients between the share of agricultural workers using the Internet for work and the job satisfaction level Показатели Доля пользователей Интернета для работы Доля удовлетворенных работой в целом -0,63062 Доля удовлетворенных условиями труда -0,45347 Доля удовлетворенных оплатой труда -0,72488 Доля удовлетворенных возможностями профес- сионального роста -0,78174 Составлено автором по материалам исследования / Compiled by the author on the materials of the study Составлено автором по материалам исследования / Compiled by the author on the materials of the study Очевиден высокий уровень обратной взаи- мосвязи между долей работников, пользующих- ся Интернетом в производственных целях, и по- казателями уровня удовлетворенности трудом. Наиболее высокую тесноту обратной связи де- монстрируют показатели уровня удовлетворен- ности возможностями профессионального ро- ста и оплаты труда с уровнем использования средств ИКТ для работы. Рост доли работни- ков сельского хозяйства, обладающих цифро- Все полученные модели и их коэффициенты статистически значимы на 5 % уровне. Однако, очевидно, что лишь зависимость доли удовлет- воренных возможностями профессионального роста от доли использующих Интернет в про- фессиональных целях описывает 61,8 % наблю- дений, все остальные зависимости охватывают ме- нее 50 % случаев. Следовательно, степень владе- ния средствами ИКТ в производственных целях 47 Цифровая социология / / Т. 6 , № 2 2023 г. и, следовательно, к снижению степени удовлет- воренности трудом в целом. На основе получен- ных результатов можно предположить, что в на- стоящий момент сельскохозяйственная отрасль не может в полной мере удовлетворить уровень притязаний специалистов с высокими цифро- выми компетенциями. Как показали результа- ты исследования, существенное влияние на уро- вень удовлетворенности трудом, хотя и с времен- ным лагом, оказывают экономические факторы. В первую очередь инвестиции в основной капи- тал сельского, лесного хозяйства, позволяющие внедрять новые технологии. Рис. 1. Динамика использования Интернета работниками сельского хозяйства вносит значительный вклад лишь в уровень удов- летворенности от открывающихся возможностей профессионального роста. Для оплаты труда данный фактор имеет значение лишь в 47,3 % случаев, а для удовлетворенности работой в це- лом – лишь для 34,3 % наблюдений. Что каса- ется условий труда, то степень владения сред- ствами ИКТ оказалась незначимым фактором для удовлетворенности условиями труда в сель- ском хозяйстве. ЗАКЛЮЧЕНИЕ / CONCLUSION В результате проведенного исследования на- чальная гипотеза подтвердилась в следующих моментах. Уровень удовлетворенности различ- ными аспектами своей трудовой деятельности работников сельского хозяйства, владеющих средствами ИКТ и использующих их в произ- водственных целях, выше, чем у тех, кто не ис- пользует Интернет для работы. Уровень циф- ровых компетенций для работников сельского хозяйства является весьма значимым фактором, влияющим на возможности профессионально- го роста и, соответственно, на уровень их удов- летворенности трудом. Удовлетворенность трудом – один из важ- нейших социальных факторов, позволяющих закрепить работника в сельском хозяйстве, по- этому необходим многоаспектный подход, вклю- чающий всевозможные механизмы социальной и экономической поддержки, способствующие увеличению степени удовлетворенности тру- дом специалистов, обладающих необходимым уровнем знаний для работы в условиях цифро- вой трансформации АПК. Однако более высокий уровень цифровых ком- петенций приводит к росту притязаний работни- ка к различным аспектам трудовой деятельности Каримов А.Г., Фаткуллина Г.Р. (2022). Современные информационные технологии как инструмент сокращения бедности работающего населения // Цифровая социология. Т. 5, № 1. С. 44–53. Цифровая социология: направления исследований Цифровая социология: направления исследований Anastasiou S., Karipoglou K., Nathanailides C. (2014). Participation in Decision Making, Productivity and Job Satisfaction among Managers of Fish Farms in Greece // International Business Research. V.7, no. 12. Pp.10–22. https://doi.org/10.5539/ibr.v7n12p92 Asiedu-Darko E., Amanor M. (2016). Factors affecting job satisfaction of agricultural sector workers in Ghana // American Journal of Business, Economics and Management. V. 4, no 4. Pp. 89–93. Bayona J.A., Caballer A., Peiró, J.M. (2020). The Relationship between Knowledge Characteristics’ Fit and Job Satisfaction and Job Performance: The Mediating Role of Work Engagement.// Sustainability. V.12, no 6. 20 p. https://doi.org/10.3390/su12062336 Blanchflower D., Oswald A. (2004). Weil-Being over Time in Britain and the USA // Journal of Public Economics. 004). Weil-Being over Time in Britain and the USA // Journal of Public Economics. V. 88, no 7–8. Pp. 1359–1386 DiMaggio P., Bonikowski B. (2008). Make Money Surfing the Web? The Impact of Internet Use on the Earnings of U.S. Workers // American Sociological Review. V. 73, no 2. Pp. 227–250. https://doi.org/10.1177/000312240807300203 Freeman R. (1978). Job Satisfaction as an Economic Variable // The American Economic Review. V. 68, no 2. Pp. 135–141. Jantsch A., Weirowski T., Hirschauer N. (2019). (Dis-)Satisfaction in Agriculture? An Explorative Analysis of Job and Life Satisfaction in East Germany // German Journal of Agricultural Economics. V. 68, no 4. Pp. 263–274. Hobbs M., Klachky E., Cooper M. (2020). Job satisfaction assessments of agricultural workers help employers improve the work environment and reduce turnover // California agriculture. V. 74, nо 1, https://doi.org/10.3733/ ca.2020a0002 Maican S.Ș., Muntean A.C., Paștiu C.A. et al (2021). Motivational Factors, Job Satisfaction, and Economic Performance in Romanian Small Farms // Sustainability. Vol. 13, no 11. https://doi.org/10.3390/su13115832 Wit C.D., Reed D.B., Rayens M.K., Hunsucker S. (2020). Predictors of Job Satisfaction in Female Farmers Aged 50 and Over: Implications for Occupational Health Nurses // Workplace Health & Safety. V. 68, no 11. Pp. 526–532. REFERENCES Anastasiou S., Karipoglou K., Nathanailides C. (2014), “Participation in Decision Making, Productivity and Job Satisfaction among Managers of Fish Farms in Greece”, International Business Research, vol.7, no.12, pp.10–22, https://doi.org/10.5539/ibr.v7n12p92 Asiedu-Darko E., Amanor M. (2016), “Factors affecting job satisfaction of agricultural sector workers in Ghana”, American Journal of Business, Economics and Management, vol. 4, no 4, pp. 89–93. Bayona J.A., Caballer A., Peiró, J.M. (2020), “The Relationship between Knowledge Characteristics’ Fit and Job Satisfaction and Job Performance: The Mediating Role of Work Engagement”, Sustainability, vol.12, no 6. 20 p, https://doi.org/10.3390/su12062336 Blanchflower D., Oswald A. (2004), “Weil-Being over Time in Britain and the USA”, Journal of Public Economics, Blanchflower D., Oswald A. (2004), “Weil-Being over Time in Britain and the USA”, Journal of Public Economics, vol. 88, no 7–8, pp. 1359–1386. Chabelkova I., Kiseleva L.S., Strelkovskij V. (2015), “Study of the influence of socio-demographic characteristics of workers on job satisfaction (on the example of the Czech Republic)”, Human Ecology, no 4, pp. 39–46, https://doi.org/10.17816/humeco17077 (In Russian). DiMaggio P., Bonikowski B. (2008), “Make Money Surfing the Web? The Impact of Internet Use on the Earnings of U.S. Workers”, American Sociological Review, vol. 73, no 2, pp. 227–250, https://doi.org/10.1177/000312240807300203 ob Satisfaction as an Economic Variable”, The American Economic Review, vol. 68, no 2, pp. 135–141. Freeman R. (1978), “Job Satisfaction as an Economic Variable”, The American Economic Review, vol. 68, no 2, pp Hagur F.R. (2011), “Category “job satisfaction” in the sociological dimension”, Theory and practice of social development, no. 8, pp. 82–84. (In Russian). Hobbs M., Klachky E., Cooper M. (2020), “Job satisfaction assessments of agricultural workers help employers improve the work environment and reduce turnover”, California agriculture, vol. 74, nо 1, https://doi.org/10.3733/ ca.2020a0002 Jantsch A., Weirowski T., Hirschauer N. (2019), “(Dis-)Satisfaction in Agriculture? An Explorative Analysis of Job and Life Satisfaction in East Germany”, German Journal of Agricultural Economics, vol. 68, no 4, pp. 263–274. Karimov A.G., Fatkullina G.R. (2022), “Modern information technologies as a tool to reduce the poverty of the working population”, Digital so- ciology, vol. 5. no. 1, pp. 44–53. (In Russian). Lukyanova A.L. (2021), “Digitalization and the Gender Pay Gap”, Economic Policy, vol. 16, no. 2, pp. 88–117, https://doi.org/10.18288/1994- 5124-2021-2-88-117 (In Russian). Maican S.Ș., Muntean A.C., Paștiu C.A. et al (2021), “Motivational Factors, Job Satisfaction, and Economic Performance in Romanian Small Farms”, Sustainability, vol. 13, no 11. https://doi.org/10.3390/su13115832 Monusova G. СПИСОК ЛИТЕРАТУРЫ Каримов А.Г., Фаткуллина Г.Р. (2022). Современные информационные технологии как инструмент сокращения бедности работающего населения // Цифровая социология. Т. 5, № 1. С. 44–53. Каримов А.Г., Фаткуллина Г.Р. (2022). Современные информационные технологии как инструмент сокращения бедности работающего населения // Цифровая социология. Т. 5, № 1. С. 44–53. Лукьянова А.Л. (2021). Цифровизация и гендерный разрыв в оплате труда // Экономическая политика. Т. 16, № 2. С. 88–117. https://doi. org/10.18288/1994-5124-2021-2-88-117 Лукьянова А.Л. (2021). Цифровизация и гендерный разрыв в оплате труда // Экономическая политика. Т. 16, № 2. С. 88–117. https://doi. org/10.18288/1994-5124-2021-2-88-117 Монусова Г. (2008). Удовлетворенность трудом: межстрановые сопоставления // Мировая экономика и международные отношения. № 12. С. 74–83. Смирнова А.С. (2021). Сетевые факторы профессиональной дифференциации // ИНАБ. № 4. Структурные аспекты цифрового неравенства. С. 52–73. https://doi.org/10.19181/ INAB.2021.4.4 Соболева Н.Э. (2020). Факторы, влияющие на вклад удовлетворенности работой в удовлетворенность жизнью в России // Мониторинг общественного мнения: Экономические и социальные перемены. № 1. С. 368–390. https://doi.org/10.14515/monitoring.2020.1.15 Старкова О.Я. (2020). Особенности условий труда в сельских территориях // Экономика сельского хозяйства России. № 6. С. 105–108. https://doi.org/10.32651/206-105 Федеральная служба государственной статистики. Российский статистический ежегодник. (2017). Стат.сб./Росстат. М. 686 с. Хагур Ф.Р. (2011). Категория «удовлетворенность трудом» в социологическом измерении // Теория и практика общественного развития. № 8. С. 82–84.Чабелкова И., Киселева Л.С., Стрелковский В. (2015). Исследование влияния социально-демографических характеристик работников на удовлетворенность трудом (на примере Чешской Республики) // Экология человека. № 4. С. 39–46. https://doi.org/10.17816/ humeco17077 Юсупова С.М. (2020). Кластерный анализ удовлетворенности работой по группам занятий в РФ в гендерном аспекте // Электронный научный журнал «Вектор экономики». № 11. Юсупова С.М. (2020). Кластерный анализ удовлетворенности работой по группам занятий в РФ в гендерном аспекте // Электронный научный журнал «Вектор экономики». № 11. Янч А. (2021). Анализ удовлетворенности работой и качеством жизни в сельской местности Германии // Никоновские чтения. №26. С. 220–224. 48 REFERENCES (2008), “Satisfaction with work: cross-country comparisons”, Mirovaya ekonomika i mezhdunarodnye otnosheniya, no. 12, pp. 74–83 (In Russian). Federal State Statistics Service (2017), Russian Statistical Yearbook Statistical collection, Rosstat, Moscow, Russia 49 Smirnova A.S. (2021), “Network factors of professional differentiation”, INAB, no. 4, Structural aspects of the digital divide, pp. 52–73. https://doi.org/10.19181/INAB.2021.4.4 (In Russian). Soboleva N.E. (2020), “Factors affecting the contribution of job satisfaction to life satisfaction in Russia”, Public opinion monitoring: Economic and social changes, no. 1, pp. 368–390, https://doi.org/10.14515/monitoring.2020.1.15 (In Russian). Цифровая социология / / Т. 6 , № 2 2023 г. Smirnova A.S. (2021), “Network factors of professional differentiation”, INAB, no. 4, Structural aspects of the digital divide, pp. 52–73. https://doi.org/10.19181/INAB.2021.4.4 (In Russian). Soboleva N.E. (2020), “Factors affecting the contribution of job satisfaction to life satisfaction in Russia”, Public opinion monitoring: Economic and social changes, no. 1, pp. 368–390, https://doi.org/10.14515/monitoring.2020.1.15 (In Russian). Starkova O.Ya. (2020), “Peculiarities of working conditions in rural areas”, Agricultural Economics of Russia, no. 6, pp. 105–108. https://doi.org/10.32651/206-105 (In Russian). Wit C.D., Reed D.B., Rayens M.K., Hunsucker S. (2020), “Predictors of Job Satisfaction in Female Farmers Aged 50 and Over: Implications for Occupational Health Nurses”, Workplace Health & Safety, vol. 68, no 11, pp. 526–532. Yanch A. (2021), “Analysis of satisfaction with work and quality of life in rural areas of Germany”, Nikon Readings, no Yusupova S.M. (2020), “Cluster analysis of job satisfaction by occupational groups in the Russian Federation in the gender aspect”, Electronic scientific journal “Vector of Economics”, no. 11 (In Russian). 50
https://openalex.org/W4299854197	http://etheses.lse.ac.uk/584/1/Bonina_public_values_information_2012.pdf	English	null	On Public Values and Information Technology in Government: A Critical Discourse Analysis of Trade Regulations in Mexico	Zenodo (CERN European Organization for Nuclear Research)	2,012	cc-by	92,755	The London School of Economics and Political Science The London School of Economics and Political Science ON PUBLIC VALUES AND INFORMATION TECHNOLOGY IN GOVERNMENT: A CRITICAL DISCOURSE ANALYSIS OF TRADE REGULATIONS IN MEXICO Carla M. Bonina A thesis submitted to the Department of Management of the London School of Economics and Political Science for the degree of Doctor of Philosophy London, December2012 DECLARATION I certify that the thesis I have degree of the London School of Economics and Political Science is solely my own work other than where I have clearly indicated that it is the work of others. I certify that the thesis I have presented for examination for the PhD degree of the London School of Economics and Political Science is solely my own work other than where I have clearly indicated that it is the work of others. presented for examination for the PhD degree of the London School of Economics and Political Science is solely my own work other than where I have clearly indicated that it is The copyright of this thesis rests with the author. permitted, provided that full acknowledgement is made below). I warrant that this authorisation does not, to the best of my belief, infringe the rights of any third party. The copyright of this thesis rests with the author. Quotation from it is permitted, provided that full acknowledgement is made (see license . I warrant that this authorisation does not, to the best of my belief, infringe the rights of any third party. Quotation from it is (see license . I warrant that this authorisation does not, to the best of my ACKNOWLEDGEMENTS The completion of this dissertation has been part of a great journey and I am very grateful to many wonderful people that supported me intellectually, professionally and emotionally. I am indebted to Chrisanthi Avgerou, who supervised me in the final phase of this project. Her continuous tenacity and extraordinary talent, both as researcher and supervisor, have been invaluable sources of inspiration and support to my work. I am very grateful to her. I am also grateful to staff, friends and colleagues in the Information Systems and Innovation Group at the LSE for making such an amazing and vibrant academic atmosphere. In particular, I thank Antonio Cordella and Jannis Kallinikos for advice, professional opportunities and support during these years. I am also very grateful to Edgar Whitley and Susan Scott for precious help and guidance in times of uncertainty. Thanks also to Shirin Madon, Jonathan Liebenau, Carsten Sorensen, Allen Lee, and David Arellano-Gault for their appreciation, guidance and encouragement in different phases of my PhD project. I want to express my gratitude to Lilie Chouliaraki for the conversations and valuable suggestions about a fascinating area I’ve just discovered— critical discourse analysis. I am also grateful to my PhD examiners Ela Klecun and Niall Hayes for helpful criticisms to improve this work. I am very thankful to the members of the Mexican government who were so kind in opening the doors to my research. Special thanks to Lorenza Martinez, Cesar Hernandez, Javier Anaya and the team in the Mexican Ministry of the Economy. Their support, trust and confidence made this project possible. Many friends proved to be invaluable resources to complete this project—from holding discussions and checking my writing in my non- native language, to offering me emotional support and even lodging during fieldwork. Special thanks to Carrie Paris, Roser Pujadas, Philippa Kaina, Cecilia Bustamante, Valeria Valle, Ana Ruth Solano, Savita Bailur, Aaron Martin, Magda Hercheui, Silvia Elaluf-Calderwood, Patrik Karrberg, Wifak Houij Gueddana, Paula Cutuli, Julia Vignoli, Georgina Dellacha, Veronica Rappoport, Carolina Aguerre, Sergio San Sebastian and Alejandro Campos. And to many other friends and family that were there for me no matter the distance or how my mood was. I benefited from generous financial support from the European Union under Programme Alban (scholarship No. E07D402325MX), and various research grants from the LSE—I am very grateful for that. My biggest gratitude goes to Cristobal for helping me to never give up. This work is licensed under the Creative Commons Attribution 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0/ This work is licensed under the Creative Commons Attribution 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0/. This work is licensed under the Creative Commons Attribution 3.0 This work is licensed under the Creative Commons Attribution 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0/ This work is licensed under the Creative Commons Attribution 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0/. This work is licensed under the Creative Commons Attribution 3.0 This work is licensed under the Creative Commons Attribution 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0/ This work is licensed under the Creative Commons Attribution 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0/. This work is licensed under the Creative Commons Attribution 3.0 ACKNOWLEDGEMENTS His love, endless patience and encouragement are treasures that I keep forever. I’m also extremely grateful to my parents who continue to support me unconditionally, wherever I go. It is to Cristobal and my growing family that I dedicate this thesis. Gracias! - Page 3 - ON PUBLIC VALUES AND INFORMATION TECHNOLOGY IN GOVERNMENT: A CRITICAL DISCOURSE ANALYSIS OF TRADE REGULATIONS IN MEXICO ABSTRACT The use of the internet and related information and communication technologies (ICT) in public administration (known as ‘e-government’) has gained notable space within processes of public sector reform. Arguably, ICT provide an attractive strategy to reorganize internal government tasks, routines and processes, and to make them more efficient, responsive as well as accountable to citizens. Yet, the linkages between public values and e-government programmes remain understudied or taken for granted. My research focuses on this particular aspect of public sector reforms and organising. It engages with the debates towards modernisation of central government services while contributing to discussions of the relation between technologically induced programmes and public values over time. Using critical discourse analysis, I trace the discourses on public values and technology within a longitudinal case of a technology-enabled platform to facilitate foreign trade regulations in Mexico—the Mexican Single Window for Foreign Trade. In my empirical analysis, I examine a combination of key government texts and extensive data from fieldwork to address two related questions: what public values are presented, enacted or marginalized during the trajectory of the case, and how these values are enacted and operationalised into technology over time. The analysis reveals four distinctive discourses on public values and technology: ‘technical efficiency’, ‘legality and honesty’, ‘robustness’ and ‘(forced) cooperation’. The analysis shows that while the technical efficiency cluster—commonly associated to the new public management ethos—is dominating, it cohabits with and is reinforced by other values more broadly related to traditional public administration and the bureaucratic ethos—that is, legality and honesty. In addition, the analysis shows that these four distinctive discourses have been materialised in technology in different degrees, giving rise to tensions and contestation over time. In light of the findings, I draw implications for theorizing public values and technology innovation within public sector reforms in a given context. - Page 4 - TABLE OF CONTENTS ABSTRACT ................................................................................................................ 4 TABLES .................................................................................................................... 8 FIGURES .................................................................................................................. 9 ABBREVIATIONS ..................................................................................................... 10 1 Chapter One: Introduction .............................................................................. 12 1.1 On public values, modernisation reforms and e-government .............................. 14 1.1.1 New Public Management and the rise of the economic individualism ....... 15 1.1.2 E-government and the values of reform ......................................................... 16 1.2 A discursive lens to study public values and e-government ................................. 18 1.3 Conclusion and Structure of the Thesis .................................................................. 21 2 Chapter Two: On Public Values and E-government ..................................... 24 2.1 The rise of public value as counterbalance of market-driven modernisation discourses ............................................................................................................................... 25 2.1.1 Public values: three streams of research .......................................................... 25 2.1.2 Why public values are important in public sector debates ............................ 28 2.2 Normative public values: approaches from the literature ..................................... 30 2.2.1 Preliminary inventory of public values ............................................................. 30 2.2.2 Value clusters in public administration ............................................................ 33 2.2.3 Making sense of public value in context .......................................................... 35 2.3 Public values implicated in e-government research ............................................... 35 2.4 Inventory of e-government public values in procedural oriented agencies ....... 40 2.5 Conclusions .................................................................................................................. 46 3 Chapter Three: Theorising public values and technology innovation through critical discourse analysis ..................................................................................... 47 3.1 Discourse and organising: the case for Critical Discourse Analysis .................... 48 3.1.1 Critical discourse analysis: introduction and assumptions ............................ 49 3.1.2 Text, discourse and context in CDA ................................................................ 51 3.1.3 CDA and organisational change ....................................................................... 55 3.2 Transdisciplinary work: theorising technology ....................................................... 57 3.2.1 Technology innovation and properties of technology ................................... 60 3.2.2 Bureaucracy and Technology ............................................................................ 62 3.2.3 Technology enactment and public sector studies ........................................... 64 3.3 Theorising technology innovation and public values through CDA .................. 68 3.4 Conclusions .................................................................................................................. 71 4 Chapter Four: Methodological underpinnings: case study research design . 73 4.1 Philosophical assumptions......................................................................................... 74 4.2 Strategy of inquiry: longitudinal case study and CDA ........................................... 76 4.2.1 Longitudinal case study ...................................................................................... 76 4.2.2 Reasons for the selection of the case study ..................................................... 78 4.2.3 Data collection and data sources ...................................................................... 79 4.3 Method of analysis: critical discourse analysis in practice ..................................... 82 4.3.1 From data to coding discourses ........................................................................ 82 4.3.2 CDA tools: selections of key texts, intertextuality and genre analysis ......... 85 4.3.3 A note on translation .......................................................................................... 91 4.4 On the relevance, quality and limits of my research design .................................. 92 4.4.1 On case study quality .......................................................................................... 92 - Page 5 - 4.4.2 Reflecting on CDA . 4.4.2 Reflecting on CDA ............................................................................................. TABLE OF CONTENTS 93 4.5 Conclusions .................................................................................................................. 96 5 Chapter Five: The Research Site. Building the Single Window for Foreign Trade in Mexico ................................................................................................... 97 Part 1: Mexico’s economic and political context .............................................................. 97 5.1 Mexico’s key facts ....................................................................................................... 97 5.2 The economic and political context: Mexico from 1982 to 2010 ...................... 101 5.2.1 International trade, openness to trade and economic reforms in Mexico 102 5.2.2 The public sector in Mexico: democratisation and changes in the bureaucracy since 1982 .................................................................................................. 106 5.3 Summary of Part One of the Chapter.................................................................... 110 Part 2: The Case: on technology, trade and trade regulations in Mexico ................... 111 5.4 Introduction: Foreign Trade regulations in Mexico ............................................ 111 5.4.1 Foreign trade procedures and Mexico’s regulatory activities ...................... 111 5.4.2 Mexico’s facilitation trade body: the Ministry of the Economy ................. 112 5.5 From SICEX to the Digital Single Window for Foreign Trade ........................ 113 5.5.1 Period 1: from multiple to a single foreign trade platform ......................... 115 5.5.2 Period 2: Working with SICEX (2000-2008) ................................................ 120 5.5.3 Period 3: From SICEX to the Single Window (2008-2012) ....................... 124 5.6 Conclusions of Part Two of the Chapter .............................................................. 131 5.7 Postscript .................................................................................................................... 133 6 Chapter Six: Eliciting discourses on public values and technology in Mexico 134 6.1 The nodal discourse on competitiveness and free trade ..................................... 135 6.2 Eliciting discourses on public values, technology and their trajectory .............. 138 6.3 Four clusters of public values ................................................................................. 141 6.3.1 Technical efficiency and business-like approach: the economic rationality and the productivity discourse ..................................................................................... 141 6.3.2 Legality, justice and accountability .................................................................. 149 6.3.3 Resilience and robustness ................................................................................ 163 6.3.4 (Forced) Cooperation ....................................................................................... 168 6.4 Conclusions and summary of the findings ............................................................ 175 7 Chapter Seven. Implications: the interdependent, political and dialectical relation between public values and e-government ............................................. 179 7.1 Public values and technology: from dichotomy to interdependence ................ 179 7.1.1 Multiple public values in e-government ......................................................... 182 7.1.2 Implications of public values interdependence ............................................. 186 7.2 The dynamic and political relation between public values and technology...... 188 7.2.1 Context, political imperatives and negotiations ............................................ 188 7.2.2 The type of change: continuity instead of radical transformation ............. 191 7.3 The dialectic relation between public values and technology ............................. 193 7.4 Conclusions ................................................................................................................ 195 8 Chapter Eight: Conclusions .......................................................................... 197 8.1 Main themes and overview of the thesis ............................................................... 197 8.1.1 Theoretical underpinnings: public values, e-government and critical discourse analysis ............................................................................................................ TABLE OF CONTENTS 198 8.1.2 Strategy of inquiry: longitudinal single case in light of CDA ...................... 200 8.1.3 On context and the case ................................................................................... 200 8.1.4 Findings and implications ................................................................................ 201 8.2 Research Contributions ............................................................................................ 203 8.3 Reflection on my research approach ...................................................................... 207 - Page 6 - 8.4 Future research .......................................................................................................... 209 References ........................................................................................................... 212 Appendix I: Detailed sources and significance of data collection ..................... 227 Appendix II: List of Interviews ........................................................................... 234 Appendix III: Sample of news articles ................................................................ 238 Appendix III: Data analysis ................................................................................ 240 Appendix IV: Published work and work in progress (2007-2012) ....................... 241 - Page 7 - TABLES Table 1: Objects of research and main theoretical underpinnings used ........................... 20 Table 2: Inventory of public values ........................................................................................ 31 Table 3: Three sets of core values in public management .................................................. 34 Table 4: Inventory of public values, administrative values and values of e-government 43 Table 5: Comparison of Weberian and Virtual Bureaucracies ........................................... 63 Table 6: Summary of data collections and sources .............................................................. 81 Table 7: Case study quality checks from according to Myers (2009) ................................. 92 Table 8: Methodological rigour according to Leitch and Palmer (2010) protocols for CDA. .................................................................................................................................. 95 Table 9: Mexico key facts ......................................................................................................... 99 Table 10: Mexico’s main modernisation reforms programmes by presidential administrations ............................................................................................................... 107 Table 11: Examples of broader institutional forces in the trajectory of the case .......... 144 Table 12: Quantification of simplification of procedures. Trade regulations administered by the Ministry of Economy ......................................................................................... 147 Table 13: Estimated gains in efficiency and productivity - selected trade procedures .... 148 Table 14: Change in the number of rules-Selected procedures of the ME .................... 154 Table 15: Summary of findings: four discourses on public values and technology innovation ....................................................................................................................... 178 Table 16: A dialectical relation between public values and technology ........................... 179 Table 17: Summary of discourses and associated public values ....................................... 180 - Page 8 - FIGURES FIGURES Figure 1: E-government stages and models of technological change ............................... 39 Figure 2: Dimensions of discourse and discourse analysis ................................................. 52 Figure 3: Technology Enactment Framework (Fountain 2001) ......................................... 66 Figure 4: Theorising public values and technology innovation through CDA ................ 70 Figure 5: Map of Mexico .......................................................................................................... 98 Figure 6: Mexico’s trade as % of GDP in comparison to other economies .................. 100 Figure 7: Phases of trade liberalisation in Mexico .............................................................. TABLE OF CONTENTS 103 Figure 8: Timeline of the case: from SICEX to the Mexican Single Window for Foreign Trade ................................................................................................................................ 114 Figure 9: Mexican Single Window for Foreign Trade (VUCEM): Before and after scenario ............................................................................................................................ 127 Figure 10: Screenshot of the Mexican Single Window for Foreign Trade as February 2012 .................................................................................................................................. 131 Figure 11: Excerpt from the Mexican Decree that establishes the Single Window for Foreign Trade ................................................................................................................. 136 - Page 9 - 1 I use the term ‘technology innovation’ and ‘e-government’ interchangeably throughout my dissertation to refer to the use of the internet and related ICT in government. I expand on this term later on the chapter. ABBREVIATIONS ABBREVIATIONS AMECAFE Asociación Mexicana de la Cadena Productiva del Café (Mexican Coffee Association) APEC Asia-Pacific Economic Cooperation CAAAREM Confederación de Agentes Aduanales de México (Association of Custom Agents, Mexico) CDA Critical Discourse Analysis CEPAL The United Nations Economic Commission for Latin America and the Caribbean CFC Comisión Federal de Competencia (Mexican Competition Authority) COCEX Comisión de Comercio Exterior (Foreign Trade Commission) DGCE Dirección General de Comercio Exterior (General Direction of Foreign Trade, Ministry of Economy) DOF Diario Oficial de la Federación (Official Diary of the Federation) FIEL Firma Electrónica Avanzada (Advanced Electronic Signature) GATT General Agreement on Tariffs and Trade IADB Inter-American Development Bank ICT Information and communication technologies INAH Instituto Nacional de Antropología e Historia (National Institute of Anthropology and History) INBAL Instituto Nacional de Bellas Artes (Institute for Fine Arts) INEGI Instituto Nacional de Estadística y Geografía (National Institute for Statistics and Geography) ISO International Organization for Standardisation NAFTA North American Free Trade Agreement NPM New Public Management OECD Organisation for Economic Cooperation and Development PAN Partido de Acción Nacional (National Action Party) PND Plan Nacional de Desarrollo (National Development Plan) PPP Public–private partnership PRD Partido de la Revolución Democrática (Democratic Revolutionary Party) PRI Partido Revolucionario Institucional (Institutional Revolutionary Party) PROMAP Programa de Modernización Administrativa (Modernisation programme for the Public Administration) SAGARPA Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación (Ministry of Agriculture) SALUD Secretaría de Salud (Ministry of Health) SAT Servicio de Administración Tributaria (Tax Administration Authority) SCHP Secretaría de Hacienda y Crédito Público (Ministry of Finance and Public Credit) SE/ME Secretaría de Economía (Ministry of Economy) SEDENA Secretaría de la Defensa Nacional (Ministry of Defense) SELA Latin American and Caribbean Economic System SEMARNAT Secretaría del Medio Ambiente y Recursos Naturales (Ministry of the Environment) SENER Secretaría de Energía (Ministry of Energy) SEP Secretaría de Educación Pública (Ministry of Public Education) SICEX Sistema Integral de Comercio Exterior (Integral System of Foreign Trade) SMEs Small and medium enterprises SRE Secretaría de Relaciones Exteriores (Ministry of Foreign Affairs) - Page 10 - TPA Towards a paperless administration programme TQM Total Quality Management UN United Nations US$ US dollars VUCEM Ventanilla Unica de Comercio Exterior Mexicana (Mexican Single Window for Foreign Trade) WB World Bank WEF World Economic Forum WTO World Trade Organisation - Page 11 - 1 Chapter One: Introduction Questions of public values and their effects have gained greater scholarly and social prominence in recent years. With growing budget constraints for governments and increasing income inequalities, the search for mechanisms that can deliver greater value for society is more urgent than ever. The use of internet and related information and communication technologies (ICT) in government may be one of these mechanisms. This thesis broadly addresses these concerns and seeks to explore the connections between public values and ICT over time. Government use of ICT has become of critical importance. Given the procedural nature of many government tasks and the central place that information storage, manipulation and communication occupy within the activities of public sector bureaucracies (Dunleavy et al. 2006, pp. 10–12), the use of the internet and related ICT provides an attractive strategy to reorganize tasks, routines and internal processes, as well as a low cost medium to reorganize interactions with citizens (Fountain 2001, Chadwick 2003, 2007, Chadwick and May 2003, Bhatnagar 2009, Cordella and Iannacci 2010, Cordella 2007). Arguably, ICT adoption in the public sector (known as ‘e- government’) offers new potential avenues for making government action more efficient and responsive as well as accountable to citizens. Yet, e-government is a complex undertaking; research in the field suggests there have been overly optimistic claims regarding the actual benefits that ICT have delivered in and for governments (Coursey and Norris 2008, Heeks and Bailur 2007, Yildiz 2007, Norris 2010). My research examines the connections between technology innovation1 in the remaking of public sector organisations. Early writings about e-government—from the 1990s to mid-2000s—have provided descriptive accounts of what is happening, while being less explicit in unpacking relations between processes, actors’ interactions and broader institutional contexts (Norris and Lloyd 2006, Heeks and Bailur 2007, Yildiz 2007, - Page 12 - Harris 2011). A considerable amount of empirical research has been conducted in e- government recently, but the drivers, causes and contingencies as well as a theorisation of the role of ICT in the reconstruction of public organisations remain poorly understood. In particular, despite its normative prescriptions, it is not empirically clear what sorts of public values are given priority in processes of e-government and how the linkages between public values and technology unfold over time. This thesis contributes to this last area. 1 Chapter One: Introduction It seeks to engage with the debates on the modernisation of central government services while contributing to understanding and explaining the relation between public values and technology innovation. It offers a detailed empirical case of ICT and institutional change that relates to broader public sector modernisation reforms in Mexico. More precisely, the case focuses on the processes of design and implementation of technology innovation in foreign trade regulations. The case looks at the developments towards building the Mexican Single Window for Foreign Trade (“Ventanilla Digital de Comercio Exterior”) that was officially initiated in 2008 based on previous efforts within government. The experience is grounded within a long process of economic liberalization that Mexico started during the early eighties, in which the fostering of the country’s international trade has been remarkably important for its economic model. Today, international trade represents almost 60% of the total GDP in Mexico, and giving its importance, any gains from trade, such as the facilitation of commerce, are considered crucial for improving the country’s overall welfare. I claim that the significance of addressing public values and technology innovation needs to be understood in relation to the discourses of public sector reforms that have pervaded public sector organising in the last three decades. Thus, a concern with these points—technology innovation and public values—requires looking at different bodies of knowledge to bring them into conversation with one another. I refer particularly to theoretical insights on public values emerging from public administration research, the role of discourse in organising and the sociotechnical tradition within information systems research. I argue that, by bringing these seemingly disparate bodies of work together, the complex relation between public values and technology innovation can be unfolded and better understood. - Page 13 - To present the grounds for the research, the next sections introduce the main themes and key terms that I use in the dissertation. Section 1.1 focuses on the broad context and background that motivates my research questions. That is, pervasive modernisation reforms, a growing concern with public values and the ways these have been addressed—implicitly or explicitly—in e-government research. Then, section 1.2 offers the overarching details of my research approach and conceptual framework to provide grounding for the thesis. 1 Chapter One: Introduction The goal of this section is to dive into the discursive lens that I build upon—critical discourse analysis—and its relation with the other dimensions of my study—public values and technology innovation in government. Finally, in section 1.3 I outline the structure of the thesis. 1.1 On public values, modernisation reforms and e-government Public value as a research object is subject to different understandings, meanings and views across disciplines (Bozeman 2009, Williams and Shearer 2011, de Graaf et al. 2012). While I explore these issues in detail in Chapter Two, for the moment I want to clarify how I use the term public values in this dissertation. Based on the work of Barry Bozeman, public values are understood as those providing normative consensus on the principles on which governments and policies should be based (Bozeman 2007, p. 13). In this sense, efficiency, effectiveness, fairness and equality are common examples of public values that one can find in government. In part, the reason why public values in government organising have received increasing academic attention is a response to concerns based on the spread of market fundamentalism in social life (Somers and Block 2005), and within governments in particular (Bozeman 2007, chap. 4). The latter can be best regarded as a consequence of the New Public Management (NPM) paradigm and related processes of reform in the public sector. As a means of bringing together the common grounds that this dissertation is based on, below I elaborate on the characteristics of NPM, its claimed associated values and the ways it relates to e-government research. - Page 14 - 2 On the idea of citizen as customer, chapter 6 of their book proposes a clear message from its heading: , p p p g g “Customer-Driven Government: Meeting the Needs of Customer, Not the Bureaucracy”. 3 The “Washington Consensus” was a series of policy reforms promoted for crisis-wracked developing countries by international bodies based in Washington (the International Monetary Fund (IMF), the World Bank, and the USA Treasury Department). The policies were based on the idea that “less government is better”, and it is in this sense that the programme can be broadly linked to the NPM spirit of market fundamentalism or neoliberalism. The Washington consensus was particularly powerful in the region and is perhaps less known in the European context. 1.1.1 New Public Management and the rise of the economic individualism 1.1.1 E-government programmes flourished within an already complex phenomenon: a global wave of modernisation reforms in the public sector—New Public Management, as coined by Christopher Hood in 1991 (Hood 1991, Dunsire 1995, Gruening 2001, Pollitt and Bouchaert 2004, 2011, Diefenbach 2009). In response to the crisis of welfare state economies, NPM was initiated during the 1980s in leading reforming counties such as the UK and New Zealand, reaching a global scale during the 1990s (Aucoin 1995). The advent of NPM resulted in ambitious targets: making governments more responsive, accountable, transparent and results-driven, as well as decentralized, disaggregated and efficient (Gruening 2001, Batley and Larbi 2004). Another characteristic of NPM has been the separation of politics and administration, an emphasis on business management practices and its reliance on individual rationalities and market-type mechanisms (such as outsourcing, privatization, customer orientation, incentivisation, competition and personnel management) in restructuring the operation of the public sector (Hood 1989, Gruening 2001, Batley and Larbi 2004). In a recent compilation, Christensen and Laegreid (2011, p. 4) summarize the above characteristics into four aspects of NPM: the efficiency drive; downsizing and decentralization; the search for excellence; and public service orientation. “Reinventing government” has been another common phrase associated with these modernisation reforms. Coined by the famous work of Osborne and Gaebler (1992), “reinventing American government” meant making a more efficient, cheaper and effective government by means of entrepreneurial government. The way forward, the authors suggest, was a complete change in outmoded and inefficient bureaucratic processes. Among several normative prescriptions proposed by the authors, the most salient included a clear focus on results, a decentralized authority, and a notable reduction of bureaucracy with a promoted competition attitude in which citizens are redefined as customers.2The modernisation reforms have been germane to Mexico as well. In the - Page 15 - context of Latin American countries, the “Washington Consensus” remained a powerful reformist agenda broadly based in strong market-based approaches to economic and policy life.3 Despite the enumerated characteristics, NPM still means many things (Barzelay 1999, 2001, Bozeman 2007, pp. 76–8); it has appeared as a multifaceted concept that is difficult to define in non-ambiguous terms (Lodge and Gill 2011, pp. 142-3, Salskov- Iversen et al 2000). Yet, it is possible to discern a broad agenda: the “common point... is a deep respect for the use of market discipline in governance” (Bozeman 2007, p. 76). 1.1.1 New Public Management and the rise of the economic individualism In sum, these broader discourses—and the accompanying changes in government action—provide a basis to sustain that NPM has shifted value priorities in the following way: a departure from universalism, equity, fairness and resilience toward efficiency and individualism (Hood 1991, p. 15, Pollitt 2003, p. 28, Bozeman 2007, p. 78). Its influence on global modernisation reform agendas has been argued to have effects on e- government as well, as I shall review in the next section. 1.1.2 E-government and the values of reform These global reforms spread in parallel with another important social phenomenon: the diffusion of increasingly affordable networked technologies in the modern world (Castells 2001, 2004). The common NPM motto of a government that “works better and costs less” sat easily with the rise of e-government (Mayer-Schonberger and Lazer 2007, chap. 1). The Organisation for Economic Cooperation and Development (OECD) crystallised these ideas in its first book on e-government in 2003: Reform of the public administration has been on the agendas of most OECD governments well before the advent of the term “e-government”. But e- government is an important component of today’s reform agendas because it: 1) serves as a tool for reform; 2) renews interest in public management reform; 3) - Page 16 - highlights internal inconsistencies; 4) underscores commitment to good governance objectives. (OECD 2003, p. 41) E-government can help administrations do their job better by reinforcing good governance objectives and administrative reforms are necessary if e-government is to be successful. E-government and reform are therefore mutually reinforcing. (OECD 2003, p. 25) Arguably, most research on e-government is understood as a tool to reinforce the objectives of public administration reforms in many countries (OECD 2003, 2005), although the connection between broader institutional reforms and e-government is less explicit. E-government has been studied from many perspectives and viewpoints, from means to achieve something else (i.e. administrative reforms) or as an end in itself (Yildiz 2007). That is why e-government has been described as a catchall tag for the practice of using ICT in the public sector(Mayer-Schonberger and Lazer 2007, p. 5). The most common view of e-government is the generalized use of new ICT to provide better public services, to improve public administration performance and to broaden citizen participation and democratic processes (Fountain 2001, Heeks 2002, Gil-Garcia and Pardo 2005, Cordella 2007, Yildiz 2007, Bhatnagar 2009). Arguably, most research on e-government is understood as a tool to reinforce the objectives of public administration reforms in many countries (OECD 2003, 2005), although the connection between broader institutional reforms and e-government is less explicit. E-government has been studied from many perspectives and viewpoints, from means to achieve something else (i.e. administrative reforms) or as an end in itself (Yildiz 2007). That is why e-government has been described as a catchall tag for the practice of using ICT in the public sector(Mayer-Schonberger and Lazer 2007, p. 5). 1.1.2 E-government and the values of reform The most common view of e-government is the generalized use of new ICT to provide better public services, to improve public administration performance and to broaden citizen participation and democratic processes (Fountain 2001, Heeks 2002, Gil-Garcia and Pardo 2005, Cordella 2007, Yildiz 2007, Bhatnagar 2009). Thus, from a normative perspective, there have been many claims that e-government will make citizen-government transactions easier, cheaper and faster. Implicitly, the e- government vision embodies public values that are claimed to be similar to those of NPM: a business-like approach, the push for productivity and timeliness, responsiveness and a customer focus based on citizen’s individual choices (Fountain 2001, Curthoys and Crabtree 2003, OECD 2003, p. 25, West 2005a, Cordella 2007). However, claims about the universality of these public values are difficult to sustain. First, empirical research in e-government has shown many shapes, forms and evolutions: national and local appropriations of e-government programmes exhibit an important degree of variance that suggest (if not demonstrate) that context matters (see for example Fountain 2007, Madon et al. 2007, Ahn and Bretschneider 2011, Lee et al. 2011). The definition of public values itself suggests that they should be assessed in their social context. Second, findings from longitudinal case studies suggest that models are not simply linear and that political, institutional and organisational forces mediate the enactment of goals, values and political agendas (Fountain 2001, 2007, Dunleavy et al 2006, Bloomfield and Hayes 2009). Third, while we have gained important insights on - Page 17 - the relation between certain public values and e-government (i.e. on discretion, accountability, productivity), there is a dearth of empirical research that broadly study the types of public values associated with e-government programmes over time. Thus, if e-government is assumed to be part of broader plans of reform, what is the connection between technology innovation and the values of reform? Despite the importance of discourses on modernisation reform and concerns with their effects and value shifts, very few authors have provided detailed empirical accounts of technology innovation in the public sector and its linkages with broader political and ideological agendas (but see McNulty and Ferlie 2004, Bloomfield and Hayes 2009, and Harris 2011 for notable exceptions). Moreover, the particular case of mapping public values and unfolding their relation with technology innovation has not been empirically assessed. 1.1.2 E-government and the values of reform In other words, given a context of reforms that privileges certain clusters of public values, the evidence on which ones are actually followed in different contexts remains elusive as well as the implications on different aspects of government design and action. This is precisely what I attempt to explore in my research. 1.2 A discursive lens to study public values and e-government In line with the above observations, this thesis is concerned with understanding and explaining two related issues, comprised in the main research questions: What sorts of public values underlie a central e-government initiative in Mexico? How does the relation between public values and e-government unfold over time? What can we learn from Mexico? My study begins with the premise that there is a relational link between public values and technology innovation; that is, public values shape, and are shaped by, technology. As mentioned, I work with a narrow definition of public values provided in the work of Bozeman (2007). However, the definition does not eliminate the complexity of studying public values empirically (see for example Rutgers 2008). Public values—as values in general—are elusive and intangible and researchers need to find ways to sort them out empirically. To deal with these issues, I adopt a discursive lens—in particular, critical discourse analysis—to address my research questions, which I apply to the longitudinal case in Mexico. I deal with these issues in depth in Chapter Three, although I briefly outline them here to provide the groundwork for my theoretical lens. - Page 18 - - Page 18 - Critical discourse analysis (hereafter CDA) is a theoretical approach to study how discourse figures within processes of social change (Fairclough 2003, p. 205, 2010). My elaboration of CDA draws on a particular stream developed by the work of the sociolinguist Norman Fairclough and colleagues (Chouliaraki and Fairclough 1999, Fairclough 2003, 2010). CDA has become a well-established framework to examine the use of language in the text and talk that underlie the dynamics of organising. This, in part, has been a consequence of a more general linguistic turn in social science research (Van Dijk 1997, Alvesson and Kärreman 2000a, Vaara 2010). Despite its popularity in organisation and management studies, this version of CDA remains largely unknown within IS research (but see Thompson 2004). I claim that CDA can offer useful insights in addressing my research questions and conducting my empirical work. CDA emphasises the importance of a close and detailed analysis of texts and at the same time, the integration of the analysis into broader social processes (Fairclough 2005, p. 924). From this perspective, my approach to studying discourse in organising is therefore three-dimensional: it links key government texts to broader discourses on public values, locating them within a particular historical and social context. 1.2 A discursive lens to study public values and e-government Discourse analysis is, then, suitable to consider context seriously, and to elaborate on the necessary contextual features of public values, as provided in the definition that I use in my dissertation. In addition, a consistent theorisation of technology is provided, based on a sociotechnical perspective within e-government studies and organisation studies in general (Fountain 2001, Kallinikos 2006a, Cordella and Iannacci 2010, Bloomfield and Hayes 2009, Danziger et al. 1982, Avgerou 2002), which is compatible with my overall premise: that technology shapes and is shaped by public values. On this basis, my work aims at extending the sociotechnical tradition within information systems in the public sector while accounting for public values. A summary of the three different research streams that I bring together in my dissertation is provided in table 1. - Page 19 - Object of research Conceptual concerns Theoretical underpinnings and main authors Public values What are public values? How are they defined? Where does one find them? Normative public values Bozeman 2007, Beck Jørgensen and Bozeman 2007, Hood 1991 E-government (technology innovation in the public sector) What changes do ICT bring in public sector organising? Sociotechnical perspectives in e- government Fountain 2001, Danziger et al 1982, Bloomfield and Hayes 2009 Discourse in organising How does language influence organising? How are discourses produced, interpreted and materialised in organisations? What power struggles emerge from dominating discourses? Critical Discourse Analysis Fairclough 2003, 2005, 2010, Chouliaraki and Fairclough 1999, Vaara 2010, Phillips et al 2004 Table 1: Objects of research and main theoretical underpinnings used Critical Discourse Analysis Fairclough 2003, 2005, 2010, Chouliaraki and Fairclough 1999, Vaara 2010, Phillips et al 2004 able 1: Objects of research and main theoretical underpinnings used Table 1: Objects of research and main theoretical underpinnings used I argue that bringing together such wide range of theoretical sources is possible given the flexible and open character of CDA, which advocates for a transdisciplinary approach; that is, to work in dialogue with different disciplines to theorise particular objects of research (Fairclough 2010, chap. 9). I argue that CDA is a valuable aid to study how claims about public values and technology innovation draw from broader institutional discourses and national priorities, and how they are made to work at specific organisational levels and micro productions of discourses (texts and talk). There are further terms that need clarification. 4 Some scholars tend to associate the idea of organizing to Karl Weick’s work and a particular lens to study organising processes. This is not the way I understand it here. 1.2 A discursive lens to study public values and e-government In my thesis, I talk about organising to emphasise the dynamic nature of the processes underlying public values and e- government as well as discursive practices. This builds not only on the dynamic and emergent nature of ICT that I use in my theorisation of technology (Orlikowski and Iacono 2001, pp. 131-132) but also on the dynamic nature of discourse in organisational processes (Fairclough 2005).4 In addition, I use the term ‘technology innovation’ and ‘e- government’ interchangeably throughout my dissertation to refer to the use of the internet and related ICT in government. In turn, I define ICT as those technologies for the - Page 20 - processing, storage, and transmission of digital material, which consist of ensembles of hardware and software with distinctive feature sets, that allow physical storage and logical representations of different forms of data (Mutch 2010, p. 507). 1.3 Conclusion and Structure of the Thesis Building on public values concerns and the questions that I state in this thesis, the goal of my research is to bring different bodies of knowledge into conversation in order to analyse and explain a set of related issues: which public values are dominant in a particular technologically enabled programme in Mexico, how this happens and what implications can be addressed from the case. The remainder of the dissertation is organised across seven additional chapters. Chapter Two, as anticipated, elaborates on the issues that I outlined briefly in this introduction. That is, it provides the theoretical underpinnings of public values research, its different streams and ways to study them empirically. The second part of the chapter develops a focused literature review on the types of public values that can be found in e-government research. Overall, Chapter Two focuses on the key areas of literature that provide the background for the research inquiry—public values and the current state of the art in e-government research. Chapter Three presents the research framework. The aim of this chapter is not only to provide the theoretical foundations of the version of critical discourse analysis that I use in my dissertation, but also to elaborate on the ways it accounts for technology and public values. To do so, the chapter develops an understanding of the relation of technology innovation in public sector administration that, in conjunction with public values research, is incorporated in the CDA framework in a transdisciplinary manner. I place emphasis on theory that can help to describe, analyse and explain the content of the relation between public values and technology innovation as well as its dynamics over time and in a given context. The particular choice of a discursive lens to study the relation between public values and technology implicates a series of methodological decisions as well. In Chapter Four I provide details on the strategy of inquiry that I followed to conduct the - Page 21 - longitudinal case of a technology-enabled platform for the administration of foreign trade regulations in Mexico. In particular, the chapter elaborates on the philosophical assumptions underlying my theoretical choices, the data sources and data collection as well as the specific CDA tools that I applied to analyse the case. I also place emphasis on explaining the reflexive character of the research methodology that I follow. 5 Following Bresser-Pereira, by ‘neo-liberal’ this dissertation means simply market-oriented reforms, in which competition and individual choice play a central role; as such, it allows for different types of reforms, ranging from the ‘ultra-liberal’ ones as those implemented in New Zealand since the 1980s, to the ‘social-democratic’ reforms of Brazil (Bresser-Pereira 2004, p. 120). 1.3 Conclusion and Structure of the Thesis Taken together, Chapters Three and Four lay the foundations to study the longitudinal case that I present later on. Chapter Five elaborates on the research site and the case and is divided in two parts. Part One offers a detailed account of the economic, political and administrative reforms that Mexico has carried out in the last 25 years. I pay particular attention to economic reforms on trade and other salient contextual factors that are helpful to pin down the implications of global discourses of reform. The significance of the case relies on the fact that is congruently framed within the neo-liberal spirit of modernisation reforms that I briefly commented on at the beginning of this introduction.5Part Two of Chapter Five documents the process of constructing the Single Window for Foreign Trade (2008-2012), building on the previous technological developments that the government had conducted since 1995. The story is presented in three different phases, and it provides technical details as well as important surrounding factors in light of what I elaborated in Part One. On June 2012, the Single Window started operating, making digital transactions mandatory. To account for these latest developments, the chapter includes a postscript written in September 2012. In Chapter Six, the case study is analysed using critical discourse analysis. Focusing on the trajectory of key texts of the Single Window, the chapter unveils a series of clusters of public values and technology, and elaborates on their structural characteristics. Based on extensive empirical material from my fieldwork, I offer an in-depth elaboration of each of the discourses on public values and technology innovation in a systematic way: I present the common discursive features of each cluster, I provide evidence on where they emanate from, and show the ways—and the extent to which—these discourses are - Page 22 - consumed and materialised in technology. The chapter ends with a table that summarises the main findings. Chapter Seven focuses the reader at a different level in order to explore the implications raised by the analysis. In terms of CDA, I claim that while broader discourses on public values do have an influence on technology design and implementation, technology internalises and is internalised by discourses on public values. Building on the findings from the case, in this chapter I derive more general implications for an increased understanding of the relation between public values and technology. 2 Chapter Two: OnPublic Values and E-government 2 Chapter Two: OnPublic Values and E-government There is no more important topic in public administration and policy than public values. (...)—the topic is so fundamental as to be unmanageable. (...) [I]f researchers can advance, even incrementally, the study of public values beyond its current ambiguous and unbounded status, then those advances could serve many different theory developments and even practical purposes. Beck Jørgensen and Bozeman 2007, p. 355 There is no more important topic in public administration and policy than public values. (...)—the topic is so fundamental as to be unmanageable. (...) [I]f researchers can advance, even incrementally, the study of public values beyond its current ambiguous and unbounded status, then those advances could serve many different theory developments and even practical purposes. Beck Jørgensen and Bozeman 2007, p. 355 Beck Jørgensen and Bozeman’s quote illustrate well one of the aims of this dissertation: contributing not only to identifying dominant public values in a technologically induced public sector reforms but also to assess how the relation between public values and technology unfolds over time. The chapter builds substantially on public administration literature, as it is the field that compiles more generally the debates and developments on public values. It also draws substantively on literature on e-government to present the strengths and some shortcomings, specifically, the lack of an extensive, longitudinal study of discourses on a wide-range public values and technology in a specific context. In this chapter I seek to convince the reader that studying the linkages between public values and technology offers a fruitful niche for research in e-government studies. In addition, I claim that while e-government research has advanced knowledge in many ways, it usually takes the public values of reform for granted, or studies them with limited attention to potential value conflicts from a holistic perspective. The evidence that I offer is based on a substantial and focused review on these issues. The chapter is divided in five sections. Section 2.1 expands on the notions and different streams of work associated to public value research. From there and taking my research concerns into account, section 2.2 develops on normative public values as a means to start with a list of values that can be mapped in the literature and serve as the starting point of empirical work. 1.3 Conclusion and Structure of the Thesis I particularly elaborate on the notions of interdependence, political and dialectical character of the relation between my objects of research. The final chapter provides concluding remarks. Chapter Eight presents an overview of the main themes covered in the thesis, its findings and contributions. In appreciation of the implications of my research journey using CDA, I include a reflection on the research approach, lessons learned and limitations. The conclusions close with a number of ideas for follow-on research that arose during the course of my research project. - Page 23 - 2.1 The rise of public value as counterbalance of market-driven modernisation discourses As I anticipated in the introduction, in part, the growing interest on public value research appeared as a response to concerns about the over-dominance of the economic individualism and neoliberal ideas in public life and government organising (Bozeman 2007). Globally, the influence of market logics into public sector reforms was acknowledged in scholarly debates as the New Public Management (NPM). Despite its ambiguity (Barzelay 1999, 2000, 2001, Bozeman 2007, pp. 76–8) and multiple meanings (Salskov-Iversen et al. 2000, Lodge and Gill 2011, pp. 142–3), the advent of NPM brought in a new global discourse which implied “a break with a century-old tradition of distinguishing sharply between the values and practices of the public and private sector” (Salskov-Iversen et al. 2000, p. 184). Reforms that emphasises market types of governance, privatisation and contracting out make increasingly important to consider classic public values too (Beck Jørgensen 1999, p. 580). This has opened the recent debate towards alternative visions of public value in government. 2 Chapter Two: OnPublic Values and E-government I therefore introduce two works: an inventory of public values distilled from scholarly literature in public management, and the clusters of administrative values in public administration. The section 2.2 concludes with the importance to account for context when studying public values. In section 1.3, I expand - Page 24 - on the general ways e-government research has approached public values of modernisation reforms. I review critically the general trends of the literature on the matter. Then, section 2.4 combines the list of public values from public management research and links it to specific studies produced in e-government that can be related to different categories of values. The last section finishes with concluding remarks. 2.1.1 Public values: three streams of research Of course, that notions such as public interest or public values are not new is a non- controversial statement. For example, Easton suggested in 1965 that “public policies are the means through which politics allocate values” (cited in de Graaf and van der Wal 2010, p. 624). Bozeman (2007, p. 1), in turn, states that notions of ‘public interest’ or ‘common good’ have already been discussed in Aristotle’s Politics. Despite not being new, a growing body of literature on public value has gained place in public management and political science debates that has suggested a new positioning, leading to different stream with diverse understandings and focus (Beck Jørgensen 1999, Davis - Page 25 - - Page 25 - and West 2008, West and Davis 2011, Williams and Shearer 2011). I shall review as follows, the main works that have inspired my research and my concern with the acclaimed shift in values that the NPM has ‘imposed’. One comes from an on-going debate within the field of organisation studies discussing several aspects of the institutions and values of modern society, in which bureaucracy and post-bureaucracy debates are at centre (Courpasson and Reed 2004). The particular debate that is relevant for the research I propose here relates to the ethos of bureaucracy, and to the extent that the focus on efficiency and business-like values proposed by the advocates of the “Entrepreneurial Government” (and NPM) compromises the distinctive role of democratic governments as custodian of the constitutional procedures, equality, fairness and so on. Key contributors in this area include the work of du Gay (2000, 2003, 2004, 2005), Willmott (2011), Arellano-Gault (2010), and Olsen (2006), among others. Although this debate is not directly ‘speaking’ about public values, it has important implications for my research, and I will deal more in depth with some of these arguments in chapter 3. In particular, an important theoretical account from this perspective —and for the discussion of e-government— relies on the inadequacy of referring to NPM and bureaucracy as ‘absolute opposites’ as it has been assumed elsewhere (i.e. Persson and Goldkul 2010). A second stream that has gained a dominant place in the public management literature is around the seminal book of Mark Moore, Creating Public Value. Moore’s work, initially published in 1995 in the USA, and updated recently, offers a (re)freshing perspective on the role of the state (Moore 1995, Benington and Moore 2011a). 2.1.1 Public values: three streams of research Moore’s initial piece (1995) was mainly concerned with creating a normative theory of what public managers should do to create public value given the particular circumstances they are immersed in. The concept “public value” relates to that of a public philosophy of governance that a society’s collective aspirations determine where public value lies (Moore 1995, p. 52, Barzelay 2000, p. 246). In this sense, public value proponents share the view of NPM in “looking for a more responsive, more innovative, more effective government” (Benington and Moore 2011a, p. 10), but unlike the NPM, they place the focus on the collective aspirations rather than the economic individualism of the neoliberal agenda. - Page 26 - In general terms, within this second stream, public value covers three main issues: what the role of government is in society, what roles public managers have to play within governments, and what techniques and practices public managers need in order to develop their roles. A growing literature on public value includes several positions, disperse understandings and fierce critics—i.e. public value as a new paradigm in public administration, as a heuristic device, or as an underlying theory for ‘networked community governance’ (Stoker 2006, O’Flynn 2007, Rhodes 2007, Alford and O’Flynn 2009, Rhodes and Wanna 2009, Benington 2011, West and Davis 2011). The fact that some of the ideas are still quite open (if not ambiguous) the current debate offers an opportunity to enhance its on-going developments in theoretical terms. The idea of public value in Moore’s work, therefore, is more about managerial aspects of the public sector than about providing a stable definition. An implied corollary that is relevant for my work relies on the importance of widening the scope to democratic values (political, social, and economic) in public sector problems. Moore’s work has recently appeared in e-government research, showing some cross-fertilisations and potential avenues for future research (i.e. Cordella and Willcocks 2010, Grimsley and Meehan 2007, Center for Technology in Government 2011, Cordella and Bonina 2012). The third stream of research refers to public values (in plural) and public interest. This research builds on the work of Bozeman (1987) and colleagues on publicness, and more precisely, on its normative part.6 A useful conceptualisation that emerges from this work is a stable definition of public values—that I adopt in my research. 6 Normative publicness is defined as ‘‘an approach to values analysis assuming that a knowledge of the political and economic authority of institutions and policies is a prerequisite of understanding the potential of institutions and policies to achieve public values and to work toward public interest ideals’’ (Bozeman 2007, 18). 7 The framework combines developments of two scholarly traditions within publicness (normative and empirical) and offers a heuristic device for strategic management of organizations—whether public, private or non-profit. It bases its analysis on public value mapping, an effort to elicit different public values in context. 2.1.1 Public values: three streams of research In his book entitled Public Value and Public Interest, Bozeman defines public values as follows (2007, p. 132, emphasis added): A society’s ‘public values’ are those providing normative consensus about (a) the rights, benefits, and prerogatives to which citizens should (and should not) be entitled; (b) the obligations of citizens to society, the state, and one another; and (c) the principles on which governments and policies should be based. 6 Normative publicness is defined as ‘‘an approach to values analysis assuming that a knowledge of the political and economic authority of institutions and policies is a prerequisite of understanding the potential of institutions and policies to achieve public values and to work toward public interest ideals’’ (Bozeman 2007, 18). - Page 27 - - Page 27 - As I anticipated in the introduction, for the purpose of my work, I rely on the third part of the definition. From this view, public values can be considered as those basic building blocks of the public sector (Beck Jørgensen 1999, p. 581). Also, because my analysis is focused from the organisational perspective of government; that is, assessing which public values are actually pursued in the public sector, how specific values emerge and how they ‘travel’ over time. Then, considering public values in this way enables me to trace public values analytically and empirically. Within this third stream of work, public values have been studied as clusters or inventories in both public and private organisations (Beck Jørgensen and Bozeman 2007, Bozeman 2007, van der Wal et al. 2008). This stream has also informed the latest developments within publicness theory and the integrative publicness framework for public management strategy and performance(Bozeman and Moulton 2011, Walker and Bozeman 2011).7 Although there have been some recent cross-dialogues with the work of Moore, this line of research seems to be have emerged and developed decoupled from it. As per e-government research, this stream of work does not seem to have been picked up yet. These three streams, although quite disconnected in the literature, have the potential to offer a counterbalancing argument to the claim of value shifts in public sector reforms and their connections with technology induced programmes. Let me add why studying public values is important in government affairs, which e-government is implicated. 8 I have adapted these paragraphs slightly; also, in its original the fourth idea appeared in third place. See Bozeman (2007, p. 64). 2.1.2 Why public values are important in public sector debates Following Bozeman (2007, p.64), the rationale for incorporating public values into debates of public management—in which processes of e-government are certainly part of—includes four justifications:8 8 I have adapted these paragraphs slightly; also, in its original the fourth idea appeared in third place. See Bozeman (2007, p. 64). - Page 28 - 1. Public value is something more than collective private value; 2. market-based expressions of public value are limiting; In line with the first three ideas, Bozeman offers a succinct discussion on the limits of economic theory (market failure and related economic efficiency approaches) to choosing sector roles (Bozeman 2007, p.63). “Often economic values are literally irrelevant to public choices. In many instances markets are highly competitive, pricing is efficient, and enormous public problems remain. (...) When considering public value, market failure is often simply not the point. Nevertheless, market-based reasoning often dominates public policy deliberation” (Bozeman 2007, p.65, emphasis in the original). These ideas are echoed in the work of Benington and Moore (2011a, p.10), who differentiate themselves from the economic individualism by suggesting that “the proper arbiter of public value is society as a whole acting as best as it can through the imperfect processes of representative government” (emphasis added). In turn, du Gay reminds us (2004, p.54) that, “because a system of representative government requires officials to act as custodians of the constitutional procedures and values it embodies, it cannot frame the role of public administrators simply in terms of economy, performance, audit, responsiveness and delivery.” Summing up, despite the importance and growing interest in public value research, analytical and empirical analysis of public values remain a challenging area. It is been claimed that current debates on public value rarely focus on identifying a set of common values or in discussing their meanings (Rutgers 2008, Bozeman 2007, Beck Jørgensen and Bozeman 2007). Recall that my research is concerned with distilling and assessing public values in a given context. Moore’s work (Moore 1995, Benington and Moore 2011a) provides useful insights on effective public management to achieve a given public values. However, their work is somehow agnostic about public values content and more focused on the need to improve government operations (Benington and Moore 2011a, p. 17). 2.2 Normative public values: approaches from the literature 2.2 2.1.2 Why public values are important in public sector debates For the purposes of my research, starting with normative lists of public values is useful as to provide insights on what types of goals governments follow when designing and implementing and e-government platform, and why so is. I do so in the following sections. - Page 29 - 2.2.1 Preliminary inventory of public values Eliciting a given society’s public values cannot be universal or univocal. The indeterminate character of general consensus or aggregated preferences is well known in the literature of economics and political science (Arrow 1963). This is not to say that a set of values of reform cannot be approximated. As I anticipated as part of the stream of public values (in plural), there have been scholarly attempts to distilling and assessing public values based on a variety of approaches, such as literature reviews on what other scholars have said on the matter (Beck Jørgensen and Bozeman 2007), public opinion polls (work on cultural values from Tuffs University), case studies (i.e. Jørgensen and Bozeman 2002, Feeney and Bozeman 2007) and surveys to public servants, businesses and the citizenry (Van der Wal and Huberts 2008, van der Wal et al. 2008, Berman and West 2012). The following list is based on the work of Beck Jørgensen and Bozeman (2007), who provide a preliminary inventory of public values based on an extensive review of the literature in the topic. The authors review 230 studies in the literature of public administration, organisation theory and political science, published in the USA, UK and Scandinavia, during the period 1990-2003. Their survey of the literature resulted in 72 registered values, categorised in seven ‘constellations’ based on different types of relation between them. The table below depicts the list of public values and their categorisation into these constellations of values. As the authors warned (2007, p.357), the main disadvantage of their approach is that “values are quoted out of context”. However, the authors offer a useful interim approach that illustrates the possibility of relating different clusters of values, which can serve as a guideline for further empirical analysis (p. 357). In particular, the inventory provides a very useful attempt to map a galaxy of public values that can later be analysed using robust methods for text and content analysis (or critical discourse - Page 30 - - Page 30 - analysis, as I shall discuss in the next chapter). This is, therefore, the main reason why their approach is useful for my research purposes. 2.2.1 Preliminary inventory of public values Value Category Value Set Public sector’s contribution to society (general social values) Common good Public interest Social cohesion Altruism Sustainability Regime of dignity Transformation of interests to decisions (how opinions are channelled) Majority rule Democracy Will of people Collective choice User democracy Local governance Citizen involvement Protection of minorities Relationship between public administrators and politicians Political loyalty Accountability Responsiveness Relationship between public administrators and their environment Openness-secrecy Responsiveness Listening to public opinion Advocacy-neutrality Compromise Balancing of interests Competitiveness- cooperativeness Stakeholder or shareholder value Intraorganisational aspects of public administration (organisational values) Robustness Adaptability Stability Reliability Timeliness Innovation Enthusiasm Risk readiness Productivity Effectiveness Parsimony Business-like approach Self-development of employees Good working environment Behaviour of public-sector employees Accountability Professionalism Honesty Moral standards Ethical consciousness Integrity Relationship between public administration and the citizens Legality Protection of rights of the individual Equal treatment Rule of law Justice Equity Reasonableness Fairness Professionalism Dialogue Responsiveness User democracy Citizen involvement Citizen’s self-development User orientation Timeliness Friendliness Table 2: Inventory of public values Source: adapted from Beck Jørgensen and Bozeman (2007 p 360 361) - Page 31 - Beck Jørgensen and Bozeman (2007) offer a further conceptual device and suggest that it is possible to relate values by proximity (how close a value is to another), hierarchy (a relative primacy of values), and causality (analysis of means to an end). For example, robustness may be closely related to values such as adaptability, and reliability, but also to legality, responsiveness, and the rule of law. Whilst this further classification can be subjective and controversial, the authors provide useful examples on how these relations may be analytically grasped. A particular useful distinction is to refer to prime (ends in itself) and instrumental (conditions that affect) values. The distinction is of importance because it leads to a particular type of analysis: instrumental values can be portrayed as causal hypothesis that are subject to empirical inquiry (p.376); in addition, instrumental values are means to something else, the prime values (i.e. an agency mission). This causal inquiry is particularly useful to unpack complex types of relations among values and technology in my research, and I will be drawing on them. 9 From the list, it seems reasonable to suspect that, even when more traditional public values associated with the bureaucratic ethos may have been marginalised, they are still quite prominent in the public sector. 2.2.1 Preliminary inventory of public values The underlying assumption that the authors pose—and that I share in my research—suggest that there are no inherent prime public values; that is, efficiency may be considered as the ultimate (prime) public value in a particular situation, but this does not make the value as universal, inherent desired end. Overall, In addition, my assumption is that “complete consensus about public values is not required to make head-way toward public interest considerations” (Bozeman 2007, p.143, italics in the original). Hence, a preliminary inventory serves the purpose of identifying public values in several domains of public administration life. Finally, a note of caution needs to be made in relation to this inventory of public values and the scope of my study. The list has been built from literature in public administration and political science covering Anglo-Saxon and Scandinavian countries, which may be biased to value notions that may be less germane to Mexico. In addition, the review covers a period when the NPM trend was ‘dominant’ and the results may have benefited a bias against more traditional values in public administration.9 Both reservations are important but do not constitute ‘substantive’ limitations to conduct the empirical research. That is, from a normative point of view, the list is indicative rather 9 From the list, it seems reasonable to suspect that, even when more traditional public values associated with the bureaucratic ethos may have been marginalised, they are still quite prominent in the public sector. - Page 32 - than exhaustive, and subject to empirical scrutiny. I now turn the attention to another attempt that can be combined, based on a seminal work in public management. 2.2.2 Value clusters in public administration Back in 1991, Christopher Hood coined the term New Public Management in his widely cited article of 1991, entitled “A public Management for all seasons”. A main point was to reflect on the challenges of a new form of public administration that was initiated by the drastic reforms in the UK and the USA since the early 1980s. On a much less explore part of his core argument, he presents an argumentation device—developed more extensively on Hood and Jackson (1991)—as the right tool to argue about the NPM and their potential effects. Hood focused on the notion of administrative values, which targets conventional ideas about ‘good administration’ instead of broader ideals on the role of the overall role of the state (Hood 1991). Hood identifies three families of values: the sigma values, which assign priority to lean government with a focus on economy and parsimony; the theta values, which relates to honesty and fairness; and the lambda values, those related to integrity, flexibility and security. Sigma-type values concentrate on matching resources to defined tasks, avoiding waste, and defining measurable outputs as the currency for its success and failure (i.e. time and money, costs of resources). Theta-type values embody the pursuit of 'rectitude', in which trust, legitimacy and political entitlements are the currency of success. The last group of values relates to resilience and reliability, and the capacity to avoid system failures, breakdowns and collapses. Hood (1991, p.15) asserts that “NPM can be understood as primarily an expression of sigma-type values” as its elements embodied primarily different aspects of efficiency and effectiveness, while equity, fairness or communal values are ‘side effects’ (Christensen and Laegreid 2011, p. 11). 2.2.3 Making sense of public value in context From the previous sections, I suggested that public values shall make sense in context. The very definition of a society’s consensus suggests so. In other words, public values cannot be defined universally or in absolute terms (Alford and Hughes 2008), a view shared by most authors of the recent volume on the subject (Benington and Moore 2011b). “The problem is not finding public values but understanding them in some analytically useful form” (Bozeman 2007, p. 142). I shall deal with these issues more in depth in Chapter Three—that is, how to study public values and e-government analytically and empirically. For the moment, is it useful to bring to attention an idea posed by Willmott (2010); the author suggest that questions of values shall be studied as part of ethico-political complexes: the meanings attributed to value should be recognised as articulations of a particular, culturally meaningful context, in which social practices take place. “Particular notions of ‘value’ become meaningful, achieve ‘grip’ and exert their effects though processes of contestation.” (Willmott 2010, p. 520). This note reinforces why studying public values in context is paramount to conduct my empirical research. As a means of summing up my discussion on public values, I argue that the preliminary list produced by Beck Jørgensen and Bozeman (2007) combined with the work of Hood (1991) provide a good starting point, by no means exhaustive, complete or univocal. In other words, these approaches are useful to my research because they provide the initial basis for studying complex value relations, their prominence and potential conflicts. I shall use them accordingly to trace notions of public values in the literature of e- government. 2.2.2 Value clusters in public administration - Page 33 - Sigma-type values KEEP IT LEAN PURPOSE Theta-type values KEEP IT HONEST AND FAIR Lambda-type values KEEP IT ROBUST AND RESILIENT STANDARD OF SUCCESS Frugality (matching of resources to tasks for given goals) Rectitude (achievement of fairness, mutuality, the proper discharge of duties) Resilience (achievement of reliability, adaptivity, robustness) STANDARD OF FAILURE Waste (muddle, confusion, inefficiency) Malversation (unfairness, bias, abuse of office) Catastrophe (risk, breakdown, collapse) CURRENCY OF SUCCESS AND FAILURE Money and time (resource costs of producers and consumers) Trust and entitlements (consent, legitimacy, due process, political entitlements) Security and survival (confidence, life and limb) CONTROL EMPHASIS Output Process Input/Process SLACK Low Medium High GOALS Fixed/Single Incompatible 'Double bind' Emergent/Multiple INFORMATION Coasted, segmented (commercial assets) Structured Rich exchange, collective asset COUPLING Tight Medium Loose Table 3: Three sets of core values in public management Source: Hood (1991, p. 11) Table 3: Three sets of core values in public management Source: Hood (1991, p. 11) The work of Hood (1991) and Hood and Jackson (1991) on administrative values and public administration is helpful to my research in two specific ways. First, it offers a simple categorisation that can help to match (and map) some of the value categories that Beck-Jørgensen and Bozeman present in their inventory. Because I focus particularly on the administrative design and the machine of government, their concepts can travel well. In addition, Hood (1991) states that it may be possible to align these clusters in a given administrative design, although this entails contestation. In particular, Hood suggests that, while it may be possible to align values pertaining to different clusters, the most probably scenario would entail privileging only one or two of the clusters at the expense to the others. Yet, this is a matter of empirical scrutiny that has so far been scant in the literature (but sees Lodge and Gill 2011 for a remarkable exception). Therefore, the empirical proof about the fact that NPM’s efficiency drive will come necessarily at the expense of fairness, honesty or effectiveness remains elusive (Hood 1991, p. 15). - Page 34 - 2.3 Public values implicated in e-government research E-government research has not focused directly on studying the linkages between public values and technology, and empirical studies that aim at looking at broader picture in this area are needed. In this section, I review the most influential works on e- government and I elaborate on those public values that seem to appear in the dominant research. - Page 35 - E-government has been studied from many perspectives and viewpoints, and there is a growing—if not immense—literature on the matter. E-government seemed to have emerged as a field in itself (Heeks and Bailur 2007), with a growing number of specialised journals in the topic. At the same time, there seem to be a disconnection between public administration literature and political science, which usually marginalised the role of the internet and ICT within public sector reforms (Dunleavy et al. 2006, Dunleavy and Margetts 2010). As far as public values are concerned, usually they appear as given and as part of the values of reform. For example, Heeks (2002, p. 16) explains that “reinventing government is a continuation of existing new public management reforms” (see also Cordella 2007). Accordingly, from this perspective it can be argued that the role of information systems in government is to support a set of values given in the Reinventing Government Agenda (p. 14). In line with the spirit of the NPM drivers discussed earlier on, the Agenda of reform comprises five main components: increased efficiency, decentralisation, increased accountability, improved resource management and marketisation. Although the author does not refer to values of government directly, the following ones can be identified: - Efficiency (improving the output input ratio, mainly in economic terms, cost savings); - More flexible and responsive decision making; more efficient and effective (as a consequence of decentralisation); - Accountability, productivity and performance of staff and democratisation (increased involvement of citizens in decisions and actions); - Effectiveness, parsimony and business-like approach (as part of improved resource management); - User (customer) orientation (as part of the marketisation component of reforms). Heeks and Davies (2002)reinforce the point that “public sector organisations undertaking their initial computerisations will typically see IT as a means of increasing efficiency; automating the human effort (...) thereby attempting to cut staff costs”. 2.3 Public values implicated in e-government research Heeks and Davies (2002)reinforce the point that “public sector organisations undertaking their initial computerisations will typically see IT as a means of increasing efficiency; automating the human effort (...) thereby attempting to cut staff costs”. - Page 36 - Countries with more advanced computerised public sector invest in e-government projects to pursue efficiency but also to improve the delivery of public services as means of becoming more customer-responsive (p. 24). Adding to these two clusters of values (productivity and responsiveness), Ciborra (2005) reminds that, in the context of developing countries, e-government is introduced to achieve better accountability and transparency. Both are the characteristics of good governance, and “the conditio sine qua non for the rich states and international agencies to supply aid to (...) economically less developed countries” (p. 261). Bellamy and Taylor (1998, p. 65) reflect on the normative component of e-government and public sector reforms in the USA and UK. They suggest that a key promise of the new informational capabilities in government has been the potential for a changing relationship between government and individuals (either understood as citizens or customers). In line with Heeks and others (i.e. Fountain 2001), they also share the view that the dominant vision within the Reinvention Agenda has been to consider newer technology as the catalyst (and imperative) for enhancing efficiency, effectiveness, quality and democracy. Yet, they remark that at the core of this vision was the unrealistic “belief that these new (ICT) capabilities will permit wider, more inclusive access, greater choice and more flexible, responsive public services capable of being tailored to the increasingly disparate needs of consumers.” (Bellamy and Taylor 1998, p. 65) The work of Bellamy and Taylor (1998) is perhaps the exception amongst early e- government studies that reflect on the contested role of ICT within modernisation reforms. Bhatnagar (2004, p. 19)offers a view on e-government that is also useful to highlight the prominence of certain (instrumental) values within the debate. He remarks that: e-government is understood as the use of ICT to promote more efficient and cost-effective government, facilitate more convenient government services, allow greater public access to information, and make government more accountable to citizens. The value cluster of productivity, user/consumer orientation, as well as dialogue and accountability are implicated in this definition. 2.3 Public values implicated in e-government research These examples suggest that, from a normative perspective, there have been many claims that e-government will make - Page 37 - citizen-government transactions easier, cheaper and faster. These claims have been mainly supported by models that build on stages of development and different levels of e-government maturity, originally developed for e-business processes (i.e. Layne and Lee 2001, Andersen and Henriksen 2006). Usually, the models depict a transaction- oriented view in which e-government development occurs in three general maturity phases: the early, middle, and later stages. These phases start from an initial publication of information on a webpage, evolve into more complex two-way citizen-government interactions and transactions to the integration of public services across government agencies (both vertically and horizontally), and ultimately to a potential transformation of democracy. The more mature stages entail further organisational automation, integration and complexity, as agencies usually have to work in collaboration or become integrated under a new organisational form (i.e. policy networks). Overall, the idea is that, as the stages evolve, the levels of interaction, collaboration and management increase in complexity both internally and with citizens (Bellamy and Taylor 1998, Fountain 2001, Layne and Lee 2001, Heeks and Davies 2002, Moon 2002, West 2005a, United Nations 2008, 2010, Bhatnagar 2009). These models implied an inevitable sequence towards more e-government, and assume that more e-government is better. This transaction-oriented view of e-government resulted in widespread intents to offering public services online and quite recently incorporating the use of social media such as in open government initiatives. Implicitly, this vision of e-government embodies values that can be regarded as similar to those of NPM: a business-like approach, the idea that the easier and faster, the better, and that citizens demand services to be built around their individual choices (customer focus). West (2005a) offers a good illustration to these relations (see figure below). - Page 38 - - Page 38 - Figure 1: E-government stages and models of technological change Source: West 2005a, p. 11. Figure 1: E-government stages and models of technological change Source: West 2005a, p. 11. Chadwick and May (2003) present a case in point from a citizen-government interaction. Looking at experiences in the US, Britain and the European Union, the authors study the shifts from traditional government to e-government in citizen participation. 2.3 Public values implicated in e-government research Their findings show that in spite of the potential of technology as an instrument of democratic liberation, an executive-driven, managerial model of interaction marginalized the potentials of broader “consultative” and “participatory” possibilities. Their findings are highly congruent with that of Hood’s on the dominance of sigma- type of values. Even though most of these studies highlight some normative component (i.e. the ‘values’ or benefits promised by e-government), there are vast examples on how these components of reform were actually followed. For example, the books of Heeks (2002), Bhatnagar (2009) and Bellamy and Taylor (1998) cover numerous examples on both rich and less economically developed countries. The OCDE has also produced numerable studies on several countries, including Mexico, that show empirical evidence on how the purposes of e-government and public sector reforms outlined here were actually followed. There have also been broader empirical efforts conducted in relation to maturity models using benchmarking techniques, such as the E-Readiness Index produced by the Economist and the United Nations E-Government rankings among others. From this perspective, the evidence suggests that e-government expected impacts are generally over-optimistic (Heeks and Bailur 2007, Andersen et al. 2010), and that governments show signs of steady progress rather than the expected radical - Page 39 - - Page 39 - transformations that were predicted in late nineties (West 2005b, 2007, Coursey and Norris 2008, Norris 2010). In sum, a good deal of work has explicitly emphasised that e-government design and use would serve the goals of broader modernisation reforms. However, there is considerably less research on the connections between public values and e-government that would explicitly assess which types of values are privileged or marginalised, or to what extend technology shifts value priorities or contributes towards their agendas. Despite interesting work has been done in this area, it remains largely a valuable conceptual effort (i.e. Cordella 2007). In the following section, I bring together the inventory of public values and present the findings from the focused review on public values and e-government. 2.4 Inventory of e-government public values in procedural oriented agencies As suggested in Andersen et al. (2010), the most studied effects in e-government are in the internal capabilities of the public administration. In a recent article, Andersen et al. (2010) address the literature covering major impacts of e-government on the period 2003-2009. Consistent with previous similar literature reviews (Danziger and Andersen 2002), the authors find that the majority of the articles report impacts on managerial government capabilities (55%), indicating that e-government research focuses primarily on impacts on efficiency, information quality, and effectiveness of government policies. Impacts on interactions occupy the second largest place (31%) while the other two categories, value redistribution (11%) and orientations (3%), report the fewer number of findings. Regarding these two last categories, the authors stress that (Andersen et al. 2010, p. 576): These [value redistribution and value orientations] are domains where there has been the greatest controversy about the desirability of extensive penetration of ICT into the public sector. There is little research in our analysis that clarifies the early debates about whether there would be power shifts attributable to ICT that either reinforced the existing bases of power or would mobilize and empower groups that previously had less access to key information resources. In what follows, I expand on this concerns. In this section I map relevant e-government literature using the inventory of public values and the clusters of administrative values - Page 40 - - Page 40 - that I presented before. To do so, I conducted a focused review of the most prominent texts in e-government as well as specific e-government studies that examined referred explicitly to one or several values of government.10 To make the analysis manageable, and taking into account the particular concerns of my dissertation—examining the relation between public values and technology from inside government—,I discarded two public value constellations. The first constellation of values in the classification of Beck Jørgensen and Bozeman (2007) (public sector’s contribution to society) could be understood as an indirect outcome of any e- government programme. More precisely, while some conceptual pieces touch on this aspect in the literature of e-government (i.e. Brewer et al. 2006), there are no clear debates addressing the ethics or integrity of e-government or debates surrounding the morals of e-government. So, I removed the first category of public values from the list. 10 To select the most relevant literature on e-government, I follow two steps. I first run a general search using Harzing’s “Publish or Perish” citation tool; I used the terms “electronic government” (and its different uses, such as digital government, e-government, egovernment) and “information technology in public administration” to capture both, current literature on e-government and also those that refer broadly to ICT in government. The results were first ordered by average of citations per year and sorted in descending order to rank the more relevant publications in the topic area. The works I include in this section are amongst the top 20 most cited. Most of my results (top cited articles found) are similar to those reported recently in Belanger and Carter (Belanger and Carter 2012), who conducted a review of e- government research comparing the top ten most highly cited e-government articles with research on e- government published in leading IS articles. In addition to the most cited e-government publications, I complemented the analysis with further work that was normative or theoretically related to public values and technology in government. For example, the work of Bovens and Zouridis (2002) provides very important insights for the specific literature on accountability, discretion and e-government effects on those variables, and was not necessarily appearing in the top cited articles in the field. I selected empirical research that could offer theoretical links on public values only. 11 These studies can also be found under the term “e-democracy”; lately, the term internet politics seems to be more widely used (Chadwick and Howard 2009). 2.4 Inventory of e-government public values in procedural oriented agencies The second constellation—transformation of interest to decisions—contains highly relevant values that pertain to a rapidly growing field of research, often referred to as internet politics11 (Agre 2002, Chadwick 2006, Chadwick and Howard 2009). The internet and related ICT offer new avenues for assessing how opinions from society are channelled into government actions, and values such as user democracy, protection of minorities and the citizen involvement are deemed as highly important in studies of government and technology. Yet, given the focus of my research, which deals with the machinery of government and procedural based agencies, public values such as user 11 These studies can also be found under the term “e-democracy”; lately, the term internet politics seems to be more widely used (Chadwick and Howard 2009). - Page 41 - democracy, majority rule and citizen involvement have been excluded from the categorisation. The constellation of values in the third category (relationship between public administration and their environment) has been maintained, especially because of a growing attention to issues on openness and cooperation within the studies of public administration and technology (i.e. Bertot et al. 2010). However, this cluster may not be a clear-cut one. For the purpose of my own classification of the literature, I gave particular attention to research that dealt more explicitly with issues of openness, responsiveness and transparency. The rest of the value constellations appear very relevant within e-government studies although with differences of degree. The following table summarises the analysis of e-government literature in light of the constellations of public values. The following table summarises the analysis of e-government literature in light of the constellations of public values. The following table summarises the analysis of e-government literature in light of the constellations of public values. 2.4 Inventory of e-government public values in procedural oriented agencies Value Category Value Set Hood’s clusters of values Values of e-government (importance; examples) Relationship between public administrators and politicians Political loyalty Accountability Responsiveness Theta-type Yes; moderate prominence* (Bellamy and Taylor 1998, Fountain 2001, Heeks 2002) Relationship between public administrators and their environment Openness-secrecy Responsiveness Listening to public opinion Yes, very low prominence (Wong and Welch 2004, Tolbert and Mossberger 2006, Smith 2011) Intraorganisational aspects of public administration (organisational values) Robustness Adaptability Stability Reliability Timeliness Innovation Enthusiasm Risk readiness Productivity Effectiveness Parsimony Business-like approach Efficiency Self-development of employees Lambda-type Sigma-type Sigma-type Yes, moderate prominence (Bellamy and Taylor 1998, Fountain 2001, Welch and Pandey 2007) Yes, moderate prominence (Tan and Pan 2003, Welch and Pandey 2007) Yes, very high prominence (Heeks 2002, OECD 2003, 2005, Tan and Pan 2003, Pan et al. 2006, Fountain 2007, Gil-Garcia et al. 2007, Srivastava and Teo 2008, 2010, Mareschal and Rudin 2011) Yes, very low prominence (Fountain 2001) Yes, very low prominence (Fountain 2001) - Page 42 - Behaviour of public- sector employees Accountability Professionalism Honesty Moral standards Ethical consciousness Integrity Theta-type Yes, moderate to high prominence (Bovens and Zouridis 2002, Wong and Welch 2004, Jorna and Wagenaar 2007, Wenger and Wilkins 2009, Smith et al. 2010, Bellamy and Taylor 1998, Fountain 2001) Relationship between public administration and the citizens Legality Protection of rights of the individual Equal treatment Rule of law Justice Equity Fairness Professionalism User/customer orientation Timeliness Friendliness Theta-Type Theta-type Sigma-type Yes, low to moderate prominence* (Bovens and Zouridis 2002, Welch and Pandey 2007, Wenger and Wilkins 2009, Smith 2011) Yes, low prominence (Smith 2011) Yes, very high prominence (Bellamy and Taylor 1998, Fountain 2001, Heeks 2002, OECD 2003, 2005, Bhatnagar 2004, 2009, Pan et al. 2006, Tolbert and Mossberger 2006) Table 4: Inventory of public values administrative values and values of e government Table 4: Inventory of public values, administrative values and values of e-government b b d d ff d b Table 4: Inventory of public values, administrative values and values of e-government Source: own elaboration based on different sources. Notes: column 1 reports the clusters described by Beck Jørgensen and Bozeman (2007, p.360-361); column two corresponds to the category according to Hood’s types of administrative values (1991). Sigma-type values relate to matching of resources to tasks; theta-type, relates to fairness, neutrality and accountability, and lambda-type relates to adaptivity, learning and resilience. 2.4 Inventory of e-government public values in procedural oriented agencies Notes: Accountability is referred many times in the literature of e-governmentbut usually not in the terms discussed in Beck Jørgensen and Bozeman (political accountability). The idea of accountability needs to be revised; usually, as it appears in e- government refers broadly to public servants as being accountable to citizens, but not necessarily to any of the other sub-group of values depicted by Beck Jørgensen and Bozeman 2007. *Legality is many times referred in the literature of e-government as accountability; few times is directly referred as a value to be delivered and achieved through technology. These four more prominent categories of values are also congruent with the general lines that e-government has been conceptualised in the literature: e-government is the generalized use of ICT to provide more and better public services, to improve public administration performance and to enhance democratic values (Bellamy and Taylor 1998, Fountain 2001, Heeks 2002, Gil-Garcia and Pardo 2005, Cordella 2007, Bhatnagar 2009). These four more prominent categories of values are also congruent with the general lines that e-government has been conceptualised in the literature: e-government is the generalized use of ICT to provide more and better public services, to improve public administration performance and to enhance democratic values (Bellamy and Taylor 1998, Fountain 2001, Heeks 2002, Gil-Garcia and Pardo 2005, Cordella 2007, Bhatnagar 2009). The table summarises relevant work, mostly empirical, that has been useful to shed light on those public values that are more or less studied in the field. As I suggested, it seems apparent that public values related to the internal and organisational capabilities of the - Page 43 - public sector have been vastly studied. Usually, these studies focus on the capabilities of technology to automate and enhance data access and data quality, obtain productivity gains, timesaving and improved products and services (Andersen et al. 2010, pp. 568- 70). Far less studied aspects are, for instance, the effects of technology on values such as integrity, honesty and accountability. For example, an acclaimed potential benefit of e- government relates to their facility to report and publish results, with their increasing perception of trust or accountability. However, issues of honesty reporting and their underlying mechanisms have largely been absent in public management literature in general (Yang 2009). An area of research that has informed an understanding on the connections between e-government and more ‘classic’ public values are the studies of ICT and discretion. 2.4 Inventory of e-government public values in procedural oriented agencies Bovens and Zouridis’ (2002) seminal work argues that technology has the potential to replace the traditional street-level bureaucracy by a system-level bureaucracy. The authors find that, as a consequence of the automation processes in the clerical work, street-level bureaucrats exercise less policy discretion as they have less administrative involvement in individual cases. The authors also find that because the information between citizens and bureaucrats is primarily managed through technology, this exchange drives decision making. Wenger and Wilkings (2009) find also that automation curtails the discretion of street- level bureaucrats, this time, due to the elimination of the bias certain agents faced when they entered an office in person. In this sense, their study supports the findings of Bovens and Zouridis (2002) in the sense that technology reduces discretion, and thus may contribute to ideas of fairness and justice. As far as the role of technology is concern, Wenger and Wilkins (2009, p. 327) call attention to theorise the role of technology in future work: we use technology as an exogenous factor that influences bureaucratic discretion; however, future research could also theorize about how bureaucratic discretion influences the shape and structure of technological adoption But Regardless of the stance on technology innovation and its effects, other research on accountability and discretion in e-government has suggested mix results. Jorna and Wagenaar (2007) argue that technology may not necessarily lead to the reduction of - Page 44 - administrative discretion. Indeed, the authors suggest, technology innovation in the public sector can contribute to increase the discretionary power of public servants. Similarly, Welch and Pandey (2007) examine the effects between rule-based constraints that often define public organisations and the implementation of intranet technologies in public service agencies. The authors find that intranet reliance reduces red tape in general terms and in procurement areas but there is no evidence that red tape affects intranet implementation. As a result, the findings from Welch and Pandey (2007) do not support previous assertions in the literature of e-government, which suggested that higher levels of red tape could lead to greater innovativeness (p. 396). On issues of accountability, based on the work on ‘accountability dysfunctions’ of Bovens (2005), Smith et al.(2010) problematise the diverse value conflicts that technology introduce in key areas of public service. 2.4 Inventory of e-government public values in procedural oriented agencies Their main argument is that, while technology innovation in the public sector may contribute to the functions of accountability, it may also exacerbate its dysfunctions. Based on empirical illustrations, the authors suggest that certain public values imbedded in technological applications marginalised and dominate over others. A case in point has been the push for efficiency of services in the identity card scheme in the UK that led to serious implications for surveillance, personal autonomy and liberty (p. 12). The authors insist that a “preoccupation with technology-centred solutions distracts us from addressing which, why, and how particular accountabilities should be enforced or shifted” (Smith et al. 2010, p.13). Wong and Welch (2004) offer similar conflicts in accountabilities; the authors find that, despite the fact that the use of internet in government increases general levels of accountability, it also reinforces the differences in power among agencies. Studies on trust have also shed light on the potentials of value conflicts among stakeholders. Developing theory based on critical realist assumptions, Smith (2011, p. 237) shows that e-services can be concurrently trustworthy and not trustworthy, depending upon the interest or value perspective taken by the user. His findings reveal that, in matters of transparency, procedural fairness, efficiency and equality, technology may not just resolve tensions that are inherent to democratic values, but rather change their configurations, creating new interpretations and trust judgments. - Page 45 - - Page 45 - These studies shed light in the complex dynamics among discretion, technology and flexibility, public values most commonly associated to the ethos of bureaucracy. This serves to illustrate an opportunity to advance the field studying public values in context. Overall, to assess how potential competing public values can (or cannot) be aligned is a matter of empirical scrutiny, in a given context and concrete practices. 13 I am inspired in the work of Gregor (2006, p. 624) that uses the word “sensitising device” to refer to theories that view the world in a particular way. 12 I refer to this version of CDA in particular; critical discourse analysis in IS research has drawn mainly from Habermas’ theory of communicative action (i.e. Cukier et al. 2009). 2.5 Conclusions In this chapter, I have shown the complex and elusive character of studying public values empirically. I have also offered further conceptual clarifications regarding the definition and streams of public value research that have inspired my work. I would like to close this chapter with a summarising quote that serves to illustrate the importance of studying different dimensions of public value and the need to advance more knowledge on the subject. In 2010, de Graaf and van der Wal (2010) concluded the editorial of a symposium on Ethical and Effective Governance, with the following remarks (p. 628): Despite the valuable insights the articles bring to the fore, much remains unclear, such as how often governing good conflicts with governing well and what trade-offs in values the conflicts lead to. (...) What we have thus far is partial and varying evidence that points to an existing tradeoff between integrity and effectiveness in which effectiveness seems to prevail. However general the conclusion may be, it is a promising point of departure for important research endeavors of the future. I have shown in the chapter that studying public values, their meanings and conflict matters are even more elusive when it comes to its relation to technology. In the next chapter, I elaborate on the ways to theorise the relation between public values and technology. - Page 46 - - Page 46 - 3 Chapter Three: Theorising public values and technology innovation through critical discourse analysis Thus, later on in section 3.2, I introduce the idea of transdisciplinary work and explain the main sources that I use to theorise technology in my work. In particular, I elaborate on the type of technology innovation that I study and I introduce key theoretical concepts in the studies of technology innovation in the public sector (e- government). That is, Section 3.2 lays the groundwork for the choice of concepts and dynamics that illuminate my objects of research. Drawing on the main concepts elaborated in those sections and the public values concepts presented in Chapter Two, section 3.3 brings together a CDA-based theoretical framework to study the linkages and dynamics of public values and technology innovation. I finish the Chapter with a reflection on the role of theory in my work. 3 Chapter Three: Theorising public values and technology innovation through critical discourse analysis 3 Chapter Three: Theorising public values and technology innovation through critical discourse analysis To understand what sorts of public values are imbued in technology innovation in the public sector, a theoretical lens suitable to study them and their relations within their socio-political context is needed. Recall that a recurring theme emerging from the review on public values theory has been the need to advance their understanding and meaning in context. At the same time, the e-government literature suggests that technology innovation have great potential to enhance or change public values associated to different aspects of the internal capabilities of public administrations and their relation with citizens. In this chapter, I develop a theoretical framework that can be suitable for studying the relation between public values and technologies, its content, context and its dynamics. To do so, in the following sections, I introduce a discursive organisational lens that can be suitable for that purpose. Amongst the different theoretical perspectives on organisational discourse, I adopt a particular version known as critical discourse analysis, which has received very little attention within information systems research.12 CDA is suitable to my research interest in at least two ways: first, it provides the theoretical basis and foundational premises to study discourse within its context; second, it is flexible and open to account for the particular theorisation of my objects of study— public values and technology innovation. Therefore, the reader shall not understand CDA as the ‘sole’ theoretical domain of the dissertation. CDA is a sort of general theoretical umbrella that provides also a strong methodology to study relations of technology innovation and values over time. As such, my theoretical “device”13— referred loosely as framework in the dissertation—brings together theoretical premises of CDA with the contextual and emergent character of technology innovation and the notion of public values discussed in the previous chapter. - Page 47 - To account for such theoretical elaboration, the chapter is organised as follows. In section 3.1, I elaborate on the main facts, assumptions as well as methodological implications of the tradition of CDA that I adopt in my research. I also explain how CDA has been used in studies of organisational change. My theoretical framework is complemented with knowledge from a stream of information systems research based on social theory. 3.1 Discourse and organising: the case for Critical Discourse Analysis The discursive aspects of the different facets involved in organising processes have received increasing scholarly attention (Alvesson and Kärreman 2000b, Heracleous and Barrett 2001, Grant and Hardy 2004, Grant et al. 2004, Fairclough 2005a, Phillips and Oswick 2012). This, in part, has been a consequence of a more general linguistic turn in social science research (Van Dijk 1997, Alvesson and Kärreman 2000a, 2011, Vaara 2010). Among the different theoretical streams on organisational discourse, I shall be looking at critical discourse analysis (CDA), which has been of growing influence within social sciences in general (Fairclough 1992, 2005a, 2010, Wodak 2001, Phillips and Di Domenico 2009), and within management and organisation studies in particular (see for example Leitch and Palmer 2010, Alvesson and Kärreman 2011, and two special issues on the topic in Organization Studies and Organisation in 2004). In the following sections, I present the main theoretical points that I use in my work. More specifically, I present the basics of CDA and elaborate on three key concepts: texts, discourse and social context. My research has been largely influenced by the work of Fairclough and - Page 48 - - Page 48 - colleagues, although I also draw on related material that has been produced in fruitful debates in organisational discourse research. 3.1.1 Critical discourse analysis: introduction and assumptions Critical discourse analysis is a theoretical approach to study how discourse figures within processes of change in social life (Fairclough 2003, p. 205, 2010). In the particular field of organisation and management studies, CDA has become a well- established framework to examine the use of language in text and talk that underlie the dynamics of organising (Phillips and Hardy 2002, Phillips et al. 2004, Fairclough 2005a, Munir and Phillips 2005, Vaara et al. 2006, Vaara and Tienari 2008, Chouliaraki and Fairclough 2010, Leitch and Palmer 2010). In turn, CDA has embraced a high degree of diversity in the way scholars approach it, both theoretically and methodologically (Wodak and Meyer 2009, p. 32), giving raise to concerns. In a debate presented in the Journal of Management Studies on the subject, Leitch and Palmer (2010) argue that researchers should be precise about the particular CDA assumptions and analytical components when conducting work based on this discursive lens. Among other rules, the authors suggest that researchers should specify which CDA tradition they draw upon (Leith and Palmer 2010, p. 1207), which I do below. My elaboration of CDA draws on a particular stream developed by the work of the socio-linguistic Norman Fairclough and colleagues (Fairclough 1992, 2000, 2005, 2010, Chouliaraki and Fairclough 1999). This stream of CDA emphasises the importance of a close and detailed analysis of texts and at the same time, the integration of the analysis into broader social processes, actions and events (Fairclough 2005, p. 924). Theoretically, CDA is based upon a view in which semiosis constitutes an irreducible element of social life (Fairclough 2003, p.203). Social life, in turn, is understood in CDA as an interconnected network of social practices of many sorts, such as cultural, political, discursive, economic and so on. The concept of social practice is central for CDA purposes, which Fairclough defines as follows (2003, p. 203, emphasis added): By ‘social practice’ I mean a relatively stabilized form of social activity (examples would be classroom teaching, television news, family meals, medical - Page 49 - consultations). Every social practice is an articulation of diverse social elements within a relatively stable configuration, always including discourse These different social elements comprise activities, subjects and their social relations, technology, objects, time and place, forms of consciousness, values and discourses. This brings into focus the idea of discourse as a dialectical element of social life. 14 In the methodology chapter, I expand on these assumptions and the ways critical realism has influenced my research. 3.1.1 Critical discourse analysis: introduction and assumptions Moreover, this version of CDA distinguishes from other discourse analysis in organisation studies that are said to be anti-realist or anti-materialist (Reed 2004, p.414, Fairclough 2005, p. 922-3). A typical example of the latter is offered in Mumby and Clair (1997, p.181), who suggest: Organizations exist only in so far as their members create them through discourse. This is not to claim that organizations are ‘nothing but’ discourse, but rather that discourse is the principle means by which organization members create a coherent social reality that frames their sense of who they are. Reed (2004, p. 414) criticises the fact that in this statement, ontology and epistemology are conflated; social structure is collapsed into discursive agency and hence, it becomes impossible to discern the connections among everyday discourse, sense-making practices and larger social structures and the enactment of power relations—all paramount elements in CDA. In taking a realist view, organisational discourse analysis is based on the central argument that the social world comes previously structured (Reed 2004, p.415). In studying discourse dialectically, “discourse is necessarily dependent on a pre-constituted set of separable, yet interdependent, material conditions and social structures” (2004, p.415). But discourse, as part of this dialectical relation, has the potential to change the social world as well. In the case of organisational analyses, Fairclough summarises as follows (2005, p. 929-30): Reed (2004, p. 414) criticises the fact that in this statement, ontology and epistemology are conflated; social structure is collapsed into discursive agency and hence, it becomes impossible to discern the connections among everyday discourse, sense-making practices and larger social structures and the enactment of power relations—all paramount elements in CDA. In taking a realist view, organisational discourse analysis is based on the central argument that the social world comes previously structured (Reed 2004, p.415). In studying discourse dialectically, “discourse is necessarily dependent on a pre-constituted set of separable, yet interdependent, material conditions and social structures” (2004, p.415). But discourse, as part of this dialectical relation, has the potential to change the social world as well. In the case of organisational analyses, Fairclough summarises as follows (2005, p. 929-30): Whether or not change in discourse leads to change in beliefs or habits of action, as well as change in organizations, is a contingent matter. 3.1.1 Critical discourse analysis: introduction and assumptions Chouliaraki and Fairclough (1999, p.37) illustrate: any practice articulates together diverse elements of life (as its ‘moments’) and therefore diverse mechanisms. Discourse is one such element, with its own mechanism. (...) Practices themselves are articulated together within networks of practices... Any practice is a practice of production – people in particular social relations applying technologies to materials. Also, any practice has a reflexive element – representations of a practice are generated as part of the practice. Discourse therefore figures in two ways within practices: practices are partly discursive (talking, writing, etc. is one way of acting), but they are also discursively represented. any practice articulates together diverse elements of life (as its ‘moments’) and therefore diverse mechanisms. Discourse is one such element, with its own mechanism. (...) Practices themselves are articulated together within networks of practices... Any practice is a practice of production – people in particular social relations applying technologies to materials. Also, any practice has a reflexive element – representations of a practice are generated as part of the practice. Discourse therefore figures in two ways within practices: practices are partly discursive (talking, writing, etc. is one way of acting), but they are also discursively represented. Fairclough’s version of CDA depicts a view of language as not just abstract sign systems but as medium of social practices with constitutive effects on social life (Fairclough 2010, 2005, Chouliaraki and Fairclough 1999). Any sociocultural practice, therefore, articulates together different elements of life and diverse mechanisms, being discourse just one of those elements. Discourse is then said to be dialectical in that the relations are between social elements that are different from one another yet not totally discrete (Fairclough 2005, Chouliaraki and Fairclough 1999, 2010, Fairclough et al 2002). The relationship between power and discourse offers a good illustration: power is partly discursive as it depends on discourse to build and sustain legitimacy, but power is yet not totally discursive as it can also rely on other material properties, such as physical force. This version of CDA assumes a particular perspective on social structure, social action and agency that is based on a critical realist position.14 Thus, the concept of social practice provides theoretical means to examine the ways discourses are produced and interpreted, but rejects an extreme relativistic social construction perspective that lay an - Page 50 - assumption that everything is discourse. 3.1.1 Critical discourse analysis: introduction and assumptions (...) Changes in discourse certainly can and do contribute to change in beliefs, habits of action, and indeed organizations, but it is only by consistently regarding the difference between such social elements that one can investigate the relations between them. Having depicted the general introduction and underlying assumptions of CDA, in the next section, I introduce the three-dimensional level of analysis, which is pivotal for my research. Having depicted the general introduction and underlying assumptions of CDA, in the next section, I introduce the three-dimensional level of analysis, which is pivotal for my research. 3.1.2 Text, discourse and context in CDA A particular feature of CDA, Fairclough suggests, is establishing connections between the properties of texts and wider sociocultural practices that are mediated by discourse (Fairclough 2010, p.132). The integration of different social elements unfolds in a - Page 51 - ‘three-dimensional’ framework, which offers a succinct schematic way to visualise the relation between texts, discourse and social context (see figure 2 below). The three- dimensional framework implies that discourse should be analysed simultaneously at three levels: (i) text (spoken, or written), (ii) discourse practice (text production and interpretation), and (iii) social practice (the situational, institutional and/or societal context). The first level (micro level, level of text) requires close and detailed analysis of texts, relying on different linguistics and semiotic tools. The level of discursive practices, in turn, involves a processing analysis on how texts are interpreted, or in other words, what people do with texts, how they are produced and consumed. The level of social practice (macro level) focuses on the broader context in which texts are embedded, such as the immediate situation (space-time), the wider institutional domain or organisation, or the societal environment. Figure 2: Dimensions of discourse and discourse analysis Source: Fairclough 2010, p. 133. Figure 2: Dimensions of discourse and discourse analysis Source: Fairclough 2010, p. 133. The notions of text, discourse and context need further clarification. The views on discourse as well as text have been subject to a wide and diverse usage in the social sciences in general and also with variation within CDA studies (Wodak and Meyer 2009, p. 2, and see Leitch and Palmer 2010, p. 1196-7 for examples on diverse definitions of - Page 52 - texts within CDA).15 For my purposes here, I understand text in a broad sense, including written pieces of texts such as policy reports and legal documents, but also transcripts of spoken words like conversations or interviews as well as pictures and artifacts (Phillips et al 2004, p. 636). In addition, I understand discourse in a general, abstract sense, to refer to an element of social life that is dialectically related to other elements (Fairclough 2003, p.214), such as non-discursive ones. From this perspective, discourse is language in practice, that is, discourse is an interrelated set of texts (in the sense described before) that brings objects into being alongside their related practices of text production, dissemination and consumption (Fairclough 2003, Munir and Phillips 2005, p. it is obvious that the notions of text and discourse have been subject to a hugely proliferating number of usages in the social sciences. Almost no paper or article is to be found which does not revisit these notions, quoting Michel Foucault, Jürgen Habermas, Chantal Mouffe, Ernesto Laclau, Niklas Luhmann, or many others. Thus, discourse means anything from a historical monument, a lieu de mémoire, a policy, a political strategy, narratives (...) to language per se. This causes confusion (...) This is why each contributor to this volume was asked to define their use of the term integrated in their specific approach” (italics in the original). 15 Wodak and Meyer (2009, p.3-4), two very well-known socio-linguistic scholars, exemplifies this idea nicely: 3.1.2 Text, discourse and context in CDA 1667).16 In the dialectical-relational conception of discourse that CDA embraces, context is deliberately understood as contingent and broad; context is the articulation of semiosis with other elements of social practice (Chouliaraki and Fairclough 2010, p. 1217). Context, then, is historical, social and spatial. In my research, the way I conceptualise the role of context shares the view of Avgerou (2002, p. 8-10), who suggests that contextual studies involve a process analysis that spans multiple layers and intertwined relations between co-evolving social processes (i.e. technology innovation and organisational change). In addition, my view of context in light of CDA also entails a dynamic and generative understanding (Hayes and Westrup 2012, Salskov-Iversen et al 2000, p.190): instead of treating it as a given entity that is ‘out there’ waiting to be represented, context should be studied dynamically, as ‘generating site’. That is, I see local spaces as relational and open-ended, interacting with broader social dynamics (i.e. globalisation). 16 The term discourse will be used more specifically (as a count noun), to refer concretely to particular discourses (i.e. discourses of legality). - Page 53 - - Page 53 - This version of CDA has its own methodological implications. As explained, discourse analysis in my work accounts for a detailed analysis of text, using methods for analysing linguistic and semiotic features in some detail (Fairclough 2003).17 The methodology chapter—and particularly, the section on the method of analysis—explains in detail the type of textual analysis that I have conducted. For the moment, let me say briefly that a textually oriented discourse analysis includes paying particular attention to families of words (i.e. choice of words) as well as aspects of grammar and semantics that are present in the texts (i.e. structure of sentence). The textual oriented analysis, therefore, covers the first dimension of CDA that I have presented in the figure 2 earlier on this chapter. CDA does not stop at the level of texts: “text analysis is an essential part of discourse analysis, but discourse analysis is not merely the linguistic analysis of texts” (Fairclough 2003, p. 3). That is, texts are rather meaningless if taken individually or isolated. Thus, the second dimension involves dealing with ‘intertextuality’. The intertextuality of a text is the presence of elements of other texts that can be direct (i.e. quoted speech) or indirect (i.e. reported speech) and related in different ways (i.e. 17 Let me reiterate that it is this aspect of detailed linguistic analysis of texts that distinguishes CDA from much discourse analysis in the Foucault tradition (Fairclough 2003, p.215). 3.1.2 Text, discourse and context in CDA dialogued with, assumed, presupposed, etc.) (Fairclough 2003, pp. 39-40). The second dimension also requires revealing which genres are used, and how they are mixed, textured, and linked with other discourses. In turn, the societal dimension is unfolded by creating a meaningful context level, to understand where texts emanate from (conditions of production) and how they are used and consumed. The societal stage is therefore reconstructed based on the extensive data I have collected during my fieldwork (direct observation and interviews) as well as a number of further sources of historical, political and socioeconomic data on Mexico and on the project under study (archival data). Methodologically, this process is nicely summarised in Phillips et al. (2004, p. 636, emphasis added): Discourses cannot be studied directly –they can only be explored by examining the texts that constitute them (Fairclough, 1992; Parker, 1992). Accordingly, discourse analysis involves the systematic study of texts -including their production, - Page 54 - dissemination, and consumption- in order to explore the relationship between discourse and social reality. The centrality of the text provides a focal point for data collection, one that is relatively easy to access and is amenable to systematic analysis (...). Discourse analysis does not, however, simply focus on individual or isolated texts (...) but, rather, on bodies of texts. Discourse analysis therefore involves analysis of collections of texts, the ways they are made meaningful through their links to other texts, the ways in which they draw on different discourses, how and to whom they are disseminated, the methods of their production, and the manner in which they are received and consumed In sum, a critical analysis of texts requires an understanding and description of the surrounding social processes and structures that enable the production of a text as well as the processes by which individuals or groups understand and consume texts in a certain meaningful way (Wodak 2001, p. 3). What makes CDA distinctive is to include a detailed analysis of texts, which is not the case for much discourse analysis in social sciences (Fairclough 2003, p. 215). 3.1.3 CDA and organisational change Critical discourse analysis has been adopted in many organisational analysis and studies of organisational change (i.e. Vaara and Tienari 2002, Vaara and Monin 2010). Based on the assumptions that CDA holds and the three-dimensional framework, in this section I expand on a set of research issues that are particularly important in studying the dynamics of discourse practices in organisational settings. I have anticipated that a detailed analysis of text requires going back and forth between text, its practices of production and dissemination, and the broader social context. Fairclough (2005, p. 932-3) suggests that four sets of research issues can be addressed using CDA in studies of organisational change: emergence, hegemony, recontextualisation and operationalisation. In brief, they are defined s follows: (i) Emergence: how discourses emerge, how they are constituted and articulated with existing discourses; (ii) Hegemony: the ways particular emergent discourses (and not others) become hegemonic or dominant in particular organisations or institutions; - Page 55 - (iii) Recontextualisation: how emergently hegemonic discourses travels across different boundaries (e.g. between organisations, from local to national or international scale, or vice versa); (iii) (iv) Operationalisation: follow the ways in which discourses go from being representations to having transformative effects on organisational reality. These four ‘objects of research’ in CDA—as Fairclough defines them—can be combined in a research project, using all four or a selection of them, depending on the research interest. Broadly, all these categories entail tackling temporality seriously. In my case, the first two categories are relevant from the point of view of providing the background of my research and the way e-government initiatives are framed. These are briefly addressed in the introduction and in Chapter Two, when I discussed the foundations of the NPM and how it has travelled as well as the ways it has provided a platform for e-government development. While the category of recontextualisation will be addressed when I deal with the ways the particular public agency that I study has incorporated a series of hegemonic discourses (i.e. competitiveness and openness to trade in Mexico), a rather relevant category for my work is the one on operationalisation. It is by studying operationalisation that one can gain insights into the ways discourses are dialectically transformed into other social elements. 3.1.3 CDA and organisational change There are three main ways in which operationalisation of discourses can occur: via enactment of discourses in new ways of acting and interacting; via inculcation of new ways of being (i.e. changes in communicative style); and via materialization, as new objects and properties of the physical world take form. As Fairclough explains (2005, p.934): Operationalization includes enactment: discourses may be dialectically transformed into new ways of acting and interacting. (...) Operationalization also includes inculcation –the dialectical transformation of discourses into new ways of being, which include new styles. Finally, operationalization includes materialization: a new management system in an organization may include changes in the structuring of organizational space, e.g. in the design of office space. Through these forms of operationalization, networks of social practices and the orders of discourse which are parts of them may be transformed. - Page 56 - - Page 56 - - Page 56 - The concept of operationalisation is particularly useful for the framework, as it suggests mapping where discourses come from, how they are interpreted in their discursive and social practices, and how they eventually are operationalised in technology and new ways of acting. At this point, it is rather obvious that in order to study the discourse operationalisation, one needs to go beyond the level of text and use other methods such as fieldwork. The latter provides a further reason why this category becomes suitable for the purposes of my research. So far, I have referred mainly to discourses, and the ways CDA can help in theorising the relation between public values and technology. It is precisely in creating a dialogue between disciplines that I place this endeavour. In the next sections I introduce the idea of transdiciplinary work, and the particular premises on technology and organising in the public sector that I follow in my work. 3.2 Transdisciplinary work: theorising technology Adopting a dialectical-relational view of discourse in organisational life offers a distinct way to see how language figures in different aspects of organising. As I emphasised earlier, discourse is just one of the elements that are dialectically articulated in a given social practice. CDA offers a particular theorisation to deal with language and discourse, their properties and the specific ways that language can be deconstructed. By the same token, the other social elements (i.e. technology, public values) have their own distinct properties that are amenable to distinct forms of theorising. Within the CDA spirit, it is advocated to work in dialogue with other disciplines to theorise particular objects of research. Fairclough and colleagues refer to this as ‘trans-disciplinary’ research (Fairclough 2010, 2005, Chouliaraki and Fairclough 2010). The aim is bringing disciplines and theories together to address research issues that emerge out of ‘dialogue’ between them and as a source for theoretical and methodological development (Fairclough 2010, p.231). For example, some categories now commonly used in CDA (i.e. recontextualisation) are themselves the result of working in dialogue with other disciplines such as sociology, political economy and communication studies (Chouliaraki and Fairclough 2010, p.1217, Fairclough 2003, p.6 and 225, Fairclough 2010). - Page 57 - In this dissertation, a transdisciplinary work entails theorising public values and technology innovation accordingly. While I provided the theoretical underpinnings for public values in Chapter Two, the latter requires space here. That is, finding ways to theorise about technology in the public sector, as I shall do as follows. But first, let me introduce the current state of e-government theory and the main premises I draw upon. Theory-informed empirical studies that try to explain the interplay between technology and public organisations have shed light on critical aspects on the design, use and adoption of e-government to support processes of administrative reform (Fountain 2001, Bellamy and Taylor 1998). While within the studies of e-government the state of theory is not well developed and continues in progress (Heeks and Bailur 2007, Andersen et al 2010, Yildiz 2007), my work draws from various bodies of knowledge, mainly theories from public administration and political science, information systems and studies of organisation and technology. Such a project does not suggest, however, that there is no theory at all within e-government studies. Following others (i.e. 3.2 Transdisciplinary work: theorising technology Fountain 2001, Bellamy and Taylor 1998, Danziger et al 1982, Bloomfield and Hayes 2009), I approach the use of networked technologies in government not merely as information processing tools and communicating technologies but rather as elements of a larger socio-technical system composed of humans, technologies, politics and values, as well as knowledge and tensions, and in context. Under this approach, which can be grouped generally within the ‘ensemble view of technology’ (Orlikowski and Iacono 2001, p. 125-6), technology innovation is part of complex socio-political processes, in which technology affects and is affected by others elements of social practices— organisational structures, processes, end users, institutions, discourses. Contrary to other disciplinary views in which technology is commonly treated as a black box (i.e. most public management studies), the usefulness of this approach relies on the distinctive characterization of technology within social complex contexts. This first distinction is of importance, because it rejects the tool view of technology that assumes that technology is unproblematic and expected to do what it has been intended for. In addition, the idea that complex social and institutional as well as technical issues are embedded in the process of design and implementation of a particular technology is - Page 58 - - Page 58 - well documented among information system scholars as well as within those interested in the studies of organisation and technology (i.e. Avgerou 2002, Orlikowski 2000, 1992, Coombs et al. 1992, Fountain 2001, Zammuto et al. 2007). Given its complexity, technology innovation in the public sector (e-government) and its surrounding factors can be unique or highly contingent. Yet, this is not to say that these outcomes cannot be studied or deconstructed. On this basis, my work builds on common research trends from research in information systems and institutional change, that see technology innovation in the public sector (e-government) as immersed in combinations of political drivers and organisational changes designed to support and drive a transformation in the organisation and practices of public bureaucracies (Fountain 2001, Kallinikos 2006, Bloomfield and Hayes 2009, Cordella and Ianacci 2010, Danziger et al. 1982). The particular connection between technology and public values in the public sector may be multifaceted. For example, scholars have documented that computerized systems can embody bureaucratic rules and related actor’s agendas (Clegg 2006, p. 108, Fountain 2001, p.62, Kallinikos 2006, Danziger et al 1982). 3.2 Transdisciplinary work: theorising technology In turn, it is recognised that technology can imbue political values in complex and rather opaque ways (Introna and Nissenbaum 2000, Introna 2007). By the same token, Joerges and Czarniawska (1998, p. 382) remark that institutions can become inscribed in machines and technical norms—a concept they call ‘organisational inscriptions’. These premises suggest that, in principle, CDA offers a suitable tool to unfold the dynamics associated with a particular technology. A connecting line emerges from the fact that technology needs to be understood in context, it is dynamically constructed, and internalises as it is internalised by other elements of social practices. Despite the a priori suitability of CDA to theorise technology in such ways, the work of Fairclough and colleagues remains largely unknown within IS research (but see Thompson 2004, Klecun 2011). In the next sections, I elaborate on the sociotechnical paradigm in e- government research, and the ways I theorise the type of “IT artifact” that I will be dealing with. - Page 59 - 18 This view is usually the norm rather than the exception within management and organisation studies, as the extensive literature review of Orlikowski and Scott suggest (2008, pp. 434–6). 3.2.1 Technology innovation and properties of technology In their influential article, Orlikowski and Iacono (2001) call for an increased analytical attention to technology in information systems research, what they call the IT artifact. For the authors, IT artifacts are “those bundles of material and cultural properties packaged in some socially recognizable form such as hardware and/or software” (2001, p. 121). The authors review the literature in leading IS journals to find that the IT artifact is, within the discipline, usually taken for granted, marginalised or presumed unproblematic.18 While Orlikowski and Iacono (2001, p. 130-131) admit that theorising the IT artifact may take different forms, they offer five premises: (1) IT artifacts should not be understood as natural, neutral, universal, or given: “because [they] are designed, constructed, and used by people, they are shaped by the interests, values, and assumptions of a wide variety of communities” (p. 131) (1) IT artifacts should not be understood as natural, neutral, universal, or given: “because [they] are designed, constructed, and used by people, they are shaped by the interests, values, and assumptions of a wide variety of communities” (p. 131) y g y p p y p y the interests, values, and assumptions of a wide variety of communities” (p. 131) (2) IT artifacts are always imbued in context (spatial, discursive, temporal and social); (2) IT artifacts are always imbued in context (spatial, discursive, temporal and social); (3) IT artifacts are not uniform, unified or stable entities, but rather an array of multiple and interconnected components; (4) IT artifacts emerge from a variety of social practices; they are neither fixed nor independent; (5) “IT artifacts are not static or unchanging, but dynamic” (p. 131): any stability is conditional upon history, social processes and their own innovations. Among the premises that Orlikowski and Iacono (2001, p. 131) suggest, is the need to be specific about the type of technology that is being dealt with for a particular research. I broadly refer to technology innovation as the use of the internet and related ICT in government. In turn, recall that, as stated in the introduction, I define ICT as those technologies for the processing, storage, and transmission of digital material, which consist of ensembles of hardware and software with distinctive feature sets that allow - Page 60 - physical storage and logical representations of different forms of data (Mutch 2010, p. 507). 3.2.1 Technology innovation and properties of technology More specifically, the type of technologies I refer are somehow similar to enterprise systems, in the sense that they entail the use of hardware and application software to manipulate large bodies of data, to automate and support bureaucratic processes, information flows, and reporting across the central government. In addition, these technologies rely on networked communications such as the internet; this is usually why they are referred as networked computing (i.e. Fountain 2001, chapter 3). These technologies bring interesting potential dynamics. The first one is automation: computer based systems can replicate manually performed tasks and lead to greater efficiency of existing data processes (Bellamy and Taylor 1998, p. 38-9). In addition to automation, networked technologies bring unique capacities for informating; that is, to process, handle and storage of information in a logical and meaningful way (Fountain 2001, p. 33, Kallinikos 2006). Based on the seminal work of Zuboff (1988), Bellamy and Taylor add to this by suggesting that the way software encode information processes enables reflection upon the organisation (1998, p. 26-77). Moreover, networked technologies may offer the potential to affect coordination, communication and information flows within and across organisations (Fountain 2001, p. 32). New networked technologies allow for shared databases to be accessed in easily and retrievable form over time. “Data collected at remote filed locations, once digitized, can be easily available at headquarters as in filed locations and may be easily transferred” (Fountain 2001, p. 35). Taken together, these capacities of networked technologies can lead to rationalisation of the organisation by process integration, restructuring of tasks, decentralisation and simultaneous centralisation. For my purposes, although the characteristics of networked technologies are somehow broad, they serve the purpose to set the basis for the type of technology innovation I will be referring to. At this point, it is useful to think in terms of the operational complexities and the type of process change in light of the stages of development of e- government. Thus, the type of technology innovation that I will be referring falls into the middle and later stages of development; that is, technology innovations that require the modification or integration of the back-office within one agency and/or the - Page 61 - integration with other agencies. The integration, in turn, may require changes with effects on the internal agency structure, culture and/or operations. 3.2.1 Technology innovation and properties of technology Finally, I shall say that these capacities of networked technologies are only indicative, and by no means, these potential effects should be understood as deterministic. Technology innovation and its outcomes are subject to their interaction with other elements of the socio-technical system. Although technology innovation may fix some possibilities (Mutch 2010, p. 514-5), their implementation remains rather open and ambiguous (Bellamy and Taylor 1998, p. 31). Even though broad, these properties are particularly salient for their possible effects on bureaucratic norms, the embodiments of rules, and coordination within and across institutions and organisations, that I shall review in the next section. 3.2.2 Bureaucracy and Technology It has been commonplace to find claims suggesting that ICT changes the structure of organisations. Much has been said or written about the potential for networks to replace hierarchies, and markets to replace bureaucracies. Technology innovation may play a crucial role, even though their understanding and analysis have appeared nominal within studies in the field (Fountain 2001). In other words, few e-government studies have addressed how information and communication technologies fosters, strengthens or neglects the distinguishing character (ethos) of bureaucracy (but see Fountain 2001, Kallinikos 2006). While the issues surrounding bureaucracy either as organisation or institution of modernity are by far complex (Kallinikos 2004, Clegg 2011), the intention of this section is to revise key elements Weber’s bureaucracy and the role that networked technologies may play. Whilst Weber’s view of bureaucracy conforms to an ideal-type, his depiction of this organisational form summarizes important trends in industrial capitalism that became a central model to public administration since the early twentieth-century (Fountain 2001, p.45). Weber’s depiction of bureaucracy as an ideal-type was opposed to the personalistic, patrimonial systems characteristic of pre-modern or early modern systems - Page 62 - of governance in state and economic life in general (Fountain 2001; Kallinikos 2006). As an organising regime of modernity, modern rational-legal bureaucracy entailed key elements: jurisdiction, the principle of hierarchy and a clear system of rules, whilst at the same time bringing together qualified, well trained and impersonally selected officials. Written documents were the basis of management and general rules govern the management of the office (Weber 1978; Fountain 2001). Fountain reviews these features, and combines central elements of bureaucratic structures (supervision and standardization) and functions with the potential transformations technology could have on them (summarized on the table below). 3.2.2 Bureaucracy and Technology Elements of Weberian bureaucracy Elements of a virtual bureaucracy Functional differentiation, precise division of labour, clear jurisdictional boundaries Information structures using information technology rather than people; organisational structure based on information systems rather than people Hierarchy of offices and individual Electronic and informal communication; teams carry out the work and make decisions Files, written documents, staff to maintain and transmit files Digitized files in flexible form; IT staff maintains hardware, software and telecoms Employees are neutral, impersonal, attached to a particular office Employees are cross-functional, empowered; jobs are limited not only by expertise but also by computer mediation Office system of general rules, standard operating procedures, performance programmes Rules embedded in application and information systems; an invisible, virtual structure Slow processing time, delays, lags Rapid or real-time processing Long cycles of feedback and adjustment Constant monitoring and updating of feedback; more rapid adjustment possible Table 5: Comparison of Weberian and Virtual Bureaucracies Source: adapted from Fountain (2001, p. 61) Table 5: Comparison of Weberian and Virtual Bureaucracies Source: adapted from Fountain (2001, p. 61) Technology innovation has a potential transformative effect and, as the table suggests, networked technologies have already brought changes in the way public agencies are structured as well as in the way they perform their functions. For example, digital files have replaced old and rigid written files, making them more flexible and less based on people; the same applies to the old slow processing times versus the quicker and real- - Page 63 - time possibilities of communication and feedback that ICT has made possible. These examples, in turn, connect with the properties of automation, informating and communicating presented earlier. In other areas, the changes are less fundamental: even when the internet and ICT have shortened the chain of command, hierarchy has remained central. Networked technologies are, therefore, deeply implicated in rationalisation processes of bureaucracy: (t)he use of the Internet in bureaucracy is likely to lead to greater rationalization, standardization, and use of rule-based system. These rules may not be visible because most of them will be hidden in software and hardware (Fountain 2001, p.62). Overall, and despite the differences that emerge from the table, Fountain asserts that the increased use of ICT has changed but not diminished the importance of bureaucracy: “Bureaucracy, in either rendering, has not diminished in importance” (Fountain 2001 p. 63). 3.2.2 Bureaucracy and Technology This does not mean that new structures and ways of acting and interacting may emerge from the use of networked technologies in government. The bureaucratic capabilities, routines and roles may be conditionings for technology outcomes. This leads us to the next section, and the ways e-government research has illuminated the factors that mediate technology innovation and its outcomes in government organising. 3.2.3 Technology enactment and public sector studies Recall that my research work is built in the socio-technical tradition within e- government and organisation studies more broadly; that is, a social theory view on technology innovation. The socio-technical tradition has a longstanding history within the IS field, although its influence on technology innovation in public organisations has been more erratic. Although the majority of researchers in e-government recognise that the socio-technical features either mediate or influence the impacts of e-government, most lack an explicit use of theoretical frameworks (Heeks and Bailur 2007, p. 254). In what follows, I present the technology enactment model and related research that has contributed towards theory development in the field of e-government. The rationale for - Page 64 - including these theoretical developments here is to expand on the dynamics that underlie technology innovation within the public sector more explicitly. Back in the 1970’s, a programme of research at the University of California, Irvine conducted a project to assess the state of the art in local government computing, to evaluate their impacts on several outcomes and outputs, and to provide recommendations for local practitioners. The research programme produced a collection of cases contributing to theory development, and published as a book in 1982 (Danziger et al. 1982). In the book “Computers and Politics”, Danziger et al. (1982) touches several aspects of today’s well know socio-technical systems. First, the authors find that computing did have distinctive politics, which contradicted the dominant managerial rationalism views that considered computing as apolitical. Second, while computers seemed to have improved the work conditions of government employees, technical problems also arose leading to the interruption and inconveniences for the operation of many governments. Finally, there has been little evidence to support that computing has increased service delivery to citizens; on the contrary, the uses of computers have supported mainly existing internal administrative practices and practices of bureaucratic control (Danziger et al. 1982, p. 221-3). The authors conclude stating that the extent to which redistribution of computer access and benefits is achieved depends upon the will and interests of powerful stakeholders, such as policy makers, managers, and computer specialist (p. 244). Further research expanding on similar issues was carried out in a Symposium on Public Management Information Systems in 1986 (i.e. Kraemer and King 1986, Bozeman and Bretschneider 1986) and later commented by two authors coming from the same socio-technical tradition (Kraemer and Dedrick 1997). 19 Kraemer and Dedrick (1997) conducted a review focused on empirical research in computing in public organisations as a follow up from the 1986 Symposium on Public Management Information Systems. The article’s goal was to update empirical research dealing with the ways public organisations manage information technology, as well as the several impacts technology have on organisational variables such as effectiveness, employment, and organisational politics. In that occasion, the authors remarked that: “the most striking weakness of current research, in comparison with that of a decade ago, is its sheer paucity.” (1997 p. 106) Yet, its slow progress was not the main concern, as they proceed: “we were hard pressed to find in die leading journals of public management, information systems, or business administration many research articles about computing in public organizations.” (p. 107) My own review of the literature suggests a similar pattern. For example, for the period 1980-2010, I found only 4 articles in the MIS Quarterly that dealt with technology in public organisations seriously while there were literary no articles in ISR. On the other hand, with few exceptions, the literature within public management and political science tend to either black-box technology or consider it marginal (Dunleavy and Margetts 2010). 3.2.3 Technology enactment and public sector studies Despite the internet era, the advances that this socio-technical approaches brought in terms of theorising different impacts of computing in public organisations was somehow marginalised.19 - Page 65 - More recently, Jane Fountain developed what she called the “Technology Enactment Theory” (Fountain 2001). In her book, Building the Virtual State, the author develops a an analytical framework to understand the adoption of information technology in the public sector. Bringing together notions from institutional theory, organisational networks and Giddens’ structuration theory Fountain’s work proposes theoretical lenses for understanding how public organisations, technology and institutions interact. The analytical model considers that objective technology (this includes the internet and other networked computing systems) are adjusted and modified when it enters into a given organisational setting. Those organisational forms affect and are affected by existing institutional arrangements. Thus, enacted technology is the perception, design, and use of objective technologies, once the two processesorganisational forms and institutional arrangementhave already ‘filtered’ them (Fountain 2001, chapter 6). The next figure summarizes Fountain’s model. Figure 3: Technology Enactment Framework (Fountain 2001) Figure 3: Technology Enactment Framework (Fountain 2001) Fountain’s work “illuminates the critical role played by socio-structural mechanisms in organisational and institutional arrangements as public managers struggle to make sense of, design and use new IT” (2001, p.89). Both, information technologies and - Page 66 - organisational and institutional arrangements affect each other reciprocally. In the context of her study, this means that even though in theory e-government programmes are designed following a broader reform agenda (the USA National Performance Review), the technology enactment model predicts that its outcomes are unclear and uncertain a priori (Fountain 2001, p. 98). Because there are highly complex social processes embedded in the technology enactment, the outcomes of technology enactment are said to be multiple, unpredictable, and indeterminate; “outcomes result from technological, rational, social and political logics” (2001, p.98). Fountain’s technology enactment framework has been largely acknowledged within the field, but it has been criticised too. Some scholars neglected its originality arguing that the author failed to acknowledge existing research and findings from public administration and information technology studies (Bretschneider 2003, Garson 2003, Norris 2003). More precisely, the critics referred to the insights from socio-technical systems theory, in which end users, existing organisational processes and structures among other variables affect the ways technology is implemented inasmuch technology may affect these variables (Norris 2003, p.419). 3.2.3 Technology enactment and public sector studies Despite the critics, Fountain’s work remains a remarkable effort—both theoretically and empirically—in e-government research (Danziger 2004, Yildiz 2007). There may be aspects that remain under-theorised or not tested in Fountain’s work. For example, the technology enactment framework tells very little about how different agents involved in the process of technology enactment overcame institutional constraints (Yang 2003). In addition, the political nature of technology selection and design are not discussed in her model (Cordella and Ianacci 2010). These are important aspects that can be studied through CDA, as I show later. In sum, I draw on various theoretical aspects of Fountain’s work as well as other insights from socio-technical tradition to illuminate the dynamics and mechanisms that underlie technology innovation in government. In the next section, I bring together CDA, e-government and public values theory and present the overview of my ‘theorising device’. - Page 67 - 3.3 Theorising technology innovation and public values through CDA 3.3 My research is concerned with eliciting the underlying public values that are presented and eventually enacted in processes of e-government over time. A research concern with unfolding the relation between public values and technology innovation entails understanding how these discourses are enacted and operationalised into technology and new ways of acting and interacting (Fairclough 2005, p. 934). On the importance of studying the role of language in governing, Miller and Rose (2008, p.29) state that in order “to analyse the conceptualizations, explanations and calculations that inhabit the governmental field requires an attention to language.” They express that language and politics (and I would add ideals on public values) is a mutually constitutive relationship (Miller and Rose 2008, p. 30-31): For it is in language that programmes of government are elaborated, and through which a consonance is established between the broadly specified ethical, epistemological and ontological appeals of political discourse –to the nation, to virtue, to what is or is not possible or desirable- and the plans, schemes and objectives that seek to address specific problematizations within social, economic or personal existence. As for the purpose on my dissertation, I claim that CDA, in conjunction with the particular theorisation of technology innovation and public values that I offer, is a suitable approach to address my research questions. In other words, CDA offers both a useful theoretical framework and a practical methodology to study the relation between public values and technology within a historical context. CDA is essentially open to a transdisciplinary focus. As for matters on public values, I have already shown that there is no universal consensus on public values, as there are no universal truths (Sayer 2000, Reed 2005, p. 1623). Public values take meaning in a particular historicity, social practices, space and time that make them an outcome of ethico-political complexes (Willmott 2010). - Page 68 - As per technology innovation is concern, I have shown the particular properties of technology, as well as the particular ways of theorising the ‘IT artifact’ that are relevant to my study. As Orlikowski and Iacono suggest (2001, p. 129), there is much potential in seeing technologies and organisations as mutually dependent and dynamically emergent, as a way to move beyond relatively simple black-boxed views of technology (i.e. tool view). 3.3 Theorising technology innovation and public values through CDA NPM, competitiveness), can be invoked to support the production of bodies of knowledge inside the organisation Imbued in/draws from Discourse Level (Meso level): public values and e-government as discourse in a specific organisation Reproduces/transforms Reproduces/transforms Imbued in/draws from Text Level (Micro level): organisational members produce text and talk about public values and technology 3.3 Theorising technology innovation and public values through CDA That is, the material properties of technology innovation and the dynamics of technology enactment in the public sector suggest a particular set of conditionings as well as imaginaries on their associated public values. In turn, I have also presented that there are certain conceptions about public values and technology that emerge from the literature (Chapter Two). Hence, these normative views can be reasonably taken as part of broader discourses on public values and e-government. Based on Fairclough three-dimensional analysis, complemented with Phillips et al (2008, p. 781, and Vaara (2010, p. 7), the theorising device for studying the relation between public values and technology through CDA is presented below. In summary, I hypothesise that there is a dialectical relationship among discourse, public values and technology: “the meanings of discourses are shared and social, emanating out of actors’ actions in producing texts; at the same time, discourse gives meaning to these actions, thereby constituting the social world” (Phillips et al. 2004, p. 637). Discourses at the social level imbued meanings and practices at the level of the organisation, which in turn shape the way particular actors, produce text and talk. The schema is a simplification and does not necessarily accounts for time. I preferred to highlight how particular discourses at the societal level shape a given organisational discourse, that in turn influences the production of text and talk at the micro level. - Page 69 - Social Level (Macro level): public values, e-government and modernisation as body of knowledge: Discourses that exist outside the organisation (i.e. NPM, competitiveness), can be invoked to support the production of bodies of knowledge inside the organisation Imbued in/draws from Reproduces/transforms Discourse Level (Meso level): public values and e-government as discourse in a specific organisation Imbued in/draws from Reproduces/transforms Text Level (Micro level): organisational members produce text and talk about public values and technology Social Level (Macro level): public values, e-government and modernisation as body of knowledge: Discourses that exist outside the organisation (i.e. NPM, competitiveness), can be invoked to support the production of bodies of knowledge inside the organisation knowledge: Discourses that exist outside the organisation (i.e. Figure 4: Theorising public values and technology innovation through CDA To take account for the ways discourses on public values and technology are operationalised, requires an understanding of text production, interpretation and consumption. I will follow these concepts in the analysis to trace how discourses travel downwards and upwards accordingly. It is in the materialisation of discourses that I will draw on the particular properties of technologies, discussed previously. All in all, a CDA approach—and the particular transdisciplinary version outlined here— becomes relevant to my study in the following ways: - Page 70 - - Page 70 - - Working in dialogue with the theories of public sector reforms, public values and technology in government helps to define relevant categories of analysis (i.e. public value and e-government clusters) (transdisciplinary approach); - Following the three-dimensional approach, the conceptual categories combined with textual analysis and the extensive data gathered as part of the fieldwork (inter-discursive analysis) can help eliciting which values were represented in key texts as well as what consequences and actions they triggered within the project; - Finally, as a consequences of the two previous points, a CDA approach can illuminate the analysis on the linkages between public values and technology, in a particular context and over time, to add to our understanding of processes of technology innovation and change in government. 3.4 Conclusions As means of bringing together the choice of concepts that I comprise in my theorising device (or framework), Gregor (2006) offers a useful framing. In her essay, the author discusses the nature of theory in information systems research and addresses issues of causality, explanation, prediction and generalizability within the field. Gregor (2006, p. 620) suggests that there are multiple perspectives on the role of theory in information systems, each comprising its own set of assumptions and versions on their structural components, such as means of representation, constructs and scope (p. 620). Based on the different goals of the research, Gregor proposes a taxonomy to classify different types of theory. The author argues that there are five types of theory: theory for analysing, for explaining, for predicting, for explaining and predicting, and for design sciences. Given my research purposes, I draw on theories that fall in the first two categories. That is, I am interested in identifying and analysing what types of public values are presented in the case, but also to understand them in an analytically useful way. For this purpose, I draw on public values theory and the concepts I presented in Chapter Two. More precisely, the role of public values theory has been in framing what public values are, where they can be found and what sorts of public values are commonly found in procedural oriented agencies that deal with e-services. This second - Page 71 - - Page 71 - part has been an insight from empirical findings from e-government research. I claim that the role of CDA in my work—in conjunction with e-government theory and the theorisation of technology innovation in the public sector—not only helps to explain and analyse what public values are and how they relate to technology, but also to explain and understand how the relation is, how it evolves over time, when and in what context. For this reason, my theoretical framework falls partly in theory for explaining, which can be best regarded as “theory for understanding” in Gregor’s (2006, p. 624) terms. My objective in this chapter has been to put different sets of theoretical concepts from different fields into dialogue to better understand the linkages between public values and technology innovation in the public sector through the role of discourse in organising. 3.4 Conclusions All in all, I placed emphasis on theory that can help to describe, analyse and explain the content of the relation between public values and technology innovation in as well as its dynamics over time and in a given context. The theorising device that I presented in this chapter lays the foundations for the longitudinal case that I present later on. But before that, I shall provide an extensive description on the methodological foundations that I have followed in my research. - Page 72 - 4 Chapter Four: Methodological underpinnings: case study research design while CDA does in a sense constitute a method of analysis, the methods employed in any specific piece of research which draws upon CDA are likely to be a combination of those of CDA and others Norman Fairclough 2003, Analysing Discourse The choice of a discursive lens—CDA—to study the relation between public values and technology innovation led to particular methodological arrangements. This chapter explains the ways I have designed and implemented my empirical investigation. It is important to stress that my overall research design place CDA as part of a longitudinal case study. While some authors categorise the case study as a method (i.e. Crotty 1998, p.5), in my dissertation, I understand case study as an overall research strategy that draws on different data collection techniques and that relies on critical discourse analysis (CDA) as the main method for analysis. Therefore, in this chapter, I elaborate on the scope, data collection and data sources and the particulars of the data analysis that I have conducted. To give structure to explain my research design, in section 4.1, I elaborate on the philosophical assumptions that have inspired my theorising effort. In section 4.2, I explain in detail my overall strategy of inquiry—that is, my longitudinal case study. The section is divided in three subsections intended to cover the relevant aspects of the reasons for the selection of the case, my data collection and data sources. Section 4.3 revises extensively on the particular linguistic and semiotic techniques that I have used to reveal the discourses on public values and technology within the case. In Section 4.4, I provide two related rigour checks for my research strategy: on case study quality and protocols for conducting CDA. Finally, I include a final reflection in section 4.5. - Page 73 - 4.1 Philosophical assumptions 1 Philosophical assumptions 4.1 The previous chapters offer some general ideas of the relevance of critical realism as the underlying philosophy of the critical discourse analysis version that I use on my work. By adopting a critical realist position, this dissertation rejects the idea that organisations are just discursive constructions and cultural forms with no significance beyond their textually created and mediated existence (Reed 2005, p.1622)—or in extreme terms, that everything is discourse. The underlying philosophy adopted here sees discursive practices as formations that, while being socially constructed in one part (i.e. linguistic arrangements), are subject to historical and structural variations related to the particular temporal and spatial contexts in which they are constituted and developed (Fairclough et al. 2002, Fairclough 2005a, Reed 2005, p. 1623). In other words, as Reed (2005, p.1623) suggests, this philosophy rejects the empiricist and positivist quest for universal principles, and favours a more relativistic conception of knowledge production and dissemination. In words of the author: All theoretical descriptions, explanations and evaluations are grounded in knowledge generating and diffusion processes that are temporally and spatial located in historical and social settings that makes them fallible, contested and revisable. But this does not legitimate a postmodernist discursive reductionism in which scientific research, theory generation and explanation are reduced to rhetorical or linguistic constructions that have no reference to or anchoring in an independently existing world. Such an idea calls for a realist stance, instead of an extreme social constructionist view, in which social structures are collapsed into discursive agency. Critical realism assumes a ‘stratified ontology’, which sees processes, events and structures as different layers of social reality with distinctive properties. This implies seeing life as an ‘open system’, with various dimensions and social elements that are governed by simultaneous generative mechanisms (Bhaskar 1986 as appears in Chouliaraki and Fairclough 1999, chap. 2). In turn, no mechanism has determinate effects on events because the operation of any mechanism is always mediated by the operations of others; events are complex and no predictable in any simple way (Chouliaraki and Fairclough 1999, p. 19). These mediating instances are the social practices that are central to CDA. “The reason for centring the concept of ‘social practice’ is that it allows and oscillation between the - Page 74 - - Page 74 - perspective of social structure and the perspective of social action and agency—both necessary perspectives in social research” (Fairclough 2003, p. 205). 21 Reed (2005, p. 1631) offers a succinct definition on this concept: “[abduction] aims to reconstruct, re- interpret and deconstruct, the tacit meanings and motives through which relationships and actions are discursively constructed and represented.” This idea is broadly used in empirical studies on strategy, such as in Vaara et al. (2008), Vaara and Monin (2010), Vaara et al. (2010). 20 Usually, references to constructionism and constructivism are far from consistent in the literature of social science research (Crotty 1998, p. 57-58). Constructionism (as the epistemology I am using) is the view that “all knowledge, and therefore all meaningful reality as such, is contingent upon human practices, being constructed in and out of interaction between human beings and their world, and developed and transmitted within an essentially social context” (Crotty 1998, p. 42, italics in the original). This is different to what Reed (2005, p.1622) mentions as a ‘radical social constructionist’ philosophy (post-structuralist-postmodernist informed), which views organizations as discursive constructions and cultural forms that have no ontological status beyond their textually created and mediated existence. The latter is usually associated to extreme constructivist views on discourse in organising that are not adopted in my research. 1 Philosophical assumptions Critical realism has increasingly inspired research in organising and management studies (Ackroyd and Fleetwood 2000, Reed 2001, 2005, 2009, Ackroyd 2009, O’Mahoney 2011) as well as in information systems research (Mingers 2004, Smith 2006, Volkoff et al. 2007, Zachariadis and Scott 2007, Hevner et al. 2010, Mingers and Walsham 2010, Mutch 2010). Yet, the potentials to realise a critical realism ‘turn’ are far from being fulfilled(Mingers et al. 2011 in their call for papers). In my research, critical realism is better acknowledged as a philosophical platform that accepts elements of interpretivism and constructionism (not constructivism, which is different)20, such as the ‘abductive’21 data analysis method that I will describe below (Mingers et al. 2011 in their call for papers). As Sayer (2000, p. 32) suggests, critical realism “has a relatively open or permissive stance towards epistemology” (that is, the nature and scope of knowledge). Accordingly, Crotty (1998, p. 63) provides a clear stance to clarify for point further: “constructionism in epistemology is perfectly compatible with a realism in ontology—and in more ways than one”. In other words, when I suggest that a meaningful reality is socially constructed, I do not mean that it is not real. My main point here is to show that critical realism is the philosophical platform that underlies the specific research design and the theoretical constructs that I bring together in my dissertation. More specifically, I refer to the dialectic role of discourse and semiosis in organising processes (Fairclough et al. 2002, Fairclough 2005, 2010), the relational character of technology (i.e. Volkoff 2007, Mutch 2010), and the contextual and relative nature of public values (Bozeman 2007, Beck Jørgensen and Bozeman - Page 75 - 2007, Willmott 2010). For the particular uses in the CDA approach that I follow, Fairclough (2005, p. 935) crystallises the value of relying on a critical realist perspective to study aspects of organisational change: a commitment to discourse analysis in organizational studies entails neither a reduction of organizations to organizational discourse, nor a reduction of organizational analysis to the ‘organizing’ (...) Discourse analysis is consistent with a realist approach to organizational research which distinguishes organizational process and agency from organizational structures, and focuses research on the relations and tensions between them. Incorporating discourse analysis into a realist approach both ensures that questions of discourse are properly attended to in organizational studies, and avoids these forms of reductionism. 1 Philosophical assumptions By suggesting the compatibility between constructionism in epistemology and realism in ontology, I do not intent to conflate different epistemologies and assumptions. Rather, my aim is to be transparent about my inspirational theoretical and methodological sources and to show the ways I have made use of them in my dissertation. In what follows, I present the particular methodological choices that govern my strategy of inquiry. 4.2.1 Longitudinal case study My dissertation builds upon empirical fieldwork conducted in Mexico and follows a single, in-depth longitudinal case study strategy, which is informed by a critical discourse analysis approach. In general terms, a case study strategy is chosen because of its appropriateness for asking in-depth how, and why questions, in which no experimental control is possible (Yin 2003, p. 5, Myers 2009, p. 73). In his book on qualitative research, Myers (2009, p. 76) defines case study research as one that “uses empirical evidence from one or more organisations where an attempt is made to study the subject matter in context” (emphasis added). Given my interest in analysing discourses on public values and technology, choosing a qualitative case study allowed me to conduct a detailed and multifaceted analysis of various texts and talk that happened within a given context of social practices (Fairclough 2005a, Vaara and Monin 2010, Vaara et al. 2010). - Page 76 - - Page 76 - Myers (2009, pp. 72-3) explains that case studies can be used in exploratory research (to discover) and in explanatory research (to test, to explain, or to compare). Defined as an overall research strategy for my dissertation, I use my case study in both ways: to discover what discourses on public values and technology can be traced in a particular context, and also to explain how these discourses are produced, consumed and enacted in the process of organising, and why this happens.22 The next chapter (Chapter Five) presents a detailed account of the Mexican case, its context and process features, which I elaborate in narrative terms. In brief—and as I already anticipated in the introduction—the study is situated in the Mexican central government sector and in the area of foreign trade regulations. Mexico’s central government is comprised of several agencies that are coordinated in a Cabinet, which is accountable to the President. My entry point to the case is the Ministry of the Economy, a federal government unit that is responsible for trade policies in the country, their operation and administration. The Ministry of the Economy is, in broad terms, the main unit of analysis. 24 Within the category of single-case study designs, Yin (2003, p.39-40) makes the distinction between holistic and embedded cases. The former involves a single unit of analysis, while the latter comprises more than one unit, or subunits. According to this, the process of design and implementation of the Single Window and its previous project —SICEX— could be interpreted as single case design with embedded units of analysis. However, Yin (2003, p.25-26) itself deals with the difficulty of defining the unit of analysis. Thus, in broad terms, my unit of analysis can be defined as the Ministry of the Economy, and in particular, the part of the Ministry that deals with trade regulation. From this perspective, both SICEX and the Single Window are developing cases within the same unit of analysis., and that is why I do not consider them as separate embedded units. 23 In the literature, single windows are also referred as one-stop-shops. 22 This links with the conclusions of Chapter Three, where I discuss the role of theory based in my research based on Gregor (2006). 23 In the literature, single windows are also referred as one-stop-shops. 4.2.1 Longitudinal case study Within this Ministry, I study the processes underlying the design and pre-implementation of a technologically enabled Single Window for Foreign Trade regulations (hereafter Single Window)—in Spanish, Ventanilla Digital Mexicana de Comercio Exterior.23 The design and pre-implementation phases of the Single Window started formally in March 2008 and inaugurated by the President in January 2012. I therefore pay special attention to this four-year period (2008-2012). In addition, I elaborate on a previous and smaller technological platform for the internal operation of trade regulations that the Ministry of the Economy designed and implemented since 1996 called SICEX. I do not understand SICEX as a separate unit of analysis, but rather as the background for the Single Window.24 Given the timeframe covered— - Page 77 - retrospectively and in real time analysis—I refer broadly to a longitudinal case study, which maps out key events within a historical context to reveal the essential discourses and social dynamics underlying the case. As I shall demonstrate in the analysis chapter (Chapter Six), I will be using data and events from the implementation of SICEX systematically to analyse the trajectories of the discourses on public values and technology, and their production and consumption over time. In sum, in temporal terms, the case covers a 16-year period (1997-2012). 25 This is what Flyvbjerg (2006, p. 231) describes with his own experience studying urban politics and planning in Denmark. What it first started as a critical case (theory testing type), his research findings led him to end up with an extreme case. 4.2.2 Reasons for the selection of the case study Flyvbjerg (Flyvbjerg 2006, p. 229) suggests that the selection of a case may play a significant role for theory development, theory testing and generalizability. In either case, the author suggests, the case selection should be consistent with the purpose of the research. A general distinction is based on the type of selection; that is, depending on whether the objective of the selection is to find average or typical cases on one hand, or to achieve very rich accounts on a particular problem or phenomenon on the other, the researcher may go for random or information-oriented selection respectively. Yin (2003) offers a similar rationale for single case studies selection. According to the author (Yin 2003, pp. 40-2), a single case design can be: (a) critical, (b) extreme or unique, (c) representative or typical, (d) revelatory or (e) longitudinal. Each of the designs, in turn, provides different types of exemplars for theory testing, theory building or exploration. Although the selection criteria seem useful, researchers must also count on their intuition and pragmatics. For example, gaining and maintaining access to the site requires good social skills (Walsham 2006, p. 322), regardless of the type of study to conduct (i.e. interpretive, positivistic or critical). In addition, scholars have also suggested that even when choices may be deliberately made at the beginning (i.e. selecting an extreme case), the researcher’s involvement and understanding of the case may lead to a change at the end (Flyvbjerg 2006, p. 231, Yin 2003, Myers 2009).25 - Page 78 - - Page 78 - The way I selected the case in Mexico was mainly based on the potentials that, at that time, the case offered. Reflecting backwards, the case I study is a combination of what Yin defines as longitudinal and revelatory. It is longitudinal as it entailed looking at a series of historical events to map how discourses were produced, enacted and transformed over a long period of time. It is also revelatory from the point of view that the processes were not previously studied. Perhaps more closely, the case falls into significant case in the sense of what Yin (2003, p. 162) identifies as important in the construction of an exemplary case study: “the underlying issues are nationally important, either in theoretical terms or in policy or practical terms”. 4.2.2 Reasons for the selection of the case study As I shall explain in more detail in Chapter Five, the Mexican Single Window is exemplar in the sense that (i) public values imbedded in the case are, in theoretical and practical terms, of national importance; (ii) foreign trade in Mexico is the engine for economic growth, and thus, of national importance. The selection of the case, by all means, represents only the first stage. Below, I provide details on the data sources and data collection techniques that I have followed. 4.2.3 Data collection and data sources I am interested in reconstructing actual episodes and processes in the case study that can be helpful to reveal the presence and trajectory of public values and its relation to technology. Immersion in context, in turn, has been a hallmark of my study (Kaplan and Duchon 1988, p. 572). For this purpose, I followed data collection strategies that are complementary (Langley 1999, 2009) and particularly useful for critical discourse analysis purposes. These include observations in vivo (meetings, informal conversations, and recording events), collecting memories and interpretations (semi-structured interviews, news articles) and collecting temporally-embedded artifacts (several internal plans and reports, internal presentations and documents). As in other studies (i.e. Pettigrew 1990), time is captured as a combination of retrospective and real time analysis. Real time analysis included an initial 8 month- exploratory study that I conducted from August 2006 to March 2007 in the Ministry of the Economy, which mainly served as the basis for understanding the complexities of trade regulation and the ideals behind the construction of the Single Window. Most data - Page 79 - collection relevant for the current research took place during a fieldwork trip in spring of 2009 (March to June), and then again in the spring of 2010 (May to June). Follow up interviews were conducted over the phone since then. All materials were recorded and kept in their original language (Spanish). Part of the fieldwork entailed conducting 42 in depth interviews with 35 single individuals26, most of them face to face, with an average time of one hour to one hour and half per interview. Appendix I and II provide extensive supporting data on the type of informants, length, and moment in the case that I have conducted the interviews. Whil it I k t d il f th i fl ti i f th Part of the fieldwork entailed conducting 42 in depth interviews with 35 single individuals26, most of them face to face, with an average time of one hour to one hour and half per interview. Appendix I and II provide extensive supporting data on the type of informants, length, and moment in the case that I have conducted the interviews. While on site, I kept a daily summary of the main reflections emerging from the empirical work and then used when recapping the case study. Direct observation also provided a further resource to my research inquiry. 27 Texts are seen as part of social events; some social events are textual in character (an interview, a recording of a litigation) (Fairclough 2003). To make sense of discourse, using CDA, the researcher needs to rely on very diverse forms of semiosis. Therefore, while in some disciplines text may be treated as secondary data, in my work, they became almost the primary data of research. Therefore, the distinction does not hold. What I see as important is to be transparent on my data sources and collection methods, and the ways I have analysed the data in my work (Leitch and Palmer 2010). 26 I interviewed the champions of the project more than once and in some instances and there were more than one person in the interview. Please see Appendix II for a detailed list of the interviews conducted as part of the research. 4.2.3 Data collection and data sources During fieldwork I spent most of the time working from an office within the Ministry of Economy. I participated in three key meetings during spring 2009, where the plans and design of the Single Window were under way. On average, these meetings lengthened two hours and a half. While no recordings could be made, I collected internal power-point presentations and kept written notes of key discussions, issues and questions as part of my fieldwork diary. Given the fact that my focus is on the organising processes of government and how their actors produce, interpret and enact discourses on public values and technology, most of my data sources come from documents and talks with the government officials directly involved with the project. In addition, I also use media articles from local newspapers and private sector associations as a way to approximate the public’s perceptions on the subject. The next table summarises the main sources of data that were collected and used in my research inquiry.27In Appendix I, II and III, I cover in detail the sources of my data and the reader shall refer to those in order to complement this section. 4.2.3 Data collection and data sources - Page 80 - Key texts analysed in depth (textually oriented analysis) The Presidential Decree that establishes the creation of the Mexican Single Window for foreign trade (January 2011) Memoirs of ‘Towards a paperless administration 2000-2006’ (an internal document reporting the progress on the previous IT platform from 2000-2006) History of SICEX (1999): an internal document of a previous and similar project in the Ministry The Presidential Decree that establishes the creation of the Mexican Single Window for foreign trade (January 2011) Memoirs of ‘Towards a paperless administration 2000-2006’ (an internal document reporting the progress on the previous IT platform from 2000-2006) History of SICEX (1999): an internal document of a previous and similar project in the Ministry Interviews and meetings 42 semi-structured interviews (of 1 and half hour on average), with 35 single individual 3 meetings with the Single Window team in 2009 (early phases of the design; 3 hours on average each) Other documents (selected examples) Terms of reference of the open public tender for the Single Window sponsored by Customs (September 2010) 20 internal power-point presentations in electronic format that supports the official strategy and objectives of setting up the Single Window 15 official presentations and speeches from the leaders of the Single Window that emphasise goals, benefits and plans (mainly from ME and Customs, from 2010 and 2012) 75 informative sheets downloaded from November 2011 until June 2012 from the Single Window website 95 news articles from local newspapers, covering the period from January 2009 to June 2012 Mexico’s National Development Plans (1995-2012) Mexico’s Modernisation programmes (several texts) Other data Pilots and evaluations conducted by the researcher in 2006 as part of a broader study (Telecom-CIDE 2007) Table 6: Summary of data collections and sources See Appendix I, II and III for a detailed description of data collection sources and data corpus. 4.3 Method of analysis: critical discourse analysis in practice 4.3 The aim of this section is to explain the main strategies used for coding my data. For clarity purposes, I first explain the main phases I have gone through and I then elaborate on the particularities of the main linguistic features and relevant tools of critical discourse analysis that I have drawn upon. The aim of this section is to explain the main strategies used for coding my data. For clarity purposes, I first explain the main phases I have gone through and I then elaborate on the particularities of the main linguistic features and relevant tools of critical discourse analysis that I have drawn upon. 28 Later on, I elaborate in detail the specific CDA tools that I have applied in the coding phase. Other data Pilots and evaluations conducted by the researcher in 2006 as part of a broader study (Telecom-CIDE 2007) Table 6: Summary of data collections and sources Partly because of the way I apply CDA on the case but also because of my concern with the case quality, my research design included multiple data collection methods and sources of data. For some scholars, case study research does not normally involve participant observation or extensive fieldwork (Myers 2009, p. 77). As far as CDA is concerned, a serious analysis requires going beyond the level of texts to include further empirical sources to make sense of the discourses, their production and ways to interpret them (Fairclough 2003, 2005, 2010, Chouliaraki and Fairclough 1999).Given the emphasis on the operationalisation of discourses as well as in constructing a meaningful context, I draw heavily on the fieldwork I have conducted in Mexico. For example, in addition to the analysis of key government texts, the interviews offered many insights on the aims, drivers, and motivations to drive technology innovation in government. That is, interviews proved to be crucial in interpreting discourses on public - Page 81 - - Page 81 - values and technology innovation through CDA. I shall offer a more detailed use of the data in the following section 4.3. 4.3.1 From data to coding discourses Even though I kept a diary and made analytical reflections while on site, most analysis happened after the main data collection was finished. Following Fairclough (2003, 2010) and the application of his approach elsewhere (i.e. Vaara et al. 2010, Vaara and Monin 2010, Vaara and Tienari 2008), the analysis I have conducted proceeded in stages, in an overall reflexive manner. The first stage involved the identification of the empirical sources to build the data (textual) corpus. As a result, the main empirical material included several key official documents from the government, the narratives from my fieldwork diary, interviews and the notes from the meetings that I attended. With the corpus of data already being built, in the second stage I constructed a detailed timeline of the main events, episodes, project features and contextual factors covering the period 1995-2012. The construction of the timeline—which I introduce in the next chapter—was particularly helpful as a visual mapping tool (Langley 1999, 2009) to prepare the narrative of the case as well as to identify the main textual materials to trace relevant discourses. As discourses are historically located (Parker 1992), the timeline was the first strategy used to examine how and where they emerged from. Then, I carried out substantive coding, both on the individual texts as well as the aggregate compilation of materials.28 - Page 82 - I used Atlas.ti qualitative analysis software to code my materials as it helped ensuring a systematic analysis of representative instances of data (Seale and Silverman 1997, p. 380).29 I initially approached the texts following an open coding strategy (Titcher et al. 2000, Corbin and Strauss 2008, p. 59), and identified those recurring themes related to the linkages between technology and public values, including common lexical terms (i.e. efficiency) as well as common empirical patterns that repeated across the corpus. As in the case of research studies inspired in grounded theory methodology, I went line by line and coded all the relevant texts from my corpus. While the initial coding was useful to familiarise myself with the evidence and main themes, I re-coded my materials (particularly, the key texts) using the list of public values that I presented in Chapter Two based on the work of Bozeman and Beck Jørgensen (2007) and Hood (1991). 29 I include in the Appendix a screen shot of a sample coded material. 4.3.1 From data to coding discourses This made me redefine and adjust my coding accordingly, usually, by dropping some values from the list, and defining their meaning as I was interpreting the discourses (see later my sections on CDA tools). This re-coding strategy served to reflect on the categories of public values that would bring more order to my data. In terms of CDA, a further stage meant scrutinizing key texts in-depth while coming back to other data from the fieldwork (i.e. interviews) to reflect on the process of production, dissemination and consumption of discourses over time. In sum, the coding phase meant going back and forth between the theoretical ideas—public values, relational character of technology and CDA—and the empirical analysis to produce an elaborate understanding of the discursive (and material) features involved in the case. The coding phase also guided the incorporation of further data into the corpus, such as news from local media and follow-up interviews conducted in later stages. A final stage consisted of writing up the findings and reflecting on the basis of the theoretical underpinnings of my research. All in all, the steps that I followed on my data analysis led to the identification of three complementary analytical instances that are presented in Chapters Five, Six and Seven respectively: (i) the construction of the narrative of the case, its historical, political and institutional context; (i) - Page 83 - (ii) the analysis of the dominant discourses on public values and technology over time and in a particular context; (ii) the analysis of the dominant discourses on public values and technology over time and in a particular context; (ii) the analysis of the dominant discourses on public values and technology over time and in a particular context; (iii) the broader discussion of the findings in light of theory. In light of my data analysis strategy, Chapter Five presents more than a simple description of the case; in building the historical socioeconomic background of Mexico, I found myself better informed to understand and elucidate the most prominent discourses, and how they were produced. In addition, it was by writing Chapter Five that the Single Window initiative turned out to be about producing and consuming the context dynamically, instead of being just the result of a given context (i.e. Hayes and Westrup 2012, Salskov-Iversen et al. 2000, Chouliaraki and Fairclough 1999 on recontextualisation, Avgerou 2002, p. 10). 30 Fairclough wrote this book for social science scholars that are not experts in linguistic analysis. As the author says (Fairclough 2003, p. 1), “I find that research students in Social Sciences often see the need to say more detailed things about their language data than they feel equipped to do”. His book has been particularly useful for guiding the textual oriented part of my discourse analysis. 31 I have anticipated this idea in the elaboration on theory (Chapter Three), which is also important in methodological terms. 4.3.1 From data to coding discourses In light of my data analysis strategy, Chapter Five presents more than a simple description of the case; in building the historical socioeconomic background of Mexico, I found myself better informed to understand and elucidate the most prominent discourses, and how they were produced. In addition, it was by writing Chapter Five that the Single Window initiative turned out to be about producing and consuming the context dynamically, instead of being just the result of a given context (i.e. Hayes and Westrup 2012, Salskov-Iversen et al. 2000, Chouliaraki and Fairclough 1999 on recontextualisation, Avgerou 2002, p. 10). Reporting my findings on discourses (Chapter Six) has been extremely important in terms of the internal validity of the case as well as for theoretical reflection. While writing, I had to decide on the most important and prominent discourses and leave aside a great number of interesting but disparate details. The findings, thus, present the most prominent discourses, and the tensions and relations that I found between them that were significant to answer my research questions. This chapter draws heavily from the analysis of key text presented in table 6 earlier, as well as on interviewing material and related sources to make sense of the dynamics of the discursive features in the case. As I expressed earlier, CDA does not rely solely on texts—fieldwork material is crucial for an understanding of text production, interpretation and consumption in the case context. Finally, while described as stages, the reader should not understand them as linear. As it is common in CDA, these stages are reflexive and involve an on-going process of going back and forth between empirical and theoretical concepts, which end when there is sufficient evidence to pull out a series of findings. The process of coding and re-coding and searching for meanings using CDA tools have played the most substantial part in reporting my findings and inspiring the discussion chapter (Chapter Seven). In sum, the richness of the data and the multiplicity of sources that I draw upon are vital for the reflexive process that a serious CDA effort requires. Overall, as in similar cases, analysing materials resulted in a complex interplay between observing perceived patterns - Page 84 - - Page 84 - and theorizing, a research process that goes beyond the application of critical discourse analysis (Townley et al. 2003). 32 In legal terms, a Presidential Decree in Mexico is similar to the Executive Orders in the United States. Decrees in Mexico are issued by the head of the executive branch at some level of government; commonly, the Decrees are issues by the president, but they may also be issued by a head of Ministry or at state level by a state's governor. The text that I analyse has been issued by the President and endorsed by all Ministries. 4.3.2 CDA tools: selections of key texts, intertextuality and genre analysis Scholars have noted that there are many versions of discourse analysis (see for example Phillips and Di Domenico 2009). One of the notable differences between them has to do with those approaches that include detailed analysis of text to those that pay little attention to it. Critical discourse analysis is grouped within the former, and it entails at least certain type of ‘textually oriented discourse analysis’ (Fairclough 2003). My dissertation includes a textual, detailed analysis of texts related to the project I study in Mexico. While my aim is not to depict a detailed linguistic analysis of texts (not that it would even be possible to do that), I draw extensively on the guidelines exposed in Fairclough (2003).30 Let me reiterate that the detailed textually oriented analysis was performed in several texts, though some of them were used in “more intensely”, and that is why I include a section clarifying so. In any case, the number of texts that I have included in the analysis is large in order to increase the reflexivity process that CDA entails (see my previous data collection table and Appendix I and II for further details). It is worth recalling that individual or isolated texts are rather useless to make sense of social reality in general and in discourse analysis in particular (Phillips et al. 2004, p. 636, Fairclough 2003). An important rationale for selecting certain analytical tools among all the diverse analysis that CDA may enable relies on the transdisciplinary spirit of the approach (Fairclough 2010, p. 231, 2003, p. 6).31 As I am interested in eliciting and discovering discourses on public values and technology, I shall draw upon certain linguistic features - Page 85 - that are helpful for this purpose. This is the main reason why I include three sections below that explain in depth how I have worked on my analysis of text and discourses. 32 In legal terms, a Presidential Decree in Mexico is similar to the Executive Orders in the United States. On the selection of specific government texts While a detailed textual analysis of the entire data corpus is far from being possible (see my later comments on the limits and scope of CDA), I selected a key text in the case to anchor my textually oriented analysis: - The Presidential Decree that establishes the creation of the Mexican Single Widow for Foreign Trade, published in January 2011. - The Presidential Decree that establishes the creation of the Mexican Single Widow for Foreign Trade, published in January 2011. This text is selected for three main reasons. The first one has to do with its relevance. The text of the Decree figures as a key moment of the case that is triggered within a particular history and context. The Presidential Decree has a particular functionality; it establishes the Single Window, and as such, it sets forth the conditions, particularities, policy agreements and even the obligations that the agencies (public bureaucracies) involved have to comply with.32 In this respect, the text is particularly important in terms of its textualagency, that is, the power of specific texts to produce effects in organising(Cooren 2004, Hardy 2004). Presidential Decrees are well-known for setting up specific objectives and priorities in presidential regimes, therefore becoming extremely powerful to guide government action. The Decree is also relevant as a good proxy for mapping public values as well. As Bozeman suggests (2007, p. 15), a nation’s more fundamental laws, the public speeches of national or subnational public officials as well as mission statements of public agencies provide excellent sources of public values. A second reason relies on the comprehensiveness of the text. The text of the Decree, somehow “contains” other important texts of the project in it. As I shall demonstrate in the analysis chapter, the selected text is linked to other fundamental textual productions, such as Mexico’s National Development Plans and previous laws and regulations, which - Page 86 - make it a good basis for broader intertextual and interdiscursive analysis. This, in turn, reinforces the point of relevance: other fundamental laws and ideals on public values are included in the text. A third and related reason has to do with its temporality. The text of the Decree represents a particular important moment in the lifecycle of the project. Anchoring the analysis in the moment that the Single Window is established helps to trace back the reasons of its production (i.e. On the selection of specific government texts where are the discourse coming from) as well as the ways in which these discourses have been consumed and materialised since then. The intertextual analysis, in turn, also helps to trace textual artifacts that were particularly useful for a retrospective analysis of the moments of the project. To gain order and structure in my analysis, I usually started in the text of the Decree, and systematically compared it to other key texts in the project, mainly: - ‘SICEX: a successful case for administrating trade procedures’ (dated 1999): an internal document from the SE that describes the origins, main drivers and component of the platform that was launched in 1996 and won the national public administration prize in 1999; - Memoirs of ‘Towards a paperless administration 2000-2006’: an internal, 450 pages-long document that covers the progress and evolution of SICEX and related ICT infrastructure for foreign trade procedures, its main drivers, logics and actions taken in relation to the project; - Terms of Reference, International Public Tender of the Mexican Single Window for Foreign Trade: a public document that established the aims, objectives and types of services open to tender, issued by the Servicio de Administracion Tributaria (SAT) in May 2010; - Mexico’s National Development Plans. Let me summarise the idea of this section. The CDA tools that I have applied on my analysis and the way I preceded with my coding entailed paying attention to certain semiotic structures and verbal features. The analysis I present, thus, is not on a single text; the text of the Decree is simply used as an anchoring point for the reasons I outlined above. And as an anchor, it brings in other important texts that I compare and analyse using linguistic tools. This is why the reader will find different sources of other texts and data(i.e. references to interviews and news articles) in the analysis chapter. - Page 87 - Intertextuality, assumptions and value assumptions Fairclough (2003, chap. 3) offers a number of linguistic tools that are applicable for different purposes when analysing texts. I mainly focused on intertextual analysis—how texts draw upon, incorporate and interact or dialogue with other texts. An obvious example of intertextuality is the use of quotations within a text. But quotations are rather explicit forms of intertextuality that ‘echo’ particular discourses. Besides their direct (i.e. quoted speech) or indirect (i.e. reported speech) presence of elements from other texts, intertextuality helps to identify those elements that are related in different ways, such as dialogued with, assumed or presupposed (Fairclough 2003, p. 39-40, also Parker 1992). It is precisely on the idea of assumptions that I shall be concentrating on, given my focus on eliciting discourses on public values and technology within the project. Implicitness and assumptions are a pervasive property of texts that are especially important for critical discourse analysts, given their implications in building shared meaning or the capacity to exercise power (Fairclough 2003, p.55). Assumptions, as understood here, are divided into three categories: existential, propositional and value assumptions. I am particularly interested in the analytical tools to elicit value assumptions, those assumptions about what is good and desirable. Linguistic tools to identify value assumptions entail looking for certain verbs and textual triggers in the texts. For example, the verb ‘help’ within a sentence (i.e. information technologies can help develop productivity) assumes that developing productivity is desirable (Fairclough 2003, p. 56). Yet, value assumptions may not necessarily be triggered by such explicit cases (i.e. verbs connoting desirability), and one needs to look carefully for the meaning of the sentence. In the case of the texts that I zoomed in, I draw on the idea of family resemblances of values (Rutgers 2008, pp. 106-7). This means that using linguistic tools from CDA, especially semantic relations between words, one can identify terms that, while different, denote similar values. As mentioned earlier, I started my coding following the preliminary inventory of public values of Beck Jørgensen and Bozeman (2007), complemented with the work of Hood (1991) and Hood and Jackson (1991). In CDA words, I used these lists to perform a lexical (vocabulary) analysis of public values and In the case of the texts that I zoomed in, I draw on the idea of family resemblances of values (Rutgers 2008, pp. 106-7). 33 The contextual data include, for example, the historical developments on economic liberalisation and political democratisation transition that I describe in the case chapter. As Fairclough suggests, one may argue that assumptions, and particularly value assumptions, are associated with ideology (2003, p.58). However, he warns that in order to produce such a claim, one need to go beyond the level of texts and show how a particular assumption is indeed taken for granted at societal level, or a certain actors’ level. In other words, Fairclough (2003, p.59) asserts that “one cannot simply look at a text, identify assumptions, and decide in textual evidence alone which of them are ideological” (emphasis added). 35 There are various aspects to look at in a text that are dependent upon genre, and these are: the structure of a text, the semantic relations between clauses and sentences, the type of clauses it contains, and the way other texts are incorporated in a text (intertextuality) (Fairclough 2003, p. 67). I draw on these features to understand the meanings that the texts convey to unfold the analysis chapter. Intertextuality, assumptions and value assumptions This means that using linguistic tools from CDA, especially semantic relations between words, one can identify terms that, while different, denote similar values. As mentioned earlier, I started my coding following the preliminary inventory of public values of Beck Jørgensen and Bozeman (2007), complemented with the work of Hood (1991) and Hood and Jackson (1991). In CDA words, I used these lists to perform a lexical (vocabulary) analysis of public values and - Page 88 - technology (Fairclough 2003, p. 94). Following the linguistic tools, it turned out that the value assumptions were very explicit most of the times; when otherwise, I draw on the contextual data from fieldwork33 as well as the micro-coding to identify possible alternative meanings of a term or phrase.34 I have also looked at the semantic relations between words in a sentence. Identifying discourses meant also looking at the ways words relate in a sentence, its collocations, and relations of equivalence and difference (Fairclough 2003, pp. 123-4). In complementary fashion, I followed the three basics questions that Johnstone (2002, p. 8) suggests when doing discourse analysis: Why the text is the way it is, why it is not another way, and why there are particular words in a particular order. The first question—why the text is the way it is—is precisely the theme of my next section, that is, genre analysis. 34 I refer here to the micro-analysis of texts that I have conducted following Corbin & Strauss (2008, p.59). See stage two of the general method of analysis. Genre analysis and government texts An important and related analysis to intertextuality comes from analysing how language figures as a means of government action. This entails looking at the specific genre of texts. The definition of genre varies according disciplines and the approaches to study a particular phenomenon. Within discourse analysis, genre is defined in general terms as “a way of acting in its discourse aspect” (Fairclough 2003, p. 216). For example, public speeches, interviews or news article are all examples of different genres. Thus, if we accept that genres are the discoursal aspect of ways of acting and interacting in social events (Fairclough 2003, p. 65), then a legal piece—such as an executive order or a Law—offers a particular way of using language as means of government action.35 - Page 89 - Dealing with a particular genre means that we may expect to find certain common attributes within a text. For example, the use of rhetoric and promotional claims may be well expected when interacting with government texts. The language of politics offers an interesting comparison; from a language perspective, politics is better match with markers of disagreement, dissent and polemic instead of consensus (Fairclough 2000, p. 11, see also Hoggett 2006). The reason why this relation is important is because government and politics are closely related (Fairclough 2000), but they are not the same thing. Governments, as the machines that execute policies in democratic states, depend critically on achieving a sufficient measure of consent—whether mirroring broader social realities or not. The implications from the point of view of the genre of texts are important, since I am studying government texts and not political discourses. Thus, while it should not be surprising that a text like the Presidential Decree speaks a language of consent, it is possible to study further issues from its monological tone. In particular, I study the modality of the text, that is, the particular markers of a clause or sentence that reflect what people commit themselves to when they make statements, declarations, make demands or offers (Fairclough 2003, p. 165). In simple terms, the modality of a phrase refers to the commitments, attitudes and stances that a speaker or writer makes through certain language choices. For instance, adverbs such as ‘certainly’ or ‘necessarily’ are typical examples of modalisation markers that indicate high levels of commitments within a text. Genre analysis and government texts In the analysis chapter (Chapter Six), I draw particularly on these features to assess the level of commitment from those who are supporting the text, and what can be said from the perspective of different voices in the discourse. Studying the modality of a sentence or clause is helpful to specify what types of person or thing are spoken about, and what these subjects are capable of saying in the discourse (Parker 1992). In addition, looking at the structure and class of genre is useful, particularly given its institutionalised aspect of certain government texts. For example, one should expect to find a clear and rather generic structure in a Presidential Decree or executive order as it is a more or less standardised type of text. Given my interest in elucidating relations on assumptions and ideologies, in some cases (and particularly for the Decree and the National Development Plans), I paid attention to the way the argument was constructed. - Page 90 - To do so, I looked for the three main elements in the texts: grounds, warrants and claim (Fairclough 2003, pp. 81-83 based on Toulmin 1958). The grounds are those premises or facts that form the foundation for the argument, while the warrants are what justify a movement from the ground to the claim. As in the case of value assumptions, some elements in a given argument may be very explicit while others may be hidden or assumed. The genre analysis in this way was particularly useful when applied to well- defined texts (i.e. Decree and National Development Plans) because it helped to unpack the assumptions and ideologies that are vested in texts, in a complementary fashion to the other linguistic tools that I have explained in the previous section. On the relevance, quality and limits of my research design On the relevance, quality and limits of my research design 4.4 4.3.3 A note on translation Given that the use of language is particularly important in my research, I have translated the excerpts used in the dissertation trying to maintain the integrity of the text (whether spoken or written) and the facilitation of the original text. Translation theory suggests that the translator has to develop an understanding of the language, its semiotic features and context as well as why the text was produce the way it was (Bhatia 1997). In the particular case of the legal piece that I use as my CDA anchoring point (the Presidential Decree), I checked its translation with bilingual lawyers in the policy field to avoid confusion to the reader. Whilst in order to avoid a ‘double’ interpretation of texts under scrutiny the overall analysis was conducted in Spanish (its original language), the reader may find occasionally some words in its original when relevant for interpretation. Finally, all fieldwork materials were recorded, kept and analysed in its original language (Spanish), and only translated when necessary for quotation. While some nuances may be lost in reporting findings, the richness of the case in conjunction with CDA tools allowed me to deal with specific interpretation issues by constantly comparing many examples. - Page 91 - 4.4.1 On case study quality A common misunderstanding (or critic) to case study is what Flyvbjerg defines as “conventional wisdom”; that is, that one cannot generalize from single cases, that cases are subjective, biased or arbitrary, or that the case study is most useful for generating hypothesis rather than testing them (Flyvbjerg 2006). Many scholars have demonstrated that this rather conventional wisdom is wrong if not misleading (Ragin and Becker 1992, Barzelay 1993, Klein and Myers 1999, Flyvbjerg 2001, 2006, Lee and Baskerville 2003, Sarker and Lee 2003, Yin 2003, Eisenhardt and Graebner 2007, Myers 2009), and case study research has now become a well-established and valuable method for research inquiry in social sciences in general and in the management field in particular. Of course, this does not mean that case study research should not be evaluated. It means that at least it should be evaluated accordingly. Yet, there is no single or universal approach to evaluate rigour in case study research and scholars have proceeded in different ways (for a review on the matter, see Gibbert and Ruigrok 2010). Myers (2009, pp. 83-4) offers six criteria that I complement with the ways I have addressed them in the table below (see also Yin 2003, pp. 160-165 on what makes an exemplary case study). 4.4.1 On case study quality The case study How it has been addressed in my research work must be interesting: the case tells a new story that is of national importance for Mexico; however, its judgment is done by the audience (scholars, examiners, supervisors) must display sufficient evidence this is covered by my multiple data collection methods, the many quotations and examples from the texts I have analysed, also, the appendixes should be ‘complete’ this means that the evidence should suffice to support or disprove particular theoretical points; this is covered extensively on the evidence I provide in Chapters Six and Seven must consider alternative perspectives this is at the core of my CDA approach; I elaborated already on the reflexivity of the approach; I provide further details in the section below should be written in an engaging manner I have been very enthusiastic in my writing, but as in point one, this is judged by the scholarly audience should contribute to knowledge this is addressed in my conclusions (contributions to knowledge) Table 7: Case study quality checks from according to Myers (2009) - Page 92 - In addition, to ensure quality and the reliability of the story, I shared and reviewed Chapter Five with key stakeholders of the project who provided feedback and overall approval of the case. As for the study quality, I consider that the multiple sources of data that I count on, the systematic and comprehensive way that I have collected, kept and analysed my data as well as the particular quality checks of CDA (see below) are the main strengths of my research design. 4.4.2 Reflecting on CDA CDA has its limitations and I am not claiming otherwise. One could argue that textual analysis is inevitably selective (Fairclough 2003) and incomplete. But the fact that CDA embraces heterogeneity and the possibility of multiple interpretations of reality is not just a property of this method but an intrinsic feature of interpretive research (i.e. Walsham 1995, 2006). Van Dijk asserts (2001, p. 99): in any practical sense there is no such thing as a ‘complete’ discourse analysis: a ‘full’ analysis of a short passage might take months and fill hundreds of pages. Complete discourse analysis of a large corpus of text or talk, is therefore totally out of the question. The point that I want to make is that, whilst unpacking all potential intertextual and interdiscursive aspects of a particular text is an impossible task (Keenoy and Oswick 2004, p. 140), it would also prove useless: the value of critical discourse analysis rely on illuminating a particular inquiry that is defined by the researcher. This is to say that any formal analysis of texts is not theory-free (Chouliaraki and Fairclough 1999, p. 7). The analyst’s theoretical preoccupations define what data is selected and how it is perceived; in other words, the analytical categories that emerge out of the selected texts are defined by the research questions and the research focus (theoretical preoccupations) of a particular project. In the case of my research, my theoretical preoccupations have led me to draw on those linguistic features that could help elicit the linkages between public values and technology within the case. Methodological concerns can be addressed following Leitch and Palmer (2010) suggestions and protocols about CDA. Based on their concerns about the confusing - Page 93 - and unarticulated ways that CDA has been applied in organisational and management studies, the authors suggest that a systematic and ‘protocol-based’ method should be followed. The next table presents Leith and Palmer’s nine ‘protocols’ and the ways I have addressed them in my research. They protocols are aimed to cover three methodological decisions in CDA: (i) the definitions, CDA traditions and notions on ontology carried out in the research; (ii) the implications of data selection in terms of data boundaries, social conditioning and multiple sources of data; (iii) the ways the data analysis is conducted, how interpretations are made, and the role of the researcher in the production of the findings. 4.4.2 Reflecting on CDA It is important to mention that these protocols served as sort of validity check done a posteriori, rather than as a guide to my research design. I do so in part because of practical reasons: I have come across them at a quite late stage of my research work (2011). But more importantly, I share the alternative view that Chouliaraki and Fairclough (2010) expose in their answer to the authors, in which they stress that the inherent contextual and contingent character of CDA and its theoretical versatility are their biggest strengths. In any case, I found them useful as a way to reflect on my own elaboration of CDA. Their nine protocols serve to reflect on the overall methodological (as well as theoretical) conceptions that I have used on my dissertation. - Page 94 - - Page 95 - How it has been addressed in my research work ey terms, including discourse, Addressed explicitly in Chapter Three h CDA tradition(s) your d the implications of this for Addressed explicitly in this chapter and in Chapter Three; the CDA approach I follow is based on the work of Fairclough and colleagues and as such, it entailed specific theoretical and methodological choices addressed in those chapters ently present context as itself not to do this, explain le, and the implications for your Not applicable because of my theoretical choices: “Context here [in CDA] is not conceptualized as a distinct and separable dimension of CDA epistemology (...) but as an analytical construct that emerges within specific research questions” (Chouliaraki and Fairclough 2010, p. 1215) the wider social and political research focus influenced your All these choices have been explicitly explained in sections 4.2, 4.3 and Appendix I. In respect to Protocol 2.2, my note on protocol 1.3 also applies. iteria you used in your research as associated with text and d with context your choice and availability of lluminate aspects of your limit your conclusions by interpretations h aspects of ‘what you have which parts of your analysis ns and inferences, and the basis and inferences This is extensively addressed on this chapter, in the data analysis section, but also, in chapter 6 and 7, where I make use of the evidence and suggest interpretations accordingly. terms, in DA trad he impl tly prese ot to do and the wider s search f ria you u associat with con ur choice minate a mit your erpretat spects of hich par and infe inferen spects o kely to h techniqu ons for y e as rese data rela 4.4.2 Reflecting on CDA h aspects of textual and e likely to have been lost on techniques you used to ‘tell ations for your conclusions I have addressed this partly in this section of the chapter, and more broadly in my conclusions under the section ‘limitations’ of the study. role as researcher in the of data related to text and I have made transparent, as much as possible, the method of analysis (earlier in this chapter) as well as in the section of case selection. and Palmer (2010) protocols for CDA. 05-6). s ces DA] 15) nalysi n. e method of a f case selectio possible, the he section of nsparent, as hapter) as w - I have m (earlier i s for CD - Page 95 sions he and ) protoco y ur conc cher in d to tex er (201 terms, i DA trad he impl tly prese ot to do and the wider s search f ria you u associat with con ur choice minate mit your erpretat spects o hich par and infe inferen spects o kely to h techniqu ons for y e as rese data rela rapola olation ain wh wledge a redu h i 4.5 36 This is particularly an important difference to parliamentary systems (e.g. the Westminster system), where Cabinet ministers are appointed from among sitting members of the legislature who, while serving the Cabinet, remain members of the parliament. 5 Chapter Five: The Research Site. Building the Single Window for Foreign Trade in Mexico 5 Chapter Five: The Research Site. Building the Single Window for Foreign Trade in Mexico Conclusions This chapter has covered my methodological assumptions and the devices I have used to conduct my research. I started with the ideas on critical realisms that provide foundations for my theoretical framework as well as the CDA tradition that I have chosen. Sections 4.2 and 4.3 elaborated extensively on the significance of my fieldwork as well as the reflexive data analysis that my work entailed. I want to add another reflection on my data analysis. In line with the critical theory spirit, CDA does not offer a pre-packaged step-by-step method to follow. Working with the data entailed a reflexive process of going back and forth between the theoretical ideas and the empirical analysis that ultimately led to my findings and contributions. Therefore, conducting CDA is never straightforward; it challenges the researcher to employ creativity and craftsmanship (Phillips et al. 2008, p. 786). All in all, instead of selecting and sticking to one research paradigm, my research journey meant to use different theoretical instances and methods to delineate and illustrate my own research work, in a coherent way (Crotty 1998, p. 216). I turn attention now into the details of the longitudinal case. - Page 96 - 5.1 Mexico’s key facts The United Mexican States, better known as Mexico, certainly stands out in Latin America for various reasons. Birthplace for Mayans and Aztecs, Mexico exhibits a rather complex set of cultural traditions that intertwine across different levels of its economy and society. Mexico is the 15th biggest territorial country, the 11th most populated in the world, and accounts for 112 million of inhabitants according to the Mexican national Census in 2010. Politically, Mexico is a republican and representative state, and follows a presidential system with a strong bicameral Congress. Presidential administrations are six–year long and re-election is not possible. As in other presidential systems, the President is the head of the country and is the one that appoints the Cabinet of Ministers.36 Mexico has a federal system with 31 states and a federal district, each divided into municipalities. Although it is federally constituted, central government has an important and predominant role both, in politics, economy and social aspects of the country life. 36 This is particularly an important difference to parliamentary systems (e.g. the Westminster system), where Cabinet ministers are appointed from among sitting members of the legislature who, while serving the Cabinet, remain members of the parliament. - Page 97 - Figure 5: Map of Mexico Courtesy of the University of Texas Libraries, University of Texas at Austin Figure 5: Map of Mexico Figure 5: Map of Mexico Courtesy of the University of Texas Libraries, University of Texas at Austin g p Courtesy of the University of Texas Libraries, University of Texas at Austin Courtesy of the University of Texas Libraries, University of Texas at Austin For most of the 20th century, Mexico’s political and national life was dominated by a single ruling party -Partido Revolucionario Institucional (PRI)-, which remained in power for over 70 years, until the year 2000 when an opposition party (Partido de Acción Nacional, PAN) won the presidential elections. The same party, PAN, secured another 6 year- term in power, after winning a controversial election in 2006. Despite PRI losing the 2000 and 2006 presidential elections, the party still holds a big political power in Mexico (Panizza and Philip 2005, Pardo 2009). In July 2012, PRI won the presidential elections and will come back to power after 12 years. 5.1 Mexico’s key facts - Page 98 - Formal Name United Mexican States Population 112.336.538 inhabitants (2010 Census; 11th in the world) Land Area 1.964.375 sq km (15th in the world) Major Cities Mexico City, Guadalajara, Monterrey, Puebla, Veracruz Geography Very diverse; tropical, subtropical, desert, mountains; high plateaus, 9,330 km of coastline Religion Mostly Catholic Language Spanish is the official; there are more than 60 indigenous languages also spoken mainly Náhuatl (1.2m speakers), Maya (714,000), Mixtec (387,000) and Zapotec (403,000) Government Federal, 31 states and the Federal District (Mexico City) Constitutional republican state (executive, legislative, judicial branches) Presidential system, with a heavy Congress (Bicameral, proportional representation) Current President: Felipe Calderón (PAN-right wing) 2006-2012 Main Political Parties Partido Acción Nacional (PAN), right-wing, in power since 2000; Partido Revolucionario Institucional (PRI), center-left party, in power from 1929 (foundation) to 2000; Partido de la Revolución Democrática (PRD), a left- wing party (governs Mexico City) Partido Acción Nacional (PAN), right-wing, in power since 2000; Partido Revolucionario Institucional (PRI), center-left party, in power from 1929 (foundation) to 2000; Partido de la Revolución Democrática (PRD), a left- wing party (governs Mexico City) Main socio-economic indicators (2011) Main socio-economic indicators (2011) Total GDP (USD, nominal) 1.041 trillion (13th in the world) GDP per capita (USD, nominal) 9.489 (58th in the world) Total trade (USD, nominal) 600 trillion (10th in the world; 1st Latin America) Gini Coeficient (2008) 0,516 (high inequality) Human Development Index 0.750 (56th in the world) Trade and Development Index 750 (49th in the world) Table 9: Mexico key facts Source: INEGI, World Bank, IMF, The Economist Intelligent Unit. In economic terms, Mexico occupies the 13th position among the largest economies in the world, and the second biggest economy in Latin America after Brazil. Mexico is one of the major exporters and importers in the world. Considering the classification of the International Monetary Fund, Mexico is the third major merchant of emerging and developing economies, slightly after Russia, which gained the second place only few years ago while China holds the first place. When compared to other countries, such as - Page 99 - China, Brazil, or the USA, Mexico’s international trade is undoubtedly key for its economy, as it represents almost 60% of the total GDP (see figure 6).37 Figure 6: Mexico’s trade as % of GDP in comparison to other economies Source: World Bank, World Development Indicators 2011 0 10 20 30 40 50 60 70 80 1980 1984 1988 1992 1996 2000 2004 2008 Trade as %GDP Brazil China Mexico United States Figure 6: Mexico’s trade as % of GDP in comparison to other economies Source: World Bank, World Development Indicators 2011 Mexico faces important challenges today. Notably, the war against drug cartels, which has claimed more than 40,000 lives since 2006, has been an increasing concern given its brutality and unprecedented related violence. In addition, the recent stagnation of oil production and the impact of the USA recession have increased the existent social and economic pressures within a socially uneven country. Given the fact that Mexico faces critical demands related to economic growth, income distribution and social policies, gains from international trade are considered key for improving the country’s welfare. Therefore, any potential welfare gains from trade are not trivial for an open economy like Mexico. This brief introduction of Mexico’s key facts provides an initial idea of the context in which the case takes place. 38 In Spanish is known as “Ventanilla Única de Comercio Exterior Mexicana”. 37 Data for the year 2010; based on a nominal GDP list of countries. Source: International Monetary Fund, World Economic Outlook Database, April 2011: Nominal GDP list of countries. 38 In Spanish is known as “Ventanilla Única de Comercio Exterior Mexicana”. 39 In Mexico ministries are referred to as secretariats (in Spanish, “Secretarias”). This dissertation adopts the word Ministry to all Mexican Secretariats in order to avoid confusion regarding its role within the public administration structure. Main socio-economic indicators (2011) The case focuses on the use of networked technologies to build a digitalSingle Window for Foreign Trade regulations (hereafter Single Window).38 The case constitutes an enormous back-office integration effort—still on-going— 37 Data for the year 2010; based on a nominal GDP list of countries. Source: International Monetary Fund, World Economic Outlook Database, April 2011: Nominal GDP list of countries. - Page 100 - intended to support a broader process of facilitation of international trade and based on previous efforts within the Mexican Ministry of the Economy.39 It develops within a rich story of previous leading innovations within the Ministry and as part of broader administrative modernisation reforms in the country. This chapter presents the details of the case under study, and elaborates the contextual features in which it is grounded. In terms of the latter, it provides detailed accounts of Mexico’s broader processes of modernisation in two different but complementary historical aspects: the economic transformations with an emphasis on market- orientation and trade policy, and the process of democratisation and modernisation of the public administration. As explained in the methodology section, the current chapter draws heavily on data gathered during the fieldwork as well as historical accounts and research conducted in the area of modernisation reforms in Mexico. The chapter is organized as follows. It elaborates on the contextual factors of Mexico’s reform (economic liberalisation and democratisation and reforms of bureaucracy), paying particular attention to salient features of the reforms during the last 30 years. The narrative of the case—the Single Window—follows. 41 The General Agreement on Tariffs and Trade (GATT) is a multilateral agreement regulating international trade; it was signed in 1947 and replaced by the World Trade Organisation (WTO) in 1995. An updated General Agreement is now the WTO agreement that governs trade in goods. 42 In terms of trade and globalisation, the “Washington Consensus” promoted a commitment with intense trade liberalisation (promotion of exports and an emphasis on elimination of trade restrictions) and a strong focus on promoting inward foreign direct investment. 5.2 The economic and political context: Mexico from 1982 to 2010 As Kirby (2009) suggests for Latin American countries, debates on the transformation of Mexico have followed mainly two lines: one regarding its democratization in relation to the 70 years in power of the Institutional Revolutionary Party (PRI), and a second one on institutional reforms and reformulation of economic and social policies. These are somehow inseparable when portraying the situation of Mexico’s political economy, and the reader may not see them as decoupled from each other. The next two sections present the historical background and processes of reform in two areas: the Mexican public administration (bureaucratic context) and the economic transformations. Given the time covered in this dissertation, my main focus will be in elaborating the most salient features of both strands since the 80s and particularly, within the period in which the case under study is deployed (1995-2012). - Page 101 - 40 For a detailed background of Mexico’s economic development strategy from 1950-1981 see Moreno- Brid et al (2009), pages 156-7. 5.2.1 International trade, openness to trade and economic reforms in Mexico The unavoidable starting point for the last 30 years of Mexico’s economic history goes back to 1982, a year when the country faced a critical debt crisis. The crisis meant the abandonment of Mexico’s development model based on state-led industrialisation, import substitution and universal welfare provision.40 From 1982 —better known as the episode that initiated the “lost decade” in Latin America (“la década perdida”)—a process of radical modernisation reforms started and later reinforced and accelerated under the following presidential administrations. The main components of the reforms included a clear shift towards an opening to trade and a promotion of foreign investment as an essential complement to trade, changes in the industrial policy, the privatization of public enterprises, the deregulation of domestic economic activities, and the shrinking of public expenditure and the state's intervention in the economy (Kehoe 1995, Moreno-Brid and Ros Bosch 2009, chap. 7, Moreno-Brid et al. 2009). In 1985, the period of multilateral liberalisation formally began when Mexico entered the General Agreement on Tariffs and Trade (GATT).41 In brief, soon after 1982 Mexico put an end to its welfare state model and closed economy, and embarked on a long-term process of drastic economic liberalisation. In this sense, Mexico is said to have embraced the ideas crystallised in the “Washington Consensus” programme of economic reform, along the line of the broader NPM agenda.42 Key episodes during the 1990s confirmed the focus on economic, trade and financial liberalisation. A new period of bilateral liberalisation of trade started in 1994 when Mexico, the United States and Canada launched the North America Free Trade Agreement (NAFTA), the largest free trade agreement in the world. NAFTA came into force in January 1994, which meant a progressive reduction or elimination of tariff and non-tariff barriers to intra-regional trade. Later in 1994, Mexico joined the OECD, - Page 102 - - Page 102 - becoming the first (and until 2010, the only) Latin American country in the organisation.43 In 1993 Mexico was admitted as full member of the Asia Economic Co-operation (APEC) forum, and in 1995 became full member of the newly created World Trade Organisation (WTO). 5.2.1 International trade, openness to trade and economic reforms in Mexico Since then, and even when less significant in comparison to NAFTA, Mexico continued its policy of negotiating free agreements with many c regarded as bilateral liberalisation (see figure becoming the first (and until 2010, the only) Latin American country in the In 1993 Mexico was admitted as full member of the Asia operation (APEC) forum, and in 1995 became full member of the newly created World Trade Organisation (WTO). Since then, and even when less significant in comparison to NAFTA, Mexico continued its policy of negotiating free agreements with many countries around the world. This is why this period is best regarded as bilateral liberalisation (see figure below). becoming the first (and until 2010, the only) Latin American country in the In 1993 Mexico was admitted as full member of the Asia-Pacific operation (APEC) forum, and in 1995 became full member of the newly created World Trade Organisation (WTO). Since then, and even when less significant in comparison to NAFTA, Mexico continued its policy of negotiating free-trade ountries around the world. This is why this period is best Figure 7:Phases of trade liberalisation in Mexico Y axis: Trade as % of Mexico’s GDP. trade liberalisation in Mexico Y axis: Trade as % of Mexico’s GDP. Source: WDI, and Mexican Ministry of the Economy. Ministry of the Economy. Figure 7:Phases of trade liberalisation in Mexico trade liberalisation in Mexico g Y axis: Trade as % of Mexico’s GDP. Y axis: Trade as % of Mexico’s GDP. Source: WDI, and Mexican Ministry of the Economy. Ministry of the Economy. From 2006, Mexico strengthened its efforts to achieve more openness to trade via trade facilitation, best described as unilateral liberalisation. In 2008, throughout the Ministry of the Economy, the government has set forth a stro known as the Programme for Trade Facilitation. The Programme comprises of three policy pillars: (i) tariff simplification and the revision of other measures affecting trade; (ii) customs reform and the simplification of trad institutional environment by strengthening the Foreign Trade Commission. As a result From 2006, Mexico strengthened its efforts to achieve more openness to trade via trade facilitation, best described as unilateral liberalisation. In 2008, throughout the Ministry of the Economy, the government has set forth a strong trade liberalisation agenda, known as the Programme for Trade Facilitation. 43 Chile joined the OECD in February of 2010. Chile joined the OECD in February of 2010. 43 Chile joined the OECD in February of 2010. Chile joined the OECD in February of 2010. 45 The legal foundation of trade policy in Mexico relies on Article 131 of Mexican Constitution, and article 4 of the Foreign Trade Law, which authorizes the federal government to control flows of imports and exports, and to conduct international trade negotiations. 44 “Phyto-sanitary regulations” are government rules that prohibit or restrict the importation or marketing of certain agricultural products so as to prevent the introduction of pathogens that these products may be carrying. 5.2.1 International trade, openness to trade and economic reforms in Mexico The Programme comprises of three policy pillars: (i) tariff simplification and the revision of other measures affecting trade; (ii) customs reform and the simplification of trade regulations; (iii) fostering its institutional environment by strengthening the Foreign Trade Commission. As a result From 2006, Mexico strengthened its efforts to achieve more openness to trade via trade facilitation, best described as unilateral liberalisation. In 2008, throughout the Ministry ng trade liberalisation agenda, known as the Programme for Trade Facilitation. The Programme comprises of three policy pillars: (i) tariff simplification and the revision of other measures affecting trade; e regulations; (iii) fostering its institutional environment by strengthening the Foreign Trade Commission. As a result - Page 103 - of the comprehensive liberalisation agenda that started in 1980s, by 2011, excluding phyto-sanitary regulations44, Mexico maintains very few trade restrictions and holds the biggest number of free-trade agreements with other parties in the world.45 Reforms in trade were accompanied with a series of transformation in the domestic economy too. In terms of industrial policy, the first wave of the modernisation reforms meant eliminating all sectoral development programmes and ending targeted credit and tax subsidies (Moreno-Brid et al. 2009). From the mid 1990s and the following presidential terms, there were changes in industrial policy that mainly targeted productive chains in the Mexican export-oriented manufacturing sector by allowing for some sectoral-specific programmes, as those the Ministry of the Economy administrates. The financial sector has also experienced complex cycles and reforms. After progress in economic growth and financial stability, the Mexico faced another big financial crisis in 1994-1995 but it recovered quicker than in the 1980s. Further economic measures, together with an increase of traded goods particularly as part of NAFTA, Mexico recovered the main economic indicators by the end of the 1990s. From 1996 to 2000, the country average growth rate was 5.4 per year, but then sluggish leaving open questions in terms of the impacts of reforms and specially, from openness to trade (Palma 2003). In the public finance area, neo-liberal reforms in conjunction with NAFTA resulted in sound macroeconomic fundamentals towards 2010, as Mexico's reached a good fiscal performance —low domestic inflation and a boom in participation in global markets (Moreno-Brid et al. 2005, 2009). Fiscal policy controls, however, meant a commitment on shrinking public investments and public expenditure in general. Overall, economic reforms have not been modest in Mexico. 5.2.1 International trade, openness to trade and economic reforms in Mexico Perhaps the most salient achievements of the period are the strong and sound macroeconomic indicators, the 44 “Phyto-sanitary regulations” are government rules that prohibit or restrict the importation or marketing of certain agricultural products so as to prevent the introduction of pathogens that these products may be carrying. 45 The legal foundation of trade policy in Mexico relies on Article 131 of Mexican Constitution, and article 4 of the Foreign Trade Law, which authorizes the federal government to control flows of imports and exports, and to conduct international trade negotiations. - Page 104 - general economic stability, low inflation and boom in the participation of the country in the global markets (see for example Moreno-Brid et al. 2009). Yet, there is currently a debate on why Mexico economic growth since 1985 has been so modest at bestif not disappointing. When comparing Mexico to other countries in Latin America (particularly with Chile, a country that had implemented similar liberalisation policies), Mexico’s growth rate lags behind. Let alone the modest rates of growth, Mexico’s economic neo-liberal reform agenda has been highly unbalanced in terms of social policy, wealth and income distribution (Lustig 2001, and Esquivel and Rodrı́guez-López 2003 on wage inequalities, Ramirez 2003, Moreno-Brid and Ros Bosch 2009, Moreno- Brid et al. 2009). This is reflected in Mexico’s position in the world rankings of development indicators: whilst the whole economy positions among the 15 biggest countries and traders in the world, Mexico ranks number 53 in the United Nations human development index, number 58 in terms of GDP per capita, and number 46 in the income distribution index. Research in international economics suggest that the explanations for Mexico’s stagnation and its inability to catch up with job creation and income distribution concentrate on factors such as an inefficient domestic financial sector, rigidities in the labour market and lack of contract enforcement, and a high dependence on the USA market and oil generated income (Lustig 2001, Tornell et al. 2003, Moreno-Brid et al. 2009). As Palma puts is (2003, p. 57): “Mexico may have been particularly successful in switching its ‘engine of growth’ towards the export sector, but to date [2003] it seems totally at a loss as to how best to harness the power of this new engine.” Kehoe and Ruhl (2010, p. 5.2.1 International trade, openness to trade and economic reforms in Mexico 1024) expand these findings, and suggest that further reforms are needed to eliminate the barriers to growth, and they should concentrate on increasing the efficiency of the financial system, overcoming the general lack of rule of law, and in eliminating rigidities in the labour market. These preoccupations on Mexico’s stagnation and barriers to economic growth explain why measures to reduce the frictions in foreign trade—germane to our case study— have accelerated during the hole period, and in particular, since the Programme for Trade Facilitation launched in 2008. The Programme recognizes that trade liberalisation ought to be accompanied by other measures to boost the country’s competitiveness. In other words, well known measures of trade liberalisation such as tariff reduction are not - Page 105 - enough to maximize gains from trade if they are not backed with institutional change. The Single Window for foreign trade, as I will describe more in detail shortly, is precisely one of the institutional changes that Mexico has been pushing forward. To sum up, given Mexico’s policy orientations, the role of foreign trade is highly important for the country’s economic activity and its social and economic development. Let me now turn attention to the reforms of the Mexican bureaucracy and the rule of law, as I consider it the other side of the coin. To sum up, given Mexico’s policy orientations, the role of foreign trade is highly important for the country’s economic activity and its social and economic development. Let me now turn attention to the reforms of the Mexican bureaucracy and the rule of law, as I consider it the other side of the coin. 5.2.2 The public sector in Mexico: democratisation and changes in the bureaucracy since 1982 As in the economic arena, the crisis of 1982 meant an end to the public resources that Mexico’s large national bureaucracy enjoyed for many years (Pardo 2009, Panizza and Philip 2005). The fact that the PRI dominated the political arena –and hence, party political contestation was marginal– made the bureaucratic apparatus a political one. From 1940 and at least until the 1980s, the Mexican public sector was best regarded as a traditional system of patrimonial, clientelist system in which each president in turn controlled the public bureaucracy (Gow and Pardo 1999, Arellano-Gault 2000, Arellano-Gault and Guerrero 2003). For example, besides appointing the upper level bureaucrats, the politicians could appoint around 60,000 positions in the mid-level, federal public administration (Panizza and Philip 2005). Hence, the crisis of 1982 challenged the patrimonial Mexican bureaucracy, since the neo-liberal economic transformations translated in a concomitant downsizing programme for the public sector. Gradual modernisation reforms were started, partly as a consequence of the economic, market-driven reforms (Gow and Pardo 1999, p. 541) but also as part of a process of political democratization (Cejudo 2008). Since 1982, there were several programmes to reform public administration procedures, structures and work practices aiming at reducing red tape and public costs (downsizing) as well as at achieving a more professionalized public sector (democratisation). The next table summarizes the most relevant efforts at the federal level. 46 Pardo (2009, p. 175) criticises the rhetorical efforts of President Salinas (1989-1995), and states that “in accordance to previous governments, the Salinas Administration granted no political importance to the modernisation of the public administration”. The author elaborates on how several measures that the government undertook (i.e. administrative decentralisation) were mainly targeting political negotiations and not to more responsive, professional public administration (Pardo 2009, p. 195-6). http://zedillo.presidencia.gob.mx/welcome/PAGES/library/od_publicadmon.html Accessed 22 March 2010. 47 See also Government of Zedillo’s electronic archive, available at: http://zedillo.presidencia.gob.mx/welcome/PAGES/library/od_publicadmon.html Accessed 22 March 2010. 47 See also Government of Zedillo’s electronic archive, available at: 47 See also Government of Zedillo’s electronic archive, available at: http://zedillo presidencia gob mx/welcome/PAGES/library/od publicadmon html Accessed 22 March 5.2.2 The public sector in Mexico: democratisation and changes in the bureaucracy since 1982 - Page 106 - - Page 106 - 1982-1988 1988-1994 1994-2000 2000-2006 2006-2012 • Programme for administrative decentralisation (1983-1988) • Contraloria (1983) • Programme for administrative simplification (1989-1994) • Modernisation Programme for the Public Administration( PROMAP) (1995-2000) • Strategic Model for Government Innovation(2001-2006) • Good Government Agenda (2002-2006) • Programme for transparency and against corruption (2001-2006) • Civil Service Career Law (2003) • Special Programme for Management Improvement (2008-2012) Table 10: Mexico’s main modernisation reforms programmes by presidential administrations 1982-1988 1988-1994 1994-2000 2000-2006 2006-2012 • Programme for administrative decentralisation (1983-1988) • Contraloria (1983) • Programme for administrative simplification (1989-1994) • Modernisation Programme for the Public Administration( PROMAP) (1995-2000) • Strategic Model for Government Innovation(2001-2006) • Good Government Agenda (2002-2006) • Programme for transparency and against corruption (2001-2006) • Civil Service Career Law (2003) • Special Programme for Management Improvement (2008-2012) Table 10: Mexico’s main modernisation reforms programmes by presidential administrations Table 10: Mexico’s main modernisation reforms programmes by presidential administrations The administrative reforms that were attempted before 2000 are seen as generally timid by scholars, with some exceptions. From 1980s to 1995, the programmes focused on administrative decentralization. These attempts included a series of legal changes that granted more financial resources and a greater administrative role to state and municipal authorities, aimed at giving greater autonomy for local and state governments, as well as helping with the downsizing effort (Gow and Pardo 1999, Pardo 2009). Despite their ambitious intentions, the programmes did not produce vast results, mainly because of the persistent inertia of public administration behaviour and the failure to link it to a longer-term project at best (Sanchez-Gonzalez 2009), or the absence of any real political importance granted to the modernisation efforts (Pardo 2009, p. 175)46. The “Modernisation Programme for the Public Administration” (in short, PROMAP) launched in 1995 proved a higher degree of compromise with reformist initiatives. PROMAP was regarded as an important policy to improve the efficiency and effectiveness of public services whilst fighting the opacity and corruption in the Mexican public administration (Pardo 2009).47 By means of introducing common managerial practices from the private sector, such as strategic planning, business process reengineering and total quality programs (TQM), the main goal of PROMAP was to transform public servants’ attitudes and behaviours throughout the whole public sector - Page 107 - - Page 107 - into a more effective, efficient and responsive government. 48 For a review of the political history behind the passage of the Federal Transparency and Access to Government Information Law (Ley Federal de Transparencia y Acceso a la Información Gubernamental) in 2002, see Bookman and Guerrero 2009. For a detailed description of the events and the role media played in the history of the Law, see Michener 2010, chapter 2. 5.2.2 The public sector in Mexico: democratisation and changes in the bureaucracy since 1982 The PROMAP, then, represented the first ‘infused’ ideas and jargon that were at centre of the NPM, such as the managerial focus, the emphasis on strategic planning and quality (Cejudo 2008, p. 116). While some scholars suggest that the programme did achieve some results, such as a shifting from old (and durable) forms of administrative procedures into more modern and less opaque practices (Sanchez-Gonzalez 2009, p. 90), others remain more sceptical (Cejudo 2008, Pardo 2009). In 2000, a new political scenario set the basis for new reform strategies. The year represents a milestone for the democratisation process that Mexico had started at least one decade earlier. With the National Action Party (Partido de Acción Nacional, PAN) wining the elections, the process of administrative (political and bureaucratic) reforms took momentum. For the new president, Vicente Fox, it was crucial to run an administrative apparatus that was clearly different from the one under the PRI administration (Panizza and Philip 2005, p.687). If the previous administration was successful in bringing private sector managerial practices into public administration, the government of President Fox went even beyond by giving an explicit managerial spirit to some of the reforms. Both, the “Good Government Agenda” and the “Strategic Innovation Plan” were salient examples of how the NPM visions reached Mexico fully, even though there was a ‘Mexican way’ to incorporate the features of the NPM inspired reforms (Cejudo 2008). Since 2000, Mexico consolidated a series of important transformations towards a more transparent and Weberian-type of state bureaucracy. The passage of the Transparency Law in 2001 and the Civil Service Career Law in 2003, amid their imperfections and critics, constitute landmarks in the state reforms (for detailed debates on transparency see Lopéz Ayllón and Arellano-Gault 2006, Bookman and Guerrero 2009, Michener 2010, and for civil service Panizza and Philip 2005, Cejudo 2008, Arellano-Gault and Guerrero 2003).48 - Page 108 - The Good Government Agenda was also part of the reformist discourses. It encompassed six main strategies: a government that costs less; a quality government; a professional government; an e -government; a government with improved regulation; and an honest and transparent government. The Agenda became particularly important as a driver for e-government initiatives, as its fourth pillar (the e-government one) was explicitly mentioned as both supporter and facilitator to all the other five strategies (Presidencia de la República, México 2002). 49 Mexican Law states that presidential terms are not renewable (the same applies to state governors and municipal presidents). 5.2.2 The public sector in Mexico: democratisation and changes in the bureaucracy since 1982 In 2006 the PAN won the national elections for the second time, with Felipe Calderón as the President.49 In 2007, a new “Programme for Management Improvement 2007- 2012” (PMI) appeared, just after an ‘Austerity Decree’ was put in place to help controlling fiscal numbers stable. The PMI (currently ongoing) set forth four main lines of action: to provide better public services, to increment public sector productivity, to reduce costs by streamlining resources efficiently and optimally and to focus on results and performance indicators. Sceptical scholars pointed out that the PMI is limited compared to the needs of the Mexican public administration, and somehow responded to guidelines developed by international bodies such as the OECD and the CLAD (Sánchez-González 2009). Despite the debate, the programme was discursively ambitious: to obtain a better and responsive government with a financially tough structure, as the pressures on keeping an austere budget were not abandoned. Overall, it is not surprising that Mexico’s modernisation reforms —in which many other Latin American countries were also immersed— happened at the same time when the USA and other western countries were pursuing market-oriented reforms (NPM). A word on the role of international organisations should be regarded here. Whilst it has been recognized that the most active reformers in Mexico were domestic policy scholars and political activists (Panizza and Philip 2005, p. 685, Cejudo 2008, Salskov-Iversen et al. 2000), international agencies played an important role as reform ‘promoters’. For example, the World Bank’s ‘good governance’ agenda was present in several elements of PROMAP. The fact that Mexico joined the OECD in 1995 also put the country in a more ‘exposed’ or even participatory position within the current trends of administration reforms. Yet, to claim that Mexico’s reforms are just a consequence of - Page 109 - international organisation’s agenda would be misleading or over simplistic (Salskov- Iversen et al. 2000, Cejudo 2008). 5.3 Summary of Part One of the Chapter Mexico’s modernisation reforms (administrative and economic) that started in the mid- 1980s, although with differences of degree and focus, remain ongoing. As I have reviewed in the first part of this chapter, foreign trade represents a very important part of Mexico’s economy. It is not surprising that measures for improving the administration of Customs and trade regulation are regarded as top policy priorities Whilst discussing the implications of foreign trade on social welfare is beyond the scope of this dissertation, the economic theory assumptions—trade liberalisation as an engine for economic growth—are regarded as important components in the context (and discourses) around the case. I have also presented the most relevant aspects of the democratisation and modernisation of the Mexican public bureaucracy, which set the path towards a more impartial and professionalized operation of central government agencies. Having set the general ‘scene’ (context), the next part introduces the case. 5.4 Introduction: Foreign Trade regulations in Mexico As indicated in the introduction of the chapter, the case takes place in the area of foreign trade, the engine for economic growth for an open country like Mexico. There are many aspects related to foreign trade, and one of them is its regulatory activity. This is our entrance point to the case. I shall be looking at the activities, processes and regulations that entail the use of authority of government. I shall also remind the reader that Mexico’s foreign trade is substantially large and highly complex in terms of regulatory activities and compliance (WTO 2008). 51 A common trade promotion programme is called PROSEC, and means that the government grants a tariff reduction measure to beneficiary companies to produce certain goods; the programme only applies to certain industries including textile, electronics and automotive. 52 Other parts of the ME cover related areas of international trade (i.e. agreements’ negotiations), but they do not deal directly with foreign trade procedures and services, and thus excluded from the main analysis. 5.4.1 Foreign trade procedures and Mexico’s regulatory activities Foreign trade entails the movement of goods, capital or services across borders and, as such, it involves a variety of regulatory activities that business companies have to comply with, known as trade procedures. These regulatory procedures include fiscal activities (i.e. the collection of customs duties), safety and security procedures (i.e. anti- smuggling controls) and regulations related to trade policy agreements, such as tax returns, quota restrictions or preferential access to goods. A typical foreign trade formality associated with trade policy is, for example, the certificate of origin —a document that an exporter produces and the government certifies attesting that the goods are manufactured or processed in a particular country. In Mexico, the administrative procedures related to foreign trade activities involve the issue of around 37,000 export licenses, 1 million import licenses and 10 million importation requests per year. There are more than 55,000 foreign trade users and around 30 main actors from several sectors: government agencies, exporters, importers, logistics and trade associations. The regulatory requirements and normativity of trade procedures are rather complex; for example, a common operation of foreign trade entails the presentation of at least 40 different documents that include between 7 and 14 - Page 111 - paper sheets. The government estimates that at least 65% of single data points are captured on more than one occasion (i.e. the identification of a given enterprise). The entry point to my case study is the Ministry of the Economy, which, together with Customs, oversees the biggest part of Mexico’s trade regulations. Let me review it briefly. 50 The ME constitutes the focus of this research, and thus, the main actor. See methodology chapter. 5.4.2 Mexico’s facilitation trade body: the Ministry of the Economy The Ministry of Economy (ME) of Mexico is a member of the executive cabinet of Ministries responsible generally for the competitiveness and economic growth of Mexico.50 Under these general responsibilities, the Mexican Foreign Trade Law empowered the Ministry of Economyto issue the country’s trade policy, to coordinate international trade negotiations and to issue rules for enforcing trade agreements. Since 1982 -and given the context of neo-liberal reforms in Mexico- the Ministry of Economy has occupied a key role in terms of Mexico’s economic agenda. Together with Customs, (an agency that depends on the Ministry of Finance and Public Credit), both agencies are the most important regulatory bodies in terms of trade. The ME counts on different strategic divisions to deal with different aspects of its general mission. I am particularly interested in the activities of the Vice-Ministry for Trade and Industry, which is responsible for the proposal and coordination of public policies to increase Mexico’s competitiveness, strengthening the domestic markets and promoting foreign commerce. It is also the unit that administrates foreign trade procedures and regulations, such as issuing certificates of origin, import and export licenses or trade promotion programmes for manufacturers.5152 The operative unit within the Vice-Ministry that is directly responsible for the promotion, operation and facilitation of trade is the General Direction of Foreign Trade (DGCE), which comes next in the hierarchical line. For simplicity of the narrative, I will be referring to the ME - Page 112 - - Page 112 - in general, although the reader should bear in mind that the Ministry has many other offices with no duties directly related to foreign trade. 5.5 From SICEX to the Digital Single Window for Foreign Trade The case narrative is structured into three chronological periods. Period 1 (SICEX phase I) goes back to the mid-1990s and covers the award-winning technological platform to support international trade procedures, called SICEX —short for “Integral System for Foreign Trade”. I pay particular attention to this phase’s initial problems that the technological platform came to solve as well as its main achievements. Period 2 (SICEX phase II and III) presents the main actions, developments and results from 2000 until 2008,the moment when the government announced a new initiative to replace the existing SICEX. From 2008 to 2012 (period 3), I review the most recent developments and advances towards what we know as “the Mexican Single Window for Foreign Trade”. The phases, main events and contextual factors are summarized in the next figure. - Page 113 - - Page 114 - can Single Window for Foreign Trade an S The upper part of the figure presents what I defined as the main contextual factors (navy blue colour and green). The upper part also refers to the National Development plans (PND), and a series of episodes that relate to broader reformist actions of the government. The existence of Mexico’s national planning policies goes back to early 1930s; since then, each presidential administration issues its own 6-year plan. The PND sets out a hierarchical structure of guidance and plans that covers the different levels of the government (national, regional and local planning). This is why the PND offers a good indication of Mexico’s ideals in economic and social policies. Both, PND and the particular modernisation programmes as well as relevant trade policies are connected to the first part of the chapter and are important institutional forces that were in place and with material effects within the project under study. In turn, the lower part (pink colour) clusters the process design features and main interactions during the trajectory of the project. The episodes that are portrayed in the inferior part are those that I regarded as crucial from the process point of view of the project, and I shall be referring to them in the following sections. Regarding the properties of technology, as I have anticipated in Chapter Three, they can be broadly related to enterprise systems in their functionalities, although I do not refer to these as off-the-shelf commercial software packages (i.e. as in Davenport 1998). 5.5 From SICEX to the Digital Single Window for Foreign Trade I elaborate on its technical features accordingly, as SICEX and the Single Window have their own specificity. But in general, both initiatives entailed the use of hardware and application software intended to manipulate large bodies of data to support bureaucratic processes, information flows, and reporting across the central government, relying on internet connectivity to operate. In terms of their complexity, both SICEX and the Single Window transcend building a website; they require intra or inter-agency coordination. 5.5.1 Period 1: from multiple to a single foreign trade platform The story of administrative simplification within the ME and foreign trade procedures in which the case takes place goes back to the presidential term of 1994-2000, when the government started building a new technological platform to support the administrative - Page 115 - simplification (Secretaría de Economía y Fomento Industrial (SECOFI) 1999). By mid 1990s, the scenario in terms of administrating and resolving foreign trade procedures was far from being favourable to the goals of neo-liberal reforms that aimed at increasing Mexico’s position in international trade. The initial motivation behind a collaborative, technology–supported project was the need to have clear rules and procedures to eliminate so disparate variations in decision making, performance, and results. As the former Deputy Minister illustrates: I’m going to tell you what my problem was [when I became the General Director of Trade in 1995]. The first thing I notice is that I have to sign 200 matters per day... nobody can review that. Nobody. Thus, there were several things to be done. First, to generate rules, transparent and published rules.. and we did so, we created clear rules available for all. And then, we started with process reengineering, because we realized that the same papers went around two or three times in the same Direction. So we started doing process diagrams and so on... Former Deputy Minister, Ministry of the Economy The initial motivation behind a collaborative, technology-supported project was the need to have clear rules and procedures to eliminate the disparate variations in decision- making, performance, and results of foreign trade regulation in an increasing competitive environment created by NAFTA. The absence of a clear system of rules co- existed with disparate and precarious technological platforms, each operating according to its own logic or criteria. Moreover, there was at best a weak communication infrastructure to connect databases in different locations. This means that the diverse offices of the Ministry of Economy (i.e. the central office and the 52 federal delegations in the country) analysed procedures that drew on different decision and information standards. Security was not robust (when it did exist) and, together with the different interpretations of decision standards, there were notable differences between offices when issuing administrative decisions. What did technology infrastructure mean for businesses back in 1995? 5.5.1 Period 1: from multiple to a single foreign trade platform Put it simply, if you were a foreign trade company with operations in Mexico and wanted to issue an import license, you would have had to present your company’s name, address, and telephone numbers, name of the legal representative, and all their official proving certificates, in paper, in person, face to face to the ME of your jurisdiction, even if that required traveling hours and no matter if you had done so for a different procedure (i.e. import quota) the day before. Waiting times varied, being 15 working days the quickest. - Page 116 - In the case of a rejected procedure, a company would have to review the response and check its accuracy as at least 15% of importation permits contained administrative errors, such as typos or misspelled data requirements. In the case of an error, the company would have had to start the whole process again, waiting from 15 to 25 working days to get the procedure authorized.53 53 Source: “History of SICEX”, page 5. - Page 117 - SICEX: System design, technical features and implementation With the help of its own “clients” (foreign trade companies), the government started several actions in parallel: a process redesign, a rationalisation of procedures, and the design of a unified, central database that would enable the decentralisation of the administrative operations. The team that coordinated the design and implementation of the system followed a typical information engineering methodology that started from a system analysis and design (i.e. preparing process decomposition diagrams, process dependency diagram). The first phase also encompassed defining the technological architecture to be applied and preliminary designing each of the system modules. The next step included pilot tests, the evaluation of the system performance and general training sessions across to relevant ME personnel. The system was then rolled out throughout the country. In terms of operations, the government claimed that the newly designed SICEX included four features: (i) a secure and errorless information entry that linked databases to confirm or reject certain data entries (i.e. that a given ID matched a company’s name, address and other general information); (ii) a transparent decision criteria embedded in rules and requirements to minimize possible discretionary decisions or disparate responses to a similar request; (iii) an automatic resolution mechanism that could resolve a request automatically given certain parameters (i.e. Customs’ catalogues of authorized traded goods); and (iv) a non-falsifiable response, granted by a single and unique number, printed in secured paper and recorded onto a smart card. An initial security scheme was also put in place to assign roles, authorizations and levels of responsibilities and hierarchies; the government claimed that, while far from being perfect, the security scheme made progress from the alternative paper-based system.54 The implementation phase also included the first steps towards collaboration with other federal agencies through network connectivity. Particularly, it enabled the ME and Customs to work together in a synchronised fashion over certain authorisations, particularly on preferential agreements and some non-tariff regulations. The way it worked was relatively simple: consider that the ME granted a special programme to company X to import certain inputs at a preferential rate; this information was then incorporated into SICEX as an “automatic notice”. With the automatic notice in place, Customs could verify it in situ, and authorize the importation to Company X without the need of doing additional paperwork or requesting further documentation. 55 The capabilities of the system, by 2000, where mainly reduced to streamlining the processes of reception, authorisation and resolution inside the ME. Submission or collection of procedures was vastly done face to face. SICEX: System design, technical features and implementation Although restricted to certain regulations, the internal connectivity between both agencies meant that it was no longer necessary to use secure paper or smart cards to validate the transaction in the presence of the customs agent. This initial network connectivity between the Ministry and Customs opened new channels for future developments. 54 Interview with public officials from the ME. 54 Interview with public officials from the ME. Summary and main activities of period 1 Back in 1995, the map of foreign trade services was messy and hard to manage both, internally and for companies. By the end of 2000, trade procedures were standardised, responded to newly created rules, and were broadly incorporated into a single and integrated database. By 2000 it was also possible to comply with certain regulations over the internet, mainly those that were informative such as annual reports from the beneficiaries of special export programs.55The implementation of the technological platform meant that foreign trade procedures (i.e. rules of origin certificate approvals, authorisation and management of import quotas and licenses) became integrated under a single and unique database, registered and standardised into one information system that could be accessed from decentralised points across the country. It also included - Page 118 - certain degrees of automation, like basic information validation (i.e. that the ID matches the name of the person/organisation). Overall, average response times were reduced, and also allowed the first steps towards collaboration with other government agencies. In particular, the connectivity with Customs became a salient feature. The system also facilitated the administrative decentralisation process, reaching 75% of trade procedures by the end of 2000. The fact that the rules became embedded within the technological platform translated in greater homogeneity and standardized decision criteria as well increased monitoring capabilities.56 The latter was regarded as particularly important in the implementation of a total quality system that the ME was pursuing in parallel. Particularly, SICEX entailed features such as contract reviewing, assuring the control, identification and reliability of user’s information that were necessary in order to comply with the ISO 9000 norms. The system also generated basic statistics that proved useful for archival and follow up of trade procedures. Finally, the information system reduced errors related to manual and paper based administration, another key aspect of quality systems controls. At this point, it is useful to recall that the implementation of SICEX happened within an umbrella of broader modernisation reforms, that is the design and implementation of programmes of administrative reform such as PROMAP (see section 1 and table 10 earlier on this chapter), as well as coping with enormous regulatory pressures as a result of NAFTA. 56 Source: reported results of the project in the document “History of SICEX”, and interviews with ME officers. 57 In Spanish, the Prize is called “Premio Nacional de Administracion Publica”, and it is the only one granted to the ME and to an e-government programme in Mexico. Summary and main activities of period 1 I do not contend here that any of these alone could explain the relative success of the platform created in the Ministry of Economy, but certainly that these broader institutional forces provided a basis (or operational mechanisms) that facilitated and promoted the legal and institutional transformations within the Ministry of Economy. These initial efforts may seem modest compared to the progress that other countries were making at the same time (i.e. International Trade database in the USA) or in terms of scope. After all, one may argue that the case was simply all a matter of putting things - Page 119 - in order and streamlining processes with the help of a single database. Yet, the system represented a milestone for the Mexican public administration and set a precedent for other e-government programmes. In 1999, the National Institute of Public Administration awarded the Ministry of Economy with the most prestigious national prize in public administration for its innovative achievements and possibilities of replication.57 5.5.2 Period 2: Working with SICEX (2000-2008) Towards a Paperless Administration (2000-2006) 5.5.2 Period 2: Working with SICEX (2000-2008) Towards a Paperless Administration (2000-2006) The year 2000 represented a big change in the process of democratisation in Mexico. Even when, as we reviewed in the first part of the chapter, Mexico’s bureaucracy was becoming less clientelistic, an entire new Cabinet of Ministers was appointed and with them, a series of ‘cascade’ changes occurred across the federal agencies. Recall that the year 2000 meant the end of a longstanding period of the same party in charge of the executive branch of government. Political changes, and particularly those that follow presidential elections, are precisely one of the biggest challenges for policy continuity in Mexico as well as in most Latin American countries. Even so, the team working on SICEX did not experience drastic changes in the personnel. It was indeed the case that the General Director of Foreign Trade was promoted to Deputy Minister and, with her, most middle managers remained in their duties. This is important because it enabled a smooth continuity for the award winning technological platform —SICEX. Building on the efforts I described in the previous section, the government launched a new programme to keep SICEX’s progress moving forward. Called “Towards a Paperless Administration” (TPA), the distinctive feature of the new phase was to incorporate more broadly the use of internet and magnetic discs to reduce the use of paper and to deliver better and quicker services. Strengthening the collaboration with other agencies, and particularly, giving continuation to the already initiated digital validations with Customs, was the other big aim of the project. - Page 120 - There were two complementary actions to give continuity to the programme: (i) updating the system features with advances in the elimination or simplification of the regulatory environment, and (ii) modernising the technology infrastructure that enabled SICEX to communicate across its federal offices and with other agencies (network connectivity). Both actions were identified as crucial, not only to reduce the use of paper and increase progressively the availability of web-based services, but also as means to incorporate the use of electronic data validation with a growing number of Ministries. Updating the technological platform of SICEX remained a priority for the Ministry, mainly because the volume of administrative procedures was growing and more technological capacity was needed. To do so, the government occupied a mixed strategy of in-house and outsourcing deals. 59 INFOTEC delivered three series of documents including process redesigns and manuals of procedures that followed an ISO 9000 format (SE 2006, p. 74). With that, they created the roadmap to upgrading the system in place. 58 The whole contract included the revision of 18 modules that were on SICEX. The total length of the contract was about three months. 5.5.2 Period 2: Working with SICEX (2000-2008) Towards a Paperless Administration (2000-2006) The latter entailed two failed intentions, at best. The first outsourcing deal was awarded in September 2002 and entailed a general assessment of the system features, the revision of procedures (a classic BPR exercise) and the identification of those areas in which SICEX should be upgraded.58Only three months later, the contract was rescinded as the Ministry alleged that the company lacked sufficient expertise and technical capabilities(Secretaría de Economía (SE) 2006, pp. 71– 2). After the first failed attempt, the system update was re-planned. This time, INFOTEC—a government-linked center for innovation and technology development— was in charge of designing the technological upgrade and creating a doable plan for all substantial processes and changes that were needed.59 With a clearer roadmap in hand, the Ministry set out an open tender and awarded the contract for upgrading tasks to a private company in September 2004. One year later, and with no major achievements in hand, the government cancelled the contract again (Secretaría de Economía (SE) 2006, p. 76). The arguments that the Ministry set forth were no different from before; the government officials argued that the second company had failed to meet the deliverables agreed in the contract, a claim that the private contractors challenged by taking the case to Court. While the dispute remained in the Court for - Page 121 - several years, operationally, the budget was frozen and no further upgrades were possible.60 Even though the general upgrade of the system failed, other parallel and internal actions took place, especially in the area of digital security and coordination agreements with other agencies to share the information and availability for the electronic validation of a growing number of formalities.61From 2004, the digital connectivity was extended to other non-governmental agencies such as the Mexican Federation of Customs Agents (CAAAREM), which allowed both organisations to share trade information and “cross- check” the data declared by their customs agents on a (still) limited number of international trade procedures. In broader institutional domains, the government joined efforts to adopt the use of the electronic signature in conjunction with other federal agencies as well as with other countries.62Actions taken in this line entailed several technological updates (i.e. acquiring security software and new authorisation procedures) as well as taking part in official meetings formally initiated in 2000. Despite some general progress on the use and implementation of the electronic signature in the country (i.e. 60 According to my later interviews, the case was resolved by 2011 and it favoured the Company. In the first round of interviews during 2009, voices were controversial on the issue. Some people claimed that the leader of the project at that time was too difficult and did not let the project flow, while others suggested the technological upgrade was underestimated by the company. Unfortunately, I could not get access to the case, which would have led some light on these issues. 62 By the end of 2005, the Ministry joined a foreign trade working group to facilitate the adoption of electronic signature internationally. Although no immediate results were possible, these effort marked the starting point on what it later became the digital trade scheme between Colombia and Mexico. 61 Other internal technological upgrades included the acquisition of new servers and improving the internal connectivity between the central office and the federal delegations across the country. By the year 2006, all offices operating SICEX were connected via an integrated services digital network (ISDN). (SE 2006, pags. 65-70). 5.5.2 Period 2: Working with SICEX (2000-2008) Towards a Paperless Administration (2000-2006) two federal Agreements on the subject), it was not until 2009 that the advanced electronic signature was approved and implemented across the federal government. 63 The number represented a fair one, given the fact that the legal framework in place limited the possibilities of electronic submission to only few services and regulations. In addition, these figures only represent the proportion of initiated procedures over the internet (or magnetic discs) and not to the total cycle of trade procedures. 64 The Mexican Law of Administrative Procedures establishes that responses to foreign trade regulations require the signature of a responsible public servant. Before the electronic signature was approved in 2009, public servant had to sign in the forms in paper, hence, limiting the administrative capacity to fully process procedures online. Implementation and mixed results From 2000 to 2006, the system (SICEX) processed more than 1.1 million applications from 45 thousands of users (foreign trade companies). By 2006, the administrative - Page 122 - - Page 122 - decentralisation efforts were concluded and all trade procedures appeared in charge of the ME personnel distributed in the 52 federal delegations. Most processing times were reduced and in some cases, there were savings in operational costs such as paper (Telecom-CIDE 2007). From the business’ user perspective, the system increased the number of trade procedures that could be initiated over the internet. In 2000, only 6.2% of trade procedures were submitted through digital means, and by the end of 2006, the number reached 30%.63 The absence of an approved legal framework for the use of electronic signatures in place signified that it was only possible to receive trade procedures in digital means, but not to respond to them over the internet.64 In practical terms, while the submission channel was facilitated, in most cases, businesses had to attend a government’s office in person to collect any trade regulation. 65 Interview with public officials from the ME (#12, #16, #21, #22). Turning into slow motion (2006-2008) With the arrival of a new administration in December 2006, the e-government initiative got frozen for some time. Failed outsourcing deals to upgrade SICEX (particularly the later one) ended in a legal dispute that restricted the availability of financial resources to further invest in the system.65 During 2007 and the first 2 quarters of 2008, major advances in relation to the project concentrated on the simplification and elimination of foreign trade procedures, the revision and changes to trade regulation and in carrying out the adjustments that the technological platform required accordingly. A salient example has been the passage of a Presidential Decree in March 2008, which set the basis for further trade simplification procedures and formally included the establishment of a Digital Single Window for Foreign Trade. In other words, the main - Page 123 - - Page 123 - achievements concentrated on the regulatory and policy side rather than on technological advances. 66 “Decreto por el que se otorgan facilidades administrativas en materia aduanera y de comercio exterior” (Decree that grants administrative facilities concerning customs and foreign trade), published in the Federal Official Diary, 31 of March 2008. Summary of Period 2 (2000-2008) During the years covered in this phase (2000-2006 and 2006-2008) the project was strengthened in terms of continuity, although its achievements may be modest in comparison to the changes that occurred during the previous period. By the first quarters of 2008, most results concentrated on updating and maintaining the technological capabilities internally (both the platform and the digital connectivity with Customs), continuing the efforts towards simplification and elimination of red tape (such as unnecessary trade regulations), and building the basis for international collaborations with other countries. In technological terms, there were neither major changes in the ways in which the system worked or was operated nor was there a notable change in the main features the system could support. Another change in the administration was coming, and the project was going to envisage changes as well. 5.5.3 Period 3: From SICEX to the Single Window (2008-2012) The opening of this new phase starts with a new norm that the government approved in March 2008. Concomitantly with previous changes in SICEX, the government envisaged the creation of a digital Single Window to process all trade procedures in a single entry point.66 Later in August 2008, a new Minister was appointed and further changes occurred in the next hierarchical lines. A new Vice-Minister of Trade and Industry was appointed in 2008, which later appointed a new General Director for the DGCE. The new team followed the idea of seriously building a Single Window for foreign trade, and pushed the project on the competitiveness agenda even further. - Page 124 - - Page 124 - A number of institutional forces contributed to this vision. The first one came from a ‘technical opinion’67 that the Mexican Competition Authority (Comisión Federal de Competencia, in short CFC) produced in May 2008 regarding issues affecting Mexico’s performance in foreign trade. In this opinion, which was grounded in economic language and backed with international statistics, the CFC identified that Mexico could achieve a significantly better performance of economic growth and competitiveness by investing in foreign trade facilitation policies (Comisión Federal de Competencia 2008, pp. 4–5). The CFC recommended reducing the levels and dispersion of tariff regulations, to simplify and deregulate customs clearance procedures and to build institutional capabilities for government agencies that regulate foreign trade (Comisión Federal de Competencia 2008). The use of new ICT to support processes of simplification of trade procedures, especially for customs clearance, was also highlighted. The opinion was particularly opportune as Mexico’s volume of exports to the USA was superseded for the first time by China. This episode was a non-trivial concern considering Mexico’s geographical position and its natural comparative advantage to trade not only with North but also Central and South American countries. The Ministry of Economy took this opinion —in conjunction with others coming from international bodies (i.e. Worl Bank, CEPAL, OECD see section one of this chapter)— to support an ambitious trade reform that included a massive reduction in tariffs, and a more strategic use of networked technologies to support the trade facilitation process. These policies are precisely those I commented earlier on under the label of “Programme for Trade Facilitation” (see the green box in the figure of the case timeline). 67 In Mexico, certain bodies are empowered to provide advice on given aspects of the economic and public life. They are called ‘technical opinions’ to reflect the expertise and neutrality of the opinion. One of these bodies that do so is the Federal Competition Commission (CFC), Mexico’s Competition Authority. Besides its mandate to promote competition policies, the CFC also issues opinions on current regulatory framework and public policies regarding competition and free access to markets (see CFC’s main website: http://www.cfc.gob.mx/index.php/en/%C2%BFQUE-HACEMOS/opinions.html). In this case, the opinion was no-binding and issued by the Chairman of CFC. 5.5.3 Period 3: From SICEX to the Single Window (2008-2012) A further fact, also related to the CFC’s opinion, had to do with Mexico’s poor performance in world competitiveness and trade facilitation indexes as indicated at the beginning of this chapter. The World Bank’s report Doing Business positioned Mexico number 41 across 183 economies for doing business in 2010. Yet, in terms of trading - Page 125 - - Page 125 - across borders (a sub category in which the total score is built), Mexico scored number 69. Given the complexity of the current system of trade procedures, according to ME’s data, Mexico could advance at least 40 positions with the one-stop-shop put in place, ceteris paribus all other countries’ progress (Secretaría de Economía (SE) 2010). Not surprisingly, escalating positions in a world ranking such as the World Bank Doing Business or the WEF Competitiveness Report translates in a strong political incentive, given their publicity and visibility both nationally and in global trade forums. Within this context, after a series of informal meetings between Customs, the Ministry of Economy and other members of the Foreign Trade Commission, a working group for the simplification of trade procedures was set up in July 2009. The people participating in the sub-group did know each other well—at least in their majority. In some cases, they have worked together in setting up the first steps towards coordination mechanisms between Customs and the ME, and certainly kept working together during period 2. In other words, there was a history of collaboration or coordination that started several years. Yet, it was not until 2009 that this sub-group gained a rather more formal status. The following figures offer a better grasp of the main policy ideas in designing the Single Window. The left hand side of figure 9 depicts the complexity of foreign trade procedures as per the year 2010. It involves at least 30 different actors (exporters, importers, custom clearance agents, public agencies such as the ME and Customs, etc.), up to approximately 40 different documents in a single process chain, up to 200 data points of which over 60% are captured more than once, and more than 165 procedures to comply with all Mexico’s trade regulations. More than 10 thousands documents are processed per day by Customs. On average, a single customs clearance lasts 5 days (maritime trade operation). 5.5.3 Period 3: From SICEX to the Single Window (2008-2012) - Page 126 - - Page 126 - Figure 9: Mexican Single Window for Foreign Trade (VUCEM): Before and after scenario Source: Secretaria de Economia (SE) 2010 Figure 9: Mexican Single Window for Foreign Trade (VUCEM): Before and after scenario Source: Secretaria de Economia (SE) 2010 Source: Secretaria de Economia (SE) 2010 The right-side figure portrays the government’s adopted model for the Single Window. It is based on the United Nations Centre for Trade Facilitation and E-Business (UN/CEFACT), and exhibits a much cleaner, transparent and parsimonious view on trading across borders. Based on the recommendations of UN/CEFAC, the Mexican Single Window is defined as follows: The Mexican Single Window is a facility that allows parties involved in trade to send electronic information via a single entry point, submitted only once, to fulfil all import, export, and transit-related regulatory requirements. This is possible through the simplification, standardisation and automation of management processes. (Source: VUCEM website, own translation) In practical terms, the Single Window includes two main components: (i) the inter- agency coordination; (ii) the contract of services, including the technological platform (catalogue of services). Interagency coordination. The single entry point requires identifying and aligning the work of the relevant agencies with jurisdiction in international trade regulation. Besides the ME and Customs, these include: the Ministry of Agriculture (SAGARPA), the Ministry of Foreign Affairs (SRE), the Ministry of Health (SALUD), the Ministry of the Environment (SEMARNAT) and the Tax Administration Authority (SAT). Together with SHCP, all of them met in the International Commerce Commission, which main role was suggesting and negotiating changes to the existent legal framework. Other - Page 127 - agencies to be incorporated in the Single Window include: the Ministry of National Defence (SEDENA), the Ministry of Energy (SENER), the Ministry of Education (SEP), the National Institute of Anthropology and History (INAH), the National Institute of Fine Arts (INBAL), and the Mexican Council of Coffee (AMECAFE). Since 2009, the interagency working group for trade facilitation held more than 30 coordination meetings. Their main activities entailed working together to harmonize processes, obtain consensus on required data elements and to work on proposals to simplify (or eliminate) trade procedures. These coordination meetings “entail reaching an agreement on synonyms, terms of foreign trade, definitions, and data fields with a strong component of de-regulation and reengineering of procedures” (interview with ME official, 2012, #40). 5.5.3 Period 3: From SICEX to the Single Window (2008-2012) In sum, the Single Window effort represents the “biggest effort of inter-agency coordination at the Federal level in Mexico” (interview with ME official, 2012, #40), supported with a web-based technological platform. Catalogue of services. In June 2010, Customs set forth an open tender to outsource the project, which was awarded later in October (same year). The contract —of 811 million Mexican pesos (US$ 67 million)—follows a typical public-private partnership (PPP) initially awarded for 60 months with a possibility of renewal subject to performance. The contract also put forth tough deadlines and goals to be achieved before the end of 2012. The following are the main services that have been outsourced: 1. Consultancy: an in depth review and modelling of the flows of trade procedures in government agencies and the private sector; 2. The provision of the information technology infrastructure and the Single Window solution, based on web (design of information management, implementation of modules and the automation of procedures). The technological infrastructure also includes the provision of a mobile network in the 49 customs in the country, tablets for customs clearance and digital clearance procedures, secure communication infrastructure between agencies, the operation, maintenance and support for the Single Window; 3. The digitalisation of 5 years of paper-based procedures (archival information of Customs daily operations, about 150 millions of sheets of paper); 3. The digitalisation of 5 years of paper-based procedures (archival information of Customs daily operations, about 150 millions of sheets of paper); - Page 128 - 4. Disaggregated services: provision of training and e-learning modules (for distance learning) available for trade regulators, and organisational change management. The services contracted require public servants working in close association with external consultants in an effort that goes beyond developing a technological platform. The project means an exhaustive revision of trade procedures, the design and implementation of the technological platform and the digitalisation of archives in paper- based format to be available for digital queries, as well as transforming customs clearance into a fully mobile operation. Soon after the contract was awarded, another visible milestone for the project leaders has been the passage of the Presidential Decree on January 2011. In order to avoid relying on the “good will” of the governmental agencies involved in the project, the passage of a Presidential Decree gave a strong political and legal support to the Single Window for Foreign Trade. 68 The phases of implementation were described according to the actors that should be operating in different points in time. Thus, phase I establishes that the ME and Customs start operating no later than 30 September 2011; by phase II SEDENA, SEMARNAT, SAGARPA and SALUD should be on the platform (by 30 January 2012) and phase III, included SENER and SEP (no later than 30 June 2012). 5.5.3 Period 3: From SICEX to the Single Window (2008-2012) - Page 129 - - Page 129 - ACTOR TYPE OF PARTICIPATION Presidency of Mexico Political and legal support Ministry of Economy Vice-Ministry of Trade and Industry DGCE COCEX: International Trade Commission Project coordination and leadership Ministry of Finance and Public Credit Tax Administration (SAT) Customs Administration Operation and budget Ministry of Public Administration Unit of Internal control Facilitator and supervisor of procedures Other Federal Ministries SAGARPA (Agriculture) SENASICA (Food safety agency) SALUD (Health) COFEPRIS (Protection against Sanitary Risk) SEMARNAT (Environment) SEDENA (Defence) SENER (Energy) SEP (Education) INAH and INBAL (Anthropology and Fine Arts) Direct participation on trade procedures and inter- agency coordination External IT provider Integradores de Tecnologia SA Building and operating the technical platform, providing consulting services (5 year contract) Custom agents, brokers and private sector CAAAREM (Association of Custom agents) Private sector; AMECAFE (Coffee Association) Direct users of e-services Table5.3: Main Participants in the Single Window project and key responsibilities ACTOR TYPE OF PARTICIPATION Presidency of Mexico Political and legal support Ministry of Economy Vice-Ministry of Trade and Industry DGCE COCEX: International Trade Commission Project coordination and leadership Ministry of Finance and Public Credit Tax Administration (SAT) Customs Administration Operation and budget Ministry of Public Administration Unit of Internal control Facilitator and supervisor of procedures Other Federal Ministries SAGARPA (Agriculture) SENASICA (Food safety agency) SALUD (Health) COFEPRIS (Protection against Sanitary Risk) SEMARNAT (Environment) SEDENA (Defence) SENER (Energy) SEP (Education) INAH and INBAL (Anthropology and Fine Arts) Direct participation on trade procedures and inter- agency coordination External IT provider Integradores de Tecnologia SA Building and operating the technical platform, providing consulting services (5 year contract) Custom agents, brokers and private sector CAAAREM (Association of Custom agents) Private sector; AMECAFE (Coffee Association) Direct users of e-services Table5.3: Main Participants in the Single Window project and key responsibilities TYPE OF PARTICIPATION Project coordination and leadership Operation and budget Facilitator and supervisor of procedures Facilitator and supervisor of procedures Direct participation on trade procedures and inter- agency coordination Building and operating the technical platform, providing consulting services (5 year contract) Two further events in regulatory compliance helped the progress of the Single Window. From 2009, the Mexican government made the final steps towards the implementation of the Advanced Electronic Signature (FIEL for its acronym in Spanish) for tax compliance and other administrative services. 5.5.3 Period 3: From SICEX to the Single Window (2008-2012) The legal tool, entitled “Decree that establishes the Mexican Digital Single Window for Foreign Trade” put firm deadlines for interagency integration and explicitly required that each authority must facilitate the necessary infrastructure required. In addition, it established three phases for the project implementation (article III) on a progressive calendar of integration, due to be finished by the end of 2012.68 The Decree also gave legal support to the above-mentioned working group for trade facilitation, by settling it officially as an Inter-Secretariat Commission for the Single Window. The Commission meets once a month, and has been the basis for negotiating, debating and coordinating the tight deadlines that the project set forth. The Ministry of the Economy chairs the Committee, which is also a salient feature: even when the funds for building and contracting out the Single Window are coming from the budget of Customs, the effective leadership comes from the team in the Ministry of the Economy. 5.5.3 Period 3: From SICEX to the Single Window (2008-2012) In June 2009, the Mexican Tax Authority made the compliance of patrimonial tax declaration mandatory in digital format for all public servants. That was the starting point for making the use of FIEL a reality and yet another example of the public sector relying on the monopoly of the use of authority (Moore 1995). For this case, it meant a strategic step; from 2009, the rollout of authorized digital signatures amongst public servants became pervasive. The second event came from a Presidential directive. In 2010 the President announced a big step towards regulatory clearance, which set forth to automate 70% of all government - Page 130 - services by 2012. These two episodes in conjunction with other measures (such as the increasing importance of the Digital Government Agenda and the Austerity Decree), gave political and material support to the ME-Customs Single Window initiative. In January 2012, the President formally inaugurated the Single Window for foreign trade, which has been on a trial period since October 2011 and still limited to only some locations and 21 trade procedures. The next picture shows the Single Window as it is operating. Figure 10: Screenshot of the Mexican Single Window for Foreign Trade as February 2012 Figure 10: Screenshot of the Mexican Single Window for Foreign Trade as February 2012 By March 2012, more than 24 thousand companies have registered as potential users of the Single Window (half of the entire universe of users). There have been around 25 thousands of electronic value vouchers (COVE for its acronym in Spanish), even when they are optional until the 1st of June 2012. The process of digitalisation of the paper- based archive of ME and customs has reached around 70% of completion while the website has had more than 280 thousands visits since its launch in October 2011. From the 1st of June, the use of the digital Single Window became obligatory for all trade operations with Customs, being no longer possible to make foreign trade transactions using other channels (i.e. paper based). 5.6 Conclusions of Part Two of the Chapter While at first glance the progress and results that the project achieved from 2009 to 2012 look impressive, a more detailed understanding shows that the Single Window - Page 131 - project has been immersed within a long history of broader institutional dynamics. For example, the constant work towards simplifying and eliminating unnecessary regulation has been a salient feature within the Ministry of the Economy during the whole period. The modernisation reforms oriented to achieve more openness to trade, and particularly, the enforcement of NAFTA from 1995, changed remarkably the scenario of foreign trade in Mexico. As a high-level Mexican official of foreign affairs puts it: the changes that NAFTA brought into the economy of Mexico were impressive... it made mandatory to adapt trade practices to the new pressures of competing in international markets... You could not explain Mexico’s trade today [2012] without considering NAFTA and all the concomitant changes that it meant for trade regulation Even when the case may seem smooth, there have been notable tensions and over time and particularly, in the latest three years. A salient and visible feature is the strong political support that the Single Window project achieved. But many other more ‘invisible’ factors contributed to what at first glance could be seen as a miracle. The strong process of knowing each other, the leadership position of certain figures, together with a strong political mandate that pushed for more open and competitive trade are very important contextual factors. The presence of international bodies, whether providing technical support (i.e. OECD, World Bank) or by opening platforms for sharing knowledge (SELA, IADB), has also shaped the trajectory of the project. This is not to say that the international bodies were imposing their views and agendas. I rather claim, in light of the project history, that Mexico’s government embraced some of their views to achieve their own policy on trade. The conclusions of the 3rd Latin American and Caribbean Regional Meeting on International Trade Single Windows held in November 2011 summarized what the Mexican project seemed to have achieved so far: The plans to establish International Trade Single Windows form an integral part of the structure of public services and promotion of competitiveness... 5.6 Conclusions of Part Two of the Chapter Such projects should take due account of fundamental variables, such as: support from governments, formulation of public policies on the matter, the consolidation of a legal and regulatory framework, the linkage of the agencies involved with trade processes, and the establishment of the Single Windows architecture, based on an all-encompassing vision of the institutional framework for foreign trade and its stakeholders (SELA 2011, p. 3) - Page 132 - - Page 132 - Although we are yet to see the implementation effects on trade flows and on competitive indexes, the steps that the Mexican federal government put forward are remarkable. In the next chapter, I analyse the main discourses on public values and technology innovation. Although we are yet to see the implementation effects on trade flows and on competitive indexes, the steps that the Mexican federal government put forward are remarkable. In the next chapter, I analyse the main discourses on public values and technology innovation. 70 See section 4.3 of my methodology chapter. 69 Interview with a senior government official (Area director, Ministry of the Economy, #40). 5.7 Postscript On Friday 1 June 2012, the Single Window became mandatory for all trade operations. This meant that, from that day onwards, every Customs clearance shall be done on digital means through the digital platform. Just one day before, the government released the contingency plans in case of failure. On that Friday, the Single Window crashed several times, and the contingency plan—using the old means—was authorised accordingly. The atmosphere was tense. On the night of 1 June 2012, top government officials and project leaders held a meeting with the consultants. In that meeting, they discussed whether they should extend the transition period for some more weeks. The logic was simple: if the electronic platform was amenable to crashes, then the whole project would be at risk. Despite the tensions, the government was very clear: there would be no extensions. On July 1, 2012 a general election was held in Mexico. The incumbent party lost the elections, and PRI would return to power in December. The political risks were therefore anticipated too. By September 2012, the Single Window continues operating. Apart from the crash on 1 June, there were only two more instances in which the system crashed and the contingency plan was put to work. Despite the system is far from being ‘optimal’, it keeps working and accommodating from one day to the other. There are no signs for the Single Window to go away. - Page 133 - - Page 133 - 69 Interview with a senior government official (Area director, Ministry of the Economy, #40). 6 Chapter Six: Eliciting discourses on public values and technology in Mexico 6 The aim of this chapter is to assess empirically what discourses on public values and technology can be traced over time, and to elaborate on their structural characteristics. The latter means an analysis on their meanings, historicity and relation of the discourses and the different ways it has been enacted and operationalized. It does so by focusing on the trajectory of key texts of the design and pre-implementation of the Mexican Digital Single Window for Foreign Trade (hereafter Single Window). Context should be also taken into account: as I have already discussed, Mexico’s foreign trade occupies a fundamental place in economic and political terms, a priority that has been materialised over the last 25 years through the design and implementation of policies to achieve greater gains from trade. These policies include the numerous free trade agreements as well as improvements in trade regulatory policy, being the Single Window its “most important one”.69 Reforms in foreign trade were also accompanied with a series of broader institutional changes aimed at creating a more professional, transparent and less burdensome public administration, that serve to emphasise and put the discussion of public values in context. This chapter is organized as follows. I present an excerpt of the presidential Decree that establishes the Single Window for Foreign Trade—the key text that I have selected as my anchoring point for the case analysis.70 Based on the role of texts as a mediating instance of reality (Chouliaraki and Fairclough 1999, Fairclough 2005a, 2010) and drawing on analysis on genre and intertextuality, I identify the general discourse within the case—competitiveness—. From there, I present the four different discourses that I find in the case, and elaborate on their structural characteristics. The rest of the chapter offers an in-depth elaboration of each of the discourses on public values and technology in a systematic way: I present the common discursive features of each cluster (content), I offer evidence on where they emanate from (conditions of production), and show how these discourses are consumed and materialised in technology (operationalisation) - Page 134 - (Fairclough 2005, p. 932-3, Phillipset al. p. 646, Munir and Phillips 2005, p. 1667). I focus my analysis on the specific discourses that emerge from the Single Window project. 6 Chapter Six: Eliciting discourses on public values and technology in Mexico In addition, and in order to tackle temporality, the previous and smaller technological system that the government developed from 1995—SICEX—is used systematically to trace how these discourses were conceived, preserved, or marginalised over time. Further material from fieldwork (interviews, news and archival information) complements the analysis. At the end of the chapter, I present a summary of the main findings. 71 As a whole, the Decree comprises three sections: the expert in itself is the opening part and contains what it is usually known in the legal jargon as “legal reasons” (“considerandos” in Spanish) of the order. The second part includes twelve articles that set forth definitions and the responsibilities and actions to be executed by the agencies involved. The final and remaining part adds two “transitory” articles and the signatures of the President and ten Ministers from his Cabinet. I have drawn on these twelve plus two articles in the empirical chapter to explain the responsibilities, phases and specifies of the project and how they were actually produced, consumed and met (or otherwise). 6.1 The nodal discourse on competitiveness and free trade A key achievement for the development of the Mexican Single Window has been the passage of the Presidential Decree in January 2011. To facilitate the presentation of my analysis, I bring here an excerpt from the first part of this bill, which is particularly useful for unfolding a series of value assumptions and the main discourses within the case.71 Felipe de Jesus Calderon Hinojosa, President of the United Mexican States, in exercise of the authority vested in me by the Article 89, fraction I, of the National Constitution (...) and, Whereas 1 The 2007-2012 National Development Plan indicates that a more competitive national economy will bring better investment conditions and job creation, for which it is indispensable to consolidate and enhance the institutional framework; 2 These days, foreign trade demands measures of trade facilitation and commercial simplification that grant legal certainty and the reduction of transaction costs for Mexican enterprises, measures that will increase competitiveness; 3 With the aim of supporting enterprise development in our country, it is necessary that the Federal Public Administration drives the use of information technologies, as they optimize the interaction between individuals ((particulares)) and government, together with the fact that they constitute a fundamental tool for the development of trade facilitation and offer a viable opportunity to achieve an effective and efficient foreign trade The 2007-2012 National Development Plan indicates that a more competitive national economy will bring better investment conditions and job creation, for which it is indispensable to consolidate and enhance the institutional framework; 2 These days, foreign trade demands measures of trade facilitation and commercial simplification that grant legal certainty and the reduction of transaction costs for Mexican enterprises, measures that will increase competitiveness; 3 With the aim of supporting enterprise development in our country, it is necessary that the Federal Public Administration drives the use of information technologies, as they optimize the interaction between individuals ((particulares)) and government, together with the fact that they constitute a fundamental tool for the development of trade facilitation and offer a viable opportunity to achieve an effective and efficient foreign trade 71 As a whole, the Decree comprises three sections: the expert in itself is the opening part and contains what it is usually known in the legal jargon as “legal reasons” (“considerandos” in Spanish) of the order. 6.1 The nodal discourse on competitiveness and free trade - Page 135 - regulation; 4 In addition to the paragraph above, the use of information technologies in the operation of foreign trade reduces costs substantially, promotes the incorporation of small and medium enterprises to export activities, offers a transparent and equitable service, and strengthens the security for the users; 5 It is convenient that the competent authorities concerning foreign trade coordinate and implement actions of simplification, automation and improvement of foreign trade processes that lead to, among other improvements, the reduction of time and costs of international trade operations; 6 In conformity with the Foreign Trade Law, it is the responsibility of the Federal Executive, through the Secretary of the Economy, to coordinate the participation of the other agencies of the Federal Public Administration in the activities of foreign trade promotion, as well as to coordinate those [agencies] that administrate or control non-tariff regulations, with the aim of interconnecting them electronically with the aforementioned Secretary and with the Secretary of Public Finance and Public Credit; 7 The Decree that grants administrative facilities concerning Customs and Foreign Trade, published in the Federal Official Diary the 31 of March 2008, foresees the establishment of a digital window for electronic procedures, to which the dependencies of the Federal Public Administration that operate instruments, programmes and (...) to be interconnected electronically with the Secretary of the Economy and the Secretary of Public Finance and Public Credit; 8 Based on the trade simplification and facilitation policy that has already been instrumented inside the Federal Public Administration, the Federal Executive Branch under my duties considers necessary to issue, throughout the present Decree, the general dispositions for the establishment of the Mexican Digital Window of Foreign Trade (...); 9 For the above effects, it is considered convenient to establish stages of implementation for the referred Digital Window, to which the individuals ((particulares)) will be able to carry out promotions or applications related to imports, exports or transit of foreign trade goods, as well as to comply with non-tariff regulations referred in this Decree; 10 The present instrument counts with the favourable opinion of the Foreign Trade Commission, I hereby issue this Decree Figure 11: Excerpt from the Mexican Decree that establishes the Single Window for Foreign Trade DOF, 14 January 2011 In addition to the paragraph above, the use of information technologies in the operation of foreign trade reduces costs substantially, promotes the incorporation of small and medium enterprises to export activities, offers a transparent and equitable service, and strengthens the security for the users; It is convenient that the competent authorities concerning foreign trade coordinate and implement actions of simplification, automation and improvement of foreign trade processes that lead to, among other improvements, the reduction of time and costs of international trade operations; In conformity with the Foreign Trade Law, it is the responsibility of the Federal Executive, through the Secretary of the Economy, to coordinate the participation of the other agencies of the Federal Public Administration in the activities of foreign trade promotion, as well as to coordinate those [agencies] that administrate or control non-tariff regulations, with the aim of interconnecting them electronically with the aforementioned Secretary and with the Secretary of Public Finance and Public Credit; The Decree that grants administrative facilities concerning Customs and Foreign Trade, published in the Federal Official Diary the 31 of March 2008, foresees the establishment of a digital window for electronic procedures, to which the dependencies of the Federal Public Administration that operate instruments, programmes and (...) to be interconnected electronically with the Secretary of the Economy and the Secretary of Public Finance and Public Credit; Based on the trade simplification and facilitation policy that has already been instrumented inside the Federal Public Administration, the Federal Executive Branch under my duties considers necessary to issue, throughout the present Decree, the general dispositions for the establishment of the Mexican Digital Window of Foreign Trade (...); For the above effects, it is considered convenient to establish stages of implementation for the referred Digital Window, to which the individuals ((particulares)) will be able to carry out promotions or applications related to imports, exports or transit of foreign trade goods, as well as to comply with non-tariff regulations referred in this Decree; In light of the contextual features of Mexico and its trade policy, the text of the Decree offers an important first indication towards the main discursive grounds—nodal or general discourse—on which the Mexican Single Window has been built and planned. 6.1 The nodal discourse on competitiveness and free trade The second part includes twelve articles that set forth definitions and the responsibilities and actions to be executed by the agencies involved. The final and remaining part adds two “transitory” articles and the signatures of the President and ten Ministers from his Cabinet. I have drawn on these twelve plus two articles in the empirical chapter to explain the responsibilities, phases and specifies of the project and how they were actually produced, consumed and met (or otherwise). The 2007-2012 National Development Plan foresees that a more competitive national economy will bring better investment conditions and job creation, for which is indispensable to consolidate and enhance the institutional framework; 6.1 The nodal discourse on competitiveness and free trade The general discourse can be best regarded as one on global competitiveness in which countries must compete for investment and growth in order to reach development (see for example Moreno-Brid et al. 2009 and Part One of chapter Five). The grounds of the general discourse are based on the country’s commitment to free trade and the path that - Page 136 - Mexico chose as its route for economic development since the early 1980s.72The increase in the participation of foreign trade as percentage of Mexico’s GBP during the last 25 years, for instance, illustrates how this general discourse has been materially promoted and materially enacted. That is, the push for an increasing participation in the global markets has not just been a discourse but a discourse that has been accompanied with specific actions—such as joining NAFTA, signing multiple free trade agreements with other countries, and several industrial policies to promote foreign trade and foreign investment. By the same token, building technological platforms for trade procedures—whether SICEX from 1995 to 2008 or the current Single Window project—are also actions that have materialised the overall argument for greater competitiveness. Mexico chose as its route for economic development since the early 1980s.72The increase in the participation of foreign trade as percentage of Mexico’s GBP during the last 25 years, for instance, illustrates how this general discourse has been materially promoted and materially enacted. That is, the push for an increasing participation in the global markets has not just been a discourse but a discourse that has been accompanied with specific actions—such as joining NAFTA, signing multiple free trade agreements with other countries, and several industrial policies to promote foreign trade and foreign investment. By the same token, building technological platforms for trade procedures—whether SICEX from 1995 to 2008 or the current Single Window project—are also actions that have materialised the overall argument for greater competitiveness. Examples of the presence of the general discourse can be traced back to middle 1990s when the previous and smaller project SICEX was being designed and implemented. Hence, competitiveness and trade liberalisation were central among the reasons to established SICEX, as it is shown below: The increasing economic globalisation has created the imperative need among countries to count with more competitive markets, which has translated into an increasing requirement for foreign trade efficiency. 73 The opening paragraphs of the refereed Decree of 2008 Decree are indeed very similar to those in the text under study. This fact provides further support to the established trajectory of the overall strategy in Mexico. These are the actual paragraphs (DOF, 31 March 2008, p. 1) 72 See specially the first two sections of the previous chapter. 72 See specially the first two sections of the previous chapter. 6.1 The nodal discourse on competitiveness and free trade Mexico has opted for a greater participation in global world trade flows for which has negotiated trade agreements with several countries in the world; the present administration has captured this strategy in the National Development Plan 1995-2000. SICEX: A history of Success in foreign Trade Regulations, Ministry of the Economy, 1999 p. 1-2 (Emphasis added) Paragraph 7 of the excerpt of the Decree serves as a connecting line as well: [Whereas] The Decree that grants administrative facilities concerning Customs and Foreign Trade, published in the Federal Official Diary the 31 of March 2008, foresees the establishment of a digital [Single] Window for electronic procedures [Whereas] The Decree that grants administrative facilities concerning Customs and Foreign Trade, published in the Federal Official Diary the 31 of March 2008, foresees the establishment of a digital [Single] Window for electronic procedures The presence of this reference suggests that the establishment of the Single Window was foreseen earlier and appears as a result of previous planning (an explicit claim).73 - Page 137 - Taken together, these two quotes illustrate a historical trajectory and rather common finding in the texts: increasing competitiveness and promoting concomitant changes in trade regulation—including the use of technology—have been at the core of the government’s trade strategy for the last 25 years. All in all, the general discourse is not about technology or e-government but rather about competitiveness, openness to trade and modernisation reforms. In other words, competitiveness is the general discourse that “subsume[s] and articulate[s] in a particular way a great many other discourses” (Fairclough 2005b, p. 10). I turn now the attention to elicit these instrumental discourses, their meanings and their trajectory over time. 74 As I have commented in chapter 2, Beck Jorgensen and Bozeman (2007, p. 373) offer a common distinction in social sciences between prime and instrumental values. While the former are ends in themselves, the latter functions as tools to achieve other values. Instrumental values, in turn, may themselves be prime values, depending on the socio-political complex they are immersed in. Recall that this dissertation shares the view that there are no indisputable prime values, as there are no indisputable or self-evident truths. Whereas with the aim to support the development of the enterprises, it is considered convenient to provide administrative facilities to foreign trade users, so those operations [of trade] become more agile and simplified. 6.2 Eliciting discourses on public values, technology and their trajectory In connection with the debate offered in Beck Jørgensen and Bozeman (2007, p.376-7), the general discourse—increase competitiveness to contribute to job creation and economic growth—can be reasonably assumed as the prime value in the case, which suggests an end in itself.74 Identifying competitiveness as the prime value, or general discourse, is useful as it opens up space to deal with the clusters of instrumental public values, their relationships and implications with technology in greater detail. I turn attention again to paragraphs 2 to 5of the Decree (see below for the emphasis on words). From a closer screening, a family of words refers quite explicitly to the ideas on competitiveness and the economic rationale that motivated the Single Window project. The use of the words reduction of costs, whether explicitly as transaction costs or more implicitly in terms of time and other costs, appears several times in the four paragraphs. Other terms that can be clustered as part of the same vocabulary are the use of the verbs ‘demand’ and ‘optimize’ or the adverb ‘substantially’ to qualify the - Page 138 - - Page 138 - reduction of costs. 6.2 Eliciting discourses on public values, technology and their trajectory In conjunction, the use of technology is portrayed as the ‘viable’ and most cost effective tool to support these goals: 2 These days, foreign trade demands measures of trade facilitation and commercial simplification that grant legal certainty and the reduction of transaction costs for Mexican enterprises, measures that will increase competitiveness; 3 With the aim of supporting enterprise development in our country, it is necessary that the Federal Public Administration drives the use of information technologies, as they optimize the interaction between individuals and government, together with the fact that they constitute a fundamental tool for the development of trade facilitation and offer a viable opportunity to achieve an effective and efficient foreign trade regulation; 4 In addition to the paragraph above, the use of information technologies in the operation of foreign trade reduces costs substantially, promotes the incorporation of small and medium enterprises to export activities, offers a transparent and equitable service, and strengthens the security for the users; 5 It is convenient that the competent authorities concerning foreign trade coordinate and implement actions of simplification, automation and improvement of foreign trade processes that lead to, among other improvements, the reduction of time and costs of international trade operations; A first read of these paragraphs would suggest that public values usually associated to NPM are very dominant in the text and, as an extension, within the project. Paragraph 5, however, offers a less explicit reading. When the text refers to “reduction of time and costs”, it largely implies economic costs and technical efficiency, although not exclusively. For example, there were many instances in which actors suggested less tangible costs, such as a loss in public trust or government legitimacy as a result of abuse of office or dishonesty when dealing with regulatory procedures. In addition, the use of the phrase “among other improvements” leaves itself space for ambiguity. These other improvements may refer to different value assumptions, such as transparency and internal accountabilities political loyalty, the rule of law, robustness, and so on (I have underlined the words accordingly). In sum, the fifth paragraph shows the possibility of a variety of linkages between public values and technology in the design and implementation of the Single Window. 6.2 Eliciting discourses on public values, technology and their trajectory Building on these examples, the analysis I have conducted reveals four discourses on public values and technology that, with differences of degree, are apparent in the trajectory of the Single Window: - Page 139 - 1. Technical efficiency: This cluster consists of typical new public management values: productivity, parsimony, effectiveness and references to business-like approach. It also consists of timeliness and user orientation. Usually, references of assumed values within this cluster are associated with economic thinking, a cost reduction as driving force and a focus on ratios of resource and results. The link of this cluster to the nodal discourse of competitiveness is very strong. 2. Legality, justice and accountability: This cluster includes three subgroups of related values. First, legality, as the overall umbrella to other similar and related values, such as the protection of the rights of the individual, equal treatment and the rule of law. In particular, it discusses ideas on procedural fairness. The second subgroup includes notions of distributive justice. The third one, accountability, refers to the behaviour of public sector employees; it includes references to other values, such as professionalism, honesty and integrity. Overall, discourses in this cluster relate to values more traditionally associated to public bureaucracies. 2. Legality, justice and accountability: This cluster includes three subgroups of related values. First, legality, as the overall umbrella to other similar and related values, such as the protection of the rights of the individual, equal treatment and the rule of law. In particular, it discusses ideas on procedural fairness. The second subgroup includes notions of distributive justice. The third one, accountability, refers to the behaviour of public sector employees; it includes references to other values, such as professionalism, honesty and integrity. Overall, discourses in this cluster relate to values more traditionally associated to public bureaucracies. 3. Robustness and resilience: within this cluster, common public values are robustness, adaptability and reliability. In turn, these are public values captured by the machine metaphor (Beck Jørgensen and Bozeman 2007, p.366). Related values are timeliness and security, and usually, the main concerns in this cluster centres in keeping the system up and running. 4. (Forced) cooperation: within this cluster, I define cooperation as the opposite to competition amongst public agencies (another typical mechanism that the NPM promoted);traditionally, because of lack of market forces within the public administration, the agencies have to be coordinated by cooperation (Beck Jørgensen and Bozeman 2007, p.366). 75 Allocative efficiency is characterised as “the most efficient allocation of resources to reflect the wishes of recipients” (Andrews 2011, p.284), while distributive efficiency refers to the most efficient processes to distribute goods or services amongst those that need them most. 6.2 Eliciting discourses on public values, technology and their trajectory An emphasis within this cluster relates to the use of authority to drive cooperation; an implicated value within this cluster is political loyalty, and I shall discuss it accordingly. I claim that these discourses on families of values are analytically separable, although they should not be understood as neat separations. At first glance, public values pertaining to - Page 140 - technical efficiency (i.e. parsimony, productivity, user orientation) are quite prominent in the text of the Decree, a claim that goes in line with the focus of the general discourse on competitiveness. Yet, achieving parsimony and reduction of costs may depend crucially on the enforcement of legality. In sum, the relations between discourses are multifaceted and dialectical (Fairclough 2003, 2005, 2010), and while different, they are not totally discrete. In the following sections, I elaborate on each of these four prominent discourses, their trajectories and historicity, and the ways they have been interpreted and consumed over time. 6.3 Four clusters of public values 6.3.1 Technical efficiency and business-like approach: the economic rationality and the productivity discourse Content/Discursive features Usually, NPM and its influence on e-government has been criticised for its shift of focus from effectiveness to efficiency, and market type mechanisms over equality and impartiality (Cordella 2007, p. 267-9, Persson and Goldkhul 2010). However, as Andrews (2011) suggests, very little research has gone beyond theoretical speculation about the relation and effects of NPM on efficiency and cost reduction more generally. Scholars in public administration have shown that the term efficiency is in itself ambiguous and many times confused with other values such as effectiveness or economy (i.e. Stokes and Clegg 2002, pp. 226–7 also Andrews 2011).Based on the work of Musgrave (1959), Andrews (2011, p. 283-4) proposes three different types of public sector efficiency: allocative, distributive and productive.75 While the first two may relate to responsiveness and equity respectively, only the productive efficiency reflects the classic notion of the maximization of outputs and minimisation of inputs that NPM research seem to have focused on (Andrews 2011). In a similar idea to what Andrews defines as productive efficiency, Rutgers and van der Meer(2010) suggest that within the last decades, the notion of efficiency has been predominately understood narrowly as - Page 141 - technical efficiency. Technical efficiency can be characterised as “a technical term concerning the relation between resources and results”, (Rutgers and van der Meer 2010, p. 757).76 Drawing on the idea of technical efficiency is useful here, as it represents the main spirit within this cluster of values. References to reduction of economic and transaction costs, including those derived from all sort of activities related to trade procedure compliance, are very common during the whole trajectory of the project. If we accept that the nodal discourse on competitiveness is strongly related to economic thinking, it could be reasonable to expect that a great number of the discursive features in this cluster will relate to technical efficiency. I have already shown that terms like reduction of costs, and similar references to economic thinking (i.e. to demand, to optimize, to promote) are salient expressions in the text of the Decree. In general, gains in technical efficiencies, such as an increase in the ratio of trade procedures processed and resolved per day, have a central place in the trajectory of the case. In the same line, references to actions to reduce excessive burdensome regulation (also known as “red tape”) are also significant within the materials analysed. 76 Rutgers and van der Meer (2010) elaborate on the historical origins of the word “efficiency” and discuss how it has changed within public administration thinking. They differentiate the notion of “technical efficiency” to “substantial efficiency”, the latter comprising of those professional actions and organisation of the public sector to ensuring compliance with the execution of legal rules and regulations (Rutgers and van der Meer 2010, p.757). Conditions of Production Content/Discursive features Technology, in turn, is related in the cluster by linking its potential to generate cost savings in two related areas: the back and the front office (i.e. paragraph 3 of the Decree). The argument relies on the capabilities of new networked technologies to allow the automation of services in highly process-oriented agencies, in manners that were not possible before (Fountain 2001, chap. 3, Dunleavy et al. 2006, pp.10-2, Yildiz 2007, p.655). In turn, the internet and related technologies can lower costs and facilitate access and service provision to citizens, a well-known feature in the e-government literature (i.e. Fountain 2001, Bellamy and Taylor 1998, Andersen et al 2010). In sum, within this cluster, the choice of wording is rather explicit in the official texts and talks covering the project. Conditions of Production - Page 142 - 77 In the section that presents the advantages of SICEX, the text outlines six main categories: efficiency, transparency, security, control, flexibility and homogeneity; all categories are given a similar space in the text (i.e. same number of pages). Later, in the ‘results section’, however, there is a notorious emphasis on Conditions of Production - Page 142 - The discourse on technical efficiency, and its associated values (i.e. user orientation, parsimony and productivity), has been very prominent not only during the current Single Window phase but also in earlier phases of SICEX. Let me go back to the initial text of SICEX. Back then, in order to deal with the imperative need to make trade regulations more efficient, the government claimed that the use of technology was mandatory to cope with a number of problems: The problematisation (...) in 1994 was that users’ requirements were not served adequately in terms of efficiency, simplification and quality mainly because [exiting] information systems did not conduce to achieving a better internal administration of trade procedures (...) In sum, we were not able to satisfy our clients with efficacy in terms of their demands. (...) The decentralisation, deregulation and simplification of trade procedures cannot be achieved optimally when the administrative procedures are managed without information systems that administer, control, store, and report the necessary information for the correct operation of trade procedures. SICEX: A history of Success in Foreign Trade Regulations, Ministry of the Economy, 1999 p. 13(emphasis added) A similar efficiency and economic rationale of public values is also apparent in the 2006 text, when the government explains why more trade regulations should be dealt with over the internet: A similar efficiency and economic rationale of public values is also apparent in the 2006 text, when the government explains why more trade regulations should be dealt with over the internet: The recent world trend has marked a rapid integration of governmental services over the internet, through which government seek to facilitate citizen access via more efficient channels to comply with procedures, that are available in the most convenient place and time, and with lower administrative costs. Memoirs ‘Towards a Paperless Administration’, Ministry of the Economy, 2006, p.11 (emphasis added) The recent world trend has marked a rapid integration of governmental services over the internet, through which government seek to facilitate citizen access via more efficient channels to comply with procedures, that are available in the most convenient place and time, and with lower administrative costs. 78 In addition to analysis of intertextuality (see methodological chapter), I have conducted a number of word cruncher analyses of key government texts that, although preliminary, are useful to illustrate this point. The qualitative software that I use for my data analysis--Atlas.ti-- offers a Word Cruncher tool, which counts all words in a selected document. I paid particular attention to the results of the word cruncher of the 1999 document that explains SICEX (“SICEX: A history of success”). The analysis shows that the word efficiency (and related adverbs or verbs) appeared at the top of the list of words resembling public values (25 times), followed by the word security (18 times); the word quality appears in third place (14 times). The other documents (i.e. Memoirs 2006, the Presidential Decree) did not yield to such obvious results and will need further analysis. the achievements in terms of reduction of compliance costs (i.e. travelling) for businesses, and gains in internal efficiency measured in reduction of processing and response times. Conditions of Production Memoirs ‘Towards a Paperless Administration’, Ministry of the Economy, 2006, p.11 (emphasis added) An emphasis on user orientation, offering timely and cheaper service to clients (businesses in trade) is visible from this quote, which in turn resonates with paragraph 2 and 3 of the Decree. This offers an ambiguity in what the government means by efficiency and productivity: organisational aspects of public administration are intertwined with the effects on the relationship between government and citizens (or clients). The latter, often includes other less tangible aspects of that relationship, such as legality, flexibility and the rule of law.77All in all, the prevalence of technical efficiency— An emphasis on user orientation, offering timely and cheaper service to clients (businesses in trade) is visible from this quote, which in turn resonates with paragraph 2 and 3 of the Decree. This offers an ambiguity in what the government means by efficiency and productivity: organisational aspects of public administration are intertwined with the effects on the relationship between government and citizens (or clients). Conditions of Production The latter, often includes other less tangible aspects of that relationship, such as legality, flexibility and the rule of law.77All in all, the prevalence of technical efficiency— - Page 143 - approximated by references to reduction of costs as well as parsimony in the form of simplification of procedures—has been present since the days SICEX was designed and implemented, and constantly reproduced during the Single Window phase.78 1994-2000 2000-2006 2008-2012 • National Development Plan (p.158): established that to promote economic growth Mexico “requires (...) simple administrative procedures” • Modernisation Programme for the Public Administration(PROMAP): “To transform the Federal Public Administration into an effective and efficient organisation to fully meet society’s needs” • National Development Plan: set the objective to increase Mexico’s competitiveness and included two specific strategies for foreign trade regulations: to create public services of quality, and to promote the use of information technology • Good Government Agenda: the use of ICT is presented as the fourth out of six strategies; ICT are seen as tools to support broader modernisation strategies, such as achieving more efficiency, quality, transparency and honesty • National Development Plan (chapter 5, objective 5): established the need for improving government’s efficacy and efficiency to meet citizens needs via better regulation, administration and results of the federal civil service • Special Programme for Management Improvement(PMI): included four lines of action: to make better public services, to increment public sector productivity, to reduce costs by streamlining resources efficiently and optimally, with a strong focus on results and performance indicators Table 11: Examples of broader institutional forces in the trajectory of the case 2008-2012 • National Development Plan (p.158): established that to promote economic growth Mexico “requires (...) simple administrative procedures” • Modernisation Programme for the Public Administration(PROMAP): “To transform the Federal Public Administration into an effective and efficient organisation to fully meet society’s needs” • National Development Plan (chapter 5, objective 5): established the need for improving government’s efficacy and efficiency to meet citizens needs via better regulation, administration and results of the federal civil service • National Development Plan (chapter 5, objective 5): established the need for improving government’s efficacy and efficiency to meet citizens needs via better regulation, administration and results of the federal civil service • National Development Plan: set the objective to increase Mexico’s competitiveness and included two specific strategies for foreign trade regulations: to create public services of quality, and to promote the use of information technology • Good Government Agenda: the use of ICT is presented as the fourth out of six strategies; ICT are seen as tools to support broader modernisation strategies, such as achieving more efficiency, quality, transparency and honesty • Special Programme for Management Improvement(PMI): included four lines of action: to make better public services, to increment public sector productivity, to reduce costs by streamlining resources efficiently and optimally, with a strong focus on results and f i di Table 11: Examples of broader institutional forces in the trajectory of the case These discourses—as I have suggested in the theory chapters and the narrative of the case— do not appear in a vacuum. In addition to the pressures to do better in international rankings (i.e the World Bank Doing Business ranking), broader institutional discourses at national level (i.e. Mexico’s National Development Plans, and modernisation programmes) use vocabulary that promoted and inculcated public values related to technical efficiency, parsimony and productivity as well as a business-like approach. These discourses—as I have suggested in the theory chapters and the narrative of the case— do not appear in a vacuum. In addition to the pressures to do better in international rankings (i.e the World Bank Doing Business ranking), broader institutional discourses at national level (i.e. Mexico’s National Development Plans, and modernisation programmes) use vocabulary that promoted and inculcated public values related to technical efficiency, parsimony and productivity as well as a business-like approach. - Page 144 - I refer here to the institutional programmes covered in the previous chapter, such as PROMAP, the Good Government Agenda, and the Special Programme for Management Improvement. For example, the fact that SICEX pursued the decentralisation of procedures is concomitant with PROMAP’s objectives for “decentralizing functions and not concentrating powers, so that regional development and economic decentralization is promoted, public services are improved, costs are lowered, and decisions are made where required, in order to provide a better service for the population” (SECODAM 1995, pp N/A). I refer here to the institutional programmes covered in the previous chapter, such as PROMAP, the Good Government Agenda, and the Special Programme for Management Improvement. For example, the fact that SICEX pursued the decentralisation of procedures is concomitant with PROMAP’s objectives for “decentralizing functions and not concentrating powers, so that regional development and economic decentralization is promoted, public services are improved, costs are lowered, and decisions are made where required, in order to provide a better service for the population” (SECODAM 1995, pp N/A). The importation of business sector techniques within the public administration—a typical claimed characteristic of NPM—has been also apparent since the early days of SICEX. • Special Programme for Management Narratives from the field confirm the emphasis of the technical efficiency discourses (emphasis added): For the current Minister [year 2009], who has worked for the private sector for many years, the use of technology is a constant to increase productivity and competitiveness. These are the objectives we want to achieve in the Ministry. Those are the motivators and the actual reasons to use technology in the public sector. Main IT Advisor to the Minister We need to be more efficient in our duties. We need to be able to perform as e-business companies do. That’s what is crucial for competitiveness. Area Director, Ministry of the Economy This vocabulary is also echoed by representatives of companies in the trade sector. When asked about the overall benefits that the Single Window would bring to Mexico, a partner from Deloitte in Mexico said: There are a number of different benefits from the Single Window... we can say that the overall benefit is an increase in Mexico's competitiveness... But then, you have a varied range of benefits... For example, the reduction of costs to comply with trade procedures... now, some procedures are going to be cheaper or even free in some cases... (...) you also have more agility and speed... times of response are reduced, because many trade procedures will have been eliminated or simplified, which translates in a slimmer bureaucratic structure for the government. Foreign trade Partner, Deloitte Mexico; interview in Radio Formula, March 2012 (Emphasis added) - Page 145 - - Page 145 - Other references to business-like vocabulary within government have been prominent over the years of both projects—SICEX and the Single Window. For instance, references to business process reengineering (BPR) exercises go back to early days of SICEX (1995), and continue to be a recurring theme within the developments of the digital Single Window. In a similar fashion, total quality management systems (TQM) were in vogue during the early phases of the project, although lately slightly abandoned.79 So far, I have showed examples of what constitute general findings in the data; a common and prominent vocabulary on technical efficiency that has been promoted over time. I now turn attention to the ways these discourses have been materialised and consumed. 79 See my previous footnote; references to quality and total quality systems were very explicit in the early phases of SICEX. They then become abandoned. I expand more under the cluster of robustness. Consumption and materialisation One of the most salient features of introducing technologies in the case—from the novel database in 1995 to the current Single Window—has been the facilities and gains from the automation of tasks often classified as ‘information work’ (Fountain 2001, p.38). The analysis of the texts suggests a direction towards well-known functions of ICT and networked technologies within public sector organising. The Mexican government seemed quite aware of the “viable” opportunities for collaboration and coordination at distance that new ICT could bring for the administration of trade procedures and trade regulations more in general. In the case of SICEX, the use of technology proved helpful for reducing waiting times, and accelerating processes via its automation features (see section 5.5.1 of chapter 5). The design and preparation of the Single Window offer further examples on the ways the discourses on technical efficiency, productivity and parsimony internalise (and are internalised by)technology. The Single Window has a number of technical features that aim at addressing directly the quest for costs reduction, parsimony and productivity of trade regulations. Bearing in mind that the technological artifact—Single Window—is still under way, one example should suffice to illustrate how technology has served to push the values associated with technical efficiency much further. The next table presents a comparison - Page 146 - - Page 146 - across relevant productivity indicators of trade procedures before and after the Single Window. Indicator Old (SICEX) New (Single Window) Change Number of procedures 54 22 -60% Data entry points 770 435 -44% Average waiting time 15-20 days 1-5 days Average number of requirements per procedure 104 61 -41% Total number of activities 127 25 -80% Total number of tasks 867 282 -67% Table 12: Quantification of simplification of procedures. Trade regulations administered by the Ministry of Economy Source: ME. Note: trade regulations of ME represents 55% of all procedures in foreign trade. These numbers suggest that, on average, the Single Window eliminates several trade procedures, allowing also for a great deal of automation. This means that, all other things held constant, the Single Window reduces substantially waiting and processing times of trade regulations, as well as decreasing their associated compliance costs (i.e. travel costs).A case in point are the new and automated procedures to obtain a certificate of origin, which represents the largest group of trade regulations that the Ministry of Economy administrates. 81 On average, pilot tests reported that it takes about 25 seconds to load a screen and from 2 to 5 minutes to upload a document. For a COO, it may take up to 20 minutes to complete the whole process. Data: webinar March 2012. 80 As Mexico has signed a great number of trade agreements, the COO are very important to get trade benefits such as a tariff exemption or a quota. 82 Due to regulations, for example, the EU requires the COO in printed form; since 2010, Mexico has been issuing and accepting fully digital COO with its Colombian partners. 84 I draw here on concerns raised in blogs and newspaper articles, as I could not get access to further data from the government yet. I am not suggesting the government is unwilling to provide this data; they have been particularly helpful and collaborative with all my data inquiries. Consumption and materialisation A certificate of origin (hereafter COO) is a document used in international trade to get the duty free benefits for a trade agreement (i.e. within NAFTA).80 In its current format—before entering into the Single Window processing—in order to obtain a COO, businesses are required to fill in different forms and attend the government office at least four times. The Single Window reduces the number of visits to only one, which potentially indicates a first reduction of costs (transportation costs). In addition, by automating the majority of the processes, the Single Window potentially reduces the waiting time from days to the actual time it takes to fill in the data on the web portal.81 Furthermore, some countries have already signed agreements with Mexico to accept COOs in digital format (i.e. Colombia), which means - Page 147 - - Page 147 - that the use of paper is eliminated completely. When otherwise, the government decided to cover the costs of issuing a paper-based COO.82 The next table provides detailed examples on COO simplification and other similar trade procedures that the Ministry of Economy administers. Trade procedure Response time (days) Old New Type Reduction 1 Questionnaire registry 2 1 Manual 50% Certificate of origin 1 Actual data capture time Automatic N/A 2 Drawback (tax returns) 10 Actual data capture time Automatic N/A 3 Imports/exports quota (worldwide) 7 5 Manual 29% Imports/exports quota (worldwide) 7 Actual data capture time Automatic N/A 4 Import and export permit 5 3 Manual 40% Import and export permit 5 Actual data capture time Automatic N/A Table 13: Estimated gains in efficiency and productivity - selected trade procedures Source: ME 2010. Note: trade procedures may involve different activities. For example, the COO requires first submitting a completed questionnaire; and that is why the rows show two different procedures. The column “old” and “new” show the average response time (days) that before and after the use of Single Window respectively. The column “type” distinguishes between possibilities of decision-making; manual indicates that a visit or authorisation is needed from a public servant; automatic means that, as long as the submission meets the system rules, response is given automatically with no need to further assistance. 83 Reports on the “Ventanilla Unica” facebook page; http://www.facebook.com/VUCEM. This page does not belong to the government, but to a group of custom agents and trade users. Consumption and materialisation Trade procedure Response time (days) Old New Type Reduction 1 Questionnaire registry 2 1 Manual 50% Certificate of origin 1 Actual data capture time Automatic N/A 2 Drawback (tax returns) 10 Actual data capture time Automatic N/A 3 Imports/exports quota (worldwide) 7 5 Manual 29% Imports/exports quota (worldwide) 7 Actual data capture time Automatic N/A 4 Import and export permit 5 3 Manual 40% Import and export permit 5 Actual data capture time Automatic N/A Table 13: Estimated gains in efficiency and productivity - selected trade procedures Source: ME 2010. Note: trade procedures may involve different activities. For example, the COO requires first submitting a completed questionnaire; and that is why the rows show two different procedures. The column “old” and “new” show the average response time (days) that before and after the use of Single Window respectively. The column “type” distinguishes between possibilities of decision-making; manual indicates that a visit or authorisation is needed from a public servant; automatic means that, as long as the submission meets the system rules, response is given automatically with no need to further assistance. Table 13: Estimated gains in efficiency and productivity - selected trade procedures Source: ME 2010. Note: trade procedures may involve different activities. For example, the COO requires first submitting a completed questionnaire; and that is why the rows show two different procedures. The column “old” and “new” show the average response time (days) that before and after the use of Single Window respectively. The column “type” distinguishes between possibilities of decision-making; manual indicates that a visit or authorisation is needed from a public servant; automatic means that, as long as the submission meets the system rules, response is given automatically with no need to further assistance. Evidence from pilot tests shows concerns with the speed of the system. During a course offered by customs agents in Mexico City, a real time import operation crashed at least three times before it was possible to finish with the entire operation.83 While no further data is available at the moment, concerns with the speed operation and the system robustness were raised by users (I expand more on this in the robustness cluster).84 84 I draw here on concerns raised in blogs and newspaper articles, as I could not get access to further data from the government yet. 85 These numbers are based in actual human resources costs that the Ministries dedicate for the actual administration and management of trade procedures, which are 5.1 USS million for all the Ministries and 348 USS thousands for Customs. Source: Customs figures, internal report (Hernandez, 2011). The take up and usage of the Single Window (see epilogue of chapter Five) serves also as proxy for its effectiveness. 86 Mingers and Walsham (2010, pp. 848–9) touch on similar points when they review the complexities of dealing with pragmatic, moral, and ethical issues on IS research; while in theory it is possible to elaborate on the distinctive aspects of each category, in practice, things are not so clear cut. Summary of the cluster There has been an extensive and systematic use of vocabulary that is related to technical efficiency in the design and pre-implementation phases of the Single Window. I have shown that this pattern is also similar in the earlier and smaller scale project (SICEX). Even further, the discourses in this cluster are well mapped within the contextual priorities related to competitiveness and openness to trade that Mexico has promoted for the last 25 years. In terms of consumption, the discourses on efficiency, parsimony and business-like approach have been materialised in the Single Window—as well as in SICEX in due time—by using automation and changes in regulation accordingly. Automation, in turn, has been highly dependent on standardisation, formalisation of rules and an intense work on process redesign, which leads us to the next cluster. Consumption and materialisation I am not suggesting the government is unwilling to provide this data; they have been particularly helpful and collaborative with all my data inquiries. - Page 148 - In terms of internal efficiencies, the government claimed explicit gains: an optimisation of human resources of 21%, which represents about 1.25 USS million in administrative savings for all the ministries involved in the Single Window, and 43 USS thousand for Customs annually.85 85 These numbers are based in actual human resources costs that the Ministries dedicate for the actual 6.3.2 Legality, justice and accountability This cluster is perhaps the most difficult to grasp, given the ambiguous character and multiple relations of the implicated public values within this group. The notion of accountability, for example, is well known for being an elusive concept (Bovens 2005, 2007); the same applies to fairness or justice. What is fair, for example, depends on the socio-political context, value and moral judgments of a given group (i.e. Bozeman 2000, 2007).86 To deal with these issues, I elaborate on the discourses on public values and technology in this cluster into three related themes: (i) legality, the rule of law and - Page 149 - procedural fairness, (ii) internal accountability of public servants, and (iii) distributive justice. I separate the themes for a better grasp of the argument, although I will cross- reference them when necessary. Content/Discursive features In the call for tenders for the Single Window that the government put forward in October 2010, the fourth objective specified the following87: So far, our textual entry point (the Decree) is relatively brief in terms of legality and related values, but this does not mean that these values were marginal in the case. In the call for tenders for the Single Window that the government put forward in October 2010, the fourth objective specified the following87: The objectives of the Single Window are (...): To increase legal certainty: To ensure transparency in the administration; to develop a fair and equal Customs framework; to increase the effectiveness of the rule of law. Terms of Reference, International Public Tender of the Mexican Single Window for Foreign Trade, September 2010 j g ( ) To increase legal certainty: To ensure transparency in the administration; to develop a fair and equal Customs framework; to increase the effectiveness of the rule of law. Terms of Reference, International Public Tender of the Mexican Single Window for Foreign Trade, September 2010 j g ( ) To increase legal certainty: To ensure transparency in the administration; to develop a fair and equal Customs framework; to increase the effectiveness of the rule of law. Terms of Reference, International Public Tender of the Mexican Single Window for Foreign Trade, September 2010 If taking the textual object (the Decree) as its face value, legality could be seen as marginalised, but the history and origins of these discourses offer more details. 87 The document addresses four objectives: (i) to reduced time and cost, (ii) to improve logistics, (ii) to eradicate corruption, and (iv) to increase legal certainty. These objectives are also crystallised in the benefits published in the Single Window website, available here: www.ventanillaunica.gob.mx/envucem/AboutVU/Benefits/index.htm Content/Discursive features Legality generally refers to the fundamental principle that the relationship between public administration and the citizen should be regulated by law instead of the arbitrary exercise of power or discretionary administration (Beck Jørgensen and Bozeman 2007, p.368). Within this sub-cluster, values such as the rule of law and the protection of the individual rights as well as equal treatment are related in a systematic fashion. Families of words that resemble these values in the texts correspond to “legal certainty”, “transparency of procedures”, “clear rules and regulations”, “homogeneity of decision criteria”, and “adherence to law” (in Spanish, apego a la ley —a phrase that describes that a process is legal). In addition, I also use the notion of procedural fairness, a concept that is used in the management literature on trust (Culnan and Armstrong 1999, Brockner et al. 2000, Brockner 2002). The notion of procedural fairness is concerned with the way processes and rules are used by a decision-maker. Thus, a fair procedure would require a lack of bias, clear criteria to support a decision, and a hearing appropriate to the circumstances. The first paragraphs of the Decree offer a glimpse into these values, although it is rather less explicit if compared to what I have just discussed for the case on technical efficiency. For example, I have already shown that the use of the phrase “among other improvements” in paragraph 5 of the Decree is an implicit ground that may well relate to the use of technology as an opportunity to improve legality and the rule of law. In a more explicit fashion, paragraphs2 and 4, mentions legal certainty, transparency, equity and security—all words resembling the spirit of this cluster: These days, foreign trade demands measures of trade facilitation and commercial simplification that grant legal certainty and the reduction of transaction costs (...); In addition (...) the use of information technologies in the operation of foreign trade offers a transparent and equitable service, and strengthens the security for the users; - Page 150 - So far, our textual entry point (the Decree) is relatively brief in terms of legality and related values, but this does not mean that these values were marginal in the case. 87 The document addresses four objectives: (i) to reduced time and cost, (ii) to improve logistics, (ii) to eradicate corruption, and (iv) to increase legal certainty. These objectives are also crystallised in the benefits published in the Single Window website, available here: www.ventanillaunica.gob.mx/envucem/AboutVU/Benefits/index.htm 88 Source: SICEX: A history of Success in Foreign Trade Regulations, Ministry of the Economy, 1999, p. 20. 88 Source: SICEX: A history of Success in Foreign Trade Regulations, Ministry of the Economy, 1999, p. 20. p g , www.ventanillaunica.gob.mx/envucem/AboutVU/Benefits/index.htm Conditions of Production Preoccupations with enforcing legality have been very prominent in the discourses of broader modernisation reforms that have shaped both technologically induced projects (SICEX and the Single Window), as table 11has shown before. A further example of the presence of discourses on legality and associated values is offered in the starting point of the text that describes the origins of SICEX back in 1995: In conformity with the objectives of the National Development Plan of 1995- 2000, which establish the need to broaden the legal security for the companies throughout actions of deregulation and administrative simplification, oriented to improve the [administrative] efficiency (...) and to eliminate the unnecessary discretionary acts from the authorities and the excess of bureaucratic procedures, (...) several actions have been put in place, such as the implementation of a novel information system [SICEX] In the same document, the government stated that increasing transparency was the second most important benefit listed and achieved when SICEX was designed and implemented during the 1990s.88 Transparency can be understood in the text, and in this occasion, as the existence of clear and standardised rules, shared across all federal In the same document, the government stated that increasing transparency was the second most important benefit listed and achieved when SICEX was designed and implemented during the 1990s.88 Transparency can be understood in the text, and in this occasion, as the existence of clear and standardised rules, shared across all federal - Page 151 - agencies, for decision making. Narratives from the field expressed similar value associations. When asked about the most important value creation delivered by the system to administrate trade procedures, a senior official says: The most important [value] is [legal] certainty.. this is, that the dude knows he is going to seat with certain information in a certain place, and in exchange and at a given time, he is going to obtain the [foreign trade] authorization in a clear and secure way, without surprises. Director, International Trade Commission, Ministry of Economy (#8) Although the vocabulary presented here is not necessarily using the words legality or fairness, the priorities and the broader institutional context in which SICEX was launched, implemented and used, meant “leaving the albur to entering a world of clearer rules”89 (interview #15 with senior government official). In sum, concerns with legality and the rule of law as well as procedural fairness have been prominent discursive features over time. 90 Similar results (low scores in legality and the rule of law in Mexico) are reported in the Sustainable Governance Indicators in 2009 and 2011. See Philip et al (2011) and Bertelsmann Foundation (2009). Available at: http://www.sgi-network.org/index.php 89 In Mexico, an “albur” is a double entendre in which one of the possible meanings usually carries sexual undertones. In the case it is used here, it refers to the lack of a clear meaning and obligations of procedures. 91 The indicator “rule of law” measures perceptions of the extent to which agents have confidence in and abide by the rules of society, and in particular the quality of contract enforcement, property rights, the police, and the courts, as well as the likelihood of crime and violence (Kaufmann et al 2010). Conditions of Production As suggested earlier, efforts to improve legal certainty—and legality, more broadly—come from existent institutionalised discourses at national level. Since 1990s,Mexico has put forward a series of modernisation reforms to incrementally change from a society governed through the exercise of personal discretion into one based on legal norms (Pardo 2009, Philip et al 2011, p.11, also section 5.2 of this thesis). Despite the reforms, perceptions on legality and the rule of law still appear as a weak point in Mexico. According to Kaufmann et al (2010), Mexico ranks quite poorly in the rule of law indicator when compared to other counties and the Latin America average (see figure below).90,91 This is, on its face, a result that can lead us to be at least suspicious about the level of enactment of these values. In other words, the discourse on legality has been in the government texts (and especially in modernisation reform plans) for many years, without necessarily obtaining improvements. - Page 152 - - Page 152 - Despite these ambiguities, one may recall that aggregate measures may not provide the full picture. In particular, it is important to recognize that the indicator constructed by the World Bank includes perceptions on the quality of police and likelihood of crime and violence, areas that have hampered efforts to enhance the rule of law in Mexico in recent years due to the ongoing drug war (Phillip et al. 2011, p. 11). The process of enforcing the rule of law is therefore uneven, and I shall discuss in what ways the discourses on legality have been interpreted and materialised in the design of the Single Window. Despite these ambiguities, one may recall that aggregate measures may not provide the full picture. In particular, it is important to recognize that the indicator constructed by the World Bank includes perceptions on the quality of police and likelihood of crime and violence, areas that have hampered efforts to enhance the rule of law in Mexico in recent years due to the ongoing drug war (Phillip et al. 2011, p. 11). The process of enforcing the rule of law is therefore uneven, and I shall discuss in what ways the discourses on legality have been interpreted and materialised in the design of the Single Window. Consumption and materialisation Automation and standardised procedures can be considered good proxies for equal treatment and fair treatment, if fair is understood as a non-discriminatory handling of information and services. In other words, as long as rules are the same for everyone, fairness can be ensured. Initially, SICEX was a pioneer in creating baseline rules, and procedures for all. The next quote offers an explicit entry to this claim: - Page 153 - Clear and transparent criteria for administrative decisions: (...) it is indispensable that decision criteria to authorise or reject procedures are written into the system in an explicit and simple way. SICEX: A history of Success in Foreign Trade Regulations, Ministry of the Economy, 1999 p. 1 (emphasis added) Clear and transparent criteria for administrative decisions: (...) it is indispensable that decision criteria to authorise or reject procedures are written into the system in an explicit and simple way. SICEX: A history of Success in Foreign Trade Regulations, Ministry of the Economy, 1999 p. 1 (emphasis added) In turn, the design of Single Window emphasises this functionality too. In line with the efforts to automate trade regulation procedures as much as possible, data suggests that the number of rules inscribed in the system has increased significantly. The next table summarises the quantification process that the government has concluded, as part of their process redesign. In turn, the design of Single Window emphasises this functionality too. In line with the efforts to automate trade regulation procedures as much as possible, data suggests that the number of rules inscribed in the system has increased significantly. The next table summarises the quantification process that the government has concluded, as part of their process redesign. Trade procedure Number of rules Old New Difference Change 1 Questionnaire registry 115 120 5 +4% Certificate of origin 28 54 26 +93% 2 Drawback (tax returns) 34 27 -7 -21% 3 Imports and exports quota (worldwide) 34 68 34 +100% Imports and exports quota (regional) 10 68 58 +580% 4 Import and export permits 19 61 42 +221% Table 14: Change in the number of rules-Selected procedures of the ME Source: ME. The table shows that the number of rules, that is to say, the number of ‘decision steps’ inscribed in the software, are significantly greater than before. Consumption and materialisation Of course, the increase in a number of rules does not guarantee the full elimination of discretion or the complete achievement of legal certainty. The argument here is that both efforts—the reduction of the number of procedures and the concomitant increase in rules—are the consequence of the government’s longstanding efforts to assure legality via more transparent and clear rules for everyone (internal and external). As an example an Area Director from the Ministry of Economy explains during a webinar in February 2012: There are lots of advantages in terms of resolutions that you don’t see.. but I can assure you that we have set up many internal ‘locks’ [in the software] (...) the internal responsibility is much stronger (...) [the Single Window] gives parsimony and order to our internal process, but it also makes the process much more efficient. Area Director, SE (webinar, 2012) - Page 154 - Another way to see the materialisation and consumption of the discourse is by the issuing of new laws and regulations itself. As a senior public servant puts it: Another way to see the materialisation and consumption of the discourse is by the issuing of new laws and regulations itself. As a senior public servant puts it: In public administration, you can only do what you are specifically empowered to do, via a Law or a Decree. Former Deputy Minister, Ministry of the Economy (#15) The Single Window entailed a number of legal changes, being the Decree itself a good example of it. In addition, laws and regulations have been put forward to allow for changes in rules. It was sometimes technology itself the one driving changes. For example, a project leader from Customs reflects on the operation of SICEX: In general, it’s easier to change the [legal] regulations than the system... but it has happened the other way around; sometimes we were ready to make changes to the system, to upgrade it, and the regulations were not there yet... Director/Project Leader, Customs (#17) Other interviews suggested similar viewpoints. 92 Interviews with senior officials and middle managers in the Ministry’s IT Department (#9, #11 and #20). Consumption and materialisation For example, when I talked to technical areas in the Ministry of the Economy, some actors at first suggested that the main barrier was to get the regulations approved to release the technical updates, and then provided examples on the pressures they received to get things done in few hours because a new regulation was passed.92In sum, these examples show how technology internalises and is internalised by legality and further modernisation contexts. The implementation of the Single Window experienced delays precisely because of absences of legal norms supporting certain phases of its operations. 93 In projects that involve the participation of different government agencies (and contractors), Sullivan (2003) makes the distinction between vertical accountability upwards to central government, downwards to citizens, and horizontal accountabilities to partners. In the author’s terms, I would be referring mainly to the first type of accountability (vertical upwards to central government). 94 Organisational accountability refers to the relation between public managers and their superiors, the latter being those that will ask the former to account for their assignments. This, in general terms, involves compliance with the organisational rules and procedures as well as a reliance on a strong hierarchical relationships. It is usually referred as an ‘internal’ type of accountability as this form of accountability is not usually available to the public at large (Bovens 2005, p.287). (ii) ‘Internal’ Accountability: responsibility and behaviour of public managers Standardisation and rationalisation of procedures were many times mentioned as ways to eliminate arbitrary and unnecessary discretion of public servants. This second sub- group elaborates on a particular aspect of accountability and public servant’s unnecessary discretion. - Page 155 - - Page 155 - 95 In Spanish, there is no exact equivalent to accountability; responsibility is usually used as a synonym. Content/Discursive features Accountability is best regarded as one of those golden concepts that no one can be against because it conveys an image of trustworthiness and transparency (Bovens 2007, p.448). Yet, as it is the case with notions of fairness or justice, accountability remains elusive as a concept and has little substance without its institutional and historical context (Stone 1995, Mulgan 2000, Bovens 2005, 2007). For example, an actor can be accountable or responsible to a wide range of other subjects at the same time (Beck Jørgensen and Bozeman 2007, p. 364), and for different reasons. In turn, the actor can be either an individual (i.e. an official or civil servant) or an organisation, such as a public institution (Bovens 2007, p. 449). For the purpose of my analysis, I shall be looking particularly at the notion of administrative accountability (Stone 1995), that is to say, the values associated to the behaviour of public servants in their administrative functions of government. In this sense, when I coded accountability, the most common references were grouped in transparency of processes, honesty, professionalism and competent behaviour of public servants as well as ideas on vertical accountability93 of public servants to central government. These ideas would be therefore more closely associated to what Bovens (2005, p.187) refers as ‘organisational accountability’ and ‘responsibility’.94,95 In turn, this sub-cluster is very close to some ideas that I have already discussed in the group of legality and fairness, such as the formalisation of rules and transparency of criteria. While there are unavoidable overlaps, the focus here is more related to the role of public servants as administrators of formalised transactions (procedures). 95 In Spanish, there is no exact equivalent to accountability; responsibility is usually used as a synonym. - Page 156 - Conditions of production References to increasing vertical accountabilities, and explicitly in the form of “reducing discretion”, has been a common and salient discourse in both projects (SICEX and the Single Window). I have already presented some of these references within the analysis of legality. For example, the need to establish clear rules appeared very often accompanied with the idea of reducing discretion. I shall provide a further example, this time from SICEX, which illustrates this common finding: [SICEX] includes certain decision criteria, and in some cases, it can make automatic decisions. Thus, using certain parameters, it is the system rather than the public servant who decides if the procedure gets authorised. SICEX: A history of Success in Foreign Trade Regulations, Ministry of the Economy, 1999 (emphasis added) There is a common pattern that relates transparency and the need to reduce administrative discretion in trade regulations with technological functionalities, which have been present in the trajectory of the case. The reasons for its discursive production and consumption are similar to those that I have already discussed in the legality subgroup: technology provides not only automation but also a record of activities and data that can be traced and monitor over time, and therefore forms of control. Ideas on transparency and control appear, thus, intertwined. Moreover, transparency is control. For example, back in 1999, the government claimed that the electronic platform had achieved the following benefits: CONTROL. There is a log of operations to audit in the system each of the resolutions (...), that is, we know exactly who did what and at what time. That allowed us to go deeper with our decentralisation strategy (...) as well as to perform a strict management control at a national level. SICEX: A history of Success in Foreign Trade Regulations, Ministry of the Economy, 1999 p. 21(emphasis added) CONTROL. There is a log of operations to audit in the system each of the resolutions (...), that is, we know exactly who did what and at what time. That allowed us to go deeper with our decentralisation strategy (...) as well as to perform a strict management control at a national level. SICEX: A history of Success in Foreign Trade Regulations, Ministry of the Economy, 1999 p. 21(emphasis added) Consumption and materialisation There is no doubt that SICEX have conducted a big step in providing more transparency and internal visibility of certain decisions. In the initial phases, SICEX achieved mechanisms of transparency that were very innovative. Such is the case for the use of magnetic cards to clear procedures between Customs and the Ministry of Economy: - Page 157 - [The magnetic cards] worked similarly to that of a public telephone; it allocated certain credit that you could use for a given foreign trade procedure... Now it seems obvious, but at that time it was not [1998]... The card was indestructible and it had a cool ‘logbook’.. Anything that you’d have done would be recorded... even those things that you shouldn’t be doing. Former Deputy Minister, Ministry of the Economy(#15) SICEX provided a record of trade procedures in the system as well, but according to the government officials, it did not provide space for stating in detail the reasons and justifications that would justify certain approvals, rejections or authorisations. This explains why the Deputy Minister (and champion of the Single Window project) argues:96 [2009] We need to do much better. We have a great amount of [trade] programmes, and it is really complicated to know under which conditions they were granted or under which rules... Who knows where that information is... When you have this information in electronic systems, it becomes much easier to review that everything was done in adequate terms.. Deputy Minister, Ministry of the Economy (#19) [2009] We need to do much better. We have a great amount of [trade] programmes, and it is really complicated to know under which conditions they were granted or under which rules... Who knows where that information is... When you have this information in electronic systems, it becomes much easier to review that everything was done in adequate terms.. Deputy Minister, Ministry of the Economy (#19) What these quotes illustrate are a number of tensions between honesty, control, internal accountabilities and flexibility to deal with complex issues. The central vs local tension can be seen as a corollary of the discourses on internal accountabilities, and the shifting power relations. That is, SICEX ought to decentralise the administrative procedures, at least in practice, giving more control over decisions to their local offices. A case in point is the total decentralisation of procedures that the Ministry of Economy achieved in 2002. 96 From a CDA perspective, this paragraph shows why it is important to zoom in into the relation of words. Taken it in relation to the excerpt I quoted earlier from SICEX (Control), both paragraphs have very similar vocabulary and similar subjects although expressing very different suggestions. 98 I have searched and reviewed different available indexes on perception of corruption in Mexico (INEGI, The World Bank Governance indicators, and Transparency International). While useful, these indexes usually provide a general indication of corruption at national and state level. I gather additional views from relevant stakeholders, such as public servants that have worked for different agencies including the Ministry of the Economy. These informants usually suggested that corruption in trade regulation—understood generally as bribery—are not a cause for concern in the Ministry. In addition, I also reviewed available audits performed to SICEX in the period 2004-2006, with similar findings. 99 In some instances, local government officers could no longer take control over certain administrative decisions. For example, in certain Mexican borders, Customs installed a system of automated doors controlled from the Central offices as a way to avoid extortions and violence in local offices. (Interview with stakeholders of the Single Window, #39). 97 Interviews with senior officials (#15, #38, #39). 97 Interviews with senior officials (#15, #38, #39). Consumption and materialisation With the design of the Single Window under way, a shift in control “back-to- centre” becomes apparent. As far as honesty and integrity of public servants is concerned, there were conflicting perceptions about the level of corruption (usually an intentional avoided word) in the public service. Higher officials usually reported high trustworthiness to their own - Page 158 - people97, despite the fact that public perceptions about the level of corruption in government are worst in Mexico than the average for the Latin American countries (Kaufman et al 2010). It is not my aim to argue about honesty and corruption per se, but rather to emphasise that the discourses related to internal accountability and responsibility highlight the potential of electronic technologies to imbed rules and to reduce spaces for discretion as well as to shift (or enhance) the points of power in certain areas of government action. The tensions in accountability versus flexibility can be important. Let me go back to SICEX. While within the old system the decentralisation of trade procedures included rules and automated mechanisms to avoid discretion, the technological platform gave certain flexibility to the local officers of the Ministry. The new Single Window, on the other hand, takes automation and control much further (see numbers shown in table 14). Not only because the new technological system behind the Single Window automates and reduces the flexibility of actions in a considerable number of procedures, but also because it does so in a larger number of government agencies. While there is no available disaggregated data on corruption per administrative office, informants usually suggested that the perceived discretion (corruption) in the Ministry of the Economy is nominal, particularly, at the central level.98 On the other hand, for agencies like Customs—one of the worst public agencies in terms of perceptions of corruption—the expectations to reduce spaces for discretion are rather high. Of course, corruption is a dialectic activity and it may not be considered as the sole responsibility of government officials. Indeed, in many cases, insights from interviews suggested that tighter rules and transparent systems—enabled by networked technologies—protected their own public servants from the possibilities of being bribed.99 - Page 159 - The tensions within this cluster also provide useful explanations for resistances. The central government was interested in standardized and homogeneous information that they could use to control if local agencies were complying with the law. Consumption and materialisation In relation to rules, discretion and resistances, this quote serve to illustrate this point: When you do not have rules, administrators can do whatever they want and nobody will punish them... Then, to convince administrators that they need to follow rules, that with those rules they will be audited, and if they do not do things according to those rules they will be punished... well, who’s going to like that! So, we did have strong personal resistances. (…) but then, little by little, everyone was using the system. Former Deputy Minister, Ministry of the Economy (#15) This quote illustrates an interesting tension: technology and its standardised procedures bring the possibilities of centralised control and punishment; yet, even though the potential resistances and different workarounds, the system was rolled out and used. This quote illustrates an interesting tension: technology and its standardised procedures bring the possibilities of centralised control and punishment; yet, even though the potential resistances and different workarounds, the system was rolled out and used. 100 The Stanford Encyclopedia of Philosophy contains detailed review on the different conceptions, definitions and debates on distributive justice (Lamont and Favor 2008). I use a rather simpler definition—here approximated as distributing services to the underrepresented—based on what it emerges from the production of the texts and the contextual features of the case. I also want to note that, in some cases, scholars seem to assume a rather univocal understanding; for example, Andrews and Entwistle (2010, p. 686) assume that equity is precisely distributing services to disadvantaged and hard-to- reach groups. 101 Interview with senior public servants from the Ministry of Economy (#26, 40). Conditions of production The use of networked technologies to promote the participation of SMEs within trade has been explicitly cited in paragraph 4 of the Decree. This claim has not been a prominent discursive fact before the design of the Single Window. The reasons for this inclusion may be traced back to 2002, when the Mexican Congress passed a law for the promotion and development of SMEs’ competitiveness (DOF 2002). In addition, given the fact that one of the champions of the Single Window is also the agency in charge to lead the policies for SMEs competitiveness, this finding may not be surprising. According to the government, the design of the Single Window helps to include SMEs into trade by helping them to reduce material and transport costs to comply with trade procedures. But more importantly, by using a web-based platform, it puts all companies in equal terms: “You only need an internet connection”101. Content/Discursive features The last sub-group relates to the concept of justice. The Merriam Webster dictionary defines justice as “the quality of being just, impartial, or fair”. What is fair, or just, however, is subject to contestation and must be considered in the context of policy assumptions (Bozeman 2007, p.79), something I have already suggested in the legality theme. Related notions to justice are equity and equality. For example, justice may mean offering the same benefits to all (i.e. strict egalitarianism), or it can mean distributing more services or help to those groups in society that are underserved, disadvantaged or underrepresented (i.e. Andrews and Entwistle 2010, p. 686).100 In light of the empirical materials, the notion of distributive justice in the case resonates with the latter view, that is, with the efforts to include small, medium and micro enterprises (SMEs) into foreign - Page 160 - trade activities. SMEs are usually underrepresented in comparison to the big corporations that dominate the foreign trade scenario in Mexico. Consumption and materialisation Opinions of actors that know the sector suggest that the discourse on redistributive justice may be less materialised than the others. A senior government official of the Ministry provides an example of the contradictory nature of the previous statements: When you are talking about foreign trade [in Mexico], you are talking about an elitist material... they are very big companies... SMEs do not have the intention, because of lack of buying capacities or costs... so, those who enter into foreign trade are big corporations.. and these big companies were the ones asking for less discretion, more certainty and transparent rules. Former General Director, Ministry of Economy This claim is echoed in a media article, which reports that the SMEs in Mexico face a number of additional barriers to engage in foreign trade activities (Camacho 2011). These barriers are mainly related to lack of access to relevant information and market intelligence, such as proper tools to approach a foreign market, the right assessment of This claim is echoed in a media article, which reports that the SMEs in Mexico face a number of additional barriers to engage in foreign trade activities (Camacho 2011). These barriers are mainly related to lack of access to relevant information and market intelligence, such as proper tools to approach a foreign market, the right assessment of - Page 161 - available financial resources and proper knowledge on how to expand distribution channels. In turn, the article adds that “it is not lack of quality productsbut lack of information and resources to acquire market intelligence” what prevents more SMEs to get on board international trading activities. In line with concerns for SMEs, recent media reactions also pointed out that the implementation of the Single Window would produce negative effects on the SMEs operations. SMEs represent 22% of the total export companies and only 5% of the total value of exports. These companies, it is been argued, may face higher costs rather than reductions when using the Single Window. To start with, companies must have a computer with internet connection. A consultant from one of the big international firms mentions that “[the Single Window] is going to cost money (...), we foresee that costs may vary in between 7,500 to 75,000 US$”, which is a particular high burden for SMEs (Camacho 2011). Summary of the cluster: legality, accountability and justice The related public values in this cluster shares common discursive feature: an emphasis on the need of clear and transparent rules, notion of inclusion and fair treatment and implicated notions of fairness and distributive justice. The insistence on transparency as a goal of electronic information systems—either as a tool for procedural fairness or as a measure to enforce surveillance and internal accountability—has been paramount within the case. Public values such as legality and accountability of public servants are as prominent as those in technical efficiency. Matters of justice, and specifically, distribute justice, are more ambiguous and less salient, yet present in textual features. In general, the conditions of the production of these discourses are well grounded in a preoccupation with broader agendas of democratisation in Mexico, such as a shift from governing through the exercise of personal discretion to a system more based on legality and common legal norms. The materialisation and consumption of these discourses, in turn, has suggested more ambiguous interpretations. Consumption and materialisation Analyzing the different voices that are versed in the text of the Decree (and other prominent texts of the government) provides a further useful point. While it is clear that the Decree (as well as other the government texts under analysis) reflects the voice of the Executive, it also represents the position of big and multinational companies and those sectors that benefit from trade in the country, leaving SMS as a second priority at best. In sum, the effects of the Single Window are yet to be seen in this respect. What it can be argued, though, is that references to inclusion of SMEs (or other underserved actors) into trade have not been common in previous phases of SICEX. The discursive features, and the way the Decree was produced, may suggest that a genuine preoccupation with equality is put forward in the project. At the same time, the ways this discourse have been materialised suggests two interpretations: either is purely rhetoric (because SMEs won’t engage in trade anyway), or that the Single Window may indeed contribute to engaging more SMEs into foreign trade and the digital economy. At least in the short run, it appears that the Single Window creates even more inequalities: big companies are better prepared to act and pay for complying with the newer digital services (i.e. COVE). - Page 162 - Content/Discursive features Beck-Jørgensen and Bozeman (2007, p.366) suggests that a public organisation that based its organising on values such as robustness, adaptability and reliability, may not be fun but rather trustworthy. This idea is also associated to what Hood (1991, p.14) describes as lambda-type administrative values. If robustness and reliability are placed at centre stage of an administrative design, “the central concern is to avoid system failure, ‘down time’, paralysis in the face of threat or challenge” (Hood 1991, p.14).Technology, in particular, its infrastructure, security and way of functioning, is germane to this cluster. In this cluster, I pay particular attention to those measures that have been put in place—discursively and operationalized—as back-up plans, also referred in the case usually as “contingency plans”. Conditions of production Paragraph 4 of the Decree offers our first indication on the presence of a discourse about robustness: - Page 163 - (...)the use of information technologies in the operation of foreign trade (...) strengthens the security for the users; The expression entails an ambiguity: the idea of security for users is not clear from the point of view of who users are, and what type of security they are referring to. Security may be implicated in terms of legality (i.e. security as protection of the rights of the individual) as well as protecting public sector employees from abuses of imbalance power (i.e. lobbying companies). A different read could lead to understanding ‘security’ as a proxy for reliability and a robust system. The reasons to suppose so rely on the presence of concerns with system failure during the trajectory of the projects. For example, resilience, security and system robustness are contained in the following quote: Once [year 2007] we lost one hard drive, and we were off-line for a couple of hours... it only lasted couple of hours, but it was shocking... that episode made us to designed new backup procedures... and we have been working in that line. Director, Information Technology Unit, Ministry of the Economy (Emphasis as per the conversation) Once [year 2007] we lost one hard drive, and we were off-line for a couple of hours... it only lasted couple of hours, but it was shocking... that episode made us to designed new backup procedures... and we have been working in that line. Director, Information Technology Unit, Ministry of the Economy (Emphasis as per the conversation) Once [year 2007] we lost one hard drive, and we were off-line for a couple of hours... it only lasted couple of hours, but it was shocking... that episode made us to designed new backup procedures... and we have been working in that line. Director, Information Technology Unit, Ministry of the Economy (Emphasis as per the conversation) This quote illustrates that the loss of a piece of hardware—and its associated fears of bigger failure effects—triggered further actions to minimise potential future issues. My point is that even though the text of the Decree may place a minimal space for public values related to robustness and resilience, on many occasions, actors repeatedly emphasised the need to have a back-up plan and security measures. Improved information security. Using information technologies prevent damage or data loss, even in case of disaster. Single Window website102 Improved information security. Using information technologies prevent damage or data loss, even in case of disaster. Single Window website102 Improved information security. Using information technologies prevent damage or data loss, even in case of disaster. Single Window website102 The idea of information technologies as secure tools is somehow overemphasised in the quote. Protecting information from destruction or loss is only one aspect of information security, and while information technologies may help reducing potential information security breaches, they cannot eliminate them completely. Yet, the government discourse places the focus on looking at their benefits rather than risks. Conditions of production Concerns in terms of system failure were evident in interviews with public servants operating SICEX, in which potential technical problems that could emerge in the interconnectivity with Customs were emphasised. A project leader from Customs said: Every time we have to install a new update [between Economy and Customs] is problematic and we have to stabilize it. If it were off-line, it wouldn’t be a drama... But as we’re online, if that thing doesn’t work we are in a big troubles… We cannot allow that to happen. That cannot happen. Director/Project Leader, Customs (Emphasis as per the conversation) The rationale for assuring robustness and systems integrity is quite obvious: foreign trade operations depend critically on the well-functioning of its information infrastructure—whether SICEX or the Single Window. In addition, there are many reasons to think that these concerns cannot be but bigger for the Single Window, given The rationale for assuring robustness and systems integrity is quite obvious: foreign trade operations depend critically on the well-functioning of its information infrastructure—whether SICEX or the Single Window. In addition, there are many reasons to think that these concerns cannot be but bigger for the Single Window, given - Page 164 - the larger scale of the latter compared to SICEX. Within the texts of the Single Window, issues on security and resilience are presented in rather positive ways: 102 Available at https://www.ventanillaunica.gob.mx/vucem/SobreVU/Beneficios/index.htm Accessed: 22 May 2012. Consumption and materialisation From the point of view of the consumption of these discourses, a shift can be traced when comparing SICEX and the Single Window. Given the larger scale of the Single Window, the government decided to build the Single Window using a public private partnership (PPP) contract. Among other components (i.e. process redesign, consultancy services, and providing the physical technological infrastructure), the contractor is the one that administers the Single Window, and obtains a payment per procedure completed. For the government, this is the way they can “secure” the system: The contractor is interested in the money... they do have the interest in making the system work... It’s simple... If [the Single Window] doesn’t work, they don’t get paid Area Director, Ministry of the Economy The contract established clear performance indicators and tough penalties according to possible failures, which functions here as further evidence of the monetary incentives to make the system work. However, it is not yet clear who would be accountable for in case of disaster. The problem of accountabilities and outsourcing deals is far from resolved, despite the clauses that the contract may have included. - Page 165 - The concerns with resilience and system robustness has been materialised in texts in the form of “contingency plans”. The way the contingency plan for the Single Window was produced and negotiated provides further clues on two related issues: first, that robustness is considered important; but even so, robustness may have been marginalised from the government priorities at the expense of releasing the system sooner rather than later.103 By March 2012—just two months before the Single Window became mandatory—there was no agreed contingency plan yet and discussions were ongoing amongst the government and stakeholders such as customs agents and trade associations.104 In fact, the contingency plan was released on the Single Window website on May 30th 2012, just few hours before the entry into force of the digital platform. In simple terms, what the contingency plan says is that, in case of failure or too-long waiting times, the government should accept the older paper-based procedures. Concerns from the private sector were also raised: The digital [Single window] system will have serious problems because of the volume of transactions that it pretends to deal with, that is not sufficiently prepared for. 106 In brief, TQM is a management practice that focuses on continuously improving the quality of processes (or products, when applicable). It is based on the premise that quality processes and outputs are the responsibility of all actors involved in the creation or provision of services. 105 Source: Suarez, E., “La ventanilla unica sera rebasada”, El Mexicano, 22 May 2012. 104 Interview with a project leader of the Single Window, March 2012. 103 Political timings were very important for this to happen, given the upcoming election in July 2012 and changes in government that the main actors in the implementation process foresee. Consumption and materialisation President, Association of Maquiladora Industry, Mexico (in the news)105 The main shift in comparison to SICEX, I claim, relies on the outsourcing deal: from internal to external forces. If the system does not work, the private company is to blame. In SICEX, solving system failures was a matter of internal capabilities. Issues of information security such as confidentiality, integrity and prevention of data leaks are also left to the contractor. A related finding of this shift relates to the relative abandonment of a quality discourse. Back in 1995, SICEX was built in a complementary fashion to a total quality management (TQM) system106 that the government was adopting. During the 1990s, TQM systems were envisaged as useful tools for increasing government effectiveness and performance, based on spirit of the reinvention reforms (Gruening 2001, Moynihan 103 Political timings were very important for this to happen, given the upcoming election in July 2012 and changes in government that the main actors in the implementation process foresee. - Page 166 - and Pandey 2005, p. 422). In particular, the Mexican office opted for an ISO 9000 certification, which is a type of quality management system, designed to ensure the quality of organisational processes via preventive actions. SICEX was complementary in the following ways: SICEX is a basic tool to consolidate and comply with some of the ISO 9000 requirements. SICEX: A history of Success in Foreign Trade Regulations, Ministry of the Economy, 1999 p. 31 (emphasis added) Among other things, the ISO 9000 standard requires the organisation to document its procedures, to maintain a control of records and internal audits and to issue a Quality Manual with the organisation’s quality policies. The implementation of SICEX contributed to the quality system in the following ways: it helped maintaining a record of the processes, ensuring that there is available data for auditing purposes and providing some level of statistics for performance purposes (i.e. number of procedures processes, analysed, types of responses, etc.). Despite the acclaimed benefits and complementarity of SICEX and the ISO standards, the push for quality (in the sense I have described it) has diminished over the years. The reason may be found in the potential incompatibility of sustaining quality and at the same time achieving disparate goals. 107 When comparing a set of 5 administrative procedures, whilst all of them reduced the number of required activities (or steps) in the process, two of them resulted in an increase of supporting paperwork required to complete the regulation. 108 I do not suggest that the government did drop the preoccupations with providing good and better services to their clients; on the contrary, the push for simplification of trade procedures as well as with enabling competitiveness denotes the government’s effort to reduce unnecessary regulations in trade. In Summary of the cluster Along the trajectory of the project, there have been explicit and implicit references to issues on resilience, robustness and security, mostly grasped in references to different contingency plans for both, SICEX and the Single Window. The reasons of the production of this discourse are related to the importance of trade in Mexico’s economy. Thus, foreign trade operations are increasingly depending on the well- functioning of information systems. From the point of view of its consumption and materialisation, the discourse on robustness has shifted in two directions: an abandonment of a parallel yet obsolete total quality system, and the ‘outsourcing’ of robustness via a third party (the contractor). The Single Window just started operating by the time this thesis goes to press. As in the other cases, effects remain to be seen in this respect. Consumption and materialisation For example, during the period 2000-2006, while the Ministry was pushing for a paperless administration, the presence of quality checks ended in an increased number of required documents and more paperwork.107 In other words, a focus with simplifying regulations via reducing the use of paper co-existed with pressures to assure quality and error minimisation via broader information checks. This contradiction has also been documented in other e-government programmes(see for example Margetts 1999), which may explain why parsimony and efficiency may be at odds with robustness. As a matter of fact, the word quality has been dropped from more recent texts, and there is no explicit reference to quality of services in the Decree either.108 - Page 167 - - Page 167 - terms of discourse features, the abandonment of references to quality should be seen as a distance to TQM approaches. Content/Discursive features I shall begin once more with the excerpt of the Presidential Decree, and a closer look to paragraphs 5 to 9, as starting point: I shall begin once more with the excerpt of the Presidential Decree, and a closer look to paragraphs 5 to 9, as starting point: 5 It is convenient that the competent authorities concerning foreign trade coordinate and implement actions of simplification, automation and improvement of foreign trade processes (...) 5 It is convenient that the competent authorities concerning foreign trade coordinate and implement actions of simplification, automation and improvement of foreign trade processes (...) 6 In conformity with the Foreign Trade Law corresponds to the Federal Executive, through the Secretary of the Economy, to coordinate the participation of the other agencies of the Federal Public Administration (...) 6 In conformity with the Foreign Trade Law corresponds to the Federal Executive, through the Secretary of the Economy, to coordinate the participation of the other agencies of the Federal Public Administration (...) 6 In conformity with the Foreign Trade Law corresponds to the Federal Executive, through the Secretary of the Economy, to coordinate the participation of the other agencies of the Federal Public Administration (...) 8 Based on the trade simplification and facilitation policy that has already been instrumented (...) the Federal Executive Branch under my duties considers necessary to issue, throughout the present Decree, the general dispositions for the establishment of the Mexican Digital Window of Foreign Trade (...) 8 Based on the trade simplification and facilitation policy that has already been instrumented (...) the Federal Executive Branch under my duties considers necessary to issue, throughout the present Decree, the general dispositions for the establishment of the Mexican Digital Window of Foreign Trade (...) 9 For the above effects, it is considered convenient to establish stages of implementation for the referred Digital Window (...); 9 For the above effects, it is considered convenient to establish stages of implementation for the referred Digital Window (...); The use of the words “in conformity” with the Law (par. 6), “considers necessary to issue” (par. 8) and “it is convenient” (par. 5 and 9) are legal utterances that refer to rather obligatory statements. For example, instead of saying ‘it is convenient’ the text could have said ‘it is mandatory’ that the agencies coordinate actions toward trade regulation simplifications. 109 As I explained in the methodology chapter, the modality of a clause or sentence reflects what people commit themselves to when they make statements, declarations, make demands or offers (Fairclough 2003, p.165). I look at the modality of government texts to trace the discourses on consensus. Content/Discursive features In other words, although they may look subtle, the way the Decree was produced opens up some further interpretations. Let me expand. Content/Discursive features This cluster is perhaps the least obvious although it remains an important one, particularly, because it reflects on the challenges of organising technology-led processes and its implications for broader structural reforms. The idea of cooperation as a value is defined as the opposite of competition into the public administration, the latter understood as one of the market mechanism that the NPM seek to promote as a way to increase public sector performance (Andrews 2011, p.286, Christnesen and Laegreid 2011, p. 4). In turn, cooperation is traditionally associated to a mechanism for coordination within government agencies (Beck Jørgensen and Bozeman 2007, p.366). My evidence shows that in this case, while agencies do indeed cooperate, there is an - Page 168 - - Page 168 - obligatory modality109presented in the texts that suggests the co-existence of two processes: a longstanding collaboration and a forced consensus. obligatory modality109presented in the texts that suggests the co-existence of two processes: a longstanding collaboration and a forced consensus. 110 Vice-Ministry for Trade and Industry and General Direction of Foreign Trade (DGCE). Conditions of production The design and implementation of the Single Window rely on inter-agency collaboration; collaboration constitutes a necessary mechanism for the project to have any viable future. The voices that are being reported in the Decree suggest a monological, or consensual, state of affairs. I have expressed earlier on that the language of politics generally implicate a discourse of contestation (se methodological chapter). Even so, the - Page 169 - text of the Decree exposes a version from a unified voice or protagonist. It may be literally the President’s voice and the agencies involved in trade. In this case, the conditions under which Decrees and executive orders are produced reveal that the main voice in the text represents those who are leading the initiative. A unified voice is the one of the Ministry of Economy110, whose team has a long history of mutual adjustments and negotiations with their counterparts in Customs. At the same time, the Ministry of the Economy’s mission is to serve and promote the industry, that is, the companies involved in foreign trade operations, which practically means big and multinational companies. Customs, on the other hand, are better defined as guardians of the Mexican borders whose goal is to invigilate and secure lawful trade operations. On many occasions, informants suggested that the main actors of the project (Customs and the Ministry of the Economy) had goals that were at odds with the overall aim of the project. These contested goals refer to the battle between facilitation of trade versus control that appear continually in different phases of the project. In May 2010, when the project design was advanced and closer to its formalisation, an Area Director and project leader from Economy, comments: [May 2010] We are very clear where we all want to be... the point is that we [areas of Customs and Economy] are areas with very different goals... and we have realized that; it cannot be all facilitation of trade as Economy wants, nor it can all be regulation as Customs wants... we need to reach a middle point, and we are working on it. Area Director, Ministry of the Economy (#25) This quote is useful because it shows a common pattern in the conversations, and, at the same time, it allows for more reflection on how both central government agencies managed to combine these initially contested aims. Conditions of production The reasons of the production of the Decree in the way it is written (emphasizing the need for trade facilitation instead of focusing on control), and the role given to the Ministry of the Economy, responds to a larger and yet less evident mechanism of collaboration among these two main actors. The following quotes exemplify not only the existence of disagreements, but more This quote is useful because it shows a common pattern in the conversations, and, at the same time, it allows for more reflection on how both central government agencies managed to combine these initially contested aims. The reasons of the production of the Decree in the way it is written (emphasizing the need for trade facilitation instead of focusing on control), and the role given to the Ministry of the Economy, responds to a larger and yet less evident mechanism of collaboration among these two main actors. The following quotes exemplify not only the existence of disagreements, but more - Page 170 - subtly, a trajectory of a quite stable team of public servants working together for many years111 (emphasis in the original): [May 2009] I don’t know how we did not end up punching each other… We shout at each other over the phone... There were so many issues! [giggles] But don’t think this is personal.. it’s just work! This is about sooo much negotiation, and soo much work.. that is why we want the Single Window to start with Economy... the way we have worked together have no comparison with any other agency... Director/Project Leader, Customs (#13) I think the project has been working because my counterpart in Customs is very technical, and she really wants this project to work... and without entering into racings for medals or political credit... this maturity is transcending people... and I think that is a good achievement... we have worked very well with them... I cannot tell you we haven’t argued! Indeed, we have.. There were occasions that we didn’t speak for a week... but then, we came back and kept going. Area Director, Ministry of the Economy (#25) The conditions of the production of the Presidential Decree of 2011 can be traced back to a history of strong leadership in the Ministry of the Economy. The responsible for setting up SICEX was a visionary public servant that knew the intricacies of foreign trade in detail. 111 This point was also reinforced in several interviews with senior public officials in trade. Conditions of production She built a career in the Ministry starting from the operational levels, until taking office as Deputy Minister 35 years later. By the time SICEX was first thought of in 1996, she had to convince her political superiors about implementing a system that was not only novel but also unique in scale in Mexico’s central government. Back then, foreign trade was enjoying a particular momentum: the NAFTA was driving many changes, substantiated in strong modernisation discourses in the national economy. Because of its scale, SICEX did not need a Presidential Decree but the support from the heads of the Ministrypolitically, discursively but also in material resources—which was obtained. SICEX did produce acclaimed results in the first years of its implementation although later faded away, due, in part, to the failed upgrades that happened between 2003 and 2006. For the champion of SICEX and Deputy Minister since 2000, the potential benefits of extending the pay offs from SICEX into a bigger and more comprehensive technological platform did not vanish. In March 2008, a Presidential Decree (2008) endorsed by the Minister of the Economy and the Minister of Finance, set the basis for giving more facilities in trade regulations, including the - Page 171 - - Page 171 - design and implementation of a digital single window.112 This Decree, and the history of its negotiations, was therefore an important intermediate step. Later in 2008, a new Deputy Minister took office as part of a Cabinet reshuffle. Among other policies, she put forward an intense foreign trade facilitation program to reduce trade tariffs to levels that many analysts considered “impossible”.113 And even though there were strong initial confrontations with several industries, the policy proceeded and remains ongoing (year 2012). It comes as no surprise, then, that a strong commitment with opening to trade and concurrent policies that would increase Mexico’s competitiveness was in the Ministry’s agenda. But further factors helped the development of the Single Window and the challenges of negotiating with Customs and their seemingly different value missions. First, given her personal career, the Deputy Minister hold strong ties with the heads of the Ministry of Finance—where Customs belongs. This facilitated a negotiating phase with Customs by securing support from the Ministry of Finance. Second, the Minister of the Economy at that time (2008) was regarded as one of the closest persons to President Calderón. 113 I refer here to the trade facilitation programme highlighted in the timeline of the case as part of the context features of the case. 112 Recall that Presidential Decrees differ in levels of commitments according to the Ministers that signed the bill. In this sense, the 2008 Decree had a smaller reach in responsibilities compared to the one that established the single Window in 2011, that was signed by 11 Ministers. 112 Recall that Presidential Decrees differ in levels of commitments according to the Ministers that signed 112 Recall that Presidential Decrees differ in levels of commitments according to the Ministers that signed the bill In this sense the 2008 Decree had a smaller reach in responsibilities compared to the one that Conditions of production Once the Deputy Minister was able to convince him about the importance of the trade facilitation programmeincluding the Single Windowthe Presidential support was closer. The hierarchical nature of the Mexican Executive branch, and the strong ties between the Minister of the Economy and the President, made the realisation of the trade facilitation programme possible. In addition, it also gave the Deputy Minister an additional advantage to retain the “ownership” of the Single Window under her duties, after being negotiated with a different area of the Ministry. In terms of the negotiation with Customs, important tensions emerged to agree on different phases of the tendering process during 2010. Customs hold a stronger power to control the terms and conditions of the contract and the selection of the provider, given its expertise but also because they were the ones compromising the budget. It emerged during interviews in the field that this was perhaps the cost to face, to avoid - Page 172 - - Page 172 - delays in releasing the Single Window within sensitive political timescales; that is, before the end of the Presidential Administration in 2012. By the time the contract was open to tender in 2010, the leaders from Customs and the Ministry of Economy were already discussing the ways to backing the Single Window politically. In addition to the idea of the Presidential Decree, there were other alternatives, such as signing a Ministerial Agreement. Comparatively, a Presidential Decree is stronger than an Agreement, especially if the Decree is endorsed by all Ministers with jurisdiction in foreign trade. Negotiations reached consensus and the Decree was passed on January 2011, establishing formally the Single Window, its timeline, responsibilities and political endorsements. They [other central government agencies] will need clear instructions... the Single Window is more political than technical... Agencies simply won’t respond to me... they need formal instructions... They [Ministry of Economy] need to convince the President... without that, without being at that level, this is going nowhere. Director/Project Leader, Customs (#13) Consumption and materialisation The challenges in terms of cooperation (and its obligatory modality) are more salient when considering the overall number of agencies involved. A concrete mechanism to enhance (if not force) coordination has been obtaining the support from the highest levels possible—the President. The idea of forced cooperation is then related to the public value of political loyalty, that is, the relationship between public servants as implementers of given policies designed (and decided) by politicians. An initial cue on the need to make the Single Window a reality can be illustrated by the claim that one of the project leaders from Customs suggested in 2009, when the first ‘serious’ meeting about the project: They [other central government agencies] will need clear instructions... the Single Window is more political than technical... Agencies simply won’t respond to me... they need formal instructions... They [Ministry of Economy] need to convince the President... without that, without being at that level, this is going nowhere. Director/Project Leader, Customs (#13) The Decree itself then represents an important materialisation of this discourse and, in light of the events in 2012, reinforces the view of this piece as capable of triggering - Page 173 - - Page 173 - actions.114 It serves, for example, as a motivator in public servant’s behaviour, at least from the viewpoint of the project leaders: The project has become a presidential priority and it has been in all our policy presentations... (...) So, when you work on something that is important, that also actions.114 It serves, for example, as a motivator in public servant’s behaviour, at least from the viewpoint of the project leaders: The project has become a presidential priority and it has been in all our policy presentations... (...) So, when you work on something that is important, that also motivates you... because you are doing something that is in the Presidential Agenda. Deputy Minister, Ministry of the Economy (2010, #26) The project has become a presidential priority and it has been in all our policy presentations... (...) So, when you work on something that is important, that also motivates you... because you are doing something that is in the Presidential Agenda. Deputy Minister, Ministry of the Economy (2010, #26) While there may be spaces for resistances and workarounds, technology reinforces the ‘forced’ cooperation discourse. 114 This is better reflected in the theory of CDA as textual agency, and I shall come back to this point in the discussion chapter (Chapter Seven). Consumption and materialisation In part, because the Single Window pushes for data standardisation across agencies, but also, because roles and routines are inscribed into software in such a way that it structures what may be possible and what may be not. From our internal comparison case, SICEX, technology did contribute to some form of forced cooperation too, by overcoming initial resistances. The Deputy Minister illustrates: While there may be spaces for resistances and workarounds, technology reinforces the ‘forced’ cooperation discourse. In part, because the Single Window pushes for data standardisation across agencies, but also, because roles and routines are inscribed into software in such a way that it structures what may be possible and what may be not. From our internal comparison case, SICEX, technology did contribute to some form of forced cooperation too, by overcoming initial resistances. The Deputy Minister illustrates: At the beginning [of SICEX], it took us lots of time to make it work... and there were many resistances. Little by little, one month went through, then a year, and so on... Today [2009], the Ministry [of the Economy] could not explain to live without SICEX Former Deputy Minister, Ministry of the Economy (#15) At the beginning [of SICEX], it took us lots of time to make it work... and there were many resistances. Little by little, one month went through, then a year, and so on... Today [2009], the Ministry [of the Economy] could not explain to live without SICEX Former Deputy Minister, Ministry of the Economy (#15) A similar argument can be put forward in the power of technology to standardise and reinforce the discourse on inter-agency cooperation. In a webinar that the government offered to businesses and consultants when the Single Window was about to be launched, a project leader addressed that a remarkable benefit of the Single Window was its level of data standardisation. In his words: We are talking about screens, resolutions, steps that are uniform.. if you learn to comply with a procedure from us [Ministry of Economy], you will then be able to do any other, because the screens and procedures will be exactly the same. Area Director, Ministry of the Economy (webinar 2012) Thus, from a design perspective, the Single Window reifies a significant rationalisation of agencies and interagency processes through data standardisation, and the uniformity of processes. Summary of the cluster Within the discourse on cooperation, common textual features (from text and talk) refer to the use of authority, obligatory statements and forcing inter-agency cooperation. The hierarchical structure of government in Mexico and the importance of getting the Presidential support have been sensible reasons behind the production of this discourse. The role of technology, even when subtly presented, has contributed to the materialisation of the discourse by setting up the rules in the software and by creating new ways of working and interacting. The single entry point and inter-agency operation becomes a reality in the Single Window, yet it is early to address the potential effects on inter-agency collaboration. This is recall as one of the main achievements of the initiative. The Single Window is said to go beyond political timings, such as a change in the Presidential Administration. This is recall as one of the main achievements of the initiative. The Single Window is said to go beyond political timings, such as a change in the Presidential Administration. This is recall as one of the main achievements of the initiative. The Single Window is said to go beyond political timings, such as a change in the Presidential Administration. Consumption and materialisation By the time this thesis goes to press, only Customs and Economy were operating procedures; the other agencies involved are expected to start releasing theirs Thus, from a design perspective, the Single Window reifies a significant rationalisation of agencies and interagency processes through data standardisation, and the uniformity of processes. By the time this thesis goes to press, only Customs and Economy were operating procedures; the other agencies involved are expected to start releasing theirs - Page 174 - by the end of 2012.A further change in the operationalisation of this discourse (and the others more generally), is the disappearance of SICEX as technical platform. By the time new procedures are released in the Single Window, the functionalities in SICEX are turned off. After the 1st of June, SICEX becomes only a historic database for organisational memory. As one of the leaders of the project puts it: we have secure the future of the project [Single Window]... now, there is no way back to continue operating with the legacy system General Director (#42) we have secure the future of the project [Single Window]... now, there is no way back to continue operating with the legacy system General Director (#42) 6.4 Conclusions and summary of the findings Using a discursive lens based on critical discourse analysis, in this chapter I have elaborated on the dominant discourses on public values and technology innovation in the process of change in trade regulations in Mexico. I have followed a systematic approach to elicit these discourses: elaborating on their content (discursive characteristics), tracing their origins and reasons of production, and spelling out the ways they have been consumed and operationalised over time. Many overlaps emerged, although I kept the four clusters separated for analytical purposes. Finally, a number of tensions—whether intended or not—were found in the trajectories of production, - Page 175 - interpretation and consumptions of the discourses. The central findings are summarised in the table below. They demonstrate the complex, dynamic and conflicting character of the relation between public values and technology innovation in government. The analysis of the case shows that the production and negotiation of the meaning of public values and technology innovation in the public sector, as well as their operationalisation, draw from broader discourses and imperatives that shape the dynamics of the case. But also, that technology materialised theses discourses in different degrees, reinforcing them over time. In the next chapter, I elaborate on these findings to reflect more generally on the lessons that can be learnt. - Page 177 - ons of production do they stem from) Examples on consumption and materialisation Salient Tensions ourse of ness as driving force n making trade less burdensome m better on world n competitiveness ICT streamline internal processes; reduce waiting times and compliance costs (i.e. roducti stem fro entralize d operat 6.4 Conclusions and summary of the findings transport and paper costs) Expected reduction of 21% in internal human resources Incentives in response time reductions if complying online Improved internal capabil imply staff redundancies in long-term; internal resistan Using the system to contr performance nt’s perceived need of ansparent rules to retion; (based on netelist relations in n transparency as a to obtain better control cess to foreign trade to Increase automation and number of rules embedded in the software; Reduced discretion and reduced flexibility of civil servants; Design a platform that provides equal treatment for all Redistribute tasks across f agencies giving raise to po central-local tensions Depersonalization of serv Digital platform gives mor advantage to big companie (initially) nt’s emphasis on the id failure nds critically on the oning of the digital Robustness is ‘outsourced’ via the PPP contract Monetary incentives put in place to maintain the service ongoing (monetary penalties if the service is down) No clear accountability sy place, potential increase in discretion Shift of control to a third sector) and possible loss o (fairness and justice) e loss o e) - Page ces ncreased centralized pow ecentralized operation rease entr 7 Chapter Seven. Implications: the interdependent, political and dialectical relation between public values and e-government The longitudinal case that I studied provides a rich account of the production, dissemination and consumption of discourses on public values and technology. Building on the findings from the case this chapter derives general implications for an increased understanding of the relation between public values and technology. The chapter is divided into two parts. In the first part, I discuss the different public values found in the case and their relation with technology as well as their interdependence. In the discussion I draw theoretical propositions on the relationship between public values and e- government. The second part builds on the interdependences between different public values and related tensions identified in the analysis. I discuss more broadly the implications of the case in light of what I define as a dynamic, political and dialectical relation between public values and technology. To elaborate this in more detail, I discuss the role of broader institutional agendas, the political imperatives and the language of change that underlie the design and implementation of e-government initiatives. Key themes From dichotomy to interdependence Multiple discourses on public values and technology It is not either or, public values are interdependent Possible alignments, tensions and conflicts Dynamic, political and dialectical relation Dynamic: negotiating meanings in context Political imperatives and broader context; type of change Dialectic: Technology internalises and is internalised by public values discourses Table 16: A dialectical relation between public values and technology 7.1 Public values and technology: from dichotomy to interdependence The critical discourse analysis of the case of e-government in Mexico showed that there are multiple public values linked to technology, which are related, and at times - Page 179 - contesting each other. The list of public values compiled by Beck Jørgensen and Bozeman (2007) and the clusters of administrative values posited in the work of Hood (1991) proved useful guidance to trace the most prominent public values in the case. Yet, it has been only by examining manifestations of public values in their empirical context that their relations, tensions and characterisation could be distilled and analysed. That is, critical discourse analysis provided the means not only to elicit those emergent public values but also to understand them in an analytically useful way. The analysis revealed four discourses on public values and technology: (i) technical efficiency, (ii) legality, accountability and justice, (iii) robustness, and (iv) (forced) cooperation. In turn, the analysis showed that certain discourses on public values have been more prominent that others, generating tensions over time. Discourse clusters Associated Public Values Technical efficiency Productivity, efficiency Effectiveness, parsimony, business-like approach User orientation, timeliness Legality, accountability, Justice Legality, Rule of Law, Equal Treatment, Fairness (procedural fairness) Accountability, professionalism, integrity Justice (distributive justice) Robustness Robustness Adaptability, reliability, timeliness (Forced) Cooperation Cooperativeness Political loyalty Table 17: Summary of discourses and associated public values Discourse clusters Associated Public Values Technical efficiency Productivity, efficiency Effectiveness, parsimony, business-like approach User orientation, timeliness Legality, accountability, Justice Legality, Rule of Law, Equal Treatment, Fairness (procedural fairness) Accountability, professionalism, integrity Justice (distributive justice) Robustness Robustness Adaptability, reliability, timeliness (Forced) Cooperation Cooperativeness Political loyalty Table 17: Summary of discourses and associated public values The most salient and explicit set of public values—approximated by their discourses— have been those associated with technical efficiency. Elaborating on the structural characteristics of the cluster, I found that preoccupations with cost reductions, internal efficiencies, and providing a quicker, timely and cheaper service to clients have been a hallmark in the case. These priorities, usually claimed as driven by new public management approaches, have been very explicit in the materials analysed in the case study. The role of technology, in turn, has been salient for reducing response times and resource costs for both government and clients. In sum, my case study shows how the discourse on technical efficiency takes centre stage during the trajectory of an e- government initiative. 7.1 Public values and technology: from dichotomy to interdependence - Page 180 - The second cluster includes three sub-groups of neighbouring public values, all related to commonly associated values of traditional public administration. The first one, legality, is used here as an umbrella of other similar public values (rule of law, equal treatment, fairness) to indicate that the relation between public administration and citizens should be regulated by law instead of the arbitrary exercise of power. The second subgroup mapped public values related to the behaviour of public servants, such as accountability, rectitude and honesty. Put together, these two sub-groups of public values (legality and accountability) appeared in this case to be as important as the discourses on technical efficiency. Concerns with reducing unnecessary discretion, promoting transparent and clear rules and legal norms, were salient preoccupations and at the same time, motivators to use technology within the whole trajectory of the case. The reasons of their prominence were found in a longstanding history of broader agendas on democratisation and the elimination of clientelism in the public sector in Mexico. Even though in my textual oriented analysis the discourses on legality and accountability were not as explicit as those of technical efficiency, these two groups of public values were very salient and emerged stronger over time. Distributive justice, the third sub-group of values in the second cluster, was approximated in my analysis by the role of government in distributing more services to underserved groups—small, medium and micro enterprises (SMEs) in foreign trade activities. In contrast to the findings on technical efficiency and legality, this discourse proved not only ambiguous but less materialized or non-existent during the early phases of the case study. Moreover, at least in the short run, the implementation of the Single Window appeared to create more disadvantages for SMEs. That is, the implementation of the Single Window created higher comparative costs to smaller firms, as they have to invest in new technologies or new resources. The general lesson that can be derived from this is that certain public values, although crystallised in the discourses of e- government and held by several stakeholders, may lead to opposite results in the course of e-government action. Robustness and related public values such as adaptability, resilience and timeliness also proved to have their space in the project. During the trajectory of the case, concerns with ensuring that the digital platform worked effectively appeared repeatedly. 7.1 Public values and technology: from dichotomy to interdependence However, the main change has been in shifting the role of government as builder and - Page 181 - - Page 181 - provider of the technological services to one of monitoring for the services contracted to a third party. An abandonment of the discourse and practices of total quality management systems inside government has also emphasised a shift from internal robustness to one that privileges cost reduction and the benefits on relying on external expertise. The last cluster, forced cooperation, was demonstrated to be more slippery than the others, yet salient. This discourse—and especially its forced consensus part—became more dominant during the phases of design and construction of the Single Window. The reasons, I claim, are rooted in the need to have strong political support when attempting to enforce a programme encompassing a large number of public agencies with diverse regulatory interests. One thing is to have a designed process, that is working, and say “I’m going to automate this”... And a rather different thing is to conduct a process reengineering to make that process an optimal one, and only after, systematise it... the latter is the way it should be... I am against systemising the inefficiency! Main IT Advisor to the Minister (LA) 116 The case also shows that, as widely studied in information systems research, conducting BPR exercises and only after applying technologies presupposes a linear and rational practice that is unrealistic (i.e. Avgerou 2002, Robey et al. 2002). This is no different in the public sector. 7.1.1 Multiple public values in e-government My study confirms the existence of multiple public values in e-government suggested in the literature of public value and related streams (Bozeman 2002, 2007, Beck Jørgensen and Bozeman 2007, Moore 1995, Hogget 2006, Rainey et al 1976, Rainey and Chun 2005). However, an important corollary of the existence of multiple public values in my case study is their interdependence. This thesis clearly illustrates that public values become intertwined in multiple dimensions: within and between their clusters, across different phases of discourse production, its interpretation and materialisation. On several occasions public values were also shown to be co-occurring. The case study showed that some public values were related to one another and appearing at the same time, an idea that is referred to in the literature as covalues (Beck Jørgensen and Bozeman 2007, p. 370). The notion of interdependence is closely related to covalues in the sense that certain public values—and the way they became materialised—reinforced or restrained the presence and materialisation of other values. To elaborate on this in more detail, I point to a number of identifiable public value co- occurrences as well as tensions that, while building on my findings, can be of more general understanding. - Page 182 - Legality and internal accountability as pre-conditions of technical efficiency The power of networked and novel ICT relies on its capacity to automate information processes allowing for rapid or real-time handling of information (Fountain 2001, p. 61). In addition, technology has the capacity not only to automate but to process information in a meaningful way, leading to potential changes in coordination and the ways clerical tasks are conducted (Fountain 2001, p. 35-40). The process of automation, therefore, requires in some extent the definition of information processes, organisational roles and responsibilities in government. In order to achieve gains in various aspects of technical efficiency, establishing clear rules through standardization and homogenization was considered as a necessary prior requirement for the implementation of technology—whether in SICEX or more intensively, for the Single Window. During the case study, a persistent phrase resonated among interviewees: “we need clear rules and processes and only after apply technology to them.”115 The capacity of technology to automate, handle and process large and complex amount of data was also seen as a valuable mechanism to improve trade regulations. 115 The main IT Advisor to the Minister provides an illustration to this persistent claim in the case: 117 These authors refer either to groupware technologies, data warehouses, or enterprise systems. The Single Window, while it is not necessarily labelled as such, in broad terms entails a comprehensive design of software to support and integrate cross-functional organisational processes (i.e. ERP as defined in Davenport 1998). In addition, similarly to data warehouses systems, it is designed to handle and store large volumes of historical data that can be retrieve for analysis. 7.1.1 Multiple public values in e-government At the same time, it seemed clear for the leaders of the project that automation alone was not going to support their complex network of regulations or reduce duplicities and redundancies in their processes. This is why I claim that standardisation and simplification of procedures can be understood as necessary preconditions to system design. In turn, this has been illustrated in the case by the series of business process reengineering (BPR) exercises over time.116 The automation and standardisation of processes have been essential features to achieve two related set of public values: technical efficiency and internal accountability. That is, automation and standardisation served as a precondition to make trade procedures less burdensome and costly (technical efficiency), and as a way to reduce discretion of public servants. Automation and standardisation are thus highly intertwined in the production One thing is to have a designed process, that is working, and say “I’m going to automate this”... And a rather different thing is to conduct a process reengineering to make that process an optimal one, and only after, systematise it... the latter is the way it should be... I am against systemising the inefficiency! Main IT Advisor to the Minister (LA) 116 The case also shows that, as widely studied in information systems research, conducting BPR exercises and only after applying technologies presupposes a linear and rational practice that is unrealistic (i.e. Avgerou 2002, Robey et al. 2002). This is no different in the public sector. - Page 183 - and materialisation of technical efficiency, legality and accountability. As such, these values can be broadly associated with the bureaucratic principles of public administration. The emphasis on standardisation resonates well with broader findings in the literature, which suggest that standardised data, structures and procedures are used to create order out of organisational ‘messiness’ through the rationalisation of agency and interagency processes (Fountain 2001, p. 27 and chaps. 8, 9, Kallinikos 2006b, chap. 2, Hasselbladh and Kallinikos 2000, Introna et al. 2009, p. 19, Townley et al. 2003, pp. 1056–7). Therefore, the actions of automation and standardisation worked as an overall mechanism to create discipline and force cooperation both amongst agencies and within them. Standardised rules, discretion and flexibility In the case of the Single Window, the idea of ‘contingency plans’ exemplifies a type of mechanism. However, given their limited scope, it also illuminates an area for future tensions. Standardised rules, discretion and flexibility Some technology innovations used in e-government can bring benefits in procedural fairness by hard-coding standardised rules and procedures (Bovens and Zouridis 2002, Reddick 2005, Reddick et al. 2011). However, standardisation and automation may bring additional tensions. Once automation of rules and procedures is in place, the computer system becomes closed and rather inaccessible to its ordinary users (Mutch 2010, p. 514, Kallinikos 2006, chap. 2). As has been demonstrated in similar cases (i.e. Volkoff et al. 2007, Fountain 2001, Mutch 2010, Ciborra and Patriotta 1996), roles, routines and responsibilities are inscribed into software in such ways that it remains difficult to work outside them.117 Foreign trade regulations in Mexico are reputed for its complexity. The more automation in place, the less flexibility employees may retain to deal with cases that need special attention (i.e. outside hours). That is, preoccupations with legality and its encapsulation in technology innovation can be either an enabler for achieving technical efficiency (the previous example), or a constraint (as in this illustration). Overall, this is in line with previous research in the public sector, which suggests that tensions between flexibility and discretion are hard to balance and tend to persist over time (Smith 2011, Feldman and Khademian 2001). - Page 184 - Robustness and accountability The Single Window, as a technological infrastructure, holds vital importance for the overall operation of foreign trade activities in Mexico. If the system fails, then the efficiency of trade operations is compromised—a rather obvious effect. An additional tension comes from the way that the technological platform has been designed and built. The model of the Single Window is based on a public-private-partnership (PPP), and it is the private sector that runs the Single Window operations for the first five years. Even though the contract includes tough penalties to avoid system failures, it is not yet clear who would be accountable in case of failed procedures or other malfunctions. Shifting the technological control to a third party complicates matters of accountability (Smith et al 2010). Tensions between the parties are related to general concerns about partnerships with the private sector, especially the changing nature of accountabilities in such partnerships. On this matter, Willems and van Dooren (2011)hypothesise that the shift from bureaucratic to collaborative arrangements with joint public and private involvement erodes traditional notions of accountability and blurs responsibilities, opening the need for new mechanisms (2011, pp. 524-5). 118 On this matter, see also (McNulty and Ferlie 2004, Olsen 2006, Bolin and Härenstam 2008, Johnson et al. 2009, Clegg et al. 2011, chap. 1). For a discussion on the artificial distinction between Bureaucracy versus Enterprise, see du Gay (2004). And for the myths of NPM versus post-NPM, see (Lodge and Gill 2011, Fattore et al. 2012). Equality, customer orientation and outsourcing Studies in e-government have noted that there is an increasing tendency towards differentiation and personalization (Bellamy and Taylor 1998, p. 88). Digital technologies provide a platform that is very appealing to that part of the public that has access and resources to use the internet in more advantageous ways (Fountain 2001, p. 201). In reinforcing the idea of citizen as customers and user orientation, tensions emerge between technical efficiency and distributive justice. While, in principle, the Single Window project offers the same service to all, it tends to favour big companies and powerful stakeholders—whether unintentional or not. In addition, a further tension could emerge when a private company is in charge of operating the Single Window: it was suggested in the case study by a public servant that the private operator sometimes showed more interest in charging for services than actually delivering them. Andrews - Page 185 - - Page 185 - and Entwistle (Andrews and Entwistle 2010, pp. 682–3) warns that public-private- partnerships may have particular negative effects on equity, which adds another value conflict between technical efficiency and distributive justice. 7.1.2 Implications of public values interdependence An important theoretical account from the multiple and interdependent relations between different clusters of public values entails the inadequacy of referring to the ethos of new public management (NPM) and bureaucracy as ‘absolute opposites’. This has been suggested in the scholarly literature dealing with e-government (i.e. Persson and Goldkulh 2010, pp. 51–2). Scholars have argued that public values usually associated with bureaucracy, democracy and traditional public administration should not be left aside in e-government designs and implementations (i.e. Brewer et al. 2006, Cordella 2007). This has been an important claim and a much-needed counter argument for the excessive attention paid to market- based principles for government organising (Bozeman 2007, du Gay 2000, 2003, 2004, Willmott 2011, Olsen 2006). My argument here is that, while value shifts occur and tensions emerge, a neat separation between public values commonly associated either to NPM or bureaucracy is artificial. Public values associated with traditional values of bureaucracy, such as a preoccupation with legality, the rule of law and procedural fairness, are not only prominent in the case but co-occurring with values more usually associated to a business-like approach, such as user orientation, a preoccupation with ‘trimming fat’ and avoiding ‘slack’ (Hood 1991, p.12). In addition, my findings in this area resonate with broader discussions in organisation studies that reject treating bureaucracy and post-bureaucracy as binary oppositions (Harris 2006, pp. 80–2, Bloomfield and Hayes 2009, pp. 480–3).118 While these discussions do not tend to focus on the role of technology (but see Harris 2006 and Bloomfield and Hayes 2009), the Mexican case supports these more general claims; that - Page 186 - is, a multiplicity of public values interacts in e-government, in positive terms (reinforcing each other) or in conflicting ways (competing for dominance). In part, the way the NPM was conceived and understood in scholarly literature has perpetuated a dichotomist vision of rather intrinsic and interdependent public management properties, such as flexibility and accountability (Feldman and Khademian 2001, p. 341). In e-government research, references to NPM are presented as if there were a coherent set of premises, types of reforms, or public values associated to them. While in general terms the NPM can be largely associated with the use of market-based principles in government with a focus on privatisation and economic individualism (i.e. Salskov-Iversen et al. 2000, p. 184, Bozeman 2007, p. 119 The discourse on forced cooperation that I elaborated in the analysis, for example, is an indication of the absence of competition. In addition, I showed in the case chapter that there were no major changes in the structures of the Ministries involved that could be comparable to those of the disaggregation movement experienced in leading NPM adopters such as the UK, New Zealand or Canada (Dunleavy et al 2006). The dynamic and political relation between public values and technology I argued in the previous section that multiple public values co-exists and are interdependent, at times acting as covalues or precondition of others, and at times generating conflicts. These public values are not simple given in e-government design. They are dynamically construed and negotiated within a context marked by national imperatives (i.e. dominant discourses on trade), the visions of public managers as well as available technical expertise. In these sections, I take the argument further to reflect on two particular dynamics: the political imperatives and previous history of negotiations; and the type and language of change. The political games and imperatives that I shall discuss in the following are important. They highlight the non-deterministic character of technology within public sector organisations and have an emergent character. Fountain (2001, chaps. 6, 7 and 8) and many others (Danziger et al. 1982, Bozeman and Bretschneider 1986, Cordella and Iannacci 2010, Dunleavy et al. 2006, Bloomfield and Hayes 2009) have documented that organisational forms and different institutional forces shape the outcomes of e- government initiatives. However, the ways ideological discourses are enacted and materialised in e-government have received less attention (but see Cordella and Iannacci 2010, and Bloomfield and Hayes 2009 for exceptions). Therefore, I argue that reflections on the political imperatives and its nature of change are useful explanations to why certain public values are sustained over time, while others are marginalised or received less attention. 7.1.2 Implications of public values interdependence 76), the actual content of NPM reforms is best regarded as ambiguous, emergent and context-based (Lodge and Gill 2011, Barzelay 1999, 2000, 2001). For example, it could be argued that Mexico has embraced many principles of the Washington Consensus and NPM ideology (such as a preoccupation with efficiency, privatisation of services, and customer orientation). However, the country did not embrace central NPM components such as extreme managerialism, or a shift to disaggregation, increasing competition and incentivisation amongst public agencies as demonstrated by Cejudo (2008).119 In line with these arguments, Lodge and Gill (2011, p. 142) argue that NPM was never as purely implemented as the literature in public management and political science has led us to believe. Overall, I contend that broad labels such as NPM or bureaucracy are usually used in the literature of e-government without reference to their institutional features. The differences in contexts and institutional dimensions therefore lead to flawed generalisations. Based on my findings, I further suggest it is more fruitful to focus on clusters of public values—manifested in discourses—rather than dichotomised visions of NPM versus bureaucracy. - Page 187 - - Page 187 - 7.2 7.2.1 Context, political imperatives and negotiations Broader political and economic agendas have shown to be important factors in the case study. When portraying the four different discourses on public values, I also explained its supporting role for the broader ideals of increased competitiveness and open trade in Mexico. These ideals form a widespread and influential body of discourse that affects perceptions of society and government in general (Mutch 2010, p. 516). From a discourse perspective “it is the texts that reproduce widely held conceptions and ideological assumptions—or create new ones” (Vaara 2010, p. 18). - Page 188 - This thesis provides several examples on the influence of the competitiveness discourse as an overall value mission behind the Single Window initiative. More specifically, the way I elaborated on the production of the different discourses on public values and technology in the case study, shed light on these dynamics. For example, the pursuit of improving Mexico’s position in global rankings, such as the World Bank’s Doing Business or the Global Competitiveness Index, framed the design and particular uses of technology innovation among stakeholders. That is, broader concerns with strengthening its global position in trade contributed to the goals of building a Single Window that would reduce the costs for Mexico’s firms as well as provide more transparent, efficient and controlled operations of foreign trade. The particular ways in which the Presidential Decree of 2011 was produced is useful in order to elaborate on the power influences behind the salient discourses on public values. The Presidential Decree and the specific technological choices made for the Single Window may be seen as the new version of an organisational truth (Introna et al. 2010, p. 22, Mutch 2010, p. 512). However, this is only ex-post; leadership capabilities, the processes of building political support and a history of previous collaborations help to unfold these dynamics. I shall elaborate on this below. The role of the leaders of the project and their success in building political support is helpful to explain why certain public values are sustained over time. Elaborating on the discourse of forced cooperation, it emerged that once the Single Window became more a concrete possibility than rhetoric, the government looked for mechanisms to endorse the initiative in stronger ways. In the work of Michael Moore and colleagues (Moore 1995, Benington and Moore 2011b), this process is understood as building and sustaining support from the authorizing environment. 121 I am drawing on Lindblom’s (1965) idea here, although in a more relaxed way. In his book, The intelligence of Democracy, Lindblom develops the concept of ‘Partisan mutual adjustments’ to address how coordination can be achieved in absence of a central coordinator. The idea of mutual adjustments entails adaptive adjustments “in which a decision-maker simply adapts to decisions around him” (p. 33). 120 It is worth to stress that Moore (1995) refers to the authorizing environment as part of a broader managerial tool called the ‘strategic triangle’ for public value creation. In this sense, I do not claim his ideas are not original, on the contrary. My point is to stress that in public affairs, backing initiatives politically is a rather common condition for policy effectiveness. 123 Most public servants (middle-level) dealing with the practicalities of the Single Window, have worked together since SICEX, and although on smaller scale, did have experience with technology, trade regulation and coordination among the agencies. 122 Recall that one of the features of SICEX was its interconnectivity with Customs. 7.2.1 Context, political imperatives and negotiations The latter is conceptualized as “a place of contestation where many different views and values struggle for acceptance and hegemony”(Benington and Moore 2011b, p. 6). In order to build stakeholder support, public servants and decision makers can seek for assistance in political superiors, overhead agencies, legislative oversees, the media and interests groups. While the leaders of the Single Window initiative draw on these mechanisms—mainly from political superiors and interest groups—the concrete way to back the Single Window - Page 189 - politically was by obtaining support from the highest political level possible: the President. It is commonplace that initiatives without strong political support usually tend to fade away in public affairs. As an example the OECD (2011, p. 13) stresses that lack of effective coordination and strong political leadership are the main barriers to e- government initiatives in Mexico in general. Even in the cases of thought-through and well-planned projects. That building a strong authorizing environment is important then, is not precisely novel.120 But it is helpful for understanding that gaining political support was, in part, fuelled by the prominence of a national imperative on trade competitiveness that has been pervasive in Mexico for the last two decades. In addition, leadership capabilities and strong commitment among project champions also played a role in persuading the authorities upwards. A related factor is the history of previous collaborations and negotiation between the two leading agencies of the project. On many occasions interviewees suggested that the champions of the Single Window initiative hold different value missions; that is, while Customs emphasises a preoccupation with control, the Ministry of the Economy promotes trade facilitation. A history of mutual adjustments121 between both agencies helps to explain why SICEX made progress and enabled collaborating mechanisms between both agencies.122 As a consequence, it turned out that public servants in charge of the design and operation of the Single Window did know each other for a long time, assisting (and sometimes constraining) the process of collaboration.123 The literature on collaboration in the public sector suggests that organisations that engaged in collaboration successfully in the past are likely to continue engaging in inter- 123 Most public servants (middle-level) dealing with the practicalities of the Single Window, have worked together since SICEX, and although on smaller scale, did have experience with technology, trade regulation and coordination among the agencies. - Page 190 - organisational projects (Bryson et al. 2006). 7.2.1 Context, political imperatives and negotiations In addition, Ansell and Gash (2008, p. 11) indicate that a history of successful past cooperation can create social capital and high levels of trust and therefore facilitating further collaboration. This thesis argues that organisations working together, sharing a history of cooperation, will tend to build and sustain similar public values over time. Summing up, the text of the Decree can be seen as a consequence of previous inter- agency negotiations and political imperatives. It is also a result of a previous history of technological developments and collaborations between the champions of the project (Customs and the Ministry of the Economy). The way the Decree was negotiated, and written, was in part because of concerns with broader institutional agendas. However, it was also a result of potential benefits of that technology. In other words, a longstanding history of mutual adjustments and a capability to negotiate contested value missions proved an important mechanism to enforce the general discourse on competitiveness. It also proved important for its associated dominant public values of technical efficiency, internal accountability and legality. 7.2.2 The type of change: continuity instead of radical transformation The reflections on the history of previous cooperation relates to the type of change implicitly inscribed in the initiative. In part, the discourses of the Single Window suggested a continuous movement towards the incremental changes that both Customs and the Ministry of the Economy have attained. The observed evolution in the vocabulary of the project cannot be described as radical change, transformation or even revolution. Instead, the many examples that I portrayed in the previous chapter are better described as a continuation from previous successes. A history of cooperation as well as utilizing the political environment favoured this path towards more openness to trade through incremental changes. Of course, the Single Window changes ways of acting and interacting as well as introducing new objects into the physical world (i.e. technology). However, the point here is to emphasise that the ‘continuation’ discourse of competitiveness seat with the political nature and power structures underlying the case. - Page 191 - My findings are in agreement with those who conceptualise design and implementation of e-government initiatives as characterized by the coexistence of a mixed of distinct factors rather than by fundamental or radical transitions (Harris 2011, p. 116, Introna et al. 2010, pp. 22-3, Bloomfield and Hayes 2009, pp. 482-3, McNulty and Ferlie 2004, p. 1389). Unlike these studies, my findings show a discourse of continuation rather than radical transformation as is the case in some other contexts (i.e. the UK).124 Using a language of continuation instead of transformation may be, precisely, another factor contributing to sustaining certain core public values alongside the Mexican initiative. More specifically, resistances are perhaps more likely to happen when a new discourse is inculcated and impose over previous practices. For example, the tensions emerging from the potential staff redundancies as a consequence of gains in technical efficiency may be better managed if choosing a discourse of continuity rather than transformation. I want to raise another point here. The idea of sediment and ongoing change instead of revolutionary transformation does not solely pertain to the domain of the public sector. As Suchman’s states (2002, p. 10 emphasis added): New ways of working and new technologies grow out of old ones. 124 The language of change in the UK has been about radical transformation, as some of these studies highlight (i.e. Harris 2011, Bloomfiled and Hayes 2009, Klecun 2011). While the findings point toward the same direction—the importance of taking the complex history, nature of the organization and power dynamics into account—the empirics of the Mexican case are quite different in the choices of language about the expected changes of ICT. 124 The language of change in the UK has been about radical transformation, as some of these studies 7.2.2 The type of change: continuity instead of radical transformation They do so neither through a process of simple incremental change, nor through wholesale displacement and transformation, but out of an ongoing interaction between understandings based in prior experience on the one hand, and leaps of faith inspired by imagination on the other. (...) This statement of continuity provides a corrective to heady pronouncements of so-called 'revolutionary' technological change. Its premise is that through juxtapositions and connections of existing forms new forms emerge. The idea of a sediment or ‘continuous change’ serves to illustrate why certain public values were more or less stable over time. The juxtaposition of the dominant discourses of technical efficiency and legality led to the design of a platform that supported control rather than flexibility. Such a development could be seen as a result of the previous historicity and expertise of those involved in the project. - Page 192 - - Page 192 - Reflecting back to the idea of sedimented or melded change (as Bloomfield and Hayes 2009, p. 482 suggest), brings another important factor into play to explain the trajectory of certain public values in the case. In Mexico, the discourse on SMEs has gained an increasing place in the national agenda if not yet fully materialised. However, it may in the long term contribute to the achievement and articulation of distributive justice in a national context. That is, technology innovation may contribute to distributive justice and procedural fairness. After all, the achievement and realisation of public values depends upon the political imperatives, institutional arrangements, technical expertise and organisational capabilities. Tensions are not going to disappear, and technology innovation may change, reinforce or create new centres of tensions between public values. 7.3 The dialectic relation between public values and technology Based on the previous discussions the public values associated to a given e-government initiative are resolutions of dynamically constructed discourses that are produced and internalised in different ways. That is, modernisation discourses and ideals shape the way e-government processes are designed and implemented. At the same time, technology feeds back into these processes. In words of CDA, I claim that while broader discourses on public values do have an influence on technology design and implementation, technology internalises and is internalised by discourses on public values. The literature on e-government and the socio-technical approaches on the dynamics of technology innovation within the public sector has already highlighted that the realisation of intended objectives is not guaranteed (Fountain 2001, chap. 6). That is, organisational forms and institutional arrangements as well as political and rational logics condition technology outcomes. The temporary achievements of SICEX are useful to illustrate this point. Back in 2000, an appealing feature for improving efficiency, timeliness and user orientation of trade regulations was to increase the availability of electronic channels to submit procedures. Yet, when comparing a set of specific procedures, the average process time was higher in the case of using the internet compared to paper based and face to face. With only one exception, using the - Page 193 - internet to submit a given foreign trade procedure resulted in longer waiting time.125This is not to say that technical efficiency imperatives were not materialised. I have showed that the total average waiting time for trade procedures was systematically reduced since 1999, that is, after the implementation of SICEX. To realise fully the potentials of SICEX in 2006, required government to change legislation on digital signatures. It was not until January 2012 that the Advanced Digital Signature Initiative (Ley de Firma Electronica Avanzada) was approved, giving digital signatures the same legal recognition as the written signature. The example reflects that the realisation of the intended public values is a process that continues to battle with emerging tensions, power dynamics and technological imperatives. Public organisations—and organisations in general—are not just empty containers into which technology innovations are poured (Fountain 2001, chap. 6, Mutch 2010, p. 512, Harris 2011). More attention should thus be paid to the surrounding conditions in which technology innovation happens. As suggested, technology innovation reinforces broader discourses as well. 125 Source: own elaboration based in the information compiled in Telecom-CIDE (2007). 7.3 The dialectic relation between public values and technology For example, despite some mixed results, the previous and smaller platform SICEX proved to deliver tangible benefits in terms of technical efficiency, transparency and decentralisation. In turn, the Single Window—even as an ideal before being implemented—was accompanied of imaginaries of certain public value achievements too. Recall that the Single Window has particular functionalities that support automation, standardisation and control at distance. Its operationalisation, therefore, enforces new ways of working in foreign trade, reflecting a step that, while provisional, frames new structural conditions. As Mutch notes (2010, p. 514): “there are clearly choices to be made at certain times, but those choices, once made, commit particular groups of users to something that in practical terms is given”. Therefore, the process of setting up data and information structures in the Single Window was at one point amenable to decision, but once decisions were made, those particular structures will be constraining possibilities of action. This ‘closure’ of the system affects the ability of its users to interact with the platform. What I want to emphasise here is that technology enables and constrain public value realisations. In doing so it has the potential to reinforce - Page 194 - broader ideological discourses as well as changing government’s action. Even when technology may allow for a flexible interpretation (Orlikowski 2000, Walsham 2001, p. 45), the way the technology is chosen and designed fixes some possibilities. The Single Window stresses automation, transparency and control to achieve competitiveness, leaving less room for flexibility, adaptability or participation. Of course, the realisation of the intended public values behind the Single Window is not only a political process of negotiations, but also a process of striving to make technology deliver (i.e. overcoming technical constrains, making systems work, project management, among other elements). I have not focused on the particular technical mechanisms in a salient way, but those are dialectically related in the process of internalisation and operationalisation of discourses. I argue that organisations develop discourses that suit their particular interests and advance their preferred technologies (Munir and Phillips 2005, p. 1667, Fairclough 2005, pp. 931-2). 7.3 The dialectic relation between public values and technology Although it is too early to study the effects of the Single Window on the potential tensions that it raises as well as the constrains, the fact that a new technology is already in place, materialised, and affecting ways of acting and interacting, can be seen as the result of discourses being dialectically transformed into other sociotechnical elements (Fairclough 2005, p. 934). 7.4 Conclusions I have started this chapter by discussing the types of public values found in the case and its character of interdependence. I shall add that the dominance of the technical efficiency cluster is congruent with previous findings in e-government research, which suggest that the effects of ICT on the internal capabilities of government are the most salient effect (i.e. Andersen et al. 2010). This, in part, may be explained by the nature of tasks and the associated benefits that can be traced as a consequence of government action. For example, reductions of time response, and the simplification of burdensome regulation are quite apparent goals to measure, against more ambiguous outcomes in the domain of accountability, fairness and justice. In the specific context of the case, I showed that four clusters of public values cohabit, and that despite the dominance of technical efficiency, other public values co-occur, reinforcing each other and creating - Page 195 - tensions as well. My findings, then, do not support a dichotomist division between public values associated to NPM and traditional public administration as it has been suggested. This artificial division is in part due to the ambiguous ways the NPM has been used in the literature of e-government and public administration more in general. Thus, my findings refute he proposition that e-government tends to favour NPM prescriptions. What is the NPM after all? I claim that it is more fruitful to refer to clusters of public values, and deconstructing their meanings, tensions and possible alignments with technology over time. My study shows that the way technology internalises and is internalised by public values —and its manifestations in discourse—offers a detailed account on how broader ideological currents are interwoven in technology innovation in public sector organisations. Much of the collective sensemaking surrounding public values and technology innovation in government either manifests in government texts or is reflected in them. Thus, the emergent discourses shape collective understandings of the complex phenomena. The focus of my dissertation, and the theoretical ideas applied using CDA, suggest that broader trends and discourses need to be taken into consideration when assessing the types of public values that e-government initiatives are said to achieve—regardless of the case. In turn, technology innovation feeds back this process bringing its own dynamics as well. Whether reinforcing an overall discourse—as the case shows—or transforming it. 8 Chapter Eight: Conclusions 8 Chapter Eight: Conclusions 8 This chapter presents the conclusions and key implications of my research, as a means of bringing together the dominant themes and arguments of my thesis. My main motivation to conduct this inquiry has been a preoccupation with public values and the ways they are presented and eventually incorporated in e-government initiatives over time. To facilitate the discussion, the chapter is divided into four sections. Section 8.1 provides an overview of the thesis and presents the main themes as they have been organised in my work. Section 8.2 restates the main contributions of the dissertation. It is divided into three sub-sections: contributions to theory, methodological contributions and practical implications. Building on the contributions, section 8.3 reflects on the strengths and limitations of my research approach, paying particular attention to the methodological choices and their implications for conducting critical discourse analysis. My research raised interesting issues that went beyond the scope of this thesis. The final part, section 8.4, elaborates on future research directions that I found promising and interesting for moving along related debates. 7.4 Conclusions Despite the importance of these broader discourses—as I showed in the analysis—very few authors have provided detailed empirical accounts of technology innovation in the public sector and its linkages with broader political and ideological agendas (but see McNulty and Ferlie 2004, Bloomfield and Hayes 2009, and Harris 2011 for notable exceptions). I claim that by studying public values and technology through a dialectical discursive lens my empirical accounts can contribute to this end and more generally, to our understanding of technology innovation and values. - Page 196 - - Page 196 - 8.1 Main themes and overview of the thesis 8.1 This thesis is concerned with understanding and explaining the linkages between public values and technology through critical discourse analysis. In particular, my concern has been with studying empirically which public values are presented and operationalised over time, in a longitudinal case of a technology-enabled platform to facilitate foreign trade regulations in Mexico—the Mexican Single Window for Foreign Trade (hereafter Single Window). The main objective was to bring different bodies of knowledge into conversation in order to analyse, understand and explain two related issues, comprised in the main research questions: This thesis is concerned with understanding and explaining the linkages between public values and technology through critical discourse analysis. In particular, my concern has been with studying empirically which public values are presented and operationalised over time, in a longitudinal case of a technology-enabled platform to facilitate foreign trade regulations in Mexico—the Mexican Single Window for Foreign Trade (hereafter Single Window). The main objective was to bring different bodies of knowledge into conversation in order to analyse, understand and explain two related issues, comprised in the main research questions: - Page 197 - What sorts of public values underlie a central e-government initiative in Mexico? How does the relation between public values and e-government unfold over time? What can we learn from Mexico? What sorts of public values underlie a central e-government initiative in Mexico? How does the relation between public values and e-government unfold over time? What can we learn from Mexico? The overall premise has been that public values shape, and are shaped by, technology; that is, there is a relational ontology. Based on the elusive character of public values, I adopted a discursive lens—in particular, critical discourse analysis—to address my research questions, focusing specifically on how public values are presented in key government texts, and how these texts are produced, disseminated and materialised during the trajectory of the case. The overall finding has been that there are multiple public values that are a combination of ‘classic’ and new public administration. These multiple public values compete for dominance, are dynamically construed and negotiated over time. Therefore, the relation between public values and technology can be best regarded as interdependent, political and dialectical. I shall elaborate accordingly in these sections. 8.1.1 Theoretical underpinnings: public values, e-government and critical discourse analysis 8.1.1 Theoretical underpinnings: public values, e-government and critical discourse analysis A concern with public values and e-government began with an exploration of the definitions and current debates in research on public values, presented in the first part of Chapter Two. This served the purpose of showing the complexity of public values theory, the different and sometimes disparate views on the subject as well as the challenges to study them empirically. For that reason, I selected the work of Bozeman (2000, 2007) on normative public values, who provides a stable and useful definition that is aligned with the purpose of my research: “public values are those providing normative consensus on the principles on which governments and policies should be based” (Bozeman 2007, pp. 13, 132). To facilitate the analysis of the literature on e- government, I used as a starting point a list of public values commonly found in contemporary literature of public management (Beck-Jørgensen and Bozeman 2007) and complemented it with the work of Hood (1991) and Hood and Jackson (1991) on administrative values. I then showed that e-government research either takes public values for granted or studies them in isolation. I therefore presented two related findings from the critical review on e-government literature in light of public values - Page 198 - - Page 198 - theory: (i) that empirical studies mapping multiple public values or public value shifts remain elusive in the literature; and (ii) that e-government research is silent on the relation between public value and its complex influence on technologically enabled public service provision. In sum, Chapter Two provided both the background and informed expectations about the thesis as well as key theoretical foundations of public values research. A key concern within public values theory is the difficulty of studying public values, given their intangible and elusive character. Thus, in Chapter Three, I presented the theoretical basis for studying public values and their linkages with technology through critical discourse analysis (CDA). This particular choice of theory also led my empirical design. The purpose was not just to search for public values in e-government but also to study them in an analytically useful way. 8.1.1 Theoretical underpinnings: public values, e-government and critical discourse analysis I based my work upon the version of CDA developed by Fairclough and colleagues (Fairclough 1992, 2003, 2005, 2010, Chouliaraki and Fairclough 1999, 2010), which emphasises the importance of a close and detailed analysis of texts, and at the same time, the integration of their analysis into broader social processes, actions and events as well as in relation to other texts. My approach to study discourse in organising is therefore three-dimensional: it links key government texts to broader discourses on public values, locating them within a particular historical and social context. As such, this version of CDA offers a useful theoretical framing and a practical methodology to study how discourse figures within processes of public value production and its realisation in technology. An important corollary that emerged from this particular way of theorising public values and technology is that discourses are not just discourses but materialised and operationalised into other elements of social life— such as activities, subjects and their social relations, artifacts, time and place, forms of consciousness. A consistent theorisation of technology was added to CDA drawing from the sociotechnical studies in e-government (Fountain 2001) and the relational character of technology in organising processes (Orlikowski 2000, Markus and Robey 1988, Volkoff et al. 2007, Mutch 2010). Technology, then, is said to be relational in the sense that it internalises discourses of public values but also has the capacity to constrain, reinforce or transform public values discourses. The theory chapter therefore presented a theorising device that builds from CDA, public values theory and social theory studies of technology innovation in government. - Page 199 - 8.1.2 Strategy of inquiry: longitudinal single case in light of CDA The particular theorisation of public values and technology through CDA implied important methodological choices. The empirical core of my dissertation was a 16-year longitudinal case of a technology-enabled platform for managing foreign trade regulations in Mexico. An extensive methodological chapter (Chapter Four) provided details on the philosophical assumptions underlying my choice of CDA, the way the longitudinal case study was selected and constructed as well as the specific CDA tools that I applied to analyse the data. The aim of my methodology (strategy of inquiry) was to help uncover the way in which public values were expressed and produced in texts but also to understand how they were consumed—that it, how public values were materialised in technology and new ways of acting and interacting. Methodologically, applying a CDA approach required a close analysis of key government texts and talks as well as drawing extensively on data from fieldwork to make sense of the meanings and conditions of production and consumption of those key texts. As is common in CDA and other reflexive approaches to qualitative research, the method of analysis involved an on-going process of moving back and forth between empirical and theoretical concepts. This reflexive process was, in turn, enabled by the richness of the data and the multiplicity of sources that I draw upon. 8.1.3 On context and the case A key theme that emerged from my choice of theory and methodology—public values, technology and CDA—has been the importance of elaborating context. Chapter Five began by providing a detailed history and socioeconomic context of Mexico’s modernisation reforms in the area of foreign trade and its administrative central bureaucracy (Part One). From the 1980s to 2012 Mexico has embarked in a profound economic change, from being a closed economy to depending dramatically on foreign trade. In parallel, several initiatives at the federal level have set the path towards a more impartial and professionalized operation of central government agencies. Both trade policies and public administration reforms were regarded as important institutional forces that dynamically formed the historical and economic context for the development of the Single Window for Foreign Trade. - Page 200 - - Page 200 - Part Two of Chapter Five documented the construction of the Single Window (2008- 2012) and features of the previous technological developments that went back to 1997. The entry point to my case study has been the Ministry of the Economy, which, together with Customs, oversee the biggest part of Mexico’s trade regulations. A key component of the case was the construction of a timeline that covered an 16-year period (1997-2012), that served to build the story in three different chronological phases and in light of the contextual developments exposed in Part 1 of the chapter. The timeline documented the key events (in sociotechnical terms) that serve to help understand the process of design and pre-implementation of the Single Window, based on a longstanding story of past developments in the Ministry. The case study therefore provided technical details and described the dynamics and efforts to construct the Single Window, including its technological features and its surrounding institutional forces. With mixed intermediate results and longstanding efforts to make it happen, the case concluded with a series of positive developments: in 2011, a Presidential Decree was enacted, backing the initiative politically. Contrary to initial expectations, the Single Window started operating in pilots from early 2012 and became mandatory on 1st June 2012. The chapter included a postscript written in September 2012 to briefly outline the main events that took place after June 2012 as well as future plans. 8.1.4 Findings and implications Focusing on discourse was helpful to shed light on the types of public values that were imbued in the case as well as the diverse dynamics behind the discourse production, dissemination and materialisation. Chapter Six therefore empirically assessed what discourses on public values and technology were most prominent in the trajectory of the case and elaborated on their structural characteristics (meanings). The analysis revealed four distinctive discourses on public values and technology: ‘technical efficiency’, ‘legality and honesty’, ‘robustness’ and ‘(forced) cooperation’. Using the Presidential Decree that established the Single Window in 2011 as my main textual anchoring point, the chapter followed a systematic pattern of analysis to show three related themes for each group of public values: (i) what structural characteristics are associated to each group of public values (textual characteristics), (ii) how these - Page 201 - - Page 201 - discourses are produced (where do they stem from), and (iii) how they are reproduced and consumed in technology over time (materialisation). The analysis showed that while the discourse on technical efficiency—commonly associated to the new public management ethos—is dominating, it co-occurs with and is reinforced by other values more broadly related to traditional public administration and the bureaucratic ethos— that is, legality, procedural fairness and honesty. In addition, the analysis showed that these four analytically distinctive discourses have been materialised in technology in different degrees, giving rise to tensions and contestation over time. The extensive empirical analysis presented in Chapter Six led to further implications, raised in Chapter Seven. The case demonstrates that public values are not simply ‘given’ (i.e. as in Heeks 2002). They are dynamically articulated, constructed and materialized within ongoing processes of design and implementation of technology innovation. From this point, I offered two implications: first, the relation between public values and technology is one of interdependence and not dichotomy. Therefore, concerns that have tended to dichotomise NPM versus bureaucracy, or old public administration versus new public administration, are not supported empirically. In addition, the findings also showed that key public values associated with bureaucracy were not only prominent in the case but made stronger as a result of the process of technology innovation. Second, working with abstract notions of public values is difficult as public values take meaning in a given context. 8.1.4 Findings and implications It has been only by elaborating on the structural characteristics of the discourses on public values and technology that the relation between them unfolds tensions, conflicts and negotiations. The broader discussion presented in Chapter Seven—in light of the findings of Chapter Six—reinforces the idea that technology is not simply materialising public values in a neutral way; technology also shapes the diverse discourses of public values production, consumption and operationalisation. The way technology innovation is chosen and designed fixes future possibilities. The Single Window stresses automation, transparency and control, reinforcing the broader imperative of competitiveness. In so doing it, it leaves less room for flexibility, adaptability or participation. That certain public values are sustained while others marginalised over time is, in part, a consequence of power struggles, political negotiations and national imperatives—but it is also a result of the dynamics that technology innovation puts in place. This idea led to the theme that the - Page 202 - relation between public values and technology is dynamic, political and dialectical in character. The achievement and realisation of public values depends upon the political and national imperatives, institutional arrangements, technical expertise and organisational capabilities. Tensions are not going to disappear, and technology innovation may change, reinforce or create new centres of tensions between public values. Contributions to theory The thesis has brought together different bodies of literature in dialogue: public values theory, e-government and critical discourse analysis. While weak connections were found in the literature, these bodies of knowledge have not been combined before in a systematic way. In bridging these theories together, my contributions to theory are threefold: (i) to extend e-government research by providing a theorisation of the relation between public values and technology over time and in a particular context; (ii) to extend public values theory to account for technology, context and content relations; and (iii) to offer a theoretically driven methodology to study empirically the relation between public values and technology, based on the role of discourse in organising. The first contribution relates to the knowledge gap that I presented at the beginning of this dissertation. While there have been notable advances in e-government theory, the relation between public values and technology remains particularly under-theorised. I claim that paying attention to the role of discourse in e-government organising is a valuable avenue for unfolding the relation between public values and technology innovation in government. In particular, it emphasises the political nature of the relation, an area that has received less attention in the literature of e-government. The second theoretical contribution has been to move along the public values debate and the role of context in organising more generally. The inventory of public values compiled by Beck Jørgensen and Bozeman (2007) proved useful to map the most prominent values in the case using CDA. As the authors warned, the main disadvantage - Page 203 - of their approach is that “values are quoted out of context” (Beck Jørgensen and Bozeman 2007, p. 357). Chapter Six offered an extensive elaboration on the characteristics of commonly used—but usually elusive—public values, providing not only content but context and the relations between them. In turn, the particular usage of the CDA method in the study shows a useful theoretically driven methodology to unpack relations between technology and discourse in context. The three-level interlinked approach to study discourses in the particular case of public values and technology has shown that context is dynamically represented (i.e. Hayes and Westrup 2012), or generative (Salskov-Iversen et al. 2000). The way CDA theorises context proved to be useful to extend these lines of research. Finally, the three-dimensional approach to analyse discourse offered a particular device to answer my research questions. Contributions to theory Studies that seriously consider the discursive elements in e-government are extremely rare, despite the fact that much of the collective sense- making surrounding e-government agendas either manifests in government texts and talks or is reflected in them. In particular, the discursive lens served as a valuable aid to studying how claims about public values and technology draw from broader institutional discourses and country’s priorities, and how they are made to work in specific organisational levels and micro productions of discourses. The way of theorizing public values and technology innovation through CDA provides an analytical framework that can be extrapolated for future comparisons of similar projects and in different contexts. Moreover, this thesis demonstrates that the particular discursive lens that CDA offers can be successfully utilised in the study of technology and organising, despite its low recognition within the information systems field. More specifically, CDA can shed light on the ways technology innovation emerges and evolves over time, and to account for the ways it becomes interwoven with broader socioeconomic contexts and practices more generally (Orlikowski and Iacono 2001, p. 133). While this contribution holds theoretical importance in itself, it has methodological implications, and I shall present them in more detail in the next section. - Page 204 - - Page 204 - Contributions to research methodology The main contribution of this dissertation to research methodology entails the use of critical discourse analysis (CDA) as a method for studying relations of public values and technology. CDA has become a well-established research methodology in organisation and management studies, although used in confusing and unarticulated ways (Leitch and Palmer 2010). I claim that my main methodological contribution has been in providing transparent and clear methodological criteria for practicing CDA in studies of public sector organising and technology. In addition, it is important to stress that my overall research design places CDA as part of a longitudinal case study. The latter is not usually found in CDA studies, and proved particularly useful to highlight the historical and social context of the case. Therefore, my methodological choices, in conjunction with their theoretical underpinnings, provide detailed material to inform newcomers into CDA that wish to conduct similar studies. Despite its increasing popularity, critical discourse analysis—and in particular the textually oriented version I have used in my research—remains largely unknown within the field of public administration and information systems. My study provides a well- grounded research design that can be extended to similar concerns of public sector organising, and more generally, to studies of technology innovation and values. By incorporating a theorisation of other elements that affect the way discourses are produced, consumed and operationalised (i.e. public values and technology), my research design has contributed modestly towards the integration of multidisciplinary approaches that CDA theorists have called upon (Van Dijk 2003, p. 363). Practical implications This thesis is about public policy decisions. Governments usually face challenges in several fronts: budgets, organisational capabilities, technical expertise and a pressing environment that will judge their legitimacy. Yet, ultimately, governments should be concerned about creating and delivering public value (Moore 1995, Bozeman 2007, Benington and Moore 2011a). Practically, this thesis informs public managers leading processes of e-government in a number of areas. It highlights the importance of - Page 205 - defining and reasoning about the types of public values that the government wishes to pursue. For example, ideas of transparency as well as efficiency were all raised in the discourses of government, but usually in a loose fashion. In relation, my research informs government leaders of potential inconsistencies that may emerge when trying to accommodate multiple public values. By revealing the complex, emergent and dialectical relation between public values and technology, this thesis raises awareness of the difficulty to align multiple public values in a single design. The case shows that while some public values can reinforce each other, it does not mean that they will always be aligned. Choices need to be made and trade-offs are likely to occur. My study stresses that public managers and policy makers need peripheral vision (Wright et al. 2012). Hence, a broader vision on public values and technology can help them to think more strategically and develop capabilities to help choose and protect public values more consciously. Another practical contribution comes from documenting the ways in which the Single Window was designed and built from the specific political and historical context. By the time this thesis goes to print, several countries in the region, such as Chile, Peru and Costa Rica, have embarked in similar Single Window projects for foreign trade. Thus, this case should help to reinforce the fact that technology innovation is not neutral and it will not simply lead to expected outcomes. This is not a new finding in theoretical terms, but one that needs more attention in public administration circles: building a network-enabled government is more about the process and politics of institutional change, rather than about a set of technological prescriptions to achieve predetermined results. Some specific lessons emerged can be drawn from the case. First, building a Single Window requires coordination and collaboration; starting with those agencies that hold expertise and that have a history of past collaborations demonstrates to enhance interagency collaboration. Practical implications Second, sustaining the initiative on previous achievements or technological expertise proves useful to secure the project. Third, leadership capabilities demonstrated to have played a crucial role. Investing in finding those actors that would prove to build more political support may be desirable. Finally, building and sustaining strong political support during the whole trajectory of the project is mandatory—perhaps, one of the strongest points in the case. - Page 206 - - Page 206 - Reflection on my research approach 8.3 Theorizing public values and e-government through CDA has been fruitful yet challenging. I have tried to convince the reader that CDA offers an analytically useful methodology for understanding the relation between public values and technology (see my previous sections on contributions). Yet, this research approach has its limitations too. One could argue, for instance, that textual analysis is inevitably selective and incomplete (Fairclough 2003, p. 15-6). As a matter of fact, unpacking all potential intertextual and interdiscursive aspects of a particular text is not only an impossible task (i.e. Keenoy and Oswick 2003, p. 140), but it would also prove useless: the value of critical discourse analysis relies on illuminating a particular inquiry that is defined by the researcher. This is not to say that any formal analysis of texts is theory-free (Chouliaraki and Fairclough 1999, p. 7). The analyst’s theoretical preoccupations define what data is selected and how it is perceived; in other words, the analytical categories that emerge out of the selected texts are defined by the research questions and the research focus (theoretical preoccupations) of a particular project. In the case of my research, my theoretical preoccupations have led me to draw on those linguistic features that could help elicit the linkages between public values and technology innovation, building from existing research on public values (Bozeman 2007) and e-government theory (Fountain 2001). In addition, I could have covered or selected other texts for the detail-oriented textual analysis. For example, I could have focused on reactions reported in media texts (i.e. Vaara and Tienari 2008), or on selecting a particular public speech or an interview text. However, I do draw on media commentaries as additional material to enhance the ways on how these texts are produced, consumed and interpreted in a broader sense. I shall insist that the texts I selected are only instances of a larger body of texts and social relations that are useful to study the trajectory of the discourses on public values and technology, its reasons for production and consumption over time. A specific challenge of CDA entailed placing specific texts within their social contexts. This has certainly been the most difficult part of my research inquiry. Even though I have complemented the analysis with additional material gathered in the fieldwork, the - Page 207 - reflexive and interpretative process can be too numb or relative to inform actions. Reflection on my research approach The strengths of CDA in this respect have relied on its tools for identifying interpretations that went beyond the surface level of texts. In sum, applying CDA entailed a longstanding critical and reflexive research exercise. This is, perhaps, the most powerful feature of the method as well as its main challenge (see for example Phillips et al. 2008, p. 786). Working with my empirical data entailed a reflexive process of going back and forth between the theoretical ideas and the empirical analysis, without a clear ‘step-by- step’ method. I would have ideally liked to construct a comparative analysis that went beyond a single case design. The intense and demanding effort to analyse and get access to data proved far complex. The trade-off would have been losing the richness of the case elaboration as well as its deconstruction using CDA. A further limitation has been in applying only a limited number of linguistic tools, out of the broad range of devices that are available from CDA. In particular, I would have liked to apply more extensively other computational tools to make sense of the relations and discursive features such as vocabulary. For example, specialised software packages, such as Alceste and TLab, can perform automated content analysis to check for robustness and consistency in large corpora of data (see for example Schonhardt-Bailey 2012). Although I do plan to extend my empirical work using features like these in the future, this exercise would be only complementary, and in any case, it does not imply leaving aside the role of the analyst in making sense of “automated” results. A further limitation is related to my unit of analysis and its relation with public value theory. I have deliberately centred my analysis on the central government in order to understand how it made sense and operationalised a set of public values. While I have incorporated news articles and opinions from external stakeholders to some extent (i.e. private companies in trade or customs agents), I have limited the scope of my research to observe, talk with and gather information from the champions of the project. Consequently, I have acknowledged this in my research methodology chapter. Further research should expand on the “public” side of public values (see the next sections). - Page 208 - 8.4 Future research Competing rationalities and institutional logics Public values implicated in technology innovation in the public sector may be conflicting at times. Although I have drawn on some aspects of competing rationalities and the work based on institutional theory (i.e. Fountain 2001), I have not particularly discussed interesting concepts coming from the scholarly work on institutional logics. Therefore, research on institutional logics and competing rationalities in organising(Friedland and Alford 1991, Townley 1997, 2002, Thornton and Ocasio 1999, Reay and Hinings 2009), and in particular, in studies of organising and technology (Avgerou 2002, Hayes 2008, Hayes and Rajão 2011) appears as a fruitful theoretical lens to apply to the Mexican case. For example, parallels could be traced with the work of Friedland and Alford (1991) and what they refer as “market rationality” and “bureaucratic rationality”. The discourses on technical efficiency and legality that I identified in my work seem closely related to their definitions. In light of the richness of the case I studied, future work could analyse the existence of competing logics for different purposes. For example, to trace the processes of enactment that are not easily uncovered if one places the sole attention on instrumental or market rationality. Legitimation and resistance My focus has been in understanding the visions, plans and public value constructs from the point of view of a single federal agency (the Ministry of the Economy) and to a lesser extent, of Customs. Further data could analyse how different discourses become legitimised, contested or resisted by other Ministries and stakeholders. This has been a particularly salient cue from the process of inter-agency cooperation and the discourse on ‘forced’ cooperation that has not been fully materialised among all the participants. The case leaves room to understand in more detail how people resist technologically- induced institutional changes and what power struggles are experienced by specific actors once the Single Window is fully implemented. In particular, the Mexican case offers an interesting setting to study the power struggles between central and local offices, and between government and certain stakeholders like the Customs Agents - Page 209 - Associations. My dissertation touched only briefly on these points, given my primarily interest in understanding first what and in which ways discourses on public values were articulated by the actors in charge of the design and implementation of the case. Future research could explore how power dynamics influence the enactment of certain public values over others through everyday actions, and how power relations shape institutional change from diverse angles (i.e. local versus central, between different agencies). The work of Thomas and Davies (2005) and Thomas et al.(2011) on the micro-politics of power-resistance, or the work on critical genre analysis (Levina and Orlikowski 2009) could be useful resources to complement my critical discourse analysis approach. Broader discussions on public values There are a number of current discussions on whether public values vary across countries with different political systems and national cultures (Beck Jørgensen and Vrangbæk 2011), or whether certain public values can be universal (Kamto 1997, p. 303, Kernaghan 2000, p. 100). Usually, these studies rely on the use of surveys as the main method to collect data, and point to the empirical difficulties of comparing results. CDA could be applied to other settings to compare whether one can find similarities or differences with the findings of the Mexican case. For example, a CDA approach could be applied to other similar Single Window initiatives, such as those ongoing in Latin American countries, or to more disparate ones, such as South Korea and Singapore, which have already implemented similar initiatives in the past. In doing so, one could compare if the clusters of public values hold despite the differences in context and to move along public values discussions in the field. Citizen’s perceptions Public values are ideals about the collective aspirations of citizens (Moore 1995, Benington and Moore 2010). Future studies could focus on citizens’ perceptions about the different dimensions of public values implicated in the case. For example, it would be particularly interesting to study public perceptions on the effects that technology has on discretion of street-level bureaucrats to contribute to ongoing debates on the subject (Bovens and Zouridis 2002, Jorna and Wagenaar 2007, Wenger and Wilkins 2009, Smith 2011). The tensions surrounding discretion, procedural fairness and justice in the - Page 210 - - Page 210 - Mexican case present an interesting opportunity to study the perceived effects of the programme on different sets of public values. References Ackroyd, S., 2009. Research Designs for Realist Research. In: D.A. Buchanan and A. Bryman, eds. The Sage Handbook of Organizational Research Methods. London: SAGE, 532–548. Ackroyd, S. and Fleetwood, S., eds., 2000. Realist Perspectives on Management and Organisations. London: Routledge. Agre, P.E., 2002. Real-Time Politics: The Internet and the Political Process. The Information Society, 18 (5), 311–331. Ahn, M. and Bretschneider, S., 2011. Politics of E-Government: E-Government and the Political Control of Bureaucracy. Public Administration Review, 71 (3), 414– 424. Alford, J. and Hughes, O., 2008. Public Value Pragmatism as the Next Phase of Public Management. The American Review of Public Administration, 38 (2), 130–148. Alford, J. and O’Flynn, J., 2009. Making Sense of Public Value: Concepts, Critiques and Emergent Meanings. International Journal of Public Administration, 32 (3), 171–191. Alvesson, M. and Kärreman, D., 2000a. Taking the Linguistic Turn in Organizational Research. The Journal of Applied Behavioral Science, 36 (2), 136–158. Alvesson, M. and Kärreman, D., 2000b. Varieties of Discourse: On the Study of Organizations through Discourse Analysis. Human Relations, 53 (9), 1125 –1149. Alvesson, M. and Kärreman, D., 2011. Decolonializing discourse: Critical reflections on organizational discourse analysis. Human Relations, 64 (9), 1121 –1146. Andersen, K., Henriksen, H., Medaglia, R., Danziger, J., Sannarnes, M., and Enemærke, M., 2010. Fads and Facts of E-Government: A Review of Impacts of E- government (2003-2009). International Journal of Public Administration, 33 (11), 564–579. Andersen, K.V. and Henriksen, H.Z., 2006. E-government maturity models: Extension of the Layne and Lee model. Government Information Quarterly, 23 (2), 236–248. Andersen, K.V. and Henriksen, H.Z., 2006. E-government maturity models: Extension of the Layne and Lee model. Government Information Quarterly, 23 (2), 236–248. Andrews, R., 2011. NPM and the Search for Efficiency. In: T. Christiensen and P. Lægreid, eds. The Ashgate research companion to new public management: GB: Ashgate Publishing Ltd, 281–294. Andrews, R., 2011. NPM and the Search for Efficiency. In: T. Christiensen and P. Lægreid, eds. The Ashgate research companion to new public management: GB: Ashgate Publishing Ltd, 281–294. Andrews, R. and Entwistle, T., 2010. Does Cross-Sectoral Partnership Deliver? An Empirical Exploration of Public Service Effectiveness, Efficiency, and Equity. Journal of Public Administration Research and Theory, 20 (3), 679 –701. Ansell, C. and Gash, A., 2008. Collaborative Governance in Theory and Practice. Journal of Public Administration Research and Theory, 18 (4), 543–571. Arellano-Gault, D., 2000. Mexican public sector reform: Patrimonialist values and governmental organisation culture in Mexico. Hybrids in public management The findings have revealed that, instead of dichotomies, public values are a result of a mix between traditional public administration (bureaucratic ethos), new public management or post-bureaucracy. The idea of hybridisation has been at the centre of attention in public administration circles (Olsen 2006, Clegg et al. 2011), although more work is needed to understand the consequences of hybridisation on the role of public administration in providing public services (Van Gestel et al. 2012 in their call for papers). Future research could build on recent contributions in the particular area of hybridity and e-government (Bloomfield and Hayes 2009) to account for public values. In particular, it would be interesting to study the consequences of new forms of control and accountability that are mediated through technology as well as the hybrid work practices emerging from the public-private-partnership in the case. - Page 211 - - Page 211 - References References Centro de Investigación y Docencia Económicas. Arellano-Gault, D., 2010. Economic-NPM and the Need to Bring Justice and Equity Back to the Debate on Public Organizations. Administration & Society, 42 (5), 591–612. Arellano-Gault, D. and Guerrero, J.P., 2003. Stalled Administrative Reforms of the Mexican State. In: B.R. Schneider and B. Heredia, eds. Reinventing Leviathan: the politics of administrative reform in developing countries. North-South Center Press. Arrow, K.J., 1963. Social Choice and Individual Values. Yale University Press. Aucoin, P., 1995. The new public management : Canada in comparative perspective. Québec: Ashgate : IRPP Governace. Avgerou, C., 2002. Information Systems and Global Diversity. Oxford University Press. B l M 1993 Th Si l C S d I ll ll A bi i I i J l f vgerou, C., 00 . Information Systems and Global Diversity. Oxford University Press. Barzelay, M., 1993. The Single Case Study as Intellectually Ambitious Inquiry. Journal of Public Administration Research and Theory, 3 (3), 305 –318. - Page 212 - Barzelay, M., 1999. How to argue about the new public management. International Public Management Journal, 2 (2), 183–216. Barzelay, M., 2000. The New Public Management: a bibliographical essay for Latin American (and other) scholars. International Public Management Journal, 3 (2), 229– 265. Barzelay, M., 2001. The new public management: improving research and policy dialogue. Berkeley: University of California Press. Batley, R. and Larbi, G.A., 2004. The changing role of government : the reform of public services in developing countries. Basingstoke ; New York: Palgrave Macmillan. Beck Jørgensen, T., 1999. The Public Sector In An In-Between Time: Searching For New Public Values. Public Administration, 77 (3), 565–584. Beck Jørgensen, T. and Bozeman, B., 2007. Public values - An inventory. Administration & Society, 39 (3), 354–381. Beck Jørgensen, T. and Vrangbæk, K., 2011. Value Dynamics: Towards a Framework for Analyzing Public Value Changes. International Journal of Public Administration, 34 (8), 486–496. Belanger, F. and Carter, L., 2012. Digitizing Government Interactions with Constituents: An Historical Review of E-Government Research in Information Systems. Journal of the Association for Information Systems, 13 (5). Bellamy, C. and Taylor, J.A., 1998. Governing in the information age. Buckingham: Open University Press. Benington, J., 2011. From Private Choice to Public Value? In: J. Benington and M.H. Moore, eds. Public value: theory and practice. Basingstoke: Palgrave Macmillan, 31– 51. Benington, J. and Moore, M.H., eds., 2011a. Public value: theory and practice. Basingstoke: Palgrave Macmillan. Benington, J. References and Moore, M.H., 2011b. Public value in Complex and Changing Times. In: J. Benington and M.H. Moore, eds. Public value: theory and practice. Basingstoke: Palgrave Macmillan, 1–30. Berman, E.M. and West, J.P., 2012. Public Values in Special Districts: A Survey of Managerial Commitment. Public Administration Review, 72 (1), 43–54. Bertot, J.C., Jaeger, P.T., and Grimes, J.M., 2010. Using ICTs to create a culture of transparency: E-government and social media as openness and anti-corruption tools for societies. Government Information Quarterly, 27 (3), 264–271. Bhatia, V., 1997. Translating legal genres. In: A. Trosborg, ed. Text Typology and Translation. Amsterdam: John Benjamins Publishing Company, 202–213. Bhatnagar, S., 2009. Unlocking E-Government Potential: Concepts, Cases and Practical Insights. SAGE Publications Ltd. SAGE Publications Ltd. Bhatnagar, S.C., 2004. E-government: from vision to implementation: a practical guide with case studies. New Delhi: Sage Publications. Bloomfield, B. and Hayes, N., 2009. Power and Organizational Transformation through Technology: Hybrids of Electronic Government. Organization Studies, 30 (5), 461–487. Bolin, M. and Härenstam, A., 2008. An Empirical Study of Bureaucratic and Post- Bureaucratic Characteristics in 90 Workplaces. Economic and Industrial Democracy, 29 (4), 541 –564. Bookman, Z. and Guerrero, J.P., 2009. Two Steps Forward, One Step Back: Assessing the Implementation of Mexico’s Freedom of Information Act. Mexican Law Journal, 1 (2), 1–50. - Page 213 - Bovens, M., 2005. Bureaucracy in the Twenty-First Century. In: E. Ferlie, L.E. Lynn, and C. Pollitt, eds. The Oxford handbook of public management. Oxford ; New York: Oxford University Press, 182–208. Bovens, M., 2007. Analysing and Assessing Accountability: A Conceptual Framework. European Law Journal, 13 (4), 447–468. Bovens, M. and Zouridis, S., 2002. From street-level to system-level bureaucracies: how information and communication technology is transforming administrative discretion and constitutional control. Public administration review, 62 (2), 174–184. Bozeman, B., 1987. All organizations are public: bridging public and private organizational theories. 1st ed. San Francisco: Jossey-Bass. Bozeman, B., 2000. Bureaucracy and red tape. Prentice Hall. Bozeman, B., 2007. Public values and public interest: Counterbalancing economic individualism. Washington DC: Georgetown University Press. Bozeman, B., 2009. Public values theory: three big questions. International Journal of Public Policy, 4 (5), 369–375. Bozeman, B. and Bretschneider, S., 1986. Public Management Information Systems: Theory and Prescription. Public Administration Review, 46, 475–487. Bozeman, B. and Moulton, S., 2011. Integrative Publicness: A Framework for Public Management Strategy and Performance. Journal of Public Administration Research and Theory, 21 (suppl 3), i363–i380. y ( pp ) Bresser-Pereira, L., 2004. Democracy and Public Management Reform: Building the Republican State. Oxford University Press, USA. y Bretschneider, S., 2003. Building the virtual state: Information technology and institutional change. Public Administration Review, 63 (6), 738–741. Brewer, G.A., Neubauer, B.J., and Geiselhart, K., 2006. Designing and Implementing E- Government Systems: Critical Implications for Public Administration and Democracy. Administration Society, 38 (4), 472–499. Brockner, J., 2002. Making Sense of Procedural Fairness: How High Procedural Fairness Can Reduce or Heighten the Influence of Outcome Favorability. The Academy of Management Review, 27 (1), 58–76. Brockner, J., Chen, Y.-R., Mannix, E.A., Leung, K., and Skarlicki, D.P., 2000. Culture and Procedural Fairness: When the Effects of What You Do Depend on How You Do it. SAGE Publications Ltd. Administrative Science Quarterly, 45 (1), 138 –159. Bryson, J.M., Crosby, B.C., and Stone, M.M., 2006. The Design and Implementation of Cross-Sector Collaborations: Propositions from the Literature. Public Administration Review, 66, 44–55. Camacho, E., 2011. Dificultades para el comercio exterior. Empresarios desconocen cómo armar canales de distribución. El Universal, 14 Jun. Castells, M., 2001. The Internet galaxy : reflections on the Internet, business, and society. Oxford ; New York: Oxford University Press. Castells, M., 2004. Informationalism, Networks, And The Network Society: A Theoretical Blueprint. In: The Network Society: A Cross-Cultural Perspective. Nothampton, MA: Edward Elgar. Cejudo, G., 2008. Explaining change in the Mexican public sector: the limits of New Public Management. International Review of Administrative Sciences, 74 (1), 111–127. Center for Technology in Government, 2011. Open Government and Public Value: Conceptualizing a Portfolio Assessment Tool. Center for Technology In Government, University at Albany, Albany, NY. Chadwick, A., 2003. Bringing E-Democracy Back In: Why it Matters for Future Research on E-Governance. Social Science Computer Review, 21 (4), 443–455. - Page 214 - Chadwick, A., 2006. Internet politics: states, citizens, and new communication technologies. New York: Oxford University Press. Chadwick, A., 2007. Implementing and managing e-government: An international text. Public Administration, 85 (3), 857–U1. ( ) Chadwick, A. and Howard, P.N., eds., 2009. Routledge Handbook of Internet Politics. New York: Routledge. Chadwick, A. and May, C., 2003. Interaction between states and citizens in the age of the internet: “e-government” in the United States, Britain, and the European Union. Governance-an International Journal of Policy and Administration, 16 (2), 271– 300. Chouliaraki, L. and Fairclough, N., 1999. Discourse in late modernity : rethinking critical discourse analysis. Edinburgh: Edinburgh University Press. Chouliaraki, L. and Fairclough, N., 2010. Critical Discourse Analysis in Organizational Studies: Towards an Integrationist Methodology. Journal of Management Studies, 47 (6), 1213–1218. Christensen, T. and Lægreid, P., 2011. Beyond NPM? Some development features. In: T. Christiensen and P. Lægreid, eds. The Ashgate research companion to new public management: GB: Ashgate Publishing Ltd, 391–403. Ciborra, C., 2005. Interpreting e-government and development: Efficiency, transparency or governance at a distance? Information Technology & People, 18 (3), 260–279. Ciborra, C. and Patriotta, G., 1996. Groupware and Teamwork in the New Product Development: The Case of a Consumer Goods Multinational. In: C. Ciborra, ed. Groupware and teamwork: invisible aid or technical hindrance? Chichester, UK: Wiley, 121–142. Clegg, S., 2011. Under Reconstruction: Modern Bureaucracies. In: S. Clegg, M. SAGE Publications Ltd. Harris, and H. Höpfl, eds. Managing modernity: beyond bureaucracy? Oxford: Oxford University Press. Clegg, S., Harris, M., and Höpfl, H., eds., 2011. Managing modernity: beyond bureaucracy? Oxford: Oxford University Press. Comisión Federal de Competencia, 2008. Opinión con el fin de promover la aplicación de los principios de competencia y libre concurrencia en el diseño y aplicación de políticas y regulaciones del comercio exterior de mercancías. No. Oficio PRES-10-096-2008-082. Presidencia, Mexico City. Coombs, R., Knights, D., and Willmott, H., 1992. Culture, Control and Competition; Towards a Conceptual Framework for the Study of Information Technology in Organizations. Organization Studies, 13 (1), 051–72. g g z ( ) Cooren, F., 2004. Textual Agency: How Texts Do Things in Organizational Settings. Organization, 11 (3), 373 –393. Corbin, J.M. and Strauss, A.L., 2008. Basics of Qualitative Research: Techniques and Procedures for Developing Grounded Theory. 3rd ed. Los Angeles, CA: SAGE Publications. Cordella, A., 2007. E-government: towards the e-bureaucratic form? Journal of Information Technology, 22 (3), 265–274. Cordella, A. and Bonina, C.M., 2012. A public value perspective for ICT enabled public sector reforms: A theoretical reflection. Government Information Quarterly, 29 (4), 512–520. Cordella, A. and Iannacci, F., 2010. Information systems in the public sector: The e- Government enactment framework. The Journal of Strategic Information Systems, 19 (1), 52–66. Cordella, A. and Willcocks, L., 2010. Outsourcing, Bureaucracy and Public Value: Reappraising the notion of the “contract state”. Government Information Quarterly, 27 (1), 82–88. - Page 215 - - Page 215 - Courpasson, D. and Reed, M., 2004. Introduction: Bureaucracy in the Age of Enterprise. Organization, 11 (1), 5–12. Coursey, D. and Norris, D.F., 2008. Models of E-Government: Are They Correct? An Empirical Assessment. Public Administration Review, 68 (3), 523–536. Empirical Assessment. Public Administration Review, 68 (3), 523–536. Crotty, M., 1998. The foundations of social research: meaning and perspective in the research process. London: Sage Crotty, M., 1998. The foundations of social research: meaning and perspective in the research process. London: Sage. Cukier, W., Ngwenyama, O., Bauer, R., and Middleton, C., 2009. A critical analysis of media discourse on information technology: preliminary results of a proposed method for critical discourse analysis. Information Systems Journal, 19 (2), 175–196. Culnan, M.J. and Armstrong, P.K., 1999. Information Privacy Concerns, Procedural Fairness, and Impersonal Trust: An Empirical Investigation. Organization Science, 10 (1), 104–115. Curthoys, N. and Crabtree, J., 2003. SmartGov – Renewing Electronic Government for Improved Service Delivery. London: The Works Foundation. SAGE Publications Ltd. Danziger, J.N., 2004. Innovation in Innovation?: The Technology Enactment Framework. Social Science Computer Review, 22 (1), 100–110. Danziger, J.N. and Andersen, K.V., 2002. The Impacts of Information Technology on Public Administration: An Analysis of Empirical Research from the “golden Age” of Transformation[1]. International Journal of Public Administration, 25 (5), 591–627. Danziger, J.N., Dutton, W., Kling, R., and Kraemer, K.L., 1982. Computers and politics: high technology in American local governments. New York: Columbia University Press. Davis, P. and West, K., 2008. What Do Public Values Mean for Public Action? Putting Public Values in Their Plural Place. The American Review of Public Administration, 0275074008328499. Diefenbach, T., 2009. New Public Management in Public Sector Organizations: The Dark Sides of Managerialistic “enlightenment”. Public Administration, 87 (4), 892– 909. Van Dijk, T.A., 2001. Multidisciplinary CDA: a plea for diversity. In: R. Wodak and M. Meyer, eds. Methods of critical discourse analysis. London: SAGE, 95–120. Van Dijk, T.A., 2003. Critical Discourse Analysis. In: D. Schiffrin, D. Tannen, and H.E. Hamilton, eds. The Handbook of Discourse Analysis. Blackwell Publishers Ltd, 352– 371. Van Dijk, T.A., 1997. Discourse as social interaction: Discourse studies—Volume II. Dunleavy, P. and Margetts, H., 2010. The second wave of digital era governance. In: 2010 Annual Meeting American Political Science Association. Washington DC, USA: APSA. Dunleavy, P., Margetts, H., Bastow, S., and Tinkler, J., 2006. Digital-era governance. IT Corporations, The State, and E-Government. Oxford: Oxford University Press. Dunsire, A., 1995. Administrative Theory in the 1980s - a Viewpoint. Public Administration, 73 (1), 17–40. Eisenhardt, K.M. and Graebner, M.E., 2007. Theory building from cases: opportunities and challenges. Academy of Management Journal, 50 (1), 25–32. ́ Esquivel, G. and Rodrı́guez-López, J.A., 2003. Technology, trade, and wage inequality in Mexico before and after NAFTA. Journal of Development Economics, 72 (2), 543– 565. Fairclough, N., 1992. Discourse and social change. Cambridge: Polity. Fairclough, N., 2000. New labour, new language? London: Routledge. Fairclough, N., 2003. Analysing discourse: textual analysis for social research. Abingdon, Oxon: Routledge. - Page 216 - Fairclough, N., 2005a. Peripheral Vision: Discourse Analysis in Organization Studies: The Case for Critical Realism. Organization Studies, 26 (6), 915–939. g z ( ) Fairclough, N., 2005b. Critical Discourse Analysis. Marges Linguistiques, 9, 76–94. Fairclough, N., 2010. Critical discourse analysis: the critical study of language. 2nd ed. Harlow: Longman. Fairclough, N., Jessop, B., and Sayer, A., 2002. Critical Realism and Semiosis. Journal of Critical Realism, 5 (1), 2–10. SAGE Publications Ltd. Fattore, G., Dubois, H., and Lapenta, A., 2012. Measuring New Public Management and Governance in Political Debate. Public Administration Review, 72 (2), 218–227. Feldman, M.S. and Khademian, A.M., 2001. Principles for public management practice: From dichotomies to interdependence. Governance, 14 (3), 339–362. Flyvbjerg, B., 2001. Making Social Science Matter: Why Social Inquiry Fails and How It Can Succeed Again. Cambridge University Press. Flyvbjerg, B., 2006. Five Misunderstandings About Case-Study Research. Qualitative Inquiry, 12 (2), 219 –245. Fountain, J., 2001. Building the virtual state: information technology and institutional change. Washington DC: Brookings Institution Press. Fountain, J.E., 2007. Bureaucratic Reform and E-Government in the United States: An Institutional Perspective. Friedland, R. and Alford, R., 1991. Bringing Society Back In: Symbols, Practices and Institutional Contradictions. In: The New Institutionalism in Organizational Analysis. University of Chicago Press, 232–263. Garson, G.D., 2003. Symposium on the Theory of Technology Enactment in Jane Fountain’s (2001)Building the Virtual State: An Introduction. Social Science Computer Review, 21 (4), 409–410. Du Gay, P., 2000. In Praise of Bureaucracy: Weber - Organization - Ethics (Organization Theory and Society Series). Sage Publications Ltd. Du Gay, P., 2003. The Tyranny of the Epochal: Change, Epochalism and Organizational Reform. Organization, 10 (4), 663–684. Du Gay, P., 2004. Against “Enterprise” (but not against “enterprise”, for that would make no sense). Organization, 11 (1), 37–57. ) g z ( ) Du Gay, P., ed., 2005. The values of bureaucracy. Oxford: Oxford University Press. Van Gestel, N., Denis, J.-L., and Ferlie, E., 2012. Special Issue—Call for Papers— Hybrids and Hybridisation in Public Management. Public Administration. Gibbert, M. and Ruigrok, W., 2010. The “What” and “How” of Case Study Rigor: Three Strategies Based on Published Work. Organizational Research Methods, 13 (4), 710–737. Gil-Garcia, J.R., Chengalur-Smith, I., and Duchessi, P., 2007. Collaborative e- Government: impediments and benefits of information-sharing projects in the public sector. European journal of information systems, 16 (2), 121–133. Gil-Garcia, J.R. and Pardo, T.A., 2005. E-government success factors: Mapping practical tools to theoretical foundations. Government Information Quarterly, 22 (2), 187–216. Gow, J.I. and Pardo, M. del C., 1999. Comparing different civil services: the advantages of a multi-level analysis for comparing Canada with Mexico. International Review of Administrative Sciences, 65 (4), 527–550. ( ) De Graaf, G., Nabatchi, T., and Van der Wal, Z., 2012. From Galaxy to Universe: A Multidisciplinary Meta-Analysis of Publications on Public Values from 1969 to 2012. f f De Graaf, G. SAGE Publications Ltd. and Van der Wal, Z., 2010. Managing Conflicting Public Values: Governing With Integrity and Effectiveness. The American Review of Public Administration, 40 (6), 623–630. - Page 217 - - Page 217 - Grant, D. and Hardy, C., 2004. Introduction: Struggles with Organizational Discourse. Organization Studies, 25 (1), 5 –13. Grant, D., Hardy, C., Oswick, C., and Putnam, L.L., eds., 2004. Introduction: Organizational discourse: Exploring the field. In: The SAGE handbook of organizational discourse. London: SAGE, 1–36. g z Gregor, S., 2006. The nature of theory in information systems. MIS Quarterly, 30 (3), 611–642. Grimsley, M. and Meehan, A., 2007. e-Government information systems: Evaluation- led design for public value and client trust. European journal of information systems, 16 (2), 134–148. Gruening, G., 2001. Origins and theoretical basis of New public Management. International Public Management Journal, 4 (1), 1–25. Hardy, C., 2004. Scaling up and Bearing Down in Discourse Analysis: Questions Regarding Textual Agencies and Their Context. Organization, 11 (3), 415 –425. Harris, M., 2006. Technology, innovation and post-bureaucracy: the case of the British Library. Journal of Organizational Change Management, 19 (1), 80–92. Harris, M., 2011. Network Governance and the Politics of Organizational Resistance in UK Health Care: The National Programme for Information Technology. In: S. Clegg, M. Harris, and H. Höpfl, eds. Managing modernity: beyond bureaucracy? Oxford: Oxford University Press. Hasselbladh, H. and Kallinikos, J., 2000. The Project of Rationalization: A Critique and Reappraisal of Neo-Institutionalism in Organization Studies. Organization Studies, 21 (4), 697–720. Hayes, N., 2008. Institutionalizing change in a high-technology optronics company: The role of information and communication technologies. Human Relations, 61 (2), 243 –269. Hayes, N. and Rajão, R., 2011. Competing institutional logics and sustainable development: the case of geographic information systems in Brazil’s Amazon region. Information Technology for Development, 17 (1), 4–23. g f f p Hayes, N. and Westrup, C., 2012. Context and the processes of ICT for development. Information and Organization, 22 (1), 23–36. Heeks, R., 2002. Reinventing Government in the Information Age - International practice in IT- enabled public sector reform. London: Routledge. Heeks, R. and Bailur, S., 2007. Analyzing e-government research: Perspective, philosophies, theories, methods, and practice. Government Information Quarterly, 24 (2), 243–265. Heeks, R. and Davies, A., 2002. Different approaches to information age reform. In: Reinventing Government in the Information Age - International practice in IT-enabled public sector reform. London: Routledge, 22–48. Heracleous, L. and Barrett, M., 2001. SAGE Publications Ltd. Organizational Change as Discourse: Communicative Actions and Deep Structures in the Context of Information Technology Implementation. The Academy of Management Journal, 44 (4), 755–778. Hevner, A., Chatterjee, S., and Carlsson, S.A., 2010. Design Science Research in Information systems: A Critical Realist Approach. In: A. Hevner and S. Chatterjee, eds. Design Research in Information Systems: Theory and Practice. Springer, 209–234. Hoggett, P., 2006. Conflict, ambivalence, and the contested purpose of public organizations. Human Relations, 59 (2), 175 –194. g Hood, C., 1989. Public-Administration and Public-Policy - Intellectual Challenges for the 1990s. Australian Journal of Public Administration, 48 (4), 346–358. - Page 218 - Hood, C., 1991. A Public Management for All Seasons? Public Administration, 69 (1), 3– 19. Hood, C. and Jackson, M., 1991. Administrative argument. Aldershot: Dartmouth. Introna, L., 2007. Maintaining the reversibility of foldings: Making the ethics (politics) of information technology visible. Ethics and Information Technology, 9 (1), 11–25. Introna, L., Hayes, N., and Petrakaki, D., 2009. The Working Out of Modernization in the Public Sector: The Case of an E-government Initiative in Greece. International Journal of Public Administration, 33 (1), 11–25. Introna, L.D. and Nissenbaum, H., 2000. Shaping The Web: Why The Politics Of Search Engines Matters. Information Society, 16 (3), 169–185. Joerges, B. and Czamiawska, B., 1998. The Question of Technology, or How Organizations Inscribe the World. Organization Studies, 19 (3), 363 –385. Johnson, P., Wood, G., Brewster, C., and Brookes, M., 2009. The Rise of Post- Bureaucracy. International Sociology, 24 (1), 37 –61. Johnstone, B., 2002. Discourse Analysis. Blackwell. Jorna, F. and Wagenaar, P., 2007. The “iron cage” strengthened? Discretion and digital discipline. Public Administration, 85 (1), 189–214. Kallinikos, J., 2004. The Social Foundations of the Bureaucratic Order. Organization, 11 (1), 13–36. Kallinikos, J., 2006a. The Institution of Bureaucracy: Administration, Pluralism, Democracy. Economy and Society, 35 (4), 611–627. Kallinikos, J., 2006b. The consequences of information: institutional implications of technological change. Northampton, MA: Edward Elgar. Kamto, M., 1997. Reaffirming Public-Service Values and Professionalism. International Review of Administrative Sciences, 63 (3), 295–308. Kaplan, B. and Duchon, D., 1988. Combining Qualitative and Quantitative Methods in Information Systems Research: A Case Study. MIS Quarterly, 12 (4), 571–586. Kaufmann, D., Kraay, A., and Mastruzzi, M., 2010. The Worldwide Governance Indicators: Methodology and Analytical Issues. Keenoy, T. and Oswick, C., 2004. Organizing Textscapes. Organization Studies, 25 (1), 135 –142. Kehoe, T.J., 1995. A Review of Mexico’s Trade Policy from 1982 to 1994. SAGE Publications Ltd. The World Economy, 18, 135–151. Kehoe, T.J. and Ruhl, K.J., 2010. Why Have Economic Reforms in Mexico Not Generated Growth? Journal of Economic Literature, 48 (4), 1005–27. Kernaghan, K., 2000. The Post-Bureaucratic Organization and Public Service Values. International Review of Administrative Sciences, 66 (1), 91 –104. Kirby, P., 2009. Neo-structuralism and reforming the Latin American state: lessons from the Irish case. Economy and Society, 38 (1), 132–153. Klecun, E., 2011. Weaving Discourses and Changing Organizations: The Role of ICT in the Transformation of Healthcare Towards Patient-Centered Model. ICIS 2011 Proceedings. Klein, H.K. and Myers, M.D., 1999. A Set of Principles for Conducting and Evaluating Interpretive Field Studies in Information Systems. MIS Quarterly, 23 (1), 67–93. Kraemer, K.L. and Dedrick, J., 1997. Computing and Public Organizations. Journal of Public Administration Research and Theory, 7 (1), 89–112. y ( ) Kraemer, K.L. and King, J.L., 1986. Computing and Public Organizations. Public Administration Review, 46, 488–496. Lamont, J. and Favor, C., 2008. Distributive Justice. The Stanford Encyclopedia of Philosophy. Lamont, J. and Favor, C., 2008. Distributive Justice. The Stanford Encyclopedia of Philosophy. Langley, A., 1999. Strategies for theorizing from process data. The Academy of Management Review, 24 (4), 691–710. - Page 219 - Langley, A., 2009. Studying processes in and around organisations. In: D.A. Buchanan and A. Bryman, eds. The Sage Handbook of Organizational Research Methods. London: SAGE, 409–429. Layne, K. and Lee, J.W., 2001. Developing fully functional E-government: A four stage model. Government Information Quarterly, 18 (2), 122–136. Lee, A.S. and Baskerville, R.L., 2003. Generalizing Generalizability in Information Systems Research. Information Systems Research, 14 (3), 221–243. Lee, C., Chang, K., and Berry, F., 2011. Testing the Development and Diffusion of E- Government and E-Democracy: A Global Perspective. Public Administration Review, 71 (3), 444–454. Leitch, S. and Palmer, I., 2010. Analysing Texts in Context: Current Practices and New Protocols for Critical Discourse Analysis in Organization Studies. Journal of Management Studies, 47 (6), 1194–1212. Levina, N. and Orlikowski, W., 2009. Understanding Shifting Power Relations within and Across Organizations: A Critical Genre Analysis. The Academy of Management Journal, 52 (4), 672–703. Lindblom, C.E., 1965. The intelligence of democracy: decision making through mutual adjustment. New York: Free Press. Lodge, M. and Gill, D., 2011. Toward a New Era of Administrative Reform? The Myth of Post-NPM in New Zealand. Governance-an International Journal of Policy and Administration, 24 (1), 141–166. Lopéz Ayllón, S. and Arellano-Gault, D., 2006. SAGE Publications Ltd. Estudio en materia de Transparencia de otros sujetos obligados por la Ley Federal de Transparencia y Acceso a la Información Pública Gubernamental. CIDE, Mexico City. j g p y p Información Pública Gubernamental. CIDE, Mexico City. Lustig, N., 2001. Life is not Easy: Mexico’s Quest for Stability and Growth. The Journal of Economic Perspectives, 15 (1), 85–106. Madon, S., Sahay, S., and Sudan, R., 2007. E-Government Policy and Health Information Systems Implementation in Andhra Pradesh, India: Need for Articulation of Linkages Between the Macro and the Micro. The Information Society, 23 (5), 327–345. Mareschal, P. and Rudin, J., 2011. E-Government Versus E-Business: A Comparison of Online Recruitment in the Public and Private Sectors. The American Review of Public Administration, 41 (4), 453–467. Margetts, H., 1999. Information Technology in Government Britain and America. London: Routledge. Markus, M.L. and Robey, D., 1988. Information technology and organizational change: causal structure in theory and research. Management Science, 34 (5), 583–598. Mayer-Schonberger, V. and Lazer, D., eds., 2007. Governance and information technology: from electronic government to information government. Cambridge, MA: MIT Press. McNulty, T. and Ferlie, E., 2004. Process Transformation: Limitations to Radical Organizational Change within Public Service Organizations. Organization Studies, 25 (8), 1389–1412. Michener, G., 2010. The Surrender of Secrecy: Explaining the Emergence of Strong Access to Information Laws in Latin America. University of Texas at Austin, Austin. Mingers, J., 2004. Real-izing information systems: critical realism as an underpinning philosophy for information systems. Information and Organization, 14 (2), 87–103. Mingers, J., Mutch, A., and Willcocks, L., 2011. Special Issue—Call for Papers—Critical Realism in Information Systems Research. MIS Quarterly. Mingers, J. and Walsham, G., 2010. Toward ethical information systems: the contribution of discourse ethics. MIS Quarterly, 34 (4), 833–854. - Page 220 - Moon, M.J., 2002. The Evolution of E-Government among Municipalities: Rhetoric or Reality? Public Administration Review, 62 (4), 424–433. y ( ) Moore, M., 1995. Creating Public Value: Strategic Management in Government. Cambridge, MA: Harvard University Press. Moreno-Brid, J.C., Carpizo, J.E.P., and Ros Bosch, J., 2009. Economic development and social policies in Mexico. Economy and Society, 38 (1), 154–176. Moreno-Brid, J.C. and Ros Bosch, J., 2009. Development and growth in the Mexican economy: a historical perspective. Moreno-Brid, J.C., Santamaria, J., and Rivas Valdivia, J.C., 2005. Industrialization and Economic Growth in Mexico after NAFTA: The Road Travelled. Development and Change, 36 (6), 1095–1119. Moynihan, D.P. and Pandey, S.K., 2005. SAGE Publications Ltd. Testing How Management Matters in an Era of Government by Performance Management. Journal of Public Administration Research and Theory, 15 (3), 421 –439. Mulgan, R., 2000. “Accountability”: An Ever-Expanding Concept? Public Administration, 78 (3), 555–573. Munir, K.A. and Phillips, N., 2005. The Birth of the “Kodak Moment”: Institutional Entrepreneurship and the Adoption of New Technologies. Organization Studies, 26 (11), 1665–1687. Mutch, A., 2010. Technology, Organization, and Structure—A Morphogenetic Approach. Organization Science, 21 (2), 507 –520. pp g z ( ) Myers, M.D., 2009. Qualitative Research in Business & Management. SAGE Publications Ltd. Myers, M.D., 2009. Qualitative Research in Business & Management. SAGE Publications Ltd. Norris, D.F., 2003. Building the virtual state... or not? A critical appraisal. Social Science Computer Review, 21 (4), 417–424. p ( ) Norris, D.F., 2010. E-Government 2020: Plus ca change, plus c’est la meme chose. Public Administration Review, Special Issue, S180–181. Norris, D.F. and Lloyd, B., 2006. The Scholarly Literature on E-Government: Characterizing a Nascent Field. International Journal of Electronic Government Research, 2 (4), 40–56. O’Flynn, J., 2007. From New Public Management to Public Value: Paradigmatic Change and Managerial Implications. Australian journal of public administration, 66 (3). O’Mahoney, J., 2011. Embracing essentialism: a realist critique of resistance to discursive power. Organization. OECD, 2003. The e-Government imperative. Paris: Organisation for Economic Co- operation and Development (OECD). p p ( ) OECD, 2005. E-government for better government. Paris, France: OECD. OECD, 2011. Mexican Review: Chapter on E-Government. Leveraging E-Government to Find New Approaches for Old Challenges. No. GOV/PGC/EGOV(2011)1. Organisation for Economic Co-operation and Development (OECD), Paris, France. Olsen, J.P., 2006. Maybe It Is Time to Rediscover Bureaucracy. Journal of Public Administration Research and Theory, 16 (1), 1–24. Orlikowski, W. and Iacono, S., 2001. Research Commentary: Desperately Seeking the “IT” in IT Research--A Call to Theorizing the IT Artifact. Information Systems Research, 12 (2), 121–134. Orlikowski, W.J., 1992. The Duality of Technology: Rethinking the Concept of Technology in Organizations. Organization Science, 3 (3), 398–427. Orlikowski, W.J., 2000. Using Technology and Constituting Structures: A Practice Lens for Studying Technology in Organizations. Organization Science, 11 (4), 404 –428. - Page 221 - Orlikowski, W.J. and Scott, S.V., 2008. Sociomateriality: Challenging the Separation of Technology, Work and Organization. Academy of Management Annals, 2 (1), 433– 474. Osborne, D. and Gaebler, T., 1992. Reinventing Government: How the Entrepreneurial Spirit is Transforming the Public Sector. Massachusetts: Addison-Wesley. f g y Palma, G., 2003. SAGE Publications Ltd. Trade liberalization in Mexico: its impact on growth, employment and wages. Working paper No. 2003/55. International Labour Office, Geneva. Pan, S.L., Tan, C.W., and Lim, E.T.K., 2006. Customer relationship management (CRM) in e-government: a relational perspective. Decision Support Systems, 42 (1), 237–250. Panizza, F. and Philip, G., 2005. Second Generation Reform in Latin America: Reforming the public sector in Uruguay and Mexico. Journal of Latin American Studies, 37 (04), 667–691. Pardo, M. del C., 2009. La modernización administrativa en México- 1940-2006. Mexico, DF: Colegio de Mexico. Parker, I., 1992. Discovering discourses, tackling texts. In: Discourse dynamics: critical analysis for social and individual psychology. New York: Routledge. Persson, A. and Goldkulh, G., 2010. Government Value Paradigms—Bureaucracy, New Public Management, and E-Government. Communications of the Association for Information Systems, 27 (1), 45–62. Pettigrew, A.M., 1990. Longitudinal Field Research on Change: Theory and Practice. Organization Science, 1 (3), 267–292. Phillip, G., Faust, J., and Thunert, M., 2011. Sustainable Governance Indicators 2011- Mexico Report. Bertelsmann Foundation. Phillip, G., Faust, J., and Thunert, M., 2011. Sustain Mexico Report. Bertelsmann Foundation. Phillips, N. and Di Domenico, M.L., 2009. Discourse Analysis in organizational research: methods and debates. In: D.A. Buchanan and A. Bryman, eds. The Sage Handbook of Organizational Research Methods. London: SAGE. Phillips, N. and Hardy, C., 2002. Discourse analysis: investigating processes of social construction. London: Sage Publications. Phillips, N., Lawrence, T.B., and Hardy, C., 2004. Discourse and Institutions. The Academy of Management Review, 29 (4), 635–652. Phillips, N. and Oswick, C., 2012. Organizational Discourse: Domains, Debates, and Directions. The Academy of Management Annals, 6 (1), 435–481. Phillips, N., Sewell, G., and Jaynes, S., 2008. Applying Critical Discourse Analysis in Strategic Management Research. Organizational Research Methods, 11 (4), 770–789. Pollitt, C., 2003. The Essential Public Manager. Open University Press. Pollitt, C. and Bouchaert, G., 2004. Public Management Reform: a comparative analysis. 2nd ed. Oxford: Oxford University Press. Pollitt, C. and Bouchaert, G., 2011. Public Management Reform: a comparative analysis. 3nd ed. Oxford: Oxford University Press. y Presidencia de la República, México, 2002. Agenda Presidencial de Buen Gobierno. Ragin, C.C. and Becker, H.S., eds., 1992. What Is a Case? Exploring the Foundations of Social Inquiry. Cambridge, UK: Cambridge University Press. Ramirez, M.D., 2003. Mexico under NAFTA: a critical assessment. The Quarterly Review of Economics and Finance, 43 (5), 863–892. Reay, T. and Hinings, C.R., 2009. Managing the Rivalry of Competing Institutional Logics. SAGE Publications Ltd. Organization Studies, 30 (6), 629 –652. Reddick, C.G., 2005. Citizen interaction with e-government: From the streets to servers? Government Information Quarterly, 22 (1), 38–57. - Page 222 - Reddick, C.G., Abdelsalam, H.M., and Elkadi, H., 2011. The influence of e-government on administrative discretion: the case of local governments in Egypt. Public Administration and Development, 31 (5), 390–407. p ( ) Reed, M., 2004. Getting real about organizational discourse. In: D. Grant, C. Hardy, C. Oswick, and L.L. Putnam, eds. The SAGE handbook of organizational discourse. London: SAGE, 413–420. Reed, M., 2005. Reflections on the “Realist Turn” in Organization and Management Studies. Journal of Management Studies, 42 (8), 1621–1644. Reed, M., 2009. Critical Realism: Philosophy, Method, or Philosophy in search of a Method? In: D.A. Buchanan and A. Bryman, eds. The Sage Handbook of Organizational Research Methods. London: SAGE, 430–448. Reed, M.I., 2001. Organization, Trust and Control: A Realist Analysis. Organization Studies, 22 (2), 201–228. Rhodes, R.A.W., 2007. Understanding Governance: Ten Years On. Organization Studies, 28 (8), 1243–1264. Rhodes and Wanna, J., 2009. Bringing the politics back in: Public Value in Westminster parliamentary government. Public Administration, 87 (2), 161–183. Robey, D., Ross, J., and Boudreau, M.-C., 2002. Learning to Implement Enterprise Systems: An Exploratory Study of the Dialectics of Change. Journal of Management Information Systems, 19 (1), 17–46. Rutgers, M.R., 2008. Sorting out public values? On the contingency of value classifications in public administration. Administrative Theory & Praxis, 30 (1), 92– 113. Rutgers, M.R. and Van der Meer, H., 2010. The Origins and Restriction of Efficiency in Public Administration. Administration & Society, 42 (7), 755 –779. Salskov-Iversen, D., Hansen, H.K., and Bislev, S., 2000. Governmentality, Globalization, and Local Practice: Transformations of a Hegemonic Discourse. Alternatives: Global, Local, Political, 25 (2), 183–222. ( ) Sanchez-Gonzalez, J., 2009. El cambio institucional en la reforma y modernización de la administración pública mexicana. Gestion y politica publica, XVIII (1), 67–105. Sarker, S. and Lee, A.S., 2003. Using a case study to test the role of three key social enablers in ERP implementation Information & Management 40 (8) 813–829 ( ) Sanchez-Gonzalez, J., 2009. El cambio institucional en la reforma y modernización de la administración pública mexicana. Gestion y politica publica, XVIII (1), 67–105. k d A d h f h k p y p p , ( ), Sarker, S. and Lee, A.S., 2003. SAGE Publications Ltd. Using a case study to test the role of three key social enablers in ERP implementation. Information & Management, 40 (8), 813–829. Sayer, A., 2000. Realism and social science. London: Sage. Schonhardt-Bailey, C., 2012. Looking at Congressional Committee Deliberations from Different Perspectives: Is the Added Effort Worth It? Presented at the NCRM Research Methods Festival 2012, 2nd - 5th July 2012, St. Catherine’s College, Oxford (Unpublished). Seale, C. and Silverman, D., 1997. Ensuring rigour in qualitative research. The European Journal of Public Health, 7 (4), 379 –384. Secretaría de Economía (SE), 2006. Memoria Documental Hacia la Administración Sin Papeles/SICEX (Memoirs “Towards a paperless Administration”). p ( p p ) Secretaría de Economía (SE), 2010. Ventanilla Única de Comercio Exterior Mexicana. Secretaría de Economía y Fomento Industrial (SECOFI), 1999. Sistema Integral de Comercio Exterior. Un caso de éxito en la administración de autorizaciones de comercio exterior (SICEX: A history of sucess in foreign trade regulations). SELA, 2011. Conclusions and Recommendations: III Latin American and Caribbean Regional Meeting on International Trade Single Windows. No. SP/III-ERLC- VUCE:CSPGRoc/CR-11. Latin American and Caribbean Economic System (SELA), Caracas, Venezuela. Smith, M.L., 2006. Overcoming theory-practice inconsistencies: Critical realism and information systems research. Information and Organization, 16 (3), 191–211. - Page 223 - - Page 223 - Smith, M.L., 2011. Limitations to building institutional trustworthiness through e- government: a comparative study of two e-services in Chile. Journal of Information Technology, 26 (1), 79–93. Smith, M.L., Noorman, M.E., and Martin, A.K., 2010. Automating the Public Sector and Organizing Accountabilities. Communications of the Association for Information Systems, 26 (1). Somers, M.R. and Block, F., 2005. From Poverty to Perversity: Ideas, Markets, and Institutions over 200 Years of Welfare Debate. American Sociological Review, 70 (2), 260–287. Srivastava, S.C. and Teo, T.S.H., 2008. The relationship between E-Government and National Competitiveness: The Moderating Influence of Environmental Factors. Communications of the AIS, 23. Srivastava, S.C. and Teo, T.S.H., 2010. E-Government, E-business, and National Economic Performance. Communications of the AIS, 26. Stoker, G., 2006. Public Value Management. The American Review of Public Administration, 36 (1), 41 –57. Stokes, J. and Clegg, S., 2002. Once Upon a Time in the Bureaucracy: Power and Public Sector Management. Organization, 9 (2), 225 –247. g g z ( ) Stone, B., 1995. Administrative Accountability in the “Westminster” Democracies: Towards a New Conceptual Framework. Governance, 8 (4), 505–526. Suchman, L., 2002. Located accountabilities in technology production. SAGE Publications Ltd. Scandinavian Journal of Information Systems, 14 (2). Tan, C.W. and Pan, S.L., 2003. Managing e-transformation in the public sector: an e- government study of the inland revenue authority of Singapore (IRAS). European Journal of Information Systems, 12 (4), 269–281. Telecom-CIDE, 2007. Evaluación del Proyecto “Hacia la Administración sin papeles” de la Secretaría de Economía. Reporte Final. Centro de Investigacion y Docencia Económicas-CIDE, Mexico City. Thomas, R. and Davies, A., 2005. Theorizing the Micro-politics of Resistance: New Public Management and Managerial Identities in the UK Public Services. Organization Studies, 26 (5), 683–706. Thomas, R., Sargent, L.D., and Hardy, C., 2011. Managing Organizational Change: Negotiating Meaning and Power-Resistance Relations. Organization Science, 22 (1), 22 –41. Thompson, M., 2004. Discourse, “Development” & the “Digital Divide”: ICT & the World Bank. Review of African Political Economy, 31 (99), 103–123. Thornton, P.H. and Ocasio, W., 1999. Institutional Logics and the Historical Contingency of Power in Organizations: Executive Succession in the Higher Education Publishing Industry, 1958‐1990. American Journal of Sociology, 105 (3), 801–843. Titcher, S., Meyer, M., Wodak, R., and Vetter, E., 2000. Methods of Text and Discourse Analysis. London: SAGE. Tolbert, C.J. and Mossberger, K., 2006. The Effects of E-Government on Trust and Confidence in Government. Public Administration Review, 66 (3), 354–369. Tornell, A., Westermann, F., and Martínez, L., 2003. Liberalization, Growth, and Financial Crises: Lessons from Mexico and the Developing World. Brookings Papers on Economic Activity, 2003 (2), 1–88. p y ( ) Townley, B., 1997. The Institutional Logic of Performance Appraisal. Organization Studies, 18 (2), 261 –285. Townley, B., 2002. The Role of Competing Rationalities in Institutional Change. The Academy of Management Journal, 45 (1), 163–179. - Page 224 - Townley, B., Cooper, D., and Oakes, L., 2003. Performance Measures and the Rationalization of Organizations. Organization Studies, 24 (7), 1045–1071. g United Nations, 2008. The United Nations e-Government Survey 2008: From e- Government to Connected Governance. United Nations, New York. United Nations, 2010. The 2010 United Nations e-Government Survey: Leveraging e- government at a time of financial and economic crisis. United Nations, New York. Vaara, E., 2010. Taking the linguistic turn seriously: Strategy as A multifaceted and interdiscursive phenomenon. Advances in Strategic Management, 27, 29–50. Vaara, E. and Monin, P., 2010. A Recursive Perspective on Discursive Legitimation and Organizational Action in Mergers and Acquisitions. Organization Science, 21 (1), 3 –22. Vaara, E., Sorsa, V., and Pälli, P., 2010. SAGE Publications Ltd. On the force potential of strategy texts: a critical discourse analysis of a strategic plan and its power effects in a city organization. Organization, 17 (6), 685–702. Vaara, E. and Tienari, J., 2002. Justification, Legitimization and Naturalization of Mergers and Acquisitions: A Critical Discourse Analysis of Media Texts. Organization, 9 (2), 275–304. g z ( ) Vaara, E. and Tienari, J., 2008. A discursive perspective on legitimation strategies in multinational corporations. Academy of Management Review, 33 (4), 985–993. Vaara, E., Tienari, J., and Laurila, J., 2006. Pulp and Paper Fiction: On the Discursive Legitimation of Global Industrial Restructuring. Organization Studies, 27 (6), 789 –813. Volkoff, O., Strong, D.M., and Elmes, M.B., 2007. Technological Embeddedness and Organizational Change. Organization Science, 18 (5), 832 –848. Van der Wal, Z., De Graaf, G., and Lasthuizen, K., 2008. What’s valued most? Similarities and differences between the organizational values of the public and private sector. Public Administration, 86 (2), 465–482. p ( ) Van der Wal, Z. and Huberts, L., 2008. Value solidity in government and business - Results of an empirical study on public and private sector organizational values. American Review of Public Administration, 38 (3), 264–285. f ( ) Walker, R. and Bozeman, B., 2011. Publicness and Organizational Performance. Journal of Public Administration Research and Theory, 21 (suppl 3), i279–i281. Walsham, G., 1995. Interpretive case studies in IS research: nature and method. European Journal of Information Systems, 4 (2), 74–81. Walsham, G., 2001. Making a World of Difference: IT in a Global Context. John Wiley & Sons. Walsham, G., 2006. Doing interpretive research. European Journal of Information Systems, 15 (3), 320–330. Welch, E.W. and Pandey, S.K., 2007. E-government and bureaucracy: Toward a better understanding of intranet implementation and its effect on red tape. Journal of Public Administration Research and Theory, 17 (3), 379–404. Wenger, J.B. and Wilkins, V.M., 2009. At the Discretion of Rogue Agents: How Automation Improves Women’s Outcomes in Unemployment Insurance. Journal of Public Administration Research and Theory, 19 (2), 313 –333. West, D.M., 2005a. Digital Government: Technology and Public Sector Performance. Princeton University Press. y West, D.M., 2005b. Global eGovernment, 2005. Brown University, Providence, RI. W D M 200 Gl b l G 200 B U i i P id RI West, D.M., 2005b. Global eGovernment, 2005. Brown University, Providence, RI. West, D.M., 2007. Global eGovernment, 2007. Brown University, Providence, RI. West, D.M., 2005b. Global eGovernment, 2005. SAGE Publications Ltd. Brown University, Providence, RI. West, D.M., 2007. Global eGovernment, 2007. Brown University, Providence, RI. West, D.M., 2007. Global eGovernment, 2007. Brown University, Providence, R - Page 225 - West, K. and Davis, P., 2011. What is the public value of government action? Towards a (new) pragmatic approach to values questions in public endeavours. Public Administration, 89 (2), 226–241. Willems, T. and Van Dooren, W., 2011. Lost in diffusion? How collaborative arrangements lead to an accountability paradox. International Review of Administrative Sciences, 77 (3), 505–530. ( ) Williams, I. and Shearer, H., 2011. Appraising Public Value: Past, Present and Futures. Public Administration, 89 (4), 1367–1384. ( ) Willmott, H., 2010. Creating “value” beyond the point of production: branding, financialization and market capitalization. Organization, 17 (5), 517–542. Willmott, H., 2011. Back to the Future: What does Studying Bureaucracy Tell Us? In: S. Clegg, M. Harris, and H. Höpfl, eds. Managing modernity: beyond bureaucracy? Oxford: Oxford University Press. Wodak, R., 2001. What CDA is about - a summary of its history, important concepts and its developments. In: Methods of critical discourse analysis. London: SAGE. Wodak, R. and Meyer, M., 2009. Critical Discourse Analysis: History, Agenda, Theory, and Methodology. In: R. Wodak and M. Meyer, eds. Methods for Critical Discourse Analysis. London: SAGE, 1–33. Wong, W. and Welch, E., 2004. Does E-Government Promote Accountability? A Comparative Analysis of Website Openness and Government Accountability. Governance, 17 (2), 275–297. Wright, R.P., Paroutis, S.E., and Blettner, D.P., 2012. How Useful are the Strategic Tools We Teach in Business Schools? Journal of Management Studies, no–no. WTO, 2008. Trade Policy Review-Report by the Secretariat-MEXICO. No. WT/TPR/S/195. World Trade Organization (WTO), Trade Policy Review Body, Geneva. Yang, K., 2009. Examining Perceived Honest Performance Reporting by Public Organizations: Bureaucratic Politics and Organizational Practice. Journal of Public Administration Research and Theory, 19 (1), 81 –105. Yildiz, M., 2007. E-government research: Reviewing the literature, limitations, and ways forward. Government Information Quarterly, 24 (3), 646–665. Yin, R.K., 2003. Case study research: design and methods. Third Edition. Thousand Oaks, California: Sage Publications. Zachariadis, M. and Scott, S., 2007. Diversity in IS research: Developing a Mixed Methodology Approach to Understanding the Business Value of Payment System Innovations in Financial Services. In: ICIS 2007 Proceedings. Zammuto, R.F., Griffith, T.L., Majchrzak, A., Dougherty, D.J., and Faraj, S., 2007. Information Technology and the Changing Fabric of Organization. Organization Science, 18 (5), 749–762. Zuboff, S., 1988. Zuboff, S., 1988. In the age of the smart machine: the future of work and power. Basic Books. SAGE Publications Ltd. In the age of the smart machine: the future of work and power. Basic Books. - Page 226 - - Page 226 - Appendix I: Detailed sources and significance of data collection This appendix explains the different collection methods that I have used, and their significance for the research inquiry. As such, it complements in detail the data collection section that I have included in Chapter Four. The next sections cover in detail the following categories: in-depth interviews, direct observation, and extensive document and archival collection. Let me just remind the reader that my fieldwork included the following: an initial 8 month-exploratory study that I conducted from August 2006 to March 2007. This initial study was decoupled from the current research and it was part of a project I led as research associated in Telecom-CIDE. With permission from the Mexican government, I do draw on these data as it has been useful for the research. Data collection that was relevant for my current PhD project took place during two fieldwork trips: one in spring of 2009 (March to June), and in the spring of 2010 (May and June). Follow up interviews, index of news and document collection were conducted since then. 126 Following common ethical guidelines on social science research, interviewees were provided with information and the purpose of the study the author was conducting. In depth interviews and fieldwork notes As indicated in Chapter Four, I conducted 42 in-depth interviews with 35 single individuals, most of them face to face, with an average time of one hour to one hour and half per interview. In all cases, interviewees were informed about the purposes of the project in advanced, either via email or orally at the moment of the interview (i.e. Walsham 2006, p.323).126 Interviewees were given the option of keeping the interviews confidential and/or anonymous. All interviews were conducted in Spanish, recorded with the participants consent, and transcribed maintaining its original language. Materials were only translated when necessary for quotation. The process of interviewing during fieldwork was largely an on-going one. Looking at interview materials and relevant literature at the same time was useful for adapting questions and follow up interviews. As well known for qualitative researchers, the time spent in the field was highly consuming and intense. It even included dealing with unexpected events, such as carrying out interviews wearing masks at the time the swine flu surprised Mexico in 2009. A daily summary of the main reflections emerging from - Page 227 - - Page 227 - the empirical work were kept in the fieldwork diary and then used when recapping the case study. Given the objectives and focus of the research (eliciting discourses on public values and technology on trade regulations in Mexico), the interviews focused on five key categories of informants: (i) Mexican public managers in charge of international commerce procedures and regulations at the federal (national) level, more precisely, the Vice-Ministry of Economy and the team at the General Direction of Foreign Commerce; (ii) public servants involved in the design and implementation of the Single Window at SAT/Customs; (iii) other public servants involved with the general implementation of the digital agenda in Mexico (E-government Unit at the Ministry of Public Function); (iv) external stakeholders (OECD team, IT providers); and (v) academics with expertise in e-government and technological platforms for trade. In depth interviews and fieldwork notes Ministry of Economy Vice-Ministry of Trade and Industry DGCE* General Office of Informatics* Sub Offices of Informatics* General Office of International Trade Information* Offices of Norms* General Coordinator of the Federal Agencies* 32 Federal Agencies Other sub-agencies General Direction of Informatics (DGI)* COCEX: International Trade Commission (SHCP, SAGARPA, SEMARNAT, SALUD, SRE, SAT, CFC)* Ministry of Finance and Public Credit Customs Administration* Ministry of Public Function (SFP) E-Government Unit* SAGARPA* SEMARNAT SALUD SEDENA SENER SEP INAH INBAL International Trade Associations CAAAREM* External providers INFOTEC Other IT consultants Table A.1: Participants with direct responsibility in the Single Window project. Note: * Indicates areas/organisations where I have conducted interviews. Ministry of Economy Vice-Ministry of Trade and Industry* DGCE* General Office of Informatics* Sub Offices of Informatics* General Office of International Trade Information* Offices of Norms* General Coordinator of the Federal Agencies* 32 Federal Agencies Other sub-agencies General Direction of Informatics (DGI)* COCEX: International Trade Commission (SHCP, SAGARPA, SEMARNAT, SALUD, SRE, SAT, CFC)* Ministry of Finance and Public Credit Customs Administration* Ministry of Public Function (SFP) E-Government Unit* SAGARPA* SEMARNAT SALUD SEDENA SENER SEP INAH INBAL International Trade Associations CAAAREM* External providers INFOTEC Other IT consultants Table A.1: Participants with direct responsibility in the Single Window project. Note: * Indicates areas/organisations where I have conducted interviews. Table A.1: Participants with direct responsibility in the Single Window project. Note: * Indicates areas/organisations where I have conducted interviews. - Page 228 - - Page 228 - The interviews offered key insights in a number of areas: (a) main aims, drivers, and motivations to use networked technologies in government; (b) role and specifications of the main actors involved; (c) key episodes that happened during the different phases of the project; (d) general successes and challenges; (e) main interpretations of value derived from technology according to the actors. Table A.1 summarizes the main actors involved in the project and the areas and organisations covered by the interviews. Appendix II offers a comprehensive list of the interviews carried out as part of the research. Direct Observation and meetings During fieldwork I spent most of the time working from an office within the Ministry of Economy. The insights that I gained wile being there were particularly useful for constructing the context of the case, making references to culture and also to make sense of the organisational capabilities and features that can be attributable to the Mexican bureaucracy. Informal interviews were also held with a number of public officials during that time. I also participated in three key meetings during spring 2009. These meetings entailed an in-between form of direct and participant observation. The latter, because while on the Ministry, I was invited to intervene with comments and views on different issues. Although these events occurred only in few occasions, I decided to group them under the direct observation category for simplicity. 127 The document presents a general body of 115 pages and more than 300 additional pages of annexes that expand the main issues covered in the report. Documentary and archive data Texts are particularly important for my research design and data analysis. Hence, additional data were gathered from official annual reports, official documents and speeches, government mission statements, and news from local newspapers and the internet. Documentary and archive data The following list presents the main internal sources that I count on: - History of SICEX: A 35 pages, internal document from the SE that describes the origins, main drivers and component of the platform that was launched in 1996 and won the national public administration prize in 1999; - Page 229 - - Memoirs of ‘Towards a paperless administration 2000-2006’: an internal, 450127 pages-long document that covers the progress and evolution of SICEX and related ICT infrastructure for foreign trade procedures, its main drivers, logics and actions taken in relation to the project; - The presidential Decree that establishes the creation of the Mexican single widow for foreign trade (January 2011) (5 pages long); - The Presidential Decree that gives facilities to foreign trade operation (31 March 2008); - The terms of reference of the open public tender sponsored by Customs (September 2010): a 81 pages long text that offers in detail the terms and conditions for the acquisition of the services to implement the single window; it includes more than 150 pages of technical annexes; - Mexico’s National Development Plan 1995-2000 - Mexico’s National Development Plan 1995-2000 - Mexico’s National Development Plan 2001-2006 - Mexico’s National Development Plan 2001-2006 - Mexico’s National Development Plan 2007-2012 - Mexico’s National Development Plan 2007-2012 - A report from an independent citizen witness procedure (“Testigo Ciudadano”), that evaluates the acquisition process for the single window; - 6 general texts downloaded from the single window website in January 2012 that explain what the single window is, what the main objectives and benefits are, and how it is operated https://www.ventanillaunica.gob.mx/vucem/SobreVU/SobrelaVU/index.htm; - 13 internal power-point presentations in electronic format that supports the official strategy and objectives of setting up the unique digital window for foreign trade; - 7 internal power point presentations of 50 to 80 slides each and 5 excel files containing the results and analysis of the reengineering exercise of trade procedures that a private consultant conducted for the Ministry of Economy in 2010; - Page 230 - - 15 official presentations and speeches from the leaders of the one stop-shop project that emphasise goals, benefits and plans (mainly from ME and Customs, from 2010 and 2011); - 75 informative sheets downloaded from November 2011 until February 2012 from the digital window website, informing about several themes: changes in law and regulations, user manuals, examples of digitisation, guidelines to use the content of the website, and process flows of some procedures (http://bit.ly/ybBQgR); - 5 workshop presentations conducted in the Latin American and Caribbean Regional Meeting on International Trade single Windows, organized by the Latin American and Caribbean Economic System (SELA) during 2010 and 2011 (3 from ME, 1 from Customs, and 1 from the private sector). 128 These reports include the review of the different experiences of international trade single windows in Latin America and represent a useful material for comparison purposes. p p Latin America and represent a useful material for comparison purposes. Documentary and archive data - 95 news articles downloaded from local newspapers, covering the period from January 2009 to July 2012 (see Appendix III for a list of sample articles). Further documents, data and indicators that help contextualizing the initiative include: - Ministry of the Economy sectoral plans, competitiveness agenda, and other documents that outlines the Ministry value mission and goals in general and in foreign trade; - The final reports conducted in each of the 3 Latin American and Caribbean Regional Meetings on International Trade Single Windows, organized by the Latin American and Caribbean Economic System (SELA) during 2010 and 2011;128 - The World Trade Organisation Trade Policy Review of Mexico (2009): report that outlines the country’s main policy, progress and issues of Mexico’s regulation of foreign trade; - The OECD E-Government Survey (2005): designed and conducted between 2002 and 2004, the survey covers federal public servants’ attitudes on different - Page 231 - e-government dimensions, such as objectives and main drivers, perceptions about collaboration, and role of public officials in embracing change; - The OECD chapter on E-government and modernisation reforms in Mexico (2011): study conducted during 2010 and published in 2011 on the advances of the digital government agenda in Mexico. The report also mentions the single window studied in this research and further updated data from a survey on patterns of collaboration in e-government projects in Mexico. (I have also interviewed the people that participated in this project); - Mexico E-Government Unit: survey of ICT adoption in federal agencies (2006- 2010), and within the Ministry of Economy; - Mexico E-Government Unit: survey of ICT adoption in federal agencies (2006- 2010), and within the Ministry of Economy; Whilst it has not been possible to collect first hand data of Mexican public opinion, I employ other valuable sources as a general context to institutional and political dimensions in the country. News articles constitute an important source of proxy for the public’s reaction to the single window design and implementation. In this respect, I followed to strategies. First, I read a diary newsletter that the Ministry compiled summarizing the headings and relevant info of the main local newspapers. I have done so from June 2010 to July 2011. Second, in October 2011 I scanned the main newspapers in Mexico, following a simple Google search. I collected about 15 news articles with references to the project and customs modernisation. Documentary and archive data From then on, I set up a Google alert that would scan the news. I usually clicked in all of them and only archive those referring to the Mexican case. I enclose in a separate appendix the complete list of articles. I also draw from further secondary data from surveys that compile public perceptions on democracy and governance indicators in Mexico. Particularly, the following: - The World Bank Governance Indicators, version 2010: The dataset reports on six broad dimensions of governance for over 200 countries over the period 1996- 2010; it includes six categories: Voice and Accountability, Political Stability and Absence of Violence, Government Effectiveness, Regulatory Quality, Rule of Law, Control of Corruption. - Page 232 - - Page 232 - - Mexico at the Latin American Public Opinion Project (LAPOP) -University of Vanderbilt: survey that compiles public perceptions on democracy and political values in regularly conducts on the country. Finally, I relied on the work of local academics to address the main trends of the administrative and political modernisation in Mexico from mid 1980s to the present (i.e. Arellano-Gault 2000, 2010, Guerrero and Arellano-Gault 2003, Pardo 2009, Moreno- Brid et al 2009, Moreno-Brid and Ros 2009, Cejudo 2008). - Page 233 - - Page 233 - Appendix II: List of Interviews Below is the full list of interviews carried out as part of this research, including when, with whom and for how long the interviews were conducted. This appendix complements the first part of Appendix I, and the information contained in Chapter Four. Names are not included to protect–as much as possible–the anonymity of those interviewed. The numbers in the first column are the keys used in the empirical chapters (notably Chapter Five and Six). The third column is included as in some cases there was more than one person in the interview, but that was not counted as a separate one. - Page 235 - Appendix III: Data analysis Appendix III: Data analysis Appendix III: Data analysis Appendix III: Data analysis A screen shot of my coding in Atlas (an excerpt of the Presidential Decree of 2011) A screen shot of my coding in Atlas (an excerpt of the Presidential Decree of 2011) A screen shot of my coding in Atlas (an excerpt of the Presidential Decree of 2011) Appendix II: List of Interviews Year 2009 # Date Position # of people Length of Interview (in minutes) 1 26-Mar-09 Directora Industria Automotriz, Ministry of Economy 1 90 2 30-Mar-09 Director de Industria Metalmecánica, Ministry of Economy 1 30 3 07-Apr-09 Director General de Comercio Exterior (DGCE), Ministry of Economy 1 60 4 14-4-09 to 21-04-09 (various interviews) Director de Programas de Fomento, Ministry of Economy 1 180 5 24-Apr-09 Director General de Industrias Pesadas y de Alta Tecnologia; Director de Industria Metalmecánica; Directora Industria Automotriz, Ministry of Economy 3 50 6 07-May-09 Director Industria Manufacturera Exportacion, Ministry of Economy 1 45 7 07-May-09 Directora de Información de Comercio Exterior (DG CE) 1 60 8 07-May-09 Secretario Tecnico de la COCEX, Ministry of Economy 1 67 9 12-May-09 Direccion General de Informática, Ministry of Economy 2 47 10 12-May-09 Subdirectora-Coordinación General de Delegaciones Federales, Ministry of Economy 2 40 - Page 234 - 11 12-May-09 Directora de Desarrollo Informático- Dirección General de Informática, Ministry of Economy 1 20 12 13-May-09 Director General de Comercio Exterior, Ministry of Economy 1 47 13 14-May-09 Administradora de Operación Aduanera 7, Customs 1 65 14 14-May-09 Director General de Economia Digital, Ministry of Economy 1 45 15 15-May-09 Vicepresidente, Instituto Nacional de Estadística y Geografía (INEGI) (Former Deputy Minister of Trade and Commerce in the Ministry of Economy) 1 55 16 18-May-09 Former Director General de Comercio Exterior, Ministry of Economy 1 104 17 20-May-09 Former Administrador de operación aduanera 7, Customs 1 32 18 20-May-09 Asesor Informático, Coordinacion de Asesores, Ministry of Economy 1 36 19 21-May-09 Subsecretaria de Industria y Comercio, Ministry of Economy 1 20 20 01-May-09 Subdirector de Programa de Fomento, Ministry of Economy 2 90 Year 2010 # Date Position # of people Length of Interview (in minutes) 21 12-May-10 Director General de Comercio Exterior (DGCE), Ministry of Economy 1 45 22 14-May-10 Director General de Comercio Exterior (DGCE), Ministry of Economy 1 64 23 25-May-10 Director General de Comercio Exterior (DGCE), Ministry of Economy 1 37 24 25-May-10 Directora de Información de Comercio Exterior, Ministry of Economy 1 40 25 25-May-10 Director de Programas de Fomento, Ministry of Economy 1 50 26 26-May-10 Subsecretaria de Industria y Comercio, Ministry of Economy 1 38 - Page 235 - - Page 235 - 27 26-May-10 Director Unidad de Gobierno Digital, Ministry of Public Function 1 42 28 26-May-10 Director de Promoción e Integración de Gobierno Digital, Ministry of Public Function 1 45 29 27-May-10 Subdirector de Negociaciones Multilaterales y Regionales Ministry of Economy 1 45 Year 2011 # Date Position # of people Length of Interview (in minutes) 30 8-Apr-11 Consultant in red tape and regulatory reform, Expert in Mexico, OECD 1 50 31 31-May-11 Director E-governmentUnit, OECD 1 30 32 4-May-11 Director Unidad de Gobierno Digital, Ministry of Public Function 1 45 33 6-June-11 Consejero comercial, ProMéxico, Mexican Trade Comission at the United Kingdom 1 50 34 29-Sep-11 Professor FLACSO, Argentina, expert in e-government 1 90 35 30-Sep-11 E-Government Programme Director, Universidad de San Andres 2 45 36 26-Oct-11 Coordinador de Asesores, Direccion General de Mineria, Ministry of Economy 1 60 37 07-Nov-11 Director General de Comercio Exterior, Ministry of the Economy 1 87 38 05-Dec-11 Directora de Vinculación Institucional, SAGARPA 1 120 Year 2012 # Date Position # of people Length of Interview (in minutes) 39 21-Mar-12 Mexican Ambassador to the United Kingdom 1 47 40 26-Mar-12 Director de Programas de Fomento, Ministry of Economy 1 90 41 02-Apr-12 International trader, Mexican private company 1 35 - Page 236 - 42 12-Jul-12 Director General de Comercio Exterior, Ministry of Economy 1 90 42 12-Jul-12 Director General de Comercio Exterior, Ministry of Economy 1 90 - Page 237 - - Page 237 - Appendix III: Sample of news articles Source Title Date 1 Uniradio Ventanilla Única de Comercio Exterior todavía está en proceso de adecuación 16/02/2012 2 Uniradio Aseguran que se reduce burocracia con VUCEM 14/02/2012 3 El Universal Dificultades para el CE 14/06/2011 4 El Universal SAT va tras empresas de comercio exterior 06/04/2011 5 El Universal Recibe SE propuestas en comercio exterior 04/07/2011 6 El Universal Aduanas investiga actos de corrupción 09/02/2010 7 El Universal Comercio representa 61% del PIB 26/07/2011 8 El Universal Normatividad excesiva limita comercio de México 12/01/2012 9 El Universal La regulación del comercio exterior, en vía de rediseño 11/09/2008 10 El Universal Ingreso honesto cierra la puerta a ingreso ilegal 16/01/2012 11 El Universal Aporta comercio exterior 23% de impuestos al país: Aduanas 16/10/2008 12 Noreste Tendrán los exportadores ventanilla única obligatoria 02/03/2012 13 El Universal Cuesta millones proteger aduanas del crimen 16/12/2011 14 El Universal Apoyarán a 150 Pymes para tener presencia en el exterior 12/02/2012 15 El Universal Con tan sólo cuatro pasos se agilizan trámites de comercio 20/01/2012 16 CAAAREM Gaceta AAAG: El Tema del Mes Avances de la Ventanilla Única de Comercio Exterior Mexicano (VUCEM) 01/06/2012 17 El Economista.mx Apresuran Ventanilla Única 02/04/2012 18 El Universal Promueven Comercio Exterior entra las Pymes 01/01/2006 19 El Universal Calderón envía Ley de Firma Electrónica al Senado 09/12/2010 20 CNN Preocupa el proyecto VUCEM 14/10/2010 21 La Crónica Aplazan entrada de ventanilla única CAAAREM 24/02/2012 22 El Diario Explicara SAT aquí su nuevo esquema de certificación 12/04/2012 23 El Economista.mx Gobierno Prefiere las "rudas" de Panasonic 03/04/2012 24 El Mañana Buen inicio en foro de Comce 15/03/2012 25 El Mañana Destacan beneficios de la Ventanilla Única 26/05/2012 26 La Nación El porqué del trabajo interdisciplinario 15/05/2012 27 El Universal Exigencias de exportadores 18/05/2012 28 El Universal Se desacelera el comercio exterior, dice el Comce 17/05/2012 29 El Universal PRI no apoyara el cierre de secretarias 12/11/2009 30 El Economista.mx Ven ventanilla única regional 28/05/2012 31 El Economista.mx Ventanilla única de comercio va sin contratiempos: SE 05/06/2012 32 El Siglo de Torreón Frenan a Pymes altos costos de ventanilla única 05/03/2012 33 Grupo Formula Con ventanilla única, se reduce tiempo para la gestión de trámites en 90%: SAT 22/03/2012 34 IDC Online Primeros resultados de la ventanilla única 04/06/2012 - Page 238 - 35 Latin America Data Base President Felipe Calderon Replaces interior Secretary Fernando Gomez Mont Appoints New Economy secretary 21/07/2010 36 Latin America Data Base World customs organization recognizes Mexico for efforts to combat counterfeit medicines 30/06/2010 37 Latin America Data Base President Felipe Calderon Appoints Agustin Carstens New Central Bank Head; Two Cabinet Changes Announced 16/12/2009 38 Latin America Data Base President Felipe Calderon Overhauls Cabinet 09/09/2009 39 Latin America Data Base Nobel Economist Riles President Felipe Calderon's Administration With Criticism Of Economic Policies 02/12/2009 40 El Universal La regulación del comercio exterior, en vía de rediseño 11/09/2008 41 Council on Foreign Relations Latin Americas Moment: North American Competitiveness 18/05/2012 42 ManzanilloXport Ahorrara 9 Tramites la Ventanilla Única 15/03/2012 43 MexicoXport Inicia el Viernes ventanilla única 29/05/2012 44 Linkedin Problemas en importaciones y exportaciones en Mexico 29/03/2012 45 El Golfo Daria ventanilla ventaja al País 09/04/2012 46 El Siglo de Torreón Reclaman por ventanilla única 16/03/2012 47 Reforma Aplazan inicio a junio de la ventanilla única 24/02/2012 48 El Universal Normatividad excesiva limita comercio de México 12/01/2010 49 El Economista.mx Se "cae" el sistema de las aduanas 26/01/2010 50 El Siglo de Torreón Da STA luz verde a la ventanilla única 29/03/2012 51 El Sol de Tijuana Ventanilla única del SAT, incapaz de manejar nuestros volumen, dicen 09/05/2012 52 T21.com.mx Opcional el uso de la ventanilla única hasta junio 29/02/2012 53 T21.com.mx Sin marcha atrás uso obligatorio de ventanilla única y COVE 06/05/2012 54 Tiempo Logístico Capacidad informática, vital para la ventanilla única: SDI Cointer 09/03/2012 55 La Nación Una herramienta con tinte paraarancelario 13/03/2012 56 Uniradio Continua rezago en aplicación de factura electrónica COVE 08/05/2012 57 El Universal Diversificación e inclusión de PyMES, logros en comercio exterior 28/08/2007 58 Uniradio Ventanilla única de comercio exterior todavía está en proceso de adecuación 16/02/2012 59 MexicoXport Ventanilla única implica mas tramites 30/05/2012 60 El Economista.mx Volatilidad y corrupción atrasan crecimiento: PwC 25/04/2012 61 Mexico Exporta - Secretaria de Economía A la Vanguardia con la Ventanilla única de comercio exterior 01/09/2011 62 Yucatán Noticias Merida sede de la sesión del consejo directivo nacional de la confederación de asociaciones de agencias aduanales 31/05/2012 - Page 239 - Journal Articles Cordella, A. and Bonina, C., 2012, A Public Value Perspective for ICT Enabled Public Sector Reforms: A theoretical reflection, Government Information Quarterly, Volume 29, Issue 4, Pages 512–520. Book Chapters Martin, A. and Bonina, C., Open Government and Citizen Identities: Promise, Peril, and Policy, in Open Development: Technological, Organizational, and Social Innovation in International Development, edited by M.L. Smith and K. Reilly, MIT Press, Forthcoming. Bonina, C., 2012, Building ICT Critical Infrastructures for Trade Regulations: Implications from a National Initiative in Mexico, in ICT Critical Infrastructures and Society, M. Hercheui, D. Whitehouse, W. McIver, and J. Phahlamohlaka (Eds.), International Federation for Information Processing (IFIP), Springer, pp. 22-33. Mariscal, J. and Bonina, C., 2008, Mobile Phone Usage in Mexico: Policy and Popular Dimensions, in Handbook of Mobile Communication Studies, James Katz (Ed.), MIT Press, Cambridge: MA. Mariscal, J., Bonina, C. and Luna, J., 2007, New Market Scenarios in Latin America, in Digital Poverty: Latin American and Caribbean Perspectives, Mariscal, J. and Galperin, H. (Eds.), Practical Action Publishing/IDRC, Canada. - Page 240 - - Page 240 - Conference Papers Bonina, C., 2012, Technologies are more than tools: Searching for public values within e-government and public sector reforms, 28th European Group of Organisation Studies (EGOS) Colloquium Proceedings, Sub-theme 6:Organizing the Public Sector, Helsinki, Finland, 5-8 July. Bonina, C., 2011, The Internet in Government: on bureaucracy, public value and the reorganisation of the state, The Sixth Organization Studies Summer Workshop, Abbaye des Vaux de Cernay, France, May 25–28. Bonina, C. and Cordella, A., 2010, The Internet and Public Bureaucracies: towards balancing competing values, Proceedings of Internet, Politics, and Policy 2010: An Impact Assessment, Oxford Internet Institute, University of Oxford, 16-17 September. Bonina, C. and Cordella, A., 2010, Public sector and ICT: beyond the private sector rationale, 3rd Latin and European Meeting on Organization Studies (LAEMOS) Proceedings, Sub-Theme 7, Buenos Aires, April 7-10. Martin, A., Bonina, C. and S. Goldchluk, 2010, Constructing and disrupting transparencies: ICTs and identity management in Latin America, 3rd Latin and European Meeting on Organization StudiesProceedings (LAEMOS), Sub-theme 4, Buenos Aires, Argentina, April 7-10. Bonina, C. and Cordella, A., 2009, Public sector reforms and the notion of ‘public value’: implications for e-government deployment, Proceedings of the 15th Americas Conference on Information Systems (AMCIS 2009), San Francisco, August 6-9, 2009. - Page 241 - Bonina, C. and Cordella, A., 2008, The new public management, e-government and the notion of ‘public value’: lessons from Mexico (joint with A. Cordella), Proceedings Annual Workshop of the AIS Special Interest Group for ICT in Global Development (GlobDev), Paper 11, Paris. Bonina, C. and Rivero-Illa, M., 2008, Mobile Telephony in Latin America: new opportunities to reduce poverty?, Proceedings of the 14th Americas Conference on Information Systems (AMCIS 2008), Toronto, Canada. Bonina, C. and Rivero-Illa, M., 2008, Mobile Telephony in Latin America: new opportunities to reduce poverty?, Proceedings of the 14th Americas Conference on Information Systems (AMCIS 2008), Toronto, Canada. Work in progress Public values, information technology and public sector organizing: a critical discourse analysis of trade regulations in Mexico Accounting for open data and social media: principles for protecting identity in Latin America (joint with Aaron Martin) Accounting for open data and social media: principles for protecting identity in Latin America (joint with Aaron Martin) Confronting the elusive: using critical discourse analysis to study public values in context The affordances of transparency and information technology: lessons from Mexico and Argentina - Page 242 - - Page 242 -
https://openalex.org/W3112709699	https://hal.science/hal-03261726/document	English	null	Networks of Care in Rural Madagascar for Achieving Universal Health Coverage in Ifanadiana District	Health systems and reform	2,020	cc-by	8,035	To cite this version: Laura F Cordier, Katherine Kalaris, Rado J L Rakotonanahary, Luc Rakotonirina, Justin Haruna, et al.. Networks of Care in Rural Madagascar for Achieving Universal Health Coverage in Ifanadiana District. Health Systems & Reform, 2020, 6 (2), pp.e1841437. 10.1080/23288604.2020.1841437. hal-03261726 Networks of Care in Rural Madagascar for Achieving Universal Health Coverage in Ifanadiana District Laura F Cordier, Katherine Kalaris, Rado J L Rakotonanahary, Luc Rakotonirina, Justin Haruna, Alishya Mayfield, Lanto Marovavy, Meg G Mccarty, Andritiana Tsirinomen’ny Aina, Baolova Ratsimbazafy, et al. HAL Id: hal-03261726 https://hal.science/hal-03261726v1 Submitted on 16 Jun 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Health Systems & Reform ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/khsr20 Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=khsr20 ARTICLE HISTORY ARTICLE HISTORY Received 11 August 2020 Revised 16 October 2020 Accepted 19 October 2020 KEYWORDS Networks of care (NOC); health system strengthening; community health; universal health coverage; health-care quality Health care is most effective when a patient’s basic primary care needs are met as close to home as possible, with advanced care accessible when needed. In Ifanadiana District, Madagascar, a collaboration between the Ministry of Public Health (MoPH) and PIVOT, a non-governmental organization (NGO), fosters Networks of Care (NOC) to support high-quality, patient-centered care. The district’s health system has three levels of care: community, health center, district hospital; a regional hospital is available for tertiary care services. We explore the MoPH/PIVOT collaboration through a case study which focuses on noteworthy elements of the collaboration across the four NOC domains: (I) agreement and enabling environment, (II) operational standards, (III) quality, efficiency, and responsibility, (IV) learning and adaptation. Under Domain I, we describe formal agreements between the MoPH and PIVOT and the process for engaging communities in creating effective NOC. Domain II discusses patient referral across levels of the health system and improve ments to facility readiness and service availability. Under Domain III the collaboration prioritizes communication and supervision to support clinical quality, and social support for patients. Domain IV focuses on evaluation, research, and the use of data to modify programs to better meet community needs. The case study, organized by the domains of the NOC framework, demonstrates that a collaboration between the MoPH and an NGO can create effective NOC in a remote district with limited accessibility and advance the country’s agenda to achieve universal health coverage. KEYWORDS Networks of care (NOC); health system strengthening; community health; universal health coverage; health-care quality Networks of Care in Rural Madagascar for Achieving Universal Health Coverage in Ifanadiana District Laura F. Cordier , Katherine Kalaris , Rado J. L. Rakotonanahary , Luc Rakotonirina , Justin Haruna , Alishya Mayfield , Lanto Marovavy , Meg G. McCarty , Andritiana Tsirinomen’ny Aina , Baolova Ratsimbazafy , Benedicte Razafinjato , Tara Loyd , Felana Ihantamalala , Andres Garchitorena , Matthew H. Bonds & Karen E. Finnegan To cite this article: Laura F. Cordier , Katherine Kalaris , Rado J. L. Rakotonanahary , Luc Rakotonirina , Justin Haruna , Alishya Mayfield , Lanto Marovavy , Meg G. McCarty , Andritiana Tsirinomen’ny Aina , Baolova Ratsimbazafy , Benedicte Razafinjato , Tara Loyd , Felana Ihantamalala , Andres Garchitorena , Matthew H. Bonds & Karen E. Finnegan (2020) Networks of Care in Rural Madagascar for Achieving Universal Health Coverage in Ifanadiana District, Health Systems & Reform, 6:2, e1841437, DOI: 10.1080/23288604.2020.1841437 o link to this article: https://doi.org/10.1080/23288604.2020.1841437 To link to this article: https://doi.org/10.1080/23288604.2020.1841437 © 2020 The Author(s). Published with license by Taylor & Francis Group, LLC. Published online: 14 Dec 2020. Submit your article to this journal View related articles View Crossmark data View related articles HEALTH SYSTEMS & REFORM 2020, VOL. 6, NO. 2, e1841437 (13 pages) https://doi.org/10.1080/23288604.2020.1841437 HEALTH SYSTEMS & REFORM 2020, VOL. 6, NO. 2, e1841437 (13 pages) https://doi.org/10.1080/23288604.2020.1841437 Networks of Care in Rural Madagascar for Achieving Universal Health Coverage in Ifanadiana District Laura F. Cordiera, Katherine Kalarisb, Rado J. L. Rakotonanaharya, Luc Rakotonirinaa, Justin Harunaa, Alishya Mayfielda,c,d, Lanto Marovavya, Meg G. McCartya, Andritiana Tsirinomen’ny Ainaa, Baolova Ratsimbazafya, Benedicte Razafinjatoa, Tara Loyda, Felana Ihantamalalaa, Andres Garchitorenaa,e, Matthew H. Bondsa,d, and Karen E. Finnegana,d aNGO PIVOT, Ranomafana, Madagascar; bMaternal Newborn and Reproductive Health, Clinton Health Access Initiative, Boston, Massachusetts, USA; cDepartment of Global Health Equity, Brigham and Women’s Hospital, Boston, Massachusetts, USA; dDepartment of Global Health and Social Medicine, Harvard Medical School, Boston, Massachusetts, USA; eMIVEGEC Laboratory, University of Montpellier, Centre National de la Recherche Scientifique, Institut de Recherche pour le Développement, Antananarivo, Madagascar © 2020 The Author(s). Published with license by Taylor & Francis Group, LLC. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Maternal Newborn and Reproductive Health, Clinton Health Access Initiative, CONTACT Karen E. Finnegan Karen_finnegan@hms.harvard.edu Maternal Newborn and Reproductive Health, Clinton Health Access Initiative, Boston, MA 02115, USA. © 2020 The Author(s). Published with license by Taylor & Francis Group, LLC. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT ARTICLE HISTORY Received 11 August 2020 Revised 16 October 2020 Accepted 19 October 2020 © 2020 The Author(s). Published with license by Taylor & Francis Group, LLC. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licens use, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction 2030, including through the implementation of Universal Health Coverage (UHC). Madagascar’s Ministry of Public Health (MoPH) envi sions that “by 2030, the population of Madagascar will be in good health, living in a healthy environment, leading better and productive lives.”1 The MoPH’s Health Sector Strategic Plan presents six strategic objectives to achieve this vision: improving the avail ability of quality services at all levels of care, increasing service utilization at all levels of care, strengthening governance and management of the health system, improving maternal and child health, strengthening the fight against communicable and non- communicable diseases, and promoting healthy behaviors.1 The plan is part of the MoPH’s strategy for achieving the Sustainable Development Goals by Madagascar’s GDP per capita has steadily risen since 2014—when democratic elections were held for the first time following a 2009 coup—creating an opportunity to transform the health system and improve care coordination to fulfill the vision of the MoPH. In 2017, total per capita spending on health care in Madagascar was 25 USD, an increase from 14 USD per capita in 2014.2 Notwithstanding improvements in health finan cing and health conditions in recent years, Madagascar has stark health indicators: in 2018, the under-5 mortality rate was 59 deaths per 1,000 live births and the neonatal mortality rate was 21 Maternal Newborn and Reproductive Health, Clinton Health Access Initiative, Maternal Newborn and Reproductive Health, Clinton Health Access Initiative, CONTACT Karen E. Finnegan Boston, MA 02115, USA. e1841437-2 L. CORDIER ET AL. e1841437-2 L. CORDIER ET AL. deaths per 1,000 live births.3 The national averages mask internal disparities; most health indicators are significantly worse in rural areas of the country.3 an additional more basic CSB level I. Long dis tances and travel time to a health facility greatly affect people’s ability to access care; in Ifanadiana District, 70% of the population lives at least 5 km from a health facility and 49% of the population lives more than 10 km from a health facility.4 To reduce geographic barriers to care, Ifanadiana has one village-based community health site for each of its 196 fokontany (the smallest administrative unit in Madagascar that is composed of one or several villages). Per national policy, each commu nity health site has two community health workers (CHWs), who provide basic health services and ensure a communication link between commu nities and the health system. Introduction The district of Ifanadiana, in the Vatovavy Fitovinany region, is representative of the chal lenges and opportunities for improving health out comes in Madagascar. Ifanadiana is a rural mountainous district with an estimated population of 183,000 in 2020 (Figure 1). It is comprised of 15 communes, which are administrative units ranging in population from 4,300 to nearly 18,000. Per MoPH norms, one public sector referral hospital (Centre Hospitalier de Référence de District [CHRD]) serves the district. Each commune has one primary health center level II (Centre de Santé de Base [CSB II]), and the larger communes have Various measures of mortality and care-seeking were worse in Ifanadiana than national averages. In 2014, a population-based household survey esti mated maternal mortality in Ifanadiana at 1,044 deaths per 100,000 live births; under-five mortality was 145 deaths per 1,000 live births.5 Only 18% of births occurred in a health facility.5,6 Fewer than a third of children under age five sought care for an illness and only 35% of children were fully vaccinated by 12 months.6 Approximately half (51%) of children were stunted.7 In the district, there were 1.6 health-care workers per 10,000 peo ple and only 15% of primary health centers met Ministry of Public Health (MoPH) staffing norms.8 Figure 1. Map of Ifanadiana district with CHRD (green), CSB I (blue), CSB II (white with blue outline), and community health sites (red) denoted across the district’s 15 communes. The communes which receive PIVOT support for the CHW program are shaded in gray; the commune which piloted the new approach to community health is in red. In 2014, PIVOT, an international non- governmental organization (NGO), began a partnership with the MoPH to build a district- level model health system based on integrating clinical care, system readiness, and scientific inno vation at all levels of the health system in Ifanadiana District. PIVOT provides technical and financial support at the direction of MoPH leadership. We apply the Networks of Care (NOC) framework to analyze the MoPH/PIVOT partner ship and demonstrate the creation of effective NOC with the support of this collaboration. The MoPH/PIVOT collaboration begins at the house hold level with a focus on community health, extends through primary and secondary care in the district, and on to tertiary care when needed, facilitating comprehensive quality care and lin kages between levels of care for those with com plex conditions. The MoPH/PIVOT collaboration Figure 1. Purposeful Arrangements The NOC are established through a collection of Memoranda of Understanding (MoU) with the central MoPH, and project-specific regional and district agreements. In 2020, PIVOT revised the MoU with the MoPH to better reflect its alignment with the MoPH Strategic Plan for 2020 to 2024 and to focus on UHC. The new MoU outlines specific projects and details the roles and respon sibilities of national, regional, and district colla borators to increase accountability and buy-in to the MoPH/PIVOT collaboration. In addition, PIVOT engages in annual joint planning with dis trict MoPH officials and actively participates in their monthly and quarterly reviews. The MoPH, including CHRD and CSB leadership and district health officials, establish annual priorities and seek financial and technical support from PIVOT. Introduction Map of Ifanadiana district with CHRD (green), CSB I (blue), CSB II (white with blue outline), and community health sites (red) denoted across the district’s 15 communes. The communes which receive PIVOT support for the CHW program are shaded in gray; the commune which piloted the new approach to community health is in red. HEALTH SYSTEMS & REFORM e1841437-3 e1841437-3 fosters an enabling environment for the NOC. Community engagement is continuously stew arded through support and communication with local leaders. uses new strategies and innovations to improve the availability of and access to high-quality care for the population of Ifanadiana. In this case study of the MoPH/PIVOT colla boration in Ifanadiana District, key components of the collaboration are described using the NOC framework and the following four domains: I: Agreement and Enabling Environment; II: Operational Standards; III: Quality, Efficiency, and Responsibility; and, IV: Learning and Adaptation. We focus on innovations and best practices that support NOC in a rural district with limited financial resources (Figure 2). e1841437-4 L. CORDIER ET AL. components needed for functional NOC and the achievement of the MoPH vision for health-care delivery. At the local level, the NOC require the buy-in of various civil authorities. Some health system activ ities (e.g. the election of community health work ers and construction of certain health infrastructure) fall under the purview of civil administration, not the MoPH. These relationships are governed by informal relationships. Community Engagement The MoPH posits community care as the base of the health system. In support of this, PIVOT actively engages the community in the develop ment and execution of its work. CHWs play a central role in care delivery, coordination, and community engagement. Per national norms, communities nominate their CHWs, who are respected local residents. The MoPH and PIVOT support the elected CHWs with training and supervision and require that CHWs demonstrate proficiency in providing clinical care in accor dance with protocols. In the district, the CHW program is being reformed to align with the Community Health Impact Coalition’s best prac tices in program design and care delivery and to better meet the needs of the community.9 Changes include the professionalization of CHWs, enhanced supervision, and an emphasis on disease surveillance and management through proactive care. PIVOT also collaborates with communities by providing materials for the construction of a community health site. The community health site, located in a central location in each fokon tany, is staffed by a CHW who provides basic primary care services. Community health sites are a means of fostering community engagement in health care and provide a physical location for service delivery. To initiate a referral, CSBs must call the CHRD emergency call center, staffed by the referral team, to request an ambulance transfer. The CSB com municates the patient status, vital signs, and other information necessary for the CHRD to approve or deny the transfer by ambulance. The ambulance is managed and financed by PIVOT, but the authority to dispatch it resides with the CHRD MoPH team. For urgent referrals, PIVOT has equipped two ambulances that are based at the CHRD and staffed by twelve paramedics and eight drivers. Because fewer than a third of CSBs can be reached by road, relay protocols are used; referred patients are brought to a CSB or other accessible point for pickup. The ambulance staff includes a referral nurse who remains in contact with the CHRD refer ral team regarding the patient’s condition through out the patient transfer process. In 2019, there were 2,217 referrals from CSBs to higher levels of care, 38% of which were transported by ambulance. Referral System The health system in Ifanadiana strives to deliver high-quality basic primary care as close to the community as possible, while also developing sec ondary care services for complex health-care needs. PIVOT began supporting the referral sys tem in 2014 as one of its first activities, by stan dardizing referral criteria and providing additional options for patient transport. The Ifanadiana health system manages urgent and non-urgent referrals for primary and secondary care within the district, as well as referrals to tertiary care to the regional hospital in Fianarantsoa, located approximately 85 km from the CHRD, and speci alty hospitals in the capital. Domain I: Agreement and Enabling Environment As a central part of the collaboration, PIVOT developed strong relationships with the Government of Madagascar and with local com munities in Ifanadiana. The foundation of the MoPH/PIVOT partnership is based on purposeful agreements that clarify common objectives and specify each party’s responsibilities; this clarity Figure 2. Components of the NOC in Ifanadiana district, Madagascar as described in the case study of the MoPH/PIVOT partnership. s of the NOC in Ifanadiana district, Madagascar as described in the case study of the MoPH/PIVOT partners e1841437-4 L. CORDIER ET AL. Domain II: Operational Standards PIVOT invests in health system readiness at all levels to improve quality of care and meet MoPH minimum standards. By strengthening pillars of the health system such as human resources, med ical services, infrastructure, communication, trans fer services, and information systems, the NGO supports the development of the operational For non-urgent referrals, the CHRD may deter mine that the patient can be seen during normal consultation hours and can travel independently. The CSB clinician informs the patient of the CHRD consultation hours and instructs the patient to take public transport. While public transport only costs 1000 ariary (0.26 USD), this HEALTH SYSTEMS & REFORM e1841437-5 can still be a barrier to accessing care. Therefore, in 2019, PIVOT began covering the costs asso ciated with non-urgent approved referrals via pub lic transportation for patients traveling within the district. the CHRD regarding referrals and to communi cate with the district health team for general coordination of activities. This has also proven useful during outbreaks to facilitate epidemiolo gical tracking and the dissemination of information. If a transfer is not determined to be necessary by the CHRD, the referral team provides guidance to the CSB on case management. Moreover, the CSB can also call the CHRD emergency call center if they are uncertain if a referral is urgent. The call center also provides general counseling by phone on complex cases. PIVOT has also found that communication with patients and their families is an essential step in the referral process and fosters trust in the health sys tem. Specifically, it has introduced a consent form that presents a patient’s rights. While reviewing the paper consent form with the patient and family, the provider explains the reason for the referral and ensures that they understand the referral process; this can include the mode of transport, anticipated upcoming medical care, eligibility for social cover age through PIVOT, logistics (including materials to bring), and what is expected of accompanying family members to support the patient. Because ambulances are only available to trans fer patients from CSB to CHRD, communities and CHWs must oversee the transfer of patients from their homes to the CSB. In Ifanadiana, people typically build handmade stretchers to transport the very ill to the CSB, which can delay referrals. Domain II: Operational Standards After consultation with the communities, PIVOT distributed “rolling stretchers” (a bicycle with a stretcher on top) to CSBs to ease patient trans port across difficult terrain. Patients’ family mem bers are responsible for transporting the patient in the rolling stretcher and for returning it to the CSB and completing any cleaning and maintenance. Evaluation of this pilot program showed that deploying normal stretchers without wheels to other access points in the community would be as effective as the rolling stretcher and allow increased coverage. Referral Documentation The MoPH/PIVOT have worked to strengthen the documentation which accompanies the referral and counter referral process. For both urgent and non-urgent referrals, the referring facility sends a referral form with the patient; this document includes patient identifiers, the patient’s clinical state, medical history, clinical exams completed at the facility, and the diagnosis. This information is supplemented by additional data on care adminis tered during ambulance transport. This facilitates the transfer of critical medical information to ensure a complete patient file and accelerates a patient’s care at the referral facility. Communication PIVOT has worked closely with the MoPH to improve coordination of patient care across levels of care by maximizing communication among facilities throughout the referral process (Figure 3). Given low connectivity and network coverage in Madagascar, PIVOT donated cell phones and equipped all facilities in the district with an internal communication network, enabling them to communicate with CSBs, the CHRD, and the district health office at no charge (up to 60 minutes call time and 25 SMS per month). More recently, areas in the district with poor network connectivity were equipped with a “Village Phone.” This is an antenna that ampli fies the available networks and connects to a landline telephone, enabling facilities to call Ensuring that information about the referred patient’s case is communicated back to the refer ring facility in a timely manner is equally impor tant. A system has been put in place to support the return of information throughout the referral pro cess. This counter referral process includes com pletion of a form which summarizes the patient’s care, discharge details and follow-up dates. This counter referral process typically depends on a health facility, like the CHRD, communicating the information back to a lower level facility, that then passes it on to the patient’s family or the CHW. However, relying on this manual exchange can lead to a communication breakdown. To e1841437-6 L. CORDIER ET AL. Figure 3. The MoPH/PIVOT partnership supports the movement of patients from household and community health site, to health center, and the district hospital. If necessary, patients are transferred to a regional hospital for tertiary care. The movement across levels of the health system (solid line) are facilitated by stretcher, ambulance, and other means of transport depending on urgency. Information about patient cases flows up and down levels of the health system (dotted line). e1841437-6 L. CORDIER ET AL. Figure 3. The MoPH/PIVOT partnership supports the movement of patients from household and community health site, to health center, and the district hospital. If necessary, patients are transferred to a regional hospital for tertiary care. The movement across levels of the health system (solid line) are facilitated by stretcher, ambulance, and other means of transport depending on urgency. Information about patient cases flows up and down levels of the health system (dotted line). mitigate those risks, the hospital call center acts as an intermediary in the communication chain in addition to the form. Communication construction, have been financed by PIVOT once needs have been established in consultation with the district health office. Community health sites and maternal waiting homes are constructed through community mobilization efforts sup ported by PIVOT. Once facilities are renovated, they also receive equipment and materials neces sary for primary care; support may also include solar panels or another alternative energy source depending on the availability of utilities. As of mid-2020, PIVOT has helped the MoPH to reno vate the CHRD and five health centers and has Workforce As in many remote rural districts, human resource availability is a major challenge in Ifanadiana District. MoPH staffing norms stipulate for CSB I, one nurse and one midwife and for CSB II, one each of a general doctor, nurse, and midwife. However, due to funding challenges, only four communes in the district have doctors at CSB IIs. Nurses or midwives thus commonly fill doc tors’ roles in health facilities. Clinicians’ reluctance to accept remote placements is also a challenge. Supporting human resources is a pillar of PIVOT’s health systems strengthening approach. As a short- term solution, PIVOT sometimes places its own clinical staff in health facilities to fill MoPH vacan cies. However, a more sustainable solution has been the deployment of joint recruitment pro grams, whereby PIVOT pays the salaries of clin icians who are formally integrated into the MoPH workforce after 2 years. Health-care workers are further incentivized to provide care at remote locations through the provision of necessary equipment (e.g. stethoscope, blood pressure cuff, thermometer) and by being actively included in capacity building initiatives. Results from 2016 showed that each additional health-care provider at a facility in Ifanadiana was associated with a 10% increase in service utilization.10 This increase in utilization provides evidence that when care is available and accessible (geographi cally and financially), patients seek health care. Alongside improving general infrastructure, the MoPH/PIVOT have a vision of enhanced diagnostic capacity at all levels of care. Basic diagnostic capacity has been set up at CSBs to include a glucometer, pregnancy test, and urine test strips. Moreover, PIVOT also helped the CHRD laboratory develop its capacity to run tests for prevalent infectious diseases (e.g., hepa titis B and C, and schistosomiasis), and estab lished a blood bank in 2019. Previously, a patient requiring a blood transfusion received it via patient-to-patient transfusion or was referred to tertiary level of care. PIVOT provided financial management of the laboratory at the CHRD for a year and continues to support the technical operations of the lab and blood bank. Other partners have also invested in improving diagnostics at the CHRD, for example, making a GeneXpert machine available, which is used in diagnosis of tuberculosis and COVID-19. Supply and infrastructure The MoPH/PIVOT have worked to improve the overall readiness of health facilities in Ifanadiana. Since 2014, PIVOT has provided infrastructure support to the CHRD, many CSBs, and commu nity health sites. Efforts to improve existing health facilities, either through renovations or new HEALTH SYSTEMS & REFORM e1841437-7 HEALTH SYSTEMS & REFORM e1841437-7 access to medicines at no cost to patients through out the entire district by 2022. supported the construction of 58 community health sites and two maternal waiting homes. PIVOT is currently developing plans to support some level of infrastructure improvements to all public health-care facilities in the district by 2022. Workforce g One of the major barriers to accessing care throughout Madagascar, like much of sub- Saharan Africa, is the fees for drugs and consum ables, and frequent stock outs; this plagues care at all levels of the health system. Nationally, com modities are procured through the MoPH’s pro curement agent, SALAMA, and sold through the national supply chain. PIVOT works with the MoPH to strengthen the district’s supply chain, an essential component of a NOC, and, in extreme instances, responds to stock outs with a medication donation or funds to procure addi tional medications. To eliminate financial barriers to accessing care, PIVOT reimburses health facility pharmacies for the costs of certain medications and other commodities, so patients are not charged. The removal of these point-of-care user fees led to a threefold increase in utilization for maternal and child health services in the district.10 Currently, PIVOT provides financial coverage for medicines in 6 (of 21) CSBs and in the CHRD. Following the inclusion of Ifanadiana as one of the first pilot districts for the UHC national strategy, the MoPH and PIVOT are working to achieve Quality and Clinical Guidance The MoPH/PIVOT collaboration prioritizes a culture of quality improvement. Clinical care is guided by national and international protocols on diagnosis and treatment at all levels of care. Official MoPH guidelines and checklists are used to determine if providers follow clinical protocols and to identify when support, such as refresher trainings or clinical mentorship, is needed. The MoPH leads clinical supervision activities; PIVOT accompanies them and provides financial and technical support to ensure there is routine feedback on clinical care. Monitoring The MoPH has implemented a health manage ment information system (HMIS) that captures system inputs and utilization at all facilities in the district. The HMIS includes epidemiologic tracking that varies in frequency depending on disease outbreaks. PIVOT has further developed an extensive dashboard for Ifanadiana District to measure the intervention’s effects on health system processes and health outcomes (Figure 4).8 The dashboard incorporates MoPH HMIS data and data collected by PIVOT. The interface allows e1841437-8 L. CORDIER ET AL. Figure 4. A sample of PIVOT’s dashboard, which includes more than 800 indicators and geo-located information on utilization, coverage, and population health. Figure 4. A sample of PIVOT’s dashboard, which includes more than 800 indicators and geo-located information on utilization, coverage, and population health. staff to assess change over time, hone in on one facility or a subset, and to compare facilities receiving full PIVOT support with those that receive only a basic support package. To improve the quality and timeliness of data collection, PIVOT developed electronic data collection forms for the various activities it implements at every level of the health system. The dashboard provides a comprehensive picture of the district’s health system at every point in time, showing over 800 indicators including routine data for key indi cators of every program implemented across the NOC. In addition to utilization per health service and proxy measures for quality of care, the dash board also includes information on per patient cost, stock out rates, and patients lost to follow- up.8 All program managers have access to the dashboard with core indicators for programmatic decision-making and program management targets. diseases, have resulted in significant improvements in the coverage and quality of care at facilities.12,13 Specifically, PIVOT has accompanied the MoPH in the improvement of clinical programs such as Integrated Management of Childhood Illness (IMCI) at community- and CSB-level, malnutri tion and tuberculosis, particularly through clinical skills building for health-care workers of each cadre and improved readiness, as mentioned above. Social Support PIVOT has advocated for the integration of social support as part of clinical care and as a way of strengthening the NOC. PIVOT’s social team sup ports patients through two main avenues. First, PIVOT provides material and financial support by covering the costs of transport, lodging and food when seeking health-care services. Secondly, PIVOT offers psycho-social accompaniment of patients and their families throughout their jour neys. The social team acts as patient advocates and ensures that patients understand their rights and that their needs are met during hospitalization. If patients are lost to follow up, the social team works with CHWs to find them, understand their challenges and support their reentry into the health-care system as necessary. In addition, PIVOT’s social work team pays bi-monthly visits to patients referred for follow-up at home and provides psycho-social support. MoPH and PIVOT staff conduct joint super vision at the CSBs. Supervision is specific to a program area and includes reviewing registers, consultation sheets, and the quality of HMIS data. Joint supervision is an opportunity to focus on a clinical area while in the health facility, and to give focused attention to the providers. Group quarterly meetings are held by the district with the heads of the CSBs to review data, targets, and results. Meetings with other partners working in the district are also held regularly. District leader ship is working with PIVOT to develop a new model of supportive supervision in which clinical teams are placed throughout the district and rotate among facilities to support clinicians by addres sing questions, identifying and rectifying chal lenges, and providing hands-on mentorship. Domain III: Quality, Efficiency and Responsibility High-quality health systems are essential,11 and an important component of NOC. PIVOT support to clinical programs, with a focus on child health, maternal and reproductive care, and infectious CHWs are supervised twice monthly, once in the community and once at the CSB. Theoretically, CHWs should be supervised by the head of the HEALTH SYSTEMS & REFORM e1841437-9 CSB—but in practice, time and resources for dedi cated CHW supervision are scarce. Therefore, a cadre of health workers was created by PIVOT, with approval from the MoPH, to act as an exten sion of the CSB. CHWs are supervised by ACCs (agents cliniques communautaires). This cadre does not exist in other districts in Madagascar. The ACCs observe CHWs providing care to sick chil dren under 5 years old based on the integrated community case management (iCCM) protocol. Using a protocolized checklist, the ACC provides real-time feedback on the CHW’s diagnosis, treat ment, and referral practices, as well as the quality of the counseling provided by the CHW to the caregiver. Under this supervision model, there have been improvements in the utilization and quality of care provided by the CHWs. ACCs also provide intensive supervisory support for the care of people with medically complicated malnu trition and tuberculosis. Once a month, CHWs convene as a group at their commune’s CSB; the ACCs, district health office and NGO representa tives are also present. During these meetings, par ticipants share feedback on activities, discuss difficulties encountered, and communicate infor mation that needs to be disseminated to all provi ders. Each meeting also focuses on a current clinical or management issue, for example, an anticipated rise in the expected number of malaria cases during malaria season. providers are following guidelines for clinical ser vices and if refresher training is required. Trainings are led by the CHRD Medical Director, staff from the regional hospital, or specialists. The CHRD holds maternal and perinatal death reviews (MPDSR), led by the head of the hospital. The MPDSR includes a review of deaths, factors asso ciated with the death, and areas for intervention or improvement. Clinical reviews are intended to improve care and management of cases. For exam ple, following the introduction of the reviews, the hospital has seen a reduction in c-section initiation time. Example 1—Flexibility and Extending Reach The MoPH/PIVOT collaboration uses monitoring, evaluation, and research to shape health-care delivery and envision its evolution. The routine monitoring described in Domain II is complemen ted by evaluation activities. Every 2 years, the MoPH and PIVOT jointly conduct a facility assessment using a modified version of the World Health Organization’s Service Availability and Readiness Assessment (SARA).14 The data are used to measure impact of the MoPH/PIVOT col laboration on facility infrastructure and readiness over time, while also providing information on priorities for joint work planning. Other evalua tion activities are determined by clinical and orga nizational priorities. Recent evaluations have included an assessment of the rolling stretcher pilot, a qualitative evaluation to understand care- seeking for maternity care and decision-making, and an ongoing evaluation of the community health program and the impact of care delivery model on utilization and quality of care. Although PIVOT initially focused its efforts on supporting the MOPH’s facility-based care (via the CHRD, CSBs, and the referral network), eva luations of health-care access in Ifanadiana showed that while coverage of most essential health services rapidly improved over time, major geographic barriers to care left remote commu nities with persistent unmet primary care needs.12,18 It was clear that PIVOT’s financial and technical support needed to shift to include community-based care delivery via community health sites. Yet, the geography in Ifanadiana is such that many communities have poor geo graphic access to community health sites.18 To respond to this persistent challenge, and at the request of the MoPH, an innovative pilot to more actively meet the primary care needs of clients in their homes and communities was designed. The commune of Ranomafana has piloted a two-pronged approach to service deliv ery: one CHW is available at the community health site, while other CHWs travel a circuit of households throughout the fokontany to proac tively identify sick children and provide basic care following a roster. As part of the pilot, additional CHWs were recruited and CHWs were financially compensated. Three months after implementation of the pilot, PIVOT conducted focus groups on the new approach and found that CHWs were enthusiastic about the new model and their ability to provide high-quality care where the need was greatest. Domain IV: Learning and Adaptation One of the components that sets the work in Ifanadiana apart from that being implemented by collaborations worldwide is the extensive effort to build an environment that prioritizes learning and innovation through the integration of data systems with implementation. Since its inception, PIVOT has invested in a data and analytics platform that allows for extensive monitoring and evaluation, rigorous quantitative epidemiology and operations research, technological tool development, and qua litative inquiry. These are essential for developing At the CHRD, a protocol developed by MoPH/ PIVOT has been instituted to determine whether e1841437-10 L. CORDIER ET AL. generating evidence of intervention impact. The focus on implementation science has generated rig orous evidence on the impact of health system strengthening interventions.12,13,15,16 PIVOT there fore also collaborates with a network of researchers to create a broad field-based research agenda for sustainability.7,17 Two examples demonstrate how the process of learning through monitoring, evalua tion and research is contributing to flexibility and extending the reach of the health system in Ifanadiana, as well on prioritizing patient- centeredness: a functional NOC that can constantly improve and optimize its impact on population health. PIVOT’s research answers pressing questions identified by the MoPH/PIVOT collaboration about what impact their work has, who does and does not benefit from it, and what changes are needed to improve care delivery. Rigorous research addresses questions about financial sustainability of the col laboration (i.e. through a costing analysis of the district health system) and about which programs should be scaled up due to demonstrated uptake and impact. Example 2—Client Centeredness The evolution of the MoPH/PIVOT collaboration has been shaped by the needs of the community. In the district, women tend to deliver at home due to the distance to facilities; some live 10 to 15 km away from the closest CSB. Cultural norms and perceptions of the quality of care at facilities also play roles in the decision to give birth at home. Indeed, results from the longitudinal cohort showed that improvements in the coverage of maternal health services lagged behind most other primary care services where progress had been achieved,12 and coverage in remote popula tions was particularly low (2020 Jul 16 e-mail from A Garchitorena). Beginning with a foundation of agreement and an enabling environment (Domain I), the NOC have been established within a health sys tem where the vision is understood, and roles and responsibilities are delineated across levels of care and between NGO and state actors. Second, the NOC have sought to equip each level of the system with appropriate operational standards (Domain II), including the critical infrastructure necessary for service delivery, and the creation of linkages across the conti nuum of care. Beyond this, however, delivering on UHC for a rural population requires a patient-centered strategy, one that is focused on ensuring that appropriate care happens where and when it should, and in which care is continuously evaluated for quality, efficiency, and responsibility (Domain III). This patient- centered approach requires a NOC strategy with structured engagement with the social determinants of health, and every consideration is made to promote the patient’s right to care throughout the system. This is why, in large part, the removal of user fees for all patients throughout the NOC was one of the collabora tive’s earliest initiatives.10 For the rural popula tion of Ifanadiana District, this has also meant bringing care as close to communities as possible through establishing new sites and deploying more CHWs. The NOC rest upon the principles of learning and adaptation (Domain IV) with a grounding in monitoring, evaluation, and Consequently, PIVOT instituted a mobile antenatal care (ANC) program with ACCs who offer the first two ANC appointments in the com munity and then refer pregnant women to the CSB for the remaining ANC visits and delivery. The ACCs are equipped with the same material for ANC as is available at the CSBs. Conclusion Strong NOC are essential in order to deliver on the promise of UHC in a poor and rural context. The NOC created through the MoPH/PIVOT colla boration have strived to provide care that is acces sible to all, responsive to patient needs, adaptive to system failures, and timely and efficient. During the first 2 years of the MoPH/PIVOT collaboration in Ifanadiana District, under-five mortality declined 19% and neonatal mortality by 36%.12 From 2014 to 2016, care-seeking for childhood illness increased by 51% in PIVOT-supported areas and fell slightly in the rest of the district; attendance at four or more ANC visits increased for all areas of the district.13 HEALTH SYSTEMS & REFORM e1841437-11 HEALTH SYSTEMS & REFORM e1841437-11 satisfaction are being studied to generate lessons for local and national scale-up. Example 1—Flexibility and Extending Reach The impacts of the community health pilot on utilization, quality of care, and patient PIVOT combines facility-based information with population-based and geographic information sys tem data to carry out rigorous research on the drivers of disease, health-care access, and the impact of the interventions implemented since 2014. Prior to the start of the health system strengthening intervention, PIVOT initiated a district-representative longitudi nal cohort of 1,600 households (~9000 individuals) to follow the evolution of health-care coverage, health outcomes and socio-economic information every 2 years.6 This permits a rapid feedback loop between population-based research and program implementation in Ifanadiana, while simultaneously References 1. Ministère de la Santé Publique. Plan de développement de secteur sante 2015–2019. Antananarivo (Madagascar): Ministère de la Santé Publique; 2015. 2. World Bank. World bank data. Washington (DC): The World Bank Group; [accessed 2020 June 10]. https:// data.worldbank.org/indicator/SH.XPD.CHEX.PC.CD. 3. Institute National de la Statistique (INSTAT) Madgascar. Enquête par grappes à indicateurs, 2018, resumé statistique. Antananarivo (Madagascar): INSTAT, World Bank, Unicef; 2019. 4. Ihantamalala FA, Herbreteau V, Revillion C, Randriamamihaja M, Commins J, Andréambelson T, Rafenoarimalala FH, Randrianambiniina A, Cordier LF, Bonds MH, et al. Improving geographical accessi bility modeling for operational use by local health actors. Int J Health Geogr. 2020;3. doi:10.1101/ 2020.03.09.20033100. 4. Ihantamalala FA, Herbreteau V, Revillion C, Randriamamihaja M, Commins J, Andréambelson T, Rafenoarimalala FH, Randrianambiniina A, Cordier LF, Bonds MH, et al. Improving geographical accessi bility modeling for operational use by local health actors. Int J Health Geogr. 2020;3. doi:10.1101/ 2020.03.09.20033100. The institutionalization of UHC is a major priority of the Government of Madagascar. As PIVOT works to operationalize the principles of UHC in Ifanadiana by ensuring the provision of high-quality free services, the NGO supports the government in this initiative and is actively engaged in the national UHC dialogue. Early evi dence on the implementation of fee removal in Ifanadiana District demonstrated an increase in use of services by 65% for all patients, 52% for children under 5, and over 25% for maternity consults.10 These results were achieved at an aver age cost for medicines and consumables (that patients would have otherwise had to pay directly) of 0.60 USD per patient.10 Currently, PIVOT is completing a costing study to estimate the per capita cost of delivering care in a strengthened district health system accounting for all financial actors, including MoPH, PIVOT, bilateral donors, and other partners. 5. Miller A, Ramananjato RH, Garchitorena A, Rabeza VR, Gikic D, Cripps A, Cordier L, Razanadrakato HT, Randriamanambintsoa M, Hall L, et al. Baseline popula tion health conditions ahead of a health system strength ening program in rural Madagascar. Glob Health Action. 2017;10. doi:10.1080/16549716.2017.1329961. 6. Miller AC, Garchitorena A, Rabeza VR, Randriamanambintsoa M, Razanadrakato HT, Cordier L, Ouenzar M, Murray MB, Thomson DR, Bonds MH. Cohort profile: Ifanadiana health outcomes and prosper ity longitudinal evaluation (IHOPE). Int J Epidemiol. 2018 June;1394–95. doi:10.1093/ije/dyy099. 7. Mccuskee S, Garchitorena A, Miller AC, Hall L, Ouenzar MA, Rabeza VR, Ramananjato RH, Razanadrakato HTR, Randriamanambintsoa M, Barry M. Disclosure of Potential Conflicts of Interest Disclosure of Potential Conflicts of Interest LC, RJLR, LR, JH, AM, Lm, MGM, ATA, BR, BR, TL, FI, MHB, and KF receive salary support from PIVOT. LC, RJLR, LR, JH, AM, Lm, MGM, ATA, BR, BR, TL, FI, MHB, and KF receive salary support from PIVOT. LC, RJLR, LR, JH, AM, Lm, MGM, ATA, BR, BR, TL, FI, MHB, and KF receive salary support from PIVOT. Example 2—Client Centeredness Another initiative seeks to motivate matrons, unlicensed but respected community-based maternal health pro viders, to accompany pregnant women to facilities for delivery. Maternal waiting homes at CSBs con structed in collaboration with communes, also encourage facility delivery. At the maternal waiting homes, up to two family members may accompany the pregnant woman, who stays for one to two weeks before the birth and for a minimum of 3 days (and up to a week) following the birth. The social support team provides food during stays at the maternal waiting home. PIVOT has conducted interviews with women and trusted sources of maternal health information, learning how deci sions are made about where to give birth and what factors are most important for those decisions, which the MoPH is using to improve its approach to maternal care. In addition, an evaluation is underway to understand the impact of these activ ities on the evolution of geographic access to maternal care services in the district. e1841437-12 L. CORDIER ET AL. research, which encourages the NGO and the partnership to evolve as community needs change. development of similar arrangements in other rural areas of Madagascar. The MoPH provides comprehensive care through a network of CHWs, CSBs, and the CHRD. PIVOT’s work in Ifanadiana District began with focused attention on the CHRD and four health facilities and has progressively expanded. The MoPH, championing UHC, colla borates with PIVOT to develop a district-wide strategy that brings enhanced support to all facil ities in the district. Over the course of their colla boration, MoPH/PIVOT have seen an increase in use of services, particularly among those living within 5 km of a health facility. The network of facilities within the district supported by the pro ject has been progressively expanding, with a target of reaching the entire population of the district through 15 model health centers (CSB IIs) and providing community health care by 2022. References Child malnutrition in Ifanadiana district, Madagascar: associated factors and timing of growth faltering ahead of a health system strengthening intervention. Glob Health Action. 2018;11(1):1452357. doi:10.1080/16549716.2018.1452357. The MoPH/PIVOT collaboration in Ifanadiana District has demonstrated what can and needs to be done to strengthen the health system and build a robust NOC. This case study reveals that NOC can be supported in an impoverished rural district through attention to the four NOC domains and a vision for the delivery of high quality, patient-centered health care. Findings from these NOC can be used to inform the 8. Bonds MH, Garchitorena A, Cordier L, Miller AC, McCarty M, Andiamihaja B, Ratsirarson J, Randrianambinina A, Rabeza V, Finnegan K, et al. Advancing a science for sustaining health: establishing a model health district in Madagascar. bioRxiv. 2017;1–7. doi:10.1101/141549. 9. Ballard M, Bonds M, Burey J, Forth J, Fiori K, Holeman I, Johnson A, Malaba S, Palazuelos D, HEALTH SYSTEMS & REFORM e1841437-13 Raghavan M, et al. CHW AIM: updated program func tionality matrix for optimizing community health programs. Washington (DC): USAID; 2018. initiative in rural Madagascar: A longitudinal cohort study. PLoS Med. 2019 DiD;1–23. doi:10.7910/DVN/ LSEW2L. 14. World Health Organization. Service availability and readiness assessment (SARA): an annual monitoring system for service delivery. Geneva (Switzerland): World Health Organization; 2015. 10. Garchitorena A, Miller A, Cordier LF, Ramananjato R, Rabeza VR, Murray M, Cripps A, Hall L, Farmer P, Rich M, et al. In Madagascar, use of health care services increased when fees were removed: lessons for univer sal health coverage. Health Aff. 2017;36(8):1443–51. doi:10.1377/hlthaff.2016.1419. 15. Roberts L. Sweating the small things. Science. 2019;363 (6430):918–23. doi:10.1126/science.363.6430.918. 16. Bonds MH, Rich ML. Integrated health system strength ening can generate rapid population impacts that can be replicated: lessons from Rwanda to Madagascar. BMJ Glob Health. 2018:10–12. doi:10.1136/bmjgh-2018-000976. 11. Kruk ME, Gage AD, Arsenault C, Jordan K, Leslie HH, Roder-DeWan S, Adeyi O, Barker P, Daelmans B, Doubova SV, et al. High-quality health systems in the sustainable development goals era: time for a revolution. Lan Global Health Comm. 2018;6(November):1196–252. doi:10.1016/S2214-109X(18)30386-3. 17. Miller AC, Garchitorena A, Rabemananjara F, Cordier L, Randriamanambintsoa M, Rabeza V, Razanadrakoto HT, Ramakasoa RR, Tiana OR, Ratsimbazafy BN, et al. Factors associated with risk of developmental delay in preschool children in a setting with high rates of mal nutrition : a cross-sectional analysis of data from the IHOPE study, Madagascar. BMC Pediatr. 2020;20: 1–11. 12. References Garchitorena A, Miller AC, Cordier LF, Rabeza VR, Randriamanambintsoa M, Razanadrakato HTR, Hall L, Gikic D, Haruna J, McCarty M, et al. Early changes in intervention coverage and mortality rates following the implementation of an integrated health system inter vention in Madagascar. BMJ Glob Heal. 2018;3(3): e000762. doi:10.1136/bmjgh-2018-000762. 18. Ihantamalala FA, Herbreteau V, Revillion C, Randriamihaja M, Commins J, Andréambeloson T, Rafenoarimalala FH, Randrianambinina A, Cordier LF, Bonds MH. Improving geographical accessibility model ing for operational use by local health actors. Int J Health Geogr. 2020;19(1). doi:10.1186/s12942-020-00220-6. 13. Ezran C, Bonds MH, Miller AC, Cordier LF, Haruna J, Mwanawabenea D, Randriamanambintsoa M, Razanadrakato HTR, Ouenzar MA, Razafinjato BR, et al. Assessing trends in the content of maternal and child care following a health system strengthening
https://openalex.org/W2087084897	https://scholarworks.wm.edu/cgi/viewcontent.cgi?article=2861&context=vimsarticles	English	null	Effects of terrestrial–aquatic connectivity on an estuarine turtle	Diversity and distributions	2,015	cc-by	9,327	Effects of terrestrial–aquatic connectivity on an estuarine turtle Effects of terrestrial–aquatic connectivity on an estuarine turtle Robert Isdell Follow this and additional works at: https://scholarworks.wm.edu/vimsarticles Part of the Marine Biology Commons, and the Terrestrial and Aquatic Ecology Commons This Article is brought to you for free and open access by the Virginia Institute of Marine Science at W&M ScholarWorks. It has been accepted for inclusion in VIMS Articles by an authorized administrator of W&M ScholarWorks. For more information, please contact scholarworks@wm.edu. W&M ScholarWorks W&M ScholarWorks Virginia Institute of Marine Science Virginia Institute of Marine Science Virginia Institute of Marine Science Isdell, Robert; Chambers, Randolph M.; Bilkovic, Donna M.; and Leu, Matthias, Effects of terrestrial–aquatic connectivity on an estuarine turtle (2015). Diversity and Distributions, 21(6), 643-653. DOI: 10.1111/ddi.12289 Recommended Citation Recommended Citation Isdell, Robert; Chambers, Randolph M.; Bilkovic, Donna M.; and Leu, Matthias, Effects of terrestrial–aquatic connectivity on an estuarine turtle (2015). Diversity and Distributions, 21(6), 643-653. DOI: 10.1111/ddi.12289 This Article is brought to you for free and open access by the Virginia Institute of Marine Science at W&M ScholarWorks. It has been accepted for inclusion in VIMS Articles by an authorized administrator of W&M ScholarWorks. For more information, please contact scholarworks@wm.edu. ScholarWorks. For more information, please contact scholarworks@wm.edu. Diversity and Distributions, (Diversity Distrib.) (2015) 21, 643–653 A Journal of Conservation Biogeography A Journal of Conservation Biogeography Effects of terrestrial–aquatic connectivity on an estuarine turtle BIODIVERSITY RESEARCH BIODIVERSITY RESEARCH BIO Robert E. Isdell1,2, Randolph M. Chambers1, Donna M. Bilkovic2 and Matthias Leu1* 1Biology Department, College of William & Mary, Williamsburg, VA 23187, USA, 2Virginia Institute of Marine Science, College of William & Mary, Gloucester Point, VA 23062, USA Keywords Aquatic–terrestrial ecotone, connectivity, crab pots, diamondback terrapin, salt marsh, shoreline development. Murray, 2012), biogeochemical (Carpenter et al., 1999), and biological interactions (Nakano & Murakami, 2001; Cristol et al., 2008), is less understood (Talley et al., 2006). Research primarily has focused on terrestrial effects on aquatic systems through processes such as eutrophication (Kemp et al., 2005; Worm & Lotze, 2006), shoreline armouring (Morley et al., 2012), land use (Seitz et al., 2006; Bilkovic & Roggero, 2008), and non-native invasive species (Silliman & Bertness, ABSTRACT Aim Estuaries world-wide have been modiﬁed or fragmented due to human stressors in their terrestrial and aquatic components. Estuary fragmentation often results in reductions in species richness, diversity and connectivity. Effects of human modiﬁcation on estuaries have been well studied, but less is known about how land use alters connectivity of the terrestrial–aquatic ecotone. We studied the relationship between terrestrial–aquatic connectivity and the distri- bution of an estuarine turtle, diamondback terrapin (Malaclemys terrapin). A Journal of Conse Correspondence: Matthias Leu, Biology Department, College of William & Mary, Williamsburg, VA 23187, USA. E-mail: mleu@wm.edu Location Chesapeake Bay, Virginia, USA. Methods We conducted diamondback terrapin surveys at 165 sites from late spring to mid-summer in 2012 and 2013. We evaluated associations between terrapin occurrence, land use, salt marsh, shoreline armouring and crabbing intensity in concentric–circular neighbourhoods ranging from 0.27 to 2 km to cover daily and annual terrapin movements. We used occupancy modelling and model averaging to identify key terrestrial and aquatic variables explaining het- erogeneity in terrapin occupancy. We evaluated the ﬁnal model with an independent data set and identiﬁed occurrence thresholds for key variables. Results Diamondback terrapin occupy areas with ≥10% of marsh within a 750-m neighbourhood, ≤17% armoured shoreline within a 1-km neighbour- hood, ≤20% of agriculture within a 500-m neighbourhood, ≤33% low-density housing within a 270-m neighbourhood and ≤9 active crab pots within a 270- m neighbourhood. Our model performed well when evaluated with an inde- pendent data set. Diversity and Distributions Diversity and Distribu Main conclusions We are the ﬁrst to identify thresholds and quantify negative associations between the distribution of diamondback terrapin and alterations to terrestrial–aquatic connectivity from land development, shoreline armouring, and ﬁshing activity. Because diamondback terrapin responses are reﬂective of changes in coastal habitats, especially marshes, terrapin occurrence can be used to direct wetlands conservation and restoration efforts. Correspondence: Matthias Leu, Biology Department, College of William & Mary, Williamsburg, VA 23187, USA. E il l @ d DOI: 10.1111/ddi.12289 http://wileyonlinelibrary.com/journal/ddi 643 Murray, 2012), biogeochemical (Carpenter et al., 1999), and biological interactions (Nakano & Murakami, 2001; Cristol et al., 2008), is less understood (Talley et al., 2006). Research primarily has focused on terrestrial effects on aquatic systems through processes such as eutrophication (Kemp et al., 2005; Worm & Lotze, 2006), shoreline armouring (Morley et al., 2012), land use (Seitz et al., 2006; Bilkovic & Roggero, 2008), and non-native invasive species (Silliman & Bertness, ª 2015 John Wiley & Sons Ltd 1Biology Department, College of William & Mary, Williamsburg, VA 23187, USA, 2Virginia Institute of Marine Science, College of William & Mary, Gloucester Point, VA 23062, USA Study area The Chesapeake Bay is located between the eastern and wes- tern shores of Maryland and Virginia, USA. Approximately 15,000 km of the bay’s shoreline falls within Virginia (NOAA NGDC, 2000), where land use varies from wildlife manage- ment in refuges to agriculture, housing, and commercial and industrial activities. An estimated 18% of the tidal shoreline has been armoured to prevent erosion, 32% of riparian land cover has been converted to residential or commercial devel- opment, and approximately 47,000 docks have been con- structed (Titus et al., 2009; Center for Coastal Resources Management, 2011). Commercial crabbing in the nearshore waters is extensive throughout the Chesapeake Bay, with more than 385,000 pots permitted in Virginia in 2013 (Bilkovic et al., 2014). Annually, approximately 20% of pots are lost (Havens et al., 2008), and Bilkovic et al. (2014) con- servatively estimated 50,000 derelict crab pots (those pots lost or abandoned by ﬁshermen) are added to the Virginia portion of the Bay each year. We investigated associations between the spatial distribution of the diamondback terrapin (Malaclemys terrapin; terrapin hereafter) throughout the Chesapeake Bay, Virginia, and fac- tors that can inﬂuence functional connectivity at the terres- trial–aquatic ecotone. Terrapin feed, mature and mate in tidal salt marshes and adjacent open water, but, like all reptiles, the terrapin must lay its eggs on dry land (Brennessel, 2006). Human activities can disrupt the connection between the near- shore aquatic and terrestrial ecosystems the terrapin inhabits. For example, shoreline structures (i.e. bulkheads, riprap and docks) are common throughout much of the Chesapeake Bay (Isdell, 2014). Shoreline structures and armouring block terra- pins from moving from the water to nesting habitat above the tideline (Roosenburg, 1991; J. M. Winters pers. comm.). Addi- tionally, shoreline armouring is associated with changes in spe- cies composition, and reductions in species richness and structural complexity of nekton and benthic macroinvertebrate communities (Bilkovic et al., 2006; Seitz et al., 2006; Bilkovic & Roggero, 2008). These changes may reduce the availability and diversity of prey for the terrapin. The abundance of terra- pin in Virginia has not been estimated. METHODS Of all anthropogenic modiﬁcations, landscape/seascape fragmentation has the greatest effect on estuarine connectiv- ity (Rizkalla & Swihart, 2006). The largest estuary in the United States, the Chesapeake Bay, exempliﬁes estuarine fragmentation (Jantz et al., 2005). For centuries, humans have modiﬁed both terrestrial and aquatic systems within this region by extracting resources for sustenance, defence, and social and economic gain (Bradley, 2011). The ongoing fragmentation has led to reductions in species abundance, richness, diversity, and distribution (King et al., 2005; DeLuca et al., 2008). INTRODUCTION The degree of spatial and temporal exchange of energy, material and organisms constitutes functional landscape con- nectivity (Wu, 2013). The majority of research on the connectivity of ecological processes has occurred within either terrestrial or aquatic systems, while connectivity at the terrestrial–aquatic interface, which includes physical (Ells & 643 ª 2015 John Wiley & Sons Ltd R. E. Isdell et al. 2004; Chambers et al., 2012). Disturbance in either the ter- restrial or aquatic system has the potential to decrease con- nectivity. However, beyond physical phenomena such as waves, tides, and ﬂooding, comparatively few studies have demonstrated connectivity from aquatic to terrestrial ecosys- tems (Bouchard & Bjorndal, 2000; Cristol et al., 2008). thesized that terrapin occupancy relates positively to their primary habitat (salt marsh) and negatively to elevated levels of human stressors, such as crabbing activity, shoreline armouring, and coastal development. Survey design We used a priori stratiﬁed, random sampling to select survey points (Fig. 1) across gradients of variables previously deter- mined to inﬂuence terrapin presence or absence (henceforth occurrence) either positively (marsh) or negatively (shoreline armouring and crabbing pressure; Roosenburg, 1991; Rook et al., 2010). For a detailed explanation of our survey point selection methodology, see Appendix S1 in Supporting Infor- mation. We visited each point by kayak on three occasions. We conducted surveys from early May through late July (2012) or early August (2013); we extended the ﬁeld season in 2013 because of adverse weather during May and June. We used a Garmin GPSmap 60Cx to navigate to each survey point, where we anchored our kayak 50 5 m from the shoreline. Before the start of each survey, we measured weather variables that might affect detection probabilities (see Appendix S1). We scanned the open water for 15 min, sufﬁciently long to detect any emerging terrapin during months of peak activity (Brennessel, 2006) when average div- ing time is 8.4 5.7 min (Baker et al., 2013). We recorded the time of each detection and used an 89 monocular laser- rangeﬁnder (Zeiss Victory PRF; Oberkochen, Baden-Wu¨rt- temberg, Germany) to estimate the distance between the observer and a terrapin. At the end of each survey, we recorded the distance from the observer to each crab-pot buoy and used the number of pots within 270 m as a measure of current crabbing intensity. Coastal development increases the abundance of synan- thropic terrapin-nest predators (animals that thrive in human developed areas (Johnston, 2001)) such as raccoons (Procyon lotor) and crows (Corvus spp.; Hart & Lee, 2006; Ernst & Lovich, 2009). Terrapins also die as bycatch in active or derelict blue crab (Callinectes sapidus) pots that have been placed within their home ranges. Numerous studies have shown that whether baited or unbaited, terrapins enter pots and drown, sometimes in massive numbers (Roosenburg et al., 1997; Grosse et al., 2011; Morris et al., 2011). Our objectives for this study were to use occupancy mod- elling (MacKenzie et al., 2006) to (1) determine the distribu- tion of terrapins throughout the southern Chesapeake Bay, Virginia, (2) assess which habitat and human activities affected their distribution, (3) explore possible connections between terrapin distribution and terrestrial–aquatic linkages and (4) identify management targets for restoration and con- servation of estuaries in the Chesapeake Bay. Survey design We hypo- Diversity and Distributions, 21, 643–653, ª 2015 John Wiley & Sons Ltd 644 Diamondback terrapin occupancy model Figure 1 The study area in the southern Chesapeake Bay. Black circles indicate survey points; white circles indicate evaluation points. shoreline armouring data (Center for Coastal Resources Management, 2011), we calculated the proportion of shore- line armouring for the total shoreline length in a given area. Additionally, to account for the heterogeneous distribution of armoured shoreline within an area, we generated the bio- logical distance (i.e. not the shortest distance but the distance based on area where terrapins would traverse) from each sur- vey point along the shoreline to the nearest section of armouring. All spatial analyses were conducted in ARCGIS 10.0 (ESRI, 2011). To account for aquatic threats, we included derelict and active blue crab pots in our analyses. A Marine Debris Loca- tion and Removal Program conducted in Virginia over four consecutive winters (2008–2012) recovered nearly 32,000 spa- tially referenced derelict (lost or abandoned) pots using side- scan sonar (Bilkovic et al., 2014). We used this data set as both a way to assess the potential impact of derelict crab pots on terrapin distribution as well as a proxy for crabbing pressure prior to the beginning of this study. Because no spatial data set of active pots existed, we used the mean number of pots counted at each survey point. As crabbing pressure varies throughout the season, we divided the mean number of pots per site by the SE + 1; we added one to the SE to avoid dividing by SE = 0 for sites where crab pot counts were homogenous. We used mean adjusted by SE as it weighted sites with consistent crabbing pressure higher than sites with sporadic crabbing pressure. Figure 1 The study area in the southern Chesapeake Bay. Black circles indicate survey points; white circles indicate evaluation points. All site-speciﬁc covariates (with the exception of active pots) were evaluated in concentric–circular neighbourhoods ranging in radii from 270 m to 2 km with intervals of ~ 250 m. Neighbourhood extents overlap with daily and annual terrapin habitat use. We used focal statistics to calcu- late either proportion or intensity within neighbourhoods for all survey points. Site-speciﬁc covariates We selected different landscape and seascape features to explain the observed variation in terrapin distribution. We obtained three terrestrial variables, low-intensity develop- ment, agriculture, and marsh, from SEGAP (Biodiversity & Spatial Information Center, 2010). First, because terrapin eggs and juveniles are known prey of synanthropic predators, we predicted that low-intensity development would have a negative effect on terrapin distribution. Terrapins have also been observed nesting in agricultural ﬁelds (Roosenburg, 1994; Feinberg & Burke, 2003). We hypothesized that as the amount of suitable nesting beach in an area declined due to human development, terrapins might turn to agricultural ﬁelds as an alternate nesting substrate. Therefore, we pre- dicted that agriculture might have a positive effect on terra- pin distribution. Terrapins occurring along the Atlantic coast have been shown to rely on marshes for both food and shel- ter (Roosenburg et al., 1999; Brennessel, 2006; Butler et al., 2006). We hypothesized that terrapin occupancy would be positively related to the amount of marsh in an area. Diversity and Distributions, 21, 643–653, ª 2015 John Wiley & Sons Ltd Occupancy models We used single-season occupancy models (MacKenzie et al., 2006) to estimate detection probability (P) based on survey- speciﬁc covariates and occupancy W) on the basis of site-spe- ciﬁc covariates at all neighbourhood sizes. We conducted all occupancy modelling in package ‘Unmarked’ (Fiske & Chan- dler, 2011) in R (R Development Core Team, 2011). We ﬁrst identiﬁed the best neighbourhood size for each site-speciﬁc co- variate using univariate analyses where the neighbourhood size with the lowest Akaike’s information criterion (AIC) value was retained. Any survey-speciﬁc covariate and the neighbourhood size for each site-speciﬁc covariate that received a lower AIC value than the null model was then selected for inclusion in a global model. We then used Pearson’s correlation coefﬁcient to test multicollinearity among selected variables. If two or more variables had a correlation coefﬁcient > 0.7, we retained the variable that we hypothesized had the strongest relation- ship with occupancy (Leu et al., 2011). Additionally, we plot- ted dependent and independent variables to check for linear and nonlinear associations (Zuur et al., 2010). We centred and We also included variables at the terrestrial–aquatic eco- tone to explain the observed heterogeneity in terrapin distri- bution. We included shoreline armouring because it prevents terrapins from moving from the water to land above the tide-line (Roosenburg, 1991; J. M. Winters pers. comm.). For 645 Diversity and Distributions, 21, 643–653, ª 2015 John Wiley & Sons Ltd R. E. Isdell et al. Response Calculator for ARCGIS (Hanser et al., 2011). Thresh- olds for spatial variables were determined where the pre- dicted occupancy curve intersected the predicted occupancy cut-off value derived from the sensitivity–speciﬁcity analysis (Liu et al., 2005). We calculated the predicted occupancy curve across the range of values for each spatial variable while keeping all other variables in the model at mean value. The intersection value was then used to estimate the thresh- old for each spatial predictor variable. We ﬁt a loess curve to the predicted probability of occurrence and its upper and lower 95% conﬁdence intervals. The same methods were used to derive the threshold for the active crab pots (local variable), but rather than using occupancy values derived from the entire study area, we used study site-speciﬁc values. scaled all of the variables on the basis of means and standard deviations from all points. Model evaluation To determine whether the ﬁnal model effectively predicted occurrence beyond the original data set, we used an indepen- dent data set of terrapin presence locations collected by boat surveys during summer 2011 (Bilkovic et al. 2012). Surveys were conducted across a large portion of Virginia’s middle peninsula (Fig. 1), and each of the 174 terrapin presence loca- tions was georeferenced. For each observation, we extracted predicted occupancy values from the ﬁnal model. Because our evaluation data set only included presence locations, we bin- ned the extracted and study area-wide occupancy probabilities into deciles (i.e. 0–10%, 11–20%, . . ., 91–100%) and calculated the proportion of the study area in each bin. We estimated the expected number of observations in each bin as Terrapins were susceptible to anthropogenic stressors at multiple scales, ranging from 270 to 1,000 m. Within the home range scale, terrapin occurrence was affected by the proportion of low-intensity development within a 270-m neighbourhood, the number of active crab pots within a 270-m neighbourhood, the proportion of agriculture within a 500-m neighbourhood and the number of derelict crab pots within a 500-m neighbourhood. The proportion of marsh within a 750-m neighbourhood and the proportion of armoured shoreline within a 1-km neighbourhood had an effect on terrapin occurrence at or just above the terra- pin home range scale. Cost–path distance from sites to shoreline armouring was not an important predictor of ter- rapin occupancy (see Table S2 for AIC values and variable selection). BiNWi P 10 i¼1 BiNWi N (1) (1) where Bi is proportion of the study area in the ith bin, N is number of observations in the validation data, and Wi is median value of the predicted probability of occurrence for the ith bin. We used linear regression to assess ﬁt between model predictions and observations. A perfect model would have a slope of 1 and an intercept of 0. Proportion of marsh within a 750-m neighbourhood had the strongest positive effect and proportion of agriculture within a 500-m neighbourhood had the strongest negative effect on terrapin occupancy. Both current crabbing intensity and per cent armoured shoreline within a 1-km neighbour- hood had intermediate negative effects on occupancy (Table 1). The ﬁnal, spatially applied model (Fig. 2) was based on 45 models accounting for 95% of the total AIC weight (see Table S3). RESULTS We surveyed a total of 165 sites (85 in 2012, 80 in 2013; Fig. 1), with an average distance between sites of 1,011 159 m (mean SE). Terrapins were observed at 55 of the 165 sites (na€ıve occupancy = 33%). Of sites where ter- rapins were detected, 66% had ≤3 terrapins in total (range 1–14 terrapins) across three surveys. Two detection covari- ates, starting time and precipitation, explained heterogeneity in detection probabilities. Estimates of detection probability did not differ substantially between the null model (0.49 0.05) and the averaged models (0.46 0.07). We found no nonlinear associations between the dependent and independent variables, nor any multicollinearity among vari- ables that had lower AIC values than the null model. Our model structure consisted of six site-speciﬁc and two detec- tion covariates. We spatially applied the ﬁnal model to create a predictive surface map. All predictions were restricted to within 1500 m of the shoreline based on radio telemetry estimates of terrapin movement patterns (Tulipani 2013). Occupancy models We included all statistically independent variables in a glo- bal model and ran models with all possible combinations of those variables (Doherty et al., 2012). Although we estimated detection probability as a function of survey-speciﬁc covari- ates (see Table S1), those associations were not our focus. We model-averaged parameter estimates for variables included in the set of models for which AIC model weights summed to 0.95 of the cumulative AIC weight (Burnham & Anderson, 2002). We included the model-averaged parameter estimates in a ﬁnal model to estimate probability of occur- rence at each point. We assessed model predictive capabilities by computing the area under the receiver-operator curve (AUC). We used package ‘ROCR’ (Sing et al., 2013) in R (R Development Core Team, 2011) to derive the cut-off value – the probability of occurrence at which the model predicts false positives and false negatives with equal accuracy (Metz, 1978). Although Lobo et al. (2008) questioned the use of AUC to assess model predictive capabilities, a high AUC in combination with model evaluation typically increases conﬁ- dence in the predictive capabilities of a model. Thresholds We identiﬁed thresholds for spatial predictor variables above or below which terrapins were present using the Dose Diversity and Distributions, 21, 643–653, ª 2015 John Wiley & Sons Ltd 646 Diamondback terrapin occupancy model Table 1 Model-averaged regression coefﬁcients and standard errors for variables included in the 45 models for which cumulative Akaike’s information criterion (AIC) weights summed to 0.95. Variables were centred and scaled to make the b-values directly comparable Variable Neighbourhood (m) b SE AIC cumulative weight Intercept 3.69 0.85 Proportion of marsh 750 2.64 1.05 1.00 Proportion of agriculture 500 1.37 0.67 0.89 Current number of crab pots 270 0.90 0.40 0.72 Percentage of shoreline armoured 1000 0.80 0.49 0.48 Percentage of low-intensity development 270 0.48 0.37 0.44 Number of derelict crab pots 500 0.11 0.06 0.54 study documents a strong association with marsh and identi- ﬁed a 10% threshold within a neighbourhood of 750 m, which suggests that the terrapin is an area-sensitive species. As such, we attribute the negative associations of terrapin with the terrestrial–aquatic modiﬁcations examined in this study to the effects of marsh fragmentation and subsequent altered connectivity. Further, our work suggests that terra- pins respond in predictable ways to alterations to the aquatic–terrestrial ecotone. The sensitivity–speciﬁcity analyses showed an optimal occupancy cut-off of 0.39 and AUC = 0.91, indicating a robust predictive model. Model-based conditional estimates of occupancy (points where terrapins were observed or pre- dicted probability of occurrence was > 0.39) indicated 73 of 165 sites (44%) were likely occupied (versus our 33% na€ıve occupancy estimate). Most of these occupied sites were within extensive marshes. Our model suggested that 26% of the shoreline in our study was occupied by terrapin and that the most extensively occupied areas were along the north- east sector of our study area (Fig. 2a) and along the western shore near the open waters of the Chesapeake Bay. These areas are relatively far from urban development. Our study suggests that terrestrial–aquatic connectivity is inﬂuenced at multiple scales, as has been well established separately for both terrestrial (Bergin et al., 2000; Hanser et al., 2011; Martin & Fahrig, 2012; Soranno et al., 2014) and aquatic systems (Bostr€om et al., 2011; Hitt et al., 2011; Wedding et al., 2011). We identiﬁed a number of spatial scale-dependent factors that were associated with the distri- bution of terrapin. Thresholds Both the proportion of armoured shore- line within a 1-km neighbourhood and the proportion of marsh within a 750-m neighbourhood explained heterogene- ity of terrapin occupancy at or just beyond the home range scale. Important variables within the home range scale are agricultural land cover and derelict crab pots within a 500-m neighbourhood. Proportion of low-intensity development within a 270-m neighbourhood was associated with local dis- tribution of terrapin, likely determining whether a section of shoreline within a terrapin’s home range was unsuitable. These results are consistent with the results of other studies of turtles where habitat variables were most important at the home range scale (Rasmussen & Litzgus, 2010). We found that our ﬁnal model predicted well when evalu- ated with independent terrapin presence data collected in the western portion of the study area. Predicted and observed values related positively (P < 0.001). Neither the intercept (b0 = 2.59, SE = 2.42) nor the slope (b1 = 0.85, SE = 0.08) varied signiﬁcantly (a = 0.05) from the expected values of 0 and 1, respectively. The adjusted R2 for the model was 0.92. We ran dose–response calculations to estimate terrapin occupancy thresholds for proportion of salt marsh, agricul- ture, low-intensity housing, armoured shoreline, derelict crab pots and active crab pots (Fig. 3). Terrapin were unlikely to occur in areas with < 10% (17.6 ha) of salt marsh within a 750-m neighbourhood (Fig. 3a). On the basis of this thresh- old, approximately 72% of shoreline in the study area is unsuitable for terrapin. Thresholds for the other variables, above which terrapin presence was unlikely, were ≥17% armoured shoreline within a 1-km neighbourhood, ≥19.6% (15.4 ha) of agriculture within a 500-m neighbourhood, ≥33% (7.6 ha) of low-intensity housing within a 270-m neighbourhood and ≥9 active pots within a 270-m neigh- bourhood (Fig. 3b–d, f). Because the upper conﬁdence inter- val for derelict crab pots did not cross the occupancy threshold, we were unable to deﬁne a threshold for this variable. Our study suggests that terrapins are area sensitive to marsh and that marshes in excess of 17.6 ha within a 750-m neighbourhood are most suitable. While not a focus of our study, the effect of marsh conﬁguration on terrapin occu- pancy is an important consideration and worthy of future investigation. Thresholds For example, the total area of fringing marshes along a convoluted shoreline may exceed the above threshold and even support ‘robust terrapin populations’, as observed in Maryland (W. M. Roosenburg pers. comm.). Smaller total areas of narrow, disjunct fringing marshes, however, may not be sufﬁcient for core terrapin habitat, but may still provide food and shelter for dispersing individuals or females on long-distance nesting forays. In this sense, the connectivity Diversity and Distributions, 21, 643–653, ª 2015 John Wiley & Sons Ltd DISCUSSION We are the ﬁrst to quantify multiple-scale associations between terrapin distribution and habitat variables. Our 647 Diversity and Distributions, 21, 643–653, ª 2015 John Wiley & Sons Ltd between core terrapin habitat may be mediated by narrow fringing marshes. occurrence, synanthropic predators including the American crow (Corvus brachyrhynchos), ﬁsh crow (Corvus ossifragus), (a) (b) Figure 2 Predicted probability of occupancy (Ψ) of diamondback terrapin in the Chesapeake Bay, Virginia, and illustrative sections of shoreline with high (a) and low (b) probabilities of occupancy. (a) (b) (c) (d) (e) (f) Figure 3 Dose–response relationship for variables associated strongly with probability of terrapin occurrence. Solid black lines represent predicted probability of occurrence; short-dashed lines delineate upper and lower 95% conﬁdence intervals; dotted lines indicate intersection between the upper 95% conﬁdence interval and the predicted occupancy threshold value; and horizontal long-dashed lines represent predicted occupancy threshold value. Grey bars indicate the proportion of the study area in each bin. Note that the right axis on panel (f) is proportion of study sites rather than proportion of study area.Panels a-e represent % marsh, % armoured shoreline, % agriculture, % low-intensity development, and number of derelict pots, respectively. R. E. Isdell et al. R. E. Isdell et al. (a) (b) Figure 2 Predicted probability of occupancy (Ψ) of diamondback terrapin in the Chesapeake Bay, Virginia, and illustrative sections of shoreline with high (a) and low (b) probabilities of occupancy. (a) Figure 2 Predicted probability of occupancy (Ψ) of diamondback terrapin in the Chesapeake Bay, Virginia, and illustrative sections of shoreline with high (a) and low (b) probabilities of occupancy. between core terrapin habitat may be mediated by narrow occurrence, syna (a) (b) (c) (d) (e) (f) (a) (b) (c) (d) (e) (f) (a) (b) Figure 3 Dose–response relationship for variables associated strongly with probability of terrapin occurrence. Solid black lines represent predicted probability of occurrence; short-dashed lines delineate upper and lower 95% conﬁdence intervals; dotted lines indicate intersection between the upper 95% conﬁdence interval and the predicted occupancy threshold value; and horizontal long-dashed lines represent predicted occupancy threshold value. Grey bars indicate the proportion of the study area in each bin. Note that the right axis on panel (f) is proportion of study sites rather than proportion of study area.Panels a-e represent % marsh, % armoured shoreline, % agriculture, % low-intensity development, and number of derelict pots, respectively. Diamondback terrapin occupancy model Living shorelines prevent erosion by providing protection, restoration, or enhancement of wet- land habitats through strategic placement of plants, stone, and sand while maintaining natural coastal processes (Bilko- vic & Mitchell, 2013). Many US states, including Maryland, Virginia and North Carolina, have enacted regulations to encourage or require the use of living shorelines instead of traditional armouring, such as bulkheads, for shoreline ero- sion control. Preferentially selecting a living shoreline approach over armouring to stabilize a shore may help to maintain or even enhance terrestrial–aquatic connectivity for terrapin throughout its range. Our study adds to the growing body of evidence for the importance of maintaining shore- line integrity over a large spatial extent. The strong, negative association of active crab pots with terrapin occupancy is in line with our hypothesis and other studies that have demonstrated the detrimental effects of crab pots on terrapins (Roosenburg et al., 1997; Dorcas et al., 2007; Wolak et al., 2010). Our threshold of ≥9 active pots within a 270-m neighbourhood indicates that a rela- tively high crabbing pressure is required to affect terrapin occupancy. Local terrapin abundance, however, may be affected by lower levels of crabbing pressure. Some locations with lower crabbing pressure could be occupied by a declin- ing population, possibly resulting in a lag effect on terrapin occupancy. We suspect that crab pots affect terrapin connec- tivity in much the same way as a gill net catches ﬁsh. With a set mesh size, gill nets passively capture those animals that are the correct size to be caught and those unlucky enough to become entangled. Crab pots may act similarly as a ﬁlter to connectivity. Of the adults, crab pots primarily catch male terrapin as they are small enough to ﬁt through the openings in the crab pots (Roosenburg et al., 1997). Some juveniles are small enough to escape, and mature females are often too large to ﬁt. Thus, as male terrapin have been docu- mented as the most important for dispersal (Sheridan et al., 2010), crab pots may reduce immigration/emigration success among habitat patches. One contradictory result was the positive relationship of derelict crab pots to terrapin occurrence. This variable had the smallest relative effect of all variables. One possible expla- nation for this result is that for each pot removed from the water, there may be a slight increase in terrapin survival due to the removal of those potentially deadly traps. Diamondback terrapin occupancy model loose soil near the beginning of the nesting season. However, as crops grow, roots (a common source of nest mortality on beaches (Brennessel, 2006)) and agricultural activities (e.g. application of biocides and cultivation) may destroy nests. Agricultural lands are ecological traps for other species (Hiron et al., 2012; Northrup et al., 2012), and some species of turtles avoid agricultural lands (Bodie & Semlitsch, 2000; Rizkalla & Swihart, 2006). Although agricultural run-off is often cited as a cause of estuarine eutrophication (Kemp et al., 2005), the effects of eutrophication on terrapin distri- bution are unclear. (Burdick & Short, 1999; Shafer, 1999), and boat trafﬁc is high (Liddle & Scorgie, 1980; Asplund & Cook, 1997), all of which may negatively affect terrapin occupancy. Many water- front property owners also deploy recreational crab pots, which are identical to commercial pots, from their docks. Virginia law allows two pots per person per household (VA §28.2–262). More than 47,000 docks are along Virginia’s coastline, and recreational pots may be a substantial source of terrapin mortality. Additionally, because most docks occur in the relatively shallow waters that are inhabited by terra- pins, many more recreational pots than commercial pots may overlap with terrapin habitat (Harden & Williard, 2012). Other studies have demonstrated reductions in estuarine integrity when 10–25% of the shoreline is armoured (Silli- man & Bertness, 2004; Bilkovic et al., 2006; Bilkovic & Rogg- ero, 2008; DeLuca et al., 2008). Our threshold of 17% (Fig. 3b) falls within the ranges documented by these other studies. Although shoreline armouring prevents terrapin from accessing a given location (Roosenburg, 1991; Wnek, 2010), the effect of armouring on terrapin distribution is likely from a reduction in habitat quality across extensive areas rather than a local nesting effect. Other studies have indicated that shoreline armouring is increasing rapidly (Isdell, 2014) and will continue to increase in the future (Woth et al., 2006). As more of the shoreline becomes armoured, terrapin habitat will become increasingly frag- mented, and ecosystem connectivity will be diminished. For- tunately, this is a recognized problem in the Chesapeake Bay, and for the last 20+ years, there has been a concerted effort by local institutions, agencies and private groups to restore wetlands, reduce or replace armouring with nature-based shoreline protection designs (or living shorelines), and to encourage best management practices for shoreline stabiliza- tion (Koslow et al., 2006). DISCUSSION (c) (d) (c) (e) (f) (e) between core terrapin habitat may be mediated by narrow fringing marshes. between core terrapin habitat may be mediated by narrow fringing marshes. occurrence, synanthropic predators including the American crow (Corvus brachyrhynchos), ﬁsh crow (Corvus ossifragus), raccoon and red fox (Vulpes vulpes) thrive in landscapes dominated by agriculture (Johnston, 2001; Graser et al., 2012) and in urban areas (Marzluff et al., 2001). Many agri- cultural ﬁelds are ploughed in spring, which provides open, Contrary to our hypothesis, we found a strong, negative association between per cent cover of agricultural land and terrapin occupancy. Although no studies have demonstrated that agriculture is the mechanism for decreased terrapin Diversity and Distributions, 21, 643–653, ª 2015 John Wiley & Sons Ltd 648 Diamondback terrapin occupancy model ACKNOWLEDGEMENTS We thank Bruce Pﬁrrmann and Mary Isdell for help in the ﬁeld. Timothy Russell helped with spatial shoreline delineations, and John Swaddle, Cheryl Leu and Erica Fleishman provided helpful editorial comments. This study was funded by the Common- wealth of Virginia (ML), the College of William & Mary (RI), Virginia Sea Grant (DB, RC, and ML) and NOAA (DB and RC). This paper is Contribution No. 3421 of the Virginia Institute of Marine Science, College of William & Mary. The results of this study have direct implications for man- agement throughout the terrapin’s range. Salt marsh conser- vation, the expansion of shoreline armouring and coastal development, and bycatch in ﬁshing gear are important issues not only from Cape Cod, Massachusetts to Corpus Christi, Texas, but throughout coastal regions of the world. Additionally, all of these factors are likely to be affected by rising sea level. As sea level continues to rise, the extent of salt marshes along the Atlantic coast is likely to decrease substantially (Titus et al., 2009). Much of this loss will be due to interactions between rising sea level and the preven- tion of inland migration of marshes by topography, includ- ing embankments with steep slopes and shoreline armouring. In regions where subsidence is occurring, such as the Chesapeake Bay, marshes likely will be unable to accrete vertically to keep apace of anticipated accelerated sea level rise without a signiﬁcant sediment source (Craft et al., 2009). Although we do not know how other factors such as bank erosion might provide sediment to sustain marshes (Bozek & Burdick, 2005), the cumulative effects of sea level rise, the human response to protect personal property and subsequent loss of marshes would exacerbate terrapin habitat loss and result in further decreased terrestrial–aquatic connectivity. R. E. Isdell et al. agricultural ﬁelds, limiting future shoreline armouring in areas below the 17% threshold, requiring turtle excluder devices (TEDs) on those crab pots that overlap with terrapin habitat, and a strong effort to maintain extensive salt marshes. Improving terrapin habitat quality would also have positive effects on other estuarine species, as demonstrated by Roosenburg et al. (2003). The thresholds identiﬁed in this study provide spatially explicit, data-driven conservation and restoration targets for the Chesapeake Bay, while the meth- ods provide a cost-effective and efﬁcient way to establish those targets in other systems. When examined together, the variables identiﬁed by this study signiﬁcantly affect terrestrial–aquatic connectivity in the region. Although we cannot assess the likelihood of gene ﬂow among meta-populations of terrapin, as our study focused on local factors associated with terrapin occurrence, one could imagine a dramatic reduction in gene ﬂow among meta-populations where the terrestrial–aquatic connectivity was impaired. The dispersal of male terrapin between meta- populations, for example, would be reduced by high num- bers of crab pots in which terrapin drown. In addition to reduced gene ﬂow, Wolak et al. (2010) found that terrapin in the Chesapeake Bay are signiﬁcantly larger and mature faster than other populations in the Mid-Atlantic, indicating that crab pot-related mortality may have resulted in direc- tional selection in local populations. Because of the spatial overlap between terrapin habitat, some commercial crabbing, and most recreational crabbing, implementation of measures to reduce crab pot-related terrapin mortality would be recommended. Diamondback terrapin occupancy model The small positive result could also be a statistical anomaly. Derelict crab pots were recovered with the use of larger, deeper- hulled boats than are typically used by crabbers when they set and retrieve pots. This led to an undersampling of the shallow waters where terrapins and crab pots are more likely to overlap. An alternative hypothesis is that high-quality ter- rapin habitat overlaps with high-quality blue crab habitat, and therefore, we see an increased ﬁshing effort in those areas. However, this seems unlikely as we have found a strong negative association between active crab pots and ter- rapin occupancy. The active pots that were counted at each site are, therefore, more likely to accurately reﬂect the nega- tive impact that crab pots are known to have on terrapins. Low-intensity development may have many of the same effects on terrapin distribution as agriculture. The density of synanthropic predators can be relatively high in these areas (Neatherlin & Marzluff, 2004), and terrapins lay their eggs in the low-quality habitat around houses (R. E. Isdell pers. obs.). Urban areas also have relatively high densities of recre- ational docks and piers (Isdell, 2014). Several studies have demonstrated that near docks, diversity of nekton is rela- tively low (Scheuerell & Schindler, 2004; Able & Duffy- Anderson, 2006), submerged aquatic vegetation is limited Diversity and Distributions, 21, 643–653, ª 2015 John Wiley & Sons Ltd 649 R. E. Isdell et al. Diamondback terrapin occupancy model Bilkovic, D.M., Havens, K., Stanhope, D. & Angstadt, K. (2014) Derelict ﬁshing gear in Chesapeake Bay, Virginia: spatial patterns and implications for marine fauna. Marine Pollution Bulletin, 80, 114–123. DeLuca, W.V., Studds, C.E., King, R.S. & Marra, P.P. (2008) Coastal urbanization and the integrity of estuarine water- bird communities: threshold responses and the importance of scale. Biological Conservation, 141, 2669–2678. Biodiversity and Spatial Information Center (2010) SEGAP. Available at: http://www.basic.ncsu.edu/segap/ (accessed 18 November 2013). Doherty, P.F., White, G.C. & Burnham, K.P. (2012) Compar- ison of model building and selection strategies. Journal of Ornithology, 152, 317–323. Dorcas, M.E., Willson, J.D. & Gibbons, J.W. (2007) Crab trapping causes population decline and demographic changes in diamondback terrapins over two decades. Bio- logical Conservation, 137, 334–340. Bodie, J.R. & Semlitsch, R.D. (2000) Spatial and temporal use of ﬂoodplain habitats by lentic and lotic species of aquatic turtles. Oecologia, 122, 138–146. Bostr€om, C., Pittman, S., Simenstad, C. & Kneib, R. (2011) Seascape ecology of coastal biogenic habitats: advances, gaps, and challenges. Marine Ecology Progress Series, 427, 191–217. Ells, K. & Murray, A.B. (2012) Long-term, non-local coast- line responses to local shoreline stabilization. Geophysical Research Letters, 39, 1–7. Bouchard, S.S. & Bjorndal, K.A. (2000) Sea turtles as biologi- cal transporters of nutrients and energy from marine to terrestrial ecosystems. Ecology, 81, 2305–2313. Ernst, C.H. & Lovich, J.E. (2009) Turtles of the United States and Canada. Johns Hopkins University Press, Baltimore, MD. Bozek, C.M. & Burdick, D.M. (2005) Impacts of seawalls on saltmarsh plant communities in the Great Bay Estuary, New Hampshire, USA. Wetlands Ecology and Management, 13, 553–568. ESRI (2011) ArcMap. Environmental Systems Research Insti- tute, Redlands, CA. Feinberg, J. & Burke, R. (2003) Nesting ecology and preda- tion of diamondback terrapins, Malaclemys terrapin, at Gateway National Recreation Area, New York. Journal of Herpetology, 37, 517–526. Bradley, C. (2011) The impacts of shoreline development on shallow-water benthic communities in the Patuxent River, MD. Virginia Institute of Marine Science, College of Wil- liam & Mary, Gloucester Point, VA. PhD Dissertation. Fiske, I. & Chandler, R. (2011) unmarked: an R package for ﬁtting hierarchical models of wildlife occurrence and abun- dance. Journal of Statistical Software, 43, 1–23. Brennessel, B. (2006) Diamonds in the marsh: a natural his- tory of the diamondback terrapin. UPNE, Lebanon, NH. Graser, W.H., Gehrt, S.D., Hungerford, L.L. & Anchor, C. (2012) Variation in demographic patterns and population structure of raccoons across an urban landscape. Diamondback terrapin occupancy model The Jour- nal of Wildlife Management, 76, 976–986. Burdick, D.M. & Short, F.T. (1999) The effects of boat docks on eelgrass beds in coastal waters of Massachusetts. Envi- ronmental Management, 23, 231–240. Burnham, K.P. & Anderson, D.R. (2002) Model selection and multimodel inference: a practical information-theoretic approach. Springer, New York, NY. Grosse, A.M., Maerz, J.C., Hepinstall-Cymerman, J. & Dor- cas, M.E. (2011) Effects of roads and crabbing pressures on diamondback terrapin populations in coastal Georgia. The Journal of Wildlife Management, 75, 762–770. Butler, J.A., Seigel, R.A. & Mealey, B.K. (2006) Malaclemys terrapin – diamondback terrapin. Chelonian Research Monographs, 3, 279–295. Hanser, S.E., Leu, M., Knick, S.T. & Aldridge, C.L. (2011) Sagebrush ecosystem conservation and management: ecore- gional assessment tools and models for the Wyoming Basins. Allen Press, Lawrence, KS. Carpenter, S.R., Ludwig, D. & Brock, W.A. (1999) Manage- ment of eutrophication for lakes subject to potentially irre- versible change. Ecological Applications, 9, 751–771. Harden, L.A. & Williard, A.S. (2012) Using spatial and behavioral data to evaluate the seasonal bycatch risk of dia- mondback terrapins, Malaclemys terrapin, in crab pots. Marine Ecology Progress Series, 467, 207–217. Center for Coastal Resources Management (2011) Comprehen- sive Coastal Inventory. Available at: http://ccrm.vims.edu/ gis_data_maps/shoreline_inventories/index.html (accessed 18 November 2013). Hart, K.M. & Lee, D.S. (2006) The diamondback terrapin: the biology, ecology, cultural history, and conservation sta- tus of an obligate estuarine turtle. Terrestrial vertebrates of tidal marshes: evolution, ecology and conservation (ed. by R. Greenberg, J. Maldonado, S. Droege and M.V. McDonald), pp. 206–213. Cooper Ornithological Society, Ephrata, PA. Chambers, R.M., Meyerson, L.A. & Dibble, K.L. (2012) Ecol- ogy of Phragmites australis and responses to tidal restora- tion. Tidal marsh restoration (ed. by C.T. Roman and D.M. Burdick), pp. 81–96. Island Press/Center for Resource Eco- nomics, Washington, D.C., USA. Craft, C., Clough, J., Ehman, J., Joye, S., Park, R., Pennings, S., Guo, H. & Machmuller, M. (2009) Forecasting the effects of accelerated sea-level rise on tidal marsh ecosystem services. Frontiers in Ecology and the Environment, 7, 73–78. Havens, K.J., Bilkovic, D.M., Stanhope, D., Angstadt, K. & Hershner, C. (2008) The effects of derelict blue crab traps on marine organisms in the lower York River, Virginia. North American Journal of Fisheries Management, 28, 1194–1200. Hiron, M., Berg, A. & P€art, T. (2012) Do skylarks prefer autumn sown cereals? Effects of agricultural land use, region and time in the breeding season on density. REFERENCES Able, K.W. & Duffy-Anderson, J.T. (2006) Impacts of piers in the lower Hudson River. The Hudson River estuary (ed. by J.S. Levinton and J.R. Waldman), pp. 428–440. Cam- bridge University Press, New York, NY. Asplund, T.R. & Cook, C.M. (1997) Effects of motor boats on submerged aquatic macrophytes. Lake and Reservoir Management, 13, 1–12. Baker, P.J., Thomson, A., Vatnick, I. & Wood, R.C. (2013) Estimating survival times for northern diamondback terra- pins, Malaclemys terrapin terrapin, in submerged crab pots. Herpetological Conservation and Biology, 8, 667–680. Bergin, T.M., Best, L.B., Freemark, K.E. & Koehler, K.J. (2000) Effects of landscape structure on nest predation in roadsides of a midwestern agroecosystem: a multiscale analysis. Landscape Ecology, 15, 131–143. Bilkovic, D.M., Chambers, R.M., Leu, M. & Russell, T. (2012) Diamondback terrapin bycatch reduction strategies for commercial and recreational blue crab ﬁsheries. Final Report to Virginia Sea Grant, National Oceanic and Atmospheric Administration. Report NA10OAR4170085. 21. We are the ﬁrst to identify numerous spatial scales of effect and to quantify ecological occupancy thresholds based on ﬁeld observations and models evaluated with independent data for terrapins in any segment of their range. The meth- ods developed for this study also provide an inexpensive and effective tool for monitoring terrapins. If applied over a longer time, the approach may also afford scientists the abil- ity to identify and assess the factors that lead to site-level colonization and extinction (MacKenzie et al., 2006). Our methods may also be applicable to study other aquatic turtle distributions in riverine systems that ﬂow through complex, heterogeneous landscapes. In the Chesapeake Bay, conserva- tion efforts could include increasing the riparian buffer of Bilkovic, D.M. & Mitchell, M.M. (2013) Ecological tradeoffs of stabilized salt marshes as a shoreline protection strategy: effects of artiﬁcial structures on macrobenthic assemblages. Ecological Engineering, 61, Part A, 469–481. Bilkovic, D.M. & Roggero, M.M. (2008) Effects of coastal development on nearshore estuarine nekton communities. Marine Ecology Progress Series, 358, 27–39. Bilkovic, D.M., Roggero, M., Hershner, C.H. & Havens, K.H. (2006) Inﬂuence of land use on macrobenthic communities in nearshore estuarine habitats. Estuaries and Coasts, 29, 1185–1195. Diversity and Distributions, 21, 643–653, ª 2015 John Wiley & Sons Ltd 650 Diamondback terrapin occupancy model Pro- ceedings of the National Academy of Sciences USA, 98, 166–170. D.G., Miller, W.D., Newell, R.I.E., Roman, M.R., Smith, E.M. & Stevenson, J.C. (2005) Eutrophication of Chesa- peake Bay: historical trends and ecological interactions. Marine Ecology Progress Series, 303, 1–29. Neatherlin, E.A. & Marzluff, J.M. (2004) Responses of American Crow populations to campgrounds in remote native forest land- scapes. Journal of Wildlife Management, 68, 708–718. King, R.S., Hines, A.H., Craige, F.D. & Grap, S. (2005) Regional, watershed and local correlates of blue crab and bivalve abun- dances in subestuaries of Chesapeake Bay, USA. Journal of Exper- imental Marine Biology and Ecology, 319, 101–116. NOAA NGDC (2000) Medium Resolution Digital Vector Shoreline Digital Geography. Available at: http://www.ngdc. noaa.gov/mgg/shorelines/data/gshhg/latest/ (accessed 01 April 2013). Koslow, D., Register, K., Burton, B., Priest III, W.I. & Luscher, A.E. (2006) Living shoreline case studies. Manage- ment, policy, science, and engineering of nonstructural erosion control in the Chesapeake Bay (ed. by S.Y. Erdle, J.L.D. Davis and K.G. Sellner), pp. 117–121. CRC Publication No. 08-164, Gloucester Point, VA. Northrup, J.M., Stenhouse, G.B. & Boyce, M.S. (2012) Agri- cultural lands as ecological traps for grizzly bears. Animal Conservation, 15, 369–377. R Development Core Team (2011) R: a language and envi- ronment for statistical computing. R Foundation for Statisti- cal Computing, Vienna. Leu, M., Hanser, S.E., Aldridge, C.L., Cade, B.S. & Knick, S.T. (2011) A sampling and analytical approach to develop spatial distribution models for sagebrush-associated species. Sagebrush ecosystem conservation and management: ecore- gional assessment tools and models for the Wyoming Basins (ed. by S.E. Hanser, M. Leu, S.T. Knick and C.L. Aldridge), pp. 387–409. Allen Press, Lawrence, KS. Rasmussen, M.L. & Litzgus, J.D. (2010) Habitat selection and movement patterns of Spotted Turtles (Clemmys guttata): effects of spatial and temporal scales of analyses. Copeia, 2010, 86–96. Rizkalla, C.E. & Swihart, R.K. (2006) Community structure and differential responses of aquatic turtles to agriculturally induced habitat fragmentation. Landscape Ecology, 21, 1361–1375. Liddle, M.J. & Scorgie, H.R.A. (1980) The effects of recrea- tion on freshwater plants and animals: a review. Biological Conservation, 17, 183–206. Rook, M.A., Lipcius, R.N., Bronner, B.M. & Chambers, R.M. (2010) Bycatch reduction device conserves diamondback terrapin without affecting catch of blue crab. Marine Ecol- ogy Progress Series, 409, 171–179. Liu, C., Berry, P.M., Dawson, T.P. & Pearson, R.G. (2005) Selecting thresholds of occurrence in the prediction of spe- cies distributions. Ecography, 28, 385–393. Roosenburg, W.M. Diamondback terrapin occupancy model Agricul- ture, Ecosystems & Environment, 150, 82–90. Cristol, D.A., Brasso, R.L., Condon, A.M., Fovargue, R.E., Friedman, S.L., Hallinger, K.K., Monroe, A.P. & White, A.E. (2008) The movement of aquatic mercury through terrestrial food webs. Science, 320, 335. 651 Diversity and Distributions, 21, 643–653, ª 2015 John Wiley & Sons Ltd R. E. Isdell et al. Martin, A.E. & Fahrig, L. (2012) Measuring and selecting scales of effect for landscape predictors in species–habitat models. Ecological Applications, 22, 2277–2292. Hitt, S., Pittman, S. & Nemeth, R. (2011) Diel movements of ﬁshes linked to benthic seascape structure in a Caribbean coral reef ecosystem. Marine Ecology Progress Series, 427, 275–291. Marzluff, J.M., McGowan, K.J., Donnelly, R. & Knight, R.L. (2001) Causes and consequences of expanding American Crow populations. Avian ecology and conservation in an urbanizing world (ed. by J.M. Marzluff, R. Bowman and R. Donnelly), pp. 331–363. Springer US, New York, NY. Isdell, R.E. (2014) Anthropogenic modiﬁcations of connectivity at the aquatic-terrestrial ecotone in the Chesapeake Bay. Master’s Thesis, College of William and Mary, Williams- burg, Virginia, USA. Metz, C.E. (1978) Basic principles of ROC analysis. Seminars in Nuclear Medicine, 8, 283–298. Jantz, P., Goetz, S. & Jantz, C. (2005) Urbanization and the loss of resource lands in the Chesapeake Bay watershed. Environmental Management, 36, 808–825. Morley, S.A., Toft, J.D. & Hanson, K.M. (2012) Ecological effects of shoreline armoring on intertidal habitats of a Puget Sound urban estuary. Estuaries and Coasts, 35, 774–784. Johnston, R.F. (2001) Synanthropic birds of North America. Avian ecology and conservation in an urbanizing world (ed. by J.M. Marzluff, R. Bowman and R. Donnelly), pp. 49–67. Springer US, New York, USA. Morris, A.S., Wilson, S.M., Dever, E.F. & Chambers, R.M. (2011) A test of bycatch reduction devices on commercial crab pots in a tidal marsh creek in Virginia. Estuaries and Coasts, 34, 386–390. Kemp, W.M., Boynton, W.R., Adolf, J.E., Boesch, D.F., Boi- court, W.C., Brush, G., Cornwell, J.C., Fisher, T.R., Glibert, P.M., Hagy, J.D., Harding, L.W., Houde, E.D., Kimmel, D.G., Miller, W.D., Newell, R.I.E., Roman, M.R., Smith, E.M. & Stevenson, J.C. (2005) Eutrophication of Chesa- peake Bay: historical trends and ecological interactions. Marine Ecology Progress Series, 303, 1–29. Kemp, W.M., Boynton, W.R., Adolf, J.E., Boesch, D.F., Boi- court, W.C., Brush, G., Cornwell, J.C., Fisher, T.R., Glibert, P.M., Hagy, J.D., Harding, L.W., Houde, E.D., Kimmel, Nakano, S. & Murakami, M. (2001) Reciprocal subsidies: dynamic interdependence between terrestrial and aquatic food webs. Diamondback terrapin occupancy model (1991) The diamondback terrapin: popu- lation dynamics, habitat requirements, and opportunities for conservation. New Perspectives in the Chesapeake Sys- tem: A Research and Management Partnership. Proceedings of a Conference, 227–234. Lobo, J.M., Jimenez-Valverde, A. & Real, R. (2008) AUC: a mis- leading measure of the performance of predictive distribution models. Global Ecology and Biogeography, 17, 145–151. Roosenburg, W.M. (1994) Nesting habitat requirements of the diamondback terrapin: a geographic comparison. Wet- land Journal, 6, 8–11. MacKenzie, D.I., Nichols, J.D., Royle, J.A., Pollock, K.H., Bailey, L.L. & Hines, J.E. (2006) Occupancy estimation and modeling: inferring patterns and dynamics of species occur- rence. Academic Press, Burlington, Massachusetts, USA. Roosenburg, W.M., Cresko, W., Modesitte, M. & Robbins, M.B. (1997) Diamondback terrapin (Malaclemys terrapin) mortality in crab pots. Conservation Biology, 11, 1166–1172. MacKenzie, D.I., Nichols, J.D., Lachman, G.B., Droege, S., Andrew Royle, J. & Langtimm, C.A. (2002) Estimating site occupancy rates when detection probabilities are less than one. Ecology, 83, 2248–2255. Roosenburg, W.M., Haley, K.L. & McGuire, S. (1999) Habi- tat selection and movements of diamondback terrapins, Diversity and Distributions, 21, 643–653, ª 2015 John Wiley & Sons Ltd 652 Diamondback terrapin occupancy model Diamondback terrapin occupancy model Diamondback terrapin occupancy model Malaclemys terrapin, in a Maryland estuary. Chelonian Con- servation and Biology, 3, 425–429. Wnek, J.P. (2010) Anthropogenic impacts on the reproduc- tive ecology of the diamondback terrapin, Malaclemys ter- rapin. Doctoral Thesis submitted to Drexel University, Philadelphia, Pennsylvania, USA. Roosenburg, W.M., Allman, P.E. & Fruh, B.J. (2003) Diamond- back terrapin nesting on the Poplar Island environmental res- toration project. 13th Biennial Coastal Zone Conference, Baltimore, MD, July 13–17. 2003. NOAA/CS/20322-CD. CD- ROM. Charleston, SC: NOAA Coastal Services Center. Wolak, M.E., Gilchrist, G.W., Ruzicka, V.A., Nally, D.M. & Chambers, R.M. (2010) A contemporary, sex-limited change in body size of an estuarine turtle in response to commercial ﬁshing. Conservation Biology, 24, 1268–1277. Scheuerell, M.D. & Schindler, D.E. (2004) Changes in the spatial distribution of ﬁshes in lakes along a residential development gradient. Ecosystems, 7, 98–106. Worm, B. & Lotze, H.K. (2006) Effects of eutrophication, grazing, and algal blooms on rocky shores. Limnology and Oceanography, 51, 569–579. Seitz, R.D., Lipcius, R.N., Olmstead, N.H., Seebo, M.S. & Lambert, D.M. (2006) Inﬂuence of shallow-water habitats and shoreline development on abundance, biomass, and diversity of benthic prey and predators in Chesapeake Bay. Marine Ecology Progress Series, 326, 11–27. Woth, K., Weisse, R. & von Storch, H. Diamondback terrapin occupancy model (2006) Climate change and North Sea storm surge extremes: an ensemble study of storm surge extremes expected in a changed climate projected by four different regional climate models. Ocean Dynamics, 56, 3–15. Shafer, D.J. (1999) The effects of dock shading on the sea- grass Halodule wrightii in Perdido Bay, Alabama. Estuaries, 22, 936–943. Wu, J. (2013) Landscape ecology. Ecological systems (ed. by R. Leemans), pp. 179–200. Springer, New York, NY. Zuur, A.F., Ieno, E.N. & Elphick, C.S. (2010) A protocol for data exploration to avoid common statistical problems. Methods in Ecology and Evolution, 1, 3–14. Sheridan, C.M., Spotila, J.R., Bien, W.F. & Avery, H.W. (2010) Sex-biased dispersal and natal philopatry in the diamondback terrapin, Malaclemys terrapin. Molecular Ecology, 19, 5497–5510. Silliman, B.R. & Bertness, M.D. (2004) Shoreline develop- ment drives invasion of Phragmites australis and the loss of plant diversity on New England salt marshes. Conservation Biology, 18, 1424–1434. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Sing, T., Sander, O., Beerenwinkel, N. & Lengauer, T. (2013) Visualizing the performance of scoring classiﬁers, Version 1.0-5. Available at: http://rocr.bioinf.mpi-sb.mpg.de/ (accessed 01 September 2013). Appendix S1 Detailed methodology. Table S1 Full list of statistical models and rankings. Table S2 Site-speciﬁc covariate selection. Soranno, P.A., Cheruvelil, K.S., Bissell, E.G., Bremigan, M.T., Downing, J.A., Fergus, C.E., Filstrup, C.T., Henry, E.N., Lottig, N.R., Stanley, E.H., Stow, C.A., Tan, P.-N., Wagner, T. & Webster, K.E. (2014) Cross-scale interac- tions: quantifying multi-scaled cause–effect relationships in macrosystems. Frontiers in Ecology and the Environ- ment, 12, 65–73. Table S3 Top models and parameter estimates for model averaging. BIOSKETCH Robert E. Isdell is a PhD student with the Center for Coastal Resources Management at the Virginia Institute of Marine Science, College of William & Mary, and is studying interactions between humans and the environment at multi- ple scales throughout the Chesapeake Bay. His interests include spatial ecology, coastal resource conservation and management, and the impacts of climate change. Talley, D.M., Huxel, G.R. & Holyoak, M. (2006) Connectiv- ity at the land-water interface. Connectivity conservation (ed. by K.R. Crooks and M. Sanjayan), pp. 97–129. Cam- bridge University Press, New York, NY. g y Titus, J., Hudgens, D., Trescott, D.L., Craghan, M., Nuckols, W.H., Hershner, C.H., Kassakian, J.M., Linn, C.J., Merritt, P.G., McCue, T.M., O’Connell, J.F., Tanski, J. & Wang, J. (2009) State and local governments plan for development of most land vulnerable to rising sea level along the US Atlantic coast. Environmental Research Letters, 4, 1–7. As a group, we are interested in combining principles and techniques of terrestrial and marine ecology to study the aquatic–terrestrial ecotone. Previous research efforts have involved marine debris impacts, bycatch reduction and spe- cies distribution models. Tulipani, D.C. (2013) Foraging ecology and habitat use of the northern diamondback terrapin (Malaclemys terrapin terrapin) in southern Chesapeake Bay. College of William and Mary - School of Marine Science, PhD Dissertation. 238. Author contributions: R.E.I., R.M.C., D.M.B. and M.L. con- ceived the ideas; R.E.I. collected the data; R.E.I. and M.L. analysed the data; and R.E.I., R.M.C., D.M.B. and M.L. con- tributed to the writing. Wedding, L., Lepczyk, C., Pittman, S., Friedlander, A. & Jor- gensen, S. (2011) Quantifying seascape structure: extending terrestrial spatial pattern metrics to the marine realm. Mar- ine Ecology Progress Series, 427, 219–232. Editor: David Richardson 653 Diversity and Distributions, 21, 643–653, ª 2015 John Wiley & Sons Ltd
https://openalex.org/W1971317397	https://europepmc.org/articles/pmc3445472?pdf=render	English	null	Idebenone Protects against Retinal Damage and Loss of Vision in a Mouse Model of Leber’s Hereditary Optic Neuropathy	PloS one	2,012	cc-by	10,768	Abstract Funding: Santhera Pharmaceuticals (Switzerland)funded and approved the publication of this work. Competing Interests: The authors have read the journal’s policy and have the following conflicts: During the preparation of this manuscript, all authors except VP were paid employees of Santhera Pharmaceuticals (Switzerland). Santhera is marketing CatenaH (idebenone) for the treatment of Friedreich’s Ataxia in Canada and develops Catena for several other mitochondrial and neuromuscular indications. NG is academic editor of PLOS ONE. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: nuri.guven@utas.edu.au LHON is caused by well characterized mitochondrial DNA (mtDNA) mutations, among which three so called primary mtDNA mutations account for more than 95% of all LHON cases: 11778G.A (ND4 subunit) [8], 14484T.C (ND6 subunit) [9], and 3460G.A (ND1 subunit) [10]. The 11778G.A mutation generally is the most abundant, although there is considerable variation in the relative frequency of the three primary LHON mutations worldwide. The degree of severity of the disease largely depends on the individual mutation, with the 14484T.C demonstrating a milder phenotype with a higher frequency of spontaneous recovery. All the mutations affect genes encoding mitochondrial complex I subunits (NADH dehydrogenase), which is part of this large multi-enzyme assembly that generates ATP as a cellular energy source through electron transfer and oxidative phosphorylation (OXPHOS). Therefore not surprising, defects in mitochondrial energy production have been reported for cells harboring the LHON mutations [11]. Experimental evidence connects complex I dysfunction to decreased ATP synthesis, elevated levels of oxidative stress [12,13] and impaired glutamate transport [14] leading to RGC dysfunction and ultimately to apoptotic cell death [15]. Recently developed murine animal models of LHON that mimic essential aspects of pathology of Idebenone Protects against Retinal Damage and Loss of Vision in a Mouse Model of Leber’s Hereditary Optic Neuropathy Fabrice D. Heitz1, Michael Erb1, Corinne Anklin1, Dimitri Robay1, Vincent Pernet2, Nuri Gueven1,3* 1 Santhera Pharmaceuticals, Liestal, Switzerland, 2 Brain Research Institute, Medical Faculty of the University of Zu¨rich, Zu¨rich, Switzerland, 3 School of Pharmacy, University of Tasmania, Hobart, Australia Abstract Leber’s hereditary optic neuropathy (LHON) is an inherited disease caused by mutations in complex I of the mitochondrial respiratory chain. The disease is characterized by loss of central vision due to retinal ganglion cell (RGC) dysfunction and optic nerve atrophy. Despite progress towards a better understanding of the disease, no therapeutic treatment is currently approved for this devastating disease. Idebenone, a short-chain benzoquinone, has shown promising evidence of efficacy in protecting vision loss and in accelerating recovery of visual acuity in patients with LHON. It was therefore of interest to study suitable LHON models in vitro and in vivo to identify anatomical correlates for this protective activity. At nanomolar concentrations, idebenone protected the rodent RGC cell line RGC-5 against complex I dysfunction in vitro. Consistent with the reported dosing and observed effects in LHON patients, we describe that in mice, idebenone penetrated into the eye at concentrations equivalent to those which protected RGC-5 cells from complex I dysfunction in vitro. Consequently, we next investigated the protective effect of idebenone in a mouse model of LHON, whereby mitochondrial complex I dysfunction was caused by exposure to rotenone. In this model, idebenone protected against the loss of retinal ganglion cells, reduction in retinal thickness and gliosis. Furthermore, consistent with this protection of retinal integrity, idebenone restored the functional loss of vision in this disease model. These results support the pharmacological activity of idebenone and indicate that idebenone holds potential as an effective treatment for vision loss in LHON patients. Citation: Heitz FD, Erb M, Anklin C, Robay D, Pernet V, et al. (2012) Idebenone Protects against Retinal Damage and Loss of Vision in a Mouse Model of Leber’s Hereditary Optic Neuropathy. PLoS ONE 7(9): e45182. doi:10.1371/journal.pone.0045182 Received May 17, 2012; Accepted August 14, 2012; Published September 18, 2012 Received May 17, 2012; Accepted August 14, 2012; Published September 18, 2012 Copyright: 2012 Heitz et al. This is an open-access article distributed under the terms of the Creative Commons Attr unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2012 Heitz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Santhera Pharmaceuticals (Switzerland)funded and approved the publication of this work. Citation: Heitz FD, Erb M, Anklin C, Robay D, Pernet V, et al. (2012) Idebenone Protects against Retinal Damage and Loss of Vision in a Mouse Model of Leber’s Hereditary Optic Neuropathy. PLoS ONE 7(9): e45182. doi:10.1371/journal.pone.0045182 Test Compound, Formulation and Chemicals For mouse dietary treatment, idebenone (2-(10-Hydroxy-decyl)- 5,6-dimethoxy-3-methyl- [1,4]benzoquinone) was mixed with 0.5% carboxymethyl cellulose (CMC), food powder (made from autoclaved food pellets) and sucrose (at a final concentration of 4%). Individual portions dispersed in small dishes and containing a daily dose of idebenone from 20 to 2000 mg/kg body weight were administered to single caged animals. A mixture of 0.5% CMC, food powder and sucrose (4%) was used as vehicle. In addition to the food mixture, idebenone suspension was prepared for p.o. (gavage) application by mixing idebenone with 0.5% CMC. Finally, a volume to result in a 60 mg/kg dose was applied by gavage. For cell culture experiments, idebenone was dissolved at 1 mM (stock solution) in 100% DMSO (Acros Organics, Belgium) and the final concentration of DMSO did not exceed 0.1% in the culture medium. For mouse intravitreal injection, rotenone was dissolved at 5 mM in 100% DMSO. DMSO was used as a control for the sham injection. All other chemical reagents and cell culture media and solutions, if not otherwise stated, were purchased from Sigma (Sigma-Aldrich, Buchs, Switzerland) and PAA Laboratories GmbH (Pasching, Austria). Idebenone Protects against Retinal Damage Idebenone Protects against Retinal Damage incubation). For the 1 day post-incubation period, cells were cultured in Hanks’ BSS (1 g/l glucose, PSG, idebenone or vehicle). LHON pathology can be used to test possible treatment strategies for LHON. One of these mouse models relies on intravitreal injection of the complex I inhibitor rotenone which causes loss of RGCs accompanied by reduced retinal nerve fiber layer thickness, thereby mimicking the cellular pathology of LHON patients [16,17,18,19,20]. Consistent with this, complex I inhibition by rotenone leads to ROS-mediated toxicity and apoptotic cell death in vitro, which are also observed in LHON [21]. Cell Viability Analysis As a measure of cell viability, the cellular ATP content was quantified by luminescence from the ATP-dependent enzymatic oxidation of luciferin by luciferase and using a multimode plate reader (Tecan M1000, Tecan iControl 1.6 software; Tecan, Groedig, Austria). Briefly, RGC-5 cells were washed in PBS and lysed in 100 ml lysis solution (4 mM EDTA, 0.2% Triton X-100) for five minutes at room temperature. In 96-well plates, 100 ml of ATP measurement buffer (25 mM HEPES pH 7.25, 300 mM D- luciferin, 5 mg/ml firefly luciferase, 75 mM DTT, 6.25 mM MgCl2, 625 mM EDTA and 1 mg/ml BSA) was combined with 10 ml cell lysate to start the reaction. Idebenone-mediated rescue in cell viability was defined as the percentage of idebenone- mediated increase in cell viability (based on ATP content) relative to the rotenone-induced reduction in cell viability (based on ATP content). Idebenone, a short-chain benzoquinone, is a potent antioxidant and inhibitor of lipid peroxidation [22,23,24] and thus is able to protect cell membranes and mitochondria from oxidative damage. Idebenone also interacts with the mitochondrial electron transport chain and modulates mitochondrial electron flux [25]. Specifically, it has been suggested that idebenone is effective in by-passing complex I to maintain cellular energy production. This bypass of complex I was first suggested by [26] and recently described in detail by [27] who demonstrated that idebenone is efficiently reduced in the cytoplasm by NAD(P)H quinone oxidoreductase 1 (NQO1) to the hydroquinone form. Under conditions of impaired complex I function, this reduced form of idebenone is able to transport electron equivalents from the cytoplasm to the mitochondria, where they are directly fed into the respiratory chain at the level of complex III. This mechanism of by-passing a defective complex I, which effectively restores cellular ATP levels [27,28], together with its antioxidant function, provides a strong rationale for the use of idebenone in LHON. Pharmacokinetics Mice were treated daily with dietary doses of idebenone or vehicle for 21 days or were treated orally by gavage with a single 60 mg/kg dose of idebenone. Mice were sacrificed and biological fluids were sampled 17 to 20 hours following dietary treatment and at time intervals from 5 min to 6 hours after gavage dosing. Briefly, mice were anesthetized by 5% isofluran inhalation in an induction chamber and sacrificed by cervical dislocation followed by decapitation. Blood was collected from the head into EDTA- containing tubes for plasma preparation. Individual plasma samples (Table S1) or pooled plasma samples from 4 individual mice (Table S2, S3) were analyzed. Both eyes were dissected out for the collection of aqueous and vitreous humor. Due to low individual collection volumes, aqueous humor and vitreous humors samples were pooled from 20 and 10 individual mice, respectively. Samples were transferred into 1.5 ml microcentrifuge tubes and stored frozen at -80uC until analyzed. Idebenone content was quantified by Inovalab AG Bioanalytics (Switzerland) using validated LC-MS/MS methods for plasma, aqueous humor and vitreous humor. The lower limit of detection of idebenone was determined as 2 ng/ml. Concentrations below this limit of detection were set as zero for the concentration summary statistics. Pharmacokinetic data are presented as descriptive statistics. Animals Male wild-type C57BL/6J mice (7 weeks old, 25 g body weight) obtained from Janvier (France) were single-housed in Makrolon type II cages under a regular 12-hours light-dark cycle (8 am to 8 pm) with free access to food and water. All procedures were performed in accordance with the Swiss regulation on the use and care of animals and under the required license approved from the Kantonales Veterina¨ramt Basel-Land (permission Nr. 415). Here we describe the pharmacological effects of idebenone in an RGC cell line and a well-established mouse model of LHON and demonstrate the protective effect of idebenone on retinal toxicity and visual impairment induced by complex I dysfunction. Materials and Methods Test Compound, Formulation and Chemicals Introduction Leber’s hereditary optic neuropathy (LHON) is a rare inherited mitochondrial disorder with an estimated prevalence in Europe of 1:45000 [1] characterized by rapid loss of visual acuity and color contrast sensitivity leading to blindness [2,3]. While generally this blindness persists lifelong, there is a limited rate of spontaneous recovery. LHON predominantly affects young adult males in all ethnic groups, with a peak of onset between the ages of 15–30 years. LHON starts with an acute onset of vision loss, first in one eye, followed by second eye involvement, typically within weeks to a few months. In most cases the vision loss is permanent, although a minority of patients shows spontaneous recovery of visual acuity [4,5]. LHON patients show swelling of the retinal nerve fiber layer with pseudoedema of the optic disc and a circumpapillary telangiectatic microangiopathy during the acute stage of the pathology. Retinal ganglion cell (RGC) bodies and their projecting axons forming the retinal nerve fiber layer (RNFL) gradually degenerate during the chronic phase with atrophy of the temporal optic nerve or diffuse optic atrophy [6], preferentially affecting the small-caliber fibers of the papillomacular bundle (PMB) at the center of the retina [7]. September 2012 \| Volume 7 \| Issue 9 \| e45182 1 PLOS ONE \| www.plosone.org Cell Culture Intravitreal injection of rotenone (5 and 15 mM in DMSO) or DMSO (vehicle) was performed as previously described [16,17]. Briefly, mice were anesthetized by inhalation with 1.5% to 2% isoflurane before 1 ml of rotenone was injected into the vitreous chamber of the left eye using an intraocular injection system (IO- KIT, World Precision Instruments, USA) adapted with a 35-gauge needle, and coupled to a 10-ml syringe (NanoFil, World Precision Instruments, USA). The needle tip was inserted into the superior hemisphere of the left eye, at the level of the pars plana, at a 45u RGC-5 cells were cultured under standard conditions (37uC, 5% CO2, 90% humidity) in DMEM (1 g/l glucose; 10% foetal bovine serum (FBS), Penicillin-Streptomycin-Glutamine (PSG)). For experiments, 36103 RGC-5 cells per well were seeded in DMEM (1 g/l glucose; 2% FBS, PSG) into BD FalconTM 96-well plates. RGC-5 cells were constantly exposed to either idebenone or vehicle from one day after seeding (pre-incubation), together with rotenone (co-incubation) or after the rotenone stress (post- September 2012 \| Volume 7 \| Issue 9 \| e45182 2 PLOS ONE \| www.plosone.org Idebenone Protects against Retinal Damage Figure 1. Protective effect of idebenone against complex I inhibition in RGC-5 cells in vitro. (A) Scheme of the experimental design. RGC-5 cells were pre-incubated with idebenone or vehicle (DMSO) for 1 or 2 days before the complex I inhibitor rotenone was applied to the cells for 6 hours. Cell viability was then measured after 1 day post-incubation with idebenone or vehicle. (B) Treatment with rotenone caused a 59% reduction in cell viability in vehicle-treated RGC-5 cells. (C) Idebenone pre-treatment for 1 (white bars) and 2 days (black bars) significantly rescued cell viability (expressed as % rescue). Data are expressed as mean 6 SD (n = 6 wells per group). Statistical significance relative to vehicle group: p#0.05 (), p#0.001 (*). doi:10.1371/journal.pone.0045182.g001 Figure 1. Protective effect of idebenone against complex I inhibition in RGC-5 cells in vitro. (A) Scheme of the experimental design. RGC-5 cells were pre-incubated with idebenone or vehicle (DMSO) for 1 or 2 days before the complex I inhibitor rotenone was applied to the cells for 6 hours. Cell viability was then measured after 1 day post-incubation with idebenone or vehicle. (B) Treatment with rotenone caused a 59% reduction in cell viability in vehicle-treated RGC-5 cells. Idebenone Protects against Retinal Damage Glutamine synthetase (GS) expression and the extent of gliosis was assessed by quantifying specific fluorescence signals within three regions of interest per retinal slice (background fluorescence was subtracted and signals from 3 regions of interest were averaged for each retinal slice). Quantitative analysis was performed on a minimum of two slices per individual retina for each parameter and the mean value was recorded. Image analysis of retinal slices was blinded to ensure that the person performing the analysis was not aware of the treatment received by the animal. After all images were analyzed and quantitative data were recorded, the study was unblinded and the treatment allocation list was matched to the data. Figure 3. Pharmacokinetic analysis of idebenone in eye fluids. Figure 3. Pharmacokinetic analysis of idebenone in eye fluids. Concentrations of idebenone in aqueous humor (open circle) and vitreous humor (filled triangle) following once daily administration of idebenone in the diet for 21 days (A) and following single oral administration of idebenone at 60 mg/kg (B) were determined. Concentrations of idebenone in the eye fluids are expressed as ng/ml (left y axis) and nM (right y axis). For (A), sampling time was more than 8h after last dose of idebenone, the values therefore represent trough levels. doi:10.1371/journal.pone.0045182.g003 doi:10.1371/journal.pone.0045182.g003 angle through the sclera into the vitreous body. This route of administration avoids retinal detachment or injury to eye structures, including the lens and the iris. The injection was performed using a syringe infusion pump (VIT-FIT, Lambda, USA) over a period of 2 min. The needle was kept in place for an additional 3 min, after which it was gently removed. Surgical glue (Indermill, Tyco Health Care, USA) was used to seal the site of injection and Neosporin eye drops (Neosporin, GlaxoSmithKline AG, Switzerland) were applied to prevent any infection. Idebenone Protects against Retinal Damage Figure 3. Pharmacokinetic analysis of idebenone in eye fluids. Concentrations of idebenone in aqueous humor (open circle) and vitreous humor (filled triangle) following once daily administration of idebenone in the diet for 21 days (A) and following single oral administration of idebenone at 60 mg/kg (B) were determined. Concentrations of idebenone in the eye fluids are expressed as ng/ml (left y axis) and nM (right y axis). For (A), sampling time was more than 8h after last dose of idebenone, the values therefore represent trough levels. doi:10.1371/journal.pone.0045182.g003 soaked in a PBS 0.1 M, 30% sucrose solution for 4 days at 4uC, embedded in freezing resin (OCT compound, Tissue-Tek), cryostat-cut in longitudinal sections (14-mm thick), placed on glass slides and stored at -20uC until stained. Sections were blocked and permeabilized in 0.1 M PBS, 0.3% Triton X-100 (Sigma) and 5% heat-inactivated horse serum (HS; Sigma) for 1 h at room temperature. Slices were then incubated overnight at 4uC with primary antibodies in 0.1 M PBS, 0.3% Triton X-100 and 5% HS. The following primary antibodies were used: mouse monoclonal and rabbit anti-Brn3a (1:100; Millipore, Mab1585, [29]) to stain for RGCs, rabbit anti-GFAP (1:500; Dako, Z0334) to stain for gliosis, mouse anti-beta 3 tubulin (1:500; Promega, G7121) to stain for all retinal layers to allow measurement of retinal thickness, and rabbit anti-NAD(P)H-quinone oxidoreductase 1 (NQO1, 1:100; Sigma, HPA007308) to stain for NQO1 expression in the retina. Slices were then washed three times in 0.1 M PBS and incubated 1 h at room temperature with goat anti-mouse Alexa 594, donkey anti-mouse 488, and donkey anti-rabbit Alexa 488 fluorescence- conjugated secondary antibodies (1:2000; Invitrogen) in 0.1 M PBS, 0.3% Triton X-100 and 5% HS. DAPI (Invitrogen) was used for the staining of cell nuclei. After washing three times in 0.1 M PBS, slices were mounted using FluorSave reagent (Calbiochem) and stored in the dark at 4uC. Images were acquired with a fluorescent microscope (Olympus) and analyzed using Image J software. Immunostaining analysis of the retinal sections was achieved on the contralateral side of the injection (inferior hemisphere of the eye). For RGC numbers, Brn3a-positive RGCs were counted and presented as number of RGCs per millimeter in the region of interest. Retinal thickness was determined by tubulin staining. Optomotor Testing The optomotor response was measured as previously described [30]. Mice were placed on a central platform (10 cm diameter) surrounded by a motorized drum (29.0 cm diameter) with black and white vertical stripes (1.0 cm thickness) that could rotate both clock- and counterclock-wise at different velocities (which was adjusted in the first experiment to evoke the optimal optokinetic response) under constant light levels (Video S1, S2). This test is particularly suitable for assessing visual acuity under these experimental conditions because the left eye is entirely responsible for controlling the optomotor reflex (head move- ments) when the drum is rotating clockwise, whereas the reflex is controlled exclusively by the right eye for the counterclockwise rotation. Therefore, the visual acuity of the left (injected) and of the right (non-injected) eye was tested by clock- and counter- clock-wise rotation of the drum. Mice were allowed to habituate for 5 min to the experimental setup before the drum was rotated alternately clockwise and counterclockwise, for 2 min in each direction with an interval of 30 s between the two rotations. Movements were digitally recorded using a video camera mounted above the apparatus and the number of head movements was subsequently manually scored. Head movements Cell Culture (C) Idebenone pre-treatment for 1 (white bars) and 2 days (black bars) significantly rescued cell viability (expressed as % rescue). Data are expressed as mean 6 SD (n = 6 wells per group). Statistical significance relative to vehicle group: p#0.05 (), p#0.001 (**). doi:10.1371/journal.pone.0045182.g001 doi:10.1371/journal.pone.0045182.g001 doi:10.1371/journal.pone.0045182.g001 Figure 2. NQO1 expression in RGCs and the inner segment of photoreceptors. Distribution of NQO1 in the mouse retina co-stained with rabbit anti-NQO1 and mouse monoclonal anti-Brn3a primary antibodies. Anti-rabbit Alexa 488-conjugated and anti-mouse Alexa 495-conjugated secondary antibodies were used for fluorescent visualization of NQO1 (green) and RGCs (red) in the retina. The white rectangle in (A) depicts the region of interest shown at high magnification in (B). The different retinal cell layers are shown by nuclear staining using DAPI (blue). Scale bars represent 30 mm. (OS: outer segment, IS: inner segment, ONL: outer nuclear layer, OPL: outer plexiform layer, INL: inner nuclear layer, IPL: inner plexiform layer, GCL: ganglion cell layer (incl. nerve fiber layer). doi:10.1371/journal.pone.0045182.g002 Figure 2. NQO1 expression in RGCs and the inner segment of photoreceptors. Distribution of NQO1 in the mouse retina co-stained with rabbit anti-NQO1 and mouse monoclonal anti-Brn3a primary antibodies. Anti-rabbit Alexa 488-conjugated and anti-mouse Alexa 495-conjugated secondary antibodies were used for fluorescent visualization of NQO1 (green) and RGCs (red) in the retina. The white rectangle in (A) depicts the region of interest shown at high magnification in (B). The different retinal cell layers are shown by nuclear staining using DAPI (blue). Scale bars represent 30 mm. (OS: outer segment, IS: inner segment, ONL: outer nuclear layer, OPL: outer plexiform layer, INL: inner nuclear layer, IPL: inner plexiform layer, GCL: ganglion cell layer (incl. nerve fiber layer). doi:10.1371/journal.pone.0045182.g002 September 2012 \| Volume 7 \| Issue 9 \| e45182 PLOS ONE \| www.plosone.org 3 3 Idebenone Protects against Retinal Damage Immunohistochemistry Mice were anesthetized by i.p. injection of ketamine/xylazine (2/1, 200 ml) 7 days after rotenone injection and transcardially perfused with 4% PFA in PBS 0.1 M (pH 7.4). The left eyes (injected) and the right eyes (uninjected controls) were rapidly removed and the corneas were eliminated at limbus level; vitreous humor and crystalline lens were discarded. Tissue samples were labeled with random numbers by a person not involved in the study to ensure blinding during the analyses. This person kept the ‘treatment allocation’ list to himself and persons involved in tissue processing and data analysis were blind to the treatment allocation at all times. The samples thus obtained were post-fixed with 4% PFA in PBS 0.1 M (pH 7.4) overnight at 4uC. Samples were September 2012 \| Volume 7 \| Issue 9 \| e45182 PLOS ONE \| www.plosone.org 4 Idebenone Protects against Retinal Damage Figure 4. Experimental approach to study the protective effect of idebenone in a LHON mouse model. (A) Image of a normal retinal section showing RGCs (red) and Mu¨ller glial cell projections (green) using anti-Brn3a and anti-GFAP primary antibodies respectively. The different retinal cell layers are shown by nuclear staining using DAPI (blue). (Scale bar = 25 mm). (B) To investigate the efficacy of idebenone in protecting against complex I deficiency in the retina in vivo, we performed intravitreal injection of rotenone (15 mM) in mice pre-treated for 21 days with idebenone or vehicle in diet. Retinal ganglion cell number, retinal thickness and reactive gliosis were evaluated based on histological analysis of retinal sections 7 days after rotenone challenge. doi:10.1371/journal.pone.0045182.g004 Figure 4. Experimental approach to study the protective effect of idebenone in a LHON section showing RGCs (red) and Mu¨ller glial cell projections (green) using anti Brn3a and anti GFAP Figure 4. Experimental approach to study the protective effect of idebenone in a LHON mouse model. (A) Image of a normal retinal section showing RGCs (red) and Mu¨ller glial cell projections (green) using anti-Brn3a and anti-GFAP primary antibodies respectively. The different retinal cell layers are shown by nuclear staining using DAPI (blue). (Scale bar = 25 mm). (B) To investigate the efficacy of idebenone in protecting against complex I deficiency in the retina in vivo, we performed intravitreal injection of rotenone (15 mM) in mice pre-treated for 21 days with idebenone or vehicle in diet. Exclusion Criteria Behavioral analysis of the optomotor response was achieved for 1) visual recovery by counting the number of clockwise head movements per 2 min session prior to rotenone injection and at different time intervals following rotenone injection, 2) number of mice showing clockwise head movements at different time intervals following injection and expressed as the percentage of responder mice, and 3) visual acuity of the non-injected eye used as a control by counting the number of counterclockwise head movements per 2 min session prior injection and at different time intervals following injection. The overall experimental process was blinded at each step to ensure that the persons involved in optomotor testing, counting optomotor responses and analyzing the data were not aware of the treatment given to the animals. At the end of the To correct for possible deviations in the procedure that could lead to erroneous conclusions, immunolabeled sections were evaluated for staining quality. Specifically, samples were excluded from the quantitative analysis in case of weak or unspecific fluorescence signal defined as relative fluorescence units (RFU) #20% of standard values or #120% of the background fluorescence. In addition, functional quantification of visual acuity (see Optomotor testing) was used to control for successful intraocular rotenone injection. In response to the injection of rotenone, a total loss of visual acuity was generally observed within 24h in the optomotor test system. Consequently, animals not showing complete loss of optomotor response at 24h post rotenone injection were excluded from histological and behavioral analyses. Table 1. Dose-effect of idebenone treatment on multiple endpoints in an in vivo mouse model for LHON. ble 1. Dose-effect of idebenone treatment on multiple endpoints in an in vivo mouse model for LHON Table 1. Dose-effect of idebenone treatment on multiple endpoints in an in vivo mouse model for LHON. Treatment RGC number Retinal thickness Reactive gliosis Sham (DMSO) 100.0613.7 (n = 10) 100.064.9 (n = 8) 100.069.7 (n = 7) Vehicle + rotenone 32.866.4 (n = 6) 78.163.8 (n = 6) 178.1621.6 (n = 3) IDE200 + rotenone 64.069.1 (n = 10) 92.764.9 (n = 10) 124.567.4* (n = 7) IDE400 + rotenone 71.766.7 (n = 8) 91.962.0 (n = 8) 125.069.1* (n = 6) IDE2000 + rotenone 70.865.6*** (n = 10) 90.362.3* (n = 9) 129.967.3* (n = 7) Histological data are expressed as % of Sham (DMSO) controls 6 SEM. Immunohistochemistry Retinal ganglion cell number, retinal thickness and reactive gliosis were evaluated based on histological analysis of retinal sections 7 days after rotenone challenge. doi:10.1371/journal.pone.0045182.g004 Figure 5. Idebenone prevents rotenone-induced RGC death. Analysis of RGCs in the mouse retina 7 days after intravitreal injection of rotenone or DMSO (Sham). Mice were treated with dietary idebenone (IDE 200, 400 and 2000 mg/kg body weight) or vehicle. (A) Images of retinal slices stained with anti-Brn3a primary antibody to visualize RGCs. Scale bar = 25 mm. (B) Quantification of RGCs (RGC number/mm) following idebenone treatment and rotenone injection (n = 6 to 10 per group). Data expressed as mean 6 SEM. doi:10.1371/journal.pone.0045182.g005 Figure 5. Idebenone prevents rotenone-induced RGC death. Analysis of RGCs in the mouse retina 7 days after intravitreal injection of rotenone or DMSO (Sham). Mice were treated with dietary idebenone (IDE 200, 400 and 2000 mg/kg body weight) or vehicle. (A) Images of retinal slices stained with anti-Brn3a primary antibody to visualize RGCs. Scale bar = 25 mm. (B) Quantification of RGCs (RGC number/mm) following idebenone treatment and rotenone injection (n = 6 to 10 per group). Data expressed as mean 6 SEM. doi:10.1371/journal.pone.0045182.g005 September 2012 \| Volume 7 \| Issue 9 \| e45182 PLOS ONE \| www.plosone.org 5 Idebenone Protects against Retinal Damage Figure 6. Idebenone protects against rotenone-induced decrease in retinal thickness. For analysis of retinal thickness 7 days after intravitreal injection of rotenone or DMSO (Sham), mice were treated with dietary idebenone (IDE 200, 400 and 2000 mg/kg body weight) or vehicle. (A) Images of retinal slices stained with anti-beta 3 tubulin primary antibodies to visualize inner and outer retinal layers. The arrows indicate retinal thickness measured from the pigment epithelium to the nerve fiber layer. Scale bar = 30 mm. (B) Quantification of total retinal thickness (mm) following idebenone treatment and rotenone injection (n = 6 to 10 per group). Data are expressed as mean 6 SEM. doi:10.1371/journal.pone.0045182.g006 Figure 6. Idebenone protects against rotenone-induced decrease in retinal thickness. For analysis of retinal thickness 7 days after intravitreal injection of rotenone or DMSO (Sham), mice were treated with dietary idebenone (IDE 200, 400 and 2000 mg/kg body weight) or vehicle. (A) Images of retinal slices stained with anti-beta 3 tubulin primary antibodies to visualize inner and outer retinal layers. The arrows indicate retinal thickness measured from the pigment epithelium to the nerve fiber layer. Scale bar = 30 mm. Immunohistochemistry (B) Quantification of total retinal thickness (mm) following idebenone treatment and rotenone injection (n = 6 to 10 per group). Data are expressed as mean 6 SEM. doi:10.1371/journal.pone.0045182.g006 analysis, the study was unblinded and the treatment allocation was matched to the data. were scored only when the angular speed of the head turn corresponded to that of the drum rotation. Each mouse was tested at weekly intervals throughout the experiment and the apparatus was cleaned between mice. Exclusion Criteria Numbers of retinal ganglion cells (RGC number) is based on RGCs/mm 7 days post-injection applicable to all groups; retinal thickness (mm) 7 days post-injection; gliosis is based on relative fluorescence units (RFU) of GFAP-specific immunostaining 7 days post- injection. Sham (DMSO): sham-injected with DMSO; vehicle: vehicle treated; rotenone: rotenone-injected; IDE200: idebenone treated (200 mg/kg); IDE400: idebenone treated (400 mg/kg); IDE2000: idebenone treated (2000 mg/kg). Statistical significance relative to vehicle + rotenone group: p#0.05 (), p#0.01 (), p#0.001 (*); based on raw numbers; n = number of retinas per group. doi:10.1371/journal.pone.0045182.t001 Histological data are expressed as % of Sham (DMSO) controls 6 SEM. Numbers of retinal ganglion cells (RGC number) is based on RGCs/mm 7 days post-injection applicable to all groups; retinal thickness (mm) 7 days post-injection; gliosis is based on relative fluorescence units (RFU) of GFAP-specific immunostaining 7 days post- injection. Sham (DMSO): sham-injected with DMSO; vehicle: vehicle treated; rotenone: rotenone-injected; IDE200: idebenone treated (200 mg/kg); IDE400: idebenone treated (400 mg/kg); IDE2000: idebenone treated (2000 mg/kg). Statistical significance relative to vehicle + rotenone group: p#0.05 (), p#0.01 (), p#0.001 (); based on raw numbers; n = number of retinas per group. doi:10.1371/journal.pone.0045182.t001 Histological data are expressed as % of Sham (DMSO) controls 6 SEM. Numbers of retinal ganglion cells (RGC number) is based on RGCs/mm 7 days post-injection applicable to all groups; retinal thickness (mm) 7 days post-injection; gliosis is based on relative fluorescence units (RFU) of GFAP-specific immunostaining 7 days post- injection. Sham (DMSO): sham-injected with DMSO; vehicle: vehicle treated; rotenone: rotenone-injected; IDE200: idebenone treated (200 mg/kg); IDE400: idebenone treated (400 mg/kg); IDE2000: idebenone treated (2000 mg/kg). Statistical significance relative to vehicle + rotenone group: p#0.05 (), p#0.01 (), p#0.001 (); based on raw numbers; n = number of retinas per group. doi:10.1371/journal.pone.0045182.t001 September 2012 \| Volume 7 \| Issue 9 \| e45182 6 PLOS ONE \| www.plosone.org Idebenone Protects against Retinal Damage Figure 7. Idebenone protects against rotenone-induced gliosis. For analysis of gliosis 7 days after intravitreal injection of rotenone or DMSO (Sham), mice were treated with dietary idebenone (IDE 200, 400 and 2000 mg/kg body weight) or vehicle. (A) Images of retinal slices stained with anti-GFAP primary antibody to visualize Mu¨ller glial cell projections (white arrows). White arrowheads indicate outer plexiform layer and also illustrate rotenone induced reduction in retinal thickness (i.e. distance between white arrow heads and ganglion cell layer at the top of the images). Idebenone Increases Survival of Complex I Deficient RGC- 5 Cells To assess a cytoprotective effect of idebenone, we recapitulated the situation of mutated mitochondrial complex I in LHON RGC cells by exposing the retinal ganglion cell line RGC-5 [31] with the mitochondrial complex I inhibitor rotenone [21]. RGC-5 cells were continuously incubated with 1, 10, 100 nM or 1 mM idebenone for 1 or 2 days prior to a 6 hour exposure to rotenone (1 mM). This toxic stimulus was followed by a 24h post-incubation time with or without idebenone before RGC-5 cell viability was quantified by analyzing cellular ATP content (Fig. 1A). While rotenone-mediated complex I inhibition strongly reduced the viability of RGC-5 cells by 59% (Fig. 1B), idebenone treatment dose-dependently increased cell viability (Fig. 1C). Significant protection by idebenone was already seen at concentrations of $10 nM. The protective effect was dependent on the duration of idebenone pre-incubation, since pre-incubation with 1 mM idebenone for 1 day (white bars) and 2 days (black bars) increased RGC-5 cell viability by 26% and 58%, respectively. In summary, these results demonstrate that concentrations of idebenone above 10 nM are cytoprotective against complex I inhibition in a RGC cell line in vitro. Exclusion Criteria Scale bar = 15 mm. (B) Quantification of gliosis (GFAP signal based on relative fluorescence units, RFU) following idebenone treatment and rotenone injection (n = 3 to 7 per group). Data are expressed as mean 6 SEM. doi:10.1371/journal.pone.0045182.g007 Figure 7. Idebenone protects against rotenone-induced gliosis. For analysis of gliosis 7 days after intravitreal injection of rotenone or DMSO (Sham), mice were treated with dietary idebenone (IDE 200, 400 and 2000 mg/kg body weight) or vehicle. (A) Images of retinal slices stained with anti-GFAP primary antibody to visualize Mu¨ller glial cell projections (white arrows). White arrowheads indicate outer plexiform layer and also illustrate rotenone induced reduction in retinal thickness (i.e. distance between white arrow heads and ganglion cell layer at the top of the images). Scale bar = 15 mm. (B) Quantification of gliosis (GFAP signal based on relative fluorescence units, RFU) following idebenone treatment and rotenone injection (n = 3 to 7 per group). Data are expressed as mean 6 SEM. doi:10.1371/journal.pone.0045182.g007 Statistical Analysis We previously reported that next to its antioxidant activity, one important function of idebenone is to bypass a dysfunctional complex I. This mechanism involves an alternate electron flux directly to complex III and can restore cellular ATP levels [27] and largely relies on the presence of NQO1. We thus examined whether significant levels of NQO1 were expressed in the murine retina and specifically in the RGC cells which are most affected in LHON. When untreated mouse retina sections were stained for NQO1 expression, strong immunoreactivity was found predom- inantly in the RGC cell layer and the photoreceptor layer (Fig. 2A– B). As expected NQO1 staining in RGC cells was located to the cytoplasm (Fig. 2B). At the same time, only low level immuno- reactivity for NQO1 was observed in the other retinal layers (Fig. 2A). The presence of strong NQO1-immunoreactivity in RGCs is in agreement with the proposed mode of action whereby idebenone bypasses a deficient complex I. Data are presented as mean 6 SD or mean 6 SEM as indicated in the figure legends. Paired Student’s t-test and analysis of variance (ANOVA) was used to compare non-normalized data. Statistical significance was set at p#0.05(), p#0.01() and p#0.001(). Results Idebenone Increases Survival of Complex I Deficient RGC- 5 Cells Idebenone Penetrates into the Eye Fluids after Oral Administration in the Mouse Since nanomolar concentrations of idebenone protect against complex I dysfunction and cell death in an RGC-like cell line in vitro, we next assessed whether such concentrations of idebenone could be achieved in the mouse eye following oral administration of idebenone. Following treatment with idebenone given in the diet over a period of three weeks or after a single oral administration (gavage) in the adult mouse, the concentrations of idebenone in the aqueous and vitreous humor and plasma were measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). After administration of idebenone through the diet of male mice from 20 to 2000 mg/kg of body September 2012 \| Volume 7 \| Issue 9 \| e45182 PLOS ONE \| www.plosone.org 7 Idebenone Protects against Retinal Damage Figure 8. Idebenone time-dependently restores rotenone-induced loss of vision. Visual acuity was evaluated by counting the number of head movements at a velocity of 3 rpm 7 days prior injection (day -7) and 1 to 70 days after injection (day 1, 7, 21, 35, 70) of rotenone (5 mM). Mice were treated with dietary idebenone 2000 mg/kg or vehicle for the time of the experiment. (A) Schematic representation of the experimental setup. After vehicle (DMSO) injection, head movements in both directions of drum rotation (clockwise: CW; counterclockwise CCW) remain intact. Injection of rotenone into the left eye however, affects only the CW responses (dashed arrow) whereas CCW responses remain unaffected. (B) Quantification of clockwise (CW) head movement (number of head movements/2 min) following vehicle treatment and rotenone injection (vehicle + rotenone, n = 10 animals), and idebenone 2000 mg/kg treatment and rotenone injection (IDE 2000+ rotenone, n = 11 animals). Data are expressed as mean 6 SEM. Statistical significance relative to vehicle + rotenone group: p#0.05 (); (C) Percentage of mice showing clockwise (CW) head movements for each treatment group 70 days after injection. Responder: group of mice showing head movements; Non-responder: group of mice without head movements. d i 10 1371/j l 0045182 008 Figure 8. Idebenone time-dependently restores rotenone-induced loss of vision. Visual acuity w . Idebenone time-dependently restores rotenone-induced loss of vision. Visual acuity was evaluated by cou Figure 8. Idebenone time-dependently restores rotenone-induced loss of vision. Idebenone Penetrates into the Eye Fluids after Oral Administration in the Mouse Visual acuity was evaluated by counting the number of head movements at a velocity of 3 rpm 7 days prior injection (day -7) and 1 to 70 days after injection (day 1, 7, 21, 35, 70) of rotenone (5 mM). Mice were treated with dietary idebenone 2000 mg/kg or vehicle for the time of the experiment. (A) Schematic representation of the experimental setup. After vehicle (DMSO) injection, head movements in both directions of drum rotation (clockwise: CW; counterclockwise CCW) remain intact. Injection of rotenone into the left eye however, affects only the CW responses (dashed arrow) whereas CCW responses remain unaffected. (B) Quantification of clockwise (CW) head movement (number of head movements/2 min) following vehicle treatment and rotenone injection (vehicle + rotenone, n = 10 animals), and idebenone 2000 mg/kg treatment and rotenone injection (IDE 2000+ rotenone, n = 11 animals). Data are expressed as mean 6 SEM. Statistical significance relative to vehicle + rotenone group: p#0.05 (); (C) Percentage of mice showing clockwise (CW) head movements for each treatment group 70 days after injection. Responder: group of mice showing head movements; Non-responder: group of mice without head movements. doi:10 1371/journal pone 0045182 g008 Figure 8. Idebenone time-dependently restores rotenone-induced loss of vision. Visual acuity was evaluated by counting the number of head movements at a velocity of 3 rpm 7 days prior injection (day -7) and 1 to 70 days after injection (day 1, 7, 21, 35, 70) of rotenone (5 mM). Mice were treated with dietary idebenone 2000 mg/kg or vehicle for the time of the experiment. (A) Schematic representation of the experimental setup. After vehicle (DMSO) injection, head movements in both directions of drum rotation (clockwise: CW; counterclockwise CCW) remain intact. Injection of rotenone into the left eye however, affects only the CW responses (dashed arrow) whereas CCW responses remain unaffected. (B) Quantification of clockwise (CW) head movement (number of head movements/2 min) following vehicle treatment and rotenone injection (vehicle + rotenone, n = 10 animals), and idebenone 2000 mg/kg treatment and rotenone injection (IDE 2000+ rotenone, n = 11 animals). Data are expressed as mean 6 SEM. Statistical significance relative to vehicle + rotenone group: p#0.05 (); (C) Percentage of mice showing clockwise (CW) head movements for each treatment group 70 days after injection. Responder: group of mice showing head movements; Non-responder: group of mice without head movements. Idebenone Protects Against Rotenone-induced RGC Death and Retinal Damage in vivo g Next, we investigated the efficacy of idebenone against consequences of complex I deficiency in the mouse retina in vivo. Mice were pre-treated for 3 weeks with idebenone or vehicle in the diet before rotenone was injected intravitrealy into the left eye. Retinal ganglion cell number, retinal thickness and reactive gliosis were quantitated based on immunohistochemical analysis of retinal sections 1 week after rotenone challenge (Fig. 4A–B). Animals pre-treated with dietary idebenone at 200, 400 and 2000 mg/kg/day were significantly protected against rotenone- induced RGC loss 7 days after the rotenone challenge when compared to vehicle-treated mice (Fig. 5A–B and Table 1). The injection-procedure itself caused no toxicity, as shown by comparable RGC numbers in sham-injected (6369, n = 10) compared with non-injected eyes (5066, n = 3). Consistent with this protection of RGCs, idebenone also prevented the decrease of retinal thickness 7 days after the rotenone challenge (Fig. 6A–B and Table 1). Similarly to RGCs, retinal thickness was not affected by the injection per se (sham-injected: 287614 mm, n = 8; non- injected: 296619 mm, n = 5). The extent of protection seen against rotenone-induced RGC loss and retinal thickness was comparable with the three doses of idebenone tested (Fig. 5B and 6B and Table 1). In addition, 7 days after rotenone injection, each of the idebenone doses tested significantly suppressed secondary patho- logical consequences of rotenone-mediated complex I inhibition, such as increased Mu¨ller cell gliosis (Fig. 7A–B and Table 1) and reduced glutamine synthetase expression (data not shown), which are markers of retinal pathology and cellular stress [32,33]. These findings demonstrate that idebenone selectively protects against RGC loss, reduction of retinal thickness and reactive gliosis caused by complex I dysfunction in the retina. Since intra-ocular exposure with idebenone was not determined in the recent trials in LHON patients, it is of interest to compare the intra-ocular exposure that might result from dosing in humans at 900 mg idebenone/day (the dose used by [40] in the RHODOS trial) with the exposures found efficacious in the in vitro and in vivo models of LHON described here. This can be achieved by comparing the dose effective in the prevention of rotenone- induced cell death in the in vivo mouse model and in the clinical trial on a mg/kg and/or mg/m2 basis. Idebenone Protects Against Rotenone-induced RGC Death and Retinal Damage in vivo The clinical dose of 900 mg/day idebenone (CatenaH) was administered as 300 mg three times a day (tid) which corresponds to 15 mg/kg/day or 5 mg/kg tid for an average patient weight of 60 kg [40]. Taking a conservative approach, a single dose of 5 mg/kg in man (one third of the daily dose) represents a dose equivalent to 61.5 mg/kg in the mouse (using a generally accepted factor of 12.3 for an equivalent mg/kg dose for conversion from human to mouse; [49]). Assuming dose proportionality in plasma exposure, it can be expected that a dose of 61.5 mg/kg, given by gavage in the mouse would lead to an aqueous humor exposure of around 39 nM, well within the range shown to be effective in vitro (Fig. 1). This comparison therefore suggests that following a dose of 300 mg idebenone (CatenaH) tid, the aqueous humor concentration of idebenone in man can be expected to reach levels in the same range as those shown to be effective in the in vitro and in vivo LHON animal models. Together with the LHON-like pathologies observed in the mouse model our results therefore not only support the clinical data but also validate the animal model used. It is important to note that, in addition to its antioxidant function [22,23,25,26,50], idebenone can also maintain energy production under conditions of mitochondrial complex I de- ficiency by shuttling electrons directly from the cytoplasm to Discussion Leber’s hereditary optic neuropathy (LHON) is caused by mutations in complex I of the mitochondrial respiratory chain, which leads to complex I dysfunction, decreased ATP production and oxidative stress. The pathology of LHON is characterized by swelling of the retinal nerve fiber layer (RNFL) which precedes loss of retinal ganglion cells (RGC). This dynamic process evolves in parallel with functional impairment and atrophy of the nerve [6,35]. Moreover, gliosis and decreased glutamine synthetase expression are consistent with optic nerve sheath distension [36,37,38] and glutamate transport impairment [14,39], which can be observed in LHON patients. These pathologies, including the loss of optic nerve integrity, ultimately result in blindness of the patient. Here we demonstrated that idebenone is able to protect RGCs and retinal integrity against mitochondrial dysfunction in vitro and in vivo. Even though the mouse model used in this study might not replicate the condition in LHON patients in all aspects of the disease, these results strongly support the recently published data showing evidence for a therapeutic benefit of idebenone in prospective and retrospective clinical studies in LHON [40,41,42], and in a series of case reports where LHON patients favorably responded to idebenone [43,44,45,46,47,48]. Idebenone Protects against Retinal Damage Idebenone Protects against Retinal Damage suggesting rapid uptake of idebenone into the plasma and the eye. Concentrations of idebenone in aqueous and vitreous humor generally declined over a period of 2 h post-dose (Fig. 3B and Table S2), suggesting an absence of sequestration of idebenone into the eye. The overall amount of idebenone in the aqueous humor, as measured by AUC0-6h, represented ,10% of that observed in plasma (Table S3). Similarly, the concentration of idebenone in the vitreous humor represented ,0.5% of that observed in plasma. These results indicate a rapid penetration of idebenone into the eye at concentrations shown to protect against rotenone-induced retinal ganglion cell death in vitro. clockwise head responses were similar in both idebenone- and vehicle-treated mice (Table S4). This is also evident when analyzing the ratio of clockwise and counterclockwise head responses (Fig. S1B). Moreover, sham-injected mice had no loss in clockwise head responses, suggesting no detrimental effect of the injection procedure itself (data not shown). These results demon- strate that idebenone preserves visual function in animals under conditions of impaired complex I function by protecting RGC and retinal integrity. Idebenone Penetrates into the Eye Fluids after Oral Administration in the Mouse doi:10 1371/journal pone 0045182 g008 doi:10.1371/journal.pone.0045182.g008 weight/day, concentrations of idebenone in aqueous and vitreous humor increased approximately dose proportionally, similar to the increase observed in the plasma (Fig. 3A and Table S1). The concentration ratios of aqueous humor/plasma and vitreous humor/plasma appeared to be independent of the dose, consistent with an absence of accumulation of idebenone in the eye (data not shown). Concentrations of idebenone in aqueous humor were approximately 2 to 7-fold higher than that in vitreous humor. Importantly, intra-ocular exposure to idebenone reached peak concentrations of 2.3 ng/ml (7 nM), 3.0 ng/ml (9 nM), and 12.6 ng/ml (37 nM) in the aqueous humor at the oral doses of 200, 400, and 2000 mg/kg of body weight/day, respectively (Fig. 3A and Table S1) which are within the range shown to be effective in protecting RGC-5 cells from rotenone-induced cell death in vitro (i.e. 10 nM and above, Fig. 1B). We next determined the concentration-time profile of idebenone in the mouse eye fluids. Following a single oral (gavage) administration of idebenone (60 mg/kg) to male mice, intra-ocular concentrations of idebenone reached a peak (Cmax) of 37.4 ng/ml (111nM) in the aqueous humor at the first sampling time 5 min post-dose (tmax) (Fig. 3B and Tables S2, S3). The concentration-time profile of idebenone in the eye fluids was comparable to the plasma profile (Table S2), weight/day, concentrations of idebenone in aqueous and vitreous humor increased approximately dose proportionally, similar to the increase observed in the plasma (Fig. 3A and Table S1). The concentration ratios of aqueous humor/plasma and vitreous humor/plasma appeared to be independent of the dose, consistent with an absence of accumulation of idebenone in the eye (data not shown). Concentrations of idebenone in aqueous humor were approximately 2 to 7-fold higher than that in vitreous humor. Importantly, intra-ocular exposure to idebenone reached peak concentrations of 2.3 ng/ml (7 nM), 3.0 ng/ml (9 nM), and 12.6 ng/ml (37 nM) in the aqueous humor at the oral doses of September 2012 \| Volume 7 \| Issue 9 \| e45182 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 8 September 2012 \| Volume 7 \| Issue 9 \| e45182 Idebenone Protects against Retinal Damage complex III [27,28]. The process relies on NAD(P)H:quinone oxidoreductase 1 (NQO1), a cytosolic flavoprotein that catalyzes the reduction of idebenone [27], and is likely to contribute to the efficacy of idebenone in LHON. Thus, for the effectiveness of idebenone to restore energy production, expression of NQO1 or a similar reductase appears to be a fundamental requirement. Consistent with the protective effect of idebenone on RGCs shown in the LHON mouse model, NQO1 was found to be highly expressed in RGCs in the mouse retina (Fig. 2). The reason for the cell type-specific vulnerability of RGCs in LHON is still unclear but could lie at least in part in intrinsic features of RGCs which are known to have an extremely high energy demand [51] that makes them vulnerable to failure of mitochondrial energy production. of idebenone at 60 mg/kg to male mice. Data are expressed in ng/ ml (6 SEM). Idebenone concentration was measured 5, 15, 30, 60, 120, 240, and 360 min post-dose. blq: below limit of quantification (2 ng/ml); n = number of mice used for sampling. Samples for aqueous and vitreous humor were pooled as outlined in the Material and Methods section. (DOCX) of idebenone at 60 mg/kg to male mice. Data are expressed in ng/ ml (6 SEM). Idebenone concentration was measured 5, 15, 30, 60, 120, 240, and 360 min post-dose. blq: below limit of quantification (2 ng/ml); n = number of mice used for sampling. Samples for aqueous and vitreous humor were pooled as outlined in the Material and Methods section. Table S3 Pharmacokinetic parameters of idebenone in plasma, aqueous and vitreous humor following single oral administration of idebenone at 60 mg/kg to male mice. Data are expressed as mean for plasma based on n = 20 mice per time point. Samples for aqueous and vitreous humor were pooled as outlined in the Material and Methods section. Based on our results, the potency of idebenone to alleviate symptoms and pathologies of LHON could thus be a consequence of its combined antioxidant function [22,23,24] and its ability to modulate mitochondrial electron flow [27,28]. Since the visual system is the major energy consumer of the central nervous system, with a high susceptibility towards dysregulation of the energy metabolism [51], it is not surprising that a large number of ophthalmological disorders are associated with mitochondrial defects in energy metabolism and oxidative damage. Supporting Information Figure S1 Idebenone protects against rotenone-induced loss of vision. (a) Photograph of the experimental set up used for assessing visual acuity. (b) Visual acuity was also evaluated by counting the ratio between clock-wise and counterclock-wise head movements (CW/CCW) within 2 min. 7 days prior injection (day -7) and 1 to 70 days after injection (day 1, 7, 21, 35, 70) of 5 mM rotenone. Quantification following vehicle treatment and rotenone injection (vehicle + rotenone, n = 10 animals), and idebenone 2000 mg/kg treatment and rotenone injection (IDE 2000+ rotenone, n = 11 animals). Data are expressed as mean 6 SEM. (TIF) Video S1 Normal clockwise (CW) and counter-clockwise (CCW) head movements during the optomotor response of a representative mouse, which was sham (DMSO)- injected into the eye. (WMV) Video S2 Absence of clockwise (CW) head movements during the optomotor response of a representative mouse, which was rotenone-injected into the eye. (WMV) Table S1 Concentrations of idebenone in plasma, aqueous and vitreous humor following repeated administration of idebenone in the diet to male mice. Data are expressed in ng/ml (6 SEM). Idebenone was administered at 20, 40, 200, 400, and 2000 mg/kg in the diet. Idebenone concentration was measured at least 8 hours post last dose. blq: below limit of quantification (2 ng/ml); n = number of mice used for sampling; samples were pooled as outlined in Material and Methods section. Acknowledgments We thank Martin Schwab for his support and the donation of the RGC5 cell line [52] and Thomas Meier for conceptual contribution and reviewing of the manuscript. We also acknowledge staff members of Inovalab. Idebenone Protects against Retinal Damage Table S4 Effect of idebenone (2000 mg/kg) treatment on head movements of mice with rotenone (5 mM)- and sham (DMSO)- injected eyes. Optomotor analysis of the rotenone (5 mM)-injected eye and the sham (DMSO)-injected eye in C57BL/6J mice was performed by counting clockwise (CW) and counterclockwise (CCW) head movements. Head movements were evaluated at a velocity of 3 rpm 7 days prior injection (day -7) and after injection (day 1, 7, 21, 35, 70). Optomotor data expressed as mean number of head movements/2 min. 6 SEM. Statistical signifi- cance relative to vehicle + rotenone group: p#0.05 (*) based on raw numbers; n = number of mice per group. CW: clockwise head movements; CCW: counterclockwise head movements; vehicle + rotenone: vehicle treated and injected with 5 mM rotenone; IDE2000+ rotenone: idebenone (2000 mg/kg) treated and in- jected with 5 mM rotenone. In summary, the data presented here, together with the favorable safety and tolerability profile of idebenone, provide an excellent rationale for the use of idebenone in LHON and open perspectives for future applications of idebenone in other ophthalmological indication. (DOCX) Table S2 Pharmacokinetic profile of idebenone in plasma, aqueous and vitreous humor following single oral administration Table S2 Pharmacokinetic profile of idebenone in plasma, aqueous and vitreous humor following single oral administration 8. Wallace DC, Singh G, Lott MT, Hodge JA, Schurr TG, et al. (1988) Mitochondrial DNA mutation associated with Leber’s hereditary optic neuropathy. Science 242: 1427–1430. Idebenone Protects Against Rotenone-induced Loss of Vision in the Mouse in vivo Since RGC loss is associated with visual acuity impairment [34] and idebenone protects against RGC loss and retinal damage, we investigated the functional effect of idebenone pre-treatment on visual acuity in animals with intravitreal injection of 5 mM rotenone using the optomotor head-tracking test (Fig. 8A, S1A and Video S1, S2). Intravitreal injection of 5 mM rotenone specifically abolished clockwise head responses entirely controlled by the left (injected) eye in all the treatment groups after 24 hours (Fig. 8B and Table S4). Consistent with the RGC protection previously observed, idebenone (2000 mg/kg) pre-treatment promoted a sig- nificant vision recovery 70 days after the rotenone injection when compared to vehicle-treated animals (Fig. 8B and Table S4). This protective effect of idebenone was also reflected by the increased number of mice displaying clockwise head responses (Fig. 8C). The effect of idebenone did not result from any change of vision related to the right (non-injected) eye since right eye-controlled counter- September 2012 \| Volume 7 \| Issue 9 \| e45182 PLOS ONE \| www.plosone.org 9 Idebenone Protects against Retinal Damage 7. Sadun AA, Win PH, Ross-Cisneros FN, Walker SO, Carelli V (2000) Leber’s hereditary optic neuropathy differentially affects smaller axons in the optic nerve. Trans Am Ophthalmol Soc 98: 223–232. 5. Fraser JA, Biousse V, Newman NJ (2010) The neuro-ophthalmology of mitochondrial disease. Surv Ophthalmol 55: 299–334. 4. Carelli V, Ross-Cisneros FN, Sadun AA (2004) Mitochondrial dysfunction as a cause of optic neuropathies. Prog Retin Eye Res 23: 53–89. 2. Sadun AA, La Morgia C, Carelli V (2011) Leber’s Hereditary Optic Neuropathy. Curr Treat Options Neurol 13: 109–117. 1. Mascialino B, Leinonen M, Meier T (2012) Meta-analysis of the prevalence of Leber hereditary optic neuropathy mtDNA mutations in Europe. Eur J Ophthalmol 22(3): 461–465. 3. Tonska K, Kodron A, Bartnik E (2010) Genotype-phenotype correlations in Leber hereditary optic neuropathy. Biochim Biophys Acta 1797: 1119–1123. Author Contributions Conceived and designed the experiments: FDH NG. Performed the experiments: FDH ME CA DR. Analyzed the data: FDH ME NG. Contributed reagents/materials/analysis tools: VP. Wrote the paper: FDH NG. Conceived and designed the experiments: FDH NG. Performed the experiments: FDH ME CA DR. Analyzed the data: FDH ME NG. Contributed reagents/materials/analysis tools: VP. Wrote the paper: FDH NG. Idebenone Protects against Retinal Damage 9. Johns DR, Neufeld MJ, Park RD (1992) An ND-6 mitochondrial DNA mutation associated with Leber hereditary optic neuropathy. Biochem Biophys Res Commun 187: 1551–1557. 30. Abdeljalil J, Hamid M, Abdel-Mouttalib O, Stephane R, Raymond R, et al. (2005) The optomotor response: a robust first-line visual screening method for mice. Vision Res 45: 1439–1446. 10. Huoponen K, Vilkki J, Aula P, Nikoskelainen EK, Savontaus ML (1991) A new mtDNA mutation associated with Leber hereditary optic neuroretinopathy. Am J Hum Genet 48: 1147–1153. 31. Krishnamoorthy RR, Agarwal P, Prasanna G, Vopat K, Lambert W, et al. (2001) Characterization of a transformed rat retinal ganglion cell line. Brain Res Mol Brain Res 86: 1–12. 32. Bringmann A, Iandiev I, Pannicke T, Wurm A, Hollborn M, et al. (2009) Cellular signaling and factors involved in Muller cell gliosis: neuroprotective and detrimental effects. Prog Retin Eye Res 28: 423–451. 11. Brown MD, Allen JC, Van Stavern GP, Newman NJ, Wallace DC (2001) Clinical, genetic, and biochemical characterization of a Leber hereditary optic neuropathy family containing both the 11778 and 14484 primary mutations. Am J Med Genet 104: 331–338. 33. Yu XH, Zhang H, Wang YH, Liu LJ, Teng Y, et al. (2009) Time-dependent reduction of glutamine synthetase in retina of diabetic rats. Exp Eye Res 89: 967–971. 12. Beretta S, Wood JP, Derham B, Sala G, Tremolizzo L, et al. (2006) Partial mitochondrial complex I inhibition induces oxidative damage and perturbs glutamate transport in primary retinal cultures. Relevance to Leber Hereditary Optic Neuropathy (LHON). Neurobiol Dis 24: 308–317. 34. Ellouze S, Augustin S, Bouaita A, Bonnet C, Simonutti M, et al. (2008) Optimized allotopic expression of the human mitochondrial ND4 prevents blindness in a rat model of mitochondrial dysfunction. Am J Hum Genet 83: 373–387. 13. Carelli V, Rugolo M, Sgarbi G, Ghelli A, Zanna C, et al. (2004) Bioenergetics shapes cellular death pathways in Leber’s hereditary optic neuropathy: a model of mitochondrial neurodegeneration. Biochim Biophys Acta 1658: 172–179. 35. Barcella V, Rocca MA, Bianchi-Marzoli S, Milesi J, Melzi L, et al. (2010) Evidence for retrochiasmatic tissue loss in Leber’s hereditary optic neuropathy. Hum Brain Mapp 31: 1900–1906. 14. Beretta S, Mattavelli L, Sala G, Tremolizzo L, Schapira AH, et al. (2004) Leber hereditary optic neuropathy mtDNA mutations disrupt glutamate transport in cybrid cell lines. Brain 127: 2183–2192. 36. Idebenone Protects against Retinal Damage Mashima Y, Hiida Y, Oguchi Y (1992) Remission of Leber’s hereditary optic neuropathy with idebenone. Lancet 340: 368–369. 21. Li N, Ragheb K, Lawler G, Sturgis J, Rajwa B, et al. (2003) Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochon- drial reactive oxygen species production. J Biol Chem 278: 8516–8525. p y 44. Cortelli P, Montagna P, Pierangeli G, Lodi R, Barboni P, et al. (1997) Clinical and brain bioenergetics improvement with idebenone in a patient with Leber’s hereditary optic neuropathy: a clinical and 31P-MRS study. J Neurol Sci 148: 25–31. 22. Suno M, Nagaoka A (1989) Inhibition of lipid peroxidation by idebenone in brain mitochondria in the presence of succinate. Arch Gerontol Geriatr 8: 291– 297. 45. Carelli V, Barboni P, Zacchini A, Mancini R, Monari L, et al. (1998) Leber’s Hereditary Optic Neuropathy (LHON) with 14484/ND6 mutation in a North African patient. J Neurol Sci 160: 183–188. 23. Mordente A, Martorana GE, Minotti G, Giardina B (1998) Antioxidant properties of 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone (idebenone). Chem Res Toxicol 11: 54–63. 46. Carelli V, Valentino ML, Liguori R, Meletti S, Vetrugno R, et al. (2001) Leber’s hereditary optic neuropathy (LHON/11778) with myoclonus: report of two cases. J Neurol Neurosurg Psychiatry 71: 813–816. 24. Rauchova H, Drahota Z, Bergamini C, Fato R, Lenaz G (2008) Modification of respiratory-chain enzyme activities in brown adipose tissue mitochondria by idebenone (hydroxydecyl-ubiquinone). J Bioenerg Biomembr 40: 85–93. 47. Barnils N, Mesa E, Munoz S, Ferrer-Artola A, Arruga J (2007) Response to idebenone and multivitamin therapy in Leber’s hereditary optic neuropathy. Arch Soc Esp Oftalmol 82: 377–380. 25. Sugiyama Y, Fujita T (1985) Stimulation of the respiratory and phosphorylating activities in rat brain mitochondria by idebenone (CV-2619), a new agent improving cerebral metabolism. FEBS Lett 184: 48–51. 48. Sabet-Peyman EJ, Khaderi KR, Sadun AA (2012) Is Leber Hereditary Optic Neuropathy Treatable? Encouraging Results With Idebenone in Both Pro- spective and Retrospective Trials and An Illustrative Case. J Neuroophthalmol 32: 54–7. 26. Rauchova H, Vrbacky M, Bergamini C, Fato R, Lenaz G, et al. (2006) Inhibition of glycerophosphate-dependent H2O2 generation in brown fat mitochondria by idebenone. Biochem Biophys Res Commun 339: 362–366. 49. Reagan-Shaw S, Nihal M, Ahmad N (2008) Dose translation from animal to human studies revisited. FASEB J 22: 659–661. 27. Haefeli RH, Erb M, Gemperli AC, Robay D, Courdier Fruh I, et al. References 6. Barboni P, Carbonelli M, Savini G, Ramos Cdo V, Carta A, et al. (2010) Natural history of Leber’s hereditary optic neuropathy: longitudinal analysis of the retinal nerve fiber layer by optical coherence tomography. Ophthalmology 117: 623–627. 1. Mascialino B, Leinonen M, Meier T (2012) Meta-analysis of the prevalence of Leber hereditary optic neuropathy mtDNA mutations in Europe. Eur J Ophthalmol 22(3): 461–465. 2. Sadun AA, La Morgia C, Carelli V (2011) Leber’s Hereditary Optic Neuropathy. Curr Treat Options Neurol 13: 109–117. 7. Sadun AA, Win PH, Ross-Cisneros FN, Walker SO, Carelli V (2000) Leber’s hereditary optic neuropathy differentially affects smaller axons in the optic nerve. Trans Am Ophthalmol Soc 98: 223–232. 3. Tonska K, Kodron A, Bartnik E (2010) Genotype-phenotype correlations in Leber hereditary optic neuropathy. Biochim Biophys Acta 1797: 1119–1123. 4. Carelli V, Ross-Cisneros FN, Sadun AA (2004) Mitochondrial dysfunction as a cause of optic neuropathies. Prog Retin Eye Res 23: 53–89. 5. Fraser JA, Biousse V, Newman NJ (2010) The neuro-ophthalmology of mitochondrial disease. Surv Ophthalmol 55: 299–334. September 2012 \| Volume 7 \| Issue 9 \| e45182 10 PLOS ONE \| www.plosone.org Idebenone Protects against Retinal Damage Smith JL, Tse DT, Byrne SF, Johns DR, Stone EM (1990) Optic nerve sheath distention in Leber’s optic neuropathy and the significance of the ‘‘Wallace mutation’’. J Clin Neuroophthalmol 10: 231–238. 15. Zanna C, Ghelli A, Porcelli AM, Martinuzzi A, Carelli V, et al. (2005) Caspase- independent death of Leber’s hereditary optic neuropathy cybrids is driven by energetic failure and mediated by AIF and Endonuclease G. Apoptosis 10: 997– 1007. J p 37. de Gottrau P, Buchi ER, Daicker B (1992) Distended optic nerve sheaths in Leber’s hereditary optic neuropathy. J Clin Neuroophthalmol 12: 89–93. y p p y J p 38. Carelli V, Sadun AA (2001) Optic neuropathy in Lhon and Leigh syndrome. Ophthalmology 108: 1172–1173. 16. Zhang X, Jones D, Gonzalez-Lima F (2002) Mouse model of optic neuropathy caused by mitochondrial complex I dysfunction. Neurosci Lett 326: 97–100. 39. Ishikawa M, Yoshitomi T, Zorumski CF, Izumi Y (2011) Downregulation of glutamine synthetase via GLAST suppression induces retinal axonal swelling in a rat ex vivo hydrostatic pressure model. Invest Ophthalmol Vis Sci 52: 6604– 6616. 17. Rojas JC, Saavedra JA, Gonzalez-Lima F (2008) Neuroprotective effects of memantine in a mouse model of retinal degeneration induced by rotenone. Brain Res 1215: 208–217. 18. Chadderton N, Palfi A, Millington-Ward S, Gobbo O, Overlack N, et al. (2012) Intravitreal delivery of AAV-NDI1 provides functional benefit in a murine model of Leber hereditary optic neuropathy. Eur J Hum Genet. doi:10.1038/ ejhg.2012.112. 40. Klopstock T, Yu-Wai-Man P, Dimitriadis K, Rouleau J, Heck S, et al. (2011) A randomized placebo-controlled trial of idebenone in Leber’s hereditary optic neuropathy. Brain 134: 2677–2686. p y 41. Carelli V, La Morgia C, Valentino ML, Rizzo G, Carbonelli M, et al. (2011) Idebenone treatment in Leber’s hereditary optic neuropathy. Brain 134: e188. 19. Hayworth CR, Rojas JC, Gonzalez-Lima F (2008) Transgenic mice expressing cyan fluorescent protein as a reporter strain to detect the effects of rotenone toxicity on retinal ganglion cells. J Toxicol Environ Health A.71(24): 1582–92. 42. Mashima Y, Kigasawa K, Wakakura M, Oguchi Y (2000) Do idebenone and vitamin therapy shorten the time to achieve visual recovery in Leber hereditary optic neuropathy? J Neuroophthalmol 20: 166–170. 20. Marella M, Seo BB, Thomas BB, Matsuno-Yagi A, Yagi T (2010) Successful amelioration of mitochondrial optic neuropathy using the yeast NDI1 gene in a rat animal model. PLoS One 8;5(7): e11472. p p y J p 43. Idebenone Protects against Retinal Damage (2011) NQO1-dependent redox cycling of idebenone: effects on cellular redox potential and energy levels. PLoS One 6: e17963. 50. Suno M, Nagaoka A (1984) Inhibition of lipid peroxidation by a novel compound (CV-2619) in brain mitochondria and mode of action of the inhibition. Biochem Biophys Res Commun 125: 1046–1052. gy 28. Giorgio V, Petronilli V, Ghelli A, Carelli V, Rugolo M, et al. (2011) The effects of idebenone on mitochondrial bioenergetics. Biochim Biophys Acta 1817: 363– 369. p y 51. Bristow EA, Griffiths PG, Andrews RM, Johnson MA, Turnbull DM (2002) The distribution of mitochondrial activity in relation to optic nerve structure. Arch Ophthalmol 120: 791–796. 29. Nadal-Nicolas FM, Jimenez-Lopez M, Sobrado-Calvo P, Nieto-Lopez L, Canovas-Martinez I, et al. (2009) Brn3a as a marker of retinal ganglion cells: qualitative and quantitative time course studies in naive and optic nerve-injured retinas. Invest Ophthalmol Vis Sci 50: 3860–3868. 52. Krishnamoorthy RR, Agarwal P, Prasanna G, Vopat K, Lambert W, et al. (2001) Characterization of a transformed rat retinal ganglion cell line. Brain Res Mol Brain Res. 86: 1–12. September 2012 \| Volume 7 \| Issue 9 \| e45182 PLOS ONE \| www.plosone.org 11
https://openalex.org/W2743172250	https://www.scielo.br/j/bar/a/wWZ3hhrGBTktQWMDp7RcKsG/?lang=en&format=pdf	English	null	Business-Community Relationships for Extractive Industries: A Case Study in Peru	BAR - Brazilian Administration Review	2,017	cc-by	12,262	Business-Community Relationships for Extractive Industries: A Case Study in Peru Jose Ventura1 Kety Jauregui1 Universidad ESAN1 Received 19 November 2016; received in revised form in 19 May 2017; accepted in 2 July 2017; first published online 24 July 2017. Editor’s note. Diogo Helal served as Action Editor for this article. Business-Community Relationships for Extractive Industries: A Case Study in Peru Received 19 November 2016; received in revised form in 19 May 2017; accepted in 2 July 2017; first published online 24 July 2017. Editor’s note. Diogo Helal served as Action Editor for this article. Available online at http://www.anpad.org.br/bar BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 http://dx.doi.org/10.1590/1807-7692bar2017160114 Business-Community Relationships for Extractive Industries: A Case Study in Peru Available online at http://www.anpad.org.br/bar BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 http://dx.doi.org/10.1590/1807-7692bar2017160114 Business-Community Relationships for Extractive Industries: A Case Study in Peru Available online at http://www.anpad.org.br/bar BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 http://dx.doi.org/10.1590/1807-7692bar2017160114 Available online at http://www.anpad.org.br/bar BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 http://dx.doi.org/10.1590/1807-7692bar2017160114 p y ditor’s note. Diogo Helal served as Action Editor for this article. ived 19 November 2016; received in revised form in 19 May 2017; accepted in 2 July 2017 published online 24 July 2017. Jose Ventura1 Kety Jauregui1 Universidad ESAN1 Received 19 November 2016; received in revised form in 19 May 2017; accepted in 2 July 2017; first published online 24 July 2017. Editor’s note. Diogo Helal served as Action Editor for this article. p y Editor’s note. Diogo Helal served as Action Editor for this article. J. Ventura, K. Jauregui J. Ventura, K. Jauregui 2 Introduction Over the last 15 years, extractive industries have gained momentum in Latin America (Damonte, Kuramoto, & Glave, 2014) and contributed significantly to the growth of the gross domestic product (GDP) of economies in the region. For instance, Peru’s extractive sector accounted for an average of 13.5% of its GDP over the last ten years (Banco Central de Reserva del Perú [BCRP], 2015) and made up about 62% of total exports in 2014 (Superintendencia Nacional de Aduanas y Administración Tributaria [SUNAT], 2015). Clearly, the Peruvian economy depends heavily on extractive industries; likewise, the social and environmental impacts of these industries on rural communities are also of note. Peru’s mining industry went from operating on 15 million hectares of community lands (Dorregaray, 2005) in 1993, to 81.72 million hectares in 2015 (Ministerio de Energía y Minas [MINEM], 2015a). According to Peruvian government sources, by 2013 a great deal of lands occupied by 6,436 officially recognized peasant communities in Peru had been franchised to mining companies (Superintendencia Nacional de los Registros Públicos [SUNARP], 2016). Although the Peruvian economy grew 2.4% in 2014, compared with 1.3% growth in Latin America and the Caribbean overall and 3.4% growth worldwide during the same period (International Monetary Fund [IMF], 2015), the number of social conflicts resulting from the exploitation of natural resources increased much more substantially during the same period. As of October 2007, the Peruvian Ombudsman’s Office had registered 80 social conflicts (Defensoría del Pueblo, 2007), and by November 2015 this figure had climbed to 215, of which 149 (69.3%) were socio-environmental conflicts (Defensoría del Pueblo, 2015). Thus far, a climate of distrust and a lack of cooperative relationships remain key challenges for extractive firms. The reactions of rural communities to extractive operations have become all the more delicate, thus leading to delays and/or the cancellation of projects, which in turn may compromise the producing country’s economic health and, hence, prevent the government from fulfilling its mandate to develop the countryside. For instance Davis and Franks (2014) contend that company-community conflict triggered by environmental and social impacts can involve costs arising from temporary shutdowns or delays, of up to US $20 million per week. At the same time, the greatest expenses are the opportunity costs due to planned future projects or expansion plans that stalled. Abstract Natural resource-based economies have long relied on foreign demand to fuel their growth. For instance, the extractive sectors in Peru have experienced a rapid expansion, driven by a rising demand for commodities. Alongside economic growth, extractive operations have triggered social and environmental concerns among the various stakeholders, thus resulting in either social conflict or a deterioration of the relationship between companies in the extractive industries and local communities. In this context, the purpose of this paper is to understand the relationships between companies in the extractive industries and rural families. This research uses the case-study method. The findings show that a trustful relationship is supported by a beneficiary-society approach that builds upon philanthropic and ethical types of relationships. Unlike the type of relationship based on economic or legal interests, a trust-based relationship offers avenues for managing social conflict that have yet to be explored. ey words: rural communities; extractive industries; social conflict; business-community relationships; Per BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 www.anpad.org.br/bar www.anpad.org.br/bar Business-Community Relationships for Extractive Industries 3 Business-Community Relationships for Extractive Industries Introduction For companies in the extractive industries, therefore, reconciling the needs and views of stakeholders (i.e., governments, local communities, employees, non-governmental agencies) with the company’s own obligations to investors and showing how company activities can contribute both to GDP and to meaningful outcomes for local communities have become critical concerns to the prospect of earning the “social license to operate” (Deloitte, 2015, p. 6). The literature on the relations between business and society shows two clearly different approaches. On the one hand, businesses seek to create goods and services to meet the needs of society, creating economic values for the company and satisfying the needs and wants of society. Companies are profitable as a result of the goods and services that they provide to society (Drucker, 1985; Friedman, 1962, 1970; Prahalad & Hart, 2008; Porter & Kramer, 2006). On the other hand, companies must share their profits with society. Companies must contribute directly to society because business activity negatively affects society; negative externalities (Coase, 1960) society assumes such liabilities, including environmental pollution, asymmetry of power, and social and economic inequities. In this case, society must be directly compensated by the company for its negative practices and effects on society (Ahlstrom, 2010; Bell & Emory, 1971; Du, Bhattacharya, & Sen, 2007, 2010; Freeman & Phillips, 2002; Freeman & Reed, 1983; Freeman, Wicks, & Parmar, 2004; Garriga & Melé, 2004). Focusing a little more on the interaction between business and society, the literature describes the conflicted relationship between companies in the extractive industries and rural communities. Studies show the dimensions of the problem and the negative effects of such conflicts (Bebbington, 2013; Damonte et al., 2014; Davis. & Franks, 2014; Nem Singh, 2012). However, these studies reveal little on the link between companies in the extractive industries and rural communities. The information BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 www.anpad.org.br/bar J. Ventura, K. Jauregui J. Ventura, K. Jauregui 4 available on the subject focuses on social conflicts and how they are managed but not on the underlying relationships themselves. Corporate social responsibility (CSR) seeks to bring companies in the extractive sector closer to rural communities. However, they do not study the type of relationships being built. Introduction Companies’ CSR strategies are exclusively disputes about the environment, human rights or health and safety; rather, they are better understood as the control over the community and the right of community members to control the direction of their lives (Laplante & Spears, 2008). Furthermore, information on social responsibility has yet to penetrate deeply into the kinds of relationships built between businesses and rural communities (Altman, 1998; Damonte, 2008; Laplante & Spears, 2008; McKenna, 2016). Very little is known about the type of relationship there is between companies in the extractive sector and the members of communities, especially for the cases in which the literature indicates that the rural communities are not homogenous entities. Furthermore, Andean rural communities, as well as indigenous societies, are in the process of change. Significantly, the family and the community remain very intertwined concepts. Studies affirm that family is the most stable social entity within the rural communities (Castro, 2012). In this sense, the relationship between companies in the extractive sector and rural communities ends up being a relationship between the companies and rural families. Hence, through the case study method this paper seeks to understand the relationships between companies and rural families. A review of the literature on the relationship between business and society opens the paper and includes a conceptual framework. The second part of the paper introduces the research method. The third part presents and compares findings related to two companies in the extractive sector. Finally, the fourth section discusses these findings and offers possible conclusions. Literature Review potential source of perceived social irresponsibility and, hence, the loss of trust, which can undermine consumer-related outcomes such as purchases and loyalty (Klein & Dawar, 2004). Recent research in management and ethics, however, contends that stakeholder theory, as a view of the relationship between business and society, is rather business-centric and market-oriented than stakeholder-oriented. Following this line of thought, Laczniak and Murphy (2012) and Hillebrand, Driessen and Koll (2015) are among the scholars who advocate for a view that acknowledges the multiplicity of relationships of companies with different stakeholders and who call for more prosociety and proenvironment perspectives regarding the ways companies both relate to societal members and conduct their activities. Notwithstanding the fact that the extant business-stakeholder literature addresses a progression towards more balanced kinds of relationships, for example, those between companies and groups within society (key stakeholders) are often given a short shrift, as occurs with local communities and families (the most basic unit in any society). Alternate streams of thought started in the 60s, with the seminal works by Friedman (1962, 1970) and Porter (1980), who first referred to society as a consumer of business activity, as well as the study by Bell and Emory (1971), who referred to society as a beneficiary of business activity. Specific criteria signal how a company approaches society. Thus, a company that approaches society as a consumer society pursues specific business objectives. These include (a) maximizing profits and redistributing them among its stockholders, (b) providing goods and services to meet the needs of society, and (c) perceiving society as its real and potential market (Drucker, 1985; Friedman, 1962, 1970; Hillman & Keim, 2001; Prahalad, & Hart, 2008; Porter & Kramer, 2006). In contrast, a company that approaches society as a beneficiary society possesses a different strategy. It seeks to (a) maximize its profits and simultaneously share them with select stakeholders, (b) compensate and remedy the damages it has created in society, (c) perceive society as a vulnerable entity lacking basic needs (Ahlstrom, 2010; Bell & Emory, 1971; Du et al., 2007, 2010; Freeman & Phillips, 2002; Freeman & Reed, 1983; Freeman et al., 2004; Garriga & Melé, 2004). Literature Review The relationship between business and society has been determined by the evolving conception of the business as a social entity. Companies (the basic units of the business sector) are commonly viewed as members of society; that is, the visible economic face of a social system (Steiner, 1971). In spite of this, the initial definitions of a business alluded to it as a vehicle of wealth creation that was independent from society (Friedman, 1962; Lucas & Sargent, 1981). Nowadays, nonetheless, the multiplicity of linkages with other social agents requires firms to accept greater responsibility toward society. The activities of business influence society by either inducing or preventing changes in the social system (Schumpeter, 1939). Business outcomes can contribute by providing inputs to meet social needs. They can also exert positive or negative influences on the local scenario in which they undertake their activities (neighboring communities). This latter aspect makes the social responsibility (SR) of a company a relevant issue, because the direct influence of businesses on society makes it likely for a business to accept the greater responsibilities that go along with this influence (Carroll, 1993; Garriga & Melé, 2004). In so doing, companies adopt SR activities to create accountability and to demonstrate their concern for societal members’ well-being and issues in a broader social context (Peloza & Shang, 2011; Sen, Bhattacharya, & Korschun, 2006). Likewise, social influence on companies is paramount. Society, for example, provides a firm with qualified labor resources as well as consumers, both necessary for its very existence. This argument showcases the influence of society, which has also been contemplated within the frame of the stakeholder theory, whereby different parties or interest groups (consumers, employees, suppliers, or trade unions, to name a few) have a stake in the success of failure of a business (Parmar et al., 2010). Consumers, for instance, are capable of exerting scrutiny over companies’ behavior. Over time, historical experience creates an evaluative context whereby consumers can elicit positive/negative associations that increase/decrease their sense of trust (Bhattacharya & Sen, 2003, 2004; Ellen, Webb, & Mohr, 2006). Furthermore, on a society-wide level, enhanced firms’ exposure makes scrutiny a BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 www.anpad.org.br/bar Business-Community Relationships for Extractive Industries 5 5 potential source of perceived social irresponsibility and, hence, the loss of trust, which can undermine consumer-related outcomes such as purchases and loyalty (Klein & Dawar, 2004). BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 Society as a Consumer Perspective Businesses serve the consumer society by running the business well and in accordance with both the rule of law and ethical customs. From this stance, SR is subsidiary to profit maximization as stated by Friedman, who in 1962 wrote that “businesses only have one SR: to use their resources and engage in activities designed to increase their profits so long as they stay within the rules of the game, i.e., operate in open and free competition without deception or fraud” (Friedman, 1962, p. 133). Although Friedman’s view has been repeatedly challenged, it is important to note that he is one of the main proponents of the stockholder theory; a business-ethics theory concerning businesses and the behavior of business people, which does not preclude social interest (Hasnas, 1998). As such, the long-term benefit of investors makes it possible for a company to invest in product innovation and, thus, to deliver high-value products that improve people’s standards of living, in addition to goals other than profit alone. That is, as businesses develop new goods and/or services that generate economic growth, they also deliver important benefits to society (Ahlstrom, 2010). Thus, business meets society’s needs, and society is a major consumer of goods and services produced by companies. Porter (1980) contended that companies have a duty to maximize value creation, that is, to create value for the customer. This translates into a difference between the production costs for a product or service and the price people accept to pay for that product or service. In this view, the purpose of business is not limited to making a profit but instead to search for competitiveness, which entails making more and better products for the benefit of society (Husted & Salazar-Cantú, 2006), provided that families and individuals are able to consume affordable, high-quality goods and services. BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 www.anpad.org.br/bar www.anpad.org.br/bar J. Ventura, K. Jauregui J. Ventura, K. Jauregui 6 All in all, the initial conceptualizations of SR presented the business within a context of freedom and open competition as a stockholder-centered, law-abiding entity with no social obligation beyond making a profit (Friedman, 1962). Social responsibility was regarded as a tool for accomplishing the firm’s economic goals of greater productivity and wealth creation (Carroll, 2008; Garriga & Melé, 2004; Hillman & Keim, 2001; Husted & Salazar-Cantú, 2006). Society as a Consumer Perspective Soon after, interdependence grew between business and society as consumers shed light on businesses’ responsibility for their economic and marketing functions of rendering goods and services and on the observations society’s values and customs, leaving other considerations to philanthropy (Friedman, 1962). In addition, Drucker (1985) stated that the responsibility of a company is to generate profit so it can be sustainable: A company that does not earn a profit that is at least equal to its cost of capital is irresponsible because it wastes society’s resources. Good economic performance is the foundation without which the company cannot meet any of its other responsibilities (p. 13). Note that, under the society-as-a-consumer approach, the scope of social responsibility links the economic and marketing functions of firms to law-abiding and ethical considerations (Robin & Reidenbach, 1987). Relationships between Businesses and Rural Families Both the previous approaches delve further into the business-society relationship. Neither of them, however, addresses in a straightforward manner the relationship between companies and subsets within society, like a local community or a family, the latter being not only the basic unit of society but also a key entity within the microeconomic scenario. Families constitute the demand side of the market: they consume to satisfy their needs and wants. They are also part of the supply side of the market: they produce goods and services for society and businesses alike. Hence, families play a key role in business development so that their interests cannot be viewed as contrary to those of powerful companies. To this extent, Carroll (1979) and, later on, Porter and Kramer (2002, 2006, 2011) further investigated the nature of the society-as-consumer and the society-as-beneficiary relationships, in attunement with the evolving concept of SR. To them, companies and families (or communities) can enact relationships of four types: economic, legal, ethical, and philanthropic. From a microeconomic viewpoint, Maddala and Miller (1991) have contended that, on the one hand, the business is a unit of production of goods and services that meets the needs of society and, on the other hand, they assert that the family is an economic agent that consumes, saves, invests, and provides labor. The two units are parts of society. As consumers of goods and services produced by business, families form the company’s most attractive market. Businesses meet household food, housing, health, education, mobility, and entertainment needs. Families cannot be regarded as opponents or antagonists of productive business interests but are rather one of the business’ most important partners and the ultimate beneficiaries of all business activities. To that extent, the relationships between business and families, whether the latter are regarded as consumers or beneficiaries, are of four types: economic, legal, ethical, and discretionary or philanthropic (Carroll, 2003). Business must produce the goods and services that families need or want and sell them at a reasonable price. The company thus benefits financially, meets its expectations, perpetuation of self, grows, and makes a profit for its shareholders (Carroll, 2008). Therefore, in an economic relationship, a company provides goods and services required by families, whereas families elicit positive associations which lead to consumer-related outcomes. Other SR actions, such as cause-related marketing, or basic infrastructure projects, to name a few, can be seen as strategic philanthropy. Society as Beneficiary Perspective Business also benefits societal members in ways that do not collide with obligations to shareholders. Engaging employee recruitment that live in the community, partnering with local suppliers, improving workplace conditions, and building roads and basic infrastructure are among the many activities that go beyond both legal requirements and wealth-creation for shareholders, and yet they are intertwined with profit-oriented practices. That is, to produce goods and services, a company must engage in activities likely to have a positive impact on different stakeholders or societal groups with a legitimate interest in the success or failure of the business (Parmar et al., 2010). Moreover, while doing so, the company is also ensuring the means for achieving profits (Daou & Sarkis, 2013). Subsequently, under the approach in which society is a beneficiary, a business’s main goal is profit maximization, although other stakeholder-related goals are also possible. Some such goals might be mandatory, for they are of critical importance to attaining the resources firms need to be profitable and competitive (Connelly, Ketchen, & Slater, 2011), as is the case wherein technical capabilities are rendered by qualified employees, materials and services are provided by suppliers, and so forth. Conversely, in a free-market economy, companies seek to minimize production costs to maximize profits and remain competitive (Roarty, 1997). Often, this entails shifting part of the actual production costs to the community rather than paying for them. These so-called social costs or negative externalities are what business are not willing to pay for, otherwise costs will increase and reduce profitability and competitiveness. Ultimately, such costs are paid for by society. In a way, business activity harms certain social sectors, has negative consequences including social and cultural conflicts, and may provoke social imbalances, among other undesired outcomes (Carroll, 2008). An unstated assumption is that business’ activities harm society in one way or another. Thus, SR takes on another hue that is no longer inherent to business activity, because it emerges as a collateral response of the business against the social costs it creates in its efforts to gain legitimacy from society (Melé, 2008). Figure 1 describes the business - society relationship approaches. The upper and lower vertices represent the two proposed social arrangements: (a) business-consumer society, and (b) business-beneficiary society. BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 www.anpad.org.br/bar 7 7 Business-Community Relationships for Extractive Industries Figure 1. Society as Beneficiary Perspective Business – Society Perspective Source: Elaborated by the authors. Society Business Beneficiary society Perspective Consumer society Perspective Consumer society Perspective Society Business Beneficiary society Perspective Figure 1. Business – Society Perspective Source: Elaborated by the authors. Figure 1. Business – Society Perspective BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 Relationships between Businesses and Rural Families The business’ second responsibility is legal (Carroll, 1979, 2003). Having a legal responsibility implies that business and families abide by a body of existing laws. If a rift appears between families and business, both parties can resort to legal procedures to resolve their dispute (Carroll, 2003). Porter and Kramer (2006, p. 5) call it the license to operate: “The notion of license to operate is derived from the fact that every company needs tacit or explicit governments, communities and several other stakeholders’ authorization to do business”. Therefore, in a legal relationship, governments establish a legal and regulatory environment that companies must comply with. However, that is not the extent of their social responsibility. Companies can abide by other mechanisms: the attainment of the social license to operate, commonly found in extractive industries (Moffat & Zhang, 2014); the adoption of BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 www.anpad.org.br/bar J. Ventura, K. Jauregui 8 SR practices championed at the industry level and voluntary concern with international endeavors (e.g. the Global Reporting Initiative, the UN Global Compact), among other social causes. Ethical responsibilities include decisions, practices and duties for which societal members (business agents included) are accountable, and these must be validated in accordance to the moral values that society at large has agreed upon. Thus, ethical responsibilities are to reflect the values and emerging standards that society expects from businesses. Consequently, a company’s ethical responsibilities are always under scrutiny and subject to public debate because their legitimacy depends on society. From the business standpoint, the extent of SR depends on voluntary commitments determined by business’ understanding of their impacts, either positive or negative, on society (Maon, Lindgreen, & Swaen, 2010; Safwat, 2015). Therefore, a relationship based on ethical responsibilities includes the non-statutory activities and practices that members of society allow or prohibit (Carroll, 1991, 2003). A clear distinction exists between relationships based on legal responsibilities and those based on ethical responsibilities. Legal responsibilities are required and mandatory, whereas ethical responsibilities involve value-guided acts (Carroll, 1993, 2003) and imply a moral obligation (Porter & Kramer, 2006). Regarding discretionary responsibilities, Carroll (2003) mentions those that are subject to a business’ discretionary decisions. Such responsibilities are completely voluntary and are guided solely by the desire of entrepreneurs to engage in social activities. Relationships between Businesses and Rural Families These are actions that are neither required by law nor expected by society but that the company undertakes out of a sense of charity or philanthropy, nonetheless. Although philanthropy is often regarded as the oldest form of corporate SR, nowadays companies consider their SR activities to be an investment that should yield a social return (Godfrey, 2005); in fact, Porter and Kramer (2006) argued that philanthropy should be strategic and support the competitive environment of the company. All in all, managing to build up a good corporate reputation reinforces the company’s brand image (Kapelus, 2002). In this context, Hillman and Keim (2001) and Du, Bhattacharya and Sen (2007, 2010) pointed out how social action may lead to relational outcomes such as customer loyalty, identification with the company and its products, and even brand advocacy, particularly if consumers or families are aware of a company’s SR actions and interpret them as being driven more by intrinsic motives and less by business serving or extrinsic motives. However, in a non- strategic philanthropic relationship, companies voluntarily commit themselves to give of their own resources (or those that they can leverage) to support a certain action in order to achieve a social aim (Mihaljevic & Tokic, 2015) without being concerned about promoting business objectives (Diener, 2013), as is the case with strategic philanthropy. Figure 2 describes the relationship between companies in the extractive sector and families. The company and family appear at the left and right side of the figure. Carroll’s four social relations (Economic, Statutory, Ethical, Philanthropic) are represented. Extractive Company Economic Statutory Ethical Philanthropic Rural Family Figure 2. Company – Family Relationship Source: Elaborated by the authors. The conceptual framework was introduced by combining the aforementioned perspective with the types of relationships existing between business and society. Figure 3 describes the company-family relationship. The business appears on the far left and families on the right. The approaches to the relationship between the company and the family are (a) business consumer society (top of the figure) and (b) business beneficiary society (bottom of the figure). The types of relationship between companies and families are represented within the figure. Extractive Company Economic Statutory Ethical Philanthropic Rural Family Figure 2. Company – Family Relationship Source: Elaborated by the authors. Figure 2. Company – Family Relationship Source: Elaborated by the authors. Relationships between Businesses and Rural Families The conceptual framework was introduced by combining the aforementioned perspective with the types of relationships existing between business and society. Figure 3 describes the company-family relationship. The business appears on the far left and families on the right. The approaches to the relationship between the company and the family are (a) business consumer society (top of the figure) and (b) business beneficiary society (bottom of the figure). The types of relationship between companies and families are represented within the figure. BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 www.anpad.org.br/bar 9 Business-Community Relationships for Extractive Industries Figure 3. Business-Family Relationship Source: Elaborated by the authors. Actor: Rural Family Actor: Company Beneficiary society Perspective Consumer society Perspective Economic Ethical Philanthropic Statutory Consumer society Perspective Actor: Rural Family Philanthropic Actor: Company Beneficiary society Perspective Figure 3. Business-Family Relationship Source: Elaborated by the authors. Method This study applies a qualitative research method that seeks to approach as much as possible the viewpoint of the participants under study (Creswell, 2013; Norman & Lincoln, 2005; Strauss, 1987). The case-study method is used because of the following reasons: (a) it can easily be used to ask how companies and families relate to each other (Yin, 1994); (b) it has a specific character; for example, it can be used to study the company-family relationship (Stake, 1995); (c) it is limited to a defined context (Peru, in this case) (Creswell, 2013; Stake, 1995); and (d) it can describe behaviors that can be included in conceptual frameworks, in this case, the company-family relationship (Stake, 1995). Setting Moreover, the rate of poverty reaches 70% of rural population in Cajamarca and 50% of the same population in Ucayali. of Cajamarca (Andean highlands), which hosts gold-mining operations. Moreover, the rate of poverty reaches 70% of rural population in Cajamarca and 50% of the same population in Ucayali. Setting The study was conducted in Peru, an emerging economy largely reliant on two extractive industries (mining and natural gas). In Peru, there is a long history of social conflict in relation to extractive operations conducted in areas where numerous rural communities were engaged in traditional economic activities (mainly agriculture and livestock). Although surrounded by a richness of natural landscapes and hydrocarbon deposits (in the forest) and minerals (in the Andean region), the vast majority of communities either live below the poverty line or have their basic needs unmet. Cajamarca presents an incidence of extreme poverty of 15 to 23% and Ucayali of 1.1 to 3%; in contrast, Lima, capital of Peru, presents an incidence less than 0.8% (Instituto Nacional de Estadística e Informática [INEI], 2015). Peru ranks first for gold production and second for copper and silver production in Latin America. It ranks third for copper and silver production worldwide (MINEM, 2015b). Investment in extractive industries has been a key driver of economic growth: the industry grew from US $ 1609.9 million in 2006 to US $ 8893.7 million in 2014 (MINEM, 2015a). Exports rose from US $ 17.2 billion in 2005 to US $ 38.3 billion in 2014 (BCRP, 2015), and mining exports rose from 81.79% of total exports in 2005 to 82.06% in 2014 (MINEM, 2015a). In Peru, natural gas is the third-largest source of energy, after oil and coal. Natural gas reserves currently grow at a rate of 5% a year (Organismo Supervisor de la Inversión en Energía y Minería [OSINERGMIN], 2008). Main deposits are located in the departments of Cusco, Piura, and Ucayali. Output increased from 1376.5 bcf in 2001 to 418 794.8 bcf in 2012 (INEI, 2015). Also, in Lima gas consumption rose from 318 connected users in 2008 to 395,000 connected users in 2016. These figures depict not only exponential growth but also the growing importance of this source of energy for consumers and industry alike. Thus, it was chosen to conduct the study in both departments of Peru: Ucayali and Cajamarca. The department of Ucayali (Amazon rainforest), which hosts natural gas operations, and the department BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 www.anpad.org.br/ba BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 www.anpad.org.br/bar J. Ventura, K. Jauregui J. Ventura, K. Jauregui 10 of Cajamarca (Andean highlands), which hosts gold-mining operations. Table 1 Company Characteristics Company Yanacocha Aguaytía Sector Extractive Extractive Location Rural Andes Cajamarca Rural rainforest Ucayali Size of company Large Intermediate Products or services Gold mining Natural gas energy Shareholders Newmont Buenaventura Maple Energy Industry ranking Largest Latin American Exporter in 2014 Largest National Producer in 2014 Second largest national producer in 2012 Note. Source: Prepared by the authors. Company Characteristics Company Characteristics Note. Source: Prepared by the authors. The families interviewed were chosen according to certain criteria from the databases of the selected companies. Families were prioritized that (a) had worked and/or had direct relations with the given companies for over three years, and (b) lived in poverty (income of less than two dollars per day) prior to having initiated a relationship with the company. Were identified 60 families in total, 30 from each company. Next, each family filled out a brief questionnaire on their socioeconomic condition (family income, composition, formal education, basic needs met). The frame of reference for the questionnaire was the National Household Survey (ENAHO, http://www.ipe.org.pe/enaho, retrieved September, 1, 2015). Afterward, descriptive statistics were utilized to process the data collected and to classify the rural families according to their socioeconomic situation. This procedure allowed us to control for (a) information incomplete by more than 40% and (b) families that were not in a poverty situation at the time they initiated their relationship with the given company. As a result, were selected a total of 30 families (15 in Cajamarca and 15 in Ucayali) that had similar socioeconomic conditions and an enduring relationship with the company. Of the 15 selected, only 10 families were interviewed for each company for a total of twenty families. It should be noted that sample size is related to data collection; as the interviews are carried out, codes are identified and it is decided whether more interviews are needed or not. The saturation is achieved after ten interviews, because no new information emerges. In addition, two managers from each company were interviewed for a total of four in-depth interviews with managers. Were prepared two interviewing guides: one for families and another for company managers or community relations. Data gathering For the purpose of this research, companies were selected in the extractive sector whose main characteristic was that they had a direct relationship with rural families through their SR programs. Four criteria were used to select the companies: (a) the company should operate in an area where the population is mostly rural, (b) the company should be involved in social conflicts, (c) the company should operate in the rural highlands or the rural jungle, and (d) the company should operate in the mining and/or hydrocarbon-extraction industry. The Table 1 identifies some characteristics of these companies. Table 1 The interviewing guide for families was about (a) family background, (b) general perceptions of the company’s extractive operations as well as of the role played out by private investment, (c) the relationship with the company, (d) the perceived socioeconomic changes up to the BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 www.anpad.org.br/bar Business-Community Relationships for Extractive Industries 11 date, and (e) an appraisal of basic needs that had been met up to the date. In the case of the interviewing guide for managers or community relations, the questions were in regards to (a) the activities of the company and its area of socio-environmental influence, (b) key stakeholders, (c) the relationship with social stakeholders, mainly local communities, and (d) an appraisal of the role played out by the company to improve the socioeconomic conditions of local communities within the company’s direct area of influence. date, and (e) an appraisal of basic needs that had been met up to the date. In the case of the interviewing guide for managers or community relations, the questions were in regards to (a) the activities of the company and its area of socio-environmental influence, (b) key stakeholders, (c) the relationship with social stakeholders, mainly local communities, and (d) an appraisal of the role played out by the company to improve the socioeconomic conditions of local communities within the company’s direct area of influence. Finally, an adult informant was identified in each family through prior contacts and with the help of the company family-liaison managers. The interview was scheduled at the homes of the families, because it was wanted to build trust with the families. Interviews took two hours, on average. After interviewing the families, were scheduled two interviews with the companies’ SR managers. On average, the interviews with the companies’ SR managers too lasted 2 hours. Data collection was triangulated to determine the consistency of the results, namely, by using socioeconomic surveys, in-depth interviews, secondary information provided by companies, and the managers’ views of the families interviewed. The interviews were recorded and transcribed. A description of the characteristics of the informants, the location of the interviews, the process of selecting the interviewed families were all logged to demonstrate the reliability and validity of the qualitative research, as per Guba and Lincoln (1989). Data analysis For data analysis, Corbin and Strauss’s (2008) grounded theory was used. First, the interviews were transcribed and then organized by category and code type. To identify the codes, each paragraph of the interview was read and the codes emerged. For example, from the phrase, “The company builds roads and provides health services…” and the phrase, “The company has helped us with loans…” emerged the code, The company becomes the main source of financing. In addition, the literature was reviewed each time a code was identified. For example, the code that emerges from literature review is Family meets its needs. In summary, codes are identified from both literature review and interviews. Then, the codes were grouped in order to identify categories. For example, Company affects people and Family meets its needs were grouped into the category of beneficiary society. Next, the categories were integrated in the interest of explain the business-community relationships for extractive industries. Atlas TI software was used as support. Finally, each case study (Yanacocha and Aguaytia) was reported individually, following which was made a comparative (cross-case) analysis. Table 2  Families regard themselves as beneficiaries of the company’s interventions.  “They have given us a loan for a business …. I hope they will keep on helping me” (Family 1, Yanacocha).  “We carry out programs and projects that go beyond the Yanacocha operation” (Manager 2, Yanacocha). Note. Source: Prepared by the authors. Findings This section presents the results obtained using the method described above. In addition, it includes an analysis of the findings for both cases studied. For both cases, the types of relationships between the company and the rural families within the company’s influence are identified. The relationships are analyzed through the lenses of Carroll’s hierarchical pyramid: economic, legal, ethical and philanthropic relationships (Carroll, 2003). Finally, findings from both cases are compared and analyzed. BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 www.anpad.org.br/bar J. Ventura, K. Jauregui 12 Table 2 Relationship between Yanacocha and Rural Families Category Code (framework) Code (evidence) Evidence Beneficiar y society Company affects people  The company perceives its own influence on rural communities  Management perceives families’ demands for compensation from the company  “If you divert a channel and no longer has access to the water, the company artifici access to water. The co resettlement plan and a package” (Manager 1, Yanaco  “People have a ‘give me’ at company and the compensatio families affected by the min Yanacocha). Families meet their needs  The company becomes the main source of financing.  “The company builds roa health services in the comm (Manager 1, Yanacocha).  “The company has helped The first loan was two thousan was a loan of four thousand great support” (Family 8, Yan Families get support  SR seeks to prevent social conflict.  Families regard themselves as beneficiaries of the company’s interventions.  “For example, the guinea p helps to maintain good relation can keep your operations goin Yanacocha).  “They have given us a loa …. I hope they will keep o (Family 1, Yanacocha). Collateral to business activities  Promoting development is a necessary collateral of company’s activities.  “We carry out programs and beyond the Yanacocha operati Yanacocha). Note. Source: Prepared by the authors. Relationship between Yanacocha and Rural Families Evidence  “If you divert a channel and the community no longer has access to the same amount of water, the company artificially replenishes access to water. The company has a resettlement plan and a compensation package” (Manager 1, Yanacocha).  “People have a ‘give me’ attitude toward the company and the compensation it provides to families affected by the mine” (Manager 2, Yanacocha).  “The company builds roads and provides health services in the community affected” (Manager 1, Yanacocha).  “The company builds roads and provides health services in the community affected” (Manager 1, Yanacocha).  “The company builds roads and provides health services in the community affected” (Manager 1, Yanacocha).  “The company has helped us with loans .... The first loan was two thousand soles, after that was a loan of four thousand soles. This was great support” (Family 8, Yanacocha).  “For example, the guinea pig (farm) project helps to maintain good relations. The company can keep your operations going” (Manager 2, Yanacocha). Relationship between Yanacocha Mining Company and Rural Families The relationship between the Yanacocha mining company and rural families can be described as Beneficiary society. The interviewees identified the following characteristics: (a) the company perceives its own influence on rural communities, (b) the managers perceive families’ demands for compensation from the company, (c) the company is the main source of financing for the rural communities, (d) SR helps to prevent social conflicts, (e) families perceive themselves as beneficiaries of the company’s interventions, and (f) contributing to rural community development is a necessary collateral of mining. Table 2 outlines the relationship between Yanacocha and rural families based on the information gathered in the interviews.  “We carry out programs and projects that go beyond the Yanacocha operation” (Manager 2, Yanacocha). Type of relationship between Yanacocha mining company and families Yanacocha established a philanthropic, ethical, legal relationship with rural families (see Figure 4 Yanacocha established a philanthropic, ethical, legal relationship with rural families (see Figure 4). BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 www.anpad.org.br/bar Business-Community Relationships for Extractive Industries 13 Figure 4. Type of Relationship between Yanacocha and Rural Families Source: Prepared by the authors. Rural Families Yanacocha Mining Company Beneficiary society EthicalPhilanthropicStatutory Yanacocha Mining Company Rural Families Beneficiary society EthicalPhilanthropicStatutory Figure 4. Type of Relationship between Yanacocha and Rural Families Source: Prepared by the authors. The interviewees argued that (a) the company follows the rules set forth by the stakeholders, (b) its SR policy contributes to sustainable development, (c) the company provides discretionary and voluntary support, and (d) the company seeks to improve its reputation. Table 3 outlines the type of relationship that exists between Yanacocha and rural families based on the information gathered in the interviews. Relationship between Yanacocha Mining Company and Rural Families Category Code (framework) Code (evidence) Evidence Legal  Social license  Company follows stakeholders’ principles “The company seeks to respect existing standards of its stakeholders: its owners” (Newmont, World Bank). (Manager 2, Yanacocha). Ethical  Voluntary respect for the social and environmental context  SR as a contribution to sustainable development “The company meets more than it is committed to the environmental impact study” (Manager 1, Yanacocha). Philanthro pic  Discretionary, voluntary  Company provides discretionary and voluntary support “They have helped us .... The first loan was two thousand (Peruvian currency) soles …. That helped us” (Family 3, Yanacocha).  Seeks to enhance corporate reputation  Company seeks to enhance its reputation “When we created ALAC, we said we should do right by doing our best and not brag” (Manager 2, Yanacocha). Note. Source: Prepared by the authors. Relationship between Yanacocha Mining Company and Rural Families Note. Source: Prepared by the authors. Relationship between Aguaytía Energy and Rural Families The relationship between Aguaytía Energy and rural households combines the approaches of the consumer society and the beneficiary society. After analyzing the interviews with managers and families, was characterized the company-family relationship as follows: (a) the relationship with the population serves two objectives: profitability and sustainable development; (b) the company co- finances projects (to which beneficiaries are also asked to contribute); (c) social responsibility is a way to protect the company’s operations; (d) families bring their needs to the company; (e) the company co- BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 www.anpad.org.br/bar J. Ventura, K. Jauregui 14 finances or supports the population; (f) the people perceive themselves as beneficiaries. Table 4 outlines the relationship between Aguaytía and rural families based on the information gathered in the interviews. Table 4 Relationship between Aguaytía Energy Company and Rural Families Category Code (framework) Code (evidence) Evidence Consumer society Company profits Two-pronged relationship with the people: returns and sustainable development “We have donated around 50,000 stoves to housewives who previously used firewood. That has to do with our business” (Manager 2, Aguaytía). Company provides services or products Natural and liquefied gas “We sell fuel. But there is an environmental and social side: housewives use firewood with the side effects of pollution and lung disease” (Manager 2, Aguaytía). Company perceives families as attractive market (clients) Company co- finances projects and requires contributions from beneficiaries “The farmer has a water well and I say: Look, I’m going to help you buy a power pump. I’ll buy the conversion kit that will cost me $1150” (Manager 2, Aguaytía). Beneficiary society Company affects people SR to prevent harm to company’s operations “A company principle is to ‘live and let live.’ The people live in peace and they let the company operate” (Manager 1, Aguaytía). Family meets its needs Families share their needs with companies “They give us technical advice. When I started raising animals, they died because there wasn’t a veterinarian, but an engineer of the mine came to talk and the fish farms now have come true” (Family 4, Aguaytía). Families get support Companies become a source of financing “They had no money to buy the whole system, so we donated the entirety” (Manager 2, Aguaytía). No returns The company perceives people as beneficiaries. Relationship between Aguaytía Energy and Rural Families “The programs we have developed are for them; the transportation program has benefitted forty to fifty percent (of the people), and stove donations, another twenty five percent” (Manager 1, Aguaytía). Relationship between Aguaytía Energy Company and Rural Families Note. Source: Prepared by the authors. Type of Relationship between Aguaytía energy group and rural families The relationship between the Aguaytía Energy Group and families can best be described as ethical and economic (see Figure 5). BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 www.anpad.org.br/bar Business-Community Relationships for Extractive Industries 15 Figure 5. Type of Relationship between Aguaytía-Energy Maple Gas and Rural Families Source: Prepared by the authors. Philanthropic Aguaytia Energy Beneficiary society Consumer society Rural Family Economic Ethical Consumer society Aguaytia Energy Rural Family Figure 5. Type of Relationship between Aguaytía-Energy Maple Gas and Rural Families Source: Prepared by the authors. The analysis of the data reveals the following characteristics of the relationship: (a) The relationship with families is integrated into the company’s strategy. (b) The relationship with families allows the company to stay in the market and expand its consumer market. (c) The relationship with the company affects the short-term revenues of households. (d) Social responsibility is inherent to the company’s policy. (e) Social responsibility contributes to sustainable development. (f) The company deliberately supports the families because of its commitment to society. Table 5 outlines this relationship in greater detail. Table 5 Type of Relationship between Aguaytía Energy Company and Rural Families Category Code (framework) Code (evidence) Evidence Economic Included in the company’s strategy: returns/cost reduction Relationship with families included in corporate strategy “We are producing one hundred seventy- three megawatts. Before the electricity was sold to the national grid but now it stays in the local market” (Manager 2, Aguaytía). Stay in the market Relationship with families allows companies to remain in markets and expand its consumer market “The local mass market for liquefied gas includes flat ferries and taxi cabs. People who joined the program improved their livelihoods” (Manager 1, Aguaytía). Income promoted Impact on families’ short- term revenues “When they switched to gas, they could save, on average, eight to ten extra soles (Peruvian currency) a day” (Manager 2, Aguaytía). Ethical Company values and rules (doing what is right) SR is inherent to company policy “We have always engaged in SR. We never were a company that delegated SR to lower third management. So we have to make a contribution to our society” (Manager 1). C ti Type of Relationship between Aguaytía Energy Company and Rural Families Evidence “We are producing one hundred seventy- three megawatts. Before the electricity was sold to the national grid but now it stays in the local market” (Manager 2, Aguaytía). “We are producing one hundred seventy- three megawatts. Before the electricity was sold to the national grid but now it stays in the local market” (Manager 2, Aguaytía). Continues BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 www.anpad.org.br/bar 16 J. Ventura, K. Jauregui Table 5 (continued) Category Code (framework) Code (evidence) Evidence Ethical Voluntary respect for the social and environmental context SR as a contribution to sustainable development “We started with communities near the operations area in Curimaná, Horizonte. It takes several hours to reach Curimaná. No power. People used to use candles. And now they have electricity” (Manager 1, Aguaytía). Philanthro pic Discretionary, voluntary The company deliberately provides support out of its commitment to society “I was in Ayacucho in 1990, helping orphanages …. I am convinced that SR is a way of life, not for showing off” (Manager 1, Aguaytía). Note. Source: Prepared by the authors. Table 5 (continued) Evidence Evidence Note. Source: Prepared by the authors. Comparing the Two Companies in the Extractive Sector In both cases these companies in the extractive sector have built a relationship with rural families under the beneficiary-society approach because their relationship is considered compensation for the damage caused or that may be caused to families by the companies’ activity. In this sense, the relationship involves a collateral cost to the company because the relationship with the families is not part of the company’s business. It is a relationship that benefits the families so as to avoid social conflicts. In turn, the families perceive themselves as beneficiaries of the companies in the extractive sector. A similarity exists between these two companies, which have both adopted a beneficiary society approach. However, only in the case of Aguaytía there is a consumer society approach to the relationship. The company seeks to earn a profit, change the pattern of energy consumption among families, and transform them into consumers of its products. Both extractive companies engage in a philanthropic type of relationship because they make discretionary and voluntary contributions. There is also an ethics-driven company-family relationship that reflects the organizations’ institutional values. Table 6 compares the approaches of Yanacocha and Aguaytía. Business-Community Relationships for Extractive Industries From a conceptual standpoint, the company and the families engage in four types of relationships: economic, legal, ethical, and philanthropic. In the cases reviewed here, the philanthropic relationship prevails. Companies in the extractive sector see the relationship between company and families as voluntary, discretionary, and driven by their strong sense of support to families who feel affected by the companies’ operations. The companies’ social initiatives address the families’ main problems of poverty, unemployment, and poor health and education. Even when managers are aware that these issues are not part of the companies’ business, they think that these issues must be considered. As a result, the company improves or maintains its legitimacy and image, and society benefits from corporate initiatives. The second type of relationship found in the cases reviewed here is the ethical relationship that emerges when a company incorporates its relationship with families into its values, principles, and business rules. As a matter of principle, companies respect social, economic, and environmental rights. Aguaytía’s respect for the community and the environment is manifested in the words of the SR manager: “Our economic initiatives seek to provide clean energy to communities, encourage environmental stewardship, care for people’s health, and at the same time increase profit margins through reduced costs”. Yanacocha clearly also follows the principle of financially sustainable investments. The miner’s project manager said, “Our investments in the areas impacted by the company should last beyond the company’s period of intervention and operation”. This kind of relationship has been tested by triangulating using the opinion of managers, official documents, and the views of rural families. The economic relationship occurred only with one company; however, the interviews with managers allow us to deduce that the relationship with rural families has in both cases an indirect economic effect: a good relationship with rural families protects the company’s operations in the areas where families live, thus reducing the eventual risk and cost of an interruption of business resulting from social conflicts. The triangulation method did not confirm the legal type of relationship, which was only taken into consideration for Yanacocha. Thus, even if national regulations require social investments (e.g., mining and gas canon royalties, solidarity funds, development trust funds, income tax breaks, social license, prior consultation with local communities), they are not considered to be the main reason for the relationship by either the managers or the rural families in the cases reviewed here. Table 6 BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 www.anpad.org.br/bar Comparison of Categories between Yanacocha and Aguaytía Energy Category Code (framework) Minera Yanacocha Grupo Aguaytía Pattern Consumer society Company profits X X X Company perceives families as attractive market (clients) X Beneficiary society Company affects people X X X Family meets its needs X X X Families get support X X X Collateral to business activities X Note. Source: Prepared by the authors. Comparison of Categories between Yanacocha and Aguaytía Energy BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 17 Discussion and Conclusions The conceptual framework uses two approaches: one in which society is a consumer of the companies’ goods and services, and the other in which society is a beneficiary of the companies. The relationship between the extractive sector companies and rural families is that of a beneficiary society. Families are seen as victims of corporate action, contrary to what might expected from the literature (Husted & Salazar-Cantú, 2006; Prahalad & Hart, 2008). Therefore, the main form of company intervention in their communities is through their SR programs to support social projects - not through considering society as a consumer of company goods and services. In the contrast, in the case of Aguaytía Energy managers do tend to see rural families as consumers of their product, natural gas. This finding is explained by the fact that families use natural gas for domestic purposes. The results show that the relationship between the companies and rural families is ethical and traditional philanthropic, the latter being non-strategic philanthropic, through the approach wherein society is a beneficiary of business. This finding partially contradicts the statement of Porter and Kramer (2006), according to whom the relations between companies and society leading to social actions are motivated by strategic ethical and philanthropic considerations. Porter and Kramer (2002) hold that “philanthropy is increasingly used as a form of public relations or advertising to promote the image of a company” (p. 56). Although this approach indeed seems to be the case with the companies studied here, no strategic philanthropic relationship has yet been created. Companies are unable to improve their “competitive context, i.e., the quality of the business environment in the localities where they operate” (Porter & Kramer, 2002, p. 59). This study also shows that, through a number of SR programs, the extractive sector companies and the local rural families have built a philanthropic- and ethical-type relationship (as opposed to an economic- or legal-type of relationship). This finding contradicts Carroll’s hierarchy. Carroll (1979, 1991, 2003, 2008) posited that the main reasons for corporate social initiatives are, in order of importance, economic, legal, ethical, and philanthropic. This study shows that ethical and philanthropic relationships are more notable in both cases addressed. Provided that philanthropy has no limit, this kind of relationship can be deeply unsatisfactory, and hence can lead to a permanent situation of latent conflict. Business-Community Relationships for Extractive Industries Two reasons account for this: the limited capacity of the state to enforce its own rules (weak enforcement) and the low effectiveness and impact of these social investments that, although required by law, are not perceived by the people. Table 7 compares the relationship types between Yanacocha and Aguaytía. BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 www.anpad.org.br/bar Comparison of Type of Relationships between Yanacocha and Aguaytía Energy Category Code Framework Minera Yanacocha Grupo Aguaytía Pattern Economic Included in the company’s strategy X Stay in the market X Legal License, company follows stakeholders’ principles X Ethical Company values and rules (doing what is right) X Voluntary respect for the social and environmental context X X X Note. Source: Elaborated by the authors. Comparison of Type of Relationships between Yanacocha and Aguaytía Energy BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 J. Ventura, K. Jauregui 18 J. Ventura, K. Jauregui Discussion and Conclusions The traditional philanthropy does not solve the main problems of the conflicts between extractive companies and rural communities. It is only hiding them. Both the mining and hydrocarbon extractive industries have created social and environmental impacts that affect rural communities in many places, resulting in social conflicts that harm the communities in many ways, including loss of human life, the trampling of citizen rights, and irreparable environmental pollution (Bebbington, 2013; Davis & Franks, 2014; Zarsky & Stanley, 2013). For companies, the consequences were production overruns, declining investment, and bad corporate image. To manage social conflicts between extractive sector companies and rural communities, the companies have typically adopted the Harvard negotiation method (Fisher, Ury, & Patton, 1991), which is based essentially on satisfying the plaintiffs’ economic interests and expectations. Most schemes propounded by the government, such as trust funds, mining and energy canon (i.e., royalties), and compensation funds, assume an economic relationship between enterprises and rural families, as posited by Carroll (1979). Other mechanisms provided by the government, such as prior consultation and social license, are legal instruments in the second rung of Carroll’s pyramid. Finally, the discussion above clarifies the contribution of this research, which is two-fold. First, Carroll’s pyramid of hierarchical types of relationships remains partially valid in that economic, legal, ethical and philanthropic relational aspects which are identified in the context of extractive operations taking place in territories wherein local communities live and carry out traditional socioeconomic activities. However, the relationships’ order of importance differs from what Carroll proposed. For business managers, economic and legal motives drive their SR actions, whereas for families and communities at large, ethical and philanthropic motives are perceived as the basis for the very existence of a relationship. Both parties, therefore, hold an opposite view that leads to a conflictive relationship, BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 www.anpad.org.br/bar www.anpad.org.br/bar 19 Business-Community Relationships for Extractive Industries 19 which forces companies to adopt a society-as-beneficiary approach regarding their SR commitment and prevents companies from supporting activities that contribute to communities’ long-term well-being and prosperity. Second, the results of this research give us a different perspective of the relationship between extractive sector companies and rural families: if the relationships the companies hope to create with families are to be based on philanthropic and ethical principles, then government conflict-management strategies should consider the potential of these elements. Discussion and Conclusions The solution to a conflict is not only to meet economic interests, which often results in a zero sum scenario, but also to construct a robust relationship based on trust, in other words, an ethical strategic-philanthropy approach that allows for a healthy transition from a society that perceives itself as a victim that must be compensated through pure philanthropy, to a society that develops the ability to partner with its business neighbor in order to leverage opportunities to improve well-being and prosperity. Limitations and future research The study includes some limitations that should be addressed in future research. The first shortcoming is that the study was conducted with only two extractive sector companies. The extractive industry is complex and dissimilar. The mining sector has a long history in Latin America, mainly in the Andean region. However, the hydrocarbon industry, especially natural gas, is a recent participant. It operates mainly in areas where native communities dwell in the Amazon jungle. This industry in the jungle is going through a learning process. In future research, studies could include additional extractive industries (i.e., mining, renewable energy, timber and hydrocarbons). The second limitation is that the rural families interviewed were not equally adapted to the recent changes spurred on by globalization and the market economy. Some rural families still maintain their traditions and values. Others have already adapted to the global market economy. In future research, the study could control for these differences among families. The third limitation is that more people from each of the families have to be interviewed in order to gain more points of view. For example, future research could include informants who were not the heads of household, in other words, women and children, in order to obtain a more comprehensive perspective from within the same family. The fourth limitation was that third parties external to the relationship between the business and family were not taken into consideration. The role and influence of social organizations, environmental movements and human rights watchdog groups were not taken into account. In future research the role of such external agents should be considered. Finally, the management of social conflicts was not directly investigated. However, the results throw new perspectives on social conflicts. In future research, it would be expedient to examine the social conflicts between mining companies and the rural and indigenous communities near their operations. The issue of conflict management using an ethical approach deserves further exploration. References Ahlstrom, D. (2010). Innovation and growth: How business contributes to society. Academy of Management Perspectives, 24(3), 11-24. https://doi.org/10.5465/amp.2010.52842948 Altman, B. W. (1998). Transformed corporate community relations: A management tool for achieving corporate citizenship. Business and Society Review, 102/103(1), 43-51. https://doi.org/10.1111/0045-3609.00024 Banco Central de Reserva del Perú. (2015). Memoria 2015. Retrieved from http://www.bcrp.gob.pe/docs/Publicaciones/Memoria/2015/memoria-bcrp-2015.pdf Bhattacharya, C. B., & Sen, S. (2003). Consumer-company identification: A framework for understanding consumers’ relationships with companies. Journal of Marketing, 67(2), 76-88. https://doi.org/10.1509/jmkg.67.2.76.18609 BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 www.anpad.org.br/bar J. Ventura, K. Jauregui 20 Bhattacharya, C. B., & Sen, S. (2004). Doing better at doing good: When, why, and how consumers respond to corporate social initiatives. California Management Review, 47(1), 9-24. https://doi.org/10.2307/41166284 Bebbington, A. (2013). Industrias extractivas: Conflict social y dinámicas institucionales en la Región Andina. Lima, Peru: Instituto de Estudios Peruanos. Bell, M. L., & Emory, C. W. (1971). The faltering marketing concept. Journal of Marketing, 35(4), 37- 42. https://doi.org/10.2307/1250455 Carroll, A. B. (1979). A three-dimensional conceptual model of corporate social performance. Academy of Management Review, 4(4), 497-505. https://doi.org/10.5465/amr.1979.4498296 Carroll, A. B. (1991). The pyramid of corporate social responsibility: Toward the moral management of organizational stakeholders. Business Horizons, 34(4), 39-48. https://doi.org/10.1016/0007- 6813(91)90005-g Carroll, A. B. (1993). Business and society: Ethics and stakeholder management (2nd ed.). Cincinnati: South-Western Publishing. Carroll, A. B. (2003). The four faces of corporate citizenship. Business and Society Review, 100(101), 1-7. https://doi.org/10.1111/0045-3609.00008 Carroll, A. B. (2008). A history of corporate social responsibility: Concepts and practices. In A. Crane, A. McWilliams, D. Matten, J. Moon, & D. Siegel (Eds.), The Oxford handbook of corporate social responsibility (pp. 19-46). Oxford: Oxford University Press. Castro, A. (2012). Familias rurales y sus procesos de transformación: Estudio de casos en un escenario de ruralidad en tensión. Psicoperspectivas, 11(1), 180-203. Retrieved from http://www.psicoperspectivas.cl/index.php/psicoperspectivas/article/viewFile/172/188. http://dx.doi.org/10.5027/psicoperspectivas-Vol11-Issue1-fulltext-172. Coase, R. H. (1960). The problem of social cost. Journal of Law and Economics, 3(1), 1-44. Retrieved from http://www.jstor.org/stable/724810 Connelly, B. L., Ketchen, D. J., & Slater, S. F. (2011). Toward a “theoretical toolbox” for sustainability research in marketing. Journal of the Academy of Marketing Science, 39(1), 86-100. https://doi.org/10.1007/s11747-010-0199-0 Corbin, J., & Strauss, A. (2008). Basics of qualitative research: Techniques and procedure for developing grounded theory (3rd ed.). Thousand Oaks: Sage Publications. Creswell, J. (2013). Research design. Qualitative, quantitative and mixed methods approach (4th ed.). Los Angeles: Sage Publications. Damonte, G. (2008). References The transnationalization of the mining industry in Peru and Bolivia. In G. Damonte, The constitution of political actors: Peasant communities, mining and mobilization in Bolivian and Peruvian Andes (pp. 135-174). Saarbrücken-Berlin: VDM Verlag. Damonte, G., Kuramoto, J., & Glave, M. (2014). Industrias extractivas y manejo de conflictos [Manual]. Peru: Grupo de Análisis para el Desarrollo (GRADE). Daou, L., & Sarkis, N. (2013). Giving back to the community, an obligation or an option today? Case of the educational sector in Lebanon. International Strategic Management Review, 1(1/2), 59-64. https://doi.org/10.1016/j.ism.2013.09.001 BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 www.anpad.org.br/b BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 www.anpad.org.br/bar Business-Community Relationships for Extractive Industries 21 Davis, R., & Franks, D. (2014). Cost of company-community conflict in the extractive sector (Corporate Social Responsibility Initiative Report 66). Retrieved from https://www.hks.harvard.edu/m- rcbg/CSRI/research/Costs%20of%20Conflict_Davis%20%20Franks.pdf Defensoría del Pueblo. (2007, Noviembre 30). Conflictos sociales conocidos por la defensoría del pueblo (Unidad de Conflictos Sociales - Reporte de conflictos sociales Nº 45). Retrieved from http://www.defensoria.gob.pe/modules/Downloads/conflictos/2007/conflictos_sociales45.pdf Defensoría del Pueblo. (2015, Noviembre). Reporte de conflictos sociales Nº 141. Adjuntía para la Prevención de los Conflictos Sociales y la Gobernabilidad. Retrieved from http://www.defensoria.gob.pe/modules/Downloads/conflictos/2015/Reporte-Mensual-de- Conflictos-Sociales-N--141---Noviembre-2015.pdf Deloitte. (2015). Tracking the trends 2016. The top 10 issues mining companies will face in the coming year. Retrieved from https://www2.deloitte.com/content/dam/Deloitte/global/Documents/Energy-and-Resources/gx- er-tracking-the-trends-2016.pdf Diener, K. W. (2013, June). The charitable responsibilities model of corporate social responsibility. Journal of Academic and Business Ethics, 7, 1-13. Retrieved from http://www.aabri.com/manuscripts/121341.pdf Dorregaray, P. F. P. (2005). Visión panorámica de las actividades mineras en el Perú – 2005. Peru: CARE Perú Programa Fortalece. Retrieved from http://www.care.pe/pdfs/cinfo/libro/minera%20final.pdf Drucker, P. (1985). Innovation and entrepreneurship. New York: Harper & Row. Drucker, P. (1985). Innovation and entrepreneurship. New York: Harper & Row. Du, S., Bhattacharya, C. B., & Sen, S. (2007). Reaping relational rewards from corporate social responsibility: The role of competitive positioning. International Journal in Research Marketing, 24(3), 224-241. https://doi.org/10.1016/j.ijresmar.2007.01.001 Du, S., Bhattacharya, C. B., & Sen, S. (2010). Maximizing business returns to corporate social responsibility (CSR): The role of CSR communication. International Journal of Management Review, 12(1), 8-19. https://doi.org/10.1111/j.1468-2370.2009.00276.x Ellen, P. S., Webb, D. J., & Mohr, L. A. (2006). Building corporate associations: consumer attributions for corporate socially responsible programs. Journal of the Academy of Marketing Science, 34(2), 147-157. https://doi.org/10.1177/0092070305284976 Fisher, R., Ury, W., & Patton, B. (1991). Getting to yes: Negotiating agreement without giving in (2nd ed.). Sydney: Century Business. Freeman, R. E., & Phillips, R. A. (2002). Stakeholder theory: A libertarian defense. Business Ethics Quarterly, 12(3), 331-350. https://doi.org/10.2307/3858020 Freeman, R. E., & Reed, D. L. (1983). Stockholders and stakeholders: A new perspective on corporate governance. California Management Review, 25(3), 88-106. https://doi.org/10.2307/41165018 Freeman, R. E., Wicks, A. C., & Parmar, B. (2004). Stakeholder theory and “the corporate objective revisited”. Organization Science, 15(3), 364-369. https://doi.org/10.1287/orsc.1040.0066 Friedman, M. (1962). Capitalism and freedom. Chicago: University of Chicago Press. Friedman, M. (1970, September 13). The social responsibility of business is to increase its profits. The New York Times Magazine, 33, 122-126. BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 www.anpad.org.br/b BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 www.anpad.org.br/bar J. Ventura, K. Jauregui 22 Garriga, E., & Melé, D. (2004). Corporate social responsibility theories: Mapping the territory. Journal of Business Ethic, 53(1), 51-71. https://doi.org/10.1023/b:busi.0000039399.90587.34 Godfrey, P. C. (2005). The relationship between corporate philanthropy and shareholder wealth: A risk management perspective. Academy of Management Review, 30(4), 777-798. https://doi.org/10.5465/amr.2005.18378878 Guba, E., & Lincoln, Y. (1989). Fourth generation evaluation. Newbury Park: Sage Publications. Hasnas, J. (1998). The normative theories of business ethics: A guide for the perplexed. Business Ethics Quarterly, 8(1), 19-42. https://doi.org/10.2307/3857520 Hillebrand, B., Driessen, P. H., & Koll, O. (2015). Stakeholder marketing: Theoretical foundations and required capabilities. Journal of the Academy of Marketing Science, 43(4), 411-428. https://doi.org/10.1007/s11747-015-0424-y Hillman, A. J., & Keim, G. D. (2001). Shareholder value, stakeholder management; and social issues: What’s the bottom line? Strategic Management Journal, 22(2), 125-139. https://doi.org/10.1002/1097-0266(200101)22:2<125::aid-smj150>3.0.co;2-h Husted, B. W., & Salazar-Cantú, J. (2006). Taking Friedman seriously: Maximizing profits and social performance. Journal of Management Studies, 43(1), 75-91. https://doi.org/10.1111/j.1467- 6486.2006.00583.x Instituto Nacional de Estadística e Informática. (2015a). Drucker, P. (1985). Innovation and entrepreneurship. New York: Harper & Row. Perú: Anuario de estadísticas ambientales 2014. Retrieved from https://www.inei.gob.pe/media/MenuRecursivo/publicaciones_digitales/Est/Lib1197/ Instituto Nacional de Estadística e Informática. (2015b, Septiembre). Perú: Síntesis estadística 2015. Retrieved from https://www.inei.gob.pe/media/MenuRecursivo/publicaciones_digitales/Est/Lib1292/libro.pdf International Monetary Fund. (2015). Perspectivas económicas. Las Américas: el norte se recupera y el sur aún espera. Retrieved from: https://www.imf.org /external/spanish/pubs/ft/reo/2015/whd/pdf/wreo0415s.pdf Kapelus, P. (2002). Mining, corporate social responsibility and the community: The case of Rio Tinto Richards Bay Minerals and the Mbonambi. Journal of Business Ethics, 39(3), 296-375. http://dx.doi.org/10.1023/A:1016570929359 Klein, J., & Dawar, N. (2004). Corporate social responsibility and consumers’ attributions and brand evaluations in a product-harm crisis. International Journal of Research in Marketing, 21(3), 203- 217. https://doi.org/10.1016/j.ijresmar.2003.12.003 Laczniak, G. R., & Murphy, P. E. (2012). Stakeholder theory and marketing: Moving from a firm-centric to a societal perspective. Journal of Public Policy & Marketing, 31(2), 284-292. https://doi.org/10.1509/jppm.10.106 Laplante, L. J., & Spears, S. A. (2008). Out of the conflict zone: The case for community consent processes in the extractive sector. Yale Human Rights and Development Law Journal, 11(1), 69- 116. Retrieved from http://digitalcommons.law.yale.edu/yhrdlj/vol11/iss1/6 Lucas, R., & Sargent, T. (1981). After Keynesian macroeconomics. In R. Lucas & T. Sargent (Eds.), Rational expectations and econometric practice (pp. 295-319). Minnesota: The University of Minnesota Press. Maddala, G., & Miller, E. (1991). Microeconomics: Theory and applications. New York: McGraw-Hill. BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 www.anpad.org.br/bar Business-Community Relationships for Extractive Industries 23 Maon, F., Lindgreen, A., & Swaen, V. (2010). Organizational stages and cultural phases: A critical review and a consolidative model of corporate social responsibility development. International Journal of Management Reviews, 12(1), 20-38. https://doi.org/10.1111/j.1468- 2370.2009.00278.x McKenna, K. (2016). Corporate social responsibility and natural resource conflict (Routledge Research in Sustainability and Business). London and New York: Routledge. Melé, D. (2008). Corporative social responsibility theories. In A. Crane, D. Matten, A. McWilliams, J. Moon, & D. S. Siegel (Eds.), The Oxford handbook of corporative social responsibility (pp. 47- 82). Oxford: Oxford University Press. Mihaljevic, M., & Tokic, I. (2015). Ethics and philanthropy in the field of corporate social responsibility pyramid. Interdisciplinary Management Research, 15, 799-807. Ministerio de Energía y Minas. (2015a). Boletín estadístico del subsector minero. Retrieved from http://www.minem.gob.pe/_publicacion.php?idSector=1&idPublicacion=500 Ministerio de Energía y Minas. (2015b). Perú 2014: Anuario minero. Reporte estadístico. Retrieved from http://www.minem.gob.pe/_publicacion.php?idSector=1&idPublicacion=501 Moffat, K., & Zhang, A. (2014, March). The paths to social licence to operate: An integrative model explaining community acceptance of mining. Resources Policy, 39, 61-70. https://doi.org/10.1016/j.resourpol.2013.11.003 Nem Singh, J. Drucker, P. (1985). Innovation and entrepreneurship. New York: Harper & Row. Qualitative analysis for social scientists. Cambridge: Cambridge University Press. Superintendencia Nacional de Aduanas y de Administración Tributaria. (2015, Febrero 23). Informe mensual de exportaciones (Nota de Prensa N° 039-2015). Retrieved from http://p.se- todo.com/pravo/22025/index.html Superintendencia Nacional de los Registros Públicos. (2016). Directorio Nacional de personas jurídicas. Retrieved from https://www.sunarp.gob.pe/dn-personas-juridicas.asp Yin, R. K. (1994). Case study research: design and methods (Applied Social Research Methods). Newbury Park: Sage Publications. Zarsky, L., & Stanley. L. (2013). Can extractive industries promote sustainable development? A net benefits framework and a case study of the marlin mine in Guatemala. Journal of Environment & Development, 22(2), 131-154. https://doi.org/10.1177/1070496513483131 Drucker, P. (1985). Innovation and entrepreneurship. New York: Harper & Row. (2012, January 9). The resource curse: Governing extractive industries in the global south. E-International Relations. Retrieved from http://www.e-ir.info/2012/01/09/the-myth-of-the- resource-curse-governing-extractive-industries-in-the-global-south/ Norman, D., & Lincoln, Y. (2005). The SAGE handbook of qualitative research. California: SAGE Publications. Organismo Supervisor de la Inversión en Energía y Minería. (2008). Regulación del gas natural en el Perú. Estado del Arte. Retrieved from http://www2.osinerg.gob.pe Infotec/GasNatural/pdf/Regulacion_Gas_Natural_Peru.pdf Parmar, B. L., Freeman, R. E., Harrison, J. S., Wicks, A. C., Colle, S., de, & Purnell, L. (2010). Stakeholder theory: The state of the art. The Academy of Management Annals, 4(1), 403-445. https://doi.org/10.1080/19416520.2010.495581 Peloza, J., & Shang, J. (2011). How can corporate social responsibility activities create value for stakeholders? A systematic review. Journal of the Academy of Marketing Science, 39(1), 117- 135. https://doi.org/10.1007/s11747-010-0213-6 Prahalad, C., & Hart, S. (2008). The fortune at the bottom of the pyramid. Revista Eletrônica de Estratégia & Negócios, 1(2), 1-23. http://dx.doi.org/ 10.19177/reen.v1e220081-23 Porter, M. (1980). Competitive strategy: Techniques for analyzing industries and competitors. New York: Free Press. Porter, M., & Kramer, M. (2002). The competitive advantage of corporate philanthropy. Harvard Business Review, 80(12), 56-69. Retrieved from https://hbr.org/2002/12/the-competitive- advantage-of-corporate-philanthropy Porter, M., & Kramer, M. (2006). Strategy and society: The link between competitive advantage and corporate social responsibility. Harvard Business Review, 84(12), 56-68. Retrieved from https://hbr.org/2006/12/strategy-and-society-the-link-between-competitive-advantage-and- corporate-social-responsibility BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 www.anpad.org.br/bar BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 www.anpad.org.br/bar J. Ventura, K. Jauregui 24 Porter, M., & Kramer, M. (2011). Creating shared value. Harvard Business Review, 89(1/2), 62-77. Retrieved from https://hbr.org/2011/01/the-big-idea-creating-shared-value Roarty, M. (1997). Greening business in a market economy. European Business Review, 97(5), 244- 254. https://doi.org/10.1108/09555349710179898 Robin, D. P., & Reidenbach, R. E. (1987). Social responsibility, ethics, and marketing strategy: Closing the gap between concept and application. Journal of Marketing, 51(1), 44-58. https://doi.org/10.2307/1251143 Safwat, A. M. (2015). Corporate social responsibility: Rewriting the relationship between business and society. International Journal of Social Sciences, 4(1), 85-97. https://doi.org/10.20472/ss2015.4.1.006 Schumpeter, J. (1939). Business cycles: A theoretical, historical and statistical analysis of the capitalist process. New York: McGraw Hill. Sen, S., Bhattacharya, C. B., & Korschun, D. (2006). The role of corporate social responsibility in strengthening multiple stakeholder relationships: A field experiment. Journal of the Academy of Marketing Science, 34(2), 158-166. https://doi.org/10.1177/0092070305284978 Stake, R. (1995). The art of case research. Thousand Oaks: Sage Publications. einer, G. (1971). Business and society. New York: Random House. Strauss, A. (1987). BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 Kety Jauregui Universidad ESAN, Alonso de Molina, 1652, Monterrico, Surco, Lima, Peru. E-mail address: kjauregui@esan.edu.pe Jose Ventura Universidad ESAN, Alonso de Molina, 1652, Monterrico, Surco, Lima, Peru. E-mail address: jventura@esan.edu.pe Authors’ Profiles BAR, Rio de Janeiro, v. 14, n. 2, art. 4, e160114, 2017 www.anpad.org.br/bar
https://openalex.org/W3182723848	https://www.biorxiv.org/content/biorxiv/early/2021/07/08/2021.07.08.451617.full.pdf	English	null	Mosaic cis-regulatory evolution drives transcriptional partitioning of HERVH endogenous retrovirus in the human embryo	bioRxiv (Cold Spring Harbor Laboratory)	2,021	cc-by	39,377	1 Mosaic cis-regulatory evolution drives transcriptional partitioning of 2 HERVH endogenous retrovirus in the human embryo 3 4 Thomas A. Carter1, Manvendra Singh1, Gabrijela Dumbović2,3, Jason D. Chobirko1, 5 John L. Rinn2, Cédric Feschotte1* 6 : corresponding author 7 8 1) Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14850, USA 9 2) Department of Biochemistry, University of Colorado Boulder, Boulder, CO 80309, USA 10 3) Present address: Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany 11 1 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint 1 Mosaic cis-regulatory evolution drives transcriptional partitioning of 2 HERVH endogenous retrovirus in the human embryo 3 4 Thomas A. Carter1, Manvendra Singh1, Gabrijela Dumbović2,3, Jason D. Chobirko1, 5 John L. Rinn2, Cédric Feschotte1 6 : corresponding author 7 8 1) Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14850, USA 9 2) Department of Biochemistry, University of Colorado Boulder, Boulder, CO 80309, USA 10 3) Present address: Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany 11 Mosaic cis-regulatory evolution drives transcriptional partitioning of 2 HERVH endogenous retrovirus in the human embryo 3 1 1 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: ioRxiv preprint Introduction 32 53 2020; Urusov et al., 2011). But how does this precise control of TE expression evolve? 39 Many endogenous retroviruses (ERVs) are known to exhibit highly stage-specific 40 expression during early embryonic development (Chang et al., 2021; Göke et al., 2015; 41 Hermant and Torres-Padilla, 2021; Peaston et al., 2004; Svoboda et al., 2004). ERVs 42 are derived from exogenous retroviruses with which they share the same prototypical 43 structure with two long terminal repeats (LTRs) flanking an internal region encoding 44 products promoting their replication (Eickbush and Malik, 2002). There are hundreds of 45 ERV families and subfamilies in the human genome, each associated to unique LTR 46 sequences (Kojima, 2018; Vargiu et al., 2016). Each family has infiltrated the germline 47 at different evolutionary timepoints and have achieved various levels of genomic 48 amplification (Bannert and Kurth, 2004; Vargiu et al., 2016). One of the most abundant 49 families is HERVH, a family derived from a gamma retrovirus that first entered the 50 genome of the common ancestor of apes, Old World monkeys, and New World 51 monkeys more than 40 million years ago (mya) (Goodchild et al., 1993; Izsvák et al., 52 2016; Mager and Freeman, 1995). 53 There are four subfamilies of HERVH elements currently recognized in the Dfam (Storer 54 et al., 2021) and Repbase (Bao et al., 2015; Kojima, 2018) databases and annotated in 55 the reference human genome based on distinct LTR consensus sequences: LTR7 56 (formerly known as Type I), 7b (Type II), 7c, and 7y (Type Ia) (Bao et al., 2015; 57 Goodchild et al., 1993; Jern et al., 2005, 2004). Additional subdivisions of HERVH 58 elements were also proposed based on phylogenetic analysis and structural variation of 59 their internal gene sequences (Gemmell et al., 2019; Jern et al., 2005, 2004). However, 60 all HERVH elements are currently annotated in the human genome using a single 61 There are four subfamilies of HERVH elements currently recognized in the Dfam (Storer 54 et al., 2021) and Repbase (Bao et al., 2015; Kojima, 2018) databases and annotated in 55 the reference human genome based on distinct LTR consensus sequences: LTR7 56 (formerly known as Type I), 7b (Type II), 7c, and 7y (Type Ia) (Bao et al., 2015; 57 Goodchild et al., 1993; Jern et al., 2005, 2004). Abstract 12 The human endogenous retrovirus type-H (HERVH) family is expressed in the 13 preimplantation embryo. A subset of these elements are specifically transcribed in 14 pluripotent stem cells where they appear to exert regulatory activities promoting self- 15 renewal and pluripotency. How HERVH elements achieve such transcriptional 16 specificity remains poorly understood. To uncover the sequence features underlying 17 HERVH transcriptional activity, we performed a phyloregulatory analysis of the long 18 terminal repeats (LTR7) of the HERVH family, which harbor its promoter, using a wealth 19 of regulatory genomics data. We found that the family includes at least 8 previously 20 unrecognized subfamilies that have been active at different timepoints in primate 21 evolution and display distinct expression patterns during human embryonic 22 development. Notably, nearly all HERVH elements transcribed in ESCs belong to one of 23 the youngest subfamilies we dubbed LTR7up. LTR7 sequence evolution was driven by 24 complex mutational processes, including multiple recombination events between 25 subfamilies, that led to transcription factor binding motif modules characteristic of each 26 subfamily. Using a reporter assay, we show that one such motif, a predicted SOX2/3 27 binding site unique to LTR7up, is essential for robust promoter activity in induced 28 pluripotent stem cells. Together these findings illuminate the mechanisms by which 29 HERVH diversified its expression pattern during evolution to colonize distinct cellular 30 niches within the human embryo. 31 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint Introduction 32 Transposable elements (TEs) are genomic parasites that use the host cell machinery 33 for their own propagation. To propagate in the host genome, they must generate new 34 insertions in germ cells or their embryonic precursors, as to be passed on to the next 35 generation (Charlesworth and Langley, 1986; Cosby et al., 2019; Haig, 2016). To this 36 end, many TEs have evolved stage-specific expression in germ cells or early embryonic 37 development (Faulkner et al., 2009; Fort et al., 2014; Göke et al., 2015; Miao et al., 38 2020; Urusov et al., 2011). But how does this precise control of TE expression evolve? 39 Transposable elements (TEs) are genomic parasites that use the host cell machinery 33 for their own propagation. To propagate in the host genome, they must generate new 34 insertions in germ cells or their embryonic precursors, as to be passed on to the next 35 generation (Charlesworth and Langley, 1986; Cosby et al., 2019; Haig, 2016). To this 36 end, many TEs have evolved stage-specific expression in germ cells or early embryonic 37 development (Faulkner et al., 2009; Fort et al., 2014; Göke et al., 2015; Miao et al., 38 2020; Urusov et al., 2011). But how does this precise control of TE expression evolve? 39 Many endogenous retroviruses (ERVs) are known to exhibit highly stage-specific 40 expression during early embryonic development (Chang et al., 2021; Göke et al., 2015; 41 Hermant and Torres-Padilla, 2021; Peaston et al., 2004; Svoboda et al., 2004). ERVs 42 are derived from exogenous retroviruses with which they share the same prototypical 43 structure with two long terminal repeats (LTRs) flanking an internal region encoding 44 products promoting their replication (Eickbush and Malik, 2002). There are hundreds of 45 ERV families and subfamilies in the human genome, each associated to unique LTR 46 sequences (Kojima, 2018; Vargiu et al., 2016). Each family has infiltrated the germline 47 at different evolutionary timepoints and have achieved various levels of genomic 48 amplification (Bannert and Kurth, 2004; Vargiu et al., 2016). One of the most abundant 49 families is HERVH, a family derived from a gamma retrovirus that first entered the 50 genome of the common ancestor of apes, Old World monkeys, and New World 51 monkeys more than 40 million years ago (mya) (Goodchild et al., 1993; Izsvák et al., 52 2016; Mager and Freeman, 1995). consensus sequence for the internal region (HERVH_int) and the aforementioned four 62 LTR subfamilies. 63 consensus sequence for the internal region (HERVH_int) and the aforementioned four 62 LTR subfamilies. 63 LTR subfamilies. 63 HERVH has been the focus of extensive genomic investigation for its high level of RNA 64 expression in human embryonic stem cells (ESCs) and induced pluripotent stem cells 65 (iPSCs) (Fort et al., 2014; Gemmell et al., 2015; Izsvák et al., 2016; Kelley and Rinn, 66 2012; Loewer et al., 2010; Römer et al., 2017; Santoni et al., 2012; Zhang et al., 2019). 67 Several studies showed that family-wide HERVH knockdown results in the loss of 68 pluripotency of human ESC and reduced reprogramming efficiency of somatic cells to 69 iPSC (Lu et al., 2014; Ohnuki et al., 2014; Wang et al., 2014). Others reported similar 70 phenotypes with the knockdown of individual HERVH-derived RNAs such as those 71 produced from the lincRNA-RoR and ESRG loci (Loewer et al., 2010; Wang et al., 72 2014) or the deletion of individual HERVH loci acting as boundaries for topological 73 associated domains (Zhang et al., 2019). These results converge on the notion that 74 HERVH products (RNA or proteins) exert some modulatory effect on the cellular 75 homeostasis of pluripotent stem cells. However, it is important to emphasize that 76 different HERVH knockdown constructs produced variable results and inconsistent 77 phenotypes (Lu et al., 2014; Wang et al., 2014; Zhang et al., 2019), and a recent 78 knockout experiment of the most highly transcribed locus (ESRG) failed to recapitulate 79 its previous knockdown phenotype (Takahashi et al., 2021). Despite intense study, 80 which expressed HERVH loci, if any, are necessary for the maintenance of pluripotency 81 remain unclear. 82 The mechanisms regulating the transcription of HERVH also remain poorly understood. 83 RNA-seq analyses have established that HERVH expression in human ESCs, iPSCs, 84 and the pluripotent epiblast can be attributed to a relatively small subset of loci 85 (estimated between 83 and 209) driven by LTR7 (sensu stricto) sequences (Göke et al., 86 2015; Wang et al., 2014; Zhang et al., 2019). The related 7y sequences are known to 87 be expressed in the pluripotent epiblast of human embryos (Göke et al., 2015) and a 88 distinct subset of elements associated with 7b and 7y sequences are expressed even 89 earlier in development at the onset of embryonic genome activation (Göke et al., 2015). Introduction 32 Additional subdivisions of HERVH 58 elements were also proposed based on phylogenetic analysis and structural variation of 59 their internal gene sequences (Gemmell et al., 2019; Jern et al., 2005, 2004). However, 60 all HERVH elements are currently annotated in the human genome using a single 61 3 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint consensus sequence for the internal region (HERVH_int) and the aforementioned four 62 LTR subfamilies. 63 90 These observations suggest that the HERVH family is composed of subsets of elements 91 4 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint expressed at different timepoints during embryonic development and that these 92 expression patterns reflect, at least in part, the unique cis-regulatory activities of their 93 LTRs. While it has been reported that several transcription factors (TFs) bind and 94 activate HERVH LTRs, including the pluripotency factors OCT4, NANOG, SP1, and 95 SOX2 (Göke et al., 2015; Ito et al., 2017; Kelley and Rinn, 2012; Kunarso et al., 2010; 96 Ohnuki et al., 2014; Pontis et al., 2019; Santoni et al., 2012), it remains unclear how TF 97 binding contributes to the differential expression of HERVH subfamilies and why only a 98 minority of HERVH are robustly transcribed in pluripotent stem cells and embryonic 99 development. 100 To shed light on these questions, we focused this study on the cis-regulatory evolution 101 of LTR7 elements. We use a “phyloregulatory” approach combining phylogenetic 102 analyses and regulatory genomics to investigate the sequence determinants underlying 103 the partitioning of expression of HERVH/LTR7 subfamilies during early embryonic 104 development. 105 expressed at different timepoints during embryonic development and that these 92 expression patterns reflect, at least in part, the unique cis-regulatory activities of their 93 LTRs. While it has been reported that several transcription factors (TFs) bind and 94 activate HERVH LTRs, including the pluripotency factors OCT4, NANOG, SP1, and 95 SOX2 (Göke et al., 2015; Ito et al., 2017; Kelley and Rinn, 2012; Kunarso et al., 2010; 96 Ohnuki et al., 2014; Pontis et al., 2019; Santoni et al., 2012), it remains unclear how TF 97 binding contributes to the differential expression of HERVH subfamilies and why only a 98 minority of HERVH are robustly transcribed in pluripotent stem cells and embryonic 99 development. consensus sequence for the internal region (HERVH_int) and the aforementioned four 62 LTR subfamilies. 63 100 To shed light on these questions, we focused this study on the cis-regulatory evolution 101 of LTR7 elements. We use a “phyloregulatory” approach combining phylogenetic 102 analyses and regulatory genomics to investigate the sequence determinants underlying 103 the partitioning of expression of HERVH/LTR7 subfamilies during early embryonic 104 development. 105 5 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: ioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint LTR7 consists of 8 previously undefined subfamilies 107 We began our investigation by examining the sequence relationships of the four LTR7 108 subfamilies currently recognized in the human genome: LTR7 sensu stricto (748 proviral 109 copies; 711 solo LTRs), 7b (113; 524), 7c (24; 223), and 7y (77; 77). We built a 110 maximum likelihood phylogenetic tree from a multiple sequence alignment of a total of 111 781 5’ LTR and 1073 solo LTR sequences of near complete length (>350 bp) 112 representing all intact LTR subfamilies extracted from the RepeatMasker output of the 113 hg38 human reference assembly. While 7b and 7y sequences cluster, as expected, into 114 clear monophyletic clades with relatively short internode distances and little subclade 115 structure, sequences from the 7c and LTR7 subfamilies were much more 116 heterogeneous and formed many subclades (Fig. 1A). Notably, sequences annotated 117 as LTR7 were split into distinct monophyletic clades indicative of previously 118 unrecognized subfamilies within that group. The branch length separating some of 119 these LTR7 subclades were longer from one another than they were from those falling 120 within the 7b, 7c, and 7y clades, indicating that they represent subfamilies as different 121 from each other as those previously recognized (Fig. 1A). 122 We next sought to classify LTR7 elements more finely by performing a phylogenetic 123 analysis using a multiple sequence alignment of all intact LTR7 sequences (>350 bp) 124 along with the consensus sequences for the other LTR7 subfamilies for reference. We 125 defined high-confidence subfamilies as those forming a clade supported by >95% 126 ultrafast bootstrap (UFbootstrap) and internal branches >0.015 (1.5 nucleotide 127 substitutions per 100 bp) separating subgroup nodes. Based on these criteria, LTR7 128 elements could be divided into 8 subfamilies (Fig. 1B). 129 We next sought to classify LTR7 elements more finely by performing a phylogenetic 123 analysis using a multiple sequence alignment of all intact LTR7 sequences (>350 bp) 124 along with the consensus sequences for the other LTR7 subfamilies for reference. We 125 defined high-confidence subfamilies as those forming a clade supported by >95% 126 ultrafast bootstrap (UFbootstrap) and internal branches >0.015 (1.5 nucleotide 127 substitutions per 100 bp) separating subgroup nodes. Based on these criteria, LTR7 128 elements could be divided into 8 subfamilies (Fig. 1B). LTR7 consists of 8 previously undefined subfamilies 107 129 While long internal branches with high UFbootstrap support separate LTR7 subfamilies, 130 intra-subfamily internal branches with >95% UFbootstrap support were shorter (<0.015), 131 suggesting that each subfamily was the product of a rapid burst of amplification of a 132 While long internal branches with high UFbootstrap support separate LTR7 subfamilies, 130 intra-subfamily internal branches with >95% UFbootstrap support were shorter (<0.015), 131 suggesting that each subfamily was the product of a rapid burst of amplification of a 132 6 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: ioRxiv preprint common ancestor To approximate the sequence of these ancestral elements we Fig. 1: Phylogenetic analysis of LTR7 sequences. A) Unrooted phylogeny of all solo and 5’ LTR7 sequences. All nodes with UFbootstraps >0.95, >10 member insertions, and >1.5 substitutions / 100 bp (~6 base pairs) are grouped and colored (see methods). Previously listed consensus sequences from 7b/c/y were included in the alignment and are shown in black. B) Unrooted phylogeny of all solo and 5’ LTR7 subfamilies from 1a, 7b, 7c, and 7y. Colors denote clades consisting of previously annotated 7b, 7c, and 7y with >95% concordance. C) Median joining network analysis of all LTR7 and related majority rule consensus sequences. Ticks indicate the number of SNPs at non-gaps between consensus sequences. The size of circles is proportional to the number of members in each subfamily. Only LTR7 insertions that met filtering requirements (see methods) are included while 7b/c/y counts are from dfam. . LTR7 consists of 8 previously undefined subfamilies 107 CC-BY 4.0 International license available under a as not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is 7 common ancestor. To approximate the sequence of these ancestral elements we 133 generated majority-rule consensus sequence for each of the 8 newly defined LTR7 134 subfamilies (7o, 7bc, 7up, etc.). The consensus sequences were deposited at 135 www.dfam.org. 136 To investigate the evolutionary relationships among the newly defined and previously 137 known LTR7 subfamilies, we conducted a median-joining network analysis (Leigh and 138 Fig. 1: Phylogenetic analysis of LTR7 sequences. A) Unrooted phylogeny of all solo and 5’ LTR7 sequences. All nodes with UFbootstraps >0.95, >10 member insertions, and >1.5 substitutions / 100 bp (~6 base pairs) are grouped and colored (see methods). Previously listed consensus sequences from 7b/c/y were included in the alignment and are shown in black. B) Unrooted phylogeny of all solo and 5’ LTR7 subfamilies from 1a, 7b, 7c, and 7y. Colors denote clades consisting of previously annotated 7b, 7c, and 7y with >95% concordance. C) Median joining network analysis of all LTR7 and related majority rule consensus sequences. Ticks indicate the number of SNPs at non-gaps between consensus sequences. The size of circles is proportional to the number of members in each subfamily. Only LTR7 insertions that met filtering requirements (see methods) are included while 7b/c/y counts are from dfam. Fig. 1: Phylogenetic analysis of LTR7 sequences. A) Unrooted phylogeny of all solo and 5’ LTR7 sequences. All nodes with UFbootstraps >0.95, >10 member insertions, and >1.5 substitutions / 100 bp (~6 base pairs) are grouped and colored (see methods). Previously listed consensus sequences from 7b/c/y were included in the alignment and are shown in black. B) Unrooted phylogeny of all solo and 5’ LTR7 subfamilies from 1a, 7b, 7c, and 7y. Colors denote clades consisting of previously annotated 7b, 7c, and 7y with >95% concordance. C) Median joining network analysis of all LTR7 and related majority rule consensus sequences. Ticks indicate the number of SNPs at non-gaps between consensus sequences. The size of circles is proportional to the number of members in each subfamily. Only LTR7 insertions that met filtering requirements (see methods) are included while 7b/c/y counts are from dfam. common ancestor. LTR7 consists of 8 previously undefined subfamilies 107 To approximate the sequence of these ancestral elements we 133 generated majority-rule consensus sequence for each of the 8 newly defined LTR7 134 subfamilies (7o, 7bc, 7up, etc.). The consensus sequences were deposited at 135 df 7 To investigate the evolutionary relationships among the newly defined and previously 137 known LTR7 subfamilies, we conducted a median-joining network analysis (Leigh and 138 To investigate the evolutionary relationships among the newly defined and previously 137 known LTR7 subfamilies, we conducted a median-joining network analysis (Leigh and 138 7 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint Bryant, 2015) of their consensus sequences (Fig. 1C). The network analysis provides 139 additional information on the relationships between subfamilies and approximates the 140 shortest and most parsimonious paths between them (Bandelt et al., 1999; Cordaux et 141 al., 2004; Posada and Crandall, 2001). The results place 7o in a central position from 142 which two major lineages are derived. One lineage led to two sub-lineages, formed by 143 7up1, 7up2, and 7u1 (with 7up1 and 7up2 being most closely related) and by 7d1 and 144 7d2. The other lineage emanating from 7o rapidly split into two sub-lineages; one gave 145 rise to 7u2 and then to 7y and the other gave rise to 7bc which is connected to the two 146 more diverged subfamilies 7b and 7c (Fig. 1C). Together these results indicate that the 147 LTRs of HERVH elements can be divided into additional subfamilies than those 148 previously recognized. 149 150 The age of LTR7 subfamilies suggests three major waves of HERVH propagation 151 The genetic differences between LTR7 subfamilies suggest that they may have been 152 active at different evolutionary timepoints. To examine this, we used reciprocal liftover 153 analysis to infer the presence/absence of each human LTR7 locus across five other 154 primate genomes. LTR7 consists of 8 previously undefined subfamilies 107 Insertions shared at orthologous genomic position across a set of 155 species are deemed to be ancestral to these species and thus can be inferred to be at 156 least as old as the divergence time of these species (Johnson, 2019). 157 The results of this cross-species analysis indicate that LTR7 subfamilies have been 158 transpositionally active at different timepoints in the primate lineage (Fig. 3A). The 159 Bryant, 2015) of their consensus sequences (Fig. 1C). The network analysis provides 139 additional information on the relationships between subfamilies and approximates the 140 shortest and most parsimonious paths between them (Bandelt et al., 1999; Cordaux et 141 al., 2004; Posada and Crandall, 2001). The results place 7o in a central position from 142 which two major lineages are derived. One lineage led to two sub-lineages, formed by 143 7up1, 7up2, and 7u1 (with 7up1 and 7up2 being most closely related) and by 7d1 and 144 7d2. The other lineage emanating from 7o rapidly split into two sub-lineages; one gave 145 rise to 7u2 and then to 7y and the other gave rise to 7bc which is connected to the two 146 more diverged subfamilies 7b and 7c (Fig. 1C). Together these results indicate that the 147 LTRs of HERVH elements can be divided into additional subfamilies than those 148 previously recognized. 149 The age of LTR7 subfamilies suggests three major waves of HERVH propagation 151 The genetic differences between LTR7 subfamilies suggest that they may have been 152 active at different evolutionary timepoints. To examine this, we used reciprocal liftover 153 analysis to infer the presence/absence of each human LTR7 locus across five other 154 primate genomes. Insertions shared at orthologous genomic position across a set of 155 species are deemed to be ancestral to these species and thus can be inferred to be at 156 least as old as the divergence time of these species (Johnson, 2019). 157 The results of this cross-species analysis indicate that LTR7 subfamilies have been 158 transpositionally active at different timepoints in the primate lineage (Fig. 3A). The 159 The results of this cross-species analysis indicate that LTR7 subfamilies have been 158 transpositionally active at different timepoints in the primate lineage (Fig. 3A). The 159 8 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint Fig. 2: Age analysis of LTR7 subfamilies. A) Proportion of a given subfamily that have 1:1 orthologous insertions between human and other primate species. LTR7 subfamilies are from trees in Figs. 1a and 2a; 7b/c/y subfamilies are from RepeatMasker annotations. Non-human primates are spaced out on the X axis in accordance with their approximate divergence times to the human lineage. B) Terminal branch lengths of all LTR7 insertions from Fig. 1a. Groups with similar liftover profiles were merged for statistical testing (see methods). Differences with padj<1e-15 are denoted with (Wilcox rank-sum test with Bonferroni correction). Fig. 2: Age analysis of LTR7 subfamilies. A) Proportion of a given subfamily that have 1:1 Fig. 2: Age analysis of LTR7 subfamilies. A) Proportion of a given subfamily that have 1:1 orthologous insertions between human and other primate species. LTR7 subfamilies are from trees in Figs. 1a and 2a; 7b/c/y subfamilies are from RepeatMasker annotations. The age of LTR7 subfamilies suggests three major waves of HERVH propagation 151 183 The 7up1/2, 7y, and 7u2 subfamilies represent the youngest in the human genome, with 176 most of their proliferation occurring between ~10 and ~14 mya, in the ancestor of 177 African great apes (Fig. 3A). Based on these results, these subfamilies seem to have 178 experienced a burst of transposition after the divergence of African and Asian great 179 apes but before the split of the pan/homo and gorilla lineages. For example, only 14 of 180 the 208 (6.7%) human 7up1 elements can be retrieved in orangutan, but 178 (85.6%) 181 can be found in gorilla. These data indicate that the three youngest LTR7 subfamilies 182 mostly expanded in the ancestor of African great apes (Fig. 2C). 183 As an independent dating method, we used the terminal branch length separating each 184 insertion from its nearest node in Fig. 1B (Fig. 2B). Here, the terminal branch lengths 185 are proportional to nucleotide divergence accumulated after insertion and can thus 186 approximate each insertion’s relative age. This method largely corroborated the results 187 of the liftover analysis and revealed three age groups among LTR7 subfamilies 188 characterized by statistically different mean branch lengths (p(adj)< 1e-15; Wilcox rank- 189 sum test). By contrast, we found no statistical difference between the mean branch 190 length of the subfamilies within these three age groups, suggesting that they were 191 concomitantly active. Taken together, our dating analyses distinguish 3 major waves of 192 HERV propagation: an older wave 25-40 mya involving 7c, 7o, and 7bc elements, an 193 intermediate wave 9-20 mya involving 7b, 7d1/2 and 7u1, and a most recent wave 4-10 194 mya implicating primarily 7up1/2, 7u2 and 7y elements. 195 The age of LTR7 subfamilies suggests three major waves of HERVH propagation 151 Non-human primates are spaced out on the X axis in accordance with their approximate divergence times to the human lineage. B) Terminal branch lengths of all LTR7 insertions from Fig. 1a. Groups with similar liftover profiles were merged for statistical testing (see methods). Differences with padj<1e-15 are denoted with * (Wilcox rank-sum test with Bonferroni correction). subfamilies 7o, 7bc, and 7c are the oldest since the majority of their insertions are found 160 at orthologous position in rhesus macaque, an Old World Monkey (OWM). These three 161 subfamilies share similar evolutionary trajectories, with most of their proliferation 162 occurring prior to the split of OWM and hominoids, ~25 mya (Fig. 2a). Members of the 163 7b subfamily (the most numerous, 637 solo and full-length insertions) appear to be 164 overall younger, since only 22% of the human 7b elements could be lifted over to 165 rhesus macaque and the vast majority appeared to have inserted between 10 and 20 166 mya (Fig. 2A, Figure supplement 1). Only 5 of the 550 elements in the 7d1 and 7d2 167 subfamilies could be retrieved in rhesus macaque, but ~30% were shared with gibbon 168 and ~75% were shared with orangutan. Thus, these two subfamilies are largely 169 hominoid-specific and achieved most of their proliferation prior to the split of African and 170 Asian great apes ~14 mya (Fig. 3a). Members of the 7u1 subfamily also emerged in the 171 hominoid ancestor, but the majority (55%) of 7u1 elements present in the human 172 genome inserted after the split of gibbons in the great ape ancestor, between 14 and 20 173 mya. Thus, the 7b, 7d1/2, and 7u1 subfamilies primarily amplified during the same 174 evolutionary window, 14 to 20 mya. 175 9 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. The age of LTR7 subfamilies suggests three major waves of HERVH propagation 151 ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint The 7up1/2, 7y, and 7u2 subfamilies represent the youngest in the human genome, with 176 most of their proliferation occurring between ~10 and ~14 mya, in the ancestor of 177 African great apes (Fig. 3A). Based on these results, these subfamilies seem to have 178 experienced a burst of transposition after the divergence of African and Asian great 179 apes but before the split of the pan/homo and gorilla lineages. For example, only 14 of 180 the 208 (6.7%) human 7up1 elements can be retrieved in orangutan, but 178 (85.6%) 181 can be found in gorilla. These data indicate that the three youngest LTR7 subfamilies 182 mostly expanded in the ancestor of African great apes (Fig. 2C). 183 As an independent dating method, we used the terminal branch length separating each 184 insertion from its nearest node in Fig. 1B (Fig. 2B). Here, the terminal branch lengths 185 are proportional to nucleotide divergence accumulated after insertion and can thus 186 approximate each insertion’s relative age. This method largely corroborated the results 187 of the liftover analysis and revealed three age groups among LTR7 subfamilies 188 characterized by statistically different mean branch lengths (p(adj)< 1e-15; Wilcox rank- 189 sum test). By contrast, we found no statistical difference between the mean branch 190 length of the subfamilies within these three age groups, suggesting that they were 191 concomitantly active. Taken together, our dating analyses distinguish 3 major waves of 192 HERV propagation: an older wave 25-40 mya involving 7c, 7o, and 7bc elements, an 193 intermediate wave 9-20 mya involving 7b, 7d1/2 and 7u1, and a most recent wave 4-10 194 mya implicating primarily 7up1/2, 7u2 and 7y elements. 195 The 7up1/2, 7y, and 7u2 subfamilies represent the youngest in the human genome, with 176 most of their proliferation occurring between ~10 and ~14 mya, in the ancestor of 177 African great apes (Fig. 3A). Based on these results, these subfamilies seem to have 178 experienced a burst of transposition after the divergence of African and Asian great 179 apes but before the split of the pan/homo and gorilla lineages. For example, only 14 of 180 the 208 (6.7%) human 7up1 elements can be retrieved in orangutan, but 178 (85.6%) 181 can be found in gorilla. These data indicate that the three youngest LTR7 subfamilies 182 mostly expanded in the ancestor of African great apes (Fig. 2C). Only LTR7up shows robust transcription in human ESC and iPSC 197 Our data thus far indicate that LTR7 is composed of genetically and evolutionarily 198 distinct subfamilies. Because a subset of HERVH elements linked to LTR7 were 199 previously reported to be transcribed in pluripotent stem cells (human ESCs and 200 iPSCs), we wondered whether this activity was restricted to one or several of the LTR7 201 subfamilies newly defined herein. To investigate this, we performed a “phyloregulatory” 202 analysis, where we layered locus-specific regulatory data obtained from publicly 203 available genome-wide assays in ESCs (mostly from the H1 cell line, see methods) for 204 each LTR insertion on top of a phylogenetic tree depicting their evolutionary 205 10 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint relationship. We called an individual LTR7 insertion as positive for a given feature if 206 there is overlap between the coordinates of the LTR and that of a peak called for this 207 mark (see methods). We predicted that if transcriptional activity was an ancestral 208 property of a given subfamily, evidence of transcription and “activation” marks should be 209 clustered within the cognate clade. Alternatively, if transcription and activation marks 210 were to be distributed throughout the tree, it would indicate that LTR7 transcriptional 211 activity in pluripotent cells was primarily driven by post-insertional changes or context- 212 specific effects. Differences in the proportion of positive insertions for a given mark 213 between LTR7 subfamilies were tested using a chi-square test with Bonferroni 214 correction. Unless otherwise noted, all proportions compared thereafter were 215 significantly different (padj< 0.05). 216 The results (Fig. 3A) show that HERVH elements inferred to be “highly expressed” 217 (fpkm > 2) based on RNA-seq analysis (Wang et al., 2014) were largely confined to two 218 closely related subfamilies, 7up1 and 7up2, together referred to as 7up hereafter. Only LTR7up shows robust transcription in human ESC and iPSC 197 219 Indeed, we estimated that 33% of 7up elements (88 loci) are highly expressed 220 according to RNA-seq compared with only 2% of highly expressed elements from all 221 other subfamilies combined (17 loci). Nascent RNA mapping using GRO-seq data 222 (Estarás et al., 2015) recapitulated this trend with 22% of 7up loci with visible signal 223 (Figure supplement 2), compared with only 4% of other LTR7 loci (Fig. 3D, Figure 224 supplement 2). Half of the loci displaying GRO-seq signal (53/96) also showed evidence 225 of mature RNA product (supp. file 1). Thus, HERVH transcriptional activity in H1 ESCs 226 is largely limited to loci driven by 7up sequences. 227 As previously noted from ChIP-seq data (Ohnuki et al., 2014), we found that KLF4 228 binding is a strong predictor of transcriptional activity: KLF4 ChIP-seq peaks overlap 229 91% of 7up loci and KLF4 binding is strongly enriched for the 7up subfamilies relative to 230 other subfamilies (Fig. 3A,B,D). NANOG binding is also enriched for 7up (97.7% of loci 231 overlap ChIP-seq peaks) but is observed to varying degrees at other LTR7 loci that do 232 not show evidence of active transcription based on GRO-seq and/or RNA-seq (85% of 233 7u1 loci, 32% 7d1, 45% 7d2, 13% 7o, 8.7% 7bc, and 0% of 7u2). Other TFs with known 234 roles in pluripotency are also enriched at 7up loci, such as SOX2 (32% LTR7up, 1-3% 235 11 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint all other LTR7), FOXP1(49%, 0-4.3%), and FOXA1(28%, 0-1.4%). In fact, FOXA1 bind 236 only a single non-7up insertion in our dataset, making it the most exclusive feature of 237 7up loci among the TFs examined in this analysis. In contrast, OCT4 binds merely 12% 238 of 7up loci (see supp. file 8 for full statistical analysis of all marks). Only LTR7up shows robust transcription in human ESC and iPSC 197 239 Congruent with having generally more TF binding and transcriptional activity, 7up loci 240 also have a propensity to be decorated by H3K4me3, a mark of active promoters (76% 241 LTR7up vs 19% all others) and the broader activity mark H3K27ac (89% vs 48%) (Fig. 242 3A,B). By contrast, H3K4me1, a mark typically associated with low POLII loading as 243 seen at enhancers as opposed to promoters, is spread rather evenly throughout the tre 244 of LTR7 sequences (26% vs. 18%) (Fig. 2A,B). Thus, promoter marks are primarily 245 restricted to 7up loci, but a broader range of 7up loci display putative enhancer marks. 246 Taken together, our phyloregulatory analysis suggests that strong promoter activity in 247 ESCs is restricted to 7up elements. 248 249 Differential activation, rather than repression, explain the differential 250 transcriptional activity of LTR7 subfamilies in ESCs 251 The pattern described above could be explained by two non-mutually exclusive 252 hypotheses: (i) 7up elements (most likely their progenitor) have acquired unique 253 sequences (TF binding sites, TFBS) that promote Pol II recruitment and active 254 transcription, and/or (ii) they somehow escape repressive mechanisms that actively 255 target the other subfamilies, preventing their transcription. For instance, 7up elements 256 may lack sequences targeted by transcriptional repressors such as KRAB-Zinc Finger 257 proteins (KZFP) that silence the other subfamilies in ESCs. KZFP are well-known for 258 binding TEs in a subfamily-specific manner where they nucleate inheritable epigenetic 259 silencing (Ecco et al., 2017; Jacobs et al., 2014; Wolf et al., 2020; Yang et al., 2017) 260 and several KZFPs are known to be capable of binding LTR7 loci (Imbeault et al., 261 2017). To examine whether KZFPs may differentially bind to LTR7 subfamilies, we 262 analyzed the loading of the corepressor KAP1 and the repressive histone mark 263 all other LTR7), FOXP1(49%, 0-4.3%), and FOXA1(28%, 0-1.4%). In fact, FOXA1 binds 236 only a single non-7up insertion in our dataset, making it the most exclusive feature of 237 7up loci among the TFs examined in this analysis. In contrast, OCT4 binds merely 12% 238 of 7up loci (see supp. file 8 for full statistical analysis of all marks). 239 all other LTR7), FOXP1(49%, 0-4.3%), and FOXA1(28%, 0-1.4%). In fact, FOXA1 binds 236 only a single non-7up insertion in our dataset, making it the most exclusive feature of 237 7up loci among the TFs examined in this analysis. Only LTR7up shows robust transcription in human ESC and iPSC 197 In contrast, OCT4 binds merely 12% 238 of 7up loci (see supp. file 8 for full statistical analysis of all marks). 239 Taken together, our phyloregulatory analysis suggests that strong promoter activity in 247 ESCs is restricted to 7up elements. 248 Differential activation, rather than repression, explain the differential 250 transcriptional activity of LTR7 subfamilies in ESCs 251 The pattern described above could be explained by two non-mutually exclusive 252 hypotheses: (i) 7up elements (most likely their progenitor) have acquired unique 253 sequences (TF binding sites, TFBS) that promote Pol II recruitment and active 254 transcription, and/or (ii) they somehow escape repressive mechanisms that actively 255 target the other subfamilies, preventing their transcription. For instance, 7up elements 256 may lack sequences targeted by transcriptional repressors such as KRAB-Zinc Finger 257 proteins (KZFP) that silence the other subfamilies in ESCs. KZFP are well-known for 258 binding TEs in a subfamily-specific manner where they nucleate inheritable epigenetic 259 silencing (Ecco et al., 2017; Jacobs et al., 2014; Wolf et al., 2020; Yang et al., 2017) 260 and several KZFPs are known to be capable of binding LTR7 loci (Imbeault et al., 261 2017). To examine whether KZFPs may differentially bind to LTR7 subfamilies, we 262 analyzed the loading of the corepressor KAP1 and the repressive histone mark 263 12 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint Fig. 3: Phyloregulatory analysis of LTR7. A) “Phyloregulatory” map of LTR7. The phylogenetic analysis to derive the circular tree is the same as for the tree in Fig. 1A but rooted on the 7b consensus. Subfamilies defined in Fig. 1 are denoted with dotted colored tips. Positive regulatory calls for each insertion are shown as tick marks of different colors and no tick mark indicates a negative call. All marks are derived from ESC except for ZNF90 and ZNF534, which are derived from ChIP-exo data after overexpression of these factors in HEK293 cells (see methods) B) Heatmap of major activation and repression profiles. Proportions indicate the proportion of each group positive for a given characteristic. Trees group LTR7 subfamilies on regulatory signature, not sequence similarity. Asterisks denote statistical differences between given group and 7up1 (padj> 0.05 Wilcox rank-sum with Bonferroni correction). . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint Differential activation, rather than repression, explain the differential 250 transcriptional activity of LTR7 subfamilies in ESCs 251 These observations could simply reflect the fact that ZNF534 itself is not highly 288 expressed in ESCs (Figure supplement 3) and do not preclude that ZNF534 represses 289 7up in other cellular contexts or cell types. Collectively these data suggest that 290 differential LTR binding of KZFP/KAP1 across subfamilies cannot readily explain their 291 differential regulatory activities in ESCs. Thus, differential activation is the most likely 292 driver for the promoter activity of 7up elements in ESCs. 293 To determine which factors are associated and potentially determinant for 7up promoter 294 activity, we compared the set of “highly expressed” 7up loci to 7up loci which are 295 depletion for 7bc and 7o compared to all other subfamilies including 7up (14% vs. 35% - 270 padj< 0.05 chi-square Bonferroni correction). Furthermore, KAP1 and H3K9me3 loading 271 were found in similar proportions in expressed and unexpressed 7up elements (padj> 272 0.05) (Fig. 2C). This was also the case for CpG methylation, whose presence was not 273 differential between subfamilies (padj> 0.05 Wilcox rank-sum with Bonferroni correction) 274 (Figure supplement 2). Thus, KAP1 binding and repressive marks at LTR7 in ESCs 275 poorly correlate with their transcriptional activity and differential repression is unlikely to 276 explain the differential promoter activity of LTR7 subfamilies in ESCs. 277 We also examined the binding profile of ZNF534 and ZNF90, two KZFPs previously 278 reported to be enriched for binding LTR7 elements using ChIP-exo data in human 279 embryonic kidney 293 cells (Imbeault et al., 2017), in order to examine whether they 280 bind a particular subset of elements in our LTR7 phylogeny. We found that while ZNF90 281 bound all LTR7 subfamilies to a similar extent, ZNF534 preferentially bound members of 282 the 7up subfamily (72% of LTR7up vs. 34-53% of non-LTR7up). However, ZNF534 283 binding in 293 cells did not correlate with transcriptional activity of 7up elements in 284 ESCs nor with KAP1 binding or H3K9me3 deposition in these cells (Fig. 3A,D). In other 285 words, there was no significant enrichment for ZNF534 binding within untranscribed 7up 286 elements nor depletion within the 7up elements we inferred to be highly transcribed in 287 ESCs. These observations could simply reflect the fact that ZNF534 itself is not highly 288 expressed in ESCs (Figure supplement 3) and do not preclude that ZNF534 represses 289 7up in other cellular contexts or cell types. Differential activation, rather than repression, explain the differential 250 transcriptional activity of LTR7 subfamilies in ESCs 251 C) Heatmap done in similar fashion to Fig. 3B but for repression marks. D) Heatmap of transcribed (>2 fpkm) and untranscribed 7up1/2 (<2 fpkm) and all 7d1/2. Red asterisks denote statistical differences between 7d1/2 and 7up1 (padj< 0.05 chi-square Bonferroni correction). White asterisks denote differences between transcribed and untranscribed LTR7up. Fig. 3: Phyloregulatory analysis of LTR7. A) “Phyloregulatory” map of LTR7. The phylogenetic analysis to derive the circular tree is the same as for the tree in Fig. 1A but rooted on the 7b consensus. Subfamilies defined in Fig. 1 are denoted with dotted colored tips. Positive regulatory calls for each insertion are shown as tick marks of different colors and no tick mark indicates a negative call. All marks are derived from ESC except for ZNF90 and ZNF534, which are derived from ChIP-exo data after overexpression of these factors in HEK293 cells (see methods) B) Heatmap of major activation and repression profiles. Proportions indicate the proportion of each group positive for a given characteristic. Trees group LTR7 subfamilies on regulatory signature, not sequence similarity. Asterisks denote statistical differences between given group and 7up1 (padj> 0.05 Wilcox rank-sum with Bonferroni correction). C) Heatmap done in similar fashion to Fig. 3B but for repression marks. D) Heatmap of transcribed (>2 fpkm) and untranscribed 7up1/2 (<2 fpkm) and all 7d1/2. Red asterisks denote statistical differences between 7d1/2 and 7up1 (padj< 0.05 chi-square Bonferroni correction). White asterisks denote differences between transcribed and untranscribed LTR7up. H3K9me3 typically deposited through the KZFP/KAP1 complex, across the LTR7 264 phylogeny using ChIP-seq data previously generated for ESCs (Imbeault et al., 2017; 265 Theunissen et al., 2016). We found that KAP1 and H3K9me3 loading were neither 266 enriched nor depleted for 7up elements relative to other subfamilies (Fig. 3A,C). Overall, 267 there were no significant differences in the level of H3K9me3 marking across 268 subfamilies and the only difference in KAP1 binding was a slight but significant 269 H3K9me3 typically deposited through the KZFP/KAP1 complex, across the LTR7 264 phylogeny using ChIP-seq data previously generated for ESCs (Imbeault et al., 2017; 265 Theunissen et al., 2016). We found that KAP1 and H3K9me3 loading were neither 266 enriched nor depleted for 7up elements relative to other subfamilies (Fig. 3A,C). Differential activation, rather than repression, explain the differential 250 transcriptional activity of LTR7 subfamilies in ESCs 251 Overall, 267 there were no significant differences in the level of H3K9me3 marking across 268 subfamilies and the only difference in KAP1 binding was a slight but significant 269 13 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint depletion for 7bc and 7o compared to all other subfamilies including 7up (14% vs. 35% - 270 padj< 0.05 chi-square Bonferroni correction). Furthermore, KAP1 and H3K9me3 loading 271 were found in similar proportions in expressed and unexpressed 7up elements (padj> 272 0.05) (Fig. 2C). This was also the case for CpG methylation, whose presence was not 273 differential between subfamilies (padj> 0.05 Wilcox rank-sum with Bonferroni correction) 274 (Figure supplement 2). Thus, KAP1 binding and repressive marks at LTR7 in ESCs 275 poorly correlate with their transcriptional activity and differential repression is unlikely to 276 explain the differential promoter activity of LTR7 subfamilies in ESCs. 277 We also examined the binding profile of ZNF534 and ZNF90, two KZFPs previously 278 reported to be enriched for binding LTR7 elements using ChIP-exo data in human 279 embryonic kidney 293 cells (Imbeault et al., 2017), in order to examine whether they 280 bind a particular subset of elements in our LTR7 phylogeny. We found that while ZNF90 281 bound all LTR7 subfamilies to a similar extent, ZNF534 preferentially bound members of 282 the 7up subfamily (72% of LTR7up vs. 34-53% of non-LTR7up). However, ZNF534 283 binding in 293 cells did not correlate with transcriptional activity of 7up elements in 284 ESCs nor with KAP1 binding or H3K9me3 deposition in these cells (Fig. 3A,D). In other 285 words, there was no significant enrichment for ZNF534 binding within untranscribed 7up 286 elements nor depletion within the 7up elements we inferred to be highly transcribed in 287 ESCs. Differential activation, rather than repression, explain the differential 250 transcriptional activity of LTR7 subfamilies in ESCs 251 Collectively these data suggest that 290 differential LTR binding of KZFP/KAP1 across subfamilies cannot readily explain their 291 differential regulatory activities in ESCs. Thus, differential activation is the most likely 292 driver for the promoter activity of 7up elements in ESCs. 293 To determine which factors are associated and potentially determinant for 7up promoter 294 activity, we compared the set of “highly expressed” 7up loci to 7up loci which are 295 apparently poorly expressed, using 7d1/d2 as outgroups (Fig. 3D). While known 296 regulators of LTR7 transcription, KLF4 and NANOG, are enriched for binding to 7up 297 elements, their occupancy alone cannot distinguish transcribed from untranscribed 7up 298 loci (Fig. 3D). Thus, other factors must contribute to the transcriptional activation of 7up 299 14 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint elements. Our analysis of pluripotent transcriptional activators SOX2, FOXA1, FOXP1, 300 OCT4, TCF4, and SMAD1 (Boyer et al., 2005; Chambers and Smith, 2004; Niwa, 2007) 301 binding profiles show that all of these TFs are enriched in robustly transcribed 7up loci 302 compared to non-transcribed loci (Fig. 3D). Intriguingly, when overexpressed in HEK293 303 cells, the potential KZFP repressor ZNF534 preferably binds ESC-transcribed 7up over 304 untranscribed 7up, suggesting that ZNF534 may suppress transcription-competent 7up 305 in cellular contexts where this factor is expressed. 306 Together these data suggest that differential repression cannot explain the differential 307 promoter activity of LTR7 subfamilies in ESCs but rather that highly expressed LTR7up 308 loci are preferentially bound by a cocktail of transcriptional activators that are less 309 prevalent on poorly-expressed loci. 310 311 Inter-element recombination and intra-element duplication drove LTR7 sequence 312 evolution 313 The data presented above suggest that the transcriptional activity of 7up in ESCs 314 emerged from the gain of a unique combination of TFBS. Differential activation, rather than repression, explain the differential 250 transcriptional activity of LTR7 subfamilies in ESCs 251 To identify sequences unique 315 to 7up relative to its closely related subfamilies, we aligned the consensus sequences of 316 the newly defined LTR7 subfamilies and those of 7b/c/y consensus sequences. This 317 multiple sequence alignment revealed blocks of sequences that tend to be highly 318 conserved across subfamilies, only diverging by a few SNPs, while other regions 319 showed insertion/deletion (indel) segments specific to one or a few subfamilies (Fig. 320 4A). These indels resulted in substantial gain and loss of DNA between closely related 321 subfamilies, with the longest consensus (7y) having a length of 472-bp and the shortest 322 (7o) a mere 365-bp. These observations suggest that segmental rearrangements have 323 played an important role in the evolution of LTR7 sequences. 324 Upon closer scrutiny, we noticed that the indels characterizing some of the subfamilies 325 were at odds with the evolutionary relationship of the subfamilies defined by overall 326 phylogenetic and network analyses This was particularly obvious in segments we 327 elements. Our analysis of pluripotent transcriptional activa 300 OCT4, TCF4, and SMAD1 (Boyer et al., 2005; Chambers a 301 binding profiles show that all of these TFs are enriched in 302 compared to non-transcribed loci (Fig. 3D). Intriguingly, wh 303 cells, the potential KZFP repressor ZNF534 preferably bind 304 untranscribed 7up, suggesting that ZNF534 may suppress 305 in cellular contexts where this factor is expressed. 306 Together these data suggest that differential repression ca 307 promoter activity of LTR7 subfamilies in ESCs but rather th 308 loci are preferentially bound by a cocktail of transcriptional 309 prevalent on poorly-expressed loci. 310 311 Inter-element recombination and intra-element duplica 312 evolution 313 The data presented above suggest that the transcriptional 314 emerged from the gain of a unique combination of TFBS. T 315 to 7up relative to its closely related subfamilies, we aligned 316 the newly defined LTR7 subfamilies and those of 7b/c/y co 317 multiple sequence alignment revealed blocks of sequence 318 conserved across subfamilies, only diverging by a few SNP 319 showed insertion/deletion (indel) segments specific to one 320 4A). These indels resulted in substantial gain and loss of D 321 subfamilies, with the longest consensus (7y) having a leng 322 (7o) a mere 365-bp. Differential activation, rather than repression, explain the differential 250 transcriptional activity of LTR7 subfamilies in ESCs 251 These observations suggest that segm 323 played an important role in the evolution of LTR7 sequenc 324 Upon closer scrutiny, we noticed that the indels characteriz 325 were at odds with the evolutionary relationship of the subfa 326 elements. Our analysis of pluripotent transcriptional activators SOX2, FOXA1, FOXP1, 300 OCT4, TCF4, and SMAD1 (Boyer et al., 2005; Chambers and Smith, 2004; Niwa, 2007) 301 binding profiles show that all of these TFs are enriched in robustly transcribed 7up loci 302 compared to non-transcribed loci (Fig. 3D). Intriguingly, when overexpressed in HEK293 303 cells, the potential KZFP repressor ZNF534 preferably binds ESC-transcribed 7up over 304 untranscribed 7up, suggesting that ZNF534 may suppress transcription-competent 7up 305 in cellular contexts where this factor is expressed. 306 elements. Our analysis of pluripotent transcriptional activators SOX2, FOXA1, FOXP1, 300 OCT4, TCF4, and SMAD1 (Boyer et al., 2005; Chambers and Smith, 2004; Niwa, 2007) 301 binding profiles show that all of these TFs are enriched in robustly transcribed 7up loci 302 compared to non-transcribed loci (Fig. 3D). Intriguingly, when overexpressed in HEK293 303 cells, the potential KZFP repressor ZNF534 preferably binds ESC-transcribed 7up over 304 untranscribed 7up, suggesting that ZNF534 may suppress transcription-competent 7up 305 in cellular contexts where this factor is expressed. 306 Together these data suggest that differential repression cannot explain the differential 307 promoter activity of LTR7 subfamilies in ESCs but rather that highly expressed LTR7up 308 loci are preferentially bound by a cocktail of transcriptional activators that are less 309 prevalent on poorly-expressed loci. 310 Inter-element recombination and intra-element duplication drove LTR7 sequence 312 The data presented above suggest that the transcriptional activity of 7up in ESCs 314 emerged from the gain of a unique combination of TFBS. To identify sequences unique 315 to 7up relative to its closely related subfamilies, we aligned the consensus sequences of 316 the newly defined LTR7 subfamilies and those of 7b/c/y consensus sequences. This 317 multiple sequence alignment revealed blocks of sequences that tend to be highly 318 conserved across subfamilies, only diverging by a few SNPs, while other regions 319 showed insertion/deletion (indel) segments specific to one or a few subfamilies (Fig. 320 4A). These indels resulted in substantial gain and loss of DNA between closely related 321 subfamilies, with the longest consensus (7y) having a length of 472-bp and the shortest 322 (7o) a mere 365-bp. These observations suggest that segmental rearrangements have 323 played an important role in the evolution of LTR7 sequences. 324 Upon closer scrutiny, we noticed that the indels characterizing some of the subfamilies 325 were at odds with the evolutionary relationship of the subfamilies defined by overall 326 phylogenetic and network analyses. This was particularly obvious in segments we 327 15 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint Fig. 4: Modular block evolution of LTR7 subfamilies. A) A multiple sequence alignment of LTR7 subfamily consensus sequences. The phylogenetic topology from Figure 1 is shown on the left. The MSA is broken down into sequence blocks (red lines) with differential patterns of relationships. B) Parsimony trees from Fig. 4a sequence blocks. Subfamilies whose blocks do not match the overall phylogeny are highlighted in red. Bootstrap values >0 are shown. C) Blastn alignment of LTR7up1 block 2a and 2b. D) A multiple sequence alignment of majority-rule consensus sequences from each LTR7 subfamily detailing shared structure. Blocks show aligned sequence; gaps represent absent sequence. Colored sections identify putative phylogeny-breaking events. Inter-element recombination and intra-element duplication drove LTR7 sequence 312 Recombination events whose directionality can be inferred (via Fig. 4: Modular block evolution of LTR7 subfamilies. A) A multiple sequence alignment of LTR7 subfamily consensus sequences. The phylogenetic topology from Figure 1 is shown on the left. The MSA is broken down into sequence blocks (red lines) with differential patterns of relationships. B) Parsimony trees from Fig. 4a sequence blocks. Subfamilies whose blocks do not match the overall phylogeny are highlighted in red. Bootstrap values >0 are shown. C) Blastn alignment of LTR7up1 block 2a and 2b. D) A multiple sequence alignment of majority-rule consensus sequences from each LTR7 subfamily detailing shared structure. Blocks show aligned sequence; gaps represent absent sequence. Colored sections identify putative phylogeny-breaking events. Recombination events whose directionality can be inferred (via aging) are shown with blocks and arrows on the cladogram. Recombination events with multiple possible routes are denoted with “?”. The deletion of 2b is denoted on the cladogram with a red “X”; the duplication of 2a is denoted with 2 red rectangles. Fig. 4: Modular block evolution of LTR7 subfamilies. A) A multiple sequence alignment of LTR7 subfamily consensus sequences. The phylogenetic topology from Figure 1 is shown on the left. The MSA is broken down into sequence blocks (red lines) with differential patterns of relationships. B) Parsimony trees from Fig. 4a sequence blocks. Subfamilies whose blocks do not match the overall phylogeny are highlighted in red. Bootstrap values >0 are shown. C) Blastn alignment of LTR7up1 block 2a and 2b. D) A multiple sequence alignment of majority-rule consensus sequences from each LTR7 subfamily detailing shared structure. Blocks show aligned sequence; gaps represent absent sequence. Colored sections identify putative phylogeny-breaking events. Recombination events whose directionality can be inferred (via aging) are shown with blocks and arrows on the cladogram. Recombination events with multiple possible routes are denoted with “?”. The deletion of 2b is denoted on the cladogram with a red “X”; the duplication of 2a is denoted with 2 red rectangles. Fig. 4: Modular block evolution of LTR7 subfamilies. A) A multiple sequence alignment of LTR7 subfamily consensus sequences. The phylogenetic topology from Figure 1 is shown on the left. The MSA is broken down into sequence blocks (red lines) with differential patterns of relationships. B) Parsimony trees from Fig. 4a sequence blocks. Subfamilies whose blocks do not match the overall phylogeny are highlighted in red. Bootstrap values >0 are shown. Inter-element recombination and intra-element duplication drove LTR7 sequence 312 However, block 2b 342 from 7b, 7c, 7u2, and 7y aligned poorly with block 2b from 7up and 7u1. Instead, block 343 2b from 7up/u1 2b was closely related (~81% nucleotide similarity) to block 2a from the 344 same subfamilies (Fig. 4D), suggestive that it arose via tandem duplication in the 345 common ancestor of these subfamilies. To further clarify the evolutionary history of the 346 2a-2b duplication, we aligned all 2a and 2b blocks from all subfamilies and generated a 347 parsimony tree (Figure supplement 4). This analysis indicated that the 2b block from 348 7up/u1 most closely resembles the 2a block from 7d. 349 The results above suggest that the evolution of HERVH was characterized by extensive 350 diversification of LTR sequences through a mixture of point mutations, indels, and 351 recombination events. 352 353 patterns of inter-subfamily relationships. Based on this analysis, we defined seven 331 sequence blocks shared by a different subset of subfamilies, pointing at relationships 332 that were at odds with the overall phylogeny of the LTR7 subfamilies (Fig. 4A-B). These 333 observations suggested that some of the blocks have been exchanged between LTR7 334 subfamilies through recombination events. 335 17 sequence blocks shared by a different subset of subfamilies, pointing at relationships 332 that were at odds with the overall phylogeny of the LTR7 subfamilies (Fig. 4A-B). These 333 observations suggested that some of the blocks have been exchanged between LTR7 334 subfamilies through recombination events. 335 To systematically test if recombination events between elements drove the evolution of 336 LTR7 subfamilies, we generated parsimony trees for each block of consensus 337 sequences and looked for incongruences with the overall consensus phylogeny. We 338 found a minimum of 6 instances of clades supported in the block parsimony trees that 339 were incongruent with those supported by the overall phylogeny (Fig. 4B,D). 340 We also found some blocks evolved via tandem duplication. Notably, block 2b was 341 absent from 7d1/2 and 7bc/o but present in all other subfamilies. However, block 2b 342 from 7b, 7c, 7u2, and 7y aligned poorly with block 2b from 7up and 7u1. Instead, block 343 2b from 7up/u1 2b was closely related (~81% nucleotide similarity) to block 2a from the 344 same subfamilies (Fig. 4D), suggestive that it arose via tandem duplication in the 345 common ancestor of these subfamilies. Inter-element recombination and intra-element duplication drove LTR7 sequence 312 C) Blastn alignment of LTR7up1 block 2a and 2b. D) A multiple sequence alignment of majority-rule consensus sequences from each LTR7 subfamily detailing shared structure. Blocks show aligned sequence; gaps represent absent sequence. Colored sections identify putative phylogeny-breaking events. Recombination events whose directionality can be inferred (via aging) are shown with blocks and arrows on the cladogram. Recombination events with multiple possible routes are denoted with “?”. The deletion of 2b is denoted on the cladogram with a red “X”; the duplication of 2a is denoted with 2 red rectangles. termed block 2b (where 7y and 7u2 share a large insertion with 7b and 7c) and block 3 328 (where 7y and 7b share a large insertion). This led us to carefully examine the multiple 329 sequence alignment of the LTR7 consensus sequences to identify indels with different 330 16 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint patterns of inter-subfamily relationships. Based on this analysis, we defined seven 331 sequence blocks shared by a different subset of subfamilies, pointing at relationships 332 that were at odds with the overall phylogeny of the LTR7 subfamilies (Fig. 4A-B). These 333 observations suggested that some of the blocks have been exchanged between LTR7 334 subfamilies through recombination events. 335 To systematically test if recombination events between elements drove the evolution of 336 LTR7 subfamilies, we generated parsimony trees for each block of consensus 337 sequences and looked for incongruences with the overall consensus phylogeny. We 338 found a minimum of 6 instances of clades supported in the block parsimony trees that 339 were incongruent with those supported by the overall phylogeny (Fig. 4B,D). 340 We also found some blocks evolved via tandem duplication. Notably, block 2b was 341 absent from 7d1/2 and 7bc/o but present in all other subfamilies. Inter-element recombination and intra-element duplication drove LTR7 sequence 312 To further clarify the evolutionary history of the 346 2a-2b duplication, we aligned all 2a and 2b blocks from all subfamilies and generated a 347 parsimony tree (Figure supplement 4). This analysis indicated that the 2b block from 348 7up/u1 most closely resembles the 2a block from 7d. 349 The results above suggest that the evolution of HERVH was characterized by extensive 350 diversification of LTR sequences through a mixture of point mutations, indels, and 351 recombination events. 352 353 HERVH subfamilies show distinct expression profiles in the preimplantation 354 embryo 355 We hypothesized that the mosaic pattern of LTR sequence evolution described above 356 gave rise to TFBS combinations unique to each family that drove shifts in HERVH 357 expression during early embryogenesis. To test this, we aimed to reanalyze the 358 expression profiles of newly defined LTR7 subfamilies during early human 359 To systematically test if recombination events between elements drove the evolution of 336 LTR7 subfamilies, we generated parsimony trees for each block of consensus 337 sequences and looked for incongruences with the overall consensus phylogeny. We 338 found a minimum of 6 instances of clades supported in the block parsimony trees that 339 were incongruent with those supported by the overall phylogeny (Fig. 4B,D). 340 We also found some blocks evolved via tandem duplication. Notably, block 2b was 341 absent from 7d1/2 and 7bc/o but present in all other subfamilies. However, block 2b 342 from 7b, 7c, 7u2, and 7y aligned poorly with block 2b from 7up and 7u1. Instead, block 343 2b from 7up/u1 2b was closely related (~81% nucleotide similarity) to block 2a from the 344 same subfamilies (Fig. 4D), suggestive that it arose via tandem duplication in the 345 common ancestor of these subfamilies. To further clarify the evolutionary history of the 346 2a-2b duplication, we aligned all 2a and 2b blocks from all subfamilies and generated a 347 parsimony tree (Figure supplement 4). This analysis indicated that the 2b block from 348 7up/u1 most closely resembles the 2a block from 7d. 349 The results above suggest that the evolution of HERVH was characterized by extensive 350 diversification of LTR sequences through a mixture of point mutations, indels, and 351 recombination events. 352 17 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Inter-element recombination and intra-element duplication drove LTR7 sequence 312 It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint embryogenesis and correlate these patterns with the acquisition of embryonic TF 360 binding motifs within each of the subfamilies. 361 embryogenesis and correlate these patterns with the acquisition of embryonic TF 360 binding motifs within each of the subfamilies. 361 ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint These shifts can be largely explained by the fact that 7u2 and 7y are closely related 373 (Fig. 1A-C) and 7y and 7b share a great deal of sequence through recombination 374 events (Fig. 4B-C). 375 Next we used the newly generated Repeatmasker annotations to examine the RNA 376 expression profiles of the different LTR7 subfamilies using scRNA-seq data from human 377 pre-implantation embryos and RNA-seq data from human ESCs (Blakeley et al., 2015; 378 Tang et al., 2010) (see methods). 379 As expected, we found that the 7up subfamilies were highly expressed in the pluripotent 380 epiblast and in ESCs (Fig. 5). 7up expression was highly specific to these pluripotent 381 cell types, with little to no transcription at earlier developmental time points. As 382 previously observed (Göke et al., 2015), the 7b subfamily exhibited expression at the 8- 383 cell and morula stages, coinciding with EGA (Fig. 5). Another remarkable expression 384 pattern was that of 7u2 which was restricted to the pluripotent epiblast (Fig. 5). 385 Interestingly, the 7y subfamily combined the expression of 7b and 7u2 (8-cell and 386 morula plus epiblast), perhaps reflecting the acquisition of sequence blocks from both 387 subfamilies (Fig. 4B-C). Despite very similar sequence and age (Fig. 1, Fig. 2, Fig. 4A), 388 7bc and 7o elements show stark contrast in their expression profiles. 7o elements show 389 no significant transcription at any time point in early development, while 7bc elements 390 display RNA expression throughout the blastocyst, including trophectoderm and inner 391 cell mass, primitive endoderm, and pluripotent epiblast (Fig. 5). Previous expression 392 analysis of the oldest LTR7 subfamily, 7c, did not find robust stage-specific expression 393 (Göke et al., 2015). Our analysis revealed that some 7c elements display moderate 394 RNA expression at various developmental stages (Fig. 5). This pattern may reflect the 395 relatively high level of sequence heterogeneity within this subfamily (Fig. 1). embryogenesis and correlate these patterns with the acquisition of embryonic TF 360 binding motifs within each of the subfamilies. 361 Fig. 5: Expression profile of LTR7 subfamilies in human preimplantation embryonic lineages and ESCs. The solid dots and lines encompassing the violins represent the median and quartiles of single cellular RNA expression. The color scheme is based on embryonic stages, defined as maternal control of early embryos (Oocytes, Zygote, 2-cell and 4-cell stage), EGA (8-cell and Morula), inner cell mass (ICM), trophectoderm (TE), epiblast (EPI) and primitive endoderm (PE) from the blastocyst, and ESCs at passages 0 and 10. Fig. 5: Expression profile of LTR7 subfamilies in human preimplantation embryonic lineages and ESCs. The solid dots and lines encompassing the violins represent the median and quartiles of single cellular g. 5: Expression profile of LTR7 subfamilies in human preimplantation embryonic lineages and ESCs Fig. 5: Expression profile of LTR7 subfamilies in human preimplantation embryonic lineages and ESCs. The solid dots and lines encompassing the violins represent the median and quartiles of single cellular RNA expression. The color scheme is based on embryonic stages, defined as maternal control of early embryos (Oocytes, Zygote, 2-cell and 4-cell stage), EGA (8-cell and Morula), inner cell mass (ICM), trophectoderm (TE), epiblast (EPI) and primitive endoderm (PE) from the blastocyst, and ESCs at passages 0 and 10. To perform this analysis, we first reannotated the hg38 reference genome assembly 362 using Repeatmasker with a custom library consisting of the consensus sequences for 363 the 8 newly defined LTR7 subfamilies plus newly generated consensus sequences for 364 7b, 7c, and 7y subfamilies redefined from the phylogenetic analysis presented in Fig. 1B 365 (Figure supplement 5) (see methods). Our newly generated Repeatmasker annotations 366 (supp. file 2) did not drastically differ from previous annotations of LTR7 and 7c, where 367 90% and 86% of insertions, respectively, were concordant with the old Repeatmasker 368 annotations (though LTR7 insertions were now assigned to one of the 8 newly defined 369 subfamilies). 7y and 7b annotations, however, shifted significantly. Only 33% of 370 previously annotated 7y reannotated concordantly with 53% now being annotated as 371 7u2 and only 52% of 7b reannotated concordantly, with 22% now annotated as 7y. 372 18 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. embryogenesis and correlate these patterns with the acquisition of embryonic TF 360 binding motifs within each of the subfamilies. 361 Note this motif (and insertion) was also present 418 in 7u1, the closest relative to 7up (Fig. 4C), but absent in all other subfamilies (Fig. 6B). 419 We hypothesized that the 8-bp insertion provided a binding motif for SOX2 and/or SOX3 420 contributing to 7up promoter activity in ESCs. Indeed, SOX2 and SOX3 bind a highly 421 similar motif (Bergsland et al., 2011; Heinz et al., 2010), activate an overlapping set of 422 genes and play a redundant function in pluripotency (Corsinotti et al., 2017; Niwa et al., 423 2016; Wang et al., 2012). In addition, we observed that both SOX2 and SOX3 are 424 expressed in human ESCs but SOX3 was more highly and more specifically expressed 425 in ESCs (Figure supplement 6A,C). While SOX3 binding has not been profiled in human 426 ESCs, ChIP-seq data available for SOX2 indicated that it binds preferentially 7up in a 427 region coinciding with the 8-bp motif (Fig. 6B). Together these observations suggest 428 that 7up promoter activity in ESCs might be conferred in part by the gain of a SOX2/3 429 motif in block 2b. 430 embryogenesis and correlate these patterns with the acquisition of embryonic TF 360 binding motifs within each of the subfamilies. 361 396 In summary, our analysis indicates that LTR7 subfamilies have distinct but partially 397 overlapping expression profiles during human early embryonic development that appear 398 to mirror their complex history of sequence diversification. 399 In summary, our analysis indicates that LTR7 subfamilies have distinct but partially 397 overlapping expression profiles during human early embryonic development that appear 398 to mirror their complex history of sequence diversification. 399 400 19 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint A predicted SOX2/3 motif unique to 7up is required for transcriptional activity in 401 pluripotent stem cells 402 We hypothesized that differences in embryonic transcription among LTR7 subfamilies 403 were driven by the gain and loss of TF binding motifs, and that one or more of these 404 mutations led to 7up’s pluripotent-specific transcription. To find TF motifs enriched 405 within each LTR7 subfamily relative to the others, we performed an unbiased motif 406 enrichment analysis using the program HOMER to calculate enrichment scores of 407 known TF motifs within each segmental block defined in Fig. 4A in a pairwise 408 comparison of each subfamily against each of the other subfamilies (see methods). The 409 results yielded a slew of TF motifs enriched for each subfamily relative to the others 410 (see Fig. 6A for 7up1 and enrichment for all HERVH subfamilies in supp. files 3,4). 411 These results suggested that each LTR7 subfamily possesses a unique repertoire of TF 412 binding motifs, which could explain their differential expression during embryonic 413 development. 414 Next, we sought to pinpoint mutational events responsible for the gain of TF motifs 415 responsible for the unique expression of 7up in ESC. The single most striking motif 416 distinguishing the 7up clade from the others was a SOX2/3 motif which coincided with 417 an 8-bp insertion in block 2b (Fig. 6A,B). A predicted SOX2/3 motif unique to 7up is required for transcriptional activity in 401 pluripotent stem cells 402 CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: ioRxiv preprint Fig. 6: An 8-bp insertion, SOX2/3 binding site necessary for LTR7up transcription. A) (log) p-values >500 for HOMER motifs enriched in 7up1 insertion’s sequence blocks vs the same blocks from other insertions from other HERVH subfamilies are shown. B) Line plots show SOX2 ChIP-seq signal at LTR7 subfamily loci in human ESCs. Signal from genomic loci was compiled relative to position 0. The 7up/u1 8bp insertion position is shown with a dotted line. Region 2b harboring SOX2/3 TFBS is detailed below. C) Scheme of DNA fragments cloned into pGL3-basic vector driving luciferase gene expression (LUC) with identified SOX2/3 motifs. 3 constructs were analyzed: Entire LTR7up (7up1), 7d1/2 consensus sequence (approximate ancestral sequence for all LTR7d) and LTR7up with 8 nucleotides deleted (LTR7up (Δ8bp - AAAAGAAG)) (see panel B). D) Normalized relative luciferase activity of tested fragments compared to LTR7 down; n = 4 measurements; bars, means across replicates; error bars, standard deviation of the mean, dots, individual replicates. Fig. 6: An 8-bp insertion, SOX2/3 binding site necessary for LTR7up transcription. A) (log) p-values >500 for HOMER motifs enriched in 7up1 insertion’s sequence blocks vs the same blocks from other insertions from other HERVH subfamilies are shown. B) Line plots show SOX2 ChIP-seq signal at LTR7 subfamily loci in human ESCs. Signal from genomic loci was compiled relative to position 0. The 7up/u1 8bp insertion position is shown with a dotted line. Region 2b harboring SOX2/3 TFBS is detailed below. C) Scheme of DNA fragments cloned into pGL3-basic vector driving luciferase gene expression (LUC) with identified SOX2/3 motifs. 3 constructs were analyzed: Entire LTR7up (7up1), 7d1/2 consensus sequence (approximate ancestral sequence for all LTR7d) and LTR7up with 8 nucleotides deleted (LTR7up (Δ8bp - AAAAGAAG)) (see panel B). A predicted SOX2/3 motif unique to 7up is required for transcriptional activity in 401 pluripotent stem cells 402 We hypothesized that differences in embryonic transcription among LTR7 subfamilies 403 were driven by the gain and loss of TF binding motifs, and that one or more of these 404 mutations led to 7up’s pluripotent-specific transcription. To find TF motifs enriched 405 within each LTR7 subfamily relative to the others, we performed an unbiased motif 406 enrichment analysis using the program HOMER to calculate enrichment scores of 407 known TF motifs within each segmental block defined in Fig. 4A in a pairwise 408 comparison of each subfamily against each of the other subfamilies (see methods). The 409 results yielded a slew of TF motifs enriched for each subfamily relative to the others 410 (see Fig. 6A for 7up1 and enrichment for all HERVH subfamilies in supp. files 3,4). 411 These results suggested that each LTR7 subfamily possesses a unique repertoire of TF 412 binding motifs, which could explain their differential expression during embryonic 413 development. 414 Next, we sought to pinpoint mutational events responsible for the gain of TF motifs 415 responsible for the unique expression of 7up in ESC. The single most striking motif 416 distinguishing the 7up clade from the others was a SOX2/3 motif which coincided with 417 an 8-bp insertion in block 2b (Fig. 6A,B). Note this motif (and insertion) was also present 418 in 7u1, the closest relative to 7up (Fig. 4C), but absent in all other subfamilies (Fig. 6B). 419 We hypothesized that the 8-bp insertion provided a binding motif for SOX2 and/or SOX3 420 contributing to 7up promoter activity in ESCs. Indeed, SOX2 and SOX3 bind a highly 421 similar motif (Bergsland et al., 2011; Heinz et al., 2010), activate an overlapping set of 422 genes and play a redundant function in pluripotency (Corsinotti et al., 2017; Niwa et al., 423 2016; Wang et al., 2012). In addition, we observed that both SOX2 and SOX3 are 424 expressed in human ESCs but SOX3 was more highly and more specifically expressed 425 in ESCs (Figure supplement 6A,C). While SOX3 binding has not been profiled in human 426 ESCs, ChIP-seq data available for SOX2 indicated that it binds preferentially 7up in a 427 region coinciding with the 8-bp motif (Fig. 6B). Together these observations suggest 428 that 7up promoter activity in ESCs might be conferred in part by the gain of a SOX2/3 429 motif in block 2b. 430 20 . A predicted SOX2/3 motif unique to 7up is required for transcriptional activity in 401 pluripotent stem cells 402 D) Normalized relative luciferase activity of tested fragments compared to LTR7 down; n = 4 measurements; bars, means across replicates; error bars, standard deviation of the mean, dots, individual replicates. To experimentally test this prediction, we used a luciferase reporter to assay promoter 431 activity of three different LTR7 sequences in iPSCs (see methods). The first consisted 432 of the full-length 7d consensus sequence (predicted to be inactive in iPSCs), the second 433 contained the full-length 7up1 consensus (predicted to be active) and the third used the 434 same 7up1 consensus sequence but lacking the 8-bp motif unique to 7up1/2 and 7u1 435 elements overlapping the SOX2/3 motif (Fig. 6B,C). The results of the assays revealed 436 To experimentally test this prediction, we used a luciferase reporter to assay promoter 431 activity of three different LTR7 sequences in iPSCs (see methods). The first consisted 432 of the full-length 7d consensus sequence (predicted to be inactive in iPSCs), the second 433 contained the full-length 7up1 consensus (predicted to be active) and the third used the 434 same 7up1 consensus sequence but lacking the 8-bp motif unique to 7up1/2 and 7u1 435 elements overlapping the SOX2/3 motif (Fig. 6B,C). The results of the assays revealed 436 To experimentally test this prediction, we used a luciferase reporter to assay promoter 431 activity of three different LTR7 sequences in iPSCs (see methods). The first consisted 432 of the full-length 7d consensus sequence (predicted to be inactive in iPSCs), the second 433 contained the full-length 7up1 consensus (predicted to be active) and the third used the 434 same 7up1 consensus sequence but lacking the 8-bp motif unique to 7up1/2 and 7u1 435 elements overlapping the SOX2/3 motif (Fig. 6B,C). The results of the assays revealed 436 21 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. Discussion 446 Discussion 446 The HERVH family has been the subject of intense investigation for its transcriptional 447 and regulatory activities in human pluripotent stem cells. These studies often have 448 treated the entire family as one homogenous, monophyletic entity and it has remained 449 generally unclear which loci are transcribed and potentially important for pluripotency. 450 This is in part because HERVH/LTR7 is an abundant and young family which poses 451 technical challenges to interrogate the activity of individual loci and design experiments 452 targeting specific members of the family (Chuong et al., 2017; Lanciano and Cristofari, 453 2020). Here, we applied a ‘phyloregulatory’ approach that integrates regulatory 454 genomics data to a phylogenetic analysis of LTR7 sequences to reveal several new 455 insights into the origin, evolution, and transcription of HERVH elements. In brief, our 456 results show that: (i) LTR7 is a polyphyletic group composed of at least eight 457 monophyletic subfamilies; (ii) these subfamilies have distinct evolutionary histories and 458 transcriptional profiles in human embryos and a single and relatively small subgroup 459 (~264 loci), LTR7up, exhibits robust promoter activity in ESC; (iii) LTR7 evolution is 460 characterized by the gain, loss, and exchange of cis-regulatory modules likely 461 underlying their transcriptional partitioning during early embryonic development. 462 463 Phyloregulatory analysis of LTR7 disentangles the cis-regulatory evolution of 464 HERVH 465 Previous studies have treated LTR7 sensu stricto insertions as equivalent 466 representatives of their subfamilies (Bao et al., 2015; Gemmell et al., 2019; Göke et al. 467 2015; Izsvák et al., 2016; Storer et al., 2021; Wang et al., 2014; Zhang et al., 2019). 468 While some of these studies were able to detect differential transcriptional partitioning 469 between LTR7, LTR7y, and LTR7b (Göke et al., 2015), the amalgamating of LTR7 loci 470 limited the ability to detect transcriptional variations among LTR7 and to identify key 471 sequence differences responsible for divergent transcription patterns. Our granular 472 parsing of LTR7 elements and their phyloregulatory profiling has revealed striking 473 genetic, regulatory, and evolutionary differences amongst these sequences. 474 The HERVH family has been the subject of intense investigation for its transcriptional 447 and regulatory activities in human pluripotent stem cells. These studies often have 448 treated the entire family as one homogenous, monophyletic entity and it has remained 449 generally unclear which loci are transcribed and potentially important for pluripotency. A predicted SOX2/3 motif unique to 7up is required for transcriptional activity in 401 pluripotent stem cells 402 ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint that the 7d construct exhibited, as predicted, only weak promoter activity in iPSC 437 compared to the empty vector (Fig. 6D), while the 7up1 construct had much stronger 438 promoter activity, driving on average 7.8-fold more luciferase expression than 7d and 439 100-fold more than the empty vector (Fig. 6D). Strikingly, the promoter activity was 440 essentially abolished in the 7up1 construct lacking the 8-bp motif, which drove minimal 441 luciferase expression (on average, 3-fold less than LTR7d and 20-fold less than the 442 intact LTR7up sequence). These results demonstrate that the 8-bp motif in 7up1 is 443 necessary for robust promoter activity in iPSCs, likely by providing a SOX2/3 binding 444 site essential for this activity. 445 22 22 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: ioRxiv preprint Discussion 446 450 This is in part because HERVH/LTR7 is an abundant and young family which poses 451 technical challenges to interrogate the activity of individual loci and design experiments 452 targeting specific members of the family (Chuong et al., 2017; Lanciano and Cristofari, 453 2020). Here, we applied a ‘phyloregulatory’ approach that integrates regulatory 454 genomics data to a phylogenetic analysis of LTR7 sequences to reveal several new 455 insights into the origin, evolution, and transcription of HERVH elements. In brief, our 456 results show that: (i) LTR7 is a polyphyletic group composed of at least eight 457 monophyletic subfamilies; (ii) these subfamilies have distinct evolutionary histories and 458 transcriptional profiles in human embryos and a single and relatively small subgroup 459 (~264 loci), LTR7up, exhibits robust promoter activity in ESC; (iii) LTR7 evolution is 460 characterized by the gain, loss, and exchange of cis-regulatory modules likely 461 underlying their transcriptional partitioning during early embryonic development. 462 Phyloregulatory analysis of LTR7 disentangles the cis-regulatory evolution of 464 23 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint Importantly, a phylogeny based on the coding sequence (RVT domain) of HERVH 475 provided less granularity to separate the subfamilies than the LTR sequences (Figure 476 supplement 7). The classification of new subfamilies within LTR7 enabled us to discover 477 that they have distinct expression profiles during early embryonic development (Fig. 5) 478 that were previously obscured by their aggregation into a single group of elements. For 479 example, the 7u2 subfamily is, to our knowledge, the first subfamily of human TEs 480 reported to have preimplantation expression exclusively in the epiblast. 481 It has been observed for some time that only a small subset of HERVH elements are 482 expressed in ESCs (Gemmell et al., 2019; Göke et al., 2015; Ohnuki et al., 2014; 483 Santoni et al., 2012; Schön et al., 2001; Wang et al., 2014; Zhang et al., 2019). Some 484 have attributed this property to variation in the internal region of HERVH, context- 485 dependent effects (local chromatin or cis-regulatory environment) and/or age (Gemmell 486 et al., 2019; Zhang et al., 2019). Our results provide an additional, perhaps simpler 487 explanation: we found that HERVH elements expressed in ESCs are almost exclusively 488 driven by two closely related subfamilies of LTR7 (7up) that emerged most recently in 489 hominoid evolution. We identified one 8-bp sequence motif overlaps a predicted 490 SOX2/3 binding site unique to 7up that is required for promoter activity in pluripotent 491 stem cells. These results highlight that the primary sequence of the LTR plays an 492 important role in differentiating and diversifying HERVH expression during human 493 embryonic development. 494 Importantly, a phylogeny based on the coding sequence (RVT domain) of HERVH 475 provided less granularity to separate the subfamilies than the LTR sequences (Figure 476 supplement 7). Phyloregulatory analysis of LTR7 disentangles the cis-regulatory evolution of 464 The coincidence 528 of recombination events with changes in expression profiles (Fig 7) suggests that these 529 enrichment was driven by a relatively small subset of loci within each family. We 505 suspect that some of the intrafamilial differences in TF binding and cis-regulatory 506 activity may be caused by unrecognized subfamily structure and subfamily-specific 507 combinations of TFBS, much like we observe for LTR7. 508 25 509 Recombination as a driver of LTR cis-regulatory evolution 510 Recombination is a common and important force in the evolution of exogenous RNA 511 viruses (Jetzt et al., 2000; Pérez-Losada et al., 2015; Simon-Loriere and Holmes, 2011) 512 and endogenous retroviruses (Vargiu et al., 2016). Traditional models of recombination 513 describe recombination occurring due to template switching during reverse transcription, 514 a process that requires the co-packaging of RNA genomes, a feature of retroviruses 515 and some retrotransposons (Lai, 1992; Matsuda and Garfinkel, 2009). Previous studies 516 proposed that the HERVH family had undergone inter-element recombination events of 517 both its coding genes (Mager and Freeman, 1987; Vargiu et al., 2016) and LTR 518 (Goodchild et al., 1993). Specifically, it was inferred that recombination event between 519 Type I LTR (i.e., LTR7) and Type II LTR (LTR7b) led to the emergence of Type Ia 520 (LTR7y). 521 Our findings of extensive sequence block exchange between 7y and 7b (Fig. 4D) are 522 consistent with these inferences. Furthermore, our division of HERVH into at least 11 523 subfamilies, rather than the original trio (Type I, II, Ia), and systematic analysis of 524 recombination events (Fig. 4) suggest that recombination has occurred between 525 multiple lineages of elements and has been a pervasive force underlying LTR 526 diversification. We identified a minimum of six recombination events spanning 20 million 527 years of primate evolution (see Fig. 4D and summary model in Fig. 7). The coincidence 528 of recombination events with changes in expression profiles (Fig. 7) suggests that these 529 events were instrumental to the diversification of HERVH embryonic expression. The 530 hybrid origin and subsequent burst of amplification of LTR7 subfamilies (Fig. 1,2) 531 suggest they expanded rapidly after shifting their transcriptional profiles. The 532 coincidence of niche colonization with a burst in transposition leads us to speculate that 533 these shifts in expression were foundational to the formation and successful expansion 534 Phyloregulatory analysis of LTR7 disentangles the cis-regulatory evolution of 464 The classification of new subfamilies within LTR7 enabled us to discover 477 that they have distinct expression profiles during early embryonic development (Fig. 5) 478 that were previously obscured by their aggregation into a single group of elements. For 479 example, the 7u2 subfamily is, to our knowledge, the first subfamily of human TEs 480 reported to have preimplantation expression exclusively in the epiblast. 481 It has been observed for some time that only a small subset of HERVH elements are 482 expressed in ESCs (Gemmell et al., 2019; Göke et al., 2015; Ohnuki et al., 2014; 483 Santoni et al., 2012; Schön et al., 2001; Wang et al., 2014; Zhang et al., 2019). Some 484 have attributed this property to variation in the internal region of HERVH, context- 485 dependent effects (local chromatin or cis-regulatory environment) and/or age (Gemmell 486 et al., 2019; Zhang et al., 2019). Our results provide an additional, perhaps simpler 487 explanation: we found that HERVH elements expressed in ESCs are almost exclusively 488 driven by two closely related subfamilies of LTR7 (7up) that emerged most recently in 489 hominoid evolution. We identified one 8-bp sequence motif overlaps a predicted 490 SOX2/3 binding site unique to 7up that is required for promoter activity in pluripotent 491 stem cells. These results highlight that the primary sequence of the LTR plays an 492 important role in differentiating and diversifying HERVH expression during human 493 embryonic development. 494 The phyloregulatory approach outlined in this study could be applied to illuminate the 495 regulatory activities of LTR elements in other cellular contexts. In addition to 496 embryogenesis, subsets of LTR7 and LTR7y elements are known to be upregulated in 497 oncogenic states (Babaian and Mager, 2016; Glinsky, 2015; Kong et al., 2019; Yu et al., 498 2013). It would be interesting to explore whether these activities can be linked to the 499 gain of specific TFBS using the new LTR7 annotations and regulatory information 500 presented herein. Other human LTR families, such as MER41, LTR12C, or LTR13 have 501 been previously identified as enriched for particular TF binding and cis-regulatory 502 activities in specific cellular contexts (Chuong et al., 2016; Deniz et al., 2020; Ito et al., 503 2017; Krönung et al., 2016; Sundaram et al., 2014). In each case, TF binding 504 24 . Phyloregulatory analysis of LTR7 disentangles the cis-regulatory evolution of 464 CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint enrichment was driven by a relatively small subset of loci within each family. We 505 suspect that some of the intrafamilial differences in TF binding and cis-regulatory 506 activity may be caused by unrecognized subfamily structure and subfamily-specific 507 combinations of TFBS, much like we observe for LTR7. 508 509 Recombination as a driver of LTR cis-regulatory evolution 510 Recombination is a common and important force in the evolution of exogenous RNA 511 viruses (Jetzt et al., 2000; Pérez-Losada et al., 2015; Simon-Loriere and Holmes, 2011) 512 and endogenous retroviruses (Vargiu et al., 2016). Traditional models of recombination 513 describe recombination occurring due to template switching during reverse transcription, 514 a process that requires the co-packaging of RNA genomes, a feature of retroviruses 515 and some retrotransposons (Lai, 1992; Matsuda and Garfinkel, 2009). Previous studies 516 proposed that the HERVH family had undergone inter-element recombination events of 517 both its coding genes (Mager and Freeman, 1987; Vargiu et al., 2016) and LTR 518 (Goodchild et al., 1993). Specifically, it was inferred that recombination event between 519 Type I LTR (i.e., LTR7) and Type II LTR (LTR7b) led to the emergence of Type Ia 520 (LTR7y). 521 Our findings of extensive sequence block exchange between 7y and 7b (Fig. 4D) are 522 consistent with these inferences. Furthermore, our division of HERVH into at least 11 523 subfamilies, rather than the original trio (Type I, II, Ia), and systematic analysis of 524 recombination events (Fig. 4) suggest that recombination has occurred between 525 multiple lineages of elements and has been a pervasive force underlying LTR 526 diversification. We identified a minimum of six recombination events spanning 20 million 527 years of primate evolution (see Fig. 4D and summary model in Fig. 7). Recombination as a driver of LTR cis-regulatory evolution 510 It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint of new HERVH subfamilies. It would be interesting to explore whether inter-element 535 recombination has also contributed to the evolution of other LTR subfamilies and the 536 diversification of their expression patterns. 537 Previous work has highlighted the role of TEs, and LTRs in particular, in donating built- 538 in cis-regulatory sequences promoting the evolutionary rewiring of mammalian 539 transcriptional networks (Chuong et al., 2017; Feschotte, 2008; Hermant and Torres- 540 Padilla, 2021; Jacques et al., 2013; Rebollo et al., 2012; Sundaram and Wysocka, 2020 541 Thompson et al., 2016). We show that recombination provides another layer to this 542 idea, where combinations of TFBS can be mixed-and-matched, then mobilized and 543 propagated, further accelerating the diversification of these regulatory DNA elements. 544 As HERVH expanded and diversified, its newly evolved cis-regulatory modules became 545 confined to specific host lineages (Fig. 2). Thus, it is possible that the formation of new 546 of new HERVH subfamilies. It would be interesting to explore whether inter-element 535 recombination has also contributed to the evolution of other LTR subfamilies and the 536 diversification of their expression patterns. 537 Previous work has highlighted the role of TEs, and LTRs in particular, in donating built- 538 in cis-regulatory sequences promoting the evolutionary rewiring of mammalian 539 transcriptional networks (Chuong et al., 2017; Feschotte, 2008; Hermant and Torres- 540 Padilla, 2021; Jacques et al., 2013; Rebollo et al., 2012; Sundaram and Wysocka, 2020; 541 Thompson et al., 2016). We show that recombination provides another layer to this 542 idea, where combinations of TFBS can be mixed-and-matched, then mobilized and 543 propagated, further accelerating the diversification of these regulatory DNA elements. 544 As HERVH expanded and diversified, its newly evolved cis-regulatory modules became 545 confined to specific host lineages (Fig. 2). Thus, it is possible that the formation of new 546 Fig. 7: Model of LTR7 subfamily evolution. Estimated LTR7 subfamily transpositional activity in mya are listed with corresponding approximate primate divergence times (bottom). The positioning and duration of transpositional activity are based on analysis from Fig. 3b. The grey connections between subfamilies indicate average tree topology which is driven by overall pairwise sequence similarity. Dashed lines indicate likely recombination events which led to the founding of new subfamilies. Stage-specific expression profiles from Fig. Recombination as a driver of LTR cis-regulatory evolution 510 Recombination is a common and important force in the evolution of exogenous RNA 511 viruses (Jetzt et al., 2000; Pérez-Losada et al., 2015; Simon-Loriere and Holmes, 2011) 512 and endogenous retroviruses (Vargiu et al., 2016). Traditional models of recombination 513 describe recombination occurring due to template switching during reverse transcription, 514 a process that requires the co-packaging of RNA genomes, a feature of retroviruses 515 and some retrotransposons (Lai, 1992; Matsuda and Garfinkel, 2009). Previous studies 516 proposed that the HERVH family had undergone inter-element recombination events of 517 both its coding genes (Mager and Freeman, 1987; Vargiu et al., 2016) and LTR 518 (Goodchild et al., 1993). Specifically, it was inferred that recombination event between 519 Type I LTR (i.e., LTR7) and Type II LTR (LTR7b) led to the emergence of Type Ia 520 (LTR7y). 521 Our findings of extensive sequence block exchange between 7y and 7b (Fig. 4D) are 522 consistent with these inferences. Furthermore, our division of HERVH into at least 11 523 subfamilies, rather than the original trio (Type I, II, Ia), and systematic analysis of 524 recombination events (Fig. 4) suggest that recombination has occurred between 525 multiple lineages of elements and has been a pervasive force underlying LTR 526 diversification. We identified a minimum of six recombination events spanning 20 million 527 years of primate evolution (see Fig. 4D and summary model in Fig. 7). The coincidence 528 of recombination events with changes in expression profiles (Fig. 7) suggests that these 529 events were instrumental to the diversification of HERVH embryonic expression. The 530 hybrid origin and subsequent burst of amplification of LTR7 subfamilies (Fig. 1,2) 531 suggest they expanded rapidly after shifting their transcriptional profiles. The 532 coincidence of niche colonization with a burst in transposition leads us to speculate that 533 these shifts in expression were foundational to the formation and successful expansion 534 25 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Recombination as a driver of LTR cis-regulatory evolution 510 5a are detailed to the right of each corresponding branch. Fig. 7: Model of LTR7 subfamily evolution. Estimated LTR7 subfamily transpositional activity in mya are listed with corresponding approximate primate divergence times (bottom). The positioning and duration of transpositional activity are based on analysis from Fig. 3b. The grey connections between subfamilies indicate average tree topology which is driven by overall pairwise sequence similarity. Dashed lines indicate likely recombination events which led to the founding of new subfamilies. Stage-specific expression profiles from Fig. 5a are detailed to the right of each corresponding branch. 26 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint LTR via recombination and their subsequent amplification catalyzed cis-regulatory 547 divergence across primate species. 548 549 LTR evolution enabled HERVH’s colonization of different niches in the human 550 embryo 551 Our evolutionary analysis reveals that multiple HERVH subfamilies were 552 transpositionally active in parallel during the past ~25 my of primate evolution (Fig. 2,7). 553 This is in stark contrast to the pattern of LINE1 evolution in primates, which is 554 characterized by a single subfamily being predominantly active at any given time (Khan 555 et al., 2006). We hypothesize that the ability of HERVH to colonize multiple cellular 556 niches underlie this difference. Indeed, we observe that concurrently active HERVH 557 subfamilies are transcribed at different developmental stages, such as 7up and 7u2 558 being transcribed in the pluripotent epiblast at the same time that 7y and the youngest 559 7b were transcribed at the 8 cell and morula stages (Fig. 7). We posit that this 560 partitioning allowed multiple HERVH subfamilies to amplify in parallel without causing 561 overt genome instability and cell death during embryonic development. 562 Niche diversification may have also enabled HERVH to evade cell-type-specific 563 repression by host-encoded factors such as KZFPs. LTR via recombination and their subsequent amplification catalyzed cis-regulatory 547 divergence across primate species. 548 27 q p y g y divergence across primate species. 548 549 LTR evolution enabled HERVH’s colonization of different niches in the human 550 embryo 551 Our evolutionary analysis reveals that multiple HERVH subfamilies were 552 transpositionally active in parallel during the past ~25 my of primate evolution (Fig. 2,7). 553 This is in stark contrast to the pattern of LINE1 evolution in primates, which is 554 characterized by a single subfamily being predominantly active at any given time (Khan 555 et al., 2006). We hypothesize that the ability of HERVH to colonize multiple cellular 556 niches underlie this difference. Indeed, we observe that concurrently active HERVH 557 subfamilies are transcribed at different developmental stages, such as 7up and 7u2 558 being transcribed in the pluripotent epiblast at the same time that 7y and the youngest 559 7b were transcribed at the 8 cell and morula stages (Fig. 7). We posit that this 560 partitioning allowed multiple HERVH subfamilies to amplify in parallel without causing 561 overt genome instability and cell death during embryonic development. 562 Niche diversification may have also enabled HERVH to evade cell-type-specific 563 repression by host-encoded factors such as KZFPs. KZFPs are thought to emerge and 564 adapt during evolution to silence specific TE subfamilies in a cell-type specific manner 565 (Bruno et al., 2019; Cosby et al., 2019; Ecco et al., 2017; Imbeault et al., 2017). For 566 example, there is evidence that the progenitors of the currently active L1HS subfamily 567 became silenced in human ESCs via KZFP targeting, but evaded that repression and 568 persisted in that niche through the deletion of the KZFP binding site (Jacobs et al., 569 2014). HERVH may have persisted through another evasive strategy: changing their 570 TFBS repertoire to colonize niches lacking their repressors. To silence all LTR7, any 571 potential HERVH-targeting KZFP would need to gain expression in multiple cellular 572 contexts. For example, one potential repressor, ZNF534, binds a wide range of LTR7 573 sequences, but is particularly enriched at 7up in HEK293 cells (Fig. 3A,D). Our analysis 574 shows that ZNF534 is most highly expressed in the morula, but dips in human ESC 575 (Figure supplement 3). Thus, ZNF534 may repress 7up at earlier stages of development 576 Niche diversification may have also enabled HERVH to evade cell-type-specific 563 repression by host-encoded factors such as KZFPs. Recombination as a driver of LTR cis-regulatory evolution 510 KZFPs are thought to emerge and 564 adapt during evolution to silence specific TE subfamilies in a cell-type specific manner 565 LTR via recombination and their subsequent amplification catalyzed cis-regulatory 547 divergence across primate species. 548 HERVH/LTR7 has been us 589 al., 2014; Santoni et al., 2012; Wang 590 HERVH/LTR7-positive cells may be 591 referred to as “primed” cells (Göke e 592 primed cells are not as promising for 593 (Nichols and Smith, 2009), which are 594 morula to epiblast, or so-called “form 595 the early post-implantation epiblast ( 596 Rossant and Tam, 2017). Of relevan 597 7u2 subfamily are highly and exclus 598 (Fig. 5), but weakly so in H1 ESC, w 599 minority of naïve or formative cells (G 600 develop a LTR7u2-driven reporter sy 601 from an heterogenous ESC populati 602 been used in mouse to purify rare 2- 603 but is apparently unable to suppress 7up transcription in pluripotent stem cells. If true, 577 this scenario would illustrate how LTR diversification facilitated HERVH persistence in 578 the face of KZFP coevolution. Further investigation is needed to explore the interplay 579 between KZFPs and HERVH subfamilies during primate evolution. 580 581 Implications for stem cell and regenerative biology 582 Lastly, our findings may provide new opportunities for stem cell research and 583 regenerative medicine. Our data on 7up reinforces previous findings (Corsinotti et al., 584 2017; Wang et al., 2012) that place SOX2/3 as central players in pluripotency. 585 Furthermore, our analysis identified a set of TFs whose motifs are uniquely enriched in 586 different LTR7 subfamilies with distinct expression patterns in early embryonic cells, 587 which may enable a functional discriminatory analysis of the role of these TFs in each 588 cell type. HERVH/LTR7 has been used as a marker for human pluripotency (Ohnuki et 589 al., 2014; Santoni et al., 2012; Wang et al., 2014), and recent work has revealed that 590 HERVH/LTR7-positive cells may be more amenable to differentiation, and are therefore 591 referred to as “primed” cells (Göke et al., 2015; Theunissen et al., 2016). However, 592 primed cells are not as promising for regenerative medicine as so-called “naïve” cells 593 (Nichols and Smith, 2009), which are less differentiated and resemble cells from late 594 morula to epiblast, or so-called “formative” cells, which most closely resemble cells from 595 the early post-implantation epiblast (Kalkan and Smith, 2014; Kinoshita et al., 2021; 596 Rossant and Tam, 2017). Of relevance to this issue is our finding that elements of the 597 7u2 subfamily are highly and exclusively expressed in the pluripotent epiblast in vivo 598 (Fig. LTR via recombination and their subsequent amplification catalyzed cis-regulatory 547 divergence across primate species. 548 5), but weakly so in H1 ESC, which consists of a majority of primed cells and a 599 minority of naïve or formative cells (Gafni et al., 2013). Thus, it might be possible to 600 develop a LTR7u2-driven reporter system to mark and purify naïve or formative cells 601 from an heterogenous ESC population. Similarly, a MERVL LTR-GFP transgene has 602 but is apparently unable to suppress 7up transcription in pluripotent stem cells. If true, 577 this scenario would illustrate how LTR diversification facilitated HERVH persistence in 578 the face of KZFP coevolution. Further investigation is needed to explore the interplay 579 between KZFPs and HERVH subfamilies during primate evolution. 580 LTR via recombination and their subsequent amplification catalyzed cis-regulatory 547 divergence across primate species. 548 KZFPs are thought to emerge and 564 adapt during evolution to silence specific TE subfamilies in a cell-type specific manner 565 (Bruno et al., 2019; Cosby et al., 2019; Ecco et al., 2017; Imbeault et al., 2017). For 566 example, there is evidence that the progenitors of the currently active L1HS subfamily 567 became silenced in human ESCs via KZFP targeting, but evaded that repression and 568 persisted in that niche through the deletion of the KZFP binding site (Jacobs et al., 569 2014). HERVH may have persisted through another evasive strategy: changing their 570 TFBS repertoire to colonize niches lacking their repressors. To silence all LTR7, any 571 potential HERVH-targeting KZFP would need to gain expression in multiple cellular 572 contexts. For example, one potential repressor, ZNF534, binds a wide range of LTR7 573 sequences, but is particularly enriched at 7up in HEK293 cells (Fig. 3A,D). Our analysis 574 shows that ZNF534 is most highly expressed in the morula, but dips in human ESC 575 (Figure supplement 3). Thus, ZNF534 may repress 7up at earlier stages of development 576 27 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint but is apparently unable to suppress 577 this scenario would illustrate how LT 578 the face of KZFP coevolution. Furthe 579 between KZFPs and HERVH subfam 580 581 Implications for stem cell and reg 582 Lastly, our findings may provide new 583 regenerative medicine. Our data on 584 2017; Wang et al., 2012) that place S 585 Furthermore, our analysis identified 586 different LTR7 subfamilies with distin 587 which may enable a functional discri 588 cell type. Implications for stem cell and regenerative biology 582 Lastly, our findings may provide new opportunities for stem cell research and 583 regenerative medicine. Our data on 7up reinforces previous findings (Corsinotti et al., 584 2017; Wang et al., 2012) that place SOX2/3 as central players in pluripotency. 585 Furthermore, our analysis identified a set of TFs whose motifs are uniquely enriched in 586 different LTR7 subfamilies with distinct expression patterns in early embryonic cells, 587 which may enable a functional discriminatory analysis of the role of these TFs in each 588 cell type. HERVH/LTR7 has been used as a marker for human pluripotency (Ohnuki et 589 al., 2014; Santoni et al., 2012; Wang et al., 2014), and recent work has revealed that 590 HERVH/LTR7-positive cells may be more amenable to differentiation, and are therefore 591 referred to as “primed” cells (Göke et al., 2015; Theunissen et al., 2016). However, 592 primed cells are not as promising for regenerative medicine as so-called “naïve” cells 593 (Nichols and Smith, 2009), which are less differentiated and resemble cells from late 594 morula to epiblast, or so-called “formative” cells, which most closely resemble cells from 595 the early post-implantation epiblast (Kalkan and Smith, 2014; Kinoshita et al., 2021; 596 Rossant and Tam, 2017). Of relevance to this issue is our finding that elements of the 597 7u2 subfamily are highly and exclusively expressed in the pluripotent epiblast in vivo 598 (Fig. 5), but weakly so in H1 ESC, which consists of a majority of primed cells and a 599 minority of naïve or formative cells (Gafni et al., 2013). Thus, it might be possible to 600 develop a LTR7u2-driven reporter system to mark and purify naïve or formative cells 601 from an heterogenous ESC population. Similarly, a MERVL LTR-GFP transgene has 602 been used in mouse to purify rare 2-cell-like totipotent cells where this LTR is 603 specifically expressed amidst mouse ESCs in culture (Hermant and Torres-Padilla, 604 2021; Macfarlan et al., 2012). 605 28 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . Implications for stem cell and regenerative biology 582 CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint In conclusion, our study highlights the modular cis-regulatory evolution of an 606 endogenous retrovirus which has facilitated its transcriptional partitioning in early 607 embryogenesis. We believe that phyloregulatory dissection of endogenous retroviral 608 LTRs has the potential to further our understanding of the evolution, impact, and 609 applications of these elements in a broad range of biomedical areas. 610 29 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint Acknowledgements: 611 We would like to thank Arian Smit and Robert Hubley for their assistance in remasking 612 the human genome with the custom LTR7 library in accordance with 613 RepeatMasker/Dfam specifications. We would also like to thank the Cornell statistical 614 consulting unit (CSCU), and Stephen Parry in particular, for their help with statistical 615 analyses. Finally, we would like to credit BioRender.com for its primate (Fig. 2,7) and 616 embryonic cell (Fig. 6,7) graphics. This work was supported by funds from the National 617 Institutes of Health to CF (GM112972; HG009391; GM122550); Cornell Center for 618 Vertebrate Genomics to T.A.C.; and the Cornell Presidential Fellow Program to M.S. 619 J.L.R. is supported by the Howard Hughes Medical Institute Faculty Scholar program 620 and NIH (GM099117). 621 30 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint HERVH LTR sequence identification: 623 This only displays 639 nodes with ultrafast (UF) bootstrap support >0.95. 640 To visualize inter-insertion relationships, the MSA was input into IQtree with options -nt 638 AUTO -m MFP -bb 6000 -asr -minsup .95 (Chernomor et al., 2016). This only displays 639 nodes with ultrafast (UF) bootstrap support >0.95. 640 Clusters of >10 insertions sharing a node with UFbootstrap support that were separated 641 from other insertions by internal branch lengths >0.015 (1.5subs / 100 bp) were defined 642 as belonging to a new bona fide LTR7 subfamily (Fig. 1B). 643 Clusters of >10 insertions sharing a node with UFbootstrap support that were separated 641 from other insertions by internal branch lengths >0.015 (1.5subs / 100 bp) were defined 642 as belonging to a new bona fide LTR7 subfamily (Fig. 1B). 643 Clusters of >10 insertions sharing a node with UFbootstrap support that were separated 641 from other insertions by internal branch lengths >0.015 (1.5subs / 100 bp) were defined 642 as belonging to a new bona fide LTR7 subfamily (Fig. 1B). 643 HERVH LTR sequence identification: 623 All HERVH-int and accompanying LTRs (LTR7, 7b, 7c, and 7y) were extracted from 624 masked (RepeatMasker version 4.0.5 repeat Library 20140131 - (Smit et al., 2013)) 625 GRCh38/hg38 (alt chromosomes removed). All annotated HERVH-int and HERVH LTR 626 were run through OneCodeToFindThemAll.pl (Bailly-Bechet et al., 2014) followed by 627 rename_mergedLTRelements.pl (Thomas et al., 2018) to identify solo and full-length 628 HERVH insertions. 5’ LTRs from full-length insertions >4kb were combined with and 629 solo LTRs. LTRs >350bp were considered for future analysis. 630 All HERVH LTRs (Fig. 1A – supp. file 5) or only LTR7s (Fig. 1B – supp. file 6) were 632 aligned with mafft –auto (Nakamura et al., 2018) strategy: FFT-NS-2/Progressive 633 method followed by PRANK (Löytynoja and Goldman, 2010) with options -showanc - 634 support -njtree -uselogs -prunetree -prunedata -F -showevents. Uninformative structural 635 variations were removed with Trimal (Capella-Gutierrez et al., 2009) with option -gt 636 0.01. 637 All HERVH LTRs (Fig. 1A – supp. file 5) or only LTR7s (Fig. 1B – supp. file 6) were 632 aligned with mafft –auto (Nakamura et al., 2018) strategy: FFT-NS-2/Progressive 633 method followed by PRANK (Löytynoja and Goldman, 2010) with options -showanc - 634 support -njtree -uselogs -prunetree -prunedata -F -showevents. Uninformative structural 635 variations were removed with Trimal (Capella-Gutierrez et al., 2009) with option -gt 636 0.01. 637 To visualize inter-insertion relationships, the MSA was input into IQtree with options -nt 638 AUTO -m MFP -bb 6000 -asr -minsup .95 (Chernomor et al., 2016). This only displays 639 nodes with ultrafast (UF) bootstrap support >0.95. 640 Clusters of >10 insertions sharing a node with UFbootstrap support that were separated 641 from other insertions by internal branch lengths >0.015 (1.5subs / 100 bp) were defined 642 as belonging to a new bona fide LTR7 subfamily (Fig. 1B). 643 LTR7 consensus generation and network analysis: 644 Majority rule (51%) was used to generate each LTR7 subfamily at nodes described in 645 Fig. 1. Positions without majority consensus are listed as “N”. Majority rule consensus 646 sequences were aligned with MUSCLE in SEAVIEW (supp. file 7) (Edgar, 2004; Gouy 647 et al., 2010). Alignment was visualized with Jalview2 (Waterhouse et al., 2009)(Fig. 4A) 648 and ggplot2 (Fig. 4). 649 To visualize inter-insertion relationships, the MSA was input into IQtree with options -nt 638 AUTO -m MFP -bb 6000 -asr -minsup .95 (Chernomor et al., 2016). Methods 622 HERVH LTR sequence identification: 623 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint LTR7 consensus generation and network analysis: 644 Majority rule (51%) was used to generate each LTR7 subfamily at nodes described in 645 Fig. 1. Positions without majority consensus are listed as “N”. Majority rule consensus 646 sequences were aligned with MUSCLE in SEAVIEW (supp. file 7) (Edgar, 2004; Gouy 647 et al., 2010). Alignment was visualized with Jalview2 (Waterhouse et al., 2009)(Fig. 4A) 648 and ggplot2 (Fig. 4). 649 31 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint Non-gap SNPs from the muscle alignment were used to construct a median-joining 650 network (Bandelt et al., 1999) with POPART (Leigh and Bryant, 2015). 651 Reverse Transcriptase Domain extraction, alignment, and tree generation: 652 The reverse transcriptase (RT) domain was extracted from HERVH-int consensus via 653 repbrowser (Fernandes et al., 2020): 654 CACCCTTACCCCGCTCAATGCCAATATCCCATCCCACAGCATGCTTTAAAAGGATT 655 AAAGCCTGTTATCACTCGCCTGCTACAGCATGGCCTTTTAAAGCCTATAAACTCTCC 656 TTACAATTCCCCCATTTTACCTGTCCTAAAACCAGACAAGCCTTACAAGTTAGTTCA 657 GGATCTGTGCCTTATCAACCAAATTGTTTTGCCTATCCACCCCATGGTGCCAAACC 658 CATATACTCTCCTATCCTCAATACCTCCCTCCACAACCCATTATTCTGTTCTGGATC 659 TCAAACATGCTTTCTTTACTATTCCTTTGCACCCTTCATCCCAGCCTCTCTTCGCTTT 660 CACTTGGA 661 This sequence was blated (best hit) against all annotated HERVH-int in the human 662 genome and matches were extracted. Corresponding LTR7 subdivision annotations 663 from figure 1 were matched with these HERVH-int RT domains. Mafft alignment and 664 IQTree generation were done identically to the Mafft and IQTree run for the LTRs (see 665 corresponding methods section). 666 Peak calling: 667 ChIP-seq datasets representing transcription factors (TFs), histone modifications, and 668 regulatory complexes in human embryonic stem cells and differentiated cells were 669 retrieved from GSE61475 (38 distinct TFs and histone modifications), GSE69647 670 (H3K27Ac, POU5F1, MED1 and CTCF), GSE117395 (H3K27Ac, H3K9Me3, KLF4, and 671 KLF17), and GSE78099 (An array of KRAB-ZNFs and TRIM28) (Imbeault et al., 2017). 672 ZNFs enriched in LTR7 binding (ZNF90, ZNF534, ZNF75, ZNF69B, ZNF257, ZNF57, 673 and ZNF101) from HEK293 peaks were all evaluated, but only ZNF90 and ZNF534 674 bound >100 LTR7 insertions (data not shown). LTR7 consensus generation and network analysis: 644 The others were dropped from the 675 Non-gap SNPs from the muscle alignment were used to construct a median-joining 650 network (Bandelt et al., 1999) with POPART (Leigh and Bryant, 2015). 651 Reverse Transcriptase Domain extraction, alignment, and tree generation: 652 The reverse transcriptase (RT) domain was extracted from HERVH-int consensus via 653 repbrowser (Fernandes et al., 2020): 654 The reverse transcriptase (RT) domain was extracted from HERVH-int consensus via 653 repbrowser (Fernandes et al., 2020): 654 CACCCTTACCCCGCTCAATGCCAATATCCCATCCCACAGCATGCTTTAAAAGGATT 655 AAAGCCTGTTATCACTCGCCTGCTACAGCATGGCCTTTTAAAGCCTATAAACTCTCC 656 TTACAATTCCCCCATTTTACCTGTCCTAAAACCAGACAAGCCTTACAAGTTAGTTCA 657 GGATCTGTGCCTTATCAACCAAATTGTTTTGCCTATCCACCCCATGGTGCCAAACC 658 CATATACTCTCCTATCCTCAATACCTCCCTCCACAACCCATTATTCTGTTCTGGATC 659 TCAAACATGCTTTCTTTACTATTCCTTTGCACCCTTCATCCCAGCCTCTCTTCGCTTT 660 CACTTGGA 661 This sequence was blated (best hit) against all annotated HERVH-int in the human 662 genome and matches were extracted. Corresponding LTR7 subdivision annotations 663 from figure 1 were matched with these HERVH-int RT domains. Mafft alignment and 664 IQTree generation were done identically to the Mafft and IQTree run for the LTRs (see 665 corresponding methods section). 666 Peak calling: 667 ChIP-seq datasets representing transcription factors (TFs), histone modifications, and 668 regulatory complexes in human embryonic stem cells and differentiated cells were 669 retrieved from GSE61475 (38 distinct TFs and histone modifications), GSE69647 670 (H3K27Ac, POU5F1, MED1 and CTCF), GSE117395 (H3K27Ac, H3K9Me3, KLF4, and 671 KLF17), and GSE78099 (An array of KRAB-ZNFs and TRIM28) (Imbeault et al., 2017). 672 ZNFs enriched in LTR7 binding (ZNF90, ZNF534, ZNF75, ZNF69B, ZNF257, ZNF57, 673 and ZNF101) from HEK293 peaks were all evaluated, but only ZNF90 and ZNF534 674 bound >100 LTR7 insertions (data not shown). The others were dropped from the 675 analysis. 676 32 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint ChIP-seq reads were aligned to the hg19 human reference genome using the Bowtie2. 677 All reads with phred score less than 33 and PCR duplicates were removed using 678 bowtie2 and Picard tools respectively. ChIP-seq peaks were called by MACS2 with the 679 parameters in “narrow” mode for TFs and “broad” mode for histone modifications, 680 keeping FDR < 1%. ENCODE-defined blacklisted regions were excluded from called 681 peaks. For phyloregulatory analysis (Fig. 2), we then converted hg19 to hg38 (no alt) 682 coordinates via UCSC liftover (100% of coordinates lifted) and intersected these peak 683 with the loci from LTR7 subfamilies using bedtools with any overlap. For ChIP-seq 684 binding enrichment on a subset of marks following motif analysis (Fig 5), 70% overlap of 685 peak and LTR was required. Enrichment of a given TF within LTR7 subfamilies was 686 calculated using enrichR package in R, using the customized in-house codes (see the 687 codes on GitHub for the detailed analysis pipelines and calculation of enrichment 688 score). 689 Phyloregulatory analysis: 690 Peaks from external ChIP-seq datasets were intersected with LTR7 insertions (Quinlan 691 and Hall, 2010). Peak calling: 667 LTR7 insertions that intersected with >1bp of peaks were counted as 692 positive for the respective mark. We repeated this analysis with a range of overlap 693 requirements from extending the LTR 500bp into unique DNA to 70% overlap and found 694 few differential calls (data not shown). The phylogenetic tree rooted on 7b (ggtree) was 695 combined with these binary data (ggheat). 696 “Highly transcribed” (fpkm >2) and “chimeric” HERVH from H1 cells (GSE54726) (Wang 697 et al., 2014) were intersected with LTR7 similarly to ChIP-seq data. Those which 698 intersected LTR7 were marked as “RNA-seq” or “chimeric” respectively. GRO-seq 699 profiles from H1 cells (Estaras et al.) (GSE64758) were created for windows 10bp 700 upstream and 8kb downstream of 5’ and solo LTR7 (Ramírez et al., 2016). The most 701 visible signal was confined to the top 7th of insertions (Figure supplement 2). All LTR7 702 were subdivided into septiles, due to visible signal being confined to the top 7th of 703 insertions; those of the top septile were labeled “GRO-seq”. 704 Peak proportion heatmap generation and statistical analysis: 705 ChIP-seq reads were aligned to the hg19 human reference genome using the Bowtie2. 677 All reads with phred score less than 33 and PCR duplicates were removed using 678 bowtie2 and Picard tools respectively. ChIP-seq peaks were called by MACS2 with the 679 parameters in “narrow” mode for TFs and “broad” mode for histone modifications, 680 keeping FDR < 1%. ENCODE-defined blacklisted regions were excluded from called 681 peaks. For phyloregulatory analysis (Fig. 2), we then converted hg19 to hg38 (no alt) 682 coordinates via UCSC liftover (100% of coordinates lifted) and intersected these peak 683 with the loci from LTR7 subfamilies using bedtools with any overlap. For ChIP-seq 684 binding enrichment on a subset of marks following motif analysis (Fig 5), 70% overlap of 685 peak and LTR was required. Enrichment of a given TF within LTR7 subfamilies was 686 calculated using enrichR package in R, using the customized in-house codes (see the 687 codes on GitHub for the detailed analysis pipelines and calculation of enrichment 688 score). 689 “Highly transcribed” (fpkm >2) and “chimeric” HERVH from H1 cells (GSE54726) (Wang 697 et al., 2014) were intersected with LTR7 similarly to ChIP-seq data. Those which 698 intersected LTR7 were marked as “RNA-seq” or “chimeric” respectively. Peak proportion heatmap generation and statistical analysis: 705 Aggregate signal heatmap generation: 714 GRO-seq (H1 cells - GSE64758), whole-genome bisulfite sequencing (WGBS-seq – H1 715 cells), and H3K9me3 ChIP-seq (H1 – primed - GSE78099) bams were retrieved from 716 (Estarás et al., 2015), (Dunham et al., 2012), and (Theunissen et al., 2016) respectively. 717 Deeptools (Ramírez et al., 2016) was used to visualize these marks by LTR7 subfamily 718 division in windows 10bp upstream and 8kb downstream of the most 5’ position in the 719 LTR (Figure supplement 2). 720 Orthologous insertion aging: 721 Human coordinates for 7b, 7c, and 7y and LTR7 used in alignments and tree generation 722 were lifted over (Kent et al., 2002; Raney et al., 2014) from GRCh38/hg38 (Miga et al., 723 2014) to Clint_PTRv2/panTro6 (Waterson et al., 2005), Kamilah_GGO_v0/gorGor6 724 (Scally et al., 2012), Susie_PABv2/ponAbe3 (Locke et al., 2011), GGSC 725 Nleu3.0/nomLeu3 (Carbone et al., 2014), or Mmul_10/rheMac10 (Gibbs et al., 2007). 726 Those that were successfully lifted over from human to non-human primate were then 727 lifted over back to human. Only those that survived both liftovers (1:1 orthologous) were 728 counted as present in non-human primates. The proportion of those orthologous to 729 human and total number of orthologous was plotted with ggplot2. 730 Terminal branch length aging: 731 Terminal branch lengths from the LTR7 phylogenetic tree (Fig. 1B) were extracted and 732 plotted with ggplot2. Similarly aged subfamilies were inferred from means here and from 733 orthologous insertion aging for statistical testing. Three total groups were tested for 734 Human coordinates for 7b, 7c, and 7y and LTR7 used in alignments and tree generation 722 were lifted over (Kent et al., 2002; Raney et al., 2014) from GRCh38/hg38 (Miga et al., 723 2014) to Clint_PTRv2/panTro6 (Waterson et al., 2005), Kamilah_GGO_v0/gorGor6 724 (Scally et al., 2012), Susie_PABv2/ponAbe3 (Locke et al., 2011), GGSC 725 Nleu3.0/nomLeu3 (Carbone et al., 2014), or Mmul_10/rheMac10 (Gibbs et al., 2007). 726 Those that were successfully lifted over from human to non-human primate were then 727 lifted over back to human. Only those that survived both liftovers (1:1 orthologous) were 728 counted as present in non-human primates. The proportion of those orthologous to 729 human and total number of orthologous was plotted with ggplot2. 730 Peak calling: 667 GRO-seq 699 profiles from H1 cells (Estaras et al.) (GSE64758) were created for windows 10bp 700 upstream and 8kb downstream of 5’ and solo LTR7 (Ramírez et al., 2016). The most 701 visible signal was confined to the top 7th of insertions (Figure supplement 2). All LTR7 702 were subdivided into septiles, due to visible signal being confined to the top 7th of 703 insertions; those of the top septile were labeled “GRO-seq”. 704 Peak proportion heatmap generation and statistical analysis: 705 33 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint Tables with the proportion of solo and 5’ LTRs from a given subfamily positive for select 706 marks (phyloregulatory analysis) were used to generate heatmaps with the R package 707 ggplot (ggheat) (Ginestet, 2011). Those with padj<0.05 (Chi-square Bonferroni 708 correction n=147 tests for a total of 21 marks examined) were considered significantly 709 enriched in 7up1. Enrichment for non-LTR7up subfamilies was not tested. While not all 710 tested marks are displayed in the main text, statistical analysis was performed with all 711 tested marks (n=147) (supp. file 8). For comparing transcribed 7up to untranscribed 712 7up, 18 pairwise comparisons were made (supp. file 9). 713 Terminal branch length aging: 731 Terminal branch lengths from the LTR7 phylogenetic tree (Fig. 1B) were extracted and 732 plotted with ggplot2. Similarly aged subfamilies were inferred from means here and from 733 orthologous insertion aging for statistical testing. Three total groups were tested for 734 34 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint differences in means (7up1/7up2/7u2 vs. 7d1/7d2/7u1 vs. 7bc/o) via Wilcox rank-sum 735 test with Bonferroni multiple testing correction. 736 differences in means (7up1/7up2/7u2 vs. 7d1/7d2/7u1 vs. 7bc/o) via Wilcox rank-sum 735 test with Bonferroni multiple testing correction. 736 Major recombination breakpoints were identified by eye from the consensus sequence 738 MSA, where SNPs and structural rearrangements seemed to have different 739 relationships between blocks. Putative block recombination events were identified by 740 looking for shared shapes in the block consensus MSA (Fig. 4A). To test if these were 741 truly recombination events and could not be explained by evolution by common 742 descent, inter-block sequence relationship differences were tested by generating 743 parsimony trees and comparing these to the overall phylogenetic structure from Fig. 1A. 744 Parsimony trees were generated in SEAVIEW, treating all gaps as unknown states 745 (except in the case of 2b, where the entire sequence is gaps and gaps were not treated 746 differently than other sequence), bootstrapped 5000 times with the option “more 747 thorough tree search”. Differences in block parsimony trees and the overall phylogeny 748 that had bootstrap support were marked in red and included in Fig. 4D,7. 749 7up consensus block 2a 2b alignment and parsimony tree: 750 35 7up consensus block 2a 2b alignment and parsimony tree: 750 LTR7up blocks 2a and 2b (Fig. 4) appeared to share sequence. To determine if block 751 2b was the result of a duplication of 2a, we extracted these sequences from the 752 LTR7up1 consensus and aligned them with blastn (NCBI web version) with default 753 settings. To determine the relationship of all HERVH LTR 2a and 2b blocks, we 754 performed a muscle alignment (default settings) of all 2a and 2b from all HERVH LTR 755 consensus sequences and then generated a parsimony tree with 5000 bootstraps with 756 SEAVIEW with the option “more thorough tree search”. 757 New LTR7B/C/Y consensus generation and remasking of human genome: 758 Consensus sequences for LTR7 subfamilies were generated using the tree from figure 759 1b (see above). For LTR7b/c/y, we used the alignment and tree comprising all HERVH 760 LTR (Figure supplement 5). To do this, we identified nodes with >0.95 ultrafast 761 bootstrap support that were comprised of predominately (>90%) of previously annotated 762 LTR7b, LTR7c, or LTR7y. These sequences were used to generate majority-rule 763 LTR7up blocks 2a and 2b (Fig. 4) appeared to share sequence. To determine if block 751 2b was the result of a duplication of 2a, we extracted these sequences from the 752 LTR7up1 consensus and aligned them with blastn (NCBI web version) with default 753 settings. To determine the relationship of all HERVH LTR 2a and 2b blocks, we 754 performed a muscle alignment (default settings) of all 2a and 2b from all HERVH LTR 755 consensus sequences and then generated a parsimony tree with 5000 bootstraps with 756 SEAVIEW with the option “more thorough tree search”. 757 New LTR7B/C/Y consensus generation and remasking of human genome: 758 Consensus sequences for LTR7 subfamilies were generated using the tree from figure 759 1b (see above). For LTR7b/c/y, we used the alignment and tree comprising all HERVH 760 LTR (Figure supplement 5). To do this, we identified nodes with >0.95 ultrafast 761 bootstrap support that were comprised of predominately (>90%) of previously annotated 762 Consensus sequences for LTR7 subfamilies were generated using the tree from figure 759 1b (see above). For LTR7b/c/y, we used the alignment and tree comprising all HERVH 760 LTR (Figure supplement 5). 7up consensus block 2a 2b alignment and parsimony tree: 750 Both of these subfamilies were merged into “LTR7C” 766 after remasking. 767 Parsing previously annotated LTR7 into 8 subfamilies and evidence of recurrent 768 recombination events caused concern that HERVH LTRs may be misannotated in the 769 repeat masker annotations. To compensate, we remasked (Smit et al., 2013) 770 GRCh38/hg38 (excluding alt chromosomes) with a custom library consisting of the new 771 consensus sequences for LTR7 subfamilies, new consensus sequences for 7b, 7y, and 772 7c (see above) based on the HERVH LTR tree from Fig. 4, and HERVH-int (dfam). We 773 also included annotated consensus sequences from dfam for MER48, MER39, AluYk3, 774 and MST1N2, who we found a HERVH only library also masked to a limited degree 775 (data not shown). With this library, we ran RepeatMasker with crossmatch and 776 “sensitive” settings: -e crossmatch -a -s -no_is. Changes in annotations can be found in 777 (HERVH_LTRremasking.xlsx) 778 7up consensus block 2a 2b alignment and parsimony tree: 750 To do this, we identified nodes with >0.95 ultrafast 761 bootstrap support that were comprised of predominately (>90%) of previously annotated 762 LTR7b, LTR7c, or LTR7y. These sequences were used to generate majority-rule 763 LTR7b, LTR7c, or LTR7y. These sequences were used to generate majority-rule 763 35 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint consensus sequences for their respective subfamily. We generated 2 mutually- 764 exclusive LTR7c consensus sequences (LTR7C1 and LTR7C2) due to the high 765 sequence divergence of LTR7C. Both of these subfamilies were merged into “LTR7C” 766 after remasking. 767 Parsing previously annotated LTR7 into 8 subfamilies and evidence of recurrent 768 recombination events caused concern that HERVH LTRs may be misannotated in the 769 repeat masker annotations. To compensate, we remasked (Smit et al., 2013) 770 GRCh38/hg38 (excluding alt chromosomes) with a custom library consisting of the new 771 consensus sequences for LTR7 subfamilies, new consensus sequences for 7b, 7y, and 772 7c (see above) based on the HERVH LTR tree from Fig. 4, and HERVH-int (dfam). We 773 also included annotated consensus sequences from dfam for MER48, MER39, AluYk3, 774 and MST1N2, who we found a HERVH only library also masked to a limited degree 775 (data not shown). With this library, we ran RepeatMasker with crossmatch and 776 “sensitive” settings: -e crossmatch -a -s -no_is. Changes in annotations can be found in 777 (HERVH_LTRremasking.xlsx) 778 consensus sequences for their respective subfamily. We generated 2 mutually- 764 exclusive LTR7c consensus sequences (LTR7C1 and LTR7C2) due to the high 765 sequence divergence of LTR7C. Both of these subfamilies were merged into “LTR7C” 766 after remasking. 767 consensus sequences for their respective subfamily. We generated 2 mutually- 764 exclusive LTR7c consensus sequences (LTR7C1 and LTR7C2) due to the high 765 sequence divergence of LTR7C. Embryonic HERVH subfamily expression analysis: 779 We calculated differential expression and 802 tested their significance level using Kruskal–Wallis test by comparing cell-types of 803 interest with the rest of the cells. The obtained p-values were further adjusted by the 804 Benjamini-Hochberg method to calculate the False Discovery Rate (FDR). All the 805 statistics and visualization of RNA-seq were performed on R (https://www.r-project.org/). 806 Motif Enrichment: 807 For each subfamily of LTR7 elements, all re-annotated elements were aligned against 808 the subfamily consensus sequence using MUSCLE (Edgar, 2004). These multiple- 809 sequence alignments were then split based on the recombination block positions in the 810 consensus sequence. The consensus sequence was then removed. Binding motif 811 position-weight matricies were downloaded from HOMER (Heinz et al., 2010) and were 812 used to perform pairwise motif enrichment using the command ‘homer2 find’. For 813 LTR7up1 enrichment (Fig. 6A - testing which motifs were enriched in LTR7up1 814 compared to other subfamilies), enrichment was only calculated for LTR7up1 and the 815 motifs with a -log(p-value) cutoff of 1x10-5 were kept. For enrichment in all subfamilies 816 (supp. files 3,4) – testing all subfamilies against all others), every pairwise subfamily 817 combination within each block was tested and all results are displayed. 818 819 SOX2 ChIP-seq signal on LTR7: 820 SOX2 ChIP-seq and whole-cell extract datasets from primed hESCs were downloaded 821 in fastq format from GEO ID GSE125553 (Bayerl et al., 2021). Fastq reads were 822 mapped against the hg19 reference genome with the bowtie2 parameters: –very- 823 subfamilies transcriptome and genome reference sequences using ‘salmon’ (Patro et 794 al., 2017). Finally, we aligned the trimmed sequencing reads against the curated 795 reference genome. The ‘salmon’ tool quantified the counts and normalized expression 796 (Transcripts per million (TPM)) for each single cell RNAseq sample. Overall, this 797 approach enabled us to simultaneously calculate LTR7 subfamilies and protein-coding 798 gene expression using expected maximization algorithms. Data integration of obtained 799 count matrix, normalization at logarithmic scale, and scaling were performed as per the 800 “Seurat V.3.7” (http://satijalab.org/seurat/) guidelines. The annotations of cell-types were 801 taken as it was classified in original studies. We calculated differential expression and 802 tested their significance level using Kruskal–Wallis test by comparing cell-types of 803 interest with the rest of the cells. The obtained p-values were further adjusted by the 804 Benjamini-Hochberg method to calculate the False Discovery Rate (FDR). Embryonic HERVH subfamily expression analysis: 779 All the 805 statistics and visualization of RNA-seq were performed on R (https://www.r-project.org/). 806 Motif Enrichment: 807 For each subfamily of LTR7 elements, all re-annotated elements were aligned against 808 the subfamily consensus sequence using MUSCLE (Edgar, 2004). These multiple- 809 sequence alignments were then split based on the recombination block positions in the 810 consensus sequence. The consensus sequence was then removed. Binding motif 811 position-weight matricies were downloaded from HOMER (Heinz et al., 2010) and were 812 used to perform pairwise motif enrichment using the command ‘homer2 find’. For 813 LTR7up1 enrichment (Fig. 6A - testing which motifs were enriched in LTR7up1 814 compared to other subfamilies), enrichment was only calculated for LTR7up1 and the 815 motifs with a -log(p-value) cutoff of 1x10-5 were kept. For enrichment in all subfamilies 816 (supp. files 3,4) – testing all subfamilies against all others), every pairwise subfamily 817 combination within each block was tested and all results are displayed. 818 819 Embryonic HERVH subfamily expression analysis: 779 We downloaded the raw single-cell RNA-seq datasets from early human embryos and 780 embryonic stem cells (GSE36552) and the EPI, PE, TE cells (GSE66507) in sra format. 781 Following the conversion of raw files into fastq format, the quality was determined by 782 using the FastQC. We removed two nucleotides from the ends as their quality scores 783 were highly variable compared with the rest of the sequences in RNA-seq reads. Prior 784 to aligning the resulting reads, we first curated the reference genome annotations using 785 the LTR7 classification, shown in the manuscript. We extracted the genes (genecode 786 V19), and LTR7 subfamilies (see figure 5) genomic sequences and combined them to 787 generate a reference transcriptome. These sequences were then appended, comprising 788 the coding-sequences plus UTRs of genes and locus-level LTR7 subfamilies sequences 789 in fasta format. We then annotated every fasta sequences with their respective genes or 790 LTR7 subfamilies IDs. To guide the transcriptome assembly, we also appended the 791 each of the resulting contigs and modelled them in gtf format that we utilized for the 792 expression quantification. Next, we indexed the concatenated genes and LTR7 793 36 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint subfamilies transcriptome and genome reference sequences using ‘salmon’ (Patro et 794 al., 2017). Finally, we aligned the trimmed sequencing reads against the curated 795 reference genome. The ‘salmon’ tool quantified the counts and normalized expression 796 (Transcripts per million (TPM)) for each single cell RNAseq sample. Overall, this 797 approach enabled us to simultaneously calculate LTR7 subfamilies and protein-coding 798 gene expression using expected maximization algorithms. Data integration of obtained 799 count matrix, normalization at logarithmic scale, and scaling were performed as per the 800 “Seurat V.3.7” (http://satijalab.org/seurat/) guidelines. The annotations of cell-types were 801 taken as it was classified in original studies. SOX2 ChIP-seq signal on LTR7: 820 SOX2 ChIP-seq and whole-cell extract datasets from primed hESCs were downloaded 821 in fastq format from GEO ID GSE125553 (Bayerl et al., 2021). Fastq reads were 822 mapped against the hg19 reference genome with the bowtie2 parameters: –very- 823 37 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint sensitive-local. All unmapped reads with Phred score < 33 and putative PCR duplicates 824 were removed using Picard and samtools. All the ChIP-seq narrow peaks were called 825 by MACS2 (FDR < 0.01). To generate a set of unique peaks, we merged ChIP-seq 826 peaks within 50 bp of one another using the mergeBed function from bedtools. We then 827 intersected these peak sets with LTR7 subgroups from hg19 repeat-masked 828 coordinates using bedtools intersectBed with 50% overlap. LTR7up1 and LTR7up2 829 were harboring the highest number of peaks compared with the rest of the subgroups. 830 To illustrate the enrichment over the LTR7 subgroups, we first extended 500 basepairs 831 from upstream and downstream coordinates from the left boundary of each 832 LTR7subgroups. These 1KB windows were further divided into 10 bps bins. The 833 normalized ChIP-seq signal over the local lambda (piled up bedGraph outputs from 834 MACS2) was counted in each bin. These counts were then normalized by the total 835 number of mappable reads per million in given samples and presented as signal per 836 million per 10 bps. Finally, these values were averaged across the loci for each bin to 837 illustrate the subfamilies' level of ChIP-seq enrichment. Replicates were merged prior to 838 plotting. Note: Pearson's correlation coefficient between replicates across the bins was 839 found to be r > 0.90. 840 Luciferase reporter assay: 842 Transfection efficiency and cell count was normalized with GFP. 856 1. 7down: 857 GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 2. LTR7upcons: 866 GCTAGCTGTCAGGCCTCTGAGCCCAAGCCAAGCCATCGCATCCCCTGTGACTTGC 867 ACGTATACGCCCAGATGGCCTGAAGTAACTGAAGAATCACAAAAGAAGTGAATATG 868 CCCTGCCCCACCTTAACTGATGACATTCCACCACAAAAGAAGTGTAAATGGCCGGT 869 CCTTGCCTTAAGTGATGACATTACCTTGTGAAAGTCCTTTTCCTGGCTCATCCTGGC 870 TCAAAAAGCACCCCCACTGAGCACCTTGCGACCCCCACTCCTGCCCGCCAGAGAA 871 CAAACCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAATCCTATAAAACGGCCCC 872 ACCCTTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAGCCCGCCTGCACCCAGGT 873 transfection, cell pellet was harvested and luciferase activity was measured with 854 Luciferase Reporter Assay kit (Promega) on Glomax (Promega) according to 855 instructions. Transfection efficiency and cell count was normalized with GFP. 856 transfection, cell pellet was harvested and luciferase activity was measured with 854 Luciferase Reporter Assay kit (Promega) on Glomax (Promega) according to 855 instructions. Transfection efficiency and cell count was normalized with GFP. 856 GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 Luciferase reporter assay: 842 The inserts (LTR7 variants or EF1a promoter) with restriction enzyme overhangs were 843 ordered from Genewiz and cloned into pGL3-basic plasmid upstream of the firefly 844 reporter gene (E1751, Promega). Minipreps were prepared with QIAprep Spin Miniprep 845 kit (Qiagen). Plasmids were sequenced to ensure the correct sequence and 846 directionality of the insert. 24 h before transfection, human iPSC WTC-11 (Coriell 847 Institute) cells were plated on Vitronectin (Thermo Fisher Scientific) coated 12-well 848 plates in Essential 8 Flex medium (Thermo Fisher Scientific) with E8 supplement 849 (Thermo Fisher Scientific), Rock inhibitor and 2.5% penicillin-streptomycin. Cells were 850 co-transfected with 800 ng of plasmid of interest and 150 ng plasmid containing EF1a 851 upstream of GFP for normalization with Lipofectamine Stem transfection reagent 852 (Thermo Fisher scientific) according to manufacturer’s instructions. 48 h after 853 38 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint transfection, cell pellet was harvested and luciferase activity was measured with 854 Luciferase Reporter Assay kit (Promega) on Glomax (Promega) according to 855 instructions. Transfection efficiency and cell count was normalized with GFP. 856 1. 7down: 857 GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 2. LTR7upcons: 866 GCTAGCTGTCAGGCCTCTGAGCCCAAGCCAAGCCATCGCATCCCCTGTGACTTGC 867 ACGTATACGCCCAGATGGCCTGAAGTAACTGAAGAATCACAAAAGAAGTGAATATG 868 CCCTGCCCCACCTTAACTGATGACATTCCACCACAAAAGAAGTGTAAATGGCCGGT 869 CCTTGCCTTAAGTGATGACATTACCTTGTGAAAGTCCTTTTCCTGGCTCATCCTGGC 870 TCAAAAAGCACCCCCACTGAGCACCTTGCGACCCCCACTCCTGCCCGCCAGAGAA 871 CAAACCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAATCCTATAAAACGGCCCC 872 ACCCTTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAGCCCGCCTGCACCCAGGT 873 GAAATAAACAGCCATGTTGCTCACACAAAGCCTGTTTGGTGGTCTCTTCACACGGA 874 CGCGCATGCTCGAG 875 5. LTR7upcons_AAAGAAG_deletion: 876 GCTAGCTGTCAGGCCTCTGAGCCCAAGCCAAGCCATCGCATCCCCTGTGACTTGC 877 ACGTATACGCCCAGATGGCCTGAAGTAACTGAAGAATCACAAAAGAAGTGAATATG 878 CCCTGCCCCACCTTAACTGATGACATTCCACCATTGTAAATGGCCGGTCCTTGCCT 879 TAAGTGATGACATTACCTTGTGAAAGTCCTTTTCCTGGCTCATCCTGGCTCAAAAAG 880 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whic this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint transfection, cell pellet was harvested and luciferase activity was measured with 854 Luciferase Reporter Assay kit (Promega) on Glomax (Promega) according to 855 instructions. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 GCTAGCTGTCAGGCCTCTGAGCCCAAGCCAAGCCATCGCATCCCCTGTGACTTGC 867 ACGTATACGCCCAGATGGCCTGAAGTAACTGAAGAATCACAAAAGAAGTGAATATG 868 CCCTGCCCCACCTTAACTGATGACATTCCACCACAAAAGAAGTGTAAATGGCCGGT 869 CCTTGCCTTAAGTGATGACATTACCTTGTGAAAGTCCTTTTCCTGGCTCATCCTGGC 870 TCAAAAAGCACCCCCACTGAGCACCTTGCGACCCCCACTCCTGCCCGCCAGAGAA 871 CAAACCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAATCCTATAAAACGGCCCC 872 ACCCTTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAGCCCGCCTGCACCCAGGT 873 GAAATAAACAGCCATGTTGCTCACACAAAGCCTGTTTGGTGGTCTCTTCACACGGA 874 CGCGCATGCTCGAG 875 39 . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint CACCCCCACTGAGCACCTTGCGACCCCCACTCCTGCCCGCCAGAGAACAAACCCC 881 CTTTGACTGTAATTTTCCTTTACCTACCCAAATCCTATAAAACGGCCCCACCCTTAT 882 CTCCCTTCGCTGACTCTCTTTTCGGACTCAGCCCGCCTGCACCCAGGTGAAATAAA 883 CAGCCATGTTGCTCACACAAAGCCTGTTTGGTGGTCTCTTCACACGGACGCGCAT 884 GCTCGAG 885 5'NheI highlighted in Yellow 886 3'XhoI highlighted in Cyan 887 CACCCCCACTGAGCACCTTGCGACCCCCACTCCTGCCCGCCAGAGAACAAACCCC 881 CTTTGACTGTAATTTTCCTTTACCTACCCAAATCCTATAAAACGGCCCCACCCTTAT 882 CTCCCTTCGCTGACTCTCTTTTCGGACTCAGCCCGCCTGCACCCAGGTGAAATAAA 883 CAGCCATGTTGCTCACACAAAGCCTGTTTGGTGGTCTCTTCACACGGACGCGCAT 884 GCTCGAG 885 40 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint Babaian A, Mager DL. 2016. Endogenous retroviral promoter exaptation in human cancer. Mobile DNA 888 7:24. doi:10.1186/s13100-016-0080-x 889 Bailly-Bechet M, Haudry A, Lerat E. 2014. “One code to find them all”: a perl tool to conveniently parse 890 RepeatMasker output files. Mobile DNA 5:13. doi:10.1186/1759-8753-5-13 891 Bandelt HJ, Forster P, Röhl A. 1999. Median-joining networks for inferring intraspecific phylogenies. 892 Molecular Biology and Evolution 16:37–48. doi:10.1093/oxfordjournals.molbev.a026036 893 Bannert N, Kurth R. 2004. Retroelements and the human genome: New perspectives on an old relation. 894 PNAS 101:14572–14579. doi:10.1073/pnas.0404838101 895 Bao W, Kojima KK, Kohany O. 2015. Repbase Update, a database of repetitive elements in eukaryotic 896 genomes. Mobile DNA 6:11. doi:10.1186/s13100-015-0041-9 897 Bao W, Kojima KK, Kohany O. 2015. Repbase Update, a database of repetitive elements in eukaryotic 896 y , yy , , , , , , g j , , Amir H, Sheban D, Geula S, Mor N, Weinberger L, Naveh Tassa S, Krupalnik V, Oldak B, Livnat N, Tarazi S, 899 Tawil S, Wildschutz E, Ashouokhi S, Lasman L, Rotter V, Hanna S, Ben-Yosef D, Novershtern N, Viukov S, 900 Hanna JH. 2021. Principles of signaling pathway modulation for enhancing human naive pluripotency 901 induction. Cell Stem Cell S1934-5909(21)00158–2. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 doi:10.1016/j.stem.2021.04.001 902 Amir H, Sheban D, Geula S, Mor N, Weinberger L, Naveh Tassa S, Krupalnik V, Oldak B, Livnat N, Tarazi S, 899 , , , , , , , , , Hanna JH. 2021. Principles of signaling pathway modulation for enhancing human naive pluripotency 901 induction. Cell Stem Cell S1934-5909(21)00158–2. doi:10.1016/j.stem.2021.04.001 902 Bergsland M, Ramsköld D, Zaouter C, Klum S, Sandberg R, Muhr J. 2011. Sequentially acting Sox 903 transcription factors in neural lineage development. Genes Dev 25:2453–2464. 904 d i 10 1101/ d 176008 111 905 Blakeley P, Fogarty NME, del Valle I, Wamaitha SE, Hu TX, Elder K, Snell P, Christie L, Robson P, Niakan 906 KK. 2015. Defining the three cell lineages of the human blastocyst by single-cell RNA-seq. Development 907 142:3151–3165. doi:10.1242/dev.123547 908 Boyer LA, Lee TI, Cole MF, Johnstone SE, Levine SS, Zucker JP, Guenther MG, Kumar RM, Murray HL, 909 Jenner RG, Gifford DK, Melton DA, Jaenisch R, Young RA. 2005. Core Transcriptional Regulatory Circuitry 910 in Human Embryonic Stem Cells. Cell 122:947–956. doi:10.1016/j.cell.2005.08.020 911 Jenner RG, Gifford DK, Melton DA, Jaenisch R, Young RA. 2005. Core Transcriptional Regulatory Circuitry 910 in Human Embryonic Stem Cells. Cell 122:947–956. doi:10.1016/j.cell.2005.08.020 911 Bruno M, Mahgoub M, Macfarlan TS. 2019. The Arms Race Between KRAB–Zinc Finger Proteins and 912 Endogenous Retroelements and Its Impact on Mammals. Annual Review of Genetics 53:393–416. 913 doi:10.1146/annurev-genet-112618-043717 914 Capella-Gutierrez S, Silla-Martinez JM, Gabaldon T. 2009. trimAl: a tool for automated alignment 915 trimming in large-scale phylogenetic analyses. Bioinformatics 25:1972–1973. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 916 doi:10.1093/bioinformatics/btp348 917 Carbone L, Alan Harris R, Gnerre S, Veeramah KR, Lorente-Galdos B, Huddleston J, Meyer TJ, Herrero J, 918 Roos C, Aken B, Anaclerio F, Archidiacono N, Baker C, Barrell D, Batzer MA, Beal K, Blancher A, Bohrson 919 CL, Brameier M, Campbell MS, Capozzi O, Casola C, Chiatante G, Cree A, Damert A, de Jong PJ, Dumas L, 920 Fernandez-Callejo M, Flicek P, Fuchs NV, Gut I, Gut M, Hahn MW, Hernandez-Rodriguez J, Hillier LW, 921 Hubley R, Ianc B, Izsvák Z, Jablonski NG, Johnstone LM, Karimpour-Fard A, Konkel MK, Kostka D, Lazar 922 NH, Lee SL, Lewis LR, Liu Y, Locke DP, Mallick S, Mendez FL, Muffato M, Nazareth LV, Nevonen KA, 923 O’Bleness M, Ochis C, Odom DT, Pollard KS, Quilez J, Reich D, Rocchi M, Schumann GG, Searle S, Sikela 924 JM, Skollar G, Smit A, Sonmez K, Hallers B ten, Terhune E, Thomas GWC, Ullmer B, Ventura M, Walker JA, 925 Bruno M, Mahgoub M, Macfarlan TS. 2019. The Arms Race Between KRAB–Zinc Finger Proteins and 912 Endogenous Retroelements and Its Impact on Mammals. Annual Review of Genetics 53:393–416. 913 doi:10.1146/annurev-genet-112618-043717 914 41 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint Wall JD, Walter L, Ward MC, Wheelan SJ, Whelan CW, White S, Wilhelm LJ, Woerner AE, Yandell M, Zhu 926 B, Hammer MF, Marques-Bonet T, Eichler EE, Fulton L, Fronick C, Muzny DM, Warren WC, Worley KC, 927 Rogers J, Wilson RK, Gibbs RA. 2014. Gibbon genome and the fast karyotype evolution of small apes. 928 Nature 513:195–201. doi:10.1038/nature13679 929 Chambers I, Smith A. 2004. Self-renewal of teratocarcinoma and embryonic stem cells. Oncogene 930 23:7150–7160. doi:10.1038/sj.onc.1207930 931 Chang N-C, Rovira Q, Wells JN, Feschotte C, Vaquerizas JM. 2021. A genomic portrait of zebrafish 932 transposable elements and their spatiotemporal embryonic expression. bioRxiv 2021.04.08.439009. 933 doi:10.1101/2021.04.08.439009 934 Charlesworth B, Langley CH. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 1986. THE EVOLUTION OF SELF-REGULATED TRANSPOSITION OF 935 TRANSPOSABLE ELEMENTS. Genetics 112:359–383. 936 Chernomor O, von Haeseler A, Minh BQ. 2016. Terrace Aware Data Structure for Phylogenomic 937 Inference from Supermatrices. Systematic Biology 65:997–1008. doi:10.1093/sysbio/syw037 938 Chuong EB, Elde NC, Feschotte C. 2017. Regulatory activities of transposable elements: from conflicts to 939 benefits. Nat Rev Genet 18:71–86. doi:10.1038/nrg.2016.139 940 Chuong EB, Elde NC, Feschotte C. 2016. Regulatory evolution of innate immunity through co-option of 941 endogenous retroviruses. Science 351:1083–1087. doi:10.1126/science.aad5497 942 Cordaux R, Hedges DJ, Batzer MA. 2004. Retrotransposition of Alu elements: how many sources? Trends 943 in Genetics 20:464–467. doi:10.1016/j.tig.2004.07.012 944 Corsinotti A, Wong FC, Tatar T, Szczerbinska I, Halbritter F, Colby D, Gogolok S, Pantier R, Liggat K, 945 Mirfazeli ES, Hall-Ponsele E, Mullin NP, Wilson V, Chambers I. 2017. Distinct SoxB1 networks are 946 required for naïve and primed pluripotency. eLife 6:e27746. doi:10.7554/eLife.27746 947 Corsinotti A, Wong FC, Tatar T, Szczerbinska I, Halbritter F, Colby D, Gogolok S, Pantier R, Liggat K, 945 Mirfazeli ES, Hall-Ponsele E, Mullin NP, Wilson V, Chambers I. 2017. Distinct SoxB1 networks are 946 required for naïve and primed pluripotency. eLife 6:e27746. doi:10.7554/eLife.27746 947 Cosby RL, Chang N-C, Feschotte C. 2019. Host–transposon interactions: conflict, cooperation, and 948 cooption. Genes Dev 33:1098–1116. doi:10.1101/gad.327312.119 949 Deniz Ö, Ahmed M, Todd CD, Rio-Machin A, Dawson MA, Branco MR. 2020. Endogenous retroviruses are 950 a source of enhancers with oncogenic potential in acute myeloid leukaemia. Nat Commun 11:3506. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 951 doi:10.1038/s41467-020-17206-4 952 Dunham I, Kundaje A, Aldred SF, Collins PJ, Davis CA, Doyle F, Epstein CB, Frietze S, Harrow J, Kaul R, 953 Khatun J, Lajoie BR, Landt SG, Lee B-K, Pauli F, Rosenbloom KR, Sabo P, Safi A, Sanyal A, Shoresh N, 954 Simon JM, Song L, Trinklein ND, Altshuler RC, Birney E, Brown JB, Cheng C, Djebali S, Dong X, Dunham I, 955 Ernst J, Furey TS, Gerstein M, Giardine B, Greven M, Hardison RC, Harris RS, Herrero J, Hoffman MM, 956 Iyer S, Kellis M, Khatun J, Kheradpour P, Kundaje A, Lassmann T, Li Q, Lin X, Marinov GK, Merkel A, 957 Mortazavi A, Parker SCJ, Reddy TE, Rozowsky J, Schlesinger F, Thurman RE, Wang J, Ward LD, Whitfield 958 TW, Wilder SP, Wu W, Xi HS, Yip KY, Zhuang J, Bernstein BE, Birney E, Dunham I, Green ED, Gunter C, 959 Snyder M, Pazin MJ, Lowdon RF, Dillon LAL, Adams LB, Kelly CJ, Zhang J, Wexler JR, Green ED, Good PJ, 960 Feingold EA, Bernstein BE, Birney E, Crawford GE, Dekker J, Elnitski L, Farnham PJ, Gerstein M, Giddings 961 MC, Gingeras TR, Green ED, Guigó R, Hardison RC, Hubbard TJ, Kellis M, Kent WJ, Lieb JD, Margulies EH, 962 Myers RM, Snyder M, Stamatoyannopoulos JA, Tenenbaum SA, Weng Z, White KP, Wold B, Khatun J, Yu 963 Cosby RL, Chang N-C, Feschotte C. 2019. Host–transposon interactions: conflict, cooperation, and 948 cooption. Genes Dev 33:1098–1116. doi:10.1101/gad.327312.119 949 Cosby RL, Chang N-C, Feschotte C. 2019. Host–transposon interactions: conflict, cooperation, and 948 cooption. Genes Dev 33:1098–1116. doi:10.1101/gad.327312.119 949 Deniz Ö, Ahmed M, Todd CD, Rio-Machin A, Dawson MA, Branco MR. 2020. Endogenous retroviruses are 950 a source of enhancers with oncogenic potential in acute myeloid leukaemia. Nat Commun 11:3506. 951 doi:10.1038/s41467-020-17206-4 952 Deniz Ö, Ahmed M, Todd CD, Rio-Machin A, Dawson MA, Branco MR. 2020. Endogenous retroviruses are 950 a source of enhancers with oncogenic potential in acute myeloid leukaemia. Nat Commun 11:3506. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint Y, Wrobel J, Risk BA, Gunawardena HP, Kuiper HC, Maier CW, Xie L, Chen X, Giddings MC, Bernstein BE, 964 Epstein CB, Shoresh N, Ernst J, Kheradpour P, Mikkelsen TS, Gillespie S, Goren A, Ram O, Zhang X, Wang 965 L, Issner R, Coyne MJ, Durham T, Ku M, Truong T, Ward LD, Altshuler RC, Eaton ML, Kellis M, Djebali S, 966 Davis CA, Merkel A, Dobin A, Lassmann T, Mortazavi A, Tanzer A, Lagarde J, Lin W, Schlesinger F, Xue C, 967 Marinov GK, Khatun J, Williams BA, Zaleski C, Rozowsky J, Röder M, Kokocinski F, Abdelhamid RF, Alioto 968 T, Antoshechkin I, Baer MT, Batut P, Bell I, Bell K, Chakrabortty S, Chen X, Chrast J, Curado J, Derrien T, 969 Drenkow J, Dumais E, Dumais J, Duttagupta R, Fastuca M, Fejes-Toth K, Ferreira P, Foissac S, Fullwood 970 MJ, Gao H, Gonzalez D, Gordon A, Gunawardena HP, Howald C, Jha S, Johnson R, Kapranov P, King B, 971 Kingswood C, Li G, Luo OJ, Park E, Preall JB, Presaud K, Ribeca P, Risk BA, Robyr D, Ruan X, Sammeth M, 972 Sandhu KS, Schaeffer L, See L-H, Shahab A, Skancke J, Suzuki AM, Takahashi H, Tilgner H, Trout D, 973 Walters N, Wang H, Wrobel J, Yu Y, Hayashizaki Y, Harrow J, Gerstein M, Hubbard TJ, Reymond A, 974 Antonarakis SE, Hannon GJ, Giddings MC, Ruan Y, Wold B, Carninci P, Guigó R, Gingeras TR, Rosenbloom 975 KR, Sloan CA, Learned K, Malladi VS, Wong MC, Barber GP, Cline MS, Dreszer TR, Heitner SG, Karolchik D, 976 Kent WJ, Kirkup VM, Meyer LR, Long JC, Maddren M, Raney BJ, Furey TS, Song L, Grasfeder LL, Giresi PG, 977 Lee B-K, Battenhouse A, Sheffield NC, Simon JM, Showers KA, Safi A, London D, Bhinge AA, Shestak C, 978 Schaner MR, Ki Kim S, Zhang ZZ, Mieczkowski PA, Mieczkowska JO, Liu Z, McDaniell RM, Ni Y, Rashid NU, 979 Kim MJ, Adar S, Zhang Z, Wang T, Winter D, Keefe D, Birney E, Iyer VR, Lieb JD, Crawford GE, Li G, 980 Sandhu KS, Zheng M, Wang P, Luo OJ, Shahab A, Fullwood MJ, Ruan X, Ruan Y, Myers RM, Pauli F, 981 Williams BA, Gertz J, Marinov GK, Reddy TE, Vielmetter J, Partridge E, Trout D, Varley KE, Gasper C, The 982 ENCODE Project Consortium, Overall coordination (data analysis coordination), Data production leads 983 (data production), Lead analysts (data analysis), Writing group, NHGRI project management (scientific 984 management), Principal investigators (steering committee), Boise State University and University of 985 North Carolina at Chapel Hill Proteomics groups (data production and analysis), Broad Institute Group 986 (data production and analysis), Cold Spring Harbor U of G Center for Genomic Regulation, Barcelona, 987 RIKEN, Sanger Institute, University of Lausanne, Genome Institute of Singapore group (data production 988 and analysis), Data coordination center at UC Santa Cruz (production data coordination), Duke 989 University E University of Texas, Austin, University of North Carolina-Chapel Hill group (data production 990 and analysis), Genome Institute of Singapore group (data production and analysis), HudsonAlpha 991 Institute C UC Irvine, Stanford group (data production and analysis). GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 951 doi:10.1038/s41467-020-17206-4 952 Dunham I, Kundaje A, Aldred SF, Collins PJ, Davis CA, Doyle F, Epstein CB, Frietze S, Harrow J, Kaul R, 953 Khatun J, Lajoie BR, Landt SG, Lee B-K, Pauli F, Rosenbloom KR, Sabo P, Safi A, Sanyal A, Shoresh N, 954 Simon JM, Song L, Trinklein ND, Altshuler RC, Birney E, Brown JB, Cheng C, Djebali S, Dong X, Dunham I, 955 Ernst J, Furey TS, Gerstein M, Giardine B, Greven M, Hardison RC, Harris RS, Herrero J, Hoffman MM, 956 Iyer S, Kellis M, Khatun J, Kheradpour P, Kundaje A, Lassmann T, Li Q, Lin X, Marinov GK, Merkel A, 957 Mortazavi A, Parker SCJ, Reddy TE, Rozowsky J, Schlesinger F, Thurman RE, Wang J, Ward LD, Whitfield 958 TW, Wilder SP, Wu W, Xi HS, Yip KY, Zhuang J, Bernstein BE, Birney E, Dunham I, Green ED, Gunter C, 959 Snyder M, Pazin MJ, Lowdon RF, Dillon LAL, Adams LB, Kelly CJ, Zhang J, Wexler JR, Green ED, Good PJ, 960 Feingold EA, Bernstein BE, Birney E, Crawford GE, Dekker J, Elnitski L, Farnham PJ, Gerstein M, Giddings 961 MC, Gingeras TR, Green ED, Guigó R, Hardison RC, Hubbard TJ, Kellis M, Kent WJ, Lieb JD, Margulies EH, 962 Myers RM, Snyder M, Stamatoyannopoulos JA, Tenenbaum SA, Weng Z, White KP, Wold B, Khatun J, Yu 963 Dunham I, Kundaje A, Aldred SF, Collins PJ, Davis CA, Doyle F, Epstein CB, Frietze S, Harrow J, Kaul R, 953 Khatun J, Lajoie BR, Landt SG, Lee B-K, Pauli F, Rosenbloom KR, Sabo P, Safi A, Sanyal A, Shoresh N, 954 Simon JM, Song L, Trinklein ND, Altshuler RC, Birney E, Brown JB, Cheng C, Djebali S, Dong X, Dunham I, 955 Ernst J, Furey TS, Gerstein M, Giardine B, Greven M, Hardison RC, Harris RS, Herrero J, Hoffman MM, 956 Iyer S, Kellis M, Khatun J, Kheradpour P, Kundaje A, Lassmann T, Li Q, Lin X, Marinov GK, Merkel A, 957 Mortazavi A, Parker SCJ, Reddy TE, Rozowsky J, Schlesinger F, Thurman RE, Wang J, Ward LD, Whitfield 958 TW, Wilder SP, Wu W, Xi HS, Yip KY, Zhuang J, Bernstein BE, Birney E, Dunham I, Green ED, Gunter C, 959 Snyder M, Pazin MJ, Lowdon RF, Dillon LAL, Adams LB, Kelly CJ, Zhang J, Wexler JR, Green ED, Good PJ, 960 Feingold EA, Bernstein BE, Birney E, Crawford GE, Dekker J, Elnitski L, Farnham PJ, Gerstein M, Giddings 961 MC, Gingeras TR, Green ED, Guigó R, Hardison RC, Hubbard TJ, Kellis M, Kent WJ, Lieb JD, Margulies EH, 962 Myers RM, Snyder M, Stamatoyannopoulos JA, Tenenbaum SA, Weng Z, White KP, Wold B, Khatun J, Yu 963 42 . GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 2012. An integrated encyclopedia of 992 DNA elements in the human genome. Nature 489:57–74. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 doi:10.1038/nature11247 993 Y, Wrobel J, Risk BA, Gunawardena HP, Kuiper HC, Maier CW, Xie L, Chen X, Giddings MC, Bernstein BE, 964 Epstein CB, Shoresh N, Ernst J, Kheradpour P, Mikkelsen TS, Gillespie S, Goren A, Ram O, Zhang X, Wang 965 L, Issner R, Coyne MJ, Durham T, Ku M, Truong T, Ward LD, Altshuler RC, Eaton ML, Kellis M, Djebali S, 966 Davis CA, Merkel A, Dobin A, Lassmann T, Mortazavi A, Tanzer A, Lagarde J, Lin W, Schlesinger F, Xue C, 967 Marinov GK, Khatun J, Williams BA, Zaleski C, Rozowsky J, Röder M, Kokocinski F, Abdelhamid RF, Alioto 968 T, Antoshechkin I, Baer MT, Batut P, Bell I, Bell K, Chakrabortty S, Chen X, Chrast J, Curado J, Derrien T, 969 Drenkow J, Dumais E, Dumais J, Duttagupta R, Fastuca M, Fejes-Toth K, Ferreira P, Foissac S, Fullwood 970 MJ, Gao H, Gonzalez D, Gordon A, Gunawardena HP, Howald C, Jha S, Johnson R, Kapranov P, King B, 971 Kingswood C, Li G, Luo OJ, Park E, Preall JB, Presaud K, Ribeca P, Risk BA, Robyr D, Ruan X, Sammeth M, 972 Sandhu KS, Schaeffer L, See L-H, Shahab A, Skancke J, Suzuki AM, Takahashi H, Tilgner H, Trout D, 973 Walters N, Wang H, Wrobel J, Yu Y, Hayashizaki Y, Harrow J, Gerstein M, Hubbard TJ, Reymond A, 974 Antonarakis SE, Hannon GJ, Giddings MC, Ruan Y, Wold B, Carninci P, Guigó R, Gingeras TR, Rosenbloom 975 KR, Sloan CA, Learned K, Malladi VS, Wong MC, Barber GP, Cline MS, Dreszer TR, Heitner SG, Karolchik D, 976 Kent WJ, Kirkup VM, Meyer LR, Long JC, Maddren M, Raney BJ, Furey TS, Song L, Grasfeder LL, Giresi PG, 977 Lee B-K, Battenhouse A, Sheffield NC, Simon JM, Showers KA, Safi A, London D, Bhinge AA, Shestak C, 978 Schaner MR, Ki Kim S, Zhang ZZ, Mieczkowski PA, Mieczkowska JO, Liu Z, McDaniell RM, Ni Y, Rashid NU, 979 Kim MJ, Adar S, Zhang Z, Wang T, Winter D, Keefe D, Birney E, Iyer VR, Lieb JD, Crawford GE, Li G, 980 Sandhu KS, Zheng M, Wang P, Luo OJ, Shahab A, Fullwood MJ, Ruan X, Ruan Y, Myers RM, Pauli F, 981 Williams BA, Gertz J, Marinov GK, Reddy TE, Vielmetter J, Partridge E, Trout D, Varley KE, Gasper C, The 982 ENCODE Project Consortium, Overall coordination (data analysis coordination), Data production leads 983 (data production), Lead analysts (data analysis), Writing group, NHGRI project management (scientific 984 management), Principal investigators (steering committee), Boise State University and University of 985 North Carolina at Chapel Hill Proteomics groups (data production and analysis), Broad Institute Group 986 (data production and analysis), Cold Spring Harbor U of G Center for Genomic Regulation, Barcelona, 987 RIKEN, Sanger Institute, University of Lausanne, Genome Institute of Singapore group (data production 988 and analysis), Data coordination center at UC Santa Cruz (production data coordination), Duke 989 University E University of Texas, Austin, University of North Carolina-Chapel Hill group (data production 990 and analysis), Genome Institute of Singapore group (data production and analysis), HudsonAlpha 991 Institute C UC Irvine, Stanford group (data production and analysis). GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 1015 doi:10.1038/ng.2965 1016 Gafni O, Weinberger L, Mansour AA, Manor YS, Chomsky E, Ben-Yosef D, Kalma Y, Viukov S, Maza I, 1017 Zviran A, Rais Y, Shipony Z, Mukamel Z, Krupalnik V, Zerbib M, Geula S, Caspi I, Schneir D, Shwartz T, 1018 Gilad S Amann Zalcenstein D Benjamin S Amit I Tanay A Massarwa R Novershtern N Hanna JH 2013 1019 Hume DA, Orlando V, Grimmond SM, Carninci P. 2009. The regulated retrotransposon transcriptome of 1005 mammalian cells. Nat Genet 41:563–571. doi:10.1038/ng.368 1006 Fernandes JD, Zamudio-Hurtado A, Clawson H, Kent WJ, Haussler D, Salama SR, Haeussler M. 2020. The 1007 UCSC repeat browser allows discovery and visualization of evolutionary conflict across repeat families. 1008 Mobile DNA 11:13. doi:10.1186/s13100-020-00208-w 1009 Feschotte C. 2008. Transposable elements and the evolution of regulatory networks. Nat Rev Genet 1010 9:397–405. doi:10.1038/nrg2337 1011 Fort A, Hashimoto K, Yamada D, Salimullah M, Keya CA, Saxena A, Bonetti A, Voineagu I, Bertin N, Kratz 1012 A, Noro Y, Wong C-H, de Hoon M, Andersson R, Sandelin A, Suzuki H, Wei C-L, Koseki H, Hasegawa Y, 1013 Forrest ARR, Carninci P. 2014. Deep transcriptome profiling of mammalian stem cells supports a 1014 regulatory role for retrotransposons in pluripotency maintenance. Nature Genetics 46:558–566. 1015 doi:10.1038/ng.2965 1016 Gafni O, Weinberger L, Mansour AA, Manor YS, Chomsky E, Ben-Yosef D, Kalma Y, Viukov S, Maza I, 1017 Zviran A, Rais Y, Shipony Z, Mukamel Z, Krupalnik V, Zerbib M, Geula S, Caspi I, Schneir D, Shwartz T, 1018 Gilad S, Amann-Zalcenstein D, Benjamin S, Amit I, Tanay A, Massarwa R, Novershtern N, Hanna JH. 2013. 1019 Derivation of novel human ground state naive pluripotent stem cells. Nature 504:282–286. 1020 doi:10.1038/nature12745 1021 Gemmell P, Hein J, Katzourakis A. 2019. The Exaptation of HERV-H: Evolutionary Analyses Reveal the 1022 Genomic Features of Highly Transcribed Elements. Front Immunol 10. doi:10.3389/fimmu.2019.01339 1023 Gemmell P, Hein J, Katzourakis A. 2015. Orthologous endogenous retroviruses exhibit directional 1024 selection since the chimp-human split. Retrovirology 12. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 2012. An integrated encyclopedia of 992 DNA elements in the human genome. Nature 489:57–74. doi:10.1038/nature11247 993 Estarás C, Benner C, Jones KA. 2015. SMADs and YAP Compete to Control Elongation of β-Catenin:LEF-1- 1000 Recruited RNAPII during hESC Differentiation. Molecular Cell 58:780–793. 1001 doi:10.1016/j.molcel.2015.04.001 1002 Faulkner GJ, Kimura Y, Daub CO, Wani S, Plessy C, Irvine KM, Schroder K, Cloonan N, Steptoe AL, 1003 Lassmann T, Waki K, Hornig N, Arakawa T, Takahashi H, Kawai J, Forrest ARR, Suzuki H, Hayashizaki Y, 1004 Faulkner GJ, Kimura Y, Daub CO, Wani S, Plessy C, Irvine KM, Schroder K, Cloonan N, Steptoe AL, 1003 Lassmann T, Waki K, Hornig N, Arakawa T, Takahashi H, Kawai J, Forrest ARR, Suzuki H, Hayashizaki Y, 1004 43 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint Hume DA, Orlando V, Grimmond SM, Carninci P. 2009. The regulated retrotransposon transcriptome of 1005 mammalian cells. Nat Genet 41:563–571. doi:10.1038/ng.368 1006 Fernandes JD, Zamudio-Hurtado A, Clawson H, Kent WJ, Haussler D, Salama SR, Haeussler M. 2020. The 1007 UCSC repeat browser allows discovery and visualization of evolutionary conflict across repeat families. 1008 Mobile DNA 11:13. doi:10.1186/s13100-020-00208-w 1009 Feschotte C. 2008. Transposable elements and the evolution of regulatory networks. Nat Rev Genet 1010 9:397–405. doi:10.1038/nrg2337 1011 Fort A, Hashimoto K, Yamada D, Salimullah M, Keya CA, Saxena A, Bonetti A, Voineagu I, Bertin N, Kratz 1012 A, Noro Y, Wong C-H, de Hoon M, Andersson R, Sandelin A, Suzuki H, Wei C-L, Koseki H, Hasegawa Y, 1013 Forrest ARR, Carninci P. 2014. Deep transcriptome profiling of mammalian stem cells supports a 1014 regulatory role for retrotransposons in pluripotency maintenance. Nature Genetics 46:558–566. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 doi:10.1186/s12977-015-0172-6 1025 Gibbs RA, Rogers J, Katze MG, Bumgarner R, Weinstock GM, Mardis ER, Remington KA, Strausberg RL, 1026 Venter JC, Wilson RK, Batzer MA, Bustamante CD, Eichler EE, Hahn MW, Hardison RC, Makova KD, Miller 1027 W, Milosavljevic A, Palermo RE, Siepel A, Sikela JM, Attaway T, Bell S, Bernard KE, Buhay CJ, 1028 Chandrabose MN, Dao M, Davis C, Delehaunty KD, Ding Y, Dinh HH, Dugan-Rocha S, Fulton LA, Gabisi RA, 1029 Garner TT, Godfrey J, Hawes AC, Hernandez J, Hines S, Holder M, Hume J, Jhangiani SN, Joshi V, Khan 1030 ZM, Kirkness EF, Cree A, Fowler RG, Lee S, Lewis LR, Li Z, Liu Yih-shin, Moore SM, Muzny D, Nazareth LV, 1031 Ngo DN, Okwuonu GO, Pai G, Parker D, Paul HA, Pfannkoch C, Pohl CS, Rogers Y-H, Ruiz SJ, Sabo A, 1032 Santibanez J, Schneider BW, Smith SM, Sodergren E, Svatek AF, Utterback TR, Vattathil S, Warren W, 1033 White CS, Chinwalla AT, Feng Y, Halpern AL, Hillier LW, Huang X, Minx P, Nelson JO, Pepin KH, Qin X, 1034 Sutton GG, Venter E, Walenz BP, Wallis JW, Worley KC, Yang S-P, Jones SM, Marra MA, Rocchi M, Schein 1035 JE, Baertsch R, Clarke L, Csürös M, Glasscock J, Harris RA, Havlak P, Jackson AR, Jiang H, Liu Yue, Messina 1036 DN, Shen Y, Song HX-Z, Wylie T, Zhang L, Birney E, Han K, Konkel MK, Lee J, Smit AFA, Ullmer B, Wang H, 1037 Xing J, Burhans R, Cheng Z, Karro JE, Ma J, Raney B, She X, Cox MJ, Demuth JP, Dumas LJ, Han S-G, 1038 Hopkins J, Karimpour-Fard A, Kim YH, Pollack JR, Vinar T, Addo-Quaye C, Degenhardt J, Denby A, Hubisz 1039 MJ, Indap A, Kosiol C, Lahn BT, Lawson HA, Marklein A, Nielsen R, Vallender EJ, Clark AG, Ferguson B, 1040 Hernandez RD, Hirani K, Kehrer-Sawatzki H, Kolb J, Patil S, Pu L-L, Ren Y, Smith DG, Wheeler DA, Schenck 1041 I, Ball EV, Chen R, Cooper DN, Giardine B, Hsu F, Kent WJ, Lesk A, Nelson DL, O’Brien WE, Prüfer K, 1042 Stenson PD, Wallace JC, Ke H, Liu X-M, Wang P, Xiang AP, Yang F, Barber GP, Haussler D, Karolchik D, 1043 doi:10.1038/ng.2965 1016 Gafni O, Weinberger L, Mansour AA, Manor YS, Chomsky E, Ben-Yosef D, Kalma Y, Viukov S, Maza I, 1017 Zviran A, Rais Y, Shipony Z, Mukamel Z, Krupalnik V, Zerbib M, Geula S, Caspi I, Schneir D, Shwartz T, 1018 Gilad S, Amann-Zalcenstein D, Benjamin S, Amit I, Tanay A, Massarwa R, Novershtern N, Hanna JH. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 Evolutionary and Biomedical Insights from the Rhesus 1044 Macaque Genome. Science 316:222–234. doi:10.1126/science.1139247 1045 Ginestet C. 2011. ggplot2: Elegant Graphics for Data Analysis. Journal of the Royal Statistical Society: 1046 Series A (Statistics in Society) 174:245–246. doi:https://doi.org/10.1111/j.1467-985X.2010.00676_9.x 1047 Glinsky GV. 2015. Transposable Elements and DNA Methylation Create in Embryonic Stem Cells Human- 1048 Specific Regulatory Sequences Associated with Distal Enhancers and Noncoding RNAs. Genome Biol Evol 1049 7:1432–1454. doi:10.1093/gbe/evv081 1050 Göke J, Lu X, Chan Y-S, Ng H-H, Ly L-H, Sachs F, Szczerbinska I. 2015. Dynamic Transcription of Distinct 1051 Classes of Endogenous Retroviral Elements Marks Specific Populations of Early Human Embryonic Cells. 1052 Cell Stem Cell 16:135–141. doi:10.1016/j.stem.2015.01.005 1053 Goodchild NL, Wilkinson DA, Mager DL. 1993. Recent Evolutionary Expansion of a Subfamily of RTVL-H 1054 Human Endogenous Retrovirus-like Elements. Virology 196:778–788. doi:10.1006/viro.1993.1535 1055 Gouy M, Guindon S, Gascuel O. 2010. SeaView Version 4: A Multiplatform Graphical User Interface for 1056 Sequence Alignment and Phylogenetic Tree Building. Molecular Biology and Evolution 27:221–224. 1057 doi:10.1093/molbev/msp259 1058 Haig D. 2016. Transposable elements: Self-seekers of the germline, team-players of the soma. BioEssays 1059 38:1158–1166. doi:10.1002/bies.201600125 1060 Heinz S, Benner C, Spann N, Bertolino E, Lin YC, Laslo P, Cheng JX, Murre C, Singh H, Glass CK. 2010. 1061 Simple combinations of lineage-determining transcription factors prime cis-regulatory elements 1062 required for macrophage and B cell identities. Mol Cell 38:576–589. doi:10.1016/j.molcel.2010.05.004 1063 Hermant C, Torres-Padilla M-E. 2021. TFs for TEs: the transcription factor repertoire of mammalian 1064 transposable elements. Genes Dev 35:22–39. doi:10.1101/gad.344473.120 1065 Imbeault M, Helleboid P-Y, Trono D. 2017. KRAB zinc-finger proteins contribute to the evolution of gene 1066 regulatory networks. Nature 543:550–554. doi:10.1038/nature21683 1067 Ito J, Sugimoto R, Nakaoka H, Yamada S, Kimura T, Hayano T, Inoue I. 2017. Systematic identification and 1068 characterization of regulatory elements derived from human endogenous retroviruses. PLOS Genetics 1069 13:e1006883. doi:10.1371/journal.pgen.1006883 1070 Izsvák Z, Wang J, Singh M, Mager DL, Hurst LD. 2016. Pluripotency and the endogenous retrovirus 1071 HERVH: Conflict or serendipity? BioEssays 38:109–117. doi:10.1002/bies.201500096 1072 Jacobs FM, Greenberg D, Nguyen N, Haeussler M, Ewing AD, Katzman S, Paten B, Salama SR, Haussler D. 1073 2014. An evolutionary arms race between KRAB zinc finger genes 91/93 and SVA/L1 retrotransposons. 1074 Nature 516:242–245. doi:10.1038/nature13760 1075 Jacques P-É, Jeyakani J, Bourque G. 2013. The Majority of Primate-Specific Regulatory Sequences Are 1076 Derived from Transposable Elements. PLOS Genetics 9:e1003504. doi:10.1371/journal.pgen.1003504 1077 Jacques P-É, Jeyakani J, Bourque G. 2013. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 2013. 1019 Derivation of novel human ground state naive pluripotent stem cells. Nature 504:282–286. 1020 doi:10.1038/nature12745 1021 Gemmell P, Hein J, Katzourakis A. 2019. The Exaptation of HERV-H: Evolutionary Analyses Reveal the 1022 Genomic Features of Highly Transcribed Elements. Front Immunol 10. doi:10.3389/fimmu.2019.01339 1023 Gemmell P, Hein J, Katzourakis A. 2015. Orthologous endogenous retroviruses exhibit directional 1024 selection since the chimp-human split. Retrovirology 12. doi:10.1186/s12977-015-0172-6 1025 Gibbs RA, Rogers J, Katze MG, Bumgarner R, Weinstock GM, Mardis ER, Remington KA, Strausberg RL, 1026 Venter JC, Wilson RK, Batzer MA, Bustamante CD, Eichler EE, Hahn MW, Hardison RC, Makova KD, Miller 1027 W Milosavljevic A Palermo RE Siepel A Sikela JM Attaway T Bell S Bernard KE Buhay CJ 1028 Gafni O, Weinberger L, Mansour AA, Manor YS, Chomsky E, Ben-Yosef D, Kalma Y, Viukov S, Maza I, 1017 Zviran A, Rais Y, Shipony Z, Mukamel Z, Krupalnik V, Zerbib M, Geula S, Caspi I, Schneir D, Shwartz T, 1018 Gilad S, Amann-Zalcenstein D, Benjamin S, Amit I, Tanay A, Massarwa R, Novershtern N, Hanna JH. 2013. 1019 Derivation of novel human ground state naive pluripotent stem cells. Nature 504:282–286. 1020 doi:10.1038/nature12745 1021 Gemmell P, Hein J, Katzourakis A. 2015. Orthologous endogenous retroviruses exhibit directional 1024 selection since the chimp-human split. Retrovirology 12. doi:10.1186/s12977-015-0172-6 1025 44 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint Kern AD, Kuhn RM, Smith KE, Zwieg AS. 2007. Evolutionary and Biomedical Insights from the Rhesus 1044 Macaque Genome. Science 316:222–234. doi:10.1126/science.1139247 1045 Kern AD, Kuhn RM, Smith KE, Zwieg AS. 2007. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 The Majority of Primate-Specific Regulatory Sequences Are 1076 Derived from Transposable Elements PLOS Genetics 9:e1003504 doi:10 1371/journal pgen 1003504 1077 Jern P, Sperber GO, Ahlsén G, Blomberg J. 2005. Sequence Variability, Gene Structure, and Expression of 1078 Full-Length Human Endogenous Retrovirus H. Journal of Virology 79. 1079 45 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint Jern P, Sperber GO, Blomberg J. 2004. Definition and variation of human endogenous retrovirus H. 1080 Virology 327:93–110. doi:10.1016/j.virol.2004.06.023 1081 Jetzt AE, Yu H, Klarmann GJ, Ron Y, Preston BD, Dougherty JP. 2000. High rate of recombination 1082 throughout the human immunodeficiency virus type 1 genome. J Virol 74:1234–1240. 1083 doi:10.1128/jvi.74.3.1234-1240.2000 1084 Johnson WE. 2019. Origins and evolutionary consequences of ancient endogenous retroviruses. Nat Rev 1085 Microbiol 17:355–370. doi:10.1038/s41579-019-0189-2 1086 Kalkan T, Smith A. 2014. Mapping the route from naive pluripotency to lineage specification. 1087 Philosophical Transactions of the Royal Society B: Biological Sciences 369:20130540. 1088 doi:10.1098/rstb.2013.0540 1089 Kelley D, Rinn J. 2012. Transposable elements reveal a stem cell-specific class of long noncoding RNAs. 1090 Genome Biology 13:R107. doi:10.1186/gb-2012-13-11-r107 1091 Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, Haussler and D. 2002. The Human 1092 Genome Browser at UCSC. Genome Res 12:996–1006. doi:10.1101/gr.229102 1093 Khan H, Smit A, Boissinot S. 2006. Molecular evolution and tempo of amplification of human LINE-1 1094 retrotransposons since the origin of primates. Genome Res 16:78–87. doi:10.1101/gr.4001406 1095 Kinoshita M, Barber M, Mansfield W, Cui Y, Spindlow D, Stirparo GG, Dietmann S, Nichols J, Smith A. 1096 2021. Capture of Mouse and Human Stem Cells with Features of Formative Pluripotency. Cell Stem Cell 1097 28:453-471.e8. doi:10.1016/j.stem.2020.11.005 1098 Kojima KK. 2018. Human transposable elements in Repbase: genomic footprints from fish to humans. 1099 Mobile DNA 9:2 doi:10 1186/s13100 017 0107 y 1100 Jern P, Sperber GO, Blomberg J. 2004. Definition and variation of human endogenous retrovirus H. 1080 Virology 327:93–110. doi:10.1016/j.virol.2004.06.023 1081 Jetzt AE, Yu H, Klarmann GJ, Ron Y, Preston BD, Dougherty JP. 2000. High rate of recombination 1082 throughout the human immunodeficiency virus type 1 genome. J Virol 74:1234–1240. 1083 doi:10.1128/jvi.74.3.1234-1240.2000 1084 Johnson WE. 2019. Origins and evolutionary consequences of ancient endogenous retroviruses. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 Nat Rev 1085 Microbiol 17:355–370. doi:10.1038/s41579-019-0189-2 1086 Kalkan T, Smith A. 2014. Mapping the route from naive pluripotency to lineage specification. 1087 Philosophical Transactions of the Royal Society B: Biological Sciences 369:20130540. 1088 doi:10.1098/rstb.2013.0540 1089 Kelley D, Rinn J. 2012. Transposable elements reveal a stem cell-specific class of long noncoding RNAs. 1090 Genome Biology 13:R107. doi:10.1186/gb-2012-13-11-r107 1091 Kelley D, Rinn J. 2012. Transposable elements reveal a stem cell-specific class of long noncoding RNAs. 1090 Genome Biology 13:R107. doi:10.1186/gb-2012-13-11-r107 1091 Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, Haussler and D. 2002. The Human 1092 Genome Browser at UCSC. Genome Res 12:996–1006. doi:10.1101/gr.229102 1093 Khan H, Smit A, Boissinot S. 2006. Molecular evolution and tempo of amplification of human LINE-1 1094 retrotransposons since the origin of primates. Genome Res 16:78–87. doi:10.1101/gr.4001406 1095 Khan H, Smit A, Boissinot S. 2006. Molecular evolution and tempo of amplification of human LINE-1 1094 retrotransposons since the origin of primates Genome Res 16:78 87 doi:10 1101/gr 4001406 1095 Kinoshita M, Barber M, Mansfield W, Cui Y, Spindlow D, Stirparo GG, Dietmann S, Nichols J, Smith A. 1096 2021. Capture of Mouse and Human Stem Cells with Features of Formative Pluripotency. Cell Stem Cell 1097 28:453-471.e8. doi:10.1016/j.stem.2020.11.005 1098 Kojima KK. 2018. Human transposable elements in Repbase: genomic footprints from fish to humans. 1099 Mobile DNA 9:2. doi:10.1186/s13100-017-0107-y 1100 / y Kong Y, Rose CM, Cass AA, Williams AG, Darwish M, Lianoglou S, Haverty PM, Tong A-J, Blanchette C, 1101 Albert ML, Mellman I, Bourgon R, Greally J, Jhunjhunwala S, Chen-Harris H. 2019. Transposable elemen 1102 expression in tumors is associated with immune infiltration and increased antigenicity. Nat Commun 1103 10:5228. doi:10.1038/s41467-019-13035-2 1104 Krönung SK, Beyer U, Chiaramonte ML, Dolfini D, Mantovani R, Dobbelstein M. 2016. LTR12 promoter 1105 activation in a broad range of human tumor cells by HDAC inhibition. Oncotarget 7:33484–33497. 1106 doi:10.18632/oncotarget.9255 1107 Kunarso G, Chia N-Y, Jeyakani J, Hwang C, Lu X, Chan Y-S, Ng H-H, Bourque G. 2010. Transposable 1108 elements have rewired the core regulatory network of human embryonic stem cells. Nature Genetics 1109 42:631–634. doi:10.1038/ng.600 1110 Lai MM. 1992. RNA recombination in animal and plant viruses. Microbiol Rev 56:61–79. 1111 Lanciano S, Cristofari G. 2020. Measuring and interpreting transposable element expression. Nat Rev 1112 Genet 21:721–736. doi:10.1038/s41576-020-0251-y 1113 Leigh JW, Bryant D. 2015. popart: full-feature software for haplotype network construction. Methods in 1114 Ecology and Evolution 6:1110–1116. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 doi:https://doi.org/10.1111/2041-210X.12410 1115 Kong Y, Rose CM, Cass AA, Williams AG, Darwish M, Lianoglou S, Haverty PM, Tong A-J, Blanchette C, 1101 Albert ML, Mellman I, Bourgon R, Greally J, Jhunjhunwala S, Chen-Harris H. 2019. Transposable element 1102 expression in tumors is associated with immune infiltration and increased antigenicity. Nat Commun 1103 10:5228. doi:10.1038/s41467-019-13035-2 1104 Krönung SK, Beyer U, Chiaramonte ML, Dolfini D, Mantovani R, Dobbelstein M. 2016. LTR12 promoter 1105 activation in a broad range of human tumor cells by HDAC inhibition. Oncotarget 7:33484–33497. 1106 doi:10.18632/oncotarget.9255 1107 Kunarso G, Chia N-Y, Jeyakani J, Hwang C, Lu X, Chan Y-S, Ng H-H, Bourque G. 2010. Transposable 1108 elements have rewired the core regulatory network of human embryonic stem cells. Nature Genetics 1109 42:631–634. doi:10.1038/ng.600 1110 Lai MM. 1992. RNA recombination in animal and plant viruses. Microbiol Rev 56:61–79. 1111 Lanciano S, Cristofari G. 2020. Measuring and interpreting transposable element expression. Nat Rev 1112 Genet 21:721–736. doi:10.1038/s41576-020-0251-y 1113 Leigh JW, Bryant D. 2015. popart: full-feature software for haplotype network construction. Methods in 1114 Ecology and Evolution 6:1110–1116. doi:https://doi.org/10.1111/2041-210X.12410 1115 46 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 1127 doi:10 1038/nature09687 1128 Locke DP, Hillier LW, Warren WC, Worley KC, Nazareth LV, Muzny DM, Yang S-P, Wang Z, Chinwalla AT, 1116 Minx P, Mitreva M, Cook L, Delehaunty KD, Fronick C, Schmidt H, Fulton LA, Fulton RS, Nelson JO, 1117 Magrini V, Pohl C, Graves TA, Markovic C, Cree A, Dinh HH, Hume J, Kovar CL, Fowler GR, Lunter G, 1118 Meader S, Heger A, Ponting CP, Marques-Bonet T, Alkan C, Chen L, Cheng Z, Kidd JM, Eichler EE, White S, 1119 Searle S, Vilella AJ, Chen Y, Flicek P, Ma J, Raney B, Suh B, Burhans R, Herrero J, Haussler D, Faria R, 1120 Fernando O, Darré F, Farré D, Gazave E, Oliva M, Navarro A, Roberto R, Capozzi O, Archidiacono N, Valle 1121 GD, Purgato S, Rocchi M, Konkel MK, Walker JA, Ullmer B, Batzer MA, Smit AFA, Hubley R, Casola C, 1122 Schrider DR, Hahn MW, Quesada V, Puente XS, Ordoñez GR, López-Otín C, Vinar T, Brejova B, Ratan A, 1123 Harris RS, Miller W, Kosiol C, Lawson HA, Taliwal V, Martins AL, Siepel A, RoyChoudhury A, Ma X, 1124 Degenhardt J, Bustamante CD, Gutenkunst RN, Mailund T, Dutheil JY, Hobolth A, Schierup MH, Ryder 1125 OA, Yoshinaga Y, de Jong PJ, Weinstock GM, Rogers J, Mardis ER, Gibbs RA, Wilson RK. 2011. 1126 Comparative and demographic analysis of orang-utan genomes. Nature 469:529–533. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 1127 doi:10 1038/nature09687 1128 Locke DP, Hillier LW, Warren WC, Worley KC, Nazareth LV, Muzny DM, Yang S-P, Wang Z, Chinwalla AT, 1116 Minx P, Mitreva M, Cook L, Delehaunty KD, Fronick C, Schmidt H, Fulton LA, Fulton RS, Nelson JO, 1117 Magrini V, Pohl C, Graves TA, Markovic C, Cree A, Dinh HH, Hume J, Kovar CL, Fowler GR, Lunter G, 1118 Meader S, Heger A, Ponting CP, Marques-Bonet T, Alkan C, Chen L, Cheng Z, Kidd JM, Eichler EE, White S, 1119 Searle S, Vilella AJ, Chen Y, Flicek P, Ma J, Raney B, Suh B, Burhans R, Herrero J, Haussler D, Faria R, 1120 Fernando O, Darré F, Farré D, Gazave E, Oliva M, Navarro A, Roberto R, Capozzi O, Archidiacono N, Valle 1121 GD, Purgato S, Rocchi M, Konkel MK, Walker JA, Ullmer B, Batzer MA, Smit AFA, Hubley R, Casola C, 1122 Schrider DR, Hahn MW, Quesada V, Puente XS, Ordoñez GR, López-Otín C, Vinar T, Brejova B, Ratan A, 1123 Harris RS, Miller W, Kosiol C, Lawson HA, Taliwal V, Martins AL, Siepel A, RoyChoudhury A, Ma X, 1124 Degenhardt J, Bustamante CD, Gutenkunst RN, Mailund T, Dutheil JY, Hobolth A, Schierup MH, Ryder 1125 OA, Yoshinaga Y, de Jong PJ, Weinstock GM, Rogers J, Mardis ER, Gibbs RA, Wilson RK. 2011. 1126 Comparative and demographic analysis of orang-utan genomes. Nature 469:529–533. 1127 doi:10.1038/nature09687 1128 Loewer S, Cabili MN, Guttman M, Loh Y-H, Thomas K, Park IH, Garber M, Curran M, Onder T, Agarwal S, 1129 Manos PD, Datta S, Lander ES, Schlaeger TM, Daley GQ, Rinn JL. 2010. Large intergenic non-coding RNA- 1130 RoR modulates reprogramming of human induced pluripotent stem cells. Nat Genet 42:1113–1117. 1131 doi:10.1038/ng.710 1132 Löytynoja A, Goldman N. 2010. webPRANK: a phylogeny-aware multiple sequence aligner with 1133 interactive alignment browser. BMC Bioinformatics 11:579–579. doi:10.1186/1471-2105-11-579 1134 Lu X, Sachs F, Ramsay L, Jacques P-É, Göke J, Bourque G, Ng H-H. 2014. The retrovirus HERVH is a long 1135 noncoding RNA required for human embryonic stem cell identity. Nature Structural & Molecular Biology 1136 21:423–425. doi:10.1038/nsmb.2799 1137 Macfarlan TS, Gifford WD, Driscoll S, Lettieri K, Rowe HM, Bonanomi D, Firth A, Singer O, Trono D, Pfaff 1138 SL. 2012. Embryonic stem cell potency fluctuates with endogenous retrovirus activity. Nature 487:57– 1139 63. doi:10.1038/nature11244 1140 Mager DL, Freeman JD. 1995. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 HERV-H Endogenous Retroviruses: Presence in the New World Branch but 1141 Amplification in the Old World Primate Lineage. Virology 213:395–404. doi:10.1006/viro.1995.0012 1142 Mager DL, Freeman JD. 1987. Human endogenous retroviruslike genome with type C pol sequences and 1143 gag sequences related to human T-cell lymphotropic viruses. J Virol 61:4060–4066. 1144 doi:10.1128/jvi.61.12.4060-4066.1987 1145 Matsuda E, Garfinkel DJ. 2009. Posttranslational interference of Ty1 retrotransposition by antisense 1146 RNAs. Proc Natl Acad Sci U S A 106:15657–15662. doi:10.1073/pnas.0908305106 1147 Miao B, Fu S, Lyu C, Gontarz P, Wang T, Zhang B. 2020. Tissue-specific usage of transposable element- 1148 derived promoters in mouse development. Genome Biol 21:1–25. doi:10.1186/s13059-020-02164-3 1149 Mi KH N Y J i M Al N Will d HF K WJ 2014 C f d l f 1150 Loewer S, Cabili MN, Guttman M, Loh Y-H, Thomas K, Park IH, Garber M, Curran M, Onder T, Agarwal S, 1129 Manos PD, Datta S, Lander ES, Schlaeger TM, Daley GQ, Rinn JL. 2010. Large intergenic non-coding RNA- 1130 RoR modulates reprogramming of human induced pluripotent stem cells. Nat Genet 42:1113–1117. 1131 doi:10.1038/ng.710 1132 Löytynoja A, Goldman N. 2010. webPRANK: a phylogeny-aware multiple sequence aligner with 1133 interactive alignment browser. BMC Bioinformatics 11:579–579. doi:10.1186/1471-2105-11-579 1134 Lu X, Sachs F, Ramsay L, Jacques P-É, Göke J, Bourque G, Ng H-H. 2014. The retrovirus HERVH is a long 1135 noncoding RNA required for human embryonic stem cell identity. Nature Structural & Molecular Biology 1136 21:423–425. doi:10.1038/nsmb.2799 1137 Macfarlan TS, Gifford WD, Driscoll S, Lettieri K, Rowe HM, Bonanomi D, Firth A, Singer O, Trono D, Pfaff 1138 SL. 2012. Embryonic stem cell potency fluctuates with endogenous retrovirus activity. Nature 487:57– 1139 63. doi:10.1038/nature11244 1140 Mager DL, Freeman JD. 1995. HERV-H Endogenous Retroviruses: Presence in the New World Branch but 1141 Amplification in the Old World Primate Lineage. Virology 213:395–404. doi:10.1006/viro.1995.0012 1142 Mager DL, Freeman JD. 1987. Human endogenous retroviruslike genome with type C pol sequences and 1143 gag sequences related to human T-cell lymphotropic viruses. J Virol 61:4060–4066. 1144 doi:10.1128/jvi.61.12.4060-4066.1987 1145 Matsuda E, Garfinkel DJ. 2009. Posttranslational interference of Ty1 retrotransposition by antisense 1146 RNAs. Proc Natl Acad Sci U S A 106:15657–15662. doi:10.1073/pnas.0908305106 1147 Miao B, Fu S, Lyu C, Gontarz P, Wang T, Zhang B. 2020. Tissue-specific usage of transposable element- 1148 derived promoters in mouse development. Genome Biol 21:1–25. doi:10.1186/s13059-020-02164-3 1149 Miga KH, Newton Y, Jain M, Altemose N, Willard HF, Kent WJ. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint Locke DP, Hillier LW, Warren WC, Worley KC, Nazareth LV, Muzny DM, Yang S-P, Wang Z, Chinwalla AT, 1116 Minx P, Mitreva M, Cook L, Delehaunty KD, Fronick C, Schmidt H, Fulton LA, Fulton RS, Nelson JO, 1117 Magrini V, Pohl C, Graves TA, Markovic C, Cree A, Dinh HH, Hume J, Kovar CL, Fowler GR, Lunter G, 1118 Meader S, Heger A, Ponting CP, Marques-Bonet T, Alkan C, Chen L, Cheng Z, Kidd JM, Eichler EE, White S, 1119 Searle S, Vilella AJ, Chen Y, Flicek P, Ma J, Raney B, Suh B, Burhans R, Herrero J, Haussler D, Faria R, 1120 Fernando O, Darré F, Farré D, Gazave E, Oliva M, Navarro A, Roberto R, Capozzi O, Archidiacono N, Valle 1121 GD, Purgato S, Rocchi M, Konkel MK, Walker JA, Ullmer B, Batzer MA, Smit AFA, Hubley R, Casola C, 1122 Schrider DR, Hahn MW, Quesada V, Puente XS, Ordoñez GR, López-Otín C, Vinar T, Brejova B, Ratan A, 1123 Harris RS, Miller W, Kosiol C, Lawson HA, Taliwal V, Martins AL, Siepel A, RoyChoudhury A, Ma X, 1124 Degenhardt J, Bustamante CD, Gutenkunst RN, Mailund T, Dutheil JY, Hobolth A, Schierup MH, Ryder 1125 OA, Yoshinaga Y, de Jong PJ, Weinstock GM, Rogers J, Mardis ER, Gibbs RA, Wilson RK. 2011. 1126 Comparative and demographic analysis of orang-utan genomes. Nature 469:529–533. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 1127 doi:10 1038/nature09687 1128 Locke DP, Hillier LW, Warren WC, Worley KC, Nazareth LV, Muzny DM, Yang S-P, Wang Z, Chinwalla AT, 1116 Minx P, Mitreva M, Cook L, Delehaunty KD, Fronick C, Schmidt H, Fulton LA, Fulton RS, Nelson JO, 1117 Magrini V, Pohl C, Graves TA, Markovic C, Cree A, Dinh HH, Hume J, Kovar CL, Fowler GR, Lunter G, 1118 Meader S, Heger A, Ponting CP, Marques-Bonet T, Alkan C, Chen L, Cheng Z, Kidd JM, Eichler EE, White S, 1119 Searle S, Vilella AJ, Chen Y, Flicek P, Ma J, Raney B, Suh B, Burhans R, Herrero J, Haussler D, Faria R, 1120 Fernando O, Darré F, Farré D, Gazave E, Oliva M, Navarro A, Roberto R, Capozzi O, Archidiacono N, Valle 1121 GD, Purgato S, Rocchi M, Konkel MK, Walker JA, Ullmer B, Batzer MA, Smit AFA, Hubley R, Casola C, 1122 Schrider DR, Hahn MW, Quesada V, Puente XS, Ordoñez GR, López-Otín C, Vinar T, Brejova B, Ratan A, 1123 Harris RS, Miller W, Kosiol C, Lawson HA, Taliwal V, Martins AL, Siepel A, RoyChoudhury A, Ma X, 1124 Degenhardt J, Bustamante CD, Gutenkunst RN, Mailund T, Dutheil JY, Hobolth A, Schierup MH, Ryder 1125 OA, Yoshinaga Y, de Jong PJ, Weinstock GM, Rogers J, Mardis ER, Gibbs RA, Wilson RK. 2011. 1126 Comparative and demographic analysis of orang-utan genomes. Nature 469:529–533. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 2014. Centromere reference models for 1150 human chromosomes X and Y satellite arrays. Genome Res 24:697–707. doi:10.1101/gr.159624.113 1151 Nakamura T, Yamada KD, Tomii K, Katoh K. 2018. Parallelization of MAFFT for large-scale multiple 1152 sequence alignments Bioinformatics 34:2490–2492 doi:10 1093/bioinformatics/bty121 1153 sequence alignments. Bioinformatics 34:2490–2492. doi:10.1093/bioinformatics/bty121 1153 47 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint Nichols J, Smith A. 2009. Naive and Primed Pluripotent States. Cell Stem Cell 4:487–492. 1154 doi:10.1016/j.stem.2009.05.015 1155 Niwa H. 2007. How is pluripotency determined and maintained? Development 134:635–646. 1156 doi:10.1242/dev.02787 1157 Niwa H, Nakamura A, Urata M, Shirae-Kurabayashi M, Kuraku S, Russell S, Ohtsuka S. 2016. The 1158 evolutionally-conserved function of group B1 Sox family members confers the unique role of Sox2 in 1159 mouse ES cells. BMC Evolutionary Biology 16:173. doi:10.1186/s12862-016-0755-4 1160 Ohnuki M, Tanabe K, Sutou K, Teramoto I, Sawamura Y, Narita M, Nakamura Michiko, Tokunaga Y, 1161 Nakamura Masahiro, Watanabe A, Yamanaka S, Takahashi K. 2014. Dynamic regulation of human 1162 endogenous retroviruses mediates factor-induced reprogramming and differentiation potential. PNAS 1163 111:12426–12431. doi:10.1073/pnas.1413299111 1164 Patro R, Duggal G, Love MI, Irizarry RA, Kingsford C. 2017. Salmon provides fast and bias-aware 1165 quantification of transcript expression. Nat Methods 14:417–419. doi:10.1038/nmeth.4197 1166 Peaston AE, Evsikov AV, Graber JH, Vries WN de, Holbrook AE, Solter D, Knowles BB. 2004. 1167 Retrotransposons Regulate Host Genes in Mouse Oocytes and Preimplantation Embryos. Developmental 1168 Cell 7:597–606. doi:10.1016/j.devcel.2004.09.004 1169 Pérez-Losada M, Arenas M, Galán JC, Palero F, González-Candelas F. 2015. Recombination in viruses: 1170 Mechanisms, methods of study, and evolutionary consequences. Infect Genet Evol 30:296–307. 1171 doi:10.1016/j.meegid.2014.12.022 1172 Pontis J, Planet E, Offner S, Turelli P, Duc J, Coudray A, Theunissen TW, Jaenisch R, Trono D. 2019. 1173 Hominoid-Specific Transposable Elements and KZFPs Facilitate Human Embryonic Genome Activation 1174 and Control Transcription in Naive Human ESCs. Cell Stem Cell 24:724-735.e5. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 How is pluripotency determined and maintained? Development 134:635–646. 1156 doi:10.1242/dev.02787 1157 Niwa H, Nakamura A, Urata M, Shirae-Kurabayashi M, Kuraku S, Russell S, Ohtsuka S. 2016. The 1158 evolutionally-conserved function of group B1 Sox family members confers the unique role of Sox2 in 1159 mouse ES cells. BMC Evolutionary Biology 16:173. doi:10.1186/s12862-016-0755-4 1160 Ohnuki M, Tanabe K, Sutou K, Teramoto I, Sawamura Y, Narita M, Nakamura Michiko, Tokunaga Y, 1161 Nakamura Masahiro, Watanabe A, Yamanaka S, Takahashi K. 2014. Dynamic regulation of human 1162 endogenous retroviruses mediates factor-induced reprogramming and differentiation potential. PNAS 1163 111:12426–12431. doi:10.1073/pnas.1413299111 1164 doi:10.1242/dev.02787 1157 Niwa H, Nakamura A, Urata M, Shirae-Kurabayashi M, Kuraku S, Russell S, Ohtsuka S. 2016. The 1158 evolutionally-conserved function of group B1 Sox family members confers the unique role of Sox2 in 1159 mouse ES cells. BMC Evolutionary Biology 16:173. doi:10.1186/s12862-016-0755-4 1160 Ohnuki M, Tanabe K, Sutou K, Teramoto I, Sawamura Y, Narita M, Nakamura Michiko, Tokunaga Y, 1161 Nakamura Masahiro, Watanabe A, Yamanaka S, Takahashi K. 2014. Dynamic regulation of human 1162 endogenous retroviruses mediates factor-induced reprogramming and differentiation potential. PNAS 1163 111:12426–12431. doi:10.1073/pnas.1413299111 1164 Patro R, Duggal G, Love MI, Irizarry RA, Kingsford C. 2017. Salmon provides fast and bias-aware 1165 quantification of transcript expression. Nat Methods 14:417–419. doi:10.1038/nmeth.4197 1166 Peaston AE, Evsikov AV, Graber JH, Vries WN de, Holbrook AE, Solter D, Knowles BB. 2004. 1167 Retrotransposons Regulate Host Genes in Mouse Oocytes and Preimplantation Embryos. Developmental 1168 Cell 7:597–606. doi:10.1016/j.devcel.2004.09.004 1169 Pérez-Losada M, Arenas M, Galán JC, Palero F, González-Candelas F. 2015. Recombination in viruses: 1170 Mechanisms, methods of study, and evolutionary consequences. Infect Genet Evol 30:296–307. 1171 doi:10.1016/j.meegid.2014.12.022 1172 Pontis J, Planet E, Offner S, Turelli P, Duc J, Coudray A, Theunissen TW, Jaenisch R, Trono D. 2019. 1173 Hominoid-Specific Transposable Elements and KZFPs Facilitate Human Embryonic Genome Activation 1174 and Control Transcription in Naive Human ESCs. Cell Stem Cell 24:724-735.e5. 1175 doi:10.1016/j.stem.2019.03.012 1176 Posada D, Crandall KA. 2001. Intraspecific gene genealogies: trees grafting into networks. Trends in 1177 Ecology & Evolution 16:37–45. doi:10.1016/S0169-5347(00)02026-7 1178 Niwa H, Nakamura A, Urata M, Shirae-Kurabayashi M, Kuraku S, Russell S, Ohtsuka S. 2016. The 1158 evolutionally-conserved function of group B1 Sox family members confers the unique role of Sox2 in 1159 mouse ES cells. BMC Evolutionary Biology 16:173. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 1175 doi:10.1016/j.stem.2019.03.012 1176 Posada D, Crandall KA. 2001. Intraspecific gene genealogies: trees grafting into networks. Trends in 1177 Ecology & Evolution 16:37–45. doi:10.1016/S0169-5347(00)02026-7 1178 Quinlan AR, Hall IM. 2010. BEDTools: a flexible suite of utilities for comparing genomic features. 1179 Bioinformatics 26:841–842. doi:10.1093/bioinformatics/btq033 1180 Ramírez F, Ryan DP, Grüning B, Bhardwaj V, Kilpert F, Richter AS, Heyne S, Dündar F, Manke T. 2016. 1181 deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Research 1182 44:W160–W165. doi:10.1093/nar/gkw257 1183 Raney BJ, Dreszer TR, Barber GP, Clawson H, Fujita PA, Wang T, Nguyen N, Paten B, Zweig AS, Karolchik 1184 D, Kent WJ. 2014. Track data hubs enable visualization of user-defined genome-wide annotations on the 1185 UCSC Genome Browser. Bioinformatics 30:1003–1005. doi:10.1093/bioinformatics/btt637 1186 Rebollo R, Romanish MT, Mager DL. 2012. Transposable elements: an abundant and natural source of 1187 regulatory sequences for host genes. Annu Rev Genet 46:21–42. doi:10.1146/annurev-genet-110711- 1188 155621 1189 Nichols J, Smith A. 2009. Naive and Primed Pluripotent States. Cell Stem Cell 4:487–492. 1154 doi:10.1016/j.stem.2009.05.015 1155 Niwa H. 2007. How is pluripotency determined and maintained? Development 134:635–646. 1156 doi:10.1242/dev.02787 1157 Niwa H, Nakamura A, Urata M, Shirae-Kurabayashi M, Kuraku S, Russell S, Ohtsuka S. 2016. The 1158 evolutionally-conserved function of group B1 Sox family members confers the unique role of Sox2 in 1159 mouse ES cells. BMC Evolutionary Biology 16:173. doi:10.1186/s12862-016-0755-4 1160 Ohnuki M, Tanabe K, Sutou K, Teramoto I, Sawamura Y, Narita M, Nakamura Michiko, Tokunaga Y, 1161 Nakamura Masahiro, Watanabe A, Yamanaka S, Takahashi K. 2014. Dynamic regulation of human 1162 endogenous retroviruses mediates factor-induced reprogramming and differentiation potential. PNAS 1163 111:12426–12431. doi:10.1073/pnas.1413299111 1164 Patro R, Duggal G, Love MI, Irizarry RA, Kingsford C. 2017. Salmon provides fast and bias-aware 1165 quantification of transcript expression. Nat Methods 14:417–419. doi:10.1038/nmeth.4197 1166 Peaston AE, Evsikov AV, Graber JH, Vries WN de, Holbrook AE, Solter D, Knowles BB. 2004. 1167 Retrotransposons Regulate Host Genes in Mouse Oocytes and Preimplantation Embryos. Developmental 1168 Cell 7:597–606. doi:10.1016/j.devcel.2004.09.004 1169 Pérez-Losada M, Arenas M, Galán JC, Palero F, González-Candelas F. 2015. Recombination in viruses: 1170 Mechanisms, methods of study, and evolutionary consequences. Infect Genet Evol 30:296–307. 1171 doi:10.1016/j.meegid.2014.12.022 1172 Pontis J, Planet E, Offner S, Turelli P, Duc J, Coudray A, Theunissen TW, Jaenisch R, Trono D. 2019. 1173 Nichols J, Smith A. 2009. Naive and Primed Pluripotent States. Cell Stem Cell 4:487 492. 1154 doi:10.1016/j.stem.2009.05.015 1155 Niwa H. 2007. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 How to tame an endogenous retrovirus: HERVH and the 1190 evolution of human pluripotency. Current Opinion in Virology, Animal models for viral diseases • 1191 Paleovirology 25:49–58. doi:10.1016/j.coviro.2017.07.001 1192 evolution of human pluripotency. Current Opinion in Virology, Animal models for viral diseases • 1191 Paleovirology 25:49 58 doi:10 1016/j coviro 2017 07 001 1192 Rossant J, Tam PPL. 2017. New Insights into Early Human Development: Lessons for Stem Cell Derivatio 1193 and Differentiation. Cell Stem Cell 20:18–28. doi:10.1016/j.stem.2016.12.004 1194 Santoni FA, Guerra J, Luban J. 2012. HERV-H RNA is abundant in human embryonic stem cells and a 1195 precise marker for pluripotency. Retrovirology 9:111. doi:10.1186/1742-4690-9-111 1196 Scally A, Dutheil JY, Hillier LW, Jordan GE, Goodhead I, Herrero J, Hobolth A, Lappalainen T, Mailund T, 1197 Marques-Bonet T, McCarthy S, Montgomery SH, Schwalie PC, Tang YA, Ward MC, Xue Y, Yngvadottir B, 1198 Alkan C, Andersen LN, Ayub Q, Ball EV, Beal K, Bradley BJ, Chen Y, Clee CM, Fitzgerald S, Graves TA, Gu Y 1199 Heath P, Heger A, Karakoc E, Kolb-Kokocinski A, Laird GK, Lunter G, Meader S, Mort M, Mullikin JC, 1200 Munch K, O’Connor TD, Phillips AD, Prado-Martinez J, Rogers AS, Sajjadian S, Schmidt D, Shaw K, 1201 Simpson JT, Stenson PD, Turner DJ, Vigilant L, Vilella AJ, Whitener W, Zhu B, Cooper DN, de Jong P, 1202 Dermitzakis ET, Eichler EE, Flicek P, Goldman N, Mundy NI, Ning Z, Odom DT, Ponting CP, Quail MA, 1203 Ryder OA, Searle SM, Warren WC, Wilson RK, Schierup MH, Rogers J, Tyler-Smith C, Durbin R. 2012. 1204 Insights into hominid evolution from the gorilla genome sequence. Nature 483:169–175. 1205 doi:10.1038/nature10842 1206 Schön U, Seifarth W, Baust C, Hohenadl C, Erfle V, Leib-Mösch C. 2001. Cell Type-Specific Expression and 1207 Promoter Activity of Human Endogenous Retroviral Long Terminal Repeats. Virology 279:280–291. 1208 doi:10.1006/viro.2000.0712 1209 Simon-Loriere E, Holmes EC. 2011. Why do RNA viruses recombine? Nat Rev Microbiol 9:617–626. 1210 doi:10.1038/nrmicro2614 1211 Smit AF, Hubley R, Green P. 2013. RepeatMasker Open-4.0. 1212 Storer J, Hubley R, Rosen J, Wheeler TJ, Smit AF. 2021. The Dfam community resource of transposable 1213 element families, sequence models, and genome annotations. Mobile DNA 12:2. doi:10.1186/s13100- 1214 020-00230-y 1215 Sundaram V, Cheng Y, Ma Z, Li D, Xing X, Edge P, Snyder MP, Wang T. 2014. Widespread contribution of 1216 transposable elements to the innovation of gene regulatory networks. Genome Res 24:1963–1976. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 1205 d / Scally A, Dutheil JY, Hillier LW, Jordan GE, Goodhead I, Herrero J, Hobolth A, Lappalainen T, Mailund T, 1197 Marques-Bonet T, McCarthy S, Montgomery SH, Schwalie PC, Tang YA, Ward MC, Xue Y, Yngvadottir B, 1198 Alkan C, Andersen LN, Ayub Q, Ball EV, Beal K, Bradley BJ, Chen Y, Clee CM, Fitzgerald S, Graves TA, Gu Y, 1199 Heath P, Heger A, Karakoc E, Kolb-Kokocinski A, Laird GK, Lunter G, Meader S, Mort M, Mullikin JC, 1200 Munch K, O’Connor TD, Phillips AD, Prado-Martinez J, Rogers AS, Sajjadian S, Schmidt D, Shaw K, 1201 Simpson JT, Stenson PD, Turner DJ, Vigilant L, Vilella AJ, Whitener W, Zhu B, Cooper DN, de Jong P, 1202 Dermitzakis ET, Eichler EE, Flicek P, Goldman N, Mundy NI, Ning Z, Odom DT, Ponting CP, Quail MA, 1203 Ryder OA, Searle SM, Warren WC, Wilson RK, Schierup MH, Rogers J, Tyler-Smith C, Durbin R. 2012. 1204 Insights into hominid evolution from the gorilla genome sequence. Nature 483:169–175. 1205 doi:10 1038/nature10842 1206 Simpson JT, Stenson PD, Turner DJ, Vigilant L, Vilella AJ, Whitener W, Zhu B, Cooper DN, de Jong P, 1202 Dermitzakis ET, Eichler EE, Flicek P, Goldman N, Mundy NI, Ning Z, Odom DT, Ponting CP, Quail MA, 1203 Ryder OA, Searle SM, Warren WC, Wilson RK, Schierup MH, Rogers J, Tyler-Smith C, Durbin R. 2012. 1204 Insights into hominid evolution from the gorilla genome sequence. Nature 483:169–175. 1205 doi:10.1038/nature10842 1206 Schön U, Seifarth W, Baust C, Hohenadl C, Erfle V, Leib-Mösch C. 2001. Cell Type-Specific Expression and 1207 Promoter Activity of Human Endogenous Retroviral Long Terminal Repeats. Virology 279:280–291. 1208 doi:10.1006/viro.2000.0712 1209 Simon-Loriere E, Holmes EC. 2011. Why do RNA viruses recombine? Nat Rev Microbiol 9:617–626. 1210 doi:10.1038/nrmicro2614 1211 Smit AF, Hubley R, Green P. 2013. RepeatMasker Open-4.0. 1212 Storer J, Hubley R, Rosen J, Wheeler TJ, Smit AF. 2021. The Dfam community resource of transposable 1213 element families, sequence models, and genome annotations. Mobile DNA 12:2. doi:10.1186/s13100- 1214 020-00230-y 1215 Sundaram V, Cheng Y, Ma Z, Li D, Xing X, Edge P, Snyder MP, Wang T. 2014. Widespread contribution of 1216 transposable elements to the innovation of gene regulatory networks. Genome Res 24:1963–1976. 1217 doi:10.1101/gr.168872.113 1218 Sundaram V, Wysocka J. 2020. Transposable elements as a potent source of diverse cis-regulatory 1219 sequences in mammalian genomes. Philosophical Transactions of the Royal Society B: Biological Sciences 1220 375:20190347. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 1217 doi:10.1101/gr.168872.113 1218 Sundaram V, Wysocka J. 2020. Transposable elements as a potent source of diverse cis-regulatory 1219 sequences in mammalian genomes. Philosophical Transactions of the Royal Society B: Biological Science 1220 375:20190347. doi:10.1098/rstb.2019.0347 1221 Svoboda P, Stein P, Anger M, Bernstein E, Hannon GJ, Schultz RM. 2004. RNAi and expression of 1222 retrotransposons MuERV-L and IAP in preimplantation mouse embryos. Developmental Biology 1223 269:276–285. doi:10.1016/j.ydbio.2004.01.028 1224 Takahashi K, Nakamura M, Okubo C, Kliesmete Z, Ohnuki M, Narita M, Watanabe A, Ueda M, Takashima 1225 Y, Hellmann I, Yamanaka S. 2021. The pluripotent stem cell-specific transcript ESRG is dispensable for 1226 human pluripotency. PLOS Genetics 17:e1009587. doi:10.1371/journal.pgen.1009587 1227 Rossant J, Tam PPL. 2017. New Insights into Early Human Development: Lessons for Stem Cell Derivation 1193 and Differentiation. Cell Stem Cell 20:18–28. doi:10.1016/j.stem.2016.12.004 1194 Rossant J, Tam PPL. 2017. New Insights into Early Human Development: Lessons for Stem Cell Derivation 1193 and Differentiation. Cell Stem Cell 20:18–28. doi:10.1016/j.stem.2016.12.004 1194 Santoni FA, Guerra J, Luban J. 2012. HERV-H RNA is abundant in human embryonic stem cells and a 1195 precise marker for pluripotency. Retrovirology 9:111. doi:10.1186/1742-4690-9-111 1196 Santoni FA, Guerra J, Luban J. 2012. HERV-H RNA is abundant in human embryonic stem cells and a 1195 precise marker for pluripotency. Retrovirology 9:111. doi:10.1186/1742-4690-9-111 1196 Scally A, Dutheil JY, Hillier LW, Jordan GE, Goodhead I, Herrero J, Hobolth A, Lappalainen T, Mailund T, 1197 Marques-Bonet T, McCarthy S, Montgomery SH, Schwalie PC, Tang YA, Ward MC, Xue Y, Yngvadottir B, 1198 Alkan C, Andersen LN, Ayub Q, Ball EV, Beal K, Bradley BJ, Chen Y, Clee CM, Fitzgerald S, Graves TA, Gu Y, 1199 Heath P, Heger A, Karakoc E, Kolb-Kokocinski A, Laird GK, Lunter G, Meader S, Mort M, Mullikin JC, 1200 Munch K, O’Connor TD, Phillips AD, Prado-Martinez J, Rogers AS, Sajjadian S, Schmidt D, Shaw K, 1201 Simpson JT, Stenson PD, Turner DJ, Vigilant L, Vilella AJ, Whitener W, Zhu B, Cooper DN, de Jong P, 1202 Dermitzakis ET, Eichler EE, Flicek P, Goldman N, Mundy NI, Ning Z, Odom DT, Ponting CP, Quail MA, 1203 Ryder OA, Searle SM, Warren WC, Wilson RK, Schierup MH, Rogers J, Tyler-Smith C, Durbin R. 2012. 1204 Insights into hominid evolution from the gorilla genome sequence. Nature 483:169–175. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 doi:10.1186/s12862-016-0755-4 1160 Ohnuki M, Tanabe K, Sutou K, Teramoto I, Sawamura Y, Narita M, Nakamura Michiko, Tokunaga Y, 1161 Nakamura Masahiro, Watanabe A, Yamanaka S, Takahashi K. 2014. Dynamic regulation of human 1162 endogenous retroviruses mediates factor-induced reprogramming and differentiation potential. PNAS 1163 111:12426–12431. doi:10.1073/pnas.1413299111 1164 Peaston AE, Evsikov AV, Graber JH, Vries WN de, Holbrook AE, Solter D, Knowles BB. 2004. 1167 Retrotransposons Regulate Host Genes in Mouse Oocytes and Preimplantation Embryos. Developmental 1168 Cell 7:597–606. doi:10.1016/j.devcel.2004.09.004 1169 Pontis J, Planet E, Offner S, Turelli P, Duc J, Coudray A, Theunissen TW, Jaenisch R, Trono D. 2019. 1173 Hominoid-Specific Transposable Elements and KZFPs Facilitate Human Embryonic Genome Activation 1174 and Control Transcription in Naive Human ESCs. Cell Stem Cell 24:724-735.e5. 1175 doi:10.1016/j.stem.2019.03.012 1176 Posada D, Crandall KA. 2001. Intraspecific gene genealogies: trees grafting into networks. Trends in 1177 Ecology & Evolution 16:37–45. doi:10.1016/S0169-5347(00)02026-7 1178 Quinlan AR, Hall IM. 2010. BEDTools: a flexible suite of utilities for comparing genomic features. 1179 Bioinformatics 26:841–842. doi:10.1093/bioinformatics/btq033 1180 Ramírez F, Ryan DP, Grüning B, Bhardwaj V, Kilpert F, Richter AS, Heyne S, Dündar F, Manke T. 2016. 1181 deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Research 1182 44:W160–W165. doi:10.1093/nar/gkw257 1183 Raney BJ, Dreszer TR, Barber GP, Clawson H, Fujita PA, Wang T, Nguyen N, Paten B, Zweig AS, Karolchik 1184 D, Kent WJ. 2014. Track data hubs enable visualization of user-defined genome-wide annotations on the 1185 UCSC Genome Browser. Bioinformatics 30:1003–1005. doi:10.1093/bioinformatics/btt637 1186 Rebollo R, Romanish MT, Mager DL. 2012. Transposable elements: an abundant and natural source of 1187 regulatory sequences for host genes. Annu Rev Genet 46:21–42. doi:10.1146/annurev-genet-110711- 1188 155621 1189 48 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint Römer C, Singh M, Hurst LD, Izsvák Z. 2017. How to tame an endogenous retrovir 1190 Römer C, Singh M, Hurst LD, Izsvák Z. 2017. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint Tang F, Barbacioru C, Bao S, Lee C, Nordman E, Wang X, Lao K, Surani MA. 201 1228 Tang F, Barbacioru C, Bao S, Lee C, Nordman E, Wang X, Lao K, Surani MA. 2010. Tracing the Derivation 1228 of Embryonic Stem Cells from the Inner Cell Mass by Single-Cell RNA-Seq Analysis. Cell Stem Cell 6:468– 1229 478. doi:10.1016/j.stem.2010.03.015 1230 of Embryonic Stem Cells from the Inner Cell Mass by Single-Cell RNA-Seq Analysis. Cell Stem Cell 6:468– 1229 478. doi:10.1016/j.stem.2010.03.015 1230 Theunissen TW, Friedli M, He Y, Planet E, O’Neil RC, Markoulaki S, Pontis J, Wang H, Iouranova A, 1231 Imbeault M, Duc J, Cohen MA, Wert KJ, Castanon R, Zhang Z, Huang Y, Nery JR, Drotar J, Lungjangwa T, 1232 Trono D, Ecker JR, Jaenisch R. 2016. Molecular Criteria for Defining the Naive Human Pluripotent State. 1233 Cell Stem Cell 19:502–515. doi:10.1016/j.stem.2016.06.011 1234 Imbeault M, Duc J, Cohen MA, Wert KJ, Castanon R, Zhang Z, Huang Y, Nery JR, Drotar J, Lungjangwa T, 1232 Trono D, Ecker JR, Jaenisch R. 2016. Molecular Criteria for Defining the Naive Human Pluripotent State. 1233 Cell Stem Cell 19:502–515. doi:10.1016/j.stem.2016.06.011 1234 Thomas J, Perron H, Feschotte C. 2018. Variation in proviral content among human genomes mediated 1235 by LTR recombination. Mobile DNA 9:36. doi:10.1186/s13100-018-0142-3 1236 Thompson PJ, Macfarlan TS, Lorincz MC. 2016. Long Terminal Repeats: From Parasitic Elements to 1237 Building Blocks of the Transcriptional Regulatory Repertoire. Molecular Cell 62:766–776. 1238 doi:10.1016/j.molcel.2016.03.029 1239 Urusov FA, Nefedova LN, Kim AI. 2011. Analysis of the tissue- and stage-specific transportation of the 1240 Drosophila melanogaster gypsy retrotransposon. Russ J Genet Appl Res 1:507–510. 1241 doi:10.1134/S2079059711060104 1242 Vargiu L, Rodriguez-Tomé P, Sperber GO, Cadeddu M, Grandi N, Blikstad V, Tramontano E, Blomberg J. 1243 2016. Classification and characterization of human endogenous retroviruses; mosaic forms are commo 1244 Retrovirology 13:7. doi:10.1186/s12977-015-0232-y 1245 Wang J, Xie G, Singh M, Ghanbarian AT, Raskó T, Szvetnik A, Cai H, Besser D, Prigione A, Fuchs NV, 1246 Schumann GG, Chen W, Lorincz MC, Ivics Z, Hurst LD, Izsvák Z. 2014. Primate-specific endogenous 1247 retrovirus-driven transcription defines naive-like stem cells. Nature 516:405–409. 1248 doi:10.1038/nature13804 1249 Wang Z, Oron E, Nelson B, Razis S, Ivanova N. 2012. Distinct lineage specification roles for NANOG, 1250 OCT4, and SOX2 in human embryonic stem cells. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 Cell Stem Cell 10:440–454. 1251 doi:10.1016/j.stem.2012.02.016 1252 Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ. 2009. Jalview Version 2—a multiple 1253 sequence alignment editor and analysis workbench. Bioinformatics 25:1189–1191. 1254 doi:10.1093/bioinformatics/btp033 1255 Waterson RH, Lander ES, Wilson RK, The Chimpanzee Sequencing and Analysis Consortium. 2005. Initia 1256 sequence of the chimpanzee genome and comparison with the human genome. Nature 437:69–87. 1257 doi:10.1038/nature04072 1258 Wolf G, de Iaco A, Sun M-A, Bruno M, Tinkham M, Hoang D, Mitra A, Ralls S, Trono D, Macfarlan TS. 1259 2020. KRAB-zinc finger protein gene expansion in response to active retrotransposons in the murine 1260 lineage. eLife 9:e56337. doi:10.7554/eLife.56337 1261 Yang P, Wang Y, Macfarlan TS. 2017. The role of KRAB-ZFPs in transposable element repression and 1262 mammalian evolution. Trends Genet 33:871–881. doi:10.1016/j.tig.2017.08.006 1263 Thomas J, Perron H, Feschotte C. 2018. Variation in proviral content among human genomes mediated 1235 by LTR recombination. Mobile DNA 9:36. doi:10.1186/s13100-018-0142-3 1236 Thompson PJ, Macfarlan TS, Lorincz MC. 2016. Long Terminal Repeats: From Parasitic Elements to 1237 Building Blocks of the Transcriptional Regulatory Repertoire. Molecular Cell 62:766–776. 1238 doi:10.1016/j.molcel.2016.03.029 1239 Urusov FA, Nefedova LN, Kim AI. 2011. Analysis of the tissue- and stage-specific transportation of the 1240 Drosophila melanogaster gypsy retrotransposon. Russ J Genet Appl Res 1:507–510. 1241 doi:10.1134/S2079059711060104 1242 Drosophila melanogaster gypsy retrotransposon. Russ J Genet Appl Res 1:507–510. 1241 doi:10.1134/S2079059711060104 1242 Vargiu L, Rodriguez-Tomé P, Sperber GO, Cadeddu M, Grandi N, Blikstad V, Tramontano E, Blomberg J. 1243 2016. Classification and characterization of human endogenous retroviruses; mosaic forms are common. 1244 Retrovirology 13:7. doi:10.1186/s12977-015-0232-y 1245 Wang J, Xie G, Singh M, Ghanbarian AT, Raskó T, Szvetnik A, Cai H, Besser D, Prigione A, Fuchs NV, 1246 Schumann GG, Chen W, Lorincz MC, Ivics Z, Hurst LD, Izsvák Z. 2014. Primate-specific endogenous 1247 retrovirus-driven transcription defines naive-like stem cells. Nature 516:405–409. 1248 doi:10.1038/nature13804 1249 Wang Z, Oron E, Nelson B, Razis S, Ivanova N. 2012. Distinct lineage specification roles for NANOG, 1250 OCT4, and SOX2 in human embryonic stem cells. Cell Stem Cell 10:440–454. 1251 doi:10.1016/j.stem.2012.02.016 1252 Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ. 2009. Jalview Version 2—a multiple 1253 sequence alignment editor and analysis workbench. Bioinformatics 25:1189–1191. 1254 doi:10 1093/bioinformatics/btp033 1255 Vargiu L, Rodriguez-Tomé P, Sperber GO, Cadeddu M, Grandi N, Blikstad V, Tramontano E, Blomberg J. 1243 2016. Classification and characterization of human endogenous retroviruses; mosaic forms are common. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 doi:10.1098/rstb.2019.0347 1221 Svoboda P, Stein P, Anger M, Bernstein E, Hannon GJ, Schultz RM. 2004. RNAi and expression of 1222 retrotransposons MuERV-L and IAP in preimplantation mouse embryos. Developmental Biology 1223 269:276–285. doi:10.1016/j.ydbio.2004.01.028 1224 Takahashi K, Nakamura M, Okubo C, Kliesmete Z, Ohnuki M, Narita M, Watanabe A, Ueda M, Takashima 1225 Y, Hellmann I, Yamanaka S. 2021. The pluripotent stem cell-specific transcript ESRG is dispensable for 1226 h l i i d i /j l Schön U, Seifarth W, Baust C, Hohenadl C, Erfle V, Leib-Mösch C. 2001. Cell Type-Specific Expression and 1207 Promoter Activity of Human Endogenous Retroviral Long Terminal Repeats. Virology 279:280–291. 1208 doi:10.1006/viro.2000.0712 1209 Simon-Loriere E, Holmes EC. 2011. Why do RNA viruses recombine? Nat Rev Microbiol 9:617–626. 1210 doi:10.1038/nrmicro2614 1211 F, Hubley R, Green P. 2013. RepeatMasker Open-4.0. Smit AF, Hubley R, Green P. 2013. RepeatMasker Open-4.0. 1212 Storer J, Hubley R, Rosen J, Wheeler TJ, Smit AF. 2021. The Dfam community resource of transposable 1213 element families, sequence models, and genome annotations. Mobile DNA 12:2. doi:10.1186/s13100- 1214 020-00230-y 1215 Sundaram V, Cheng Y, Ma Z, Li D, Xing X, Edge P, Snyder MP, Wang T. 2014. Widespread contribution of 1216 transposable elements to the innovation of gene regulatory networks. Genome Res 24:1963–1976. 1217 doi:10.1101/gr.168872.113 1218 Sundaram V, Wysocka J. 2020. Transposable elements as a potent source of diverse cis-regulatory 1219 sequences in mammalian genomes. Philosophical Transactions of the Royal Society B: Biological Sciences 1220 375:20190347. doi:10.1098/rstb.2019.0347 1221 Svoboda P, Stein P, Anger M, Bernstein E, Hannon GJ, Schultz RM. 2004. RNAi and expression of 1222 retrotransposons MuERV-L and IAP in preimplantation mouse embryos. Developmental Biology 1223 269:276–285. doi:10.1016/j.ydbio.2004.01.028 1224 Takahashi K, Nakamura M, Okubo C, Kliesmete Z, Ohnuki M, Narita M, Watanabe A, Ueda M, Takashima 1225 Y, Hellmann I, Yamanaka S. 2021. The pluripotent stem cell-specific transcript ESRG is dispensable for 1226 human pluripotency. PLOS Genetics 17:e1009587. doi:10.1371/journal.pgen.1009587 1227 49 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 1244 Retrovirology 13:7. doi:10.1186/s12977-015-0232-y 1245 Vargiu L, Rodriguez Tomé P, Sperber GO, Cadeddu M, Grandi N, Blikstad V, Tramontano E, Blomberg J. 1243 2016. Classification and characterization of human endogenous retroviruses; mosaic forms are commo 1244 Retrovirology 13:7. doi:10.1186/s12977-015-0232-y 1245 Wang J, Xie G, Singh M, Ghanbarian AT, Raskó T, Szvetnik A, Cai H, Besser D, Prigione A, Fuchs NV, 1246 Schumann GG, Chen W, Lorincz MC, Ivics Z, Hurst LD, Izsvák Z. 2014. Primate-specific endogenous 1247 retrovirus-driven transcription defines naive-like stem cells. Nature 516:405–409. 1248 doi:10.1038/nature13804 1249 Wang Z, Oron E, Nelson B, Razis S, Ivanova N. 2012. Distinct lineage specification roles for NANOG, 1250 OCT4, and SOX2 in human embryonic stem cells. Cell Stem Cell 10:440–454. 1251 doi:10.1016/j.stem.2012.02.016 1252 Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ. 2009. Jalview Version 2—a multiple 1253 sequence alignment editor and analysis workbench. Bioinformatics 25:1189–1191. 1254 doi:10.1093/bioinformatics/btp033 1255 Waterson RH, Lander ES, Wilson RK, The Chimpanzee Sequencing and Analysis Consortium. 2005. Initia 1256 sequence of the chimpanzee genome and comparison with the human genome. Nature 437:69–87. 1257 Wang J, Xie G, Singh M, Ghanbarian AT, Raskó T, Szvetnik A, Cai H, Besser D, Prigione A, Fuchs NV, 1246 Schumann GG, Chen W, Lorincz MC, Ivics Z, Hurst LD, Izsvák Z. 2014. Primate-specific endogenous 1247 retrovirus-driven transcription defines naive-like stem cells. Nature 516:405–409. 1248 doi:10.1038/nature13804 1249 Wang Z, Oron E, Nelson B, Razis S, Ivanova N. 2012. Distinct lineage specification roles for NANOG, 1250 OCT4, and SOX2 in human embryonic stem cells. Cell Stem Cell 10:440–454. 1251 doi:10.1016/j.stem.2012.02.016 1252 Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ. 2009. Jalview Version 2—a multiple 1253 sequence alignment editor and analysis workbench. Bioinformatics 25:1189–1191. 1254 doi:10.1093/bioinformatics/btp033 1255 Waterson RH, Lander ES, Wilson RK, The Chimpanzee Sequencing and Analysis Consortium. 2005. Initial 1256 sequence of the chimpanzee genome and comparison with the human genome. Nature 437:69–87. 1257 doi:10.1038/nature04072 1258 Wolf G, de Iaco A, Sun M-A, Bruno M, Tinkham M, Hoang D, Mitra A, Ralls S, Trono D, Macfarlan TS. 1259 2020. KRAB-zinc finger protein gene expansion in response to active retrotransposons in the murine 1260 lineage. eLife 9:e56337. doi:10.7554/eLife.56337 1261 Yang P, Wang Y, Macfarlan TS. 2017. The role of KRAB-ZFPs in transposable element repression and 1262 mammalian evolution. Trends Genet 33:871–881. doi:10.1016/j.tig.2017.08.006 1263 50 . Yu H-L, Zhao Z-K, Zhu F. 2013. The role of human endogenous retroviral long terminal repeat sequences 1264 in human cancer (Review). International Journal of Molecular Medicine 32:755–762. 1265 doi:10.3892/ijmm.2013.1460 1266 Zhang Y, Li T, Preissl S, Amaral ML, Grinstein JD, Farah EN, Destici E, Qiu Y, Hu R, Lee AY, Chee S, Ma K, Ye 1267 Z, Zhu Q, Huang H, Fang R, Yu L, Izpisua Belmonte JC, Wu J, Evans SM, Chi NC, Ren B. 2019. 1268 Transcriptionally active HERV-H retrotransposons demarcate topologically associating domains in human 1269 pluripotent stem cells. Nature Genetics 51:1380–1388. doi:10.1038/s41588-019-0479-7 1270 1271 GCTAGCTGTCAGGCCTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTGACCTGC 858 ACGTACACATCCAGATGGCCGGTTCCTGCCTTAACTGATGACATTCCACCACAAAA 859 GAAGTGAAAATGGCCTGTTCCTGCCTTAACTGATGACATTATCTTGTGAAATTCCTT 860 CTCCTGGCTCATCCTGGCTCAAAAGCTCCCCTACTGAGCACCTTGTGACCCCCACT 861 CCTGCCCGCCAGAGAACAACCCCCCTTTGACTGTAATTTTCCTTTACCTACCCAAA 862 TCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAG 863 CCCGCCTGCACCCAGGTGAAATAAACAGCTTTATTGCTCACACAAAGCCTGTTTGG 864 TGGTCTCTTCACACGGACGCGCATGCTCGAG 865 CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.08.451617 doi: ioRxiv preprint Yu H-L, Zhao Z-K, Zhu F. 2013. The role of human endogenous retroviral long terminal repeat sequences 1264 in human cancer (Review). International Journal of Molecular Medicine 32:755–762. 1265 doi:10.3892/ijmm.2013.1460 1266 Zhang Y, Li T, Preissl S, Amaral ML, Grinstein JD, Farah EN, Destici E, Qiu Y, Hu R, Lee AY, Chee S, Ma K, Ye 1267 Z, Zhu Q, Huang H, Fang R, Yu L, Izpisua Belmonte JC, Wu J, Evans SM, Chi NC, Ren B. 2019. 1268 Transcriptionally active HERV-H retrotransposons demarcate topologically associating domains in human 1269 pluripotent stem cells. Nature Genetics 51:1380–1388. doi:10.1038/s41588-019-0479-7 1270 1271 Yu H-L, Zhao Z-K, Zhu F. 2013. The role of human endogenous retroviral long terminal repeat sequences 1264 in human cancer (Review). International Journal of Molecular Medicine 32:755–762. 1265 doi:10.3892/ijmm.2013.1460 1266 Zhang Y, Li T, Preissl S, Amaral ML, Grinstein JD, Farah EN, Destici E, Qiu Y, Hu R, Lee AY, Chee S, Ma K, Ye 1267 Z, Zhu Q, Huang H, Fang R, Yu L, Izpisua Belmonte JC, Wu J, Evans SM, Chi NC, Ren B. 2019. 1268 Transcriptionally active HERV-H retrotransposons demarcate topologically associating domains in human 1269 pluripotent stem cells. Nature Genetics 51:1380–1388. doi:10.1038/s41588-019-0479-7 1270 1271 Yu H-L, Zhao Z-K, Zhu F. 2013. The role of human endogenous retroviral long terminal repeat sequences 1264 in human cancer (Review). International Journal of Molecular Medicine 32:755–762. 1265 doi:10 3892/ijmm 2013 1460 1266 Transcriptionally active HERV-H retrotransposons demarcate topologically associating domains in human 1269 pluripotent stem cells. Nature Genetics 51:1380–1388. doi:10.1038/s41588-019-0479-7 1270 51 51
https://openalex.org/W4391541228	https://www.nature.com/articles/s41599-024-02613-5.pdf	English	null	A “pre-traumatic stress syndrome”: trauma and war in Elizabeth Bowen’s The Last September and The Heat of the Day	Humanities & social sciences communications	2,024	cc-by	9,584	1 Ocean University of China, Qingdao, China. ✉email: qiong.he@foxmail.com HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2024) 11:210 \| https://doi.org/10.1057/s41599-024-02613-5 ARTICLE A “pre-traumatic stress syndrome”: trauma and war in Elizabeth Bowen’s The Last September and The Heat of the Day 1✉ https://doi.org/10.1057/s41599-024-02613-5 OPEN ARTICLE A “pre-traumatic stress syndrome”: trauma and war in Elizabeth Bowen’s The Last September and The Heat of the Day 1✉ https://doi.org/10.1057/s41599-024-02613-5 OPEN A “pre-traumatic stress syndrome”: trauma and war in Elizabeth Bowen’s The Last September and The Heat of the Day Qiong He1✉ As a modernist writer writing through the two World Wars and witnessing the decline of British imperial power through the decay of her own class, the Anglo-Irish Ascendancy, most of Elizabeth Bowen’s (1899–1973) writing is that of trauma. Many critics have suggested that modernist writing provides a response to the traumas of the era and resonates with the Freudian model of trauma, which regards the syndrome of traumatic events as post-traumatic. This essay challenges the previous studies regarding modernist writing of trauma as post- traumatic, by introducing Saint-Amour’s concept of a “pre-traumatic stress syndrome” to study Bowen’s two novels about wars, The Last September and The Heat of the Day. The essay holds that in Bowen’s writing, anticipation and anxiety arising before the war can also inﬂict psychic damage, just like the actual war. The essay demonstrates how Bowen’s writing about wars adds a new approach to literary trauma studies, which have concentrated almost solely on the aftermath of catastrophes or violence. Interpreting Bowen’s writing as pre-trauma not only provides a better understanding of the psychological condition of people during the wars but brings a new perspective to trauma studies as well. 1 HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| https://doi.org/10.1057/s41599-024-02613-5 ARTICLE Introduction The Last Sep- tember presents the traumatic psychology of the Anglo-Irish before their Big Houses are burned down during the Irish War of Independence, while The Heat of the Day concerns the traumatic conditions experienced by Londoners during the Blitz, when they are constantly traumatized by the threat of imminent bombings. To present his idea of pre-trauma, Saint-Amour juxtaposes two contradictory perspectives on traumatic psychology during the interwar period: one is Sigmund Freud’s writings on trauma, which focuses on the effects of the subject’s repressed past; the other is the psychiatrist Eugène Minkowski’s study, Le temps vécu [Lived Time], which ﬁxes its gaze in the future. Saint-Amour visually depicts Freud and Minkowski as ﬁguratively standing in an interwar present, with Freud facing the past and Minkowski looking toward the future. In his work Moses and Monotheism (1939), Freud examines the phenomenon of “traumatic neurosis” experienced by individuals who have survived a traumatic incident, such as a train collision. Freud observes that these individuals may initially appear phy- sically unharmed, but later exhibit severe psychological and physical symptoms. This leads him to the conclusion that trau- matic neurosis is characterized by a latent period, during which the effects of the trauma do not immediately manifest (84). Building on Freud’s concept, Caruth (1995) further explores the nature of trauma by focusing on the structure of the traumatic experience itself. Caruth suggests that “the [traumatic] event is not assimilated or experienced fully at the time, but only belat- edly, in its repeated possession of the one who experiences it” (4, italics in original). Trauma is characterized by its “immediacy”, for it splits the barrier of conscious protection, enabling the entrance of the outside into the inside without any mediation. Not having been fully “assimilated” as it occurred, the traumatic experience fails to be integrated into an individual’s personal history and impacts the subject belatedly, often after a period of latency, through such symptoms as ﬂashbacks, dreams, and the compulsive repetition of behavior. This Freudian perspective on trauma emphasizes its belatedness and lingering inﬂuence, viewing the syndrome and associated symptoms as post- traumatic. y y g As a modernist writer, Bowen faced a uniquely complex situation, since she not only dealt with a set of challenges during the tumultuous period of the two World Wars but also witnessed the decline of British imperial power alongside the decay of her own class, the Anglo-Irish Ascendancy. Introduction Unlike the Freudian model of trauma that concentrates almost solely on the aftermath of cat- astrophes or violence, Saint-Amour suggests that the symptoms of traumatic events arise in response to “a potentially oncoming rather than an already realized catastrophe” (8). The Last Sep- tember presents the traumatic psychology of the Anglo-Irish before their Big Houses are burned down during the Irish War of Independence, while The Heat of the Day concerns the traumatic conditions experienced by Londoners during the Blitz, when they are constantly traumatized by the threat of imminent bombings. As a modernist writer, Bowen faced a uniquely complex situation, since she not only dealt with a set of challenges during the tumultuous period of the two World Wars but also witnessed the decline of British imperial power alongside the decay of her own class, the Anglo-Irish Ascendancy. Conse- quently, scholars have identiﬁed two distinct facets of her work: the postcolonial Bowen, focused on her Irish-set works, like The Last September and A World of Love; and the modernist Bowen, exploring themes of dislocation, alienation, and betrayal in her novels and short stories from the interwar period onwards. Nevertheless, this dichotomy fails to capture the full complexity of Bowen’s artistry, as argued by Fredric Jameson, who posits that “the colonial experience is at the root of Western modernism”, and James F. Wurtz (2010), who asserts that “Bowen’s repre- sentations of Anglo-Irish Big House culture [in The Last Sep- tember in particular] are in fact focal points for understanding Bowen as a modernist” (120). Building upon these ideas, this essay proposes that Bowen’s depiction of the trauma of the Irish War of Independence is intricately linked to her portrayal of the Second World War, ultimately enhancing our understanding of her as a modernist. This essay challenges the previous studies considering mod- ernist writing of trauma as post-traumatic, by introducing Paul K. Saint-Amour’s concept of a “pre-traumatic stress syndrome”. It applies this concept to the analysis of Elizabeth Bowen’s two novels about wars, The Last September (1998a [1929]) and The Heat of the Day3(1998b [1949]). Unlike the Freudian model of trauma that concentrates almost solely on the aftermath of cat- astrophes or violence, Saint-Amour suggests that the symptoms of traumatic events arise in response to “a potentially oncoming rather than an already realized catastrophe” (8). Introduction IntI I t has been suggested that the modernist period is characterized by unprecedented catastrophes and modernist writing responds to the traumas of the era. Trauma theory, especially the Freudian model of trauma1, has been developed in response to these unprecedented atrocities. Consequently, Peters and Richards (2021) indicates that trauma is often considered “inherently linked to modernity” (3). As Baer (2007) suggests in “Modernism and Trauma”, modernist writing is “particularly apt for representing traumatic experience” and is often seen as “post- traumatic discourse” (307, emphasis added). Much of the litera- ture from this period concerned itself with trauma and the scholarship on trauma writing of this period largely concentrates on war trauma.2 As demonstrated by Rae (2007), modernist lit- erature centers on the “‘work of mourning’, or more precisely, the ‘resistance’ to this work, shaping both its themes and its formal experiments” (13). Regarded as “post-traumatic discourse”, modernist literature is often investigated through the lens of trauma theory based on Sigmund Freud’s concept. Saint Amour’s notion of “pre-traumatic stress syndrome” In his work Tense Future: Modernism, Total War, Encyclopedic Form, Saint-Amour explores the relationship between warfare and futurity. He proposes the concept of “pre-traumatic stress syndrome” to study novels written in the 1920s, a period between WWI and WWII. According to this notion, traumatic symptoms appear before, not after, the event happens: “[T]he sequentially inverted or preposterous phenomenon of traumatic symptoms— denial, dissociation, fragmentation, repression, the compulsive repetition of violence—[that] exists not in the wake of a past event, but in the shadow of a future one” (Saint-Amour 25). Saint-Amour highlights the signiﬁcance of the anticipation of war and anxiety, asserting that they can also lead to traumatic psy- chosis, as he writes, “simulated and actual disasters will inﬂict equal psychic damage, and anticipation will be a condition in which one can ‘die a thousand deaths’” (7). His understanding of trauma mediates between the repression of past traumatic memories and the anticipation of future trauma, with a greater emphasis on the latter. trauma theory based on Sigmund Freud s concept. This essay challenges the previous studies considering mod- ernist writing of trauma as post-traumatic, by introducing Paul K. Saint-Amour’s concept of a “pre-traumatic stress syndrome”. It applies this concept to the analysis of Elizabeth Bowen’s two novels about wars, The Last September (1998a [1929]) and The Heat of the Day3(1998b [1949]). Introduction Conversely, Freud believes that the past overshadows the present and should be reintegrated into the individual’s psyche through memory work. Bowen displays a pre-traumatic autobiographical revision in her novel. In the preface, Bowen (1950) confesses that “I was the child of the house from which Danielstown derives. Bowen’s Court survived—nevertheless, so often in my mind’s eye did I see it burning that the terrible last event in The Last September is more real than anything I have lived through” (204). The novel portrays the psychological state of the Anglo-Irish before their Big Houses are destroyed during the Irish War of Independence, that is, before their trauma. Furthermore, at the time of the novel’s publication in 1929, Bowen’s family house has not been destroyed. Bowen revises the fate of her own family house through the ﬁctional Big House, Danielstown, which eventually succumbs to ﬁre, unlike Bowen’s Court. In contrast to Freudian trauma studies that primarily focus on the enduring inﬂuence of past traumatic memories, Saint-Amour’s work in Tense Future directs attention toward the traumatizing power of anticipation. Despite being labeled as “pre-traumatic stress syndrome”, Saint-Amour’s theory does not reject the Freudian model altogether, nor does it apply Minkowski’s work wholesale. Rather, it accommodates both Freudian and Minkowskian approaches by addressing both “a symptom of past repression” and “a wounding anticipation” of an experience (20). Signiﬁcantly, Saint-Amour uncovers another aspect of traumatic experience, namely, traumatic anticipation, in addition to the reactivation of past memories. As noted by Saint-Amour himself, his approach to trauma in the book regards concepts such as prophecy, prolepsis, foresight, foreclosure, anticipation, and expectation, not in a magical or purely diagnostic sense, but as ways of existing in a speciﬁc present and living toward a future that seems pre- determined for identiﬁable reasons (21). Speciﬁcally, Saint-Amour’s model of pre-trauma centers on the moment that is haunted by both a previous catastrophic event and the potential for an even more catastrophic future occurrence. This model serves as a med- iator between repressed past shocks and anticipated future disasters. The Last September, in this sense, can be regarded as Bowen’s own bukimi of the ultimate downfall of the Anglo-Irish Ascen- dency, symbolized by the Big House (Blickle 2019, 138). Introduction HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2024) 11:210 \| https://doi.org/10.1057/s41599-024-02613-5 2 ARTICLE HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| https://doi.org/10.1057/s41599-024-02613-5 burning of the Big Houses from September 1920 to February 1921. The majority of the events in the novel occur in Daniels- town, a Big House owned by Sir Richard and Lady Myra Naylor, who are of Anglo-Irish descent. The house serves as a refuge for various displaced guests and family members: young Lois Farquar who is an orphan and Sir Richard’s niece; indifferent Laurence who is an undergraduate in Oxford and Lady Naylor’s nephew; deracinated Hugo and Francie Montmorency who visit the house after selling their own Big House, Rockriver; and Marda Norton, a modern woman representing an emerging trend of modern femininity. Despite their ambiguous status, the Anglo-Irish inhabitants of Danielstown remain oblivious to the resentment felt by the Catholic Irish in Ireland and employ the strategy of “not noticing” the political upheavals outside the house, in order to maintain a sense of security within their home. subject against fright and so against fright-neuroses” (Saint- Amour 13). Anxiety is seen as strengthening the psyche’s defensive mechanisms, enabling individuals to process and ulti- mately get rid of stimuli that could reactivate repressed memories and cause trauma. As Saint-Amour further concludes, in Freud’s view, “traumatic neuroses, including war neuroses, result not from a fear of death but from a near-death event’s reactivation of some ‘historical factor’ in the subject’s psychosexual past” (17–18). Although Freud occasionally links trauma to anticipa- tion, as seen in his exploration of anxiety, his focus remains on the individual’s past, which is recollected and repressed, while the future is merely a vessel for the repetition of past traumas. Minkowski’s interwar theory of the psyche in Lived Time, as demonstrated by Saint-Amour, ﬁxes its gaze in the opposite direction—the future. Traditional psychology, including Freud’s work, prioritizes memory and views the future as a projection of the past. However, Minkowski diminishes the importance of memory, stating that it “remains necessarily limited, riveted to that which has actually been” (40). Instead, he emphasizes the concept of expec- tation in his depiction of the future. Minkowski believes that “[e] xpectation penetrates the individual to his core, ﬁlls him with terror before this unknown and unexpected mass, which will engulf him in an instant” (88). Introduction He further suggests, “[p]rimary expectation is… always connected to an intense anguish” and “[i]t is always anxious expectation” (88). According to Minkowski, expectation is con- sistently associated with discomfort. In the face of imminent danger, he describes how anguish and terror immobilize us, freezing us in place as if we are paralyzed by fear (88). The Last September is closely related to Bowen’s personal life. As Bowen (1986) herself notes, out of all her works, The Last September is “nearest to my heart, [and it] had a deep, unclouded, spontaneous source” (123). Born in Dublin in 1899 to Anglo-Irish parents, Bowen was the last descendant of a Protestant Unionist family that had settled in Ireland in 1653 during Oliver Crom- well’s rule. Her family belonged to the ascendancy class whose wealth and power in Ireland were evident through their grand residences and extensive estates, commonly referred to as Big Houses. By the late nineteenth century, however, the power and authority of the ascendancy were in decline. The Irish War of Independence, which took place from 1920 to 1921 and resulted in the destruction of over two hundred Big Houses in Ireland, marked the end of the Anglo-Irish hegemony. The Bowens resi- ded in a prominent Big House called Bowen’s Court, constructed in 1775, which Elizabeth Bowen inherited as the sole heir. Bowen openly acknowledges that the ﬁctional Danielstown in The Last September is based on her own family house (126). Despite sur- viving the Irish War of Independence and enduring two world wars, Bowen’s Court was sold by Bowen herself in the 1950s due to ﬁnancial difﬁculties and eventually demolished by the new owner. This theme of “dispossessors dispossessed” (164), as expressed by Esty (2014), serves as the central focus of Bowen’s novel The Last September. Saint-Amour continues to contrast Freud’s and Minkowski’s understandings of expectation. For Freud, anxious expectation in the face of danger is not an agent of traumatization but a defense mechanism to protect the individual from traumatic neuroses. On the other hand, Minkowski views such expectation as the source of trauma, where “the image of death outside us ﬁnds its counterpart in the death within us—where, as Caruth says of traumatic neurosis, ‘the outside has gone inside without mediation’” (Saint-Amour 20). In Minkowski’s perspective, the future takes precedence over the present, dominating the overall situation. Introduction Conse- quently, scholars have identiﬁed two distinct facets of her work: the postcolonial Bowen, focused on her Irish-set works, like The Last September and A World of Love; and the modernist Bowen, exploring themes of dislocation, alienation, and betrayal in her novels and short stories from the interwar period onwards. Nevertheless, this dichotomy fails to capture the full complexity of Bowen’s artistry, as argued by Fredric Jameson, who posits that “the colonial experience is at the root of Western modernism”, and James F. Wurtz (2010), who asserts that “Bowen’s repre- sentations of Anglo-Irish Big House culture [in The Last Sep- tember in particular] are in fact focal points for understanding Bowen as a modernist” (120). Building upon these ideas, this essay proposes that Bowen’s depiction of the trauma of the Irish War of Independence is intricately linked to her portrayal of the Second World War, ultimately enhancing our understanding of her as a modernist. Despite its primary focus on the aftermath of traumatic experiences, Saint-Amour explains that the Freudian model of trauma sometimes also associates psychic wounding with antici- pation. In the interwar writing, Beyond the Pleasure Principle (1962), Freud examines the cases of shell shock or “war neurosis” and links psychic wounding to anticipation. To do this, Freud exhibits three distinct responses to danger: “anxiety” refers to a state of expecting or preparing for an unknown danger, “fear” is the expectation of a known one, and “fright” describes a state of being in immediate danger without any preparation, which leads to traumatic neurosis (12). According to Freud, traumatic neu- rosis occurs when there is only fright and no anxiety. Anxiety cannot cause traumatic neurosis; instead, it can “protect its This essay suggests that Bowen’s depiction of the characters’ psyche during the Irish War of Independence in The Last Sep- tember and during the Blitz in The Heat of the Day introduces a distinct form of psychological trauma called “pre-traumatic” as opposed to “post-traumatic”. The characters in these novels ﬁnd themselves trapped in a historical period wherein they are “traumatized by both a past conﬂagration and the prospect of a worse future one” (Saint-Amour 10). This essay contends that Bowen’s writing demonstrates how pre-war anticipation and anxiety can inﬂict comparable psychological harm akin to the real war itself. ARTICLE The novel concludes with a single sentence, “Sir Richard and Lady Naylor, not saying anything, did not look at each other, for in the light from the sky they saw too distinctly” (206). This sentence reveals the characters’ traumatic psychology since this event is beyond their comprehension and they are frozen speechless at that moment. The destruction of these houses represents the downfall of the Anglo-Irish Ascendancy, the sys- tem they symbolize, signifying the loss of their privileged position in Ireland. The abrupt ending of the novel offers no resolution for the characters, leaving them overwhelmed by the loss of their family home and uncertain about their future. The policy of “not-noticing” is particularly evident in another scene in which Lois encounters the “trench coat” whom she regards mistakenly as a ghost at the outset. After ﬁnishing a conversation with her family and guests on the porch, Lois decides to wander along the avenue of the Big House, reminiscing about the night she danced with Gerald there. After walking up a dark path through the shrubbery, she becomes overwhelmed by fears—“Her fear of the shrubberies tugged at its chain, fear behind reason, fear before her birth, fear like the earliest germ of her life that had stirred in Laura” (33). This fear intensiﬁes when she encounters a gunman, initially mistaking him for a ghost and feeling terriﬁed (33). After she realizes that he is a gunman and not a supernatural entity, however, her fear transforms into excitement, as described in the novel, “A man in a trench-coat had passed without seeing her: that was what it amounted to. She ran back to tell, in excitement” (34). Given the background of the novel, the man’s secretive actions identify him as an Irish gun- man, highlighting the proximity of the adolescent Anglo-Irish girl wandering alone at dusk to the threat and danger posed by the conﬂict. Nevertheless, she views the encounter as an exhilarating incident rather than a threat, and what frightens her is not the tangible danger surrounding her but an imagined supernatural ghost. This interaction signiﬁes that although Lois is intimately connected to the historical trauma of the war, she misinterprets it and fails to grasp its true nature. Moreover, it demonstrates that despite the tendency of the Anglo-Irish to overlook the political upheavals around them, the house and its inhabitants have become embroiled in the heart of the conﬂict. ARTICLE The last scene of the novel is incomprehensible for the Anglo- Irish because they are “oblivious” to the resentment and hostility harbored by the local Irish. In her autobiographical work centered around her family house, Bowen’s Court, Bowen asserts that the Anglo-Irish “had come to share with the [Irish] people around them sentiments, memories, interests, afﬁnities” and a wish to “see Ireland autonomous” (Bowen 2017, 160). By doing so, Bowen highlights the Anglo-Irish longing to assimilate into the Irish culture and identify as Irish themselves, as she further emphasizes in the same paragraph that “[t]themselves, they felt Irish, and acted as Irishmen” (160). Throughout the events at Bowen’s Court, there are instances of collaboration between the Anglo-Irish and Catholic Irish against English rule, although it is worth noting that the Anglo-Irish generally relied on England for support as colonizers. While the Anglo-Irish supported the notion of Home Rule and an independent Ireland in which they held authority, they failed to comprehend that this vision would never be accepted by the Catholic Irish. The Catholic Irish sought not only liberation from English rule but also the restoration of the lands seized by Cromwell that later became the inheritance of the Anglo-Irish. The Anglo-Irish community is genuinely unaware of the extent to which their presence was resented by the Catholic Irish. Considering themselves as Irish, the Anglo-Irish fail to anticipate the consequence of the Irish War of Independence, speciﬁcally, the destruction of the Big Houses. As Saint-Amour writes, “the unassimilable nature of traumatic violence would seem to depend on the impossibility of its anticipation” (1). Since they are unable to perceive the reality of the situation, they are taken by surprise by the turn of events and thus exposed to trauma’s full shock. In this regard, instead of serving as a defense mechanism from trauma, the policy of “not-noticing” before the traumatic event is itself traumatizing, for it exacerbates the effects of trauma. Speaking on the Anglo-Irish’s lack of awareness toward the animosity from the local Irish population, Bowen asserts that “if Ireland did not accept them, they did not know it—and it is in that unawareness of ﬁnal rejection, unawareness of being looked out at from some secretive, opposed life, that the Anglo-Irish naïve dignity and, even, tragedy seems to me to stand” (Bowen 2017, 160). ARTICLE ARTICLE HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| https://doi.org/10.1057/s41599-024-02613-5 fears did not materialize, the trauma persisted in her imagination to such an extent that she asserts, as mentioned earlier, that the “terrible last event in [the novel] is more real than anything I have lived through” (Bowen 1950, 204). Bowen’s bukimi of the even- tual destruction of her family house serves as a premonition that, although does not come true in reality, continues to occupy her thoughts throughout her lifetime. pretending that nothing signiﬁcant is happening. While occa- sional discussions about current political issues as well as things related to the Black and Tans and Irish rebels do arise, these conversations are quickly overshadowed by other forms of entertainment like tennis parties. As analyzed by Glendinning (1998) in the introduction to The Last September, unlike the English whose life is characterized by mobility and ﬂuidity, the Anglo-Irish lead a rather more stable life, for they live in one place, the Big House, for generations and they know each other well (5). Hence, despite the continuous rebellions and uprisings in Irish history, the Anglo-Irish families, like the Naylors, “made impotent by ambivalence”, still adhere to the belief in continuity and maintain their policy of “not noticing” (Bowen The Last September 1998a 6). The Naylors would rather stay in a perfect present—“a moment of happiness, of perfection” as depicted in the novel’s opening, hoping that they can “freeze this moment and keep it always” while failing to see what the future will bring to them (7). That is why when the British soldiers give the Naylors Lawrence’s stolen watch, they fail to perceive it as a threat. g g In The Last September, Bowen imagines this trauma as Danielstown, along with two other houses, Castle Trent and Mount Isabel, are ultimately destroyed. The transformation of the home front into the frontline is highlighted in the novel, as the country itself is described as “burning”, the neck of mountains is “frightfully outlined”, and the roads are “terriﬁed” (Bowen The Last September 1998a 206). After the executioners’ unlit car slides out, “the ﬁrst wave of a silence that was to be ultimately ﬂowed back, conﬁdently, to the steps” and “the door stood open hos- pitably upon a furnace” (206). The once familiar and comfortable house suddenly becomes terrifying, evoking a sense of the uncanny. Introduction To elucidate the concept of pre-trauma, Saint-Amour introduces the concept of bukimi, a Japanese word representing the “weird”, “ghastly”, “unearthly”, “ominous” or “uncanny” feelings experi- enced by the inhabitants of Hiroshima before the explosion of the atomic bomb. According to Saint-Amour, these “premonitions” were partly “attempts at psychic preparation, partly a form of ‘imagining the worst’ as a magical way of warding off disaster” (Saint-Amour 1–2). Bowen herself admitted to harboring a similar dread in her childhood, that is, because Bowen’s Court represents the British colonial power in Ireland, it would be burnt down eventually like other Big Houses in Ireland. Although her The policy of “not-noticing” in The Last September The Last September touches upon the political and social turmoil during the Irish War of Independence and the consequent HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2024) 11:210 \| https://doi.org/10.1057/s41599-024-02613-5 3 UMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2024) 11:210 \| https://doi.org/10.1057/s41599-024-02613-5 ARTICLE The English press publicizes the idea that Ireland is unsafe (56–57). The imminence of the war makes the Naylors anxious. As Glendinning analyses, “[t]he policy of ‘not noticing’ can be maintained only so long as the cracks in the surface of life do not open and let loose the horror and betrayal that lurks beneath” (Bowen The Last September 1998a 6). It is only when the political disturbances inﬁltrate their domestic sphere, symbolized by the house, that the characters are compelled to acknowledge and comprehend the conﬂicts surrounding them. Francie believes that times have been even “worse” at Danielstown and Lady Naylor “had a rather strained look” (18). Marda perceives a sense of “despairing optimism” in Lady Naylor’s eyes (81). Later, Sir Richard has a dream where he is riding a motorbike through the countryside, unable to detach himself from it. He is then attacked by his Irish friends and discovers that he has transformed into a Black and Tan (107). Although regarding themselves as Irish, they fear being harmed or killed by the local Irish population who may consider them to be English. In the novel, the imminence of the war, especially the execution of the Anglo-Irish house, is not only psychic but also historical in that the event has not yet, in fact, happened. Similar to the idea of “pre-trauma” which con- siders that “traumatic symptoms—denial, dissociation, fragmen- tation, repression, the compulsive repetition of violence—exist not in the wake of a past event, but in the shadow of a future one” (25), the Naylors’ symptoms of denial and anxiety appear in the shadow of a future traumatic event. In The Heat of the Day, Bowen (1986) aims to capture “war- climate” (95)—the psychological state and sensory impressions of people living in the ruins of the city during ongoing conﬂicts after the Blitz. According to Wasson (2010), “[a]ir raids (both Allied and German) were often traumatic, combining overwhelming violence with profound grief, and Bowen describes the home front experi- ence in the language of trauma” (124). Indeed, the novel centers on the characters’ traumatic psychology during and after the Blitz, as well as their compulsive efforts to revisit the past in order to fully understand the event, and reorganize it into a meaningful and coherent whole to cope with an uncertain future. ARTICLE Despite hearing about the dire situation in County Cork from residents of County Carlow, which makes Francie believe that her decision to come here is a serious mistake, the Naylors do not take it the same way—“Sir Richard would cer- tainly say, that was County Carlow all over” (23). The Naylors, like survivors of Hiroshima described by Saint-Amour in his book, think they would be exempted from the political upheavals happening around them. g y The Heat of the Day faithfully portrays the realities of everyday life in London during the war. Set between September 1942 and 1944, the novel takes place after the end of the Blitz, with the London of that time only appearing in the characters’ recollec- tions and ﬂashbacks. The story revolves around the protagonist, Stella Rodney, and her involvement in a love triangle with her lover, Robert Kelway, and a British intelligence agent named Harrison. In the aftermath of London air raids, Harrison begins to suspect Robert of being a German spy and initiates an inves- tigation, while also attempting to persuade Stella to abandon Robert and become his lover. Stella ﬁnds herself torn between two men, one a spy and the other a counter-spy. After Robert’s true identity is exposed, he attempts to escape by climbing onto the roof of Stella’s apartment but tragically falls to his death. Harrison also disappears without a trace. When London is once again attacked in 1944, Harrison unexpectedly returns to Stella’s house. The novel concludes with readers left in suspense, pondering the future of these characters. pp g As the novel develops, it can be found that despite their denial, the Naylors are deeply affected by the looming presence of war. Sir Richard acknowledges that Ireland is altogether “too full of soldiers” which unsettles the people (25). The lorry of the patrols takes pleasure in “crawling with such a menace along the boundary” (31). Lawrence worries that Lois “would get assaulted by Black and Tans if she went alone, or by sinister patriots” (55). People talk about the raid at tea between tennis matches: “a R.I.C. barracks at Ballydrum had been attacked and burnt out after a long defense” and two defenders were burnt inside it while “the others shot coming out” (47). Later, the postman delivers news of another raid in the Brittas direction, and this time “the Black and Tans had been ﬁred on” (78). ARTICLE In the introduction to Bowen’s Court and Seven Winters, Hermione Lee (2017) also suggests, “Bowen’s Court brings to consciousness a long tradition of unawareness. The Bowens were largely oblivious of their ambivalent position in Ireland” (x). The focus of The Last September lies in the examination of Anglo-Irish psychology, speciﬁcally the tendencies of denial and anxiety, before the occurrence of the traumatic event at the end of the novel, wherein the Big Houses, including Danielstown, are set ablaze by the Irish rebels. This exploration mirrors Saint-Amour’s account of “pre-traumatic stress syndrome”, which emphasizes Similarly, in The Last September, the Anglo-Irish characters identify themselves as Irish, adopting a policy of “not-noticing” and lacking a proper understanding of their position in Ireland. Despite the intense political situation during the Irish War of Independence, they continue to indulge in parties and daily life, HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2024) 11:210 \| https://doi.org/10.1057/s41599-024-02613-5 4 ARTICLE HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| https://doi.org/10.1057/s41599-024-02613-5 (2013) examines the relationship between Bowen’s portrayal of wartime England and Anglo-Ireland, suggesting that “Bowen’s stories anticipate a historical recurrence of Anglo-Ireland’s fate in post-war Britain” (31). Davis argues that Bowen’s understanding of the Second World War is “rooted in her own experience and understanding of Anglo-Ireland’s twilight years” (33). The psy- chological dislocation experienced by Londoners during the war mirrors the sense of homelessness previously encountered by the Anglo-Irish Ascendancy. Moynahan (1989) contends that “a London of houses on ﬁre from incendiary bombs and streets torn apart by high explosives was also a reliving of [Bowen’s] late teens and early twenties when country houses and whole towns in Ireland were raided and burnt out by the Black and Tans, Sinn Fein, and the IRA” (74). Therefore, Bowen’s wartime narratives create a dialog between London houses and the Big Houses of the Anglo-Irish Ascendancy. the manifestation of traumatic symptoms before, rather than after, the event transpires. The Naylors possess knowledge of the political turmoil unfolding in their surroundings, yet they habi- tually choose to dismiss it. For instance, when Francie expresses concerns about being shot at by Sinn Feiners while preparing to sit on the steps in front of the Big House at twilight, Sir Richard ﬁnds amusement in her worries. This amusement is then shared by the others, resulting in Sir Richard teasingly remarking, “You’re getting very English, Francie! Isn’t Francie getting very English?” (23). ARTICLE This narrative strategy of revi- siting the past and then returning to the present mirrors Stella’s own process of memory, as she reﬂects on her past to comprehend her current circumstances. When contemplating the possibility of Robert’s treachery, she revisits her initial memories of him, which are intermingled with recollections of London during the Blitz. Again, after Robert’s death, Stella reevaluates and analyses her relationship with him. During their ﬁnal meeting, he tells her, “You’ll have to re-read me backwards, ﬁgure me out—you will have years to do that in, if you want to” (270). Stella’s endeavor to understand the present situation is thus achieved through her ret- rospective examination of the past. p y g In The Heat of the Day, wartime London is characterized by incessant air raids that inﬁltrate every facet of daily existence. The alternating cycles of day and night embody the oscillation between hope for survival and fear of death: “[t]he night behind and the night to come met across every noon in an arch of strain” and “[t]o work or think was to ache” (91). For the Londoners, “fatigue was the one reality” and they “dared not envisage sleep” (91). “Apathetic, the injured and dying in the hospitals watched the light change on walls which might fall tonight” (91); “Strangers saying ‘Good night, good luck’, to each other at street corners, as the sky ﬁrst blanched then faded with evening, each hoped not to die that night, still more not to die unknown” (92). Londoners ﬁnd themselves caught in the fear arising from the anticipation of death, an anticipation that possesses the capacity to traumatize them during the air raids, enveloping them in anxiety and dread. y Suffering from anticipatory trauma, the characters in the novel exhibit traumatic symptoms, such as dissociating themselves from the war and appearing oblivious to the bombings occurring around them. In The Heat of the Day, Stella and Harrison are forced to acclimatize themselves to unpredictable violence. This violence becomes a recurring occurrence, ultimately transforming into a routine—a phenomenon referred to by Saint-Amour as “[weaponized] anticipation”. This anticipation makes the future seem “a predetermined site of catastrophic violence and therefore capable of inﬂicting damage in the present” (8). Stella and Har- rison regard the air raids happening around the ﬂat as a daily routine and are dissociated from the bombing and gunﬁre. As Carola M. ARTICLE Kaplan (2013) suggests, this dissociation serves as “a response to repeated trauma and a defense against retraumati- zation” (470). However, this detachment emerges due to the fear of future air raids. The novel’s sole depiction of an air raid takes place in the ﬁnal chapter when Harrison visits Stella’s new ﬂat 2 years later. Harrison stands “in the middle of a street, otherwise empty, illuminated by a chandelier ﬂare” and “[d]uring the pulse of silence between the overhead throbbing and the bark of the guns, the ﬂare made the street like a mirrored drawing-room” (Bowen The Heat of the Day 1998b 315). As the bombing ensues, “the sky to the east reﬂected ﬂamingo-pink nobody could have taken to be the dawn, the west was jagged with ﬂames” (315). In contrast to the gruesome and violent scene portrayed in Bowen’s essay, “London, 1940”, this novel describes the bombing as an ordinary facet of life. Upon entering Stella’s ﬂat, Harrison engages in conversation with her while two volleys of gunﬁre occur out- side. However, neither of them seems to pay much attention to the sounds. Only the neighbor’s cat seems restless. As Stella searches for the cat under the furniture, Harrison, silenced by the gunﬁre, “seemed at the same time to feel exonerated from making any secret of the fact that he was following her with his eyes” (317). After another burst of gunﬁre, Stella leans back against the cushions, acquiescent. Apart from the shaking of the lamp bulb and window frames, the room remains a secluded sanctuary of Robert D. Stolorow (2011) describes the disruption of time in trauma: Robert D. Stolorow (2011) describes the disruption of time in trauma: Experiences of emotional trauma become freeze-framed into an eternal present in which one remains forever trapped, or to which one is condemned to be perpetually returned through the portkeys supplied by life’s slings and arrows. In the region of trauma, all duration or stretching along collapses; past becomes present, and future loses all meaning other than endless repetition (55). For Stolorow, “Dissociation just is traumatic temporality” (61). This traumatic temporality is experienced by Stella, whose sense of helplessness is exacerbated by the war to the extent that time itself becomes an enemy that she vigorously resists. ARTICLE ARTICLE HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| https://doi.org/10.1057/s41599-024-02613-5 Illustrating the traumatic force of anticipation, Saint-Amour introduces Lewis Mumford’s work, which investigates a “collec- tive psychosis” engendered by the war. Throughout the war, fear becomes an ingrained part of everyday life, leading to a collective psychosis akin to the one that active warfare itself may foster (6). According to Mumford, the disasters that have already arrived and the disasters that might arrive have equal powers to inﬂict psychic damage and anticipation will be a condition in which one can “die a thousand deaths” (7). Mumford contends that the violence most particular to the air raid arrives along the temporal vectors of preparation and expectation and “time—and antici- pation in particular—has become a new medium for delivering injury” (7). Potential disasters and expectations of them can also inﬂict psychological harm. silence beneath the resounding sky illuminated by the ﬂare’s pale light (319). Stella’s room seems to be isolated from the rest of the world so that the bombing in the distance and the gunﬁre in the vicinity become the background—“[t]he guns, made fools of, died out again, askance” (319). Both Harrison and Stella appear unmoved by the gunﬁre outside her ﬂat. Harrison “sat through the minutes with a concentrated appearance of hearing nothing” (319). The only living being inﬂuenced by the war is the cat, whose “fur seemed to shrink and dampen as a stick of bombs fell diagonally across the middle distance” (317). g y The trauma caused by the war is also evident in the dislocation of time, which is exhibited by two instances of reversed chronology identiﬁed by Pong (2009). The novel begins in September 1942, with Harrison encountering Louie Lewis in a park and subsequently meeting Stella in her ﬂat, informing her of Robert’s espionage activities. It then shifts back to cousin Francis Morris’s funeral in May 1942, where Stella ﬁrst encounters Harrison, before the nar- rative returns to London during the peak of the Blitz in 1940, when Stella encounters Robert for the ﬁrst time. Another reversal in chronology occurs toward the end of the novel, where Bowen initially portrays the characters’ actions several months after Robert’s death, before retracing back to the inquest into his death and the consequences that follow. HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2024) 11:210 \| https://doi.org/10.1057/s41599-024-02613-5 ARTICLE Living through the Blitz, characters like Stella ﬁnd themselves caught in what Pong (2009) refers to as “a traumatic anticipation”, encompassing “both the anticipation of trauma (the bombings), as well as an antici- pation which was itself psychologically traumatic” (para. 17). They are haunted by memories of past bombings while living in fear of future air attacks. Consequently, they exist in a state of suspension, oscillating between the traumatic events that have already occurred and the anticipatory threat of future trauma. This state of suspension experienced by the characters, being caught between past bombings and the anticipation of future air raids, aligns with Saint-Amour’s concept of “pre-traumatic stress syndrome”. As mentioned earlier in this essay, Saint-Amour’s concept combines elements from both Freudian and Min- kowskian approaches and “see in the experience of an apparently inescapable future, or of a wounding anticipation, something in addition to a symptom of past repression” (Saint-Amour 20). This approach pays particular attention to the traumatizing effects of anticipatory forces, without disregarding the impact of past traumas. Similarly, in The Heat of the Day, characters during the Blitz are more traumatized by the anticipation of future air raids than by the air raids that have already taken place. “War-climate” in The Heat of the Day In her wartime ﬁction, The Heat of the Day, Bowen explores the psychological trauma experienced by Londoners during the Sec- ond World War. The traumatic sense of dispossession is remi- niscent of the experiences felt in Anglo-Irish society during the Irish War of Independence, as depicted in The Last September. Bowen transfers the class anxieties and fears of social and cultural change from the Anglo-Irish context to the British setting. Davis 5 5 HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2024) 11:210 \| https://doi.org/10.1057/s41599-024-02613-5 Notes 1 The Freudian model of trauma ﬂourished in the 1990s and was advocated by a group of critics at Yale University, including Cathy Caruth, Geoffrey Hartman (2003), Shoshana Felman and Dori Laub (1992), who were devoted to applying psychoanalytic ideas about psychological trauma, especially Freud’s, to the analysis of literary texts. 2 For scholarship on war trauma in modernist writing, see Smith’s (2000) The Second Battleﬁeld: Women, Modernism and the First World War; Rae’s (2007) Modernism and Mourning; Crosthwaite’s (2009) Trauma, Postmodernism and the Aftermath of World War II; Goodspeed-Chadwick’s (2011) Modernist Women Writers and War: Trauma and the Female Body in Djuna Barnes, HD, and Gertrude Stein; Clewell’s (2013) Modernism and Nostalgia: Bodies, Locations, Aesthetics; Saint-Amour’s (2015) Tense Future: Modernism, Total War, Encyclopedic Form; Bonikowski’s (2016) Shell Shock and the Modernist Imagination: The Death Drive in Post-World War I British Fiction; MacKay’s (2017) Modernism, War, and Violence. It is worth noting that some critics tend to study war trauma represented by male and female writers respectively. For British male writers’ writing about war trauma, see Krockel’s (2011)War Trauma and English Modernism: T. S. Eliot and D. H. Lawrence; also, Freedman (2014) studies the image of a dying man to explore the questions about modernity and masculinity. In “Modernism and Trauma”, Henke (2010) studies the historical trauma of the First World War demonstrated by some British and American female writers, who present not only shell-shock suffered by soldiers, but also trauma of noncombatants enduring “an overwhelming sense of loss, bereavement, anxiety, and emotional rupture” in “domestic isolation” (161). For British women writers writing about the Second World War, see Phyllis Lassner (1998) and Rumbarger (2006). Furthermore, Stella also demonstrates the retrospectively ampliﬁed phenomenon of “anticipatory bukimi” in her ﬂashback. Saint-Amour observes that those who survived the bombing in Hiroshima and recollected their anticipatory bukimi years after the bombing “may have retrospectively ampliﬁed their memories of weird expectation, perhaps as a way of attempting to master an incommensurable and singular event by installing it within a narrative of causality, con- tinuity, even prophecy” (2). In Chapter Five of the novel, there is a narrative detour to the ﬁrst air raids in London during September 1940, when Stella initially encounters Robert. Stella recalls a scene that anticipates Robert’s death: “That was the morning when, in the instant before opening her eyes, she saw Robert’s face with a des- pairing hallucinatory clearness. Conclusion This essay explores the ways in which Bowen’s writing about wars challenges the ﬁeld of trauma studies, which has concentrated almost solely on the aftermath of catastrophes or violence. Bowen’s portrayal of trauma differs from the Freudian model insofar as, where Freud considers symptoms as post-traumatic, that is, symptoms appear after a traumatic event happens, Bowen presents characters’ psychological trauma as pre-traumatic, namely, trau- matic conditions arise before imminent violence occurs. As dis- cussed in this essay, the characters in Bowen’s novels exhibit traumatic symptoms, such as traumatic repression, before the event happens, such as the burning of the Big House in The Last Sep- tember. Additionally, characters like Stella in The Heat of the Day are more affected by the potential bomb explosion than by the bomb that has already detonated. In both novels, characters live in a particular present—living toward a future that seems to be pre- determined by some retraceable reasons. Their traumatic symptoms like bukimi and anxiety exist in the shadow of a future event, not in the wake of a past one. Studying Bowen’s writing on trauma is of signiﬁcance to understanding trauma in modernist literature. Researching trauma in modernist literature can also be beneﬁcial to the work of mourning in the last decades of the twentieth century and the turn of the twenty-ﬁrst century, marked by such cata- clysmic events as the AIDS epidemic, racially motivated genocide, terrorist attacks, and others. Reconsidering the representations of trauma in modernist literature can facilitate understanding trau- matic events or catastrophes in our current time. y y Upon initial analysis, the disruption of time and the linear nar- ration in the text manifest a Freudian model of trauma, whereby “the traumatic past remains transgressively present as revenant, haunting, or possession, dominating the present rather than receding as it should into the past” (Saint-Amour 14–15). As Pong proposes, Stella’s compulsion to reread backward is “itself an interminable, inexhaustible exercise that emphasizes the impossi- bility of returning to, and fully elucidating, that past” (para. 16). Nevertheless, as the narrative unfolds, it becomes evident that Stella adopts the strategy of forgetting in order to survive: “Most of all there is something one has got to forget—that is, if it is to be possible to live. The more wars there are, I suppose, the more we shall learn how to be survivors” (Bowen The Heat of the Day 1998b 317). ARTICLE Throughout the nights of air raids, she tosses and turns, unable to ﬁnd solace: “Those were a series of nights in which one slept, if at all, with an abandonment in itself exhausting; but no kind of sleep accounted for the distance she felt between herself and yesterday—and, indeed, between herself and today” (Bowen The Heat of the Day 1998b 97). For Stella, there is no clear boundary between past and present, as “most of all the dead…made their anonymous presence—not as today’s dead but as yesterday’s living” (91). Stella loses her grasp on the concept of time and her surroundings, as “nothing she saw or touched gave token of even its own reality: her wrist watch seemed to belie time; she fancied it had lost hours during the night, that this might be midday, the afternoon—her ﬁrst act, as she hurried into the street, was to look about in vain for a public clock” (97). Time for Stella relinquishes its objectivity and neutrality. During the inquest, Stella’s inability to reorganize and recount her life with Robert in a coherent and chronological manner highlights the manifestation of her traumatic psychology. Her HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2024) 11:210 \| https://doi.org/10.1057/s41599-024-02613- 6 ARTICLE HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| https://doi.org/10.1057/s41599-024-02613-5 testimony is characterized by disorder and disjointedness, exem- pliﬁed by her statements such as “He was determined to leave by the roof…No, I cannot suggest any other reason, but one never knows…For two years.—Two years and two months: we met in September 1940…Yes, we saw one another frequently…I’m afraid I cannot say…No, I don’t think I remember any quarrels… I suppose we did not notice how time was passing; the war is a very inter- esting subject” (Bowen The Heat of the Day 1998b 302). By fol- lowing Stella’s account, readers are compelled to read in a non- chronological manner. Stella’s act of reproducing the past in non- chronological order, along with her strategy of not-noticing and forgetting, signiﬁes her difﬁculties in fully understanding the past and integrating it into her personal history. Failing to understand Robert’s death, Stella is compelled to revisit the past event. ARTICLE With each revisitation, she adds her own understanding of the event, as she remarks that they go on “piecing and repiecing [the events] together to try and make out something they had not had time to say—possibly even had not had time to know” (317). Conclusion Hence, her very inability to recount her story with Robert is a pre-trauma symptom, because she is anxious about the future catastrophe that might happen and this anxiety prompts her to repress past traumas in order to survive. Received: 12 September 2022; Accepted: 8 January 2024; Notes When she did open her eyes, it had been to stare around her room in the sunshine certain that he was dead. Something ﬁnal had happened, in any case” (Bowen The Heat of the Day 1998b 93). Additionally, “The non-existence of her win- dow, the church-yard hush of the square, the grit which had drifted on to her dressing-table all became ominous for the ﬁrst time” (93). The restaurant where Stella and Robert frequently meet each other is “closed—the street roped off: some nonsense about a time-bomb” (97). The unexploded bomb that could go off at any moment highlights the threat and danger of time, fostering an expectation of unexpected terror. After they sit in a strange restaurant, “[t]he gilt- faced clock in the sunburst on the restaurant’s wall had, like others in London, been shock-stopped” and “their two wrist watches—which, in the time to come, were to come at some kind of relationship of their own by never perfectly synchronizing—found it, respectively, to be a minute before and a minute after half past two” (99). During the war, Stella’s perception of time becomes distorted. Her memory is ampliﬁed with weird expectations, akin to those experienced by the survivors of the Hiroshima bombing. 3 The motif of trauma in Bowen’s work is studied by Jessica Gildersleeve, in Elizabeth Bowen and the Writing of Trauma: The Ethic of Survival (2014). Gildersleeve explores the linguistic and psychological effects of trauma in Bowen’s work and argues Bowen’s writing echoes Caruth’s understanding of trauma, which presents the symptoms of traumatic events as post-traumatic. The present essay presents a different idea from Gildersleeve’s. Reprints and permission information is available at http://www.nature.com/reprints Reprints and permission information is available at http://www.nature.com/reprints Reprints and permission information is available at http://www.nature.com/reprints G ( ) J g S ( ) Henke SA (2010) Modernism and trauma. In: Linett MT (ed) The Cambridge companion to modernist women writers, vol 2. Cambridge University Press, Cambridge, p 160–171 Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. g p Kaplan CM (2013) ‘Sudden holes in space and time’: trauma, dissociation, and the precariousness of everyday life. Psychoanal Inq 33(5):467–478 Krockel C (2011) War trauma and English modernism: T.S. Eliot and Dh Lawr- ence. Palgrave Macmillan, Hampshire, UK Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. Lassner P (1998) British women writers of World War II: battlegrounds of their own. Palgrave Macmillan, London Lee H (2017) Introduction. In: Lee H (ed) Bowen’s court and seven winters: memories of a Dublin childhood. Vintage, London g MacKay M (2017) Modernism, war, and violence. Bloomsbury Publishing, London h ( ) l b h l h acKay M (2017) Modernism, war, and violence. Bloomsbury Publi MacKay M (2017) Modernism, war, and violence. Bloomsbury Publishing, London Moynahan J (1989) Elizabeth Bowen: Anglo-Irish post-mortem. Raritan 9:68–97 Moynahan J (1989) Elizabeth Bowen: Anglo-Irish post-mortem. Raritan 9:68–97 Peters E, Richards C (2021) Early modern trauma: Europe and the Atlantic world. Ethical approval Ethical approval Ethical approval was not required as the study did not involve human participants. y p p y gy Hogarth Press and the Institute of Psycho-Analysis, London, pp 7–23 Informed consent g y y pp Gildersleeve J (2014) Elizabeth Bowen and the writing of trauma: the ethics of survival. Editions Rodopi, New York This article does not contain any studies with human participants performed by any of the authors. This article does not contain any studies with human participants performed by any of the authors. Glendinning V (1998) Introduction. In: The Last September. Random House, London, p 1–6 Goodspeed-Chadwick J (2011) Modernist women writers and war: trauma and the female body in Djuna Barnes, Hd, and Gertrude Stein. LSU Press, Baton Rouge, LA ARTICLE HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| https://doi.org/10.1057/s41599-024-02613-5 Saint-Amour PK (2015) Tense future: modernism, total war, encyclopedic form. Oxford University Press, New York Bonikowski W (2016) Shell shock and the modernist imagination: the death drive in post-World War I British ﬁction. Routledge, New York p g Bowen E (1950) Collected impressions. Longmans, London y Smith AK (2000) The second battleﬁeld: women, modernism and the First World War. Manchester University Press Bowen E (1986) The mulberry tree: writings of Elizabeth Bowen. Virago, London Stolorow RD (2011) World, affectivity, trauma: Heidegger and post-Cartesian psychoanalysis. Routledge, New York Bowen E (1998a) The last September. Random House, London Bowen E (1998b) The heat of the day. Vintage, London y y g Wasson S (2010) Urban gothic of the Second World War: dark London. Palgrave Macmillan, Basingstoke Bowen E (2017) Bowen’s court and seven winters: memories of a Dublin childhood. Vintage, London Caruth C (1995) Trauma: explorations in memory. Johns Hopkins University Press, Baltimore Wurtz JF (2010) Elizabeth Bowen, modernism, and the spectre of Anglo-Ireland. Estud Irlandeses 5:119–128 Clewell T (2013) Modernism and nostalgia: bodies, locations, aesthetics. Palgrave Macmillan, England Acknowledgements g Crosthwaite P (2009) Trauma, postmodernism and the aftermath of World War II. Palgrave Macmillan, New York This research is supported by Humanities and Social Sciences Youth Foundation of Ministry of Education of China. Funding No. 23YJC752006. g Davis TS (2013) Elizabeth Bowen’s war gothic. Textual Pract 27(1):29–47 Esty J (2014) Virgins of empire: the last September and the anti-developmental plot. Unseasonable youth: modernism, colonialism, and the ﬁction of development. Oxford University Press, New York The author solely contributed to this work. p y Felman S, Laub D (1992) Testimony: crises of witnessing in literature, psycho- analysis, and history. Routledge, New York analysis, and history. Routledge, New York References Baer U (2007) Modernism and trauma. In: Eysteinsson Astradur, Liska Vivian (eds) Modernism. John Benjamins Publishing, Philadelphia, p 301–318 Blickle B (2019) Ruined landscapes in transatlantic modernism. PhD, Stony Brook University, New York HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2024) 11:210 \| https://doi.org/10.1057/s41599-024-02613-5 COMMUNICATIONS \| (2024) 11:210 \| https://doi.org/10.1057/s41599-024-02613-5 7 The author declares no competing interests. y g Freud S (1939) Moses and monotheism. Trans. Katherine Jones. Vintage, New Yor Freud S (1939) Moses and monotheism. Trans. Katherine J Freud S (1962) Beyond the pleasure principle. In: Strachey J (ed) The standard edition of the complete psychological works of Sigmund Freud, volume XVIII (1920- 1922): Beyond the Pleasure Principle Group Psychology and Other Works. The Hogarth Press and the Institute of Psycho-Analysis, London, pp 7–23 Author contributions plot. Unseasonable youth: modernism, colonialism, and the ﬁction of development. Oxford University Press, New York Additional information Correspondence and requests for materials should be addressed to Qiong He. Correspondence and requests for materials should be addressed to Qiong He. g Hartman G (2003) Trauma within the limits of literature. Eur J Engl Stud 7(3):257–27 Reprints and permission information is available at http://www.nature.com/reprints Competing interests Freedman A (2014) Death, men, and modernism: trauma and narrative in British ﬁction from Hardy to Woolf. Routledge, London The author declares no competing interests. Reprints and permission information is available at http://www.nature.com/reprints University of Nebraska Press, Lincoln y Pong B (2009) Space and Time in the Bombed City: Graham Greene’s the Ministry of Fear and Elizabeth Bowen’s The Heat of the Day. Literary London: Interdisciplinary Studies in the Representation of London 7:24 Rae P (2007) Modernism and mourning. Bucknell University Press, Lewisburg b ( ) k d ’ d Rumbarger L (2006) Housekeeping: women modernists’ writings on war and home. Womens Stud Interdiscip J 35(1):1–15 © The Author(s) 2024 HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2024) 11:210 \| https://doi.org/10.1057/s41599-024-02613-5 8 8
https://openalex.org/W4213049494	https://hal.science/hal-03594154/file/materials-15-01463.pdf	English	null	Synthesis and Characterization of TiO2 Nanotubes (TiO2-NTs) with Ag Silver Nanoparticles (Ag-NPs): Photocatalytic Performance for Wastewater Treatment under Visible Light	Materials	2,022	cc-by	8,366	Synthesis and Characterization of TiO(2) Nanotubes (TiO(2)-NTs) with Ag Silver Nanoparticles (Ag-NPs): Photocatalytic Performance for Wastewater Treatment under Visible Light Synthesis and Characterization of TiO(2) Nanotubes (TiO(2)-NTs) with Ag Silver Nanoparticles (Ag-NPs): Photocatalytic Performance for Wastewater Treatment under Visible Light To cite this version: Achraf Amir Assadi, Sarra Karoui, Khaled Trabelsi, Anouar Hajjaji, Walid Elfalleh, et al.. Synthesis and Characterization of TiO(2) Nanotubes (TiO(2)-NTs) with Ag Silver Nanoparticles (Ag-NPs): Photocatalytic Performance for Wastewater Treatment under Visible Light. Materials, 2022, 15 (4), pp.1463. 10.3390/ma15041463. hal-03594154 Distributed under a Creative Commons Attribution 4.0 International License Abstract: In this work, we present the inﬂuence of the decoration of TiO2 nanotubes (TiO2-NTs) with Ag silver nanoparticles (Ag-NPs) on the photocatalysis of emerging pollutants such as the antibiotic diclofenac sodium. The Ag-NPs were loaded onto the TiO2-NTs by the anodization of metallic tita- nium foils. Diclofenac sodium is an emerging pollutant target of the pharmaceutical industry because of its negative environmental impact (high toxicity and conﬁrmed carcinogenicity). The obtained Ag-NP/TiO2-NT nanocomposites were characterized by X-ray diffraction (XRD), photoluminescence spectroscopy (PL), scanning electron microscopy (SEM), transmission spectroscopy (TEM), and X-ray photoelectron spectroscopy (XPS). In order to study the photocatalytic behavior of Ag-NPs/TiO2-NTs with visible cold LEDs, the possible photocatalytic mechanism of antibiotic degradation with reactive species (O2◦−and OH◦) was detailed. Moreover, the Langmuir–Hinshelwood model was used to correlate the experimental results with the optimized catalyst. Likewise, reuse tests showed the chemical stability of the catalyst. Citation: Assadi, A.A.; Karoui, S.; Trabelsi, K.; Hajjaji, A.; Elfalleh, W.; Ghorbal, A.; Maghzaoui, M.; Assadi, A.A. Synthesis and Characterization of TiO2 Nanotubes (TiO2-NTs) with Ag Silver Nanoparticles (Ag-NPs): Photocatalytic Performance for Wastewater Treatment under Visible Light. Materials 2022, 15, 1463. https://doi.org/10.3390/ma15041463 Academic Editor: Andrea Petrella Received: 9 January 2022 Accepted: 2 February 2022 Published: 16 February 2022 Citation: Assadi, A.A.; Karoui, S.; Trabelsi, K.; Hajjaji, A.; Elfalleh, W.; Ghorbal, A.; Maghzaoui, M.; Assadi, A.A. Synthesis and Characterization of TiO2 Nanotubes (TiO2-NTs) with Ag Silver Nanoparticles (Ag-NPs): Photocatalytic Performance for Wastewater Treatment under Visible Light. Materials 2022, 15, 1463. https://doi.org/10.3390/ma15041463 Keywords: Ag-NPs/TiO2 nanotubes; batch reactor; wastewater treatment; reactive species; kinetic modeling Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. hraf Amir Assadi 1,2,3,, Sarra Karoui 1, Khaled Trabelsi 4, Anouar Hajjaji 4, Walid Elfalleh 5 , hraf Ghorbal 1 , Mounir Maghzaoui 2,3 and Aymen Amin Assadi 6, Achraf Amir Assadi 1,2,3,, Sarra Karoui 1, Khaled Trabelsi 4, Anouar Hajjaji 4, Walid Elfalleh 5 , Achraf Ghorbal 1 , Mounir Maghzaoui 2,3 and Aymen Amin Assadi 6, 1 Research Unit Advanced Materials, Applied Mechanics, Innovative Processes and Environment, Higher Institute of Applied Sciences and Technology of Gabes (ISSAT), University of Gabes, Gabes 6072, Tunisia; sarra.karoui20@gmail.com (S.K.); achraf.ghorbal.issat@gmail.com (A.G.) g g g 2 Industries Chimiques du Fluor—Gabes Plant, 06 Rue Amine El Abbassi, Tunis 1002, Tunisia; m.maghzaoui@icf.ind.tn g g g 2 Industries Chimiques du Fluor—Gabes Plant, 06 Rue Amine El Abbassi, Tunis 1002, Tunisia; m.maghzaoui@icf.ind.tn 3 Industrial Zone Gabes Port, Gabes 6071, Tunisia 4 Laboratoire de Photovoltaïque, Centre de Recherches et des Technologies de l’Energie, Technopole de Borj-Cédria, BP 95, Hammam-Lif 2050, Tunisia; khaled0984@hotmail.com (K physicshajjaji@gmail.com (A.H.) gy y g g University of Gabes, Gabes 6072, Tunisia; walid.elfalleh@fst.rnu.tn 6 École Nationale Supérieure de Chimie de Rennes, CNRS, ISCR (Institut des Sciences Chimiques de Rennes)–UMR 6226, Universite de Rennes, F-35000 Rennes, France * Correspondence: achraf.assadi@gmail.com (A.A.A.); aymen.assadi@ensc-rennes.fr (A.A.A.); Tel.: +216-54-013-728 (A.A.A.); +33-22-32-38-152 (A.A.A.) p , , ( q Rennes)–UMR 6226, Universite de Rennes, F-35000 Rennes, France * Correspondence: achraf.assadi@gmail.com (A.A.A.); aymen.assadi@ensc-rennes.fr (A.A.A.); Tel.: +216-54-013-728 (A.A.A.); +33-22-32-38-152 (A.A.A.) * Correspondence: achraf.assadi@gmail.com (A.A.A.); aymen.assadi@ensc-rennes.fr (A.A.A.); Tel.: +216-54-013-728 (A.A.A.); +33-22-32-38-152 (A.A.A.) HAL Id: hal-03594154 https://hal.science/hal-03594154v1 Submitted on 2 Mar 2022 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License materials 1. Introduction Pharmaceuticals are undoubtedly one of the major advances in modern medicine. Nowadays, they are widely prescribed in humans and animals, both for curative and preventive purposes. However, excessive use can lead to the emergence of resistant bacteria through mechanisms such as (i) enzymatic degradation of antibacterial drugs, (ii) alteration of bacterial proteins that are antimicrobial targets, and (iii) modiﬁcation in membrane permeability to antibiotics [1–3]. In addition, these substances are not completely metabolized by organisms. The immediate consequence is that a certain unmetabolized amount ends up directly in the soil and in surface water [4,5]. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Thus, the sources of pharmaceutical substances in the environment are hospital and domestic discharges, which end up in wastewater treatment plants where they are not https://www.mdpi.com/journal/materials Materials 2022, 15, 1463. https://doi.org/10.3390/ma15041463 Materials 2022, 15, 1463 2 of 12 completely degraded and therefore discharged into surface water. The concentrations in the receiving environment can thus vary according to the biodegradability of the molecules as well as the capacity of the water treatment stations to eliminate or transform them, as these are designed and mainly sized to treat the so-called physicochemical parameters “conventional” (suspended solids, BOD5, DCO, nitrogen, phosphorus) [5]. The immediate consequence of such a situation is that for the past ﬁfteen years, drug residues have been found in river water because some treatment plants are still not equipped with effective processes to retain or degrade them [6–10]. To date, several emerging and promising technologies provide a solution to this problem. Among these, the heterogeneous photocatalysis with visible light reveals inter- esting prospects in terms of degradation/mineralization of compounds, with low energy consumption. Recent studies on model pharmaceutical compounds indicate that this technology has been explored and developed. In the past decade, titanium dioxide (TiO2) has been one of the most studied materials thanks to its unique chemical and physical properties, including its high chemical stability, high resistance to photo corrosion, and its low cost [11]. Nanostructured TiO2 has also been used in many applications such as sensors [12], photocatalysis [13–16], and solar energy conversion [17]. 1. Introduction TiO2 nanotubes have attracted particular attention because of their high speciﬁc surface area [18], high photoactivity [19], and the rapid transfer rate of holes and electron–hole pairs (e−/h+) photogenerated along the nanotubes [20–22]. However, two major factors limit the performance of TiO2. On the one hand, due to its wide forbidden energy band (3.2 eV for anatase TiO2), the optical absorption of TiO2 is limited to the UV spectrum. On the other hand, the photogenerated (e−/h+) pairs exhibit a high rate of recombination due to a high density of crystal defects [23]. To overcome these problems, many strategies have been established, such as doping TiO2 (metallic or non-metallic dopants) [13,17] and coupling with other semiconductors [19,20,24], which could expand absorption under visible light and improve the lifetime of the photogenerated (e−/h+) pairs. Recently, it has been shown that the decoration of TiO2 nanotubes with noble metal nanoparticles can increase their absorption in the visible range, thanks to the surface plasmon resonance (SPR) [25] induced in the metallic nanoparticles in the presence of a light wave. The SPR of the noble metal can be adjusted by controlling the size, shape, and dispersion of the nanoparticles. Nanoparticles of silver (Ag) are used to improve the photoactivity of TiO2 nanotubes due to the favorable arrangement of the energy level, which allows the electrons excited by SPR on the Ag nanoparticles to be transferred into the TiO2 nanotubes in the presence of visible light. In addition, chemical pathways have been used to decorate TiO2 nanotubes with Ag nanoparticles. However, using these techniques cannot control the size and shape of Ag nanoparticles, which can easily accumulate. In addition, the organic agents employed in the growth of Ag nanoparticles can affect the electrical and optical properties of the composite. p p p p In this work, Ag nanoparticles were deposited onto TiO2 nanotubes using the pho- toreduction process, which allows the size of nanoparticles to be controlled by adjusting the UV irradiation time. TiO2 nanotubes decorated with Ag nanoparticles were used as photoanodes in a photoelectrochemical system, showing a signiﬁcant improvement in photo-conversion efﬁciency. Moreover, nanotubes (TiO2-NTs) decorated with Ag sil- ver nanoparticles (Ag-NPs) were used for the photocatalytic degradation of emerging pollutants, such as the antibiotic diclofenac sodium, with visible cold LEDs. 2. Setup The samples were 99.99% purity metal (Ti) plates with a surface area of 2 × 1.5 cm2 and a thickness of approximately 0.5 mm. They had identical characteristics to maximize the probability of the same results. In order to achieve surface activation and to avoid any adhesion problems, the samples were ﬁrst polished using abrasive papers with different grain sizes: 320, 400, 600, 800, 1000, 1200, and 2000. Next, the samples were rinsed with acetone, ethanol, and then bi-distilled water for 10 min in an ultrasonic bath to remove Materials 2022, 15, 1463w 3 of 12 ed in impurities and foreign materials introduced in the polishing step. Finally, the samples were air-dried for some time. Electrochemical experiments were conducted in the laboratory at room temperature. All samples were prepared by anodic oxidation under identical experimental conditions. This was performed in an electrolyte cell containing 100 mL ethylene glycol, 1% ammonium ﬂuoride (NH4F), and 2% water (H2O). The anodization was 120 min in duration under a ﬁxed voltage of 60 V at a temperature maintained at approximately 25 ◦C. The samples obtained were calcined for 1 h at 400 ◦C. e pe i e ta co ditio s is was pe o ed i a e ect o yte ce co tai ethylene glycol, 1% ammonium fluoride (NH4F), and 2% water (H2O). on was 120 min in duration under a fixed voltage of 60 V at a temperat ed at approximately 25 °C. The samples obtained were calcined for 1 h at 400 photoreduction method consists of immersing the TiO2 nanotubes in a solu nitrate (AgNO3) of 0.1 M concentration for 24 h in the dark to allow the silver i orbed on the TiO2 surface Afterward the samples were rinsed with water pp y p The photoreduction method consists of immersing the TiO2 nanotubes in a solution of silver nitrate (AgNO3) of 0.1 M concentration for 24 h in the dark to allow the silver ions to be adsorbed on the TiO2 surface. Afterward, the samples were rinsed with water and then immersed in methanol under UV illumination (λ = 256 nm) for 10 and 20 min. Under the effect of UV radiation, silver ions (Ag) reduce to metallic form. By increasing the UV irradiation time, Ag nanoparticles increase in size and form agglomerates [1]. orbed on the TiO2 surface. 2. Setup Afterward, the samples were rinsed with water mersed in methanol under UV illumination (λ = 256 nm) for 10 and 20 min. Un of UV radiation, silver ions (Ag) reduce to metallic form. By increasing the on time, Ag nanoparticles increase in size and form agglomerates [1]. synthesized catalysts were placed in Petri dishes containing the antibi to be treated The catalysts were illuminated by visible cold LEDs T The synthesized catalysts were placed in Petri dishes containing the antibiotic solution to be treated. The catalysts were illuminated by visible cold LEDs. This experimental setup is shown in Figure 1. Antibiotic concentration was also monitored by spectrophotochem- istry at 270 nm. to be treated. The catalysts were illuminated by visible cold LEDs. T ental setup is shown in Figure 1. Antibiotic concentration was also monitored hotochemistry at 270 nm. The micro-reactor in a Petri dish used for the treatment of antibiotics by photocataly Figure 1. The micro-reactor in a Petri dish used for the treatment of antibiotics by photocatalysis. he micro-reactor in a Petri dish used for the treatment of antibiotics by photocatal Figure 1. The micro-reactor in a Petri dish used for the treatment of antibiotics by photocatalysis. y p y to-degradation experiments were performed in order to estimate the cata es of TiO2-NTs and Ag-NPs/TiO2-NTs at various deposition times (10 min e experimental setup consisted of the catalysts (dimensions: 1.2 × 2.5 cm) pla hotocatalytic batch reactor. An initial antibiotic concentration of 1 mg/L The lamp was lighted after reaching the adsorption–desorption equilibr Photo-degradation experiments were performed in order to estimate the catalytic efﬁciencies of TiO2-NTs and Ag-NPs/TiO2-NTs at various deposition times (10 min, 20 min). The experimental setup consisted of the catalysts (dimensions: 1.2 × 2.5 cm) placed in the photocatalytic batch reactor. An initial antibiotic concentration of 1 mg/L was studied. The lamp was lighted after reaching the adsorption–desorption equilibrium between the catalyst and the antibiotic. For this reason, the reactor containing the assembly was kept in the dark for 1 h before lighting the lamp. Scanning Electron Microscopy (SEM) g py Figure 2 shows SEM images of Figure 2 shows SEM images of TiO2 nanotubes decorated with Ag nanoparticles obtained with 10 min and 20 min pulse durations. The TiO2-NTs, without metallic nanopar- ticles, were vertically aligned on titanium substrates. These nanotubes had a diameter of around 100 nm and a length of around 15 µm (inset Figure 2a). We note the presence of small, uniformly dispersed Ag nanoparticles, and a few aggregates at the top of the nanotubes. The images show a nano-tubular structure with an average diameter of 150 nm. The Ag nanoparticles are very small in size, so they are not observable with SEM. btained with 10 min and 20 min pulse durations. The TiO2-NTs, without met anoparticles, were vertically aligned on titanium substrates. These nanotubes ha iameter of around 100 nm and a length of around 15 μm (inset Figure 2a). We note resence of small, uniformly dispersed Ag nanoparticles, and a few aggregates at the f the nanotubes. The images show a nano-tubular structure with an average diamete 50 nm. The Ag nanoparticles are very small in size, so they are not observable with S 2. Typical SEM images of TiO2-NTs (a) before and (b) after their Ag-NPs decoration at 10 min and (c) 20 min. Th h i l i i f TiO b d d i h A i l Figure 2. Typical SEM images of TiO2-NTs (a) before and (b) after their Ag-NPs decoration at 10 min and (c) 20 min. ges of TiO2-NTs (a) before and (b) after their Ag-NPs decoration at 10 min and (c) 20 min. Figure 2. Typical SEM images of TiO2-NTs (a) before and (b) after their Ag-NPs decoration at 10 min and (c) 20 min. The chemical composition of TiO2 nanotubes decorated with Ag nanoparticles stimated by EDX. Figure 3 shows the EDX spectra of TiO2 nanotubes decorated with anoparticles; the atomic percentage of silver increases with photo-reduction time: 0 for 10 min Ag/TiO2 and 0.3 at. % for 20 min Ag/TiO2. The chemical composition of TiO2 nanotubes decorated with Ag nanoparticles was estimated by EDX. Figure 3 shows the EDX spectra of TiO2 nanotubes decorated with Ag nanoparticles; the atomic percentage of silver increases with photo-reduction time: 0.2 at. % for 10 min Ag/TiO2 and 0.3 at. % for 20 min Ag/TiO2. was kept in the dark for 1 h before 3.1. Characterizations of Ag-NPs/TiO2-NTs s and Discussion acterizations of Ag-NPs/TiO2-NTs nning electron microscopy (SEM, TESCAN VEGA3) was carried out to visua structured morphologies of samples. The elemental analysis of samples w ned by energy-dispersive X-ray spectroscopy (EDS). The TEM and HRT were obtained with the FEI Tecnai G20 microscope operating at 200 kV d with the LaB6 filament. The X-ray photoelectron spectroscopy (X ments were carried out using a Jeol JPS-9200 photoelectron spectrometer with Scanning electron microscopy (SEM, TESCAN VEGA3) was carried out to visualize the nanostructured morphologies of samples. The elemental analysis of samples was determined by energy-dispersive X-ray spectroscopy (EDS). The TEM and HRTEM images were obtained with the FEI Tecnai G20 microscope operating at 200 kV and equipped with the LaB6 ﬁlament. The X-ray photoelectron spectroscopy (XPS) measurements were carried out using a Jeol JPS-9200 photoelectron spectrometer with an achromatic Mg/Al X-ray source at 500 W. The spectra excitations were performed using Mg Kα radiation (1253.6 eV). During XPS data acquisition, the C1s (285.0 eV) peak was used as a reference to correct XPS data from sample charging. The X-ray diffractometer (Cu Ka radiation, λ = 1.5406 Å, PANalytical B.V., Almelo, The Netherlands) was used to identify the crystalline structure and the Pt nanoparticles phase. The photoluminescence (PL) spectra were recorded with a Materials 2022, 15, 1463 4 of 12 minesc PerkinElmer spectrophotometer equipped with a xenon lamp at an excitation wavelength λ = 340 nm. mp at an excitation wavelength λ = 340 nm. canning Electron Microscopy (SEM) PerkinElmer spectrophotometer equipped with a xenon lamp at an excitation wavelength λ = 340 nm. amp at an excitation wavelength λ = 340 nm. canning Electron Microscopy (SEM) Scanning Electron Microscopy (SEM) g py Figure 2 shows SEM images of g g The TiO2-NTs adorned with Ag-NPs were examined by TEM (Figure 4) to verify the presence of Ag-NPs in the TiO2-NTs. A drop of Ag-NPs/TiO2-NTs dispersed in solution was applied to the TEM holey carbon grids. Figure 4a reveals the presence of arbitrary shapes of Ag-NPs on the TiO2-NTs’ surface. Figure 5 shows the X-ray diffractograms of pure TiO2 nanotubes decorated with Ag nanoparticles annealed at 400 ◦C. All samples crystallized in the anatase structure. Here, we note that the preferential orientation peak (101) characteristic of the anatase phase of titanium oxide continues to decrease with the increase in the deposition time of silver nanoparticles on TiO2 nanoparticles. In addition, it is noted that all the samples crystallize in the anatase structure, and the additional peaks characterizing Ag can only be observed from the diffractogram of the TiO2-NTs decorated with silver NPs at 20 min; they cannot be observed for the deposition time of less than 10 min. Indeed, below 20 min of UV irradiation, the amount of Ag incorporated is too low to be detected by this characterization technique. (a) (b) q As can be seen in Figure 6, the formation of Ag species was conﬁrmed by the XPS results. Figure 6a exhibits an O1s peak at 530.8 eV, attributed to the oxide. The appearance of the Ti-2p peak position at a binding energy of 459 eV (Figure 6b) proved that the main chemical valence of Ti is +4. This ﬁnding corroborates the assigned values for anatase TiO2 in the NIST database. Figure 6c shows the typical XPS signature of the Ag 3d doublet (3d5/2 and 3d3/2) taken from the surface of the TiO2-NTs decorated with Ag-NPs. The signals at 374.3 and 368.3 eV are attributed to Ag 3d3/2 and Ag 3d5/2 of Ag-NPs, respectively. Materials 2022, 15, 1463 5 of 12 d with A me: 0.2 a (a) (b) , x FOR PEER REVIEW 5 of 1 (c) Figure 3. EDX spectra of TiO2 nanotubes decorated with silver nanoparticles: (a) undecorated TiO2 NTs, (b) 10 min Ag-NPs/TiO2-NTs, and (c) 20 min Ag-NPs/TiO2-NTs. Figure 3. EDX spectra of TiO2 nanotubes decorated with silver nanoparticles: (a) undecorated TiO2-NTs, (b) 10 min Ag-NPs/TiO2-NTs, and (c) 20 min Ag-NPs/TiO2-NTs. 15, x FOR PEER REVIEW 5 of 13 (c) Figure 3. Scanning Electron Microscopy (SEM) g py Figure 2 shows SEM images of EDX spectra of TiO2 nanotubes decorated with silver nanoparticles: (a) undecorated TiO2- NTs, (b) 10 min Ag-NPs/TiO2-NTs, and (c) 20 min Ag-NPs/TiO2-NTs. The TiO2-NTs adorned with Ag-NPs were examined by TEM (Figure 4) to verify the presence of Ag-NPs in the TiO2-NTs. A drop of Ag-NPs/TiO2-NTs dispersed in solution was applied to the TEM holey carbon grids. Figure 4a reveals the presence of arbitrary shapes of Ag-NPs on the TiO2-NTs’ surface. (a) (b) ials 2022, 15, x FOR PEER REVIEW 5 o aterials 2022, 15, x FOR PEER REVIEW 5 of 1 (b) 5 5 of (a) (b) (c) (c) Figure 3. EDX spectra of TiO2 nanotubes decorated with silver nano NTs, (b) 10 min Ag-NPs/TiO2-NTs, and (c) 20 min Ag-NPs/TiO2-NT The TiO2-NTs adorned with Ag-NPs were examined by presence of Ag-NPs in the TiO2-NTs. A drop of Ag-NPs/TiO (b) (a) (c) TiO2- l igure 3. EDX spectra of TiO2 nanotubes decorated with silver nanoparticles: (a) undecorated TiO NTs, (b) 10 min Ag-NPs/TiO2-NTs, and (c) 20 min Ag-NPs/TiO2-NTs. Figure 3. EDX spectra of TiO2 nanotubes decorated with silver nanoparticles: (a) undecorated TiO2-NTs, (b) 10 min Ag-NPs/TiO2-NTs, and (c) 20 min Ag-NPs/TiO2-NTs. was applied to the TEM holey carbon grids. Figure 4a reveals the presence of arbitrary shapes of Ag-NPs on the TiO2-NTs’ surface. s were examined by TEM (Figure 4) to drop of Ag-NPs/TiO2-NTs dispersed in ids. arbitrary shapes of Ag-NPs on the T The TiO2-NTs adorned with Ag-NP resence of Ag-NPs in the TiO2-NTs. A was applied to the TEM holey carbon gr Figure 4a reveals the presence of urface. The TiO2-NTs adorned with Ag-NPs were examined by TEM (Figure 4) to verif presence of Ag-NPs in the TiO2-NTs. A drop of Ag-NPs/TiO2-NTs dispersed in solu was applied to the TEM holey carbon grids. Figure 4a reveals the presence of arbitrary shapes of Ag-NPs on the TiO2- surface. Figure 4. (a) Top view and (b) cross-section TEM images of Ag-NPs (20 min)/TiO2-NTs array. Figure 4. (a) Top view and (b) cross-section TEM images of Ag-NPs (20 min)/TiO2-NTs array. Figure 4. (a) Top view and (b) cross-section TEM images of Ag-NPs (20 min)/TiO2-NTs array. Figure 4. (a) Top view and (b) cross-section TEM images of Ag-NPs (20 min)/TiO2-NTs array. Scanning Electron Microscopy (SEM) g py Figure 2 shows SEM images of 6 of 12 Materials 2022, 15, 1463 10 20 30 40 50 60 70 80 A(112) A(301) A(220) Ag(220) A(204) A(105) A(200) Ag(200) Ti(101) A(103) TiO2 NTs-Ag 20min TiO2 NTs Intensity (a,u) 2(°) TiO2 NTs-Ag 10min A(101) A: Anatase Ti: Titanium Ag: Silver e 5. X-ray diffractogram of pure TiO2 nanotubes decorated with silver nanoparticles. Figure 5. X-ray diffractogram of pure TiO2 nanotubes decorated with silver nanoparticles. VIEW 5. X-ray diffractogram of pure TiO2 nanotubes decorated with silver nanoparticles. Figure 5. X-ray diffractogram of pure TiO2 nanotubes decorated with silver nanoparticles. As can be seen in Figure 6, the formation of Ag species was confirmed by the XP s. Figure 6a exhibits an O1s peak at 530.8 eV, attributed to the oxide. The appearan Ti-2p peak position at a binding energy of 459 eV (Figure 6b) proved that the ma cal valence of Ti is +4. This finding corroborates the assigned values for anatase Ti NIST database. Figure 6c shows the typical XPS signature of the Ag 3d doublet (3d d3/2) taken from the surface of the TiO2-NTs decorated with Ag-NPs. The signals and 368.3 eV are attributed to Ag 3d3/2 and Ag 3d5/2 of Ag-NPs, respectively. hese energies often correspond to the metal silver [26], proving the fully metal cter of the used Ag-NPs. This result confirms the effectiveness of the photoreductio ss used in the preparation step. Figure 6. XPS spectra of Ag-NPs (20 min)/TiO2-NTs: (a) O1s; (b) Ti2p; (c) Ag 3d. Figure 6. XPS spectra of Ag-NPs (20 min)/TiO2-NTs: (a) O1s; (b) Ti2p; (c) Ag 3d. can be seen in Figure 6, the formation of A Figure 6a exhibits an O1s peak at 530.8 eV, at -2p peak position at a binding energy of 459 valence of Ti is +4. This finding corroborate ST database. Figure 6c shows the typical XP ) taken from the surface of the TiO2-NTs de d 368.3 eV are attributed to Ag 3d3/2 and Ag se energies often correspond to the metal s r of the used Ag-NPs. This result confirms th used in the preparation step. ion of 8 eV a Figure 6. XPS spectra of Ag-NPs (20 min)/TiO2-NTs: (a) O1s; (b) Ti2p; (c) Ag 3d Figure 6. XPS spectra of Ag-NPs (20 min)/TiO2-NTs: (a) O1s; (b) Ti2p; (c) Ag 3d. eduction for 10 and 20 min. It is se ently affected by the different deco 3.2.1. Effect of Silver Decoration eduction for 10 and 20 min. It is se ently affected by the different deco 3.2.1. Effect of Silver Decoration ently affected by the different decoration times. In this test, it is readily seen that the ntrations of pollutants decreased over time under visible light irradiation. Moreover, ration of Ag in 10 min increased the pollutant removal from 35% to 80% of the initial ntration of 2.5 mg/L. This enhancement is related to the availability of the generated sites at the interface of the catalyst deposited at 10 min [30]. However, the pollutant dation will tend to be limited at high time values, because these values are too high m multiple layers of catalysts, thus generating different ions and charges, which o distinguished charges at the interfaces of thin films and coverage of all TiO2-NTs. 2 5 TiO2 NTs AgO-10/TiO2-NTs AgO 20/TiO2 NTs The photocatalytic performances of Ag-NP/TiO2-NT catalysts were estimated by photocatalytic degradation of the antibiotic under visible light irradiation at ambient temperature. Figure 8 shows the pollutant removal using Ag-NPs/TiO2-NTs decorated by photoreduction for 10 and 20 min. It is seen from Figure 8 that the pollutant removal was pertinently affected by the different decoration times. In this test, it is readily seen that the concentrations of pollutants decreased over time under visible light irradiation. Moreover, a decoration of Ag in 10 min increased the pollutant removal from 35% to 80% of the initial concentration of 2.5 mg/L. This enhancement is related to the availability of the generated active sites at the interface of the catalyst deposited at 10 min [30]. However, the pollutant degradation will tend to be limited at high time values, because these values are too high to form multiple layers of catalysts, thus generating different ions and charges, which leads to distinguished charges at the interfaces of thin ﬁlms and coverage of all TiO2-NTs. Scanning Electron Microscopy (SEM) g py Figure 2 shows SEM images of Materials 2022, 15, 1463 7 of 12 7 of 12 These energies often correspond to the metal silver [26], proving the fully metallic character of the used Ag-NPs. This result conﬁrms the effectiveness of the photoreduction process used in the preparation step. The photoluminescence (PL) of TiO2-NTs can inform us about the lifespan and trans- port of photogenerated charges. Figure 7 shows the photoluminescence spectra of pure TiO2-NTs decorated with Ag-NPs. The peak located at 365 nm (3.4 eV) is attributed to the electron transition between the valence and conductive bands of TiO2. The peaks present at 459 nm, 483 nm, and 531 nm are attributed to the oxygen vacancies present on the surface of TiO2 [27,28]. 8 of 13 400 450 500 550 PL (a.u)  (nm) TiO2 NT 10 min Ag/TiO2 NT 20 min Ag/TiO2 NT 7 Photoluminescence spectra of pure TiO2 nanotubes decorated with silver nanoparticles Figure 7. Photoluminescence spectra of pure TiO2 nanotubes decorated with silver nanoparticles. 7 Photoluminescence spectra of pure TiO2 nanotubes decorated with silver nanoparticles Figure 7. Photoluminescence spectra of pure TiO2 nanotubes decorated with silver nanoparticles. p p p otocatalytic Experiments Effect of Silver Decoration he photocatalytic performances of Ag-NP/TiO2-NT catalysts were estimated by catalytic degradation of the antibiotic under visible light irradiation at ambient TiO2-NTs decorated with Ag-NPs exhibit a lower PL intensity compared to that of pure TiO2, which shows that Ag nanoparticles reduce the density of radiative recombination centers (luminescent centers). This result can be explained by the migration of electrons photogenerated under UV radiation (λexcitation = 340 nm) from the conduction band towards Ag nanoparticles, thus reducing radiative recombination within TiO2 [29]. rature. Figure 8 shows the pollu eduction for 10 and 20 min It is 3.2. Photocatalytic Experiments g / 3.2.2. Effect of Inlet Concentration: Kinetic Modeling 5 A representation of the change in concentration over time for four initial concentrations is shown in Figure 9a. As was observed, the increase in the concentration generally favors the pollutant degradation. Materials 2022, 15, 1463 8 of 12 0 0.5 1 1.5 2 2.5 0 20 40 60 80 100 120 140 160 Concentration ( mg/L)) Time (min) TiO2 NTs AgO-10/TiO2-NTs AgO-20/TiO2-NTs Figure 8. Effect of decoration of Ag-NPs on the photocatalytic activity of the Ag-NP/TiO2-NT photocatalyst. VIEW 9 of 13 Figure 8. Effect of decoration of Ag-NPs on the photocatalytic activity of the Ag-NP/TiO2-NT photocatalyst. 3.2.2. Effect of Inlet Concentration: Kinetic Modeling A representation of the change in concentration over time for four initial concentrations is shown in Figure 9a. As was observed, the increase in the concentration generally favors the pollutant degradation. 0 0.5 1 1.5 2 2.5 0 20 40 60 80 100 120 140 Concentration ( mg/L)) Time (min) TiO2 NTs AgO-10/TiO2-NTs AgO-20/TiO2-NTs IEW Figure 8. Effect of decoration of Ag-NPs on the photocatalytic activity photocatalyst. 3 2 2 Eff t f I l t C t ti Ki ti M d li Figure 8. Effect of decoration of Ag-NPs on the photocatalytic activity of the Ag-NP/TiO2-NT photocatalyst. A representation of the change in concentration over time for four initial concentrations is shown in Figure 9a. As was observed, the increase in the concentration generally favors the pollutant degradation. (a) (b) Figure 9. (a) Evolution of the concentration of the diclofenac antibiotic during its photocatalytic degradation as a function of time for 4 initial concentrations. (b) Linearization of the Langmuir– Hinshelwood isotherm during the degradation of the antibiotic (calculation of kc and K constants). At low concentrations, the kinetics are faster [31]. This can be explained by the fact that more molecules are “available” in the solution and that visible light accesses the 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 0 20 40 60 80 100 120 140 160 Concentration (mg/L) Time (min) C= 1.5 mg /L C= 2.5 mg /L C= 3.5 mg /L C= 4.5 mg /L y = 33.865x + 11.726 0 5 10 15 20 25 30 35 40 45 0 0.2 0.4 0.6 0.8 1/r (L.min/mg) 1/C (L/mg) Figure 9. (a) Evolution of the concentration of the diclofenac antibiotic during its photocatalytic degradation as a function of time for 4 initial concentrations. g / 3.2.2. Effect of Inlet Concentration: Kinetic Modeling (b) Linearization of the Langmuir– Hinshelwood isotherm during the degradation of the antibiotic (calculation of kc and K constants). 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 0 20 40 60 80 100 120 140 160 Concentration (mg/L) Time (min) C= 1.5 mg /L C= 2.5 mg /L C= 3.5 mg /L C= 4.5 mg /L (a) (b) y = 33.865x + 11.726 0 5 10 15 20 25 30 35 40 45 0 0.2 0.4 0.6 0.8 1/r (L.min/mg) 1/C (L/mg) (b) Figure 9. (a) Evolution of the concentration of the diclofenac antibiotic during its photocatalytic degradation as a function of time for 4 initial concentrations. (b) Linearization of the Langmuir– Hinshelwood isotherm during the degradation of the antibiotic (calculation of kc and K constants). At low concentrations, the kinetics are faster [31]. This can be explained by the fact Figure 9. (a) Evolution of the concentration of the diclofenac antibiotic during its photocatalytic degradation as a function of time for 4 initial concentrations. (b) Linearization of the Langmuir– Hinshelwood isotherm during the degradation of the antibiotic (calculation of kc and K constants). Materials 2022, 15, 1463 9 of 12 At low concentrations, the kinetics are faster [31]. This can be explained by the fact that more molecules are “available” in the solution and that visible light accesses the surface of the catalyst more easily, which leads to an increase in the speed of photocatalytic degradation [32–35]. However, at high concentrations, molecules begin to act as a ﬁlter for incident visible light, so that light hardly reaches the surface of the TiO2, resulting in slower photocatalytic degradation [28,36–40]. This same trend has been observed by [41]. Under visible light, electron–hole pairs were produced in the valence bands (VB) of AgO and TiO2. Then, the photo-generated electrons passed from the valence bands to their conduction bands (CB). At the same time, electrons can be rapidly transferred to the semiconductor (TiO2) interface, which has been reported in Au core–Cu2O shell particles [21,25]. This could be due to the fact that the heterojunction in Ag-AgO/TiO2 assisted the transfer of photo-generated electrons and holes. Thus, these electrons can quickly be transferred into the TiO2 interface and then undergo a transformation, as described below [21]. g / 3.2.2. Effect of Inlet Concentration: Kinetic Modeling AgO/TiO2 + hV→e−+ h+ (1) e−+ h+/AgO →e−/Ag →e−/TiO2 (2) O2 + e−→O2.− (3) O2.−+ 2H2O + 2e−→3OH−+ OH· (4) H+ + H2O →H+ + OH· (5) Antibiotic+ O2.−/OH· →CO2 + H2O + other products (6) (1) (5) Antibiotic+ O2.−/OH· →CO2 + H2O + other products (6) (6) To describe the Ag (10 min)/TiO2-NTs’ catalytic performance, the Langmuir– Hinshelwood (L-H) model was used [42]: r0 = −d[pollutant] dt = kc K[pollutant]0 1 + K[pollutant]0 (7) (7) where r0 (mg·L−1 min−1) is the initial photodegradation rate, [pollutant] is the initial BUT concentration (mg/L), K is the adsorption constant (L/mg), and kc is the kinetic constant (mg·L−1 min−1) at maximum coverage of the experimental conditions. g g p The plot of 1/r0 versus 1/[pollutant]0 (Figure 9b) allows determining kc and K values. The linearized (L-H) equation is: 1 r0 = 1 kcK × 1 [pollutant]0 + 1 kC (8) (8) Table 1 illustrates the kinetic and adsorption constants of L-H. Table 1 illustrates the kinetic and adsorption constants of L-H. Table 1. L-H constants (kc and K) on the 10 min AgO-NPs/TiO2-NT catalyst. kc: Kinetic Constant of L-H (mg/(L. min)) K: Adsorption Constant of L-H (L/mg) 2.9 × 10−3 2.88 Table 1. L-H constants (kc and K) on the 10 min AgO-NPs/TiO2-NT catalyst. kc: Kinetic Constant of L-H (mg/(L. min)) K: Adsorption Constant of L-H (L/mg) 2.9 × 10−3 2.88 kc: Kinetic Constant of L-H (mg/(L. min)) K: Adsorption Constant of L-H (L/mg) 2.9 × 10−3 2.88 ns 4. Conclusions nium electrochemical anodization and the photochemical reduction of Ag- NTs were successfully used to create the Ag-NP/TiO2-NT nanocomposites. s nanotubes with a diameter of 100 nm were used to synthesize TiO2-NTs, hen crystallized in the anatase phase. The Ag-NP deposition was performed ctrodeposition times. The Ag-NPs’ adjunction to the TiO2-NTs increased the absorption until almost 500 nm. Antibiotic removal experiments were TiO2-NTs and Ag-NPs/TiO2-NTs with various photoreduction times under (380–720 nm). These tests revealed a high photocatalytic efficiency. This be explained by the combination of the energy band level positions of ors and the high visible light absorption of Ag nanoparticles. The Ag- photocatalytic system significantly improved the antibiotic degradation he TiO2-NTs alone, because of the extended photoresponse and the efficient ion of electron–hole pairs under UV/visible light. Thus, this work shows the of the Ag-NP/TiO2-NT nanocomposites under UV light irradiation. The titanium electrochemical anodization and the photochemical reduction of Ag- NPs on TiO2-NTs were successfully used to create the Ag-NP/TiO2-NT nanocomposites. Homogeneous nanotubes with a diameter of 100 nm were used to synthesize TiO2-NTs, which were then crystallized in the anatase phase. The Ag-NP deposition was performed at various electrodeposition times. The Ag-NPs’ adjunction to the TiO2-NTs increased the visible light absorption until almost 500 nm. Antibiotic removal experiments were conducted by TiO2-NTs and Ag-NPs/TiO2-NTs with various photoreduction times un- der visible light (380–720 nm). These tests revealed a high photocatalytic efﬁciency. This ﬁnding can be explained by the combination of the energy band level positions of semicon- ductors and the high visible light absorption of Ag nanoparticles. The Ag-NP/TiO2-NT photocatalytic system signiﬁcantly improved the antibiotic degradation concerning the TiO2-NTs alone, because of the extended photoresponse and the efﬁcient photogeneration of electron–hole pairs under UV/visible light. Thus, this work shows the high stability of the Ag-NP/TiO2-NT nanocomposites under UV light irradiation. ibutions: A.A.A. (Achraf Amir Assadi) and S.K.: validation, investigation, resources, writing—original draft, writing—review and editing; K.T. and A.H.: on, validation, writing—original draft, writing—review and editing, visualization, W.E. and A.A.A. (Aymen Amin Assadi): methodology, validation, investigation, w and editing; A.G. and M.M.: conceptualization, methodology, supervision. All ad and agreed to the published version of the manuscript. Author Contributions: A.A.A. (Achraf Amir Assadi) and S.K.: validation, investigation, visualiza- tion, resources, writing—original draft, writing—review and editing; K.T. and A.H.: conceptualiza- tion, validation, writing—original draft, writing—review and editing, visualization, supervision; W.E. and A.A.A. 3.3. Catalyst Reusability Catalyst reusability is a major issue to look into because it measures how well a catalyst can degrade contaminants in solution. Therefore, several visible photocatalytic experiments were performed to examine the ability of the elaborated silver sample to degrade antibiotics in four successive cycles. After each degradation cycle, the 10 min Ag-NPs/TiO2-NTs were well washed and reused. The photocatalytic ﬁndings of repetitive use (four cycles) of the 10 min Ag-NP/TiO2- NT photocatalyst are shown in Figure 10. Figure 10 shows no loss of sample photoactivity. These results prove the overall potential of the 10 min Ag-NP/TiO2-NT catalyst for water treatment applications. Generally, the same slope is noted in each cycle. Consequently, these Materials 2022, 15, 1463 10 of 12 10 of 12 results shown below prove the availability of active sites and the excellent photocatalytic stability of this catalyst. 11 of 13 results shown below prove the availability of active sites and the excellent photocatalytic stability of this catalyst. 11 of 13 results shown below prove the availability of active sites and the excellent photocatalytic stability of this catalyst. 11 of 13 . Figure 10. Four successive cycles of photocatalytic oxidations using the 10 min Ag-NP/TiO2-NT catalyst. 0 0.5 1 1.5 2 2.5 3 0 100 200 300 400 500 600 700 Concentration (mg/L) time (min) Cycle 1 cycle 2 Cycle 3 Cycle 4 Figure 10. Four successive cycles of photocatalytic oxidations using the 10 min Ag-NP/ TiO2-NT catalyst. r successive cycles of photocatalytic oxidations using the 10 min Ag-NP/TiO2-NT Figure 10. Four successive cycles of photocatalytic oxidations using the 10 min Ag-NP/ TiO2-NT catalyst. ns 4. Conclusions (Aymen Amin Assadi): methodology, validation, investigation, writing—review and editing; A.G. and M.M.: conceptualization, methodology, supervision. All authors have read and agreed to the published version of the manuscript. project was carried out under the MOBIDOC scheme, funded by the Tunisian gher Education and Scientific Research through the PromESsE project and managed Funding: This project was carried out under the MOBIDOC scheme, funded by the Tunisian Min- istry of Higher Education and Scientiﬁc Research through the PromESsE project and managed by the ANPR. sent Statement: Not applicable. Informed Consent Statement: Not applicable. lity Statement: Not applicable. Data Availability Statement: Not applicable. ity Statement: Not applicable. Data Availability Statement: Not applicable. nterest: The authors declare no conflict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 11 of 12 Materials 2022, 15, 1463 References 1. Assadi, A.A.; Bouzaza, A.; Vallet, C.; Wolbert, D. Use of DBD plasma, photocatalysis, and combined DBD plasma/photocatalysis in a continuous annular reactor for isovaleraldehyde elimination—Synergetic effect and byproducts identiﬁcation. Chem. Eng. J. 2014, 254, 124–132. [CrossRef] 2. Baaloudj, O.; Assadi, I.; Nasrallah, N.; El, A.; Khezami, L. Simultaneous removal of antibiotics and inactivation of antibiotic- resistant bacteria by photocatalysis: A review. J. Water Process Eng. 2021, 42, 102089. [CrossRef] 3. Kenfoud, H.; Nasrallah, N.; Baaloudj, O.; Meziani, D.; Chaabane, T.; Trari, M. Photocatalytic reduction of Cr(VI) onto the spinel CaFe2O4 nanoparticles. Optik 2020, 223, 165610. [CrossRef] 4. Boyjoo, Y.; Sun, H.; Liu, J.; Pareek, V.K.; Wang, S. A review on photocatalysis for air treatment: From catalyst development to reactor design. Chem. Eng. J. 2017, 310, 537–559. [CrossRef] 5. Baaloudj, O.; Nasrallah, N.; Kenfoud, H.; Algethami, F.; Modwi, A.; Guesmi, A.; Assadi, A.A.; Khezami, L. Application of Bi12ZnO20 Sillenite as an Efﬁcient Photocatalyst for Wastewater Treatment: Removal of Both Organic and Inorganic Compounds. Materials 2021, 14, 5409. [CrossRef] 6. Asadi-Ghalhari, M.; Mostafaloo, R.; Ghafouri, N.; Kishipour, A.; Usei, S.; Baaloudj, O. Removal of Ceﬁxime from aqueous solutions via proxy electrocoagulation: Modeling and optimization by response surface methodology. React. Kinet. Mech. Catal. 2021, 134, 459–471. [CrossRef] , , [ ] 7. Baaloudj, O.; Nasrallah, N.; Bouallouche, R.; Kenfoud, H. High efﬁcient Ceﬁxime removal from water by the sillenite Bi 12 TiO 20: Photocatalytic mechanism and degradation pathway. J. Clean. Prod. 2022, 330, 129934. [CrossRef] 8. Almansba, A.; Kane, A.; Nasrallah, N.; Wilson, J.M.; Maachi, R.; Lamaa, L.; Peruchon, L.; Brochier, C.; Amrane, A.; Assadi, A.A. An engineering approach towards the design of an innovative compact photo-reactor for antibiotic removal in the frame of laboratory and pilot-plant scale. J. Photochem. Photobiol. A Chem. 2021, 418, 113445. [CrossRef] g g pp g p p laboratory and pilot-plant scale. J. Photochem. Photobiol. A Chem. 2021, 418, 113445. [CrossRef] 9. Bourkeb, K.; Baaloudj, O. Facile electrodeposition of ZnO on graphitic substrate for photocatalytic application: Degradation of antibiotic in a continuous stirred-tank reactor. J. Solid State Electrochem. 2022, 26, 573–580. [CrossRef] Bourkeb, K.; Baaloudj, O. Facile electrodeposition of ZnO on graphitic substrate for photocatalytic applic antibiotic in a continuous stirred-tank reactor. J. Solid State Electrochem. 2022, 26, 573–580. [CrossRef] tibiotic in a continuous stirred-tank reactor. J. Solid State Electrochem. 2022, 26, 573–580. [CrossRef] 10. Ochiai, T.; Fujishima, A. References Photoelectrochemical properties of TiO2 photocatalyst and its applications for environmental puriﬁcation. J. Photochem. Photobiol. C Photochem. Rev. 2012, 13, 247–262. [CrossRef] J 11. Hodgson, A.T.; Destaillats, H.; Sullivan, D.P.; Fisk, W.J. Performance of ultr applications. Indoor Air 2007, 17, 305–316. [CrossRef] [PubMed] 11. Hodgson, A.T.; Destaillats, H.; Sullivan, D.P.; Fisk, W.J. Performance of ultraviolet photocatalytic oxidation for indoor air cleaning applications. Indoor Air 2007, 17, 305–316. [CrossRef] [PubMed] 11. Hodgson, A.T.; Destaillats, H.; Sullivan, D.P.; Fisk, W.J. Performance of ultraviolet photocatalytic oxida applications. Indoor Air 2007, 17, 305–316. [CrossRef] [PubMed] pp 12. Serhane, Y. Continuous air puriﬁcation by front ﬂow photocatalytic reactor: Modelling of the inﬂue under simulated real conditions. Chemosphere 2022, 295, 133809. [CrossRef] [PubMed] 13. Hou, T.; Du, H.; Yang, Z.; Tian, Z.; Shen, S.; Shi, Y.; Yang, W.; Zhang, L. Flocculation of different types of combined contaminants of antibiotics and heavy metals by thermo-responsive ﬂocculants with various architectures. Sep. Purif. Technol. 2019, 223, 123–132. [CrossRef] 14. Trabelsi, K.; Hajjaji, A.; Gaidi, M.; Bessais, B.; El Khakani, M.A. Enhancing the photoelectrochemical res through their nanodecoration by pulsed-laser-deposited Ag nanoparticles. J. Appl. Phys. 2017, 122, 064 15. Hajjaji, A.; Jemai, S.; Rebhi, A.; Trabelsi, K.; Gaidi, M.; Alhazaa, A.N.; Al-Gawati, M.A.; El Khakani, M.A.; Bessais, B. Enhancement of photocatalytic and photoelectrochemical properties of TiO2 nanotubes sensitized by SILAR—Deposited PbS nanoparticles. J. Mater. 2020, 6, 62–69. [CrossRef] 16. Hajjaji, A.; Jemai, S.; Trabelsi, K.; Kouki, A.; Ben Assaker, I.; Ka, I.; Gaidi, M.; Bessais, B.; El Khakani, M.A. Study of TiO2 nanotubes decorated with PbS nanoparticles elaborated by pulsed laser deposition: Microstructural, optoelectronic and photoelectrochemical properties. J. Mater. Sci. Mater. Electron. 2019, 30, 20935–20946. [CrossRef] 17. Wu, Y.; Chen, X.; Cao, J.; Zhu, Y.; Yuan, W.; Hu, Z.; Ao, Z.; Brudvig, G.W.; Tian, F.; Jimmy, C.Y.; et al. Photocatalytically recovering hydrogen energy from wastewater treatment using MoS2 @TiO2 with sulfur/oxygen dual-defect. Appl. Catal. B Environ. 2022, 303, 120878. [CrossRef] 18. Dimitrakopoulou, D.; Rethemiotaki, I.; Frontistis, Z.; Xekoukoulotakis, N.P.; Venieri, D.; Mantzavinos, D. Degradation, mineral- ization and antibiotic inactivation of amoxicillin by UV-A/TiO 2 photocatalysis. J. Environ. Manag. 2012, 98, 168–174. [CrossRef] [PubMed] 19. Karoui, S.; Ben Arﬁ, R.; Mougin, K.; Ghorbal, A.; Assadi, A.A.; Amrane, A. Synthesis of novel biocomposite powder for simultaneous removal of hazardous ciproﬂoxacin and methylene blue: Central composite design, kinetic and isotherm studies using Brouers-Sotolongo family models. J. Hazard. Mater. 2020, 387, 121675. References Song, L.; Luo, L.; Song, J.; Zhang, H.; Li, X.; Cheng, S.; Jin, W.; Tang, J.; Liu, L.; Wang, F. Enhanced Photodegradation Activity of Hydrogen Terminated Si Nanowires Arrays with Different Oriented Crystal Phases Catalysts 2017 7 371 [CrossRef] 28. Song, L.; Luo, L.; Song, J.; Zhang, H.; Li, X.; Cheng, S.; Jin, W.; Tang, J.; Liu, L.; Wang, F. Enhanced Photodegradation Activity of Hydrogen-Terminated Si Nanowires Arrays with Different-Oriented Crystal Phases. Catalysts 2017, 7, 371. [CrossRef] Song, J.; Zhang, H.; Li, X.; Cheng, S.; Jin, W.; Tang, J.; Liu, L.; Wang, F. Enhanced Photodegradation Activit Song, L.; Luo, L.; Song, J.; Zhang, H.; Li, X.; Cheng, S.; Jin, W.; Tang, J.; Liu, L.; Wang, F. Enhanced Photode Hydrogen-Terminated Si Nanowires Arrays with Different-Oriented Crystal Phases. Catalysts 2017, 7, 371 ydrogen-Terminated Si Nanowires Arrays with Different-Oriented Crystal Phases. Catalysts 2017, 7, 371. [C y g y y y [ ] 29. Nishanthi, S.T.; Iyyapushpam, S.; Sundarakannan, B.; Subramanian, E.; Pathinettam Padiyan, D. Plasmonic silver nanoparticles loaded titania nanotube arrays exhibiting enhanced photoelectrochemical and photocatalytic activities. J. Power Sources 2015, 274, 885–893. [CrossRef] [ ] 30. Li, C.; Lin, H.; Armutlulu, A.; Xie, R.; Zhang, Y.; Meng, X. Hydroxylamine-assisted catalytic degradation of ciproﬂoxacin in ferrate/persulfate system. Chem. Eng. J. 2019, 360, 612–620. [CrossRef] 31. Kenfoud, H.; Nasrallah, N.; Meziani, D.; Trari, M. Photoelectrochemical study of the spinel CaFe2O4 nanostructure: Application to Basic Blue 41 oxidation under solar light. J. Solid State Electrochem. 2021, 25, 1815–1823. [CrossRef] g 32. Zeghioud, H.; Assadi, A.A.; Khellaf, N.; Djelal, H.; Amrane, A.; Rtimi, S. Photocatalytic performance of CuxO/TiO2 deposited by HiPIMS on polyester under visible light LEDs: Oxidants, ions effect, and reactive oxygen species investigation. Materials 2019, 12, 412. [CrossRef] [PubMed] 33. Azzaz, A.A.; Jellali, S.; Assadi, A.A.; Bousselmi, L. Chemical treatment of orange tree sawdust for a cationic dye enhancement removal from aqueous solutions: Kinetic, equilibrium and thermodynamic studies. Desalin. Water Treat. 2016, 57, 22107–22119. [CrossRef] [ ] 34. Herrmann, J.M.; Tahiri, H.; Guillard, C.; Pichat, P. Photocatalytic degradation of aqueous hydroxy-butandioic acid (malic acid) in contact with powdered and supported titania in water. Catal. Today 1999, 54, 131–141. [CrossRef] 35. Zadi, T.; Assadi, A.A.; Nasrallah, N.; Bouallouche, R.; Tri, P.N.; Bouzaza, A.; Azizi, M.M.; Maachi, R.; Wolbert, D. Treatment of hospital indoor air by a hybrid system of combined plasma with photocatalysis: Case of trichloromethane. Chem. Eng. J. 2018, 349, 276–286. [CrossRef] 36. References [CrossRef] [PubMed] g g y 20. Assadi, A.A.; Bouzaza, A.; Lemasle, M.; Wolbert, D. Acceleration of Trimethylamine Removal Process U Photocatalytic Oxidation and Surface Discharge Plasma Reactor. Can. J. Chem. Eng. 2015, 93, 1239–124 21. Yanagida, B.S.; Yajima, T.; Takei, T.; Kumada, N. Removal of hexavalent chromium from water by Z-scheme photocatalysis using TiO2 (rutile) nanorods loaded with Au core–Cu2O shell particles. J. Environ. Sci. 2022, 115, 173–189. [CrossRef] [PubMed] p 22. Kenfoud, H.; Nasrallah, N.; Baaloudj, O.; Derridj, F.; Trari, M. Enhanced photocatalytic reduction of Cr(VI) by the novel hetero-system BaFe2O4/SnO2. J. Phys. Chem. Solids 2022, 160, 110315. [CrossRef] y y 23. Baaloudj, O.; Nasrallah, N.; Assadi, A.A. Facile synthesis, structural and optical characterizations of Bi12ZnO20 sillenite crystals: Application for Cefuroxime removal from wastewater. Mater. Lett. 2021, 304, 130658. [CrossRef] 12 of 12 12 of 12 Materials 2022, 15, 1463 24. Zeghioud, H.; Khellaf, N.; Amrane, A.; Djelal, H.; Elfalleh, W.; Assadi, A.A.; Rtimi, S. Photocatalytic performance of TiO2 impregnated polyester for the degradation of Reactive Green 12: Implications of the surface pretreatment and the microstructure. J. Photochem. Photobiol. A Chem. 2017, 346, 493–501. [CrossRef] 25. Wang, F.-Y.; Yang, Q.-D.; Xu, G.; Lei, N.g.a.i.-Y.; Tsang, Y.K.; Wong, N.-B.; Ho, J.C. Highly active and silicon nanowire arrays. Nanoscale 2011, 3, 3269–3276. [CrossRef] y , , [ ] 26. Waterhouse, G.I.N.; Bowmaker, G.A.; Metson, J.B. Oxidation of a polycrystalline silver foil by reaction with ozone. Appl. Surf. Sci. 2001, 183, 191–204. [CrossRef] , , [ ] 27. Zhang, Q.; Thrithamarassery Gangadharan, D.; Liu, Y.; Xu, Z.; Chaker, M.; Ma, D. Recent Advancements in Plasmon-Enhanced Visible Li h D i W S li i El i L d A d Th N h l d 2017 V l 3 ISBN 4509298102 27. Zhang, Q.; Thrithamarassery Gangadharan, D.; Liu, Y.; Xu, Z.; Chaker, M.; Ma, D. Recent Advancements in Plasmon-Enhanced Visible Light-Driven Water Splitting; Elsevier Ltd.: Amsterdam, The Netherlands, 2017; Volume 3, ISBN 4509298102. 27. Zhang, Q.; Thrithamarassery Gangadharan, D.; Liu, Y.; Xu, Z.; Chaker, M.; Ma, D. Recent Advancements in Light-Driven Water Splitting; Elsevier Ltd.: Amsterdam, The Netherlands, 2017; Volume 3, ISBN 450929 Driven Water Splitting; Elsevier Ltd.: Amsterdam, The Netherlands, 2017; Volume 3, ISBN 4509298102. L.; Luo, L.; Song, J.; Zhang, H.; Li, X.; Cheng, S.; Jin, W.; Tang, J.; Liu, L.; Wang, F. Enhanced Photodegradat Light-Driven Water Splitting; Elsevier Ltd.: Amsterdam, The Netherlands, 2017; Volume 3, ISBN 4509298102. 28. References Akpan, U.G.; Hameed, B.H. Parameters affecting the photocatalytic degradation of dyes using TiO2-based photocatalysts: A review. J. Hazard. Mater. 2009, 170, 520–529. [CrossRef] [PubMed] 37. Pereira, J.H.O.S.; Reis, A.C.; Queirós, D.; Nunes, O.C.; Borges, M.T.; Vilar, V.P.; Boaventura, R.A.R. Insights into solar TiO2-assisted photocatalytic oxidation of two antibiotics employed in aquatic animal production, oxolinic acid and oxytetracycline. Sci. Total Environ. 2013, 463–464, 274–283. [CrossRef] 38. Dionysiou, D.D.; Suidan, M.T.; Bekou, E.; Baudin, I.; Laîné, J.M. Effect of ionic strength and hydrogen peroxide on the photocat- alytic degradation of 4-chlorobenzoic acid in water. Appl. Catal. B Environ. 2000, 26, 153–171. [CrossRef] y g pp 39. Malato, S.; Fernández-Ibáñez, P.; Maldonado, M.I.; Blanco, J.; Gernjak, W. Decontamination and disinfection of water by solar photocatalysis: Recent overview and trends. Catal. Today 2009, 147, 1–59. [CrossRef] 39. Malato, S.; Fernández-Ibáñez, P.; Maldonado, M.I.; Blanco, J.; Gernjak, W. Decontam photocatalysis: Recent overview and trends. Catal. Today 2009, 147, 1–59. [CrossRef] 40. Gerischer, H.; Heller, A. The role of oxygen in photooxidation of organic molecules on semiconductor particles. J. Phys. Chem. 1991, 95, 5261–5267. [CrossRef] 41. Zeghioud, H.; Assadi, A.A.; Khellaf, N.; Djelal, H.; Amrane, A.; Rtimi, S. Reactive species monitoring and their contribution for removal of textile efﬂuent with photocatalysis under UV and visible lights: Dynamics and mechanism. J. Photochem. Photobiol. A Chem. 2018, 365, 94–102. [CrossRef] 42. Baaloudj, O.; Nasrallah, N.; Kebir, M.; Khezami, L.; Amrane, A.; Assadi, A.A. A comparative study of ceramic nanoparticles synthesized for antibiotic removal: Catalysis characterization and photocatalytic performance modeling. Environ. Sci. Pollut. Res. 2020, 28, 13900–13912. [CrossRef] [PubMed]
https://openalex.org/W2167364802	https://europepmc.org/articles/pmc4137096?pdf=render	English	null	Transcriptome instability as a molecular pan-cancer characteristic of carcinomas	BMC genomics	2,014	cc-by	11,862	RESEARCH ARTICLE Open Access * Correspondence: rolf.i.skotheim@rr-research.no 1Department of Cancer Prevention, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, P.O. Box 4953 Nydalen, Oslo NO-0424, Norway 2Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, P.O. Box 1078 Blindern, Oslo NO-0316, Norway Full list of author information is available at the end of the article Abstract Background: We have previously proposed transcriptome instability as a genome-wide, pre-mRNA splicing-related characteristic of colorectal cancer. Here, we explore the hypothesis of transcriptome instability being a general characteristic of cancer. Results: Exon-level microarray expression data from ten cancer datasets were analyzed, including breast cancer, cervical cancer, colorectal cancer, gastric cancer, lung cancer, neuroblastoma, and prostate cancer (555 samples), as well as paired normal tissue samples from the colon, lung, prostate, and stomach (93 samples). Based on alternative splicing scores across the genomes, we calculated sample-wise relative amounts of aberrant exon skipping and inclusion. Strong and non-random (P < 0.001) correlations between these estimates and the expression levels of splicing factor genes (n = 280) were found in most cancer types analyzed (breast-, cervical-, colorectal-, lung- and prostate cancer). This suggests a biological explanation for the splicing variation. Surprisingly, these associations prevailed in pan-cancer analyses. This is in contrast to the tissue and cancer specific patterns observed in comparisons across healthy tissue samples from the colon, lung, prostate, and stomach, and between paired cancer-normal samples from the same four tissue types. Conclusion: Based on exon-level expression profiling and computational analyses of alternative splicing, we propose transcriptome instability as a molecular pan-cancer characteristic. The affected cancers show strong and non-random associations between low expression levels of splicing factor genes, and high amounts of aberrant exon skipping and inclusion, and vice versa, on a genome-wide scale. Keywords: Alternative splicing, Carcinomas, Exon microarray, Splicing factor, Tissue specificity genome, referred to as microsatellite instability, and re- sults from a defective mismatch repair system [4-6]. Microsatellite instability has been described in several solid cancer types, including cancers of the colon, endo- metrium, stomach, and lung [7-11], and is associated with good prognosis in colorectal cancer [9,12,13]. Chro- mosomal instability, characterized by numerical and struc- tural chromosome changes, is common in solid cancers, but the causes for this molecular phenotype remain mostly unknown. Measured as aneuploidy, chromosomal instability has been found to be associated with poor prog- nosis in all four major types of carcinomas [14-17]. In addition to genomic instability, epigenome abnormality has also been described in several cancer types, although most prominent in colorectal cancer [18]. Cancers with the CpG island methylator phenotype have frequent DNA hypermethylation in gene promoter regions, often re- sulting in gene silencing [19]. Recently, we described Sveen et al. BMC Genomics 2014, 15:672 http://www.biomedcentral.com/1471-2164/15/672 Sveen et al. BMC Genomics 2014, 15:672 http://www.biomedcentral.com/1471-2164/15/672 © 2014 Sveen et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Transcriptome instability as a molecular pan-cancer characteristic of carcinomas Anita Sveen1,2, Bjarne Johannessen1,2, Manuel R Teixeira2,3,4,5, Ragnhild A Lothe1,2 and Rolf I Sk Background This transcriptome instabi- lity (TIN) was characterized by large variation in amounts of aberrant inclusion and skipping of exons in colorectal cancer samples. These amounts were shown to be strongly associated with both the expression levels of pre-mRNA splicing factors and poor prognosis for patients with colo- rectal cancer [20]. RNA splicing is a tightly regulated and highly tissue specific process that can occur by a number of different modes [21]. Alternative splicing modes include differen- tial inclusion of whole exons (cassette exons) or parts of exons (alternative 5’ or 3’ splice sites), intron retention, mutual exclusion of cassette exons, alternative ordering of exons (exon scrambling) [22], and splicing of exons encoded by different genes (trans-splicing) [23]. Such alternative splicing is a major source of functional diver- sity in the human genome [24]. Nearly all multi-exon genes are alternatively spliced, predicted to undergo on average seven alternative splicing events during tissue development and across tissue types [25,26]. In cancer, the RNA splicing pathway is commonly disrupted, as evident by aberrant, disease-specific splicing patterns [27]. There is a large collection of proteins regulating the splicing process, and genes encoding these splicing factors have been identified as cancer critical genes, as is the case with SRSF1 [28]. Splicing factors have been found to be both differentially expressed [29] and com- monly mutated in cancer [30-32]. Hence, there is tre- mendous potential for splicing variation in the cancer transcriptome. For samples in seven of the cancer datasets (breast can- cer, cervical cancer, colorectal cancer series I and II, lung cancer series II, and prostate cancer series I and II), the sample-wise TIN-estimates correlated strongly with the expression levels of pre-mRNA splicing factors (n = 280). The amount of significantly correlated splicing factor genes (Pearson correlation, P < 0.05) ranged from 41% (colorectal cancer series I) to 70% (prostate cancer series I) within the datasets (Figure 1a). There was a clear prefer- ence for negative correlation, and the average correlation coefficients for the significantly correlated genes ranged from −0.5 to −0.3 (colorectal cancer series I and II, re- spectively). In these datasets, samples were also separated according to TIN-estimates when performing unsuper- vised hierarchical clustering analyses by the expression levels of all splicing factors (Additional file 1: Figure S3). Principal components analyses based on splicing factor gene expression corroborated these results (Figure 1b). Background The four major types of cancer, lung cancer, breast can- cer, colorectal cancer, and prostate cancer, constitute ap- proximately 40% of cancer cases world-wide, with more than 5 million new diagnoses and 2.7 million related deaths every year [1]. These are all epithelial cancers and are commonly characterized by genomic instability [2]. Genomic instability is described as an enabling hallmark of cancer, generating random mutations which may also hit cancer critical genes [3]. At the nucleotide level, genomic instability can occur as frequent mutations of short nucleotide repeats dispersed throughout the Page 2 of 13 Sveen et al. BMC Genomics 2014, 15:672 http://www.biomedcentral.com/1471-2164/15/672 Sveen et al. BMC Genomics 2014, 15:672 http://www.biomedcentral.com/1471-2164/15/672 dataset were used as threshold values to designate exons as aberrantly spliced (range −2.4 to −2.0 and 1.8 to 2.1, re- spectively; log2-scale; Table S1 in Additional file 1). Sample-wise amounts of aberrant exon skipping and in- clusion were calculated as the number of exons exceeding these threshold values (Additional file 1: Table S1). These sample-wise amounts of aberrant splicing, relative to the average amount within the dataset, are referred to as TIN- estimates. The TIN-estimates and range of TIN-estimates (the difference between the sample with the highest and lowest estimate) were similar within all datasets (average range 3.4; Additional file 1: Table S1). There was no correlation in TIN-estimates between paired cancer and normal samples from the colon (n = 19), lung (n = 20), prostate (n = 29), or stomach (n = 23; Additional file 1: Figure S1). In the breast cancer dataset, there were signifi- cantly lower TIN-estimates in estrogen and progesterone receptor positive than negative samples (P = 0.04, inde- pendent samples t-test; Additional file 1: Figure S2). Also, there were significant differences in TIN-estimates bet- ween histological subtypes of both cervical cancer and lung cancer series II. In the cervical cancer dataset, adeno- carcinomas had higher TIN-estimates than squamous cell carcinomas (P = 0.002), whereas the opposite was found in lung cancer series II (P < 0.001; Additional file 1: Figure S2). There were no associations between TIN-estimates and sample characteristics in any of the other datasets (characteristics listed in Table 1). genome-wide instability acting also on the level of the transcriptome in colorectal cancer, affecting the pre- mRNA splicing process [20]. Background In general, samples with low TIN-estimates (≤−1.0) were separated from samples with high TIN-estimates (≥1.0), although not perfectly in colorectal cancer series II and prostate cancer series I. This association between TIN- estimates and expression levels of splicing factors was less clear in the other three cancer datasets (gastric cancer, lung cancer series I, and neuroblastoma; Figure 1). Here, the amount of significantly correlated splicing factor genes In the present study, we describe TIN in several major types of malignancies, including breast cancer, cervical cancer, colorectal cancer, lung cancer, and prostate cancer. By analyzing exon microarray profiles for alternative spli- cing, we characterized the individual samples within the datasets for relative amounts of aberrant exon skipping and inclusion. In cancer types with TIN, we found strong and non-random associations between these amounts and the expression levels of splicing factors. Surprisingly, this association was intact in pan-cancer analysis of the TIN- cancers, indicating that the pronounced tissue specificity found in corresponding analyses across normal tissue types and paired cancer-normal samples was lost. Strong correlations between TIN-estimates and expression levels of splicing factors Exon microarray profiles for 555 samples from ten dif- ferent cancer datasets and 93 samples from four normal tissue datasets (Table 1) were analyzed for genome-wide alternative splicing variation. The lower and upper 1st per- centiles of alternative splicing (FIRMA) scores within each Sveen et al. Strong correlations between TIN-estimates and expression levels of splicing factors BMC Genomics 2014, 15:672 http://www.biomedcentral.com/1471-2164/15/672 Page 3 of 13 Page 3 of 13 Table 1 Samples included in the study Tissue Samples Histology Other sample characteristics GEO accession number Literature references Breast cancer 84 - Hormone receptor status: HER2-positive (n = 35); ER/PR-positive (n = 25); ER/PR/ HER2-negative (n = 24) GSE16534 [45] Cervical cancer 28 Squamous cell carcinoma (n = 19); adenocarcinoma (n = 9) - GSE27388 [44] Colorectal cancer series I 26 Adenocarcinoma - GSE16534 [45] Colorectal cancer series II 101a Adenocarcinoma Stage: Stage I (n = 28); stage II (n = 34); stage III (n = 26); stage IV (n = 13) GSE24550 (n = 55); GSE29638 (n = 46) [20,49] MSI-status: MSI-high (n = 21); MSS/ MSI-low (n = 77); NA (n = 3) Gastric cancer 25 Adenocarcinoma Stage: Stage I (n = 8); stage II (n = 4); stage III (n = 5); stage IV (n = 8) GSE13195 - Lung cancer series I 20 Non-small-cell lung adenocarcinoma Stage: Stage I (n = 12); stage II (n = 3); stage III (n = 5) GSE12236 [48] Lung cancer series II 43 Non-small-cell lung adenocarcinoma (n = 21) and squamous cell carcinoma (n = 22) - GSE16534 [45] Neuroblastoma 47 - Stage: stage I (n = 10); stage IV (n = 37) GSE27608 [43] Prostate cancer series I 131 Adenocarcinoma Gleason grade: Grade 5 (n = 1); grade 6 (n = 77); grade 7 (n = 42), grade 8 (n = 7); grade 9 (n = 4) Pathologic T stage: stage 2 (n = 85); stage 3 (n = 40); stage 4 (n = 6) GSE21034 [47] Prostate cancer series II 50a Gleason grade: Grade 5 (n = 2); grade 6 (n = 15); grade 7 (n = 32); grade 9 (n = 1) Pathologic T stage: stage 2 (n = 26), stage 3 (n = 24) GSE42954 [46] Normal colonic mucosa 21a 19 samples corresponding to tumors from GSE24550 and GSE29638 GSE42690 (n = 19); GSE29638 (n = 2) [20,49] Normal lung 20 Corresponding to tumors from GSE12236 GSE12236 [48] Normal prostate 29 Corresponding to tumors from GSE21034 GSE21034 [47] Normal stomach 23b Corresponding to tumors from GSE13195 GSE13195 - aPatient samples analyzed in-house bTwo samples from the GEO entry (GSM333256 and GSM333270) were excluded due to failure of reading the raw data files. Strong correlations between TIN-estimates and expression levels of splicing factors Abbreviations: GEO Gene Expression Omnibus, MSI microsatellite instability, MSS microsatellite stable, NA not available T tumor. aPatient samples analyzed in-house bTwo samples from the GEO entry (GSM333256 and GSM333270) were excluded due to failure of reading the raw data files. Abbreviations: GEO Gene Expression Omnibus, MSI microsatellite instability, MSS microsatellite stable, NA not available T tumor. ranged from 2% (lung cancer series I) to 10% (neuroblas- toma). Analyses of the datasets with normal samples indi- cated associations between TIN-estimates and expression levels of splicing factors also in non-cancerous tissues, with strongest associations in normal colonic mucosa (Additional file 1: Figure S4). This variance between the datasets showed no associations to quality control metrics of the exon microarray data (Additional file 1: Figures S5 and Additional file 1: Figure S6). The three cancer data- sets not showing associations between TIN-estimates and expression levels of splicing factors had similar quality control metrics to the other datasets, whereas the dataset showing most deviance during quality control (cervical cancer), showed strong associations. of 280 genes; calculation of percentage of genes in each random gene set with expression levels significantly cor- related with the TIN-estimates), and permutations of the TIN-estimates in each dataset (n = 1,000 permutations; calculation of percentage of splicing factor genes with expression levels significantly correlated with each round of permutation). Corresponding with the results above, the same seven cancer datasets that showed strongest associations, also had higher amounts of splicing factor genes with expression levels significantly correlated with the TIN-estimates than was expected by chance (P < 0.001), as opposed to the other three cancer datasets (Figure 2). The same results were obtained also when analyzing correlation strengths (Additional file 1: Figure S7). Inter- estingly, all the normal tissue datasets also had higher per- centages of splicing factor genes with expression levels that were significantly correlated with the TIN-estimates than expected by chance (Additional file 1: Figure S8). To test whether the observed associations between TIN-estimates and expression levels of splicing factors were greater than expected by chance, comparisons were made with random gene sets of equal size (n = 1,000 sets Sveen et al. BMC Genomics 2014, 15:672 http://www.biomedcentral.com/1471-2164/15/672 Sveen et al. Strong correlations between TIN-estimates and expression levels of splicing factors Also in comparison with gene sets in MSigDB, as well as with the full genome, the splicing factor gene set (and other splicing-related gene sets included in MSigDB) had a strong shift towards negative correlation in the same seven cancer datasets (Additional file 1: Figure S12). Also in comparison with all gene sets included in the Molecular Signatures Database v3.1 (MSigDB; annotated with Gene Ontology terms; n = 1,454 gene sets), as well as with the full genome (n = 17,881 genes), the splicing factor gene set (and other splicing-related gene sets included in MSigDB) had high amounts of genes with expression levels significantly correlated with TIN-estimates in the same seven cancer datasets (Additional file 1: Figure S9). Transcriptome instability as a pan-cancer characteristic Although the individual normal tissue datasets showed strong correlations between the TIN-estimates and ex- pression levels of splicing factors, this association was lost in pooled analyses of the normal tissues (n = 91) of four different origins. Again, this was tested by calcu- lating the correlation between TIN-estimates and ex- pression levels of splicing factors across all the different normal tissue types, and comparing with random gene sets and permutations of the TIN-estimates (Figure 4a). This is compliant with the previously described tissue specificity in both splicing patterns [25,33] and expres- sion levels of splicing factors reviewed in [34]. The same results were found from analysis of the paired cancer- normal sample sets (except for in the colon; Additional file 1: Figure S13), again in agreement with the fact that pre-mRNA splicing is differentially regulated also bet- ween cancer and normal tissues [27]. Surprisingly, when doing the same comparisons across the cancer types with strong associations between TIN-estimates and splicing factor expression, the strong and non-random associations prevailed (P < 0.001; Figure 4b; for this ana- lysis, 20 samples were randomly selected from each of the five cancer types, to assess splicing patterns across similar numbers of samples as for the normal tissues. For cancer types represented by two datasets, samples were selected from the datasets with paired normal sam- ples, i.e. colorectal cancer series II and prostate cancer series I). Strong correlations between TIN-estimates and expression levels of splicing factors BMC Genomics 2014, 15:672 http://www.biomedcentral.com/1471-2164/15/672 (See figure on previous page.) Figure 1 TIN-estimates are associated with the expression levels of splicing factor genes in seven cancer datasets. (a) In seven of the cancer datasets (plots with grey background) there were strong and significant correlations (horizontal axes) between TIN-estimates and the expression levels of ≥41% of the totally 280 splicing factor genes. In the other three datasets, the correlations were mainly non-significant. Yellow and grey bars represent the percentages of splicing factor genes with significant and non-significant correlations, respectively. (b) In two-dimensional principal components analysis based on the expression levels of splicing factor genes (n = 280), samples were generally separated according to TIN-estimates (only samples with TIN-estimates ≥±1.0 were included, the number and percentages of samples are indicated for each dataset) in the same seven datasets (with grey background). In colorectal cancer series II and prostate cancer series I, the separation was not complete. In the gastric cancer dataset and lung cancer series I, there were few samples with TIN-estimates ≥±1.0. In the neuroblastoma dataset, there was no clear separation. PC, principal component. The association was particularly strong in normal colonic mucosa, where 56% of the splicing factors were signifi- cantly correlated. negatively vs. positively correlated splicing factor genes ranged from 4 (normal colonic mucosa) to 30 (normal stomach; Additional file 1: Figure S11). Also in comparison with all gene sets included in the Molecular Signatures Database v3.1 (MSigDB; annotated with Gene Ontology terms; n = 1,454 gene sets), as well as with the full genome (n = 17,881 genes), the splicing factor gene set (and other splicing-related gene sets included in MSigDB) had high amounts of genes with expression levels significantly correlated with TIN-estimates in the same seven cancer datasets (Additional file 1: Figure S9). The percentage of TIN-samples (samples with TIN- estimates ≥±1.0) in each dataset varied from 0 in lung can- cer series I to 48% in prostate cancer series I (Figure 1b). The datasets with the highest percentages also had higher amounts of splicing factor genes with expression levels sig- nificantly correlated with the TIN-estimates (Additional file 1: Figure S10). We consider this to strengthen the no- tion that tissue and cancer types with high variation in splicing also have a correspondingly high variation in spli- cing factor expression. Strong correlations between TIN-estimates and expression levels of splicing factors In these pan-cancer analyses, there was a much higher amount of significant negatively than positively correlated splicing factor genes (232 genes compared with 14 genes, ratio 17; also this higher than expected by chance, compared with random gene sets and permutations of the TIN-estimates, P < 0.001; Figure 4b). When limiting The percentage of TIN-samples (samples with TIN- estimates ≥±1.0) in each dataset varied from 0 in lung can- cer series I to 48% in prostate cancer series I (Figure 1b). The datasets with the highest percentages also had higher amounts of splicing factor genes with expression levels sig- nificantly correlated with the TIN-estimates (Additional file 1: Figure S10). We consider this to strengthen the no- tion that tissue and cancer types with high variation in splicing also have a correspondingly high variation in spli- cing factor expression. Strong correlations between TIN-estimates and expression levels of splicing factors BMC Genomics 2014, 15:672 Page 4 of 13 Breast cancer (n = 23; 27%) Cervical cancer (n = 5; 18%) Colorectal cancer I (n = 8; 31%) Colorectal cancer II (n = 25; 25%) Lung cancer II (n = 17; 40%) Prostate cancer I (n = 63; 48%) Prostate cancer II (n = 5; 10%) PC1 PC2 -6 12 -10 20 TIN-estimates: ≥ 1.0 ≤ -1.0 b) Breast cancer (n = 84) Cervical cancer (n = 28) Colorectal cancer I (n = 26) Colorectal cancer II (n = 101) Lung cancer II (n = 43) Prostate cancer I (n = 131) Prostate cancer II (n = 50) 0 -0.5 -1.0 0.5 1.0 Percentage of splicing factor genes Pearson correlation coefficient 0 -0.5 -1.0 0.5 1.0 0 -0.5 -1.0 0.5 1.0 0 -0.5 -1.0 0.5 1.0 0 -0.5 -1.0 0.5 1.0 0 -0.5 -1.0 0.5 1.0 0 -0.5 -1.0 0.5 1.0 5 10 15 20 25 30 35 Splicing factor genes: Significant correlation (P < 0.05) Non-significant correlation (P > 0.05) a) Gastric cancer (n = 25) Lung cancer I (n = 20) Neuroblastoma (n = 47) 0 -0.5 -1.0 0.5 1.0 0 -0.5 -1.0 0.5 1.0 0 -0.5 -1.0 0.5 1.0 Gastric cancer (n = 3; 12%) Lung cancer I (n = 0) Neuroblastoma (n = 9; 19%) Figure 1 (See legend on next page.) Breast cancer (n = 84) Cervical cancer (n = 28) Colorectal cancer I (n = 26) Colorectal cancer II (n = 101) Lung cancer II (n = 43) Prostate cancer I (n = 131) Prostate cancer II (n = 50) 0 -0.5 -1.0 0.5 1.0 Percentage of splicing factor genes Pearson correlation coefficient 0 -0.5 -1.0 0.5 1.0 0 -0.5 -1.0 0.5 1.0 0 -0.5 -1.0 0.5 1.0 0 -0.5 -1.0 0.5 1.0 0 -0.5 -1.0 0.5 1.0 0 -0.5 -1.0 0.5 1.0 5 10 15 20 25 30 35 Splicing factor genes: Significant correlation (P < 0.05) Non-significant correlation (P > 0.05) a) Lung cancer II (n = 43) Prostate cancer I (n = 131) Colorectal cancer II (n = 101) Gastric cancer (n = 25) Lung cancer I (n = 20) Neuroblastoma (n = 47) 0 -0.5 -1.0 0.5 1.0 0 -0.5 0.5 1.0 0 -0.5 -1.0 0.5 1.0 Neuroblastoma (n = 47) Lung cancer I (n = 20) Breast cancer (n = 23; 27%) Cervical cancer (n = 5; 18%) Colorectal cancer II (n = 25; 25%) Lung cancer II (n = 17; 40%) PC1 PC2 -6 12 -10 20 b) Gastric cancer (n = 3; 12%) Lung cancer I (n = 0) Figure 1 (See legend on next page.) Breast cancer (n = 23; 27%) Cervical cancer (n = 5; 18%) Colorectal cancer I (n = 8; 31%) Colorectal cancer II (n = 25; 25%) Lung cancer II (n = 17; 40%) Prostate cancer I (n = 63; 48%) Prostate canc (n = 5; 1 PC1 PC2 -6 12 -10 20 TIN-estimates: ≥ 1.0 ≤ -1.0 b) Gastric cancer (n = 3; 12%) Lung cancer I (n = 0) Neuroblastoma (n = 9; 19%) Figure 1 (See legend on next page.) Breast cancer (n = 23; 27%) Colorectal cancer II (n = 25; 25%) PC1 PC2 -6 12 -10 20 b) Gastric cancer (n = 3; 12%) Figure 1 (See legend on next page.) Colorectal cancer I (n = 8; 31%) Prostate cancer I (n = 63; 48%) Cervical cancer (n = 5; 18%) b) Lung cancer II (n = 17; 40%) Prostate cancer I (n = 63; 48%) Prostate cancer II (n = 5; 10%) Lung cancer I (n = 0) Neuroblastoma (n = 9; 19%) Gastric cancer (n = 3; 12%) Figure 1 (See legend on next page.) Figure 1 (See legend on next page.) Page 5 of 13 Sveen et al. Inverse relationship between TIN-estimates and expression levels of splicing factors In the seven cancer datasets with strong associations bet- ween TIN-estimates and expression levels of splicing fac- tors, the associations were mainly inverse. The amount of genes with expression levels that were negatively corre- lated with the TIN-estimates (≥89% of the significantly correlated splicing factor genes in all seven datasets) was much larger than the amount of positively correlated genes, with ratios ranging from 8 in prostate cancer series II to 97 in prostate cancer series I. This ratio of negative vs. positive correlations was significantly higher than ex- pected by chance (again compared with random gene sets and permutations of the amounts of aberrant exon usage; P < 0.001; Figure 3). This significant shift towards negative correlation was not found in the three cancer datasets with weak associations between TIN-estimates and ex- pression levels of splicing factors (ratios ranging from 1 to 2; Figure 3). In the normal tissue types, there was also a significant, but less pronounced shift towards negative correlation. Here, the ratios of the amounts of significant Sveen et al. BMC Genomics 2014, 15:672 http://www.biomedcentral.com/1471-2164/15/672 Page 6 of 13 Breast cancer (n = 84) Cervical cancer (n = 28) Colorectal cancer I (n = 26) Colorectal cancer II (n = 101) Lung cancer II (n = 43) Prostate cancer I (n = 131) Gastric cancer (n = 25) Lung cancer I (n = 20) Neuroblastoma (n = 47) Percentage significantly correlated genes 0 100 50 25 75 Splicing factor genes with expression levels significantly correlated with the sample-wise TIN-estimates Genes in each of 1,000 random gene sets (amount of gene sets on vertical axes) with expression levels significantly correlated with the sample-wise TIN-estimates Splicing factor genes with expression levels significantly correlated with each of 1,000 permutations of the TIN-estimates (amount of permuted datasets on vertical axes) 160 150 120 140 140 280 210 180 140 Prostate cancer II (n = 50) 120 Amount of random gene sets or permutations Percentage (horizontal axis) of: Figure 2 Correlation between TIN-estimates and splicing factor expression compared with random gene sets and permuted TIN-estimates. In seven of the cancer datasets (with grey background), high percentages of the 280 splicing factor genes had expression levels that were significantly correlated (P < 0.05; Pearson correlation) with the sample-wise TIN-estimates (41% to 70%; red dots; horizontal axis). Inverse relationship between TIN-estimates and expression levels of splicing factors In seven of the cancer datasets (with grey background), high percentages of the 280 splicing factor genes had expression levels that were significantly correlated (P < 0.05; Pearson correlation) with the sample-wise TIN-estimates (41% to 70%; red dots; horizontal axis). This was more than expected by chance, as compared with genes in each of 1,000 random sets of 280 genes (bar graphs in light blue) and with 1,000 permutations of the TIN-estimates (bar graphs in dark blue). In the other three cancer datasets (no background), the percentages of significantly correlated splicing factor genes were much lower and not higher than expected by chance. Inverse relationship between TIN-estimates and expression levels of splicing factors This was more than expected by chance, as compared with genes in each of 1,000 random sets of 280 genes (bar graphs in light blue) and with 1,000 permutations of the TIN-estimates (bar graphs in dark blue). In the other three cancer datasets (no background), the percentages of significantly correlated splicing factor genes were much lower and not higher than expected by chance. Breast cancer (n = 84) Cervical cancer (n = 28) Colorectal cancer I (n = 26) Colorectal cancer II (n = 101) Lung cancer II (n = 43) Prostate cancer I (n = 131) Percentage significantly correlated genes 0 100 50 25 75 160 150 120 140 140 280 Prostate cancer II (n = 50) 120 Amount of random gene sets or permutations Gastric cancer (n = 25) Lung cancer I (n = 20) Neuroblastoma (n = 47) 210 180 140 Prostate cancer II (n = 50) Percentage (horizontal axis) of: Percentage (horizontal axis) of: Percentage (horizontal axis) of: Splicing factor genes with expression levels significantly correlated with the sample-wise TIN-estimates Splicing factor genes with expression levels significantly correlated with the sample-wise TIN-estima Genes in each of 1,000 random gene sets (amount of gene sets on vertical axes) with expression levels significantly correlated with the sample-wise TIN-estimates Genes in each of 1,000 random gene sets (amount of gene sets on vertical axes) with expression level significantly correlated with the sample-wise TIN-estimates Splicing factor genes with expression levels significantly correlated with each of 1,000 permutations of the TIN-estimates (amount of permuted datasets on vertical axes) Figure 2 Correlation between TIN-estimates and splicing factor expression compared with random gene sets and permuted TIN-estimates. In seven of the cancer datasets (with grey background), high percentages of the 280 splicing factor genes had expression levels that were significantly correlated (P < 0.05; Pearson correlation) with the sample-wise TIN-estimates (41% to 70%; red dots; horizontal axis). This was more than expected by chance, as compared with genes in each of 1,000 random sets of 280 genes (bar graphs in light blue) and with 1,000 permutations of the TIN-estimates (bar graphs in dark blue). In the other three cancer datasets (no background), the percentages of significantly correlated splicing factor genes were much lower and not higher than expected by chance. Figure 2 Correlation between TIN-estimates and splicing factor expression compared with random gene sets and permuted TIN-estimates. Discussion This shift was higher than expected by chance, as demonstrated by comparisons with genes in each of 1,000 random sets of 280 genes (light blue), and with each of 1,000 permutations of the TIN-estimates (dark blue). This significant shift towards negative correlation was not found in the three cancer datasets with weak associations between TIN-estimates and splicing factor expression (no background). Figure 3 Negative correlation between TIN-estimates and splicing factor expression in the cancer datasets. In the seven cancer datasets (grey background) with strong associations between TIN-estimates and expression levels of splicing factors (n = 280), the relationship was inverse, with a much higher percentage of significant negatively (horizontal axes) than positively (vertical axes) correlated splicing factor genes (red). This shift was higher than expected by chance, as demonstrated by comparisons with genes in each of 1,000 random sets of 280 genes (light blue), and with each of 1,000 permutations of the TIN-estimates (dark blue). This significant shift towards negative correlation was not found in the three cancer datasets with weak associations between TIN-estimates and splicing factor expression (no background). Figure 3 Negative correlation between TIN-estimates and splicing factor expression in the cancer datasets. In the seven cancer datasets (grey background) with strong associations between TIN-estimates and expression levels of splicing factors (n = 280), the relationship was inverse, with a much higher percentage of significant negatively (horizontal axes) than positively (vertical axes) correlated splicing factor genes (red). This shift was higher than expected by chance, as demonstrated by comparisons with genes in each of 1,000 random sets of 280 genes (light blue), and with each of 1,000 permutations of the TIN-estimates (dark blue). This significant shift towards negative correlation was not found in the three cancer datasets with weak associations between TIN-estimates and splicing factor expression (no background). amounts of aberrant exon skipping and inclusion (TIN- estimates) that are significant negatively correlated with the expression levels of splicing factor genes. Such as- sociations were also found within healthy tissue types, although to less extent. This is consistent with the fact that most tissues exhibit great and tightly regulated splicing variation. However, the splicing patterns were clearly distinct between healthy tissues and their ma- lignant counterparts, as shown by discordant TIN- estimates between paired cancer and normal samples in all the four different tissue types analyzed. Discussion the analyses to cancer samples for which paired normal samples were available, the results were corresponding. Non-random associations between TIN-estimates and splicing factor expression were found only in analyses across colorectal cancer series II and prostate cancer series I (Additional file 1: Figure S13). This indicates that TIN is a shared molecular characteristic across the affected cancer types. We have previously proposed genome-wide instability of the transcriptome as a molecular, pre-mRNA splicing re- lated characteristic of colorectal cancer. Here, we show data indicating that the transcriptomes of several, but not all, other types of solid tumors also have this feature. Common cancers of the breast, cervix, large bowel, lung, and prostate all show large variation in sample-wise Sveen et al. BMC Genomics 2014, 15:672 http://www.biomedcentral.com/1471-2164/15/672 Sveen et al. BMC Genomics 2014, 15:672 Page 7 of 13 Breast cancer (n = 84) Cervical cancer (n = 28) 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 Percentage significant positively (vertical axes) vs. negatively (horizontal axes) correlated genes: Colorectal cancer I (n = 26) Colorectal cancer II (n = 101) Lung cancer II (n = 43) Prostate cancer I (n = 131) Expression levels of splicing factor genes and sample-wise TIN-estimates Expression levels of genes in each of 1,000 random gene sets and sample-wise TIN-estimates Expression levels of splicing factor genes and each of 1,000 permutations of the TIN-estimates Percentage significantly correlated genes (P < 0.05; Pearson correlation) comparing: 40 40 Gastric cancer (n = 25) Lung cancer I (n = 20) Neuroblastoma (n = 47) Prostate cancer II (n = 50) 60 30 60 30 60 30 20 20 40 40 20 20 40 40 20 20 Figure 3 Negative correlation between TIN-estimates and splicing factor expression in the cancer datasets. In the seven cancer datasets (grey background) with strong associations between TIN-estimates and expression levels of splicing factors (n = 280), the relationship was inverse, with a much higher percentage of significant negatively (horizontal axes) than positively (vertical axes) correlated splicing factor genes (red). This shift was higher than expected by chance, as demonstrated by comparisons with genes in each of 1,000 random sets of 280 genes (light blue), and with each of 1,000 permutations of the TIN-estimates (dark blue). Discussion This significant shift towards negative correlation was not found in the three cancer datasets with weak associations between TIN-estimates and splicing factor expression (no background). Breast cancer (n = 84) Cervical cancer (n = 28) 60 30 60 30 60 30 60 30 60 30 60 60 30 60 30 60 30 60 30 Percentage significant positively (vertical axes) vs. negatively (horizontal axes) correlated genes: Colorectal cancer I (n = 26) Colorectal cancer II (n = 101) Lung cancer II (n = 43) Prostate cancer I (n = 131) Prostate cancer II 60 30 60 30 60 30 Breast cancer (n = 84) 60 30 60 30 Percentage significant positively (vert 60 30 60 30 es) correlated genes: Colorectal cancer I (n = 26) 30 30 60 30 60 30 Lung cancer II (n = 43) 60 Colorectal cancer II (n = 101) Prostate cancer II ( 50) 60 30 60 30 30 30 30 Prostate cancer II (n = 50) 30 60 30 P t i ifi tl l t d (P < 0 05 P l ti ) i 40 40 Gastric cancer (n = 25) Lung cancer I (n = 20) Neuroblastoma (n = 47) (n 50) 20 20 40 40 20 20 40 40 20 20 60 30 40 40 Gastric cancer (n = 25) Lung cancer I (n = 20) Neuroblastoma (n = 47) 20 20 40 40 20 20 40 40 20 20 Lung cancer I (n = 20) Neuroblastoma (n = 47) 20 20 40 Percentage significantly correlated genes (P < 0.05; Pearson correlation) comparing: Percentage significantly correlated genes (P < 0.05; Pearson correlation) comparing: Expression levels of splicing factor genes and sample-wise TIN-estimates Expression levels of splicing factor genes and sample-wise TIN-estimates Expression levels of genes in each of 1,000 random gene sets and sample-wise TIN-estimates Expression levels of splicing factor genes and each of 1,000 permutations of the TIN-estimates Figure 3 Negative correlation between TIN-estimates and splicing factor expression in the cancer datasets. In the seven cancer datasets (grey background) with strong associations between TIN-estimates and expression levels of splicing factors (n = 280), the relationship was inverse, with a much higher percentage of significant negatively (horizontal axes) than positively (vertical axes) correlated splicing factor genes (red). Discussion BMC Genomics 2014, 15:672 http://www.biomedcentral.com/1471-2164/15/672 Page 8 of 13 Amount of random gene sets or permutations Percentage significantly correlated genes Expression levels of splicing factor genes and sample-wise TIN-estimaes Expression levels of genes in each of 1,000 random gene sets and sample-wise TIN-estimates Expression levels of splicing factor genes and each of 1,000 permutations of the TIN-estimates Percentage significantly correlated genes (P < 0.05; Pearson correlation ) comparing: All normal tissue samples (n = 91); colon (n = 19), lung (n = 20), prostate (n = 29), stomach (n = 23) Percentage significant positively correlated genes Percentage significant negatively correlated genes Amount of random gene sets or permutations Percentage significantly correlated genes 40 80 120 160 50 100 260 20 40 60 80 100 120 50 260 50 100 50 100 a) b) All cancer types with transcriptome instability (n = 5x20 samples) Percentage significant positively correlated genes Percentage significant negatively correlated genes 100 50 100 50 100 Amount of random gene sets or permutations Percentage significantly correlated genes All normal tissue samples (n = 91); colon (n = 19), lung (n = 20), prostate (n = 29), stomach (n = 23) Percentage significant positively correlated genes Percentage significant negatively correlated genes 20 40 60 80 100 120 50 260 a) 100 50 100 50 100 50 Percentage significantly correlated genes Percentage significant negatively correlated genes Percentage significantly correlated genes Percentage significant negatively correlated genes b) All cancer types with transcriptome instability (n = 5x20 samples) Amount of random gene sets or permutations Percentage significantly correlated genes 40 80 120 160 50 100 260 50 100 50 100 b) All cancer types with transcriptome instability (n = 5x20 samples) Percentage significant positively correlated genes Percentage significant negatively correlated genes 100 50 Expression levels of genes in each of 1,000 random gene sets and sample-wise TIN-estimates Expression levels of genes in each of 1,000 random gene sets and sample-wise TIN-estimates Expression levels of splicing factor genes and each of 1,000 permutations of the TIN-estimates Expression levels of splicing factor genes and each of 1,000 permutations of the TIN-estimates Figure 4 Correlation between TIN-estimates and splicing factor expression in pooled tissue types. The left and right plots correspond to plots in Figures 2 and 3, respectively. Discussion This is in compliance also with the original TIN-report, showing that colorectal cancers have higher TIN-estimates than normal colonic mucosa [20]. Furthermore, in analysis across the various healthy tissues, the correlation bet- ween TIN-estimates and expression levels of splicing factors was not greater than expected by chance. This indicates a failure to detect a common pattern of Sveen et al. Discussion (a) In pooled analyses of samples from four different normal tissues, the percentage of splicing factor genes (totally 280 genes; red dots) with expression levels that were significantly correlated with the TIN-estimates (P < 0.05; Pearson correlation), was not higher than expected by chance, as demonstrated by comparison with genes in each of 1,000 random sets of 280 genes (light blue) and with 1,000 permutations of the TIN-estimates (dark blue; left plot). The ratio between the amounts of significant negatively and positively correlated splicing factor genes was 2 (right plot). (b) Contrarily, across 20 randomly selected samples from each of five different cancer types with strong associations between TIN-estimates and splicing factor expression (breast cancer, cervical cancer, colorectal cancer series II, lung cancer series II, and prostate cancer I) the percentage of splicing factor genes with expression levels that were significantly correlated was higher than expected by chance (left plot). Also, there was a significant shift towards negative correlation (the ratio between the amounts of splicing factors genes with expression levels that were significant negatively and positively correlated was 17; right plot). regulation, and that the splicing process is predomin- antly tissue specific in healthy tissues. Interestingly, in pan-cancer analyses, the strong associations with expres- sion levels of splicing factors prevailed. These results suggest that TIN is a pan-cancer characteristic, clearly distinguished from normal splicing variation and splicing factor expression. Pan-Cancer project [35]. The recognition that cancers from separate organs may have shared molecular fea- tures, while cancers from the same organ may be dis- tinct, has great potential influence both biologically, on our understanding of the tumorigenic process, and clin- ically, for example for more personalized and targeted treatment strategies. Furthermore, pan-cancer analyses may aid in the identification of novel cancer-critical genes not disclosed in individual cancer types because of The importance of molecular pan-cancer analyses has recently been illuminated by The Cancer Genome Atlas Page 9 of 13 Sveen et al. BMC Genomics 2014, 15:672 http://www.biomedcentral.com/1471-2164/15/672 Sveen et al. BMC Genomics 2014, 15:672 http://www.biomedcentral.com/1471-2164/15/672 There are various non-biological factors that may have influenced the splicing variation observed in this study. Firstly, the analyses are sensitive to the sizes of the data- sets, and the variation in TIN-estimates increased with sample numbers (data not shown). This is an inherent consequence of the analyses, as differential exon skipping and inclusion were detected relative to all included sam- ples. Discussion Although experimental work is needed to elucidate the functional mechanisms, an underlying biological explanation for the observed va- riation is suggested by the strong and non-random asso- ciations with the expression levels of splicing factors in several cancer types. In fact, this association increased with increasing splicing variation, as indicated by the cor- relation between the amount of TIN-samples per dataset (samples with TIN-estimates ≥±1.0) and the number of splicing factor genes with expression levels that were sig- nificantly correlated with the TIN-estimates. Also worth noting is the striking inverse nature, with the great major- ity of splicing factors having expression levels that were negatively correlated with the TIN-estimates. Altogether, this suggests a mechanism in which decreased expression levels of splicing factors result in more aberrant exon skip- ping and inclusion. The fact that the majority of splicing factors are involved in several cancer types suggests a genome-wide mechanism. This is consistent with previous reports showing a coordinated regulation of RNA splicing by numerous splicing factors [38]. Splicing factor encoding genes have previously been nominated as cancer-critical based on their altered ex- pression levels [28]. Recently, individual splicing factor genes have also been found to be commonly mutated in different cancer types, and this has been shown to have important implications for carcinogenesis [30-32,39,40]. In this study, mutation analyses have not been con- ducted. Furthermore, only the splicing events aberrant skipping and inclusion of cassette exons, as well as in- tron retention, have been considered. Although these are the most common modes of splicing [41], a complete view of the genome-wide effects of splicing factor ex- pression on the splicing process should also consider the other events regulated by splicing factors (alternative 5’ and 3’ splice sites, patterns of mutual exclusion, trans- splicing, and exon scrambling). To describe TIN as a genuine molecular pan-cancer characteristic, identification of individual, aberrant splicing events and functional validation of their correlations with splicing factor expression levels are warranted. Such ana- lyses are complicated by the genome-wide nature of the described associations. No single splicing factor is likely to account for all the observed variation. This situation re- sembles the obstacles faced also when characterizing the now well established genetic and epigenetic genome-wide phenotypes (with the exception of microsatellite instability found in subgroups of certain cancer types). Discussion Secondly, technical variation in the microarray data may also have contributed. Accordingly, thorough quality control of the data was performed, and the extent of cor- relations between splicing variation and expression levels of splicing factors in each dataset, was independent of various quality control metrics. However, the failure to de- tect the strong association between splicing variation and splicing factor expression in three of the cancer datasets (gastric cancer, lung cancer series I, and neuroblastoma) may be attributable to non-biological features of the data rather than inherent characteristics of the cancer types. In particular, the two datasets from lung cancer showed conflicting results, with strong associations between the sample-wise TIN-estimates and splicing factor expression in series II, but not in series I. Both these patient series consist of non-small-cell carcinomas, but series I com- prises adenocarcinomas only, whereas series II also includes squamous cell carcinomas. Although the TIN- estimates were significantly higher in the squamous cell carcinomas than the adenocarcinomas in lung cancer series II, the subtypes individually showed strong associa- tions between the TIN-estimates and the expression levels of splicing factors (data not shown). For the other two cancer types represented by two datasets, colorectal can- cer and prostate cancer, strong associations were clearly indicated in both datasets. low mutation rates [36]. With regard to pan-cancer ana- lysis of alternative splicing, mutation of the splicing factor gene U2AF1 has been shown to result in both distinct and equal aberrant splicing events in lung adenocarcinoma and acute myeloid leukemia [37], indicating both specific and common regulation in the two cancer types. Here, from integrated expression analyses of a comprehensive collection of splicing factor genes, we have described TIN both within and across individual cancer types, suggesting that TIN is a general characteristic of cancer. Closer inspection of individual splicing factor genes revealed dif- ferences between the cancer types with respect to which genes were most closely associated with the aberrant spli- cing amounts (data not shown). No single splicing factor gene seceded in these pan-cancer analyses, suggesting that there is no dependency for the involvement of specific splicing factors in the establishment of TIN. In this study, genome-wide analyses of alternative splicing were performed by computational analyses of exon-level microarray data. Conclusions All 648 samples have been analyzed for gene expression at the exon level by the Affymetrix GeneChip Human Exon 1.0 ST Array (Affymetrix Inc, Santa Clara, CA, USA). This array contains approximately 6 million dif- ferent probes, providing genome-scale, exon-resolution expression measures. Each probe set consists of an aver- age of four probes and generally corresponds to one exon. For each sample, 284,258 probe sets belonging to the ‘core’ set of probes targeting well annotated exons were analyzed, using an annotation file custom-made for alternative splicing analysis with the Finding Isoforms using Robust Multichip Analysis (FIRMA) algorithm (HuEx-1_0-st-v2,coreR3,A20071112,EP.cdf) [50,51]. By computational analysis of alternative splicing based on exon-level microarray data, we show that common types of solid cancers (including breast, cervical, colorectal, lung, and prostate cancers) exhibit large variation among samples in amounts of aberrant skipping and inclusion that are non-randomly associated with the expression levels of splicing factors. Importantly, this type of tran- scriptome instability is a pan-cancer characteristic, as op- posed to the tissue specificity prevailing in healthy tissues. Functional evaluation of the associations between this splicing pattern and the expression levels of splicing fac- tors is needed to determine if transcriptome instability is a genuine phenotype of solid cancers. ( ) [ ] Probe cell intensity (CEL) files storing raw data for each of the samples were used as input for preprocessing and alternative splicing analysis with the FIRMA algorithm implemented in the R software environment [52] (GEO accession numbers of both public and previously unpub- lished data are indicated in Table 1). This provided the basis for calculations of sample-wise amounts of aberrant exon skipping and inclusion within the datasets, as we have also previously described [20]. The first two steps of FIRMA follow the RMA approach [53] for background correction of individual probes based on GC-content, and for inter-chip quantile normalization. Then, alternative splicing scores (FIRMA scores) are calculated for each in- dividual exon in each individual sample, as the deviance between the exon level and corresponding overall gene level expression measures. These scores represent the re- siduals from fitting gene level models to the exon level data, according to the mapping provided in the custom- made annotation file. The FIRMA scores were log2- transformed. Strong positive and negative scores reflect differential exon inclusion and skipping, respectively, compared with the rest of the samples in the dataset. Discussion However, we anticipate that ongoing RNA sequencing efforts will be highly valuable in gaining further insights into the pro- posed TIN characteristic. Although premature, the implications of TIN as a com- mon and novel, genome-wide characteristic of epithelial cancers are intriguing and warrant further investigation. Similarly to the genetic and epigenetic genome-wide phe- notypes that have been proven to be important clinical Page 10 of 13 Sveen et al. BMC Genomics 2014, 15:672 http://www.biomedcentral.com/1471-2164/15/672 Sveen et al. BMC Genomics 2014, 15:672 http://www.biomedcentral.com/1471-2164/15/672 Sveen et al. BMC Genomics 2014, 15:672 http://www.biomedcentral.com/1471-2164/15/672 boards (the Regional Committee for Medical and Health Research Ethics, number 1.2005.1629; and reference [46]), which involves that informed consent is obtained from patients being enrolled to the study. characteristics, allowing for sub-grouping of patients ac- cording to prognosis [13,16] and tumor histology [42], TIN may also have clinical potential. Previously, we have shown that TIN is associated with adverse outcome for patients with stage II and III colorectal cancer [20]. Conclusions The lower and upper 1st percentiles of FIRMA scores across each dataset (Additional file 1: Table S1) were used as thresholds for identification of deviating (aberrant) exon skipping and inclusion, respectively. Sample-wise amounts of exons with aberrant splicing patterns were summa- rized from the exons exceeding these threshold values. Amounts of aberrant exon skipping or inclusion per sam- ple are presented on a log2-scale, relative to the average sample-wise amount within the individual datasets. As a measure of TIN, aberrant exon skipping and inclusion were summarized. This TIN-estimate thus represents the total amounts of aberrant exon inclusion and skipping per sample (on a log2-scale), relative to the average amount Patient material In this study, genome-wide expression data at the exon level for a comprehensive collection of 555 tissue samples from seven types of solid tumors, including the four most common, breast, colorectal, lung, and prostate, have been analyzed. Additionally, the study comprises expression data for a total of 93 normal tissue samples, including paired samples from cancers of the colon, lung, prostate, and stomach. All exon-level microarray datasets (Affyme- trix HuEx-1_0-st-v2 arrays) with more than 20 cancer samples that have been made publically available by us and others have been included [20,43-49], except 83 colo- rectal cancer samples in GSE24549, which we previously used to describe TIN [20]. An overview of the datasets, with available clinical information of the tissue samples, is found in Table 1. From each of colorectal cancer, lung cancer and prostate cancer, two datasets have been in- cluded and are referred to as series I and II, respectively. Colorectal cancer series II is an in-house, consecutive series of stage I – IV cancers (90% inclusion rate), col- lected at Aker University Hospital, Oslo, Norway, between 2005 and 2007. This series also comprises 21 normal co- lonic mucosa samples (including 6 that have not been published before) taken from disease-free areas distant to the primary tumors (19 matched sample pairs are in- cluded). Prostate cancer series II is also an in-house series, consisting of 50 primary tumor samples from a con- secutive series of 200 clinically localized cancers treated with radical prostatectomy at the Portuguese Oncology Institute, Porto, Portugal. The analysis of the additional samples published herein has been approved by the institutional review Sveen et al. BMC Genomics 2014, 15:672 http://www.biomedcentral.com/1471-2164/15/672 Page 11 of 13 This collection of gene sets (ranging in size from 10 to 2,131 genes) comprises 8,282 unique genes, correspon- ding to 7,923 transcript clusters on the exon arrays (matched by gene symbols), thus representing 44% of the genes on the arrays. These gene sets are available from the ‘TIN’ analysis package. This collection of gene sets (ranging in size from 10 to 2,131 genes) comprises 8,282 unique genes, correspon- ding to 7,923 transcript clusters on the exon arrays (matched by gene symbols), thus representing 44% of the genes on the arrays. These gene sets are available from the ‘TIN’ analysis package. within the dataset. Availability of supporting data Availability of supporting data The data sets supporting the results of this article are available in the NCBI’s Gene Expression Omnibus re- pository, GSE42690 and GSE42954. Patient material TIN-estimates ≥1.0, indicating twice as much aberrant exon skipping and inclusion as the average sample, and ≤−1.0, indicating half as much as the average sample, are used as a thresholds for characterizing sam- ples with TIN (TIN-samples). In comparisons across data- sets, alternative splicing analyses by the FIRMA algorithm, and calculation of sample-wise TIN-estimates, were done across all the samples included in each comparison, as indicated. Previously unpublished microarray data have been de- posited to the NCBI’s Gene Expression Omnibus (GEO; accession numbers GSE42690 and GSE42954). R codes for alternative splicing analyses, statistical analyses, and plotting functions have been collected in a new Bio- conductor package called ‘TIN’. This package is available for download from http://www.bioconductor.org/. A de- scription of the package, with analysis protocols and R codes can also be found at this web site. Authors’ contributions AS and BJ carried out the data analyses. AS, MRT, RAL, and RIS participated in acquisition of the in-house and previously unpublished exon microarray data. AS drafted the manuscript. RIS conceived and directed the study. All authors participated in interpretation of results, contributed to manuscript preparation, and have read and approved the final version of the manuscript. Splicing factors and miscellaneous gene sets We have created a comprehensive list of 280 splicing factor genes by combining search results from public annotation databases, as previously described [20]. Gene-level expres- sion measures of these splicing factors were obtained from the CEL files of all samples included here. The CEL files were preprocessed across the datasets by the RMA ap- proach [53], performing background correction, inter-chip quantile normalization, and gene-level summarization. For this, the Affymetrix Expression Console™1.1 software and Affymetrix HuEx-1_0-st-v2.r2 gene-core library files were used. Acknowledgements This work was funded by the Norwegian Health Region South-East [project numbers 2011024 to R.A.L. supporting A.S. as postdoc, 2012067 to R.I.S. supporting B.J. as PhD student]; the Norwegian Cancer Society [grant numbers PR-2006-0442 to R.A.L., PR-2007-0166 to R.I.S.]; and partly supported by the Research Council of Norway through its Centres of Excellence funding scheme [project number 179571]. Competing interests The authors declare that they have no competing interests. Quality control of microarray data y y The analyses presented in this study are potentially sensitive to the quality of the microarray data. Hence, exon-level quality control of all included samples was per- formed (Supplementary Methods in Additional file 1). All cancer (Additional file 1: Figure S5) and normal (Additional file 1: Figure S6) samples were preprocessed together and compared using the quality assessment met- rics recommended by Affymetrix [55] and reported by the Affymetrix Expression Console™software. Abbreviations Abbreviations FIRMA: Finding isoforms using robust multichip analysis; GEO: Gene expression omnibus; mRNA: Messenger RNA; MSI: Microsatellite instability; MSigDB: The molecular signatures database; MSS: Microsatellite stable; NA: Not available; T: Tumor; TIN: Transcriptome instability. Additional file Additional file 1: Supplementary Methods, Table S1, Figures S1-S13. Statistical analyses Statistical analyses were conducted in the R software environment (version 2.15.1). Permutations of TIN-esti- mates across the samples, and generation of random gene sets were done using the sample() function. Pearson corre- lations and two-sided Student P-values were calculated using the corAndPvalue() function in the WGCNA pack- age [54]. Hierarchical clustering analyses with Euclidean distance metrics and complete linkage were done using J-Express 2011 (MolMine AS, Bergen, Norway). To corroborate the results from the clustering analyses, principle components analysis by singular value decom- position of the covariance matrix was performed using the prcomp() function in R. The component scores for the first two principal components are used to illustrate two- dimensional sample separation based on the input gene expression variables. References Nat Rev Mol Cell Biol 2009, 10:741–754. 35. Cancer Genome Atlas Research Network, Weinstein JN, Collisson EA, Mills GB, Shaw KR, Ozenberger BA, Ellrott K, Shmulevich I, Sander C, Stuart JM: The cancer genome atlas pan-cancer analysis project. Nat Genet 2013, 45:1113–1120. 13. Popat S, Hubner R, Houlston RS: Systematic review of microsatellite instability and colorectal cancer prognosis. J Clin Oncol 2005, 23:609–618. 14. Choi CM, Seo KW, Jang SJ, Oh YM, Shim TS, Kim WS, Lee DS, Lee SD: Chromosomal instability is a risk factor for poor prognosis of adenocarcinoma of the lung: Fluorescence in situ hybridization analysis of paraffin-embedded tissue from Korean patients. Lung Cancer 2009, 64:66–70. 36. Gonzalez-Perez A, Perez-Llamas C, Deu-Pons J, Tamborero D, Schroeder MP, Jene-Sanz A, Santos A, Lopez-Bigas N: IntOGen-mutations identifies cancer drivers across tumor types. Nat Methods 2013, 10:1081–1082. 37. Brooks AN, Choi PS, De WL, Sharifnia T, Imielinski M, Saksena G, Pedamallu CS, Sivachenko A, Rosenberg M, Chmielecki J, Lawrence MS, DeLuca DS, Getz G, Meyerson M: A pan-cancer analysis of transcriptome changes associated with somatic mutations in U2AF1 reveals commonly altered splicing events. PLoS One 2014, 9:e87361. 15. Smid M, Hoes M, Sieuwerts AM, Sleijfer S, Zhang Y, Wang Y, Foekens JA, Martens JW: Patterns and incidence of chromosomal instability and their prognostic relevance in breast cancer subtypes. Breast Cancer Res Treat 2011, 128:23–30. 38. Huelga SC, Vu AQ, Arnold JD, Liang TY, Liu PP, Yan BY, Donohue JP, Shiue L, Hoon S, Brenner S, Ares M Jr, Yeo GW: Integrative genome-wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins. Cell Rep 2012, 1:167–178. 16. Walther A, Houlston R, Tomlinson I: Association between chromosomal instability and prognosis in colorectal cancer: A meta-analysis. Gut 2008, 57:941–950. 17. Pretorius ME, Waehre H, Abeler VM, Davidson B, Vlatkovic L, Lothe RA, Giercksky KE, Danielsen HE: Large scale genomic instability as an additive prognostic marker in early prostate cancer. Cell Oncol 2009, 31:251–259. 39. Quesada V, Conde L, Villamor N, Ordonez GR, Jares P, Bassaganyas L, Ramsay AJ, Bea S, Pinyol M, Martinez-Trillos A, Lopez-Guerra M, Colomer D, Navarro A, Baumann T, Aymerich M, Rozman M, Delgado J, Gine E, Hernandez JM, Gonzalez-Diaz M, Puente DA, Velasco G, Freije JM, Tubio JM, Royo R, Gelpi JL, Orozco M, Pisano DG, Zamora J, Vazquez M, et al: Exome sequencing identifies recurrent mutations of the splicing factor SF3B1 gene in chronic lymphocytic leukemia. References Aaltonen LA, Peltomaki P, Leach FS, Sistonen P, Pylkkanen L, Mecklin JP, Jarvinen H, Powell SM, Jen J, Hamilton SR: Clues to the pathogenesis of familial colorectal cancer. Science 1993, 260:812–816. 7. Aaltonen LA, Peltomaki P, Leach FS, Sistonen P, Pylkkanen L, Mecklin JP, Jarvinen H, Powell SM, Jen J, Hamilton SR: Clues to the pathogenesis of familial colorectal cancer. Science 1993, 260:812–816. Jarvinen H, Powell SM, Jen J, Hamilton SR: Clues to the pathogenesis of familial colorectal cancer. Science 1993, 260:812–816. ferlach C, Koeffler HP, Shih LY, Haferlach T, Chiba S, Nakauchi H, et al 8. Ionov Y, Peinado MA, Malkhosyan S, Shibata D, Perucho M: Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis. Nature 1993, 363:558–561. 31. Imielinski M, Berger AH, Hammerman PS, Hernandez B, Pugh TJ, Hodis E, Cho J, Suh J, Capelletti M, Sivachenko A, Sougnez C, Auclair D, Lawrence MS, Stojanov P, Cibulskis K, Choi K, De WL, Sharifnia T, Brooks A, Greulich H, Banerji S, Zander T, Seidel D, Leenders F, Ansen S, Ludwig C, Engel-Riedel W, Stoelben E, Wolf J, Goparju C, et al: Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing. Cell 2012, 150:1107–1120. 9. Lothe RA, Peltomaki P, Meling GI, Aaltonen LA, Nystrom-Lahti M, Pylkkanen L, Heimdal K, Andersen TI, Moller P, Rognum TO: Genomic instability in colorectal cancer: relationship to clinicopathological variables and family history. Cancer Res 1993, 53:5849–5852. 10. Lothe RA: Microsatellite instability in human solid tumors. Mol Med Today 1997, 3:61–68. 32. The Cancer Genome Atlas Network: Comprehensive molecular portraits of human breast tumours. Nature 2012, 490:61–70. 11. Peltomaki P, Lothe RA, Aaltonen LA, Pylkkanen L, Nystrom-Lahti M, Seruca R, David L, Holm R, Ryberg D, Haugen A: Microsatellite instability is associated with tumors that characterize the hereditary non-polyposis colorectal carcinoma syndrome. Cancer Res 1993, 53:5853–5855. 33. Wang ET, Sandberg R, Luo S, Khrebtukova I, Zhang L, Mayr C, Kingsmore SF, Schroth GP, Burge CB: Alternative isoform regulation in human tissue transcriptomes. Nature 2008, 456:470–476. 12. Merok MA, Ahlquist T, Røyrvik EC, Tufteland KF, Hektoen M, Sjo OH, Mala T, Svindland A, Lothe RA, Nesbakken A: Microsatellite instability has a positive prognostic impact on stage II colorectal cancer after complete resection: results from a large, consecutive Norwegian series. Ann Oncol 2012, 24:1274–1282. 34. Chen M, Manley JL: Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches. References Nat Genet 2011, 44:47–52. 18. Issa JP: CpG island methylator phenotype in cancer. Nat Rev Cancer 2004, 4:988–993. 19. Toyota M, Ahuja N, Ohe-Toyota M, Herman JG, Baylin SB, Issa JP: CpG island methylator phenotype in colorectal cancer. Proc Natl Acad Sci U S A 1999, 96:8681–8686. 20. Sveen A, Ågesen TH, Nesbakken A, Rognum TO, Lothe RA, Skotheim RI: Transcriptome instability in colorectal cancer identified by exon microarray analyses: Associations with splicing factor expression levels and patient survival. Genome Med 2011, 3:32. 21. Barash Y, Calarco JA, Gao W, Pan Q, Wang X, Shai O, Blencowe BJ, Frey BJ: Deciphering the splicing code. Nature 2010, 465:53–59. 22. Salzman J, Gawad C, Wang PL, Lacayo N, Brown PO: Circular RNAs are the predominant transcript isoform from hundreds of human genes in 20. Sveen A, Ågesen TH, Nesbakken A, Rognum TO, Lothe RA, Skotheim RI: Transcriptome instability in colorectal cancer identified by exon microarray analyses: Associations with splicing factor expression levels and patient survival. Genome Med 2011, 3:32. 40. Wang L, Lawrence MS, Wan Y, Stojanov P, Sougnez C, Stevenson K, Werner L, Sivachenko A, DeLuca DS, Zhang L, Zhang W, Vartanov AR, Fernandes SM, Goldstein NR, Folco EG, Cibulskis K, Tesar B, Sievers QL, Shefler E, Gabriel S, Hacohen N, Reed R, Meyerson M, Golub TR, Lander ES, Neuberg D, Brown JR, Getz G, Wu CJ: SF3B1 and other novel cancer genes in chronic lymphocytic leukemia. N Engl J Med 2011, 365:2497–2506. and patient survival. Genome Med 2011, 3:32. 21. Barash Y, Calarco JA, Gao W, Pan Q, Wang X, Shai O, Blencowe BJ, Frey BJ: Deciphering the splicing code. Nature 2010, 465:53–59. 21. Barash Y, Calarco JA, Gao W, Pan Q, Wang X, Shai O, Blencowe BJ, Frey BJ: Deciphering the splicing code. Nature 2010, 465:53–59. 22. Salzman J, Gawad C, Wang PL, Lacayo N, Brown PO: Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types. PLoS One 2012, 7:e30733. 41. Clark F, Thanaraj TA: Categorization and characterization of transcript- confirmed constitutively and alternatively spliced introns and exons from human. Hum Mol Genet 2002, 11:451–464. 41. Clark F, Thanaraj TA: Categorization and characterization of transcript- confirmed constitutively and alternatively spliced introns and exons from human. Hum Mol Genet 2002, 11:451–464. 23. Li H, Wang J, Mor G, Sklar J: A neoplastic gene fusion mimics trans-splicing of RNAs in normal human cells. Author details 1 1Department of Cancer Prevention, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, P.O. Box 4953 Nydalen, Oslo NO-0424, Norway. 2Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, P.O. Box 1078 Blindern, Oslo NO-0316, Norway. 3Department of Genetics, Portuguese Oncology Institute, Rua Dr. António Bernardino de Almeida, Porto 4200-072, Portugal. 4Cancer Genetics Group, Research Centre of the Portuguese Oncology Institute, Rua Dr. António Bernardino de Almeida, Porto 4200-072, Portugal. 5Institute of Biomedical Sciences Abel Salazar, University of Porto, Rua de Jorge Viterbo Ferreira n.° 228, Porto 4050-313, Portugal. For comparison with the splicing factor genes, all the 1,454 Gene Ontology gene sets (the Gene Ontology Pro- ject [56]) collected in MSigDB [57] were also included. Received: 25 April 2014 Accepted: 6 August 2014 Published: 10 August 2014 Page 12 of 13 Sveen et al. BMC Genomics 2014, 15:672 http://www.biomedcentral.com/1471-2164/15/672 Sveen et al. BMC Genomics 2014, 15:672 http://www.biomedcentral.com/1471-2164/15/672 24. Blencowe BJ: Alternative splicing: new insights from global analyses. Cell 2006, 126:37–47. References 1. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM: Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 2010, 127:2893–2917. 25. Pan Q, Shai O, Lee LJ, Frey BJ, Blencowe BJ: Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet 2008, 40:1413–1415. sequencing. Nat Genet 2008, 40:1413–1415. 2. Lengauer C, Kinzler KW, Vogelstein B: Genetic instabilities in human cancers. Nature 1998, 396:643–649. 26. ENCODE Project Consortium, Bernstein BE, Birney E, Dunham I, Green ED, Gunter C, Snyder M: An integrated encyclopedia of DNA elements in the human genome. Nature 2012, 489:57–74. 3. Hanahan D, Weinberg RA: Hallmarks of cancer: the next generation. Cell 2011, 144:646–674. 27. Venables JP: Aberrant and alternative splicing in cancer. Cancer Res 2004, 64:7647–7654. 4. Fishel R, Lescoe MK, Rao MR, Copeland NG, Jenkins NA, Garber J, Kane M, Kolodner R: The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer. Cell 1993, 75:1027–1038. Karni R, De Stanchina E, Lowe SW, Sinha R, Mu D, Krainer AR: The gen 28. Karni R, De Stanchina E, Lowe SW, Sinha R, Mu D, Krainer AR: The gene encoding the splicing factor SF2/ASF is a proto-oncogene. Nat Struct Mol Biol 2007, 14:185–193. 5. Leach FS, Nicolaides NC, Papadopoulos N, Liu B, Jen J, Parsons R, Peltomaki P, Sistonen P, Aaltonen LA, Nystrom-Lahti M: Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer. Cell 1993, 75:1215–1225. 29. Skotheim RI, Nees M: Alternative splicing in cancer: noise, functional, or systematic? Int J Biochem Cell Biol 2007, 39:1432–1449. 6. Fishel R, Kolodner RD: Identification of mismatch repair genes and their role in the development of cancer. Curr Opin Genet Dev 1995, 5:382–395. 30. Yoshida K, Sanada M, Shiraishi Y, Nowak D, Nagata Y, Yamamoto R, Sato Y, Sato-Otsubo A, Kon A, Nagasaki M, Chalkidis G, Suzuki Y, Shiosaka M, Kawahata R, Yamaguchi T, Otsu M, Obara N, Sakata-Yanagimoto M, Ishiyama K, Mori H, Nolte F, Hofmann WK, Miyawaki S, Sugano S, Haferlach C, Koeffler HP, Shih LY, Haferlach T, Chiba S, Nakauchi H, et al: Frequent pathway mutations of splicing machinery in Myelodysplasia. Nature 2011, 478:64–69. 6. Fishel R, Kolodner RD: Identification of mismatch repair genes and their role in the development of cancer. Curr Opin Genet Dev 1995, 5:382–395. 7. 41. Clark F, Thanaraj TA: Categorization and characterization of transcript- confirmed constitutively and alternatively spliced introns and exons from human. Hum Mol Genet 2002, 11:451–464. Sveen et al. BMC Genomics 2014, 15:672 http://www.biomedcentral.com/1471-2164/15/672 Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, Speed TP: Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 2003, 4:249–264. 54. Langfelder P, Horvath S: WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics 2008, 9:559. 54. Langfelder P, Horvath S: WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics 2008, 9:559. 55. Affymetrix Inc: Quality assessment of exon and gene arrays. [http://www.affymetrix. com/support/technical/whitepapers/exon_gene_arrays_qa_whitepaper.pdf] 56. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G: Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 2000, 25:25–29. 57. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, Mesirov JP: Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A 2005, 102:15545–15550. 57. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, Mesirov JP: Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A 2005, 102:15545–15550. Sveen et al. BMC Genomics 2014, 15:672 http://www.biomedcentral.com/1471-2164/15/672 Sveen et al. BMC Genomics 2014, 15:672 http://www.biomedcentral.com/1471-2164/15/672 Sveen et al. BMC Genomics 2014, 15:672 http://www.biomedcentral.com/1471-2164/15/672 42. Jass JR: Classification of colorectal cancer based on correlation of clinical, morphological and molecular features. Histopathology 2006, 50:113–130. 43. Guo X, Chen QR, Song YK, Wei JS, Khan J: Exon array analysis reveals neuroblastoma tumors have distinct alternative splicing patterns according to stage and MYCN amplification status. BMC Med Genomics 2011, 4:35. 44. Hall JS, Leong HS, Armenoult LS, Newton GE, Valentine HR, Irlam JJ, Moller-Levet C, Sikand KA, Pepper SD, Miller CJ, West CM: Exon-array profiling unlocks clinically and biologically relevant gene signatures from formalin-fixed paraffin-embedded tumour samples. Br J Cancer 2011, 104:971–981. 45. Lin E, Li L, Guan Y, Soriano R, Rivers CS, Mohan S, Pandita A, Tang J, Modrusan Z: Exon array profiling detects EML4-ALK fusion in breast, colorectal, and non-small cell lung cancers. Mol Cancer Res 2009, 7:1466–1476. 46. Paulo P, Ribeiro FR, Santos J, Mesquita D, Almeida M, Barros-Silva JD, Itkonen H, Henrique R, Jeronimo C, Sveen A, Mills IG, Skotheim RI, Lothe RA, Teixeira MR: Molecular subtyping of primary prostate cancer reveals specific and shared target genes of different ETS rearrangements. Neoplasia 2012, 14:600–611. 47. Taylor BS, Schultz N, Hieronymus H, Gopalan A, Xiao Y, Carver BS, Arora VK, Kaushik P, Cerami E, Reva B, Antipin Y, Mitsiades N, Landers T, Dolgalev I, Major JE, Wilson M, Socci ND, Lash AE, Heguy A, Eastham JA, Scher HI, Reuter VE, Scardino PT, Sander C, Sawyers CL, Gerald WL: Integrative genomic profiling of human prostate cancer. Cancer Cell 2010, 18:11–22. 48. Xi L, Feber A, Gupta V, Wu M, Bergemann AD, Landreneau RJ, Litle VR, Pennathur A, Luketich JD, Godfrey TE: Whole genome exon arrays identify differential expression of alternatively spliced, cancer-related genes in lung cancer. Nucleic Acids Res 2008, 36:6535–6547. 49. Ågesen TH, Sveen A, Merok MA, Lind GE, Nesbakken A, Skotheim RI, Lothe RA: ColoGuideEx: a robust gene classifier specific for stage II colorectal cancer prognosis. Gut 2012, 61:1560–1567. 50. Purdom E, Simpson KM, Robinson MD, Conboy JG, Lapuk AV, Speed TP: FIRMA: a method for detection of alternative splicing from exon array data. Bioinformatics 2008, 24:1707–1714. 51. Purdom E: HuEx-1_0-st-v2,coreR3,A20071112,EP.cdf. http://aroma-project.org/ data/annotationData/chipTypes/HuEx-1_0-st-v2/HuEx-1_0-st-v2,coreR3, A20071112,EP.cdf.gz. 52. R Core Team: R: A language and environment for statistical computing. http://www.R-project.org/. 52. R Core Team: R: A language and environment for statistical computing. http://www.R-project.org/. j g 53. References Science 2008, 321:1357–1361. 23. Li H, Wang J, Mor G, Sklar J: A neoplastic gene fusion mimics trans-splicing of RNAs in normal human cells. Science 2008, 321:1357–1361. Page 13 of 13 Page 13 of 13 Sveen et al. BMC Genomics 2014, 15:672 http://www.biomedcentral.com/1471-2164/15/672 Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit doi:10.1186/1471-2164-15-672 Cite this article as: Sveen et al.: Transcriptome instability as a molecular pan-cancer characteristic of carcinomas. BMC Genomics 2014 15:672. Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission

Subsets and Splits

No community queries yet

The top public SQL queries from the community will appear here once available.